Near infrared fluorophores for peripheral and central nerve imaging

Abstract

The present invention provides novel near-infrared fluorophore compounds of Formula (I), formulations comprising them, and methods for their use in imaging peripheral and central nerves.

Description

[0001] NEAR INFRARED FLUOROPHORES FOR PERIPHERAL AND CENTRAL NERVE IMAGING

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns novel 3H-phenoxazine and 2,3,4,8-tetrahydro- [1 ,4]oxazino[2,3-b]phenoxazine fluorophore compounds, formulations comprising them, and methods for their use in imaging peripheral and central nerves.

[0004] STATEMENT OF GOVERNMENT SUPPORT

[0005] This invention was made with government support under R01 NS129121 and R01 CA271532 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND OF THE INVENTION

[0007] Iatrogenic nerve injury presents one of the most feared surgical complications and a major source of morbidity across all surgical specialties.1'3While crucial to maintain their vital functions, preservation of nerves remains a major challenge in surgeries, particularly in neurosurgeries. With the increased use of minimally invasive surgical approaches, difficulty with cranial nerve identification and visualization is amplified.4Cranial nerves are often intimately associated with tumors of the skull base, where surgery can be curative, but must be balanced against injury risk.5Unlike other critical tissues (e.g., blood vessels), nerve repair produces negligible, unreliable functional improvement.6Permanent motor or sensory disabilities and chronic neuropathies result, limiting patient quality of life, employability and participation in daily activities. The only known method to avoid nerve injury is to prevent its occurrence. However, no clinically approved technology sufficiently enhances intraoperative nerve visualization, where a combination of neuroanatomical knowledge, white light visualization and neurophysiological monitoring are currently used.

[0008] Fluorescence Guided Surgery (FGS) has successfully integrated into clinical medicine with only a few FDA-approved fluorophores (i.e., fluorescein, Aminolevulinic / Protoporphyin IX [ALA / PpIX], methylene blue and indocyanine green [ICG]).7 8While FGS systems for imaging outside the skull operate almost exclusively in the near infrared (NIR, 650-900 nm).9'16NIR fluorophores with cranial nerve specificity that are spectrally distinct from the visible fluorescein and ALA / PpIX commonly used for tumor enhancement, while enabling future clinical translation using existing neurosurgical FGS infrastructure, will be most promising to address the unmet need in cranial nerve identification during surgery.

[0009] SUMMARY OF THE INVENTION

[0010] Development of a NIR nerve-specific fluorophore has presented a synthetic challenge as molecules must be small enough to cross the tight blood nerve and / or blood brain barrier(s) (BNB and / or BBB, respectively), but with a sufficient degree of conjugation to reach NIR wavelengths. This is a particular challenge in neurosurgical applications where identification and visualization of structures at the interface of the peripheral and central nervous system (PNS and CNS, respectively) are required for successful surgical outcomes. To address this unmet need, we have developed NIR oxazine-based fluorophores with exquisite PNS specificity17and recently developed a new library of water-soluble oxazine fluorophores that can cross the BBB and generate high contrast for the cranial nerves.

[0011] One embodiment provides a compound of Formula (I): wherein:

[0012] X is selected from the group of a single bond and a double bond;

[0013] Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;

[0014] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0015] R2 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or R1 and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by R1 and R2 is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or R1 and R2 together with the nitrogen atom to which they are bound form a spirocyclic heterocycle of the formula: the wavy line in the Ri and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent 3-position carbon atom; n3 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n4 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n3 and n4 is not less than 2 and not greater than 4; n5 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n6 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n5 and n6 is not less than 2 and not greater than 4;

[0016] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0017] R4 is Ci-Ce alkyl when R3is Ci-Ce alkyl; and R4 is -(CH2)ni-O-(CH2)n2-CH3when R3is - (CH2)ni-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4 -(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety;

[0018] R5is H; or R5is an oxygen atom that joins with R4to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II):

[0019] R6is a ring heteroatom selected from the group of O and S; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. BRIEF DESCRIPTION OF THE MANY VIEWS OF THE DRAWINGS

[0020] FIGURE 1 presents structures of compounds in a novel oxazine fluorophore library designed and synthesized based on the first-in-class NIR nerve-specific fluorophore.

[0021] FIGURE 2A presents representative white light and fluorescence images of the eight oxazine-based fluorophores with the brightest nerve tissue fluorescence intensity for the indicated compounds after systemic administration in comparison to a vehicle-injected control group.

[0022] FIGURE 2B presents a bar graph representing quantified nerve tissue intensity values reported as A.U. / s following systemic fluorophore administration at 7.5 pmol / kg or equivalent volume of blank cosolvent formulation. Images are shown with optimal contrast.

[0023] FIGURE 2C presents a graph representing quantified N / M SBRs of the eight leading candidates and vehicle injected control group following systemic administration are arranged in ascending order. All values are presented as the mean ± SD. The ARM prefix is omitted from the x-axis for clarity. Ctrl = vehicle injected control to quantify autofluorescence. FL = Fluorescence.

[0024] FIGURE 3A presents representative fluorescence images of eight oxazine-based fluorophores with the brightest nerve tissue fluorescence intensity after systemic administration, shown in comparison to a vehicle-injected control group.

[0025] FIGURE 3B presents a graph representing quantified optical nerve tissue intensity values reported as A.U. / s following systemic fluorophore administration at 7.5 pmol / kg or equivalent volume of blank cosolvent formulation. Images are shown with optimal contrast.

[0026] FIGURE 3C presents a graph representing quantified optic nerve to background tissue SBRs of the eight leading candidates and vehicle injected control group following systemic administration are arranged in ascending order. All values are presented as the mean ± SD. The ARM prefix is omitted from the x-axis for clarity. Ctrl = vehicle injected control to quantify autofluorescence. FL = Fluorescence.

[0027] FIGURE 4 presents Table 1 listing the tabulated spectral and physical properties of the novel fluorophores.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] One embodiment provides a compound of Formula (I): wherein:

[0030] X is selected from the group of a single bond and a double bond;

[0031] Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;

[0032] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0033] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2 together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2 is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle of the formula: the wavy line in the Ri and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent 3-position carbon atom; n3 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n4 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n3 and n4 is not less than 2 and not greater than 4; n5 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n6 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n5 and n6 is not less than 2 and not greater than 4; R3 is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0034] R4 is C-i-Ce alkyl when R3is Ci-Ce alkyl; and R4is -(CH2)ni-O-(CH2)n2-CH3 when R3is - (CH2)ni-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety;

[0035] R5is H; or R5is an oxygen atom that joins with R4to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II):

[0036] R6is a ring heteroatom selected from the group of O and S; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0037] It is understood that Formula (I) includes each of its resonance structures as noted below and may be represented by each. For each embodiment herein concerning a compound of Formula (I) described herein, three additional separate embodiments exist wherein all variables (Ri , R2, R3, Rs, Rs, m , etc.) are as defined for the embodiment in question and the three additional embodiments are directed to, respectively, a compound of Formula (la), a compound of Formula (lb), and a compound of Formula (Ic), or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0038]

[0039] Non-limiting examples of heterocyclic spirocycles of the formula:

[0040] wherein the wavy line through parallel straight and dashed lines ( ) and a wavy line through a straight line each represent the optional single and double bonds through which the heterocyclic spirocycle may be bound to the adjacent carbon atom.

[0041] In view of the resonance structures for Formula (I) and the other formulas herein, it is further understood that the cyclic and spirocyclic groups formed by Ri and R2, along with the nitrogen atom they are bound to, and R3 and R4, along with the nitrogen atom to which they are bound, may be represented equally in structures herein with a single bond line ( - ), a partially dashed single / double bond ( - ), and / or a double lined double bond (^=), such as represented in the structures below.

[0042]

[0043] Another embodiment provides a compound of Formula (I): wherein:

[0044] X is selected from the group of a single bond and a double bond;

[0045] Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;

[0046] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0047] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together with the nitrogen atom to which they are bound form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of:

[0048] the wavy line through a straight line in the Ri and R2 heterocyclic spirocycle formulas represents the optional single bond or double bond through which R1 is bound to the adjacent carbon atom;

[0049] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0050] R4 is Ci-C3alkyl when R3is Ci-C3alkyl; and R4 is -(CH2)ni-O-(CH2)n2-CH3when R3is - (CH2)ni-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4 -(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; and

[0051] Rs is H; or R5is an oxygen atom that joins with R4to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II): or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. A further embodiment provides a compound of Formula (I), wherein X, Y, R3, R4, and Rs are as defined in the embodiment immediately above, and R1 and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer

[0052] (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0053] A further embodiment provides a compound of Formula (I), wherein X, Y, R3, R4, and R5are as defined in the embodiment immediately above, and R1 and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0054] Another embodiment provides a compound of Formula (I): wherein:

[0055] X is selected from the group of a single bond and a double bond;

[0056] Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;

[0057] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0058] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2 together with the nitrogen atom to which they are bound form a heterocyclic ring selected from the group of azetidinyl and pyrrolidinyl, wherein the heterocyclic ring formed by Ri and R2 is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of:

[0059] the wavy line through a straight line in the Ri and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent carbon atom;

[0060] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0061] R4is Ci-C3alkyl when R3is Ci-C3alkyl; and R4is -(CH2)ni-O-(CH2)n2-CH3when R3is - (CH2)ni-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; and

[0062] Rs is H; or R5is an oxygen atom that joins with R4to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II): or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0063] Another embodiment provides a compound of Formula (I): wherein:

[0064] X is selected from the group of a single bond and a double bond;

[0065] Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;

[0066] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0067] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2 together with the nitrogen atom to which they are bound form a heterocyclic ring selected from the group of azetidinyl and pyrrolidinyl, wherein the heterocyclic ring formed by Ri and R2 is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a heterocyclic spirocycle selected from the group of: the wavy line in the Ri and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent 3-position carbon atom;

[0068] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0069] R4 is C1-C3 alkyl when R3is C1-C3 alkyl; and R4is -(CH2)ni-O-(CH2)n2-CH3when R3is - (CH2)n1-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; and Rs is H; or Rs is an oxygen atom that joins with R4 to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II): or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0070] A further embodiment provides a compound of Formula (II): wherein:

[0071] X is selected from the group of a single bond and a double bond;

[0072] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0073] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of:

[0074] the wavy line through a straight line in the Ri and R2 heterocyclic spirocycle formulas represents the optional single bond or double bond through which R1 is bound to the adjacent carbon atom; and

[0075] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0076] A further embodiment provides a compound of Formula (II): wherein:

[0077] X is selected from the group of a single bond and a double bond;

[0078] R1 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0079] R2 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a heterocyclic spirocycle selected from the group of: the wavy line in the Ri and R2 heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent 3-position carbon atom; and

[0080] R3is selected from the group of Ci-C6alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0081] A further embodiment provides a compound of Formula (III): wherein:

[0082] X is selected from the group of a single bond and a double bond;

[0083] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0084] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or R1 and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by R1 and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or R1 and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: the wavy line through a straight line in the R1 and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent carbon atom; and

[0085] R3 is Ci-Ce alkyl; and

[0086] R4 is Ci-Ce alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0087] A further embodiment provides a compound of Formula (III): wherein:

[0088] X is selected from the group of a single bond and a double bond;

[0089] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0090] R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2 together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2 is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: the wavy line through a straight line in the R1 and R2 heterocyclic spirocycle formulas represents the optional single bond or double bond through which R1 is bound to the adjacent carbon atom;

[0091] R3is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; and

[0092] R4is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0093] A further embodiment provides a compound of Formula (III): wherein:

[0094] X is selected from the group of a single bond and a double bond;

[0095] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0096] R2 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or R1 and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by R1 and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or R1 and R2 together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: the wavy line in the R1 and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which R1 is bound to the adjacent 3-position carbon atom;

[0097] R3is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; and

[0098] R4is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0099] A further embodiment provides a compound of Formula (III): wherein:

[0100] X is selected from the group of a single bond and a double bond;

[0101] Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0102] R2 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or R1 and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by R1 and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or R1 and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of: the wavy line in the R1 and R2 heterocyclic spirocycle formulas represents the optional single bond or double bond through which R1 is bound to the adjacent 3-position carbon atom; Ra is C1-C6 alkyl; and

[0103] R4is C1-C6 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0104] A further embodiment provides a compound of Formula (III) wherein X, R1, and R2 are as defined above; n1 in each instance is an integer independently selected from the group of 1 and 2; and n2 in each instance is an integer independently selected from the group of 0 and 1 ; R3 is C1-C4 alkyl; and R4is Ci-C4alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0105] A still further embodiment provides a compound of Formula (III) wherein X, R1, and R2are as defined above; n1 in each instance is an integer independently selected from the group of 1 and 2; and n2 in each instance is an integer independently selected from the group of 0 and 1 ; R3 is C1-C3 alkyl; and R4is C1-C3 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0106] Another embodiment provides a compound of Formula (Illa): wherein:

[0107] R3 is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; and n2 is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. Another embodiment provides a compound of Formula (I I lb): wherein:

[0108] R3 is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; and n2 is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0109] Another embodiment provides a compound of Formula (I I Ic): wherein:

[0110] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; n2 is an integer independently selected from the group of 0, 1 , and 2;

[0111] R9 is selected from the group of methyl and ethyl; and

[0112] R10 is selected from the group of methyl and ethyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0113] Another embodiment provides a compound of Formula (Hid): wherein:

[0114] R3 is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; and

[0115] R7is selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0116] Another embodiment provides a compound of Formula (Hie): wherein:

[0117] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0118] R? is selected from the group of methoxy and ethoxy; and

[0119] R8is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0120] Another embodiment provides a compound of Formula (lllf): wherein:

[0121] R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; and n2 is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0122] Another embodiment provides a compound of Formula (Illg): wherein:

[0123] R3is selected from the group of Ci-C6alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; and n2 is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0124] Further separate embodiments provide a compound, respectively, of Formula (Illa), Formula (I I lb), Formula (lllc), Formula (II Id), Formula (llle), Formula (lllf), and Formula (Illg), wherein, in each separate embodiment:

[0125] R3is selected from the group of C1-C4 alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 , 2, and 3; and n2 is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0126] Further separate embodiments provide a compound, respectively, of Formula (Illa), Formula (I I lb), Formula (lllc), Formula (II Id), Formula (llle), Formula (lllf), and Formula (Illg), wherein, in each separate embodiment:

[0127] R3is selected from the group of C1-C4 alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 and 2; and n2 is an integer independently selected from the group of 0 and 1; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0128] Further separate embodiments provide a compound, respectively, of Formula (Illa), Formula (I I lb), Formula (lllc), Formula (II Id), Formula (llle), Formula (lllf), and Formula (Illg), wherein, in each separate embodiment:

[0129] R3 is selected from the group of C1-C3 alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is an integer independently selected from the group of 1 and 2; and n2 is an integer independently selected from the group of 0 and 1; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0130] Further separate embodiments provide a compound, respectively, of Formula (Illa), Formula (I I lb), Formula (lllc), Formula (II Id), Formula (llle), Formula (lllf), and Formula (Illg), wherein, in each separate embodiment:

[0131] R3 is selected from the group of C1-C2 alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 is 2; and n2 is 0; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0132] Further separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe): wherein, in each separate embodiment: R3is selected from the group of Ci-Ce alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0133] R4is selected from the group of Ci-Ce alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; with the proviso that, when R3is Ci-Ce alkyl, then R4is Ci-Ce alkyl; and when R3is - (CH2)ni-O-(CH2)n2-CH3, then R4is R3-(CH2)ni-O-(CH2)n2-CH3; and with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0134] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment:

[0135] R3is selected from the group of Ci-C4alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0136] R4is selected from the group of Ci-C4alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; with the proviso that, when R3is Ci-C4alkyl, then R4is Ci-C4alkyl; and when R3is - (CH2)ni-O-(CH2)n2-CH3, then R4is R3-(CH2)ni-O-(CH2)n2-CH3; and with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0137] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment: R3is Ci-Ce alkyl; and R4is Ci-Ce alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0138] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment: R3is C1-C4 alkyl; and R4is C1-C4 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0139] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment: R3 is C1-C3 alkyl; and R4is C1-C3 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0140] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment: R3 is C1-C2 alkyl; and R4is C1-C2 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0141] Additional separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment:

[0142] R3is a group of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0143] R4is a group of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; with the proviso that the integers of n1 and n2 in the R3 -(CH2)ni-O-(CH2)n2-CH3 moiety may be the same or different from the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3 moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. Further separate embodiments provide a compound, respectively, of each of Formula (IVa), Formula (IVb), Formula (IVc), Formula (IVd), and Formula (IVe), wherein, in each separate embodiment:

[0144] R3is a group of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0145] R4is a group of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 and 2; n2 in each instance is an integer independently selected from the group of 0 and 1 ; with the proviso that the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3 moiety may be the same or different from the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0146] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg): wherein, in each separate embodiment:

[0147] R3is selected from the group of Ci-Ce alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0148] R4is selected from the group of Ci-C6alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; R? is selected from the group of methoxy and ethoxy; and

[0149] R8, when present, is selected from the group of H, methoxy and ethoxy; with the proviso that, when R3is Ci-Ce alkyl, then R4is Ci-Ce alkyl; and when R3is - (CH2)ni-O-(CH2)n2-CH3, then R4is R3-(CH2)ni-O-(CH2)n2-CH3; and with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0150] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein:

[0151] R3is selected from the group of C1-C4 alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3;

[0152] R4is selected from the group of C1-C4 alkyl and a group of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; R? is selected from the group of methoxy and ethoxy; and

[0153] Rs, when present, is selected from the group of H, methoxy and ethoxy; with the proviso that, when R3is C1-C4 alkyl, then R4is C1-C4 alkyl; and when R3is - (CH2)ni-O-(CH2)n2-CH3, then R4is R3-(CH2)ni-O-(CH2)n2-CH3; and with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0154] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment: R3is Ci-C8alkyl; R4is Ci-C8alkyl; R7is selected from the group of methoxy and ethoxy; and R8, when present, is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0155] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment: R3is C1-C4 alkyl; R4is C1-C4 alkyl; R? is selected from the group of methoxy and ethoxy; and R8, when present, is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment: R3is C1-C3 alkyl; R4is C1-C3 alkyl; R7is selected from the group of methoxy and ethoxy; and Rs, when present, is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0156] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment: R3is C1-C2 alkyl; R4is C1-C2 alkyl; R7is selected from the group of methoxy and ethoxy; and Rs, when present, is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0157] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment:

[0158] R3is a group of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0159] R4is a group of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; R7is selected from the group of methoxy and ethoxy; and

[0160] Rs, when present, is selected from the group of H, methoxy and ethoxy; with the proviso that the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety may be the same or different from the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0161] Further separate embodiments provide a compound, respectively, of each of Formula (IVf) and Formula (IVg), wherein, in each separate embodiment:

[0162] R3is a group of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0163] R4is a group of the formula -(CH2)ni-O-(CH2)n2-CH3;

[0164] R7is selected from the group of methoxy and ethoxy; and

[0165] Rs, when present, is selected from the group of H, methoxy and ethoxy; n1 in each instance is an integer independently selected from the group of 1 and 2; n2 in each instance is an integer independently selected from the group of 0 and 1 ; with the proviso that the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3 moiety may be the same or different from the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3moiety; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0166] For each of the previous embodiments herein providing a compound of Formula (IVf), there is an additional embodiment providing a compound of Formula (IVf) in which R3and R4(including n1 and n2, when present) are as defined in the previous embodiment in question and R7is methoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0167] For each of the previous embodiments herein providing a compound of Formula (IVf), there is an additional embodiment providing a compound of Formula (IVf) in which R3and R4(including n1 and n2, when present) are as defined in the previous embodiment in question and R7is methoxy bound to the 4-position carbon atom of the azetidinyl ring; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0168] For each of the previous embodiments herein providing a compound of Formula (IVg), there is an additional embodiment providing a compound of Formula (IVg) in which R3and R4(including n1 and n2, when present) are as defined in the previous embodiment in question; R7is methoxy; and R8is methoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

[0169] For each of the previous embodiments herein providing a compound of Formula (IVg), there is an additional embodiment providing a compound of Formula (IVg) in which R3and R4(including n1 and n2, when present) are as defined in the previous embodiment in question; R7is methoxy bound to the 3-position carbon atom on the pyrrolidinyl ring; and Rs is methoxy bound to the 4-position carbon atom on the pyrrolidinyl ring; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof. For each of the embodiments provided herein directed to a compound of any of Formula (I), Formula (II), and / or Formula (III), there is a further embodiment in which all variables present (Y, Ri, R2, ni, ni, R3, R4, etc.) are as defined for the prior embodiment in question and X is a single bond. For each of the embodiments provided herein directed to a compound of any of Formula (I), Formula (II), and / or Formula (III), there is a further embodiment in which all variables are as defined for the prior embodiment in question and X is a double bond.

[0170] A limited number of fluorescent molecules exist that stain nerve and / or brain tissue in vivo, where the majority fluoresce at visible wavelengths, and are plagued with high non-specific uptake in surrounding tissues, limiting their ability to highlight the peripheral and central nervous systems (PNS and CNS, respectively).17'33These factors have resulted in characteristically shallow, surface-level nerve imaging (sub-100 pm) with nerve fluorescent intensities on par with non-specific uptake in surrounding tissues as well as variable ability to cross both the blood brain and blood nerve barriers (BBB and BNB, respectively, Table 2 / Fig. 5). However, the importance of intraoperative nerve visualization has been recognized with clinical trials of visibly-labeled, nerve-specific peptides (ALM488, NCT0442068) ongoing for head and neck cancer surgeries. This complementary technology will enable surface weighted intraoperative PNS detection, but due to tissue optical properties, buried nerve visualization will be challenging, and the fluorescent label is spectrally overlapping with Fluorescein. More importantly, due to the large size of the bioaffinity targeting agent, these nerve targeting peptides have been reported to be impermeable to the BBB31Thus, cranial nerve visualization using bioaffinity targeting agents (i.e. , peptides or proteins) in general is unlikely to be feasible. While a NIR nerve-specific probe could substantially improve nerve tissue contrast and readily integrate into the lesser used ICG channel of existing surgical microscopes / exoscopes, design and development have been a significant challenge. CNS-specific contrast agents must have a low molecular weight to cross the BNB and BBB, excluding the utility of large nerve-specific protein targets;34'36however, fluorophores require a sufficient degree of conjugation (i.e., number of double bonds) to reach NIR wavelengths, thereby increasing their molecular weight.28Due to these constraints, probes that act as both the nerve binding agent and fluorescence reporter will be most successful at generating nerve signal to all background tissue contrast after systemic administration for neurosurgery, but synthetic optimization remains challenging. To date, we have designed and synthesized novel NIR water soluble fluorophores with high specificity for both the peripheral and cranial nerves, providing utility for a variety of neurosurgical applications. Definitions

[0171] A “subject” or a “patient” or an “individual” refers to any animal. The animal may be a mammal. Examples of suitable mammals include human and non-human primates, dogs, cats, sheep, cows, pigs, horses, mice, rats, rabbits, and guinea pigs. In some embodiments the subject, patient, or individual is a human, particularly including a human undergoing or in need of a surgical procedure or examination.

[0172] The term "nerve" used herein means a bundle of neural axons. Within a nerve, each axon is surrounded by a layer of connective tissue called the endoneurium. The axons are bundled together into groups called fascicles, and each fascicle is wrapped in a layer of connective tissue called the perineurium. The entire nerve is wrapped in a layer of connective tissue called the epineurium. The term "nerve" is intended to include any tissues (e.g., the sinoatrial node or the atrioventricular node) or structures associated therewith (e.g., neuromuscular junctions).

[0173] The term “nerve-specific” or “nerve specific” herein refers to an agent that is drawn to a nerve or nerve tissue and may be used in fluorescent imaging techniques to help contrast and differentiate the nerve or nerve tissue from surrounding cells and / or tissues. The term “nerve specificity” refers to the nature or activity of an agent being nerve-specific.

[0174] The term “near infrared” or the acronym “(NIR)” refers to light at the near infrared spectrum, generally at a wavelength of about 0.65 to about 1 .4 pm (700 nm-1400 nm. It may also refer to a range designated by the International Organization for Standardization as from a wavelength of about 0.78 pm to about 3 pm. In some embodiments, the preferred near infrared spectroscopy and imaging (NI S) range is from about 650 nm to about 950 nm. In other embodiments, the preferred near infrared spectroscopy and imaging (NIRS) range is from about 650 nm to about 900 nm.

[0175] In some embodiments the agents and / or compositions comprising them are intended for direct / topical administration. Direct or topical administration are understood herein to comprise the administration of an agent or composition directly to surface of a tissue, organ, nerve bundle, or other bodily component. In some methods, the administration may be accomplished by brushing, spraying, or irrigation with the appropriate compound or composition.

[0176] In other embodiments, the agents and / or compositions may be administered systemically to the patient or subject, such as through intravenous injection or infusion.

[0177] In other embodiments, the agents and / or compositions may be administered locally to a desired tissue or organ, such as through injection. The terms “effective amount” or “medically effective amount” or like terms refers to an amount of a compound or composition as described herein to cover a target area sufficiently to complete binding to one or more nerves such that they may be identified through relevant imaging techniques, particularly near-infrared imaging techniques.

[0178] The term “imaging” herein refers to the use of fluorescent compounds in conventional medical imaging techniques, sometimes referred to as near infrared fluorescence imaging or in vivo imaging. Such techniques are sometimes categorized into fluorescence reflectance imaging (FRI) and tomographic fluorescence imaging. Exemplary uses include, but are not limited to, those related to fluorescence image-guided surgery (including minimally invasive laparoscopy or endoscopy techniques), computer-assisted surgery or surgical navigation, radiosurgery or radiation therapy, interventional radiology, fluorescence microscopy, and laser- confocal microscopy. These techniques may include near infrared wavelengths from about 650 nm to 950 nm.

[0179] The term "label" refers to a molecule that facilitates the visualization and / or detection of a targeting molecule disclosed herein. In some embodiments, the label is a fluorescent moiety. The term “labeling” refers to a successful administration of the label to a target to allow such detection.

[0180] As used herein, the terms “robotic surgery”, “robot-assisted surgery”, or “computer-assisted surgery” refer to surgical techniques involving robotic systems that control the movement of medical instruments to conduct a surgical procedure with precise, flexible, and / or minimally invasive actions designed to limit the amount of surgical trauma, blood loss, pain, scarring, and post-surgical patient recovery time and / or complications, such as infection at the surgical area. Examples of robotic surgery include those conducted using the da Vinci Surgical System (Intuitive Surgical, Sunnyvale, CA, USA) approved by the U.S. Food and Drug Administration in 2000.

[0181] The terms “surgery” or “surgical method” as used herein, refers to any method used to manipulate, change, or cause an effect by a physical intervention. These methods include, but are not limited to open surgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, robotic surgery, any procedures that may affect any neuron or nerve, such as placement of retractors during spinal surgery, electrically conducting cardiac tissue or nerve ablation, epidural injection, intrathecal injections, neuron or nerve blocks, implantation of devices such as neuron or nerve stimulators and implantation of pumps. These methods may also include biopsy or other invasive techniques for the collection of cell or tissue samples, such as for diagnostic purposes. As used herein, the term "targeting molecule" refers to any agent (e.g., peptide, protein, nucleic acid polymer, aptamer, or small molecule) that associates with (e.g., binds to) a target of interest. The target of interest may be a nerve cell or an organ or tissue associated with one or more nerve cells or nerve structures. In some embodiments, the targeting molecule is any agent that associates with (e.g., binds to) a target comprising one or more neurons, nerves, or tissues or structures associated therewith, i.e. nerve tissues, nervous system tissues, nerve bundles, etc. It is understood that nerve and nerve-related targets include those associated with the brain and spinal cord of the central nervous system (CNS) and the nerves of the peripheral nervous system (PNS).

[0182] The term “prostatectomy” refers to a surgical technique to remove all or part of a subject’s prostate gland. A “radical prostatectomy” concerns removal of a subject’s entire prostate gland, along with surrounding tissues, often including the seminal vesicles and nearby lymph nodes.

[0183] The terms “orthopedic limb repair” or “orthopedic limb repair surgeries” refer to surgical techniques performed on the limb musculoskeletal system of a subject. These techniques include limb reconstruction surgeries, joint replacement procedures, revision joint surgery, debridement, bone fusions, tendon or ligament repair, internal fixation of bone, and osteotamies.

[0184] The term “fluorophore” herein refers to any one of the compounds described herein for use in imaging techniques, particularly for nerve imaging techniques. Each of the compounds described herein as the product of a specific synthesis or described in a generic description is considered fluorophore for methods, uses, and compositions.

[0185] The term “variable” or “variables” used in the generic descriptions and claims herein refer to the entities or moieties that may, in some instances, be chosen from a specified group. Such variables may include Ri, R2, and R3, and the like.

[0186] All ranges disclosed and / or claimed herein are inclusive of the recited endpoint and independently combinable (for example, the ranges of "from 2 to 10" and “2-10” are inclusive of the endpoints, 2 and 10, and all the intermediate values).

[0187] The term “intraoperatively” as used in describing methods or uses herein refers to an activity that occurs during a surgical procedure or in immediate preparation for such procedure.

[0188] The terms “administer,” “administering”, “administration,” and the like, as used herein, refer to methods used to enable delivery of agents or compositions disclosed herein to the desired site of action, such as a site to be medically imaged. These methods include, but are not limited to parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular, intrathecal, intravitreal, infusion, or local). In some embodiments the administration is topical. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's, Pharmaceutical Sciences (current edition), Mack Publishing Co., Easton, Pa.

[0189] Methods of Use

[0190] Provided is a method of detecting nerves in a tissue or organ, the method comprising a) administering an effective amount of a composition comprising a fluorophore as described herein to the tissue or organ to form a stained tissue or a stained organ; and b) imaging the stained tissue or stained organ, thereby detecting nerves intraoperatively in the stained tissue or stained organ.

[0191] Provided is a method of detecting nerves intraoperatively in a subject undergoing surgery, the method comprising: a) administering an effective amount of a composition comprising a fluorophore as described herein to the subject before or during surgery to form a stained tissue; and b) imaging the stained tissue undergoing surgery in the subject, thereby detecting nerves intraoperatively in the subject undergoing surgery.

[0192] Also provided is a method of detecting nerves intraoperatively in a subject undergoing a prostatectomy surgery, the method comprising: a) administering an effective amount of a composition comprising a fluorophore as described herein to the subject before or during the prostatectomy surgery to form a stained tissue; and b) imaging the stained tissue undergoing surgery in the subject, thereby detecting nerves intraoperatively in the subject undergoing prostatectomy surgery.

[0193] In one embodiment is provided a method of detecting cavernous nerves intraoperatively in a subject undergoing a prostatectomy surgery, the method comprising: a) administering an effective amount of a composition comprising a fluorophore as described herein to the subject before or during the prostatectomy surgery to form a stained tissue; and b) imaging the stained tissue undergoing surgery in the subject, thereby detecting cavernous nerves intraoperatively in the subject undergoing prostatectomy surgery.

[0194] For each of the methods herein concerning a prostatectomy surgery or procedure, there is another embodiment in which the surgery or procedure is a radical prostatectomy.

[0195] For each of the methods above and herein, there is an embodiment in which the composition comprising a fluorophore is administered to the subject systemically.

[0196] For each of the methods above and herein, there is an embodiment in which the composition comprising a fluorophore is administered to the subject directly or topically, i.e. through direct administration or topical administration.

[0197] Within each of the methods herein, there is a further embodiment in which the administration of an effective amount of a composition comprising a fluorophore as described herein to the subject before or during the prostatectomy surgery to form a stained tissue can be completed in fifteen minutes or less. In a still further embodiment, the administration of an effective amount of a composition comprising a fluorophore as described herein to the subject before or during the prostatectomy surgery to form a stained tissue can be completed in ten minutes or less.

[0198] Also provided herein are methods of imaging nervous tissue tumors (neoplasms), including Gliomas, such as bliomatosis cerbri, Oligoastrocytomas, Choroid plexus papillomas, Ependymomas, Astrocytomas (Pilocytic astrocytomas and Glioblastoma multiforme), Dysembryoplastic neuroepithelial tumors, Oligodendrogliomas, Medulloblastomas, and Primitive neuroectodermal tumors; Neuroepitheliomatous tumors, such as Ganglioneuromas, Neuroblastomas, Atypical teratoid rhabdoid tumors, Retinoblastomas, and Esthesioneuroblastomas; and Nerve Sheath Tumors, such as Neurofibromas (Neurofibrosarcomas and Neurofibromatosis), Schwannomas, Neurinomas, Acoustic neuromas, and Neuromas.

[0199] Provided is a method of imaging a target area in a subject, the method comprising contacting the target area in the subject with an effective amount of a compound selected from those herein and detecting the compound in the target using fluorescence or near-infrared imaging.

[0200] Also provided is a method of imaging one or more nerves in a target area in a subject, the method comprising contacting the target area in the subject with an effective amount of a compound selected from those herein and detecting the compound in the target using fluorescence imaging. Also provided is a method of imaging one or more nerves in a target area in a subject, the method comprising contacting the target area in the subject with a compound selected from those herein and detecting the compound in the target using near-infrared imaging.

[0201] Also provided is a method of minimizing nerve damage in a target area in a subject during a medical procedure, the method comprising the steps of: a) contacting the target area in the subject with an effective amount of a compound selected from those herein; b) detecting one or more nerves bound by the compound in the target area using fluorescence imaging; and c) minimizing actions of the medical procedure that may damage one or more nerves detected.

[0202] The method above may be used to identify nerves and minimize damage to them that may be caused by a medical procedure, including traumatic, thermal, and radiological damage or that are caused by the application of therapeutic agents, anesthetics, or anesthesia in the target area.

[0203] In some embodiments, the medical procedure referenced in the method above is a surgical procedure. In other embodiments, the medical procedure is a biopsy procedure, a radiological procedure, or the application of anesthetic or anesthesia to the subject. In further embodiments, the medical procedure in the method above is the insertion or implantation of a medical device, including a medical pump, stent, pacemaker, port, artificial joints, valves, screws, pins, plates, rods, cosmetic implants, neurostimulators, and the like.

[0204] Also provided is the use of any compound disclosed herein in the preparation of a composition for use in imaging one or more nerves in a subject using near-infrared imaging.

[0205] Nerve damage plagues surgical outcomes, significantly affecting post-surgical quality of life. Despite the practice of nerve sparing techniques for decades, intraoperative nerve identification and sparing remains difficult and success rates are strongly correlated with surgeon experience level and ability to master the technique (Walsh & Donker. The Journal of urology 128, 492-497 (1982); Ficarra et al. Eur Urol 62, 405-417 (2012); Damber & Khatami. Acta oncologica 44, 599-604 (2005)). Fluorescence-guided surgery (FGS) shows promise for enhanced visualization of specifically highlighted tissue, such as nerves and tumor tissue, intraoperatively. FGS using optical imaging technology is capable of real-time, wide field identification of targeted tissues with high sensitivity and specificity from tissue targeted fluorescent probes. See, for instance: Frangioni. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 26, 4012-4021 (2008); Gibbs. Quantitative imaging in medicine and surgery 2, 177-187 (2012); Gioux et al. Molecular imaging 9, 237-255 (2010); Vahrmeijer et al. Nature reviews. Clinical oncology 10, 507-518 (2013); and Nguyen et al. Nature reviews. Cancer 13, 653-662 (2013). Operating in the near-infrared (NIR) optical window (650-900 nm wavelengths) where tissue chromophore absorbance, autofluorescence and scattering are minimal, FGS technologies have the ability to identify targeted tissues at millimeter to centimeter depths against a black background (Chance. Annals of the New York Academy of Sciences 838, 29-45 (1998); Gibbs. Quantitative imaging in medicine and surgery 2, 177-187 (2012)).

[0206] Several imaging systems have been developed for FGS applications, see, for instance: Lee et al. Plastic and reconstructive surgery 126, 1472-1481 (2010); Tummers et al. European journal of surgical oncology: the journal of the European Society of Surgical Oncology and the British Association of Surgical Oncology 40, 850-858 (2014); Troyan et al. Annals of surgical oncology 16, 2943-2952 (2009); Ashitate et al. Real-time simultaneous near-infrared fluorescence imaging of bile duct and arterial anatomy. The Journal of surgical research 176, 7-13 (2012); Verbeek etal. The Journal of urology 190, 574-579 (2013); Gibbs-Strauss et al. Molecular imaging 10, 91-101 (2011); Hirche et al. Surgical innovation 20, 516-523 (2013); Gotoh et al. Journal of surgical oncology 100, 75-79 (2009); and Kitagawa et al. Anticancer research 35, 6201-6205 (2015); Importantly, the da Vinci surgical robot, frequently used for robotic assisted radical prostatectomy (RP), can be equipped with an FDA approved fluorescence imaging channel.

[0207] Direct administration (also sometimes referred to as local administration) is an attractive alternative to systemic administration of fluorescent probes for minimizing potential toxicity and easing regulatory burdens for first in human clinical studies. By selectively labeling tissues within the surgical field, direct administration requires a significantly lower dose than systemic administration. A direct administration methodology has been developed that provides equivalent nerve signal to background (SBR) to systemic administration following a 15-minute staining protocol. Barth & Gibbs. Theranostics 7, 573-593 (2017). This methodology has been successfully applied to autonomic nerve models, which closely mimic the nerves surrounding the prostate. This method has additional benefits in the application to RP since nerve labeling via systemic administration during RP would generate high background from nerves in the prostate, which are not able to be spared, and renal fluorophore clearance, producing significant fluorescence signal in the urine within the adjacent bladder. Both of these extraneous fluorescence signals would diminish the ability to identify the cavernous nerves within the neurovascular bundle (NVB), which are responsible for continence and potency (Barth and Summer. Theranostics (2016). Tewari et al. BJU international 98, 314-323 (2006); Patel et al. Eur Urol 61 , 571-576 (2012)). Perhaps most importantly, the direct administration methodology requires 16 times lower dose than systemic administration and when scaled to humans by body surface area the dose falls within the requirements for clinical translation under an exploratory investigational new drug (eIND) application to the FDA. Studies conducted under an eIND require minimal preclinical toxicity testing, since only a microdose (<100 pg) is administered to each patient, significantly reducing the cost of first-in-human studies.

[0208] While the direct administration methodology has provided high nerve specificity and SBR with a short staining protocol in preclinical rodent models (Barth & Gibbs. Theranostics 7, 573- 593 (2017)), preliminary staining studies in large animal models generated significant background. To facilitate clinical translation, an improved formulation strategy that is FDA approved and facilitates increased application control for staining a variety of tissue surfaces, angles, and morphologies will be required.

[0209] Several classes of nerve specific fluorescence imaging probes have been studied preclinically for FGS. See, for instance: Gibbs-Strauss et al. Molecular imaging 10, 91-101 (2011); Wu et al. Journal of medicinal chemistry 51 , 6682-6688 (2008); Wang et al. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 58, 611-621 (2010); Gibbs et al. PloS one 8, e73493 (2013); Stankoff et al. Proceedings of the National Academy of Sciences of the United States of America 103, 9304-9309 (2006); Cotero et al. Molecular imaging and biology : MIB : the official publication of the Academy of Molecular Imaging 14, 708-717 (2012); Cotero et al. PloS one 10, e0130276 (2015); Bajaj et al. The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society 61 , 19-30 (2013); Gibbs-Strauss et al. Molecular imaging 9, 128-140 (2010); Meyers et al. The Journal of Neuroscience: the official journal of the Society for Neuroscience 23, 4054-4065 (2003); Wang et al. The Journal of neuroscience: the official journal of the Society for Neuroscience 31 , 2382-2390 (2011); Park et al. Theranostics 4, 823-833 (2014). Of these, oxazine fluorophores (e.g., Oxazine 4) have demonstrated the most promise for clinical translation, with high nerve specificity following both direct and systemic administration. (3-(diethyl-l4-azaneylidene)-N-ethyl-8-methyl-3H- phenoxazin-7-amine) is a particularly promising compound and was chosen as the lead compound for advancement to clinical studies. Although Comparative Example No. 1 has been shown to demonstrate high nerve specificity and adequate fluorescence signal for real time imaging, previous studies have been conducted utilizing a co-solvent formulation as a vehicle for intravenous injection (Gibbs-Strauss et al. Molecular imaging 10, 91-101 (2011); Barth & Gibbs. Theranostics 7, 573-593 (2017)). The co-solvent formulation is only stable at room temperature for <30 minutes, cannot solubilize concentrations above 5mg / mL, and requires the use of dimethyl sulfoxide and Kolliphor EL as solubilizing agents, which hampers clinical translation due to vehicle induced toxicity issues. Additionally, the co-solvent formulation is liquid based and thus not ideal for staining angled or vertical tissue surfaces. Therefore, a clinically viable formulation with FDA approval was needed for direct administration and intravenous injection of nerve-specific fluorescence for FGS.

[0210] Also provided herein are pharmaceutical or medical compositions comprising one or more fluorophore compound(s) described herein (including those of Formula (I) or any other compound formula or structure described herein) and a pharmaceutically or medically acceptable carrier or excipient. In some embodiments the composition is intended for direct / topical administration. Direct administration refers to an application of the compound or composition in question directly to a tissue or organ of interest, such as by irrigation, misting, spraying, swabbing, wiping, brushing, or other means of direct application. Systemic administration refers to administration of a compound or composition such that an entire system, organ, or tissue of interest receives compound dispersion sufficient for imaging or other medical purposes. Systemic administration includes parenteral administration, such as intravenous, intramuscular, or subcutaneous administrations by injection, infusion, or other means.

[0211] Suitable pharmaceutically-acceptable nonaqueous solvents that may be used as carriers or excipients with the present compounds include the following (as well as mixtures thereof): alcohols (these include, for example, o-glycerol formal, p-glycerol formal, 1 ,3-butyleneglycol; aliphatic or aromatic alcohols such as methanol, ethanol, propanol, isopropanol, butanol, t- butanol, hexanol, octanol, amylene hydrate, benzyl alcohol, glycerin (glycerol), glycol, hexylene, glycol, tetrahydrofuranyl alcohol, cