Process for the preparation of 2-(4-(4-(4-(4- aminostyryl)-3-methoxystyryl)phenylsulfonyl) piperazin-1-YL)ethanol

Abstract

The present technology generally relates to improved processes for the preparation of (2-(4-(4-(4-(4-aminostyryl)-3-methoxystyryl)phenyIsulfonyl)piperazin- 1 -yljethanol (11), as well as novel intermediates employed in the process, which may be useful as a nen e visualization agent for fluorescence image-guided surgery. The present technology also relates to novel radiolabeled analogs of 11 and compositions, which may have a number of uses, including use as a nerve visualization agent. Also, methods for their synthesis are also included. The present technology further relates to methods of imaging nerves in a surgical field by contacting a surgical site of a subject with a pharmaceutical composition comprising novel radiolabeled analogs of 11 and compositions.

Description

PROCESS FOR THE PREPARATION OF 2-(4-(4-(4-(4- AMINOSTYRYL)-3-METHOXYSTYRYL)PHENYLSULFONYL) PIPERAZIN-1-YL)ETHANOLCROSS-REFERENCE TO RELEATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Appl.No. 63 / 600,211, filed November 17, 2023, the contents of which are incorporated herein by reference in their entirety for any and all purposes.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under CA008748 and CA243895 awarded by National Institutes Health. The government has certain rights in the invention.TECHNICAL FIELD

[0003] The present technology is in the field of synthetic organic chemistry and pharmaceuticals. Also disclosed herein are compositions that are useful as visualization agents for fluorescence image-guided surgery.BACKGROUND

[0004] Disclosed herein are improved processes for the preparation of (2-(4-(4-(4-(4-aminostyryl)-3-methoxystyryl)phenylsulfonyl)piperazin-l-yl)ethanol, structurally represented below as Compound 11:

[0005] Compound 11 and an alternate process of preparing Compound 11 are disclosed in US Patent Appl. No. 11 / 031,349 and US Patent No. 8,658,129 which is assigned to General Electric Company and is incorporated herein by reference in itsentirety. Compound 11 may be useful as a nerve visualization agent for fluorescence image- guided surgery.

[0006] Inadvertent nerve damage is one of the leading complications associated with many surgeries occurring in both open and minimal access surgical procedures (Gray, J. E., 2017). The ability to visualize and spare nerves during surgery is critical for avoiding chronic morbidity, pain, and loss of function. The use of agents that can specifically mark nerve tissue is one approach that may be used in a clinical setting by a surgeon to distinguish nerve fibers from the surrounding tissue (Barth, C. W. & Gibbs, S. L., 2017). A number of fluorescent agents have been developed to detect nerve tissue (Gibbs-Strauss, S. L. et al. 2011; Barth, C. W., & Gibbs, S. L., 2016; Hingorani, D. V., et al. 2018; Gonzales, J. et al. 2020; Bradbury, M., et al. 2017). 11 (also referred to as GE3126 or Illuminare-1) is a nerve-selective bis-styryl fluorophore producing fluorescence in myelinated nerves has substantially improved pharmacokinetics and reduced non-specific adipose tissue fluorescence intensity, enhancing nerve visibility compared to previous analogs such as GE3082 and GE3111 (Cotero, V. E., et al. 2015). 11 can be used in fluorescence guided nerve sparing surgery and is currently undergoing clinical evaluation. However, methods currently available for the synthesis of 11 suffer from low yields and requirements for strenuous and costly purification process including reverse phase chromatography.Therefore, there is a need for improved synthetic methods for the synthesis of 11. Described herein is an improved process for the synthesis of 11 that overcomes the deficiencies of current methods.

[0007] Also described here are novel radiolabeled analogs of 11 and compositions, which may have a number of uses, including use as a nerve visualization agent. Additionally, methods for their synthesis are described.SUMMARY OF THE PRESENT TECHNOLOGY

[0008] The present disclosure generally relates to improved processes for the preparation of (2-(4-(4-(4-(4-aminostyryl)-3-methoxystyryl)phenylsulfonyl)piperazin- 1- yl)ethanol (11), as well as novel intermediates employed in the process, which may be useful as a nerve visualization agent for fluorescence image-guided surgery.

[0009] The present disclosure also relates to novel radiolabeled analogs of 11 and compositions, which may have a number of uses, including use as a nerve visualization agent. Additionally, methods for their synthesis are included.

[0010] The present disclosure further relates to methods of imaging nerves in a surgical field by contacting a surgical site of a subject with a pharmaceutical composition comprising novel radiolabeled analogs of 11 and compositions.DETAILED DESCRIPTION

[0011] The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present technology, as defined in the appended claims. Other aspects of the present technology will be apparent to those skilled in the art in view of the detailed description of the present technology as provided herein. It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0012] Definitions

[0013] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0014] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should beconstrued as indicating any non-claimed element as essential. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0015] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term — for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” — for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0016] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), or topically. Administration includes self-administration and the administration by another.

[0017] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof — for example, “A, B, and / or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”

[0018] As used herein, the terms "cancer," "neoplasm," and "tumor," are used interchangeably and refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) may be readily distinguished from non- cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. When referring to a type of cancer thatnormally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and / or which is detectable because of the expression of one or more cancer- specific antigens in a sample obtainable from a patient.

[0019] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease or condition, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0020] As used herein, an “effective amount” of a compound of the present technology includes an amount sufficient to enable detection of binding of the compound to a target of interest including, but not limited to, one or more of non-small cell lung cancer, small cell carcinoma of the lung, bladder cancer, colon cancer, gallbladder cancer, pancreatic cancer, esophageal cancer, melanoma, liver cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer (such as castration resistant prostate cancer), a neuroendocrine tumor, a pituitary tumor, a vasoactive intestinal peptide- secreting tumor, a glioma, breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, and a metastatic cancer. Another example of an effective amount includes amounts or dosages that are capable of providing a detectable gamma ray emission from positron emission and annihilation (above background) in a subject with a tissue including one or more of non-small cell lung cancer, small cell carcinoma of the lung, bladder cancer, colon cancer, gallbladder cancer, pancreatic cancer, esophageal cancer, melanoma, liver cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer (such as castration resistant prostate cancer), a neuroendocrine tumor, a pituitary tumor, a vasoactive intestinal peptide- secreting tumor, a glioma, breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a metastatic cancer, and overexpressing PSMA, such as, for example, statistically significant emission above background. Anotherexample of an effective amount includes amounts or dosages that are capable of providing a detectable Cerenkov radiation emission due to positron emission (above background) in a subject with a tissue including one or more of non-small cell lung cancer, small cell carcinoma of the lung, bladder cancer, colon cancer, gallbladder cancer, pancreatic cancer, esophageal cancer, melanoma, liver cancer, primary gastric adenocarcinoma, primary colorectal adenocarcinoma, renal cell carcinoma, prostate cancer (such as castration resistant prostate cancer), a neuroendocrine tumor, a pituitary tumor, a vasoactive intestinal peptide- secreting tumor, a glioma, breast cancer, an adrenal cortical cancer, a cervical carcinoma, a vulvar carcinoma, an endometrial carcinoma, a primary ovarian carcinoma, a metastatic ovarian carcinoma, a metastatic cancer, and overexpressing PSMA, such as, for example, statistically significant emission above background. Those skilled in the art are readily able to determine an effective amount by simply administering a compound of the present technology to a patient in increasing amounts until, for example, statistically significant resolution (via, e.g., positron emission tomography or Cerenkov luminescence imaging) of a mammalian tissue is achieved.

[0021] As used herein, the term "metastasis" or "metastatic" refers to the ability of a cancer cell to invade surrounding tissues, to enter the circulatory system and to establish malignant growths at new sites.

[0022] "Non-metastatic" refers to tumors that do not spread beyond their original site of development and specifically do not enter the circulatory system and establish malignant growths at new sites.

[0023] As used herein, “prevention,” “prevent,” or “preventing” of a disease or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disease or condition relative to the untreated control sample. As used herein, prevention includes preventing or delaying the initiation of symptoms of the disease or condition. As used herein, prevention also includes preventing a recurrence of one or more signs or symptoms of a disease or condition.

[0024] As used herein, the term “sample” refers to clinical samples obtained from a subject. Biological samples may include tissues, cells, protein or membrane extracts of cells, mucus, sputum, bone marrow, bronchial alveolar lavage (BAL), bronchial wash(BW), and biological fluids (e.g., ascites fluid or cerebrospinal fluid (CSF)) isolated from a subject, as well as tissues, cells and fluids (blood, plasma, saliva, urine, serum etc.) present within a subject.

[0025] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to an individual organism, a vertebrate, a mammal, or a human. In certain embodiments, the individual, patient or subject is a human.

[0026] “Treating”, “treat”, or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, 7.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0027] It is also to be appreciated that the various modes of treatment or prevention of medical diseases and conditions as described are intended to mean “substantial,” which includes total but also less than total treatment or prevention, and wherein some biologically or medically relevant result is achieved. The treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.

[0028] As used herein, the term “image” is understood to mean a visual display or any data representation that may be interpreted for visual display. For example, a three- dimensional image may include a dataset of values of a given quantity that varies in three spatial dimensions. A three-dimensional image (e.g., a three-dimensional data representation) may be displayed in two-dimensions (e.g., on a two-dimensional screen, or on a two-dimensional printout). The term “image” may refer, for example, to an optical image, an x-ray image, an image generated by: positron emission tomography (PET), magnetic resonance (MR), single photon emission computed tomography (SPECT), and / or ultrasound, and any combination of these.

[0029] The present technology is also intended to include all isotopes of atoms occurring on the compounds disclosed herein. Isotopes include those atoms having the same atomic number but different mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include11C,13C and14C. Isotopes of fluorine include18F.

[0030] It will also be noted that any notation of a hydrogen in structures throughout this application, when used without further notation, are intended to represent all isotopes of hydrogen, such as2H, or3H. Furthermore, any compounds containing2H or3H may specifically have the structure of any of the compounds disclosed herein.

[0031] It will be noted that any notation of a carbon in structures throughout this application, when used without further notation, are intended to represent all isotopes of carbon, such as11C,12C,13C, or14C. Furthermore, any compounds containing11C,13C or14C may specifically have the structure of any of the compounds disclosed herein.

[0032] Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non-labeled reagents employed.

[0033] The term "radiolabel", as used herein, refers to a moiety comprising a radioactive isotope of at least one element. Exemplary suitable radiolabels include but are not limited to those described herein. In some embodiments, a radiolabel is one used in positron emission tomography (PET). In some embodiments, a radiolabel is one used in single-photon emission computed tomography (SPECT). In some embodiments, the radiolabel is radiofluorine. In some embodiments, the radiofluorine is18F. In some embodiments, radioisotopes comprise2H,3H,11C,13C,14C,13N,15N,15O,18O,18F,123I,124I, and125I.

[0034] (2-(4-(4-(4-(4-aminostyryl)-3-methoxystyryl)phenylsulfonyl)piperazin-l- yl)ethanol is known as GE3126 or Illuminare-1 and has the structure of Compound 11:

[0035] TPPTS refers to 3,3',3”-phosphinidynetris(benzenesulfonic acid) trisodium salt, also known as triphenylphosphine-3,3',3''-trisulfonic acid trisodium salt, also known as tris(3-sulfophenyl)phosphine trisodium salt.

[0036] TBDMSC1 refers to tert-Butyldimethylsilyl chloride.

[0037] BOC2O refers to di-tert-butyl pyrocarbonate, also known as di-tert-butyl dicarbonate, also known as Boc anhydride.

[0038] DIPEA refers to N,N-diisopropylethylamine, also known as Hiinig’s base.

[0039] KOtBu refers to potassium / -butoxidc.

[0040] DCM refers to dichloromethane, also known as methylene chloride.

[0041] DMF refers to dimethylformamide.

[0042] EtOAc refers to ethyl acetate.

[0043] TLC refers to thin-layer chromatography.

[0044] HPLC refers to High-Performance Liquid Chromatography.

[0045] UPLC refers to Ultra-Performance Liquid Chromatography.

[0046] LCMS refers to Liquid Chromatography combined with Mass Spectroscopy.

[0047] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. 2% As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0048] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain thedesired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g, arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0049] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0050] The compounds used in the method of the present technology may be administered in various forms, including those detailed herein. The treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds. This combination therapy can be sequential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the sameroute or by two or more different routes of administration depending on the dosage forms employed.

[0051] As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human. The carrier may be liquid or solid and is selected with the planned manner of administration in mind. Liposomes are also a pharmaceutically acceptable carrier.

[0052] The dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.

[0053] The compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions. The compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.

[0054] The compounds used in the method of the present technology can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices. The unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration. The compounds can be administered alone or mixed with a pharmaceutically acceptable carrier. This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used. The active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form. Examples of suitable solid carriers include lactose, sucrose, gelatin and agar. Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegratingagents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. Examples of suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Oral dosage forms optionally contain flavorants and coloring agents. Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0055] Techniques and compositions for making dosage forms useful in the present technology are described in the following references: 7 Modern Pharmaceutics, Chapters 9 and 10 (Banker & Rhodes, Editors, 1979); Pharmaceutical Dosage Forms: Tablets (Lieberman et al., 1981); Ansel, Introduction to Pharmaceutical Dosage Forms 2nd Edition (1976); Remington's Pharmaceutical Sciences, 17th ed. (Mack Publishing Company, Easton, Pa., 1985); Advances in Pharmaceutical Sciences (David Ganderton, Trevor Jones, Eds., 1992); Advances in Pharmaceutical Sciences Vol. 7. (David Ganderton, Trevor Jones, James McGinity, Eds., 1995); Aqueous Polymeric Coatings for Pharmaceutical Dosage Forms (Drugs and the Pharmaceutical Sciences, Series 36 (James McGinity, Ed., 1989); Pharmaceutical Particulate Carriers: Therapeutic Applications: Drugs and the Pharmaceutical Sciences, Vol 61 (Alain Rolland, Ed., 1993); Drug Delivery to the Gastrointestinal Tract (Ellis Horwood Books in the Biological Sciences. Series in Pharmaceutical Technology; J. G. Hardy, S. S. Davis, Clive G. Wilson, Eds.); Modem Pharmaceutics Drugs and the Pharmaceutical Sciences, Vol 40 (Gilbert S. Banker, Christopher T. Rhodes, Eds.). All of the aforementioned publications are incorporated by reference herein.

[0056] Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents. For instance, for oral administration in the dosage unit form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like. Suitablebinders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

[0057] The compounds used in the method of the present technology may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines. The compounds may be administered as components of tissue-targeted emulsions.

[0058] The compounds used in the method of the present technology may also be coupled to soluble polymers as targetable drug carriers or as a prodrug. Such polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamidephenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.

[0059] Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.

[0060] For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. Examples of suitable liquid dosage forms includesolutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and / or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.

[0061] Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance. In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions. Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances. Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents. Also used are citric acid and its salts and sodium EDTA. In addition, parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propylparaben, and chlorobutanol. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.

[0062] The compounds used in the method of the present technology may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.

[0063] Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.

[0064] Each embodiment disclosed herein is contemplated as being applicable to each of the other disclosed embodiments. Thus, all combinations of the various elements described herein are within the scope of the present technology.

[0065] Throughout this disclosure, various publications, patents, and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of whichare provided immediately preceding the claims. The disclosures of these publications, patents, and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which the present technology pertains.

[0066] The Present Technology

[0067] Illuminare-1 is a myelin-binding fluorophore which has optimal characteristics that is currently being investigated for clinical use. A first synthesis and purification was described previously in US Patent Appl. No. 11 / 031,349, US Patent No.8,658,129 and in Cotero, V. E., et al. 2015. The present disclosure provides a synthesis and purification process which provides high quality Illuminare-1 in a reproducible as well as more economical manner. Illuminare-1 (Compound 11), which was previously described as GE 3126, with the chemical name: 2-(4-(4-(4-(4-Aminostyryl)-3-methoxystyryl) phenylsulfonyl)piperazin-l-yl)ethanol hydrochloride has the following structure:Illuminare-1 (Compound 11)

[0068] Sufficient quantities of Illuminare- 1 were required to facilitate development efforts. We found that the earlier reported GE process was encumbered with deficiencies that hindered its use for large scale production of Illuminare-1 (Scheme 1).Scheme 1. Already Reported synthesis route to produce Illuminare-lfree base

[0069] In particular, large-scale syntheses using bromomethylbenzenesulfonyl chloride 7 have proven challenging. Therefore, an alternative and preferable process was implemented that first improves the synthesis process that facilitates scale up. Second, the new synthesis process brings more characterization means to access the free base. And thirdly the purification process was changed from high-performance liquid chromatography (HPLC) to normal silica gel chromatography under conditions that lead to high quality free base. A solid alternative route for the synthesis of Illuminare-1 was achieved (Scheme 2).Scheme 2. Novel synthetic route for the synthesis of Illuminare-1 free base

[0070] A general scheme for the synthesis of Illuminare- 1 free base 11 is outlined in Scheme 2. The synthesis protocol was optimized for scale up and reproducibility. The process is convergent and brings two advanced intermediates together through a Horner- Wittig reaction between aldehyde 4 and phosphonate 9 to provide fully protected Compound 10 as a single isomer. The hydrophobic protecting groups in 10 offer a wonderful opportunity for thorough purification. Removal of both protecting groups under mild acidic conditions in a single step, followed by a basic work up and normal phase silica gel chromatography led to high quality free base 11.

[0071] The synthesis was started from 4-vinylaniline 1 which was protected as its corresponding N-Boc aniline 2 with BOC2O. This set the stage for a Heck coupling between N-Boc-4-vinylaniline 2 and bromobenzaldehyde 3 under palladium catalysis using the water-soluble ligand 3,3',3”-phosphinidynetris(benzenesulfonic acid) trisodium salt (TPPTS) at 90°C. Aldehyde 4 was obtained in 65% yield after purification. Phosphonate 9 was obtained via two different routes. The original route was based on intermediate 8 (Scheme 1). In a preferred process (Scheme 2), key Compound 9 was synthesized through chloro sulfonation of benzyl phosphonate 12 under solvent- free conditions which provided the desired para isomer 13 in a 9:1 mixture with the undesired ortho isomer. Fortunately, a simple crystallization from hexane-benzene provided the desired pure para-isomer 13. Meanwhile, TBS-protected hydroxyethyl piperazine 6 was synthesized using an improved process in refluxing dichloromethane. Chloro sulfonate 13 was then reacted with piperazine 6 to provide phosphonate 9 in excellent yield and purity. Condensation of aldehyde 4 with phosphonate 9 in the presence of potassium tert-butoxide gave the desired Compound 10 as a single isomer. Mild acid hydrolysis and mild basic work up provided Illuminare- 1 free base 11 in good yield.

[0072] Thus, in an aspect, the present disclosure provides a process for the preparation of Compound 11 where the process includes the steps of a) reacting Compound 4 of the formulawith Compound 9 of the formulain the presence of a base in a solvent, to afford Compound 10 of the formulab) reacting Compound 10 with an acid in a solvent followed by reaction with a base, to afford Compound 11.

[0073] In any embodiment herein, the base may be KOtBu. In embodiments where the base is KOtBu, the solvent may be THF. In any embodiment herein, the acid may be HC1. In embodiments where the acid is HC1, the solvent may be dioxane. In any embodiment herein, the base may be Na2CO3, NaHCO3, or both Na2CO3and NaHCO3. Inany embodiment herein of the process, Compound 11 may be afforded in greater than 97.0% trans configuration, greater than 97.5% trans configuration, greater than 98.0% trans configuration, greater than 98.5% trans configuration, greater than 99.0% trans configuration, greater than 99.5% trans configuration, and / or greater than 99.9% trans configuration. In any embodiment herein of the process, Compound 10 may be afforded in greater than 97.0% trans configuration, greater than 97.5% trans configuration, greater than 98.0% trans configuration, greater than 98.5% trans configuration, greater than 99.0% trans configuration, greater than 99.5% trans configuration, and / or greater than 99.9% trans configuration.

[0074] The earlier reported GE synthesis is shown in Scheme 1 (US Patent Appl. No. 11 / 031,349; US Patent No. 8,658,129). Key intermediate (E)-tert-butyl 4-(4-formyl-2- methoxystyryl)phenyl carbamate 4 was prepared by Heck coupling of tert-butyl 4- vinylphenylcarbamate 2 and 4-bromo-3-methoxybenzaldehyde 3 with palladium acetate (Pd(OAc)2), 3,3',3”-phosphinidynetris(benzenesulfonic acid) trisodium salt (TPPTS) and potassium carbonate in water / DMF at 95 °C for 3 h to give aldehyde 4 following silica gel chromatography in 70% yield as a yellow solid. However, condensation of 4- bromomethylbenzenesulfonyl chloride 7 with tert-butyldi methyl silyl l-(2- hydroxyethyl)piperazine 6 resulted in intermediate Compound 8 as a 9:1 mixture of chloride and bromide respectively in 51% yield after silica gel chromatography. The yield of isolated key intermediate 9 from the Michaelis Arbuzov reaction of Compound 8 with triethyl phosphite has proven challenging with tendency for lower yields and elaborate purifications with increasing scales. In further improvement to the process, Compound 10 was obtained from the Horner-Wittig olefination between phosphonate 9 and aldehyde 4 following silica gel chromatography in good yield. Final compound Illuminare-1, 11 was then obtained through deprotection of Compound 10 with 4.0 N HC1 in dioxane at room temperature, followed by basic work up and purification as a yellow solid in high yield and quality.

[0075] Aldehyde 4 and phosphonate 9 can be considered the two key intermediates used in the convergent synthesis of Illuminare-1 11 that are essential in the Homer-Wittig olefination to form Compound 10.

[0076] The synthesis of aldehyde 4 was accomplished (Scheme 2) with no significant changes to the original GE procedure (Scheme 1). However, it is important to note that the synthesis of Compound 4 resulted in the formation of 2-3% of the undesiredcis compound. It was found that lengthy multiple purifications could be avoided by thorough washing and filtration steps to remove the cis compound by extractive partitioning. Failure to remove this undesired cis impurity at this stage can result in carrying this impurity forward where it is more difficult to remove and more likely to be present in the final product. It is particularly important that the Illuminare-1 (11) obtained be of high purity and consist essentially exclusively all trans compound since its utility as a nerve visualization agent for fluorescence image-guided surgery will be adversely affected by the presence of any contaminant that may interfere and / or quench the excitation / emission of Illuminare-1 (11). In embodiments of the present technology, a new process of separation of otherwise difficult isolation of the pure trans double bond from mixture containing trace undesired cis product of the Heck coupling through differential solvent partitioning which is appreciated by the expert in the field as a much simpler method of separation than any chromatographic means.

[0077] Therefore, the importance of removing the cis compound at this stage cannot be stressed enough. The process described herein provides methods for the effective removal of the undesired cis compound that is formed in this step of the process. The presence of any cis isomer will also result in a decrease of overall fluorescence during nerve visualization.

[0078] In an aspect, the present disclosure also provides a process for the preparation of Compound 4 where the process includes the steps of a) reacting Compound 1 of the formulawith BOC2O in water to afford Compound 2 of the formulab) reacting Compound 2 with Compound 3 of the formulain presence of Pd(OAc)2, TPPTS and a base in a solvent to afford Compound 4. In any embodiment herein of the process, the base may be K2CO3. In any embodiment herein of the process, the solvent may include water, DMF, or a mixture thereof. In any embodiment herein of the process, the base may be K2CO3, and the solvent may be a mixture of DMF and water.

[0079] In contrast to the synthesis of Compound 4, many issues in the synthesis of Compound 9 were encountered, and as a result, significant effort was devoted towards improving the process. In the GE process (Scheme 1) 9 was obtained in three steps starting from reaction of 5 with tert-Butyldimethylsilyl chloride (TBDMSC1) to give O-protected Compound 6, followed by the one pot two-step reaction between piperazine 6 and 4- bromomethylbenzenesulfonyl chloride 7 to give 8 which was then treated with triethyl phosphite at 100° C for 78 h in a Michaelis Arbuzov reaction to provide key intermediate 9.

[0080] To improve the synthesis of Compound 9 (Scheme 1) amino alcohol 5 is first reacted with TBDMSC1 in order to obtain O-protected Compound 6 which was subsequently isolated in 88-99% yield. Compound 6 was then reacted with Compound 7 to give 8 which was identified as a 9:1 mixture of Cl:Br compound respectively, in near quantitative yield. This mixture 8 was not separated out further to pure Cl- or Br-analog because both are suitable for the subsequent Michaelis Arbuzov reaction to form 9. In fact, when this reaction was performed, we obtained 9 in 53 % yield after several purifications. However, this reaction showed limitations and tedious chromatographic complexity with increasing reaction scales.

[0081] To further improve the synthesis of 9 (Scheme 2) an alternative procedure was developed. In a preferred process, key Compound 9 is synthesized through chloro sulfonation of benzyl phosphonate 12 under solvent- free conditions which provided the desired para isomer 13 in a 9:1 mixture with the ortho isomer. Crystallization from hexane-benzene provided pure 13 as white needles. Meanwhile, TBDMS-protected hydroxyethyl piperazine 6 was synthesized in an improved process in refluxingdichloromethane. Chloro sulfonate 13 was then reacted with piperazine 6 to provide phosphonate 9 in excellent yield and purity.

[0082] Thus, in an aspect, the present disclosure also provides process for the preparation of Compound 9 where the process includes the steps of a) reacting Compound 12 of the formulawith CISO3H to afford Compound 13 of the formulathen b) reacting Compound 13 with Compound 6 of the formulain the presence of a base in a solvent, to afford Compound 9.In any embodiment herein of the process, the base may be DIPEA. In any embodiment herein of the process, the solvent may be DCM. In any embodiment herein of the process, the base may be DIPEA and the solvent may be DCM.

[0083] In another aspect, a process for the preparation of Compound 6 of the formulais provided where the process includes the step of reacting Compound 5 of the formulawith TBDMS-C1 in DCM at reflux.

[0084] In other embodiments of the present technology, further developments targeted the purification process. A process of chromatographic purification of the all-trans bis-(phenylvinyl)benzene moiety that avoids reverse phase chromatography by using normal phase silica gel. As is clear to the expert, the chromatography is run in the presence of varying percentages of triethylamine in the elution gradients. This not only keeps a constant protective environment but also helps in preventing band broadening of the product during the chromatographic purification process (z.e., sharpening the peaks for easier purification).

[0085] In another development, a process was introduced that secures a preparation of the free base 11 devoid of any acid which typically adds color to the final product.

[0086] The use of fluorescent nerve visualization agents during surgery can be beneficial for surgeons and patients alike. These agents help physicians visualize nerves, which can be challenging to identify during medical interventions. The all-trans isomer of a fluorescent nerve visualization agent may offer improved stability and fluorescence properties compared to other isomers which due to their configuration may not bind or bind off-target. The improved stability and fluorescence properties of the all-trans isomer can lead to better visualization and detection of nerves during surgical procedures. Therefore, it would be better to have an all-trans isomer as a fluorescent nerve visualization agent, as it may provide enhanced stability and fluorescence properties, potentially improving the standard of care in nerve visualization during surgery.

[0087] Illuminate- 1 (11) has two double bonds and the structure shown above represents the orientation of both double bonds as trans (i.e. all trans). Conceivably, the two double bonds could be trans, trans or trans, cis or cis, trans or cis, cis. The structure of Illuminaire-1 (11) refers only to the trans, trans-isomer as shown above. As such the present disclosure provides a process for the synthesis of the trans, trans-isomer Illuminare-1 (11) in exceptionally high purity essentially free from the remaining three geometric isomers. The high-quality material provided through this process ensures maximal performance as a nerve visualization agent for fluorescence image-guided surgery since it is devoid of closely related compounds (i.e., the three geometric isomers) or other contaminants that may adversely affect fluorescence properties through interference and / or quenching of the excitation / emission of Illuminare-1 (11).

[0088] Fluorescent molecules such as Illuminare-1 absorb light at one wavelength and emit it at another. The difference between the excitation and emission maxima for a given fluorophore is called the Stokes shift. Compounds with larger Stokes shifts are preferable to use as nerve visualization agents for fluorescence image-guided surgery because compounds with smaller Stokes shifts are more difficult to detect due to difficulties in distinguishing the emitted light from the compound (i.e., the signal) from the light used for excitation. Moreover, compounds with smaller Stokes shifts have more problems associated with background fluorescence that would negatively impact their use as nerve visualization agents for fluorescence image-guided surgery. Similarly, the presence of impurities would also adversely affect optical properties since these would also contribute to background fluorescence.

[0089] For these reasons it is imperative that the purity of Illuminare-1 (11) be very high in order to be useful as a nerve visualization agent for fluorescence image-guided surgery. The present technology solves this problem and provides a process for the synthesis of Illuminare-1 (11) in exceptionally high purity.

[0090] The present disclosure also describes novel radiolabeled analogs of 11 and compositions, which may have a number of uses, including use as a nerve visualization agent. Also, methods for their synthesis are also described below. Thus, in an aspect, the present disclosure provides compound having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is not

[0091] In any embodiment herein, the compound may have the structure:pharmaceutically acceptable salt thereof. In any embodiment herein, the compound may have the structure the structure:pharmaceutically acceptable salt thereof. In any embodiment herein, the compound may have the structure:pharmaceutically acceptable salt thereof. In any embodiment herein, the compound may have the structure:pharmaceutically acceptable salt thereof. In any embodiment herein, the compound may have the structure:pharmaceutically acceptable salt thereof. The present disclosure also provides a pharmaceutical composition that includes a compound of any embodiment disclosed herein as well as a pharmaceutically acceptable carrier.

[0092] Methods of imaging nerves in a surgical field are also provided. Suitable methods comprise contacting a surgical site of a subject with a pharmaceutical composition comprising one or more compounds having the structure:whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; wherein the compound is notpharmaceutically acceptable salt thereof, and detecting the presence of the compound or the pharmaceutically acceptable salt thereof.

[0093] The method of imaging nerves in a surgical field comprises detection of any one or more of the compounds, or the pharmaceutically acceptable salt thereof, as provided herein. In some embodiments, these methods preferably include applying a light source on the surgical site of the subject, wherein the light source is tuned to the spectral excitation characteristics of the compound, and observing the surgical site of the subject through an optical filter tuned to the spectral emission characteristics of the compound. In one or more embodiments, the detecting can be effected by gamma imaging, fluorescence microscopy,laser-confocal microscopy, cross-polarization microscopy, autoradiography, magnetic resonance imaging, magnetic resonance spectroscopy, or any combination thereof.

[0094] For example, in an aspect the present technology provides a method of imaging nerves in a subject, where the method includes:(i) administering to the subject an effective amount of a pharmaceutical composition comprising one or more compounds having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is not(ii) detecting a signal that is associated with the compound or the pharmaceutically acceptable salt thereof.

[0095] In any embodiment of the method herein, the compound may includepharmaceutically acceptable salt thereof. In any embodiment of the method herein, the compound may includepharmaceutically acceptable salt thereof. In any embodiment of the method herein, the compound may includepharmaceutically acceptable salt thereof. In any embodiment of the method herein, the compound may includepharmaceutically acceptable salt thereof. In any embodiment of the method herein, the compound may includepharmaceutically acceptable salt thereof.

[0096] In this regard it should also be appreciated that there exists a number of methodologies for the non-invasive and remote detection of one or more compounds as provided herein, or a portion thereof, or their pharmaceutically acceptable salts, on or within the biological tissue of the surgical field. For example, the methods provided herein canincorporate the use of any one or more of non-invasive spectroscopic and imaging techniques to determine the presence and quantity of such compounds. For example, fluorescence spectroscopy can be employed for compounds that are intrinsically fluorescent or have been conjugated to a fluorescent probe. Raman spectroscopy offers a label-free alternative, exploiting the unique vibrational signatures of compounds. Near- Infrared Spectroscopy (NIRS) leverages the near-infrared absorption spectra of compounds to analyze their presence within tissues. Positron Emission Tomography (PET) uses compounds labeled with positron-emitting isotopes to monitor and image their distribution within living tissues. Accordingly, these techniques, and equivalents thereof, can be advantageously implemented for the non-invasive and remote detection of the one or more compounds as provided herein, including their pharmaceutically acceptable salts, on or within the biological tissue of the surgical field.EXAMPLES

[0097] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology over the old one and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology. The examples should not be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further include or incorporate the variations of any or all other variations, aspects, or embodiments of the present technology. The following Examples demonstrate the improved process for the preparation of (2-(4-(4-(4-(4-aminostyryl)-3-methoxystyryl) phenylsulfonyl)piperazin- l-yl)ethanol 11, as well as novel intermediates employed in the process, which may be useful as a nerve visualization agent for fluorescence image-guided surgery. As is clear to a person of ordinary skill in the art, the structure of Compound 11 provides many opportunities for labeling with cold labels (2H,13C,15N,18O, etc.) or radiolabels (11C,14C,18F, etc.) for different applications. The following Examples also demonstrate novel radiolabeled analogs of 11 and methods for their synthesis.

[0098] Experimental Details

[0099] General Considerations. Reagents and solvents were obtained at the highest purities from commercial sources and were used without further purification unless otherwise stated. Reactions were monitored by thin layer chromatography (TLC) using solvent mixtures appropriate to each purification. Column chromatography was performed on silica gel (40 - 63 pm) or reverse phase Cl 8 silica gel (48 - 65 pm). For all compound purifications by HPLC, 0.1% by volume Trifluoroacetic Acid (TFA) was often added in the mobile phase to provide better resolution and better ionization in mass spectrometry detection when using C18-RP silica gel chromatography. Nuclear magnetic resonance spectra were recorded on Bruker Avance 500 or Avance III 600 MHz instruments, as indicated. Chemical shifts are reported as 5 values in ppm referenced to CDCl3 (1H NMR = 7.24 and13C NMR = 77.00) or methanol-3.31 and13C NMR = 49.15) and tetramethyl silane (TMS) as 0.0 ppm. Multiplicity is indicated as follows: s (singlet); d (doublet); t (triplet); dd (doublet of doublets); td (triplet of doublets); dt (doublet of triplets); dq (doublet of quartets); ddd (doublet of doublet of doublets); ddt (doublet of doublet of triplets); m (multiplet); br (broad). Analytical liquid chromatography combined with mass spectrometry (LCMS) experiments were run on a Waters ACQUIT Y Premier SQD2 with concomitant detection by mass spectrometry, diode array and evaporative light scattering (ELS). Alternatively, a Waters Autopure instrument with integrated liquid handling, and 3- way detection by mass spectrometry, diode array and ELS was also used. High-resolution mass spectra were recorded in positive / negative mode on a Waters LCT-Premier XT.

[0100] General Description of the Synthesis Process:

[0101] A general scheme for the synthesis of Illuminare- 1 free base is outlined in Scheme 2. The synthesis protocol was optimized for scale up as well as reproducibility. The process is convergent and brings two advanced intermediates together through a Horner- Wittig reaction between aldehyde 4 and phosphonate 9 to provide fully protected Compound 10 as a single isomer. The hydrophobic protecting groups in 10 offer a great opportunity for thorough purification. Removal of both protecting groups under mild acidic conditions in a single step, followed by a basic work up led to high quality free base 11.

[0102] The synthesis was started from 4-vinylaniline 1 which was protected as its corresponding N-Boc aniline 2 with BOC2O. This set the stage for a Heck coupling betweenN-Boc vinylaniline 2 and bromobenzaldehyde 3 under palladium catalysis using the water- soluble ligand TPPTS at 90°C. Aldehyde 4 was obtained in 65% yield after purification. Phosphonate 9 was obtained via two different routes. The first route was based on intermediate 8 (Method 1; Scheme 1). In a preferred process (Method 2; Scheme 2), key Compound 9 was synthesized through chloro sulfonation of benzyl phosphonate 12 under solvent-free conditions which provided the desired para isomer 13 in a 9:1 mixture with the ortho isomer. Crystallization from hexane-benzene provided pure 13. Meanwhile, TBS- protected hydroxyethyl piperazine 6 was synthesized in an improved process from reaction of 5 with TBDMSC1 in refluxing dichloromethane. Chloro sulfonate 13 was then reacted with piperazine 6 to provide phosphonate 9 in excellent yield and purity. Condensation of aldehyde 4 with phosphonate 9 in the presence of potassium tert-butoxide gave the desired Compound 10 as a single isomer following purification. Mild acid hydrolysis followed by mild basic work up provided Illuminare-1 (Compound 11) as a free base in good yield.

[0103] Example 1. Synthesis of f-Butyl-4-vinylphenylcarbamate (2)1 2

[0104] An appropriately sized flask equipped with a magnetic stirrer and a water bath was charged with 4- vinylaniline (1.0 eq., approximately IM) in water. Di-tert-butyl pyrocarbonate (BOC2O, 1.1 eq., approximately 1.1M) solid was added to the reaction mixture. The reaction mixture was heated at 35°C (water bath) for 4 h or until reaction was complete. The desired product precipitated out, which was filtered over a Buchner funnel. The solid was washed with water (amount eq. to original volume of water in reaction mixture). The precipitate was dissolved in minimum amount of dichloromethane. The DCM-insoluble solids were removed by filtration and washed with minimum amount of DCM. The combined filtrates were concentrated under reduced pressure at 20°C bath temperature to obtain an off-white solid. The solid was further washed with hexanes and further dried under high vacuum for 5 h to give the product 2 (95% yield), as an off-white solid.JH NMR (600 MHz, CDCh) 5 1.52 (s, 9H), 5.16 (dd, J = 10.9, 0.7 Hz, 1H), 5.65 (dd, J = 17.6, 0.7 Hz, 1H), 6.47 (bs, 1H), 6.65 (dd, J = 17.6, 10.9 Hz, 1H), 7.28-7.36 (m, 4H).11C NMR (125 MHz, CDCh) 8 28.4, 80.6, 112.4, 118.4, 126.9, 112.5, 116.2, 117.9, 152.6.MS (m / z): [M+H]+: 220.1.

[0105] Example 2. Synthesis of aldehyde 4

[0106] In a round bottom flask equipped with a magnetic stirrer and oil bath was charged with 4-bromo-3-methoxybenzaldehyde 3 in 1:1 DMF / water (1 eq., 0.65M). t- Butylvinylphenylcarbamate 2 (1.2 eq.) was added to the reaction mixture, followed by triphenylphosphine- 3, 3 ',3 ''-trisulfonic acid trisodium salt (0.12 eq.), palladium acetate (0.062 eq.) and potassium carbonate (3.0 eq.). The flask was heated at 95°C with an oil bath and stirring was continued at 95-100 °C for 3 h. Upon completion, the reaction mixture was diluted with EtOAc / water (3:1) and transferred to a separatory funnel and decanted. After decantation, the aqueous layer was extracted three times with EtOAc. The combined ethyl acetate extracts were dried over Na2SO4, filtered over a packed bed of celite, and the filtrate was evaporated under vacuum. The resulting solid was washed with 5:1 hexane:EtOAc, then it was dissolved in chloroform (5 mL / mmol of aldehyde) and filtered over a Buchner funnel to remove traces of insoluble material. The filtrate was dried over sodium sulfate, filtered over Celite pad and evaporated under reduced pressure to give a white solid. The solid was washed with ethyl ether and further dried under high vacuum overnight to give Compound 4 (65% yield) as a yellow solid.!H NMR (600 MHz, CDCh) 8 1.53 (s, 9H), 3.94 (s, 3H), 6.53 (bs, 1H), 7.21 (d, J = 16.4 Hz, 1H), 7.35-7.51 (m, 7H), 7.73 (d, J= 7.8 Hz, 1H), 9.95 (s, 1H).11C NMR (150 MHz, CDCh) 8 28.3, 55.7, 80.8, 109.3, 118.5, 120.9, 124.4, 126.2, 127.7, 111.9, 112.1, 113.3, 116.1, 118.4, 152.5, 157.1, 191.6. MS (m / z): [M+H]+: 354.3.

[0107] NOTE: During this step, there is formation of about 2-3% of the undesired cis isomer. To prevent carrying this undesired cis isomer forward and to avoid an otherwise cumbersome purification, it is important to follow the washing, dissolution, filtration stepsin the order indicated to be able to remove the cis isomer by extractive partitioning with binary solvent mixtures.

[0108] Example 3. Synthesis of l-(2-((tert-butyldimethylsilyl)oxy)ethyl) piperazine (6)5 6

[0109] Under argon atmosphere, a round bottom flask equipped with a mechanical stirrer and an oil bath was charged with 2-(piperazin-l-yl)ethan-l-ol 5 and anhydrous dichloromethane (molar solution). tert-Butyldimethylsilyl chloride (1.2 eq.) was added portion-wise over a period of 10 minutes. A milky precipitate appeared immediately. The resulting slurry was heated to a gentle reflux (oil bath temperature ~45°C) for 28 hours or until the solid disappeared to give a yellowish solution. The reaction mixture was monitored with TLC (SiCL), using 20% methanol in dichloro methane containing 1% triethyl amine as the eluent to ensure the piperazine starting material was completely consumed. The reaction mixture was quenched with aqueous saturated sodium carbonate (0.3 mL / mmol piperazine), then water (0.5 mL / mmol piperazine) was added to dissolve the formed solid. After decantation the aqueous layer was extracted twice with dichloromethane. The pH of the aqueous layer was monitored using pH paper and kept between pH 8-10. The combined dichloromethane extracts were washed with brine, dried briefly over sodium sulfate, and filtered over a packed bed of Celite. The volatiles were evaporated in vacuo to obtain a light-yellow oil.

[0110] Purification: The crude product was dissolved in minimum DCM and the resulting solution was applied to a sample loading cartridge, then purified by column chromatography using a silica RediSep® ISCO column.Channel A: dichloromethaneChannel B: 20% methanol in dichloro methane containing 1% triethylamineGradient elution system used: 0 to 2.5% channel B over 8 min, then up to 100% B over 25 minutes. Elution was continued until no more product observed in the eluent with TLC and UPLC.

[0111] The appropriate pure fractions were combined into a pre- weighed recovery flask and the solvents evaporated in vacuo. The product was placed under high vacuum overnight to give the desired product, 6 (66-99 % yield) as a colorless oil.!H NMR (600 MHz, CDCh) 6 0.06 (s, 6H), 0.89 (s, 9H), 1.81 (bs, 1H), 2.42-2.63 (m, 6H), 2.85-2.96 (m, 4H), 3.75 (t, J = 6.5 Hz, 2H). MS (m / z): [M+H]+: 245.2.

[0112] NOTE: Running the reaction at room temperature and using a 1:1 ratio of the starting materials, as suggested in the reference above, resulted in a lower yield of 39% of the desired product 6.

[0113] Synthesis of Diethyl (4-((4-(2-((tert- butyldimethylsilyl)oxy)ethyl)piperazin-l-yl)-sulfonyl)benzyl)phosphonate (9)

[0114] Compound 9 can be synthesized by two different routes. Method 1 was previously described (US Patent Appl. No. 11 / 031,349, US Patent No. 8,658,129 and in Cotero, V. E., et al. 2015). Method 2 (new route) is outlined below.

[0115] Scheme 3: New route to synthesize phosphonate 9 (Method 2)

[0116] Example 4. Synthesis of diethyl (4-(chlorosulfonyl)benzyl)phosphonate(13)

[0117] An appropriately sized round bottom flask equipped with a magnetic stir bar, a water condenser, a pressure equalizing addition funnel and a thermocouple, under a positive pressure of argon was charged with neat chloro sulfonic acid (11.5 eq.). This was cooled to 0-4°C before diethyl benzylphosphonate (1.0 eq., neat) was loaded into the addition funnel and added in a dropwise fashion such as to maintain an internal temperature below 15°C throughout the addition. Once addition was completed, the reaction mixture was allowed to gradually rise to room temperature and stirring was maintained while monitoring the disappearance of starting phosphonate by LCMS. Following reaction completion, the reaction mixture was carefully transferred to a large Erlenmeyer flask containing crushed ice in portion- wise fashion. The mixture was extracted with chloroform three times. The chloroform layers were combined and washed briefly and consecutively twice with both aqueous saturated sodium bicarbonate then brine. The organic layer was dried briefly over sodium sulfate under vigorous stirring, filtered over a bed of packed Celite and the filtrate was evaporated under reduced pressure to give crude 13.

[0118] Recrystallization: The residue was transferred to an appropriately sized round bottom flask equipped with a magnetic stirrer and a water condenser under argon, covered with hexanes at approximately 15 mL / gram of crude, and heated up to 60°C. Benzene was added at this temperature at approximately 4 mL / gram of initial crude. The originally cloudy reaction mixture becomes clear. The oil bath was removed, and the mixture cooled gradually to 0-4°C. This process resulted in the preferred crystallization of the para isomer, which was collected by filtration over Buchner funnel and the solid was washed with cold hexanes. The crystals are transferred to a pre-weighed recovery flask and dried under a vacuum to give 13 (40 % yield first crop). Depending on weight of first crop, additional crystallizations may be needed, in which case the recrystallization step is repeated as above. >H NMR (600 MHz, CDCh) 5 1.28 (t, J= 7.0 Hz, 6H), 3.26 (d, J = 22.4 Hz, 2H), 4.03-4.11 (m, 4H), 7.55 (dd, J = 8.3, 2.3 Hz, 2H), 7.99 (d, J = 8.3 Hz, 2H).13C NMR (150 MHz, CDCh) 5 16.4 (d, J = 5.9 Hz), 34.1 (d, J = 137 Hz), 62.5 (d, J = 6.7 Hz), 127.2 (d, J= 2.9 Hz), 131.0 (d, J= 6.3 Hz), 140.7 (d, J= 9.0 Hz), 142.8 (d, J= 3.8 Hz).31P NMR (202 MHz, CDCh) 823.8 (m). MS (m / z): [M+H]+: 327.1.

[0119] Example 5. Synthesis of Phosphonate 9

[0120] An appropriately sized round bottom flask equipped with a magnetic stir bar and a pressure equalizing addition funnel under a positive pressure of argon was charged with a solution of piperazine 6 in anhydrous DCM (1.0 eq., 2 M concentration approximately) followed by DIPEA (2.0 eq.). Chlorosulfonyl phosphonate 13 was dissolved under argon in DCM (approx. 2 M) and the resulting chlorosulfonate solution was loaded onto the addition funnel and added in a dropwise fashion such as to maintain an internal temperature below 15°C throughout the addition. An ice-water bath was used if needed. Reaction evolution was monitored at room temperature, and more piperazine 6 was added if necessary. Upon completion, the reaction was cooled to 0-4°C with an ice-water bath and quenched with excess methanol. The cold bath was removed when temperature stabilized within 10-20 minutes and the reaction mixture was concentrated under reduced pressure to give crude 9.

[0121] Purification: The residue was dissolved in minimum DCM and loaded onto sample loading SiO2 cartridge and the column was preconditioned in 10% EtOAc in Hexanes. A gradient system of EtOAc-Hexanes 10 to 90% was used before switching to 1- 10% MeOH in CH2Q2. Following TLC assessment, the appropriate pure fractions were pooled into a pre-weighed recovery flask and evaporated under vacuum to give 9 as a colorless to slightly yellow oil (90% yield).1H NMR (600 MHz, CDCl3) δ 0.02 (s, 6H), 0.86 (s, 9H), 1.26 (t, J = 7.1 Hz, 6H), 2.51 (t, J= 6.1 Hz, 2H), 2.58-2.65 (m, 4H), 2.97-3.10 (m, 4H), 3.20 (d, J = 22.2 Hz, 2H), 3.68 (t, J = 6.1 Hz, 2H), 3.99-4.10 (m, 4H), 7.44-7.50 (m, 2H), 7.70 (d, J = 8.0 Hz, 2H).11C NMR (150 MHz, CDCh) 8 -5.4, 16.4 (d, J = 5.9 Hz), 18.2, 25.9, 33.9 (d, J= 117.2 Hz), 46.0, 52.8, 60.0, 61.4, 62.3 (d, J = 6.7 Hz), 128.0 (d, J = 2.7 Hz), 110.4 (d, J= 6.4 Hz), 114.1 (d, J= 3.5 Hz), 117.5 (d, J = 8.9 Hz). MS (m / z): [M+H]+: 535.3.

[0122] Example 6. Synthesis of Compound 10

[0123] An appropriately sized round bottom flask equipped with a magnetic stirrer under positive argon atmosphere was charged with potassium Z-butoxide (1.66 eq.) and anhydrous THF (enough for a 0.4 M, solution or about 2.50 mL / mmol of KOtBu). This was cooled down to 0-5°C using ice-water bath. The resulting mixture was stirred at 0-5°C for 30 minutes before the ice bath was removed and the mixture stirred at room temperature for an additional 30 minutes to obtain a clear solution. Separately, another round bottom three- neck flask equipped with a mechanical stirrer, a reflux condenser and an appropriately sized pressure equalizing addition funnel, was flushed with a positive flow of argon, and charged with phosphonate 9 (1.11 eq.) in dry THF. The solution of potassium Z-butoxide was transferred to the addition funnel via positive pressure and transfer needle. The temperature of the reaction mixture was brought down to about 0-5°C with an ice-water bath before potassium Z-butoxide solution was added in a dropwise fashion, such that internal temperature did not exceed 10°C. The addition funnel was then quickly replaced with a solid addition funnel that was already loaded with aldehyde 4. Aldehyde 4 (1.00 eq.), in solid form was added to the reaction mixture via the solid addition funnel over a period of 15 minutes. The residue in solid addition funnel was washed with minimum dry THF into the reaction mixture. Upon completion of the aldehyde addition, the reaction mixture was stirred at room temperature for 1.5 h or until reaction completion. The reaction mixture was concentrated to a third of its original volume under reduced pressure. It was then partitioned between sat. aqueous Na2CO3(1 mL / mmol) and of ethyl acetate (2 mL / mmol), decanted,and the aqueous layer was extracted twice with ethyl acetate or until TLC indicated no product remained in aqueous phase. The combined ethyl acetate extracts were dried briefly over Na2SO4, filtered over a pad of Celite and the filtrate evaporated in vacuo to obtain crude 10 as a yellow orange solid.

[0124] Purification: The crude product was dissolved in minimum DCM and the resulting solution loaded to an appropriate size silica gel column which was pre-conditioned as a slurry in hexanes: DCM 1:1, containing 1% HPLC grade triethylamine.The gradient elution system used is as follows:• 4 L, 1:1 Hexanes: DCM• 8 L, 1: 1:0.1 Hexanes: DCM: EtOAc• 20 L, 1:1:0.25 Hexanes: DCM: EtOAc• 7.5 L, 1: 1:0.5 Hexanes: DCM: EtOAc• 8 L, 1:1:1 Hexanes: DCM: EtOAcPure fractions were collected and combined in a pre-weighed recovery flask, concentrated to dryness under reduced pressure and placed under high vacuum overnight to obtain after column chromatography, 10 as a yellow solid (61% yield).

[0125] 1H NMR (500 MHz, CDCL) 5 0.02 (s, 6H), 0.86 (s, 9H), 1.52 (s, 6H), 2.50 (t, J = 6.1 Hz, 2H), 2.58-2.63 (m, 4H), 3.00-3.05 (m, 4H), 3.66 (t, J = 6.1 Hz, 2H), 3.95 (s, 3 H), 6.49 (bs, 1 H), 7.03 (s, 1 H), 7.10 (d, J= 16.4 Hz, 1 H), 7.11 (d, J = 16.4 Hz, 1 H), 7.14 (d, 7= 7.9 Hz, 1 H), 7.18 (d, J= 16.4 Hz, 1 H), 7.34 d, 7= 8.4 Hz, 2 H), 7.36 (d, 7 = 16.4 Hz, 1 H), 7.46 (d, 7= 8.4 Hz, 2 H), 7.58 (d, 7 = 7.9 Hz, 1 H), 7.62 (d, 7= 8.4 Hz, 2 H), 7.69 (d, 7= 8.4 Hz, 2 H).13C NMR (150 MHz, CDCL) 5 -5.18, 18.41, 26.06, 28.57, 46.28, 53.00, 55.82, 60.20, 61.56, 80.86, 109.22, 118.71, 120.08, 121.71, 126.55, 126.66, 126.96, 127.42, 127.53, 128.52, 129.25, 132.33, 132.98, 133.83, 136.78, 137.97, 142.25, 152.79, 157.21. MS (m / z): [M+H]+: 734.3.

[0126] NOTES1. Sat. aqueous Na2CO3must be used to quench the reaction mixture, otherwise side products were observed that were not seen during the reaction.2. It is preferred to use a slurry of silica which is pre-conditioned in hexanes:DCM 1:1 containing 1% triethylamine in order to avoid side products not seen during the reaction.3. The column was washed with 1 column volume of 1:1 DCM:hexanes in order to stabilize the silica bed and ensure uniformity.

[0127] Example 7. Synthesis of 2-(4-((4-((E)-4-((E)-4-aminostyryl)-3- methoxystyryl)phenyl)sulfonyl)piperazin-l-yl)ethan-l-ol (11) (also known as Illuminare-1 or GE3126)10 11, Illuminare-1 Free BaseAKA as GE3126

[0128] An appropriately sized round bottom flask, equipped with argon inlet and an overhead mechanical stirrer (powerful magnetic stirrer will also work) was charged with Compound 10. Dioxane (6-10 mL per mmol of Compound 10) was added followed by the addition of 4.0 N HC1 in dioxane (18.7 mL / mmol) at room temperature. Reaction progress was monitored by TLC and LC-MS for the presence of the intermediates -NHBoc group (m / e= 633), and -OTBDMS (m / e= 619) and the starting material (m / e= 733). The reaction was stirred at room temperature for up to 7 days depending on scale and stirring power. Small amounts of 4N HC1 in dioxane could be added daily to the reaction mixture throughout reaction progress towards completion. This generates a thick yellow solution with shades of orange.

[0129] The reaction mixture was then filtered over Buchner funnel under suction and washed with cold dioxane. The solids were partitioned between dichloromethane (65 mL / 1.0 mmol) and saturated sodium carbonate (33 mL / 1.0 mmol) or until pH paper indicated a stable value of 8-10. Sonication and / or stirring may be needed to dissolve all solids. The two layers were separated, and the aqueous layer further extracted with dichloromethane until no yellow color remained (4 mL / 1 mmol). The combined dichloromethane extracts were briefly dried with Na2SO4, vacuum filtered through a packed pad of celite and the filter cake washed with dichloromethane until no yellow color remained. The volatiles were removed under vacuum to obtain a yellow-orange solid.

[0130] Purification: In a typical run approximately 70 g from the crude product was dissolved in minimum DCM (about 700 mL) then treated with 300 g of silica gel and the resulting slurry was evaporated to half the volume, before loading onto a 1.25 Kg preconditioned silica gel column. The column was pre-conditioned in DCM containing 1% triethylamine, as a slurry then packed manually with air pressure (see note). After loading the desired compound was eluted with the gradient system:• 6 L, CH2Q2 or until all top spots were out• 8 L, 0.5% MeOH in CH2CI2• 8 L, 0.75% MeOH in CH2CI2• 16L, 1.0 % MeOH in CH2CI2• Column wash with 4:5:1 MeOH: CH2Q2 : Et3N (v:v:v)Pure fractions were combined in a pre- weighed recovery flask and solvents were evaporated under vacuum to obtain Illuminare-1 free base 11(GE3126 free base), as a yellow solid (88% yield).

[0131] 1H NMR (500 MHz, CDCh) 5 2.31 (t, J= 5.1 Hz, 1H), 2.55 (t, J = 5.3 Hz, 2H), 2.56-2.62 (m, 4H), 3.05-3.11 (m, 4H), 3.55-3.61 (m, 2H), 3.75 (s, 2 H), 3.95 (s, 3 H), 6.68 (d, J= 8.4 Hz, 2 H), 7.03-7.30 (m, 6 H), 7.38 (d, J = 8.5 Hz, 2 H), 7.58 (d, J = 7.9 Hz, 1 H), 7.65 (d, J = 8.4 Hz, 2 H), 7.73 (d, J = 8.4 Hz, 2 H).13C NMR (150 MHz, CDCh) 5 46.40, 52.45, 56.05, 58.91, 60.04, 109.80, 114.41, 117.13, 120.48, 125.61, 126.01, 126.58, 127.32, 127.39, 128.12, 128.65, 130.41, 132.51, 133.27, 136.35, 142.52, 149.34, 156.67. MS (m / z): [M+H]+: 520.41.

[0132] NOTES1. The color of Illuminare-1 free base slightly varies from yellow to a light orange shade. The light orange shade may be a sign of the presence of traces of HC1 salt formed during transfer between flasks using dichloromethane. Dichloromethane carries traces of HC1 due to photochemical release of HC1 from the dichloromethane molecule. For this reason, the free base is washed with sodium carbonate in the last step to neutralize any HC1 that sticks to the molecule and brings back the very bright yellow color. If any orange color is seen after removal of the DCM upon chromatography, the saturated sodium carbonate wash should be repeated before post synthesis processing.2. Once the 1% Et3N / CH2Cl2silica slurry was loaded onto the column, the silica bed was washed with 1 column volume of CH2CH2 to stabilize the column.

[0133] Post Synthesis Processing

[0134] Microfiltration:

[0135] An alumina column with 3-6 inch diameter and 10-20 inch height was prepared. Three column volumes of dichloromethane were added and flown through without running the column to dryness. The flow through was discarded. Four liters of dichloromethane were then passed through the column and placed in a clean 4 L Erlenmeyer flask and covered. Illuminare-1 free base, 11 was dissolved in minimum alumina-filtered dichloro methane (approximately 1 g in 100 mL). A Millipore microfiltration apparatus, which includes 0.22-micron membrane filter was assembled and secured with a clamp. The apparatus was rinsed with alumina- filtered dichloromethane and the liquid discarded. The dichloromethane solution of Illuminare-1 was filtered, and the funnel washed 3 times or until no yellow color remained on filter and funnel walls. The dichloromethane was immediately evaporated under vacuum, in a pre- weighed recovery flask, then the flask placed under high vacuum overnight. The solid was weighed and placed in a vacuum oven at 45°C under vacuum (0.45 mm Hg) overnight or until constant weight is obtained andNMR indicated no solvent was present. The resulting yellow solid was ground with clean mortar and pestle to a uniform powder which was placed in certified clean amber jars which were sealed and placed in secondary containers before storage at -20

[0136] Example 8. Synthesis of tri-deuterated analog of Illuminare-1 lids

[0137] As a mass spectrometry standard, tri-deuterated 11 was prepared. Ild3 could be accessed directly from 11 through methyl ether removal by BBn in DCM (Scheme 4).Scheme 4. Synthesis of tri-deuterated analog of Illuminare-1 lids

[0138] Alternatively, lids could be preferably made following a process similar to that used for synthesizing 11 except starting from 3ds instead (Scheme 5).Scheme 5. Alternative synthesis of tri-deuterated analog of Hluminare-1 lids

[0139] Example 9. Synthesis of11C analogs of Illuminare-1 11-N-11CH3, 11-0-11CH3, and ll11CH3

[0140] The expert in surgery may also consider shorter-lived isotope-labeled Illuminare-1 which may delineate the status of myelinated nerves and tissues as means of patient selection (Beuche, S., et. al. 2023; Stankoff, B., et. al. 2006). The case for short-lived isotopes requires shorter synthesis pathways commensurate with short labeling steps and short processing post radiosynthesis. For example, a radiolabeling precursor for Positron Emission Tomography (PET) requires labeling as late as possible and involves short synthesis steps. Several possibilities are available. Ideally, such transformations would involve direct modification of Compound 11. The resulting labeled molecules as described for similar structures should still possess the myelin binding properties (Condie, A. G., et. al. 2012; Chen, X., et. al. 2023). In the case11C labeling, it is commonly accepted that direct labeling of a nitrogen or an oxygen would involve reaction with a11C - CFE-halogen under basic conditions (Scheme 6). Preferably halogen is a bromide or iodide.Scheme 6. Synthesis of11C analogs of Illuminare- 1 11-N-11CH3, 11-O-11CH3, and1111CH3

[0141] Example 10. Synthesis of fluorinated analogs of Illuminare-1 F-ll and 18F-11

[0142] As demonstrated in the art (Veach, D. R., et. al. 2007) changing the hydroxyl group into a fluoride preserves activity. This opened the opportunity to label Illuminare-1 with fluorine 18 (Scheme 7).18F has a slightly longer half-life than11C.Scheme 7. Synthesis of fluorinated analogs of Illuminare- 1 F-ll and18F-11

[0143] Starting from the fully protected Illuminare-1, 10, from which the TBDMS group was removed to liberate the hydroxyl group and set the stage for the installation of a leaving group (Scheme 6). The leaving group could be chosen from a panel of aryl sulfonate esters (benzene sulfonate, toluene sulfonate, nitrophenyl sulfonate, etc.) and alkyl sulfonate such as methane sulfonate or trifluoro methane sulfonate. In a preferred application, the sulfonate is methane sulfonate, also known as mesylate such as 10Mes.

[0144] Mesylate10 Mes could be transformed into the standard Compound F-ll and the PET probe18F-11 as needed via a 2-step protocol (Scheme 6). Displacement of the mesylate by either potassium fluoride or18F-KF under similar conditions but are more suitable for radiolabeling in the presence of a potassium chelate such as 18-crown-6 or cryptofix. The removal of the Boc group under high concentration trifluoro acetic acid (TFA) in the presence of triisopropyl silane and DCM followed by basic work up and purification provides the desired18F-11.

[0145] Example 11. Synthesis of 14C analog of Illuminare-1 14C-11

[0146] A common isotope that is used for long term biodistribution studies is14C. The design here dictates labeling rather internal carbons to avoid metabolic liabilities where the labeled carbon could be oxidized and removed by metabolic enzymes in the body.

[0147] For the14C labeling of Illuminare-1, a central carbon was chosen which follows a slightly different synthesis route compared to that which was used for Compound 11 (Scheme 8). It is based on a14C-CO2 reaction with a lithium anion resulting from the halogen-lithium exchange of bromide with tertiobutyl lithium.Scheme 8. Synthesis of14C analog of Illuminare- 114C-11

[0148] 14C-11 was synthesized at an accredited radiolabeling facility following the protocol that is similar to that used for the synthesis of Compound 11 except the radiolabeling step which required the formylation of a bromostilbene. There are two possible ways to access the bromostilbene (Scheme 7). A Heck coupling between 2- methoxy-4-bromo phenyl iodide and Boc-amino styrene was sluggish. However, in a preferred process, a bromonitrostilbene was first synthesized through a Wittig-Horner between diethyl 4-nitrobenzylphosphonate and 4-bromo-2-methoxy benzaldehyde. The resulting bromonitrostilbene (1.1 g, 3.29 mmol) was dissolved in ethanol / water (95:5, 30 mL) and SnCh (3.12 g, 16.5 mmol) was added and resulting reaction mixture was brought up to reflux and stirred at that temperature for 2 h. After cooling down to the ambient, solvents were removed under vacuum and water (50 mL) was added, the aqueous layerextracted with EtOAc (100 mL x 3). The combined organic layers were dried briefly over Na2SO4, filtered and concentrated under vacuum to provide 1.05 g of an off-white solid which was then directly submitted to the next step for Boc-protection. The crude amine (1.05 g, 3.45 mmol) in DCM (10 mL) was treated with BOC2O (1.5 g), DMAP (10 mg) and pyridine (0.2 mL). The resulting mixture was stirred at RT overnight. Concentration and purification by column chromatography, SiO2, using hexanes / ethyl acetate (9 / 1) to get the bromostilbene (900 mg, 67.7% yield for two steps).

[0149] JH NMR (500 MHz) 5 7.46-7.41 (m, 3H), 7.35-7.33 (m, 2 H), 7.27 (d, J =17.8 Hz, 1 H), 7.09 (dd, J = 1.7 Hz, J = 8.2 Hz, 1 H), 7.03 (d, J =17.8 Hz, 1 H), 7.01 (d, J =1.8 Hz, 1 H), 6.48 (bs, 1 H), 3.88 (s, 3 H), 1.53 (s, 9H). MS [M+H+]: 403.0, 405.0.

[0150] The bromide of the bromostilbene was exchanged to lithium by reaction with tBuLi, followed by reaction with14C-CO2. The resulting carboxylic acid was reduced in situ to the corresponding alcohol followed by oxidation to the aldehyde to provide14C-4.Aldehyde14C-4 was condensed with Compound 9 to provide the fully protected14C-10. Lor some reason, the removal of the Boc group under acidic conditions was not compatible with14C-10. Hence, a basic deprotection route was implemented which provided high purity14C labeled free base.

[0151] A non-radiolabeled series was tested in which the removal of the Boc group was tested under basic conditions using TBDMS triflate in excess 2,6-lutidine (Sakaitani, M. & Ohfune, Y., 1990) (Scheme 9). This unified the protecting groups into TBDMS Carbamate and TBDMS ether. Both TBDMS groups can be removed under excess (HL)x- Pyridine in pyridine.Scheme 9. Test conditions for deprotection and synthesis of 11

[0152] Subsequently, this was then repeated successfully in the radioactive series to give14C-11 (Scheme 10). LC-MS: >98%; MS (m / z): [M+H]+: 522.31.Scheme 10. Conditions for deprotection and synthesis of14C-11EQUIVALENTS

[0153] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0154] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[0155] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novelcharacteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0156] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0157] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. 2% As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0158] All publications, patent applications, issued patents, and other documents (for example, journals, articles and / or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0159] When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations, and subcombinations of ranges for specific embodiments therein are intended to be included.

[0160] Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the present technology and that such changes and modifications can be made without departing from the spirit of the present technology. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the present technology.REFERENCES

[0161] Barth, C. W., & Gibbs, S. L. (2016). Visualizing Oxazine 4 nerve- specific fluorescence ex vivo in frozen tissue sections. Proceedings of SPlE—the International Society for Optical Engineering, 9696, 96960R.

[0162] Barth, C. W., & Gibbs, S. L. (2017). Direct Administration of Nerve- Specific Contrast to Improve Nerve Sparing Radical Prostatectomy. Theranostics, 7(3), 573-593.

[0163] Beuche, S., Peyronneau, M.-A., Jego, B., Denis, C., Bourbon, P., Chauviere, C., Santerre, C., Truillet, C., Kuhnast, B., and Caille, F. Design, Radiosynthesis, and Evaluation of New Fluorinated Analogs of MeDAS for Myelin PET Imaging. Journal of Medicinal Chemistry 2023, 66 (12), 8030-8042.

[0164] Bradbury, M., et al. (2017). Imaging Systems and Methods for Tissue Differentiation, e.g., For Intraoperative Visualizaion (Patent No. WO 2017 / 106525 Al). World Intellectual Property Organization.

[0165] Chen, X., Sun, H., Du, Y., Liu, C., Liu, R. Aminophenoxazinone nearinfrared fluorescent probes for myelin- specific imaging. Tetrahedron, Volume 136, 2023.

[0166] Condie, A. G., Gerson, S. L., Miller, R. H. and Wang, Y. Two-Photon Fluorescent Imaging of Myelination in the Spinal Cord. ChemMedChem 2012, 7, 2194 - 2203.

[0167] Cotero, V. E., Kimm, S. Y., Siclovan, T. M., Zhang, R., Kim, E. M., Matsumoto, K., Gondo, T., Scardino, P. T., Yazdanfar, S., Laudone, V. P., & Tan Hehir, C.A. (2015). Improved Intraoperative Visualization of Nerves through a Myelin-Binding Fluorophore and Dual-Mode Laparoscopic Imaging. PloS one, 10(6), e0130276.

[0168] Gibbs-Strauss, S. L., Nasr, K. A., Fish, K. M., Khullar, O., Ashitate, Y.,Siclovan, T. M., Johnson, B. F., Barnhardt, N. E., Tan Hehir, C. A., & Frangioni, J. V.(2011). Nerve-highlighting fluorescent contrast agents for image-guided surgery. Molecular imaging, 10(2), 91-101.

[0169] Gonzales, J., Pirovano, G., Chow, C. Y., de Souza Franca, P. D., Carter, L. M., Klint, J. K., Guru, N., Lewis, J. S., King, G. F., & Reiner, T. (2020). Fluorescence labeling of a NaV 1.7 -targeted peptide for near-infrared nerve visualization. EJNMMI research, 10(1), 49.

[0170] Gray, J. E. (2017). Nerve injury associated with pelvic surgery. UpToDate

[0171] Hingorani, D. V., Whitney, M. A., Friedman, B., Kwon, J. K., Crisp, J. L., Xiong, Q., Gross, L., Kane, C. J., Tsien, R. Y., & Nguyen, Q. T. (2018). Nerve-targeted probes for fluorescence-guided intraoperative imaging. Theranoslics. S(15), 4226-4237.

[0172] Sakaitani, M. and Ohfune, Y. Syntheses and Reactions of Silyl Carbamates. 2. A New Mode of Cyclic Carbamate Formation from tert-Butyldimethylsilyl Carbamate. J. Am. Chem. Soc. 1990, 112, 1150-1158.

[0173] Stankoff, B., Wang, Y., Bottlaender, M., Aigrot, M. S., Dolle, F., Wu, C., Feinstein, D., Huang, G.-F., Semah, F., Mathis, C. A., Klunk, W., Gould, R. M., Lubetzki, C., and Zalc, B. Imaging of CNS myelin by positron emission tomography. PNAS, 2006, June 13, vol. 103, no. 24, 9304-9309.

[0174] Veach, D. R., Namavari, M., Pillarsetty, N., Santos, E. B., Beresten- Kochetkov, T., Lambek, C., Punzalan, B. J., Antczak, C., Smith- Jones, P. M., Djaballah, H., Clarkson, B., and Larson, S. M. Synthesis and Biological Evaluation of a Fluorine-18 Derivative of Dasatinib. J. Med. Chem. 2007, 50, 23, 5853-5857.

[0175] Incorporation by Reference

[0176] All US and PCT patent application publications and US patents cited herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0177] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A process for the preparation of Compound 11 of the formulacomprising the steps of a) reacting Compound 4 of the formulawith Compound 9 of the formulain the presence of a base in a solvent, to afford Compound 10 of the formulab) reacting Compound 10 with an acid in a solvent followed by reaction with a base, to afford Compound 11.B. The process of Paragraph A wherein the base is KOtBu, wherein the solvent is THF.C. The process of Paragraph A or Paragraph B wherein the acid is HC1, wherein the solvent is dioxane, and wherein the base is Na CO3or NaHCO3.D. The process of any one of Paragraphs A-C, wherein Compound 11 is greater than 97.0% trans configuration.E. The process of any one of Paragraphs A-D, wherein Compound 11 is greater than 97.5% trans configuration.F. The process of any one of Paragraphs A-E, wherein Compound 11 is greater than 98.0% trans configuration.G. The process of any one of Paragraphs A-F, wherein Compound 11 is greater than 98.5% trans configuration.H. The process of any one of Paragraphs A-G, wherein Compound 11 is greater than 99.0% trans configuration.I. The process of any one of Paragraphs A-H, wherein Compound 11 is greater than 99.5% trans configuration.J. The process of any one of Paragraphs A-I, wherein Compound 11 is greater than 99.9% trans configuration.K. The process of any one of Paragraphs A- J, wherein Compound 10 is greater than 97.0% trans configuration.L. The process of any one of Paragraphs A-K, wherein Compound 10 is greater than 97.5% trans configuration.M. The process of any one of Paragraphs A-L, wherein Compound 10 is greater than 98.0% trans configuration.N. The process of any one of Paragraphs A-M, wherein Compound 10 is greater than 98.5% trans configuration.O. The process of any one of Paragraphs A-N, wherein Compound 10 is greater than 99.0% trans configuration.P. The process of any one of Paragraphs A-O, wherein Compound 10 is greater than 99.5% trans configuration.Q. The process of any one of Paragraphs A-P, wherein Compound 10 is greater than 99.9% trans configuration.R. A process for the preparation of Compound 9 of the formulacomprising the steps of a) reacting Compound 12 of the formulato afford Compound 13 of the formulathen b) reacting Compound 13 with Compound 6 of the formulain the presence of a base in a solvent, to afford Compound 9.S. The process of Paragraph R, wherein the base is DIPEA, wherein the solvent is DCM.T. A process for the preparation of Compound 4 of the formulacomprising the steps of a) reacting Compound 1 of the formulawith BOC2O in water to afford Compound 2 of the formula; then b) reacting Compound 2 with Compound 3 of the formulain presence of Pd(OAc)2, TPPTS and a base in a solvent to afford Compound4.U. The process of Paragraph T wherein the base is K2CO3, wherein the solvent is a mixture of DMF and water.V. A process for the preparation of Compound 6 of the formulacomprising the step of reacting Compound 5 of the formulawith TBDMS-C1 in DCM at reflux.W. A compound having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is notX. The compound of Paragraph W having the structure:pharmaceutically acceptable salt thereof.Y. The compound of Paragraph W having the structure:or a pharmaceutically acceptable salt thereof.Z. The compound of Paragraph W having the structure:pharmaceutically acceptable salt thereof.AA. The compound of Paragraph W having the structure:or a pharmaceutically acceptable salt thereof.AB. The compound of Paragraph W having the structure:pharmaceutically acceptable salt thereof.AC. A pharmaceutical composition comprising the compound of any one of Paragraphs W- AB and a pharmaceutically acceptable carrier.AD. A method of imaging nerves in a subject, the method comprising: administering to the subject an effective amount of a pharmaceutical composition comprising one or more compounds having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is notdetecting a signal that is associated with the compound or the pharmaceutically acceptable salt thereof.AE. The method of Paragraph AD, wherein the compound comprisespharmaceutically acceptable salt thereof.AF. The method of Paragraph AD, wherein the compound comprisesor a pharmaceutically acceptable salt thereof.AG. The method of Paragraph AD, wherein the compound comprisespharmaceutically acceptable salt thereof.AH. The method of Paragraph AD, wherein the compound comprisesor a pharmaceutically acceptable salt thereof.Al. The method of Paragraph AD, wherein the compound comprisespharmaceutically acceptable salt thereof.

[0178] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.

Claims

WHAT IS CLAIMED IS:

1. A process for the preparation of Compound 11 of the formulacomprising the steps of a) reacting Compound 4 of the formulawith Compound 9 of the formulain the presence of a base in a solvent, to afford Compound 10 of the formulab) reacting Compound 10 with an acid in a solvent followed by reaction with a base, to afford Compound 11.

2. The process of claim 1 wherein the base is KOtBu, wherein the solvent is THF.

3. The process of claim 1 wherein the acid is HC1, wherein the solvent is dioxane, wherein the base is Na2CO3or NaHCO3.

4. A process for the preparation of Compound 9 of the formulacomprising the steps of a) reacting Compound 12 of the formulawith CISO3H to afford Compound 13 of the formulab) reacting Compound 13 with Compound 6 of the formulain the presence of a base in a solvent, to afford Compound 9.

5. The process of claim 4 wherein the base is DIPEA, wherein the solvent is DCM.

6. A process for the preparation of Compound 4 of the formulacomprising the steps of a) reacting Compound 1 of the formula in water to afford Compound 2 of the formulathen b) reacting Compound 2 with Compound 3 of the formulain presence of Pd(OAc)2, TPPTS and a base in a solvent to afford Compound4.

7. The process of claim 6 wherein the base is K2CO3, wherein the solvent is a mixture ofDMF and water.

8. A process for the preparation of Compound 6 of the formula f reacting Compound 5 of the formulawith TBDMS-C1 in DCM at reflux.

9. The process of claim 1, wherein Compound 11 is greater than 99.9% trans configuration.

10. The process of claim 1, wherein Compound 11 is greater than 99.5% trans configuration.

11. The process of claim 1, wherein Compound 11 is greater than 99.0% trans configuration.

12. The process of claim 1, wherein Compound 11 is greater than 98.5% trans configuration.

13. The process of claim 1, wherein Compound 11 is greater than 98.0% trans configuration.

14. The process of claim 1, wherein Compound 11 is greater than 97.5% trans configuration.

15. The process of claim 1, wherein Compound 11 is greater than 97.0% trans configuration.

16. The process of claim 1, wherein Compound 10 is greater than 99.9% trans configuration.

17. The process of claim 1, wherein Compound 10 is greater than 99.5% trans configuration.

18. The process of claim 1, wherein Compound 10 is greater than 99.0% trans configuration.

19. The process of claim 1, wherein Compound 10 is greater than 98.5% trans configuration.

20. The process of claim 1, wherein Compound 10 is greater than 98.0% trans configuration.

21. The process of claim 1, wherein Compound 10 is greater than 97.5% trans configuration.

22. The process of claim 1, wherein Compound 10 is greater than 97.0% trans configuration.

23. A compound having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is not24. The compound of claim 23 having the structure:pharmaceutically acceptable salt thereof.

25. The compound of claim 23 having the structure:

26. The compound of claim 23 having the structure:pharmaceutically acceptable salt thereof.

27. The compound of claim 23 having the structure:pharmaceutically acceptable salt thereof.

28. The compound of claim 23 having the structure:pharmaceutically acceptable salt thereof.

29. A pharmaceutical composition comprising the compound of any one of claims 23-28 and a pharmaceutically acceptable carrier.

30. A method of imaging nerves in a subject, the method comprising: administering to the subject an effective amount of a pharmaceutical composition comprising one or more compounds having the structure:or a pharmaceutically acceptable salt thereof, whereinA is CH or14CH;X is CH3, CD3,11CH3, or CF3;Y is H or11CH3; andZ is OH, O11CH3, F, or18F; and wherein the compound is notdetecting a signal that is associated with the compound or the pharmaceutically acceptable salt thereof.

31. The method of claim 30, wherein the compound comprisespharmaceutically acceptable salt thereof.

32. The method of claim 30, wherein the compound comprisespharmaceutically acceptable salt thereof.

33. The method of claim 30, wherein the compound comprisespharmaceutically acceptable salt thereof.

34. The method of claim 30, wherein the compound comprisespharmaceutically acceptable salt thereof.

35. The method of claim 30, wherein the compound comprisespharmaceutically acceptable salt thereof.

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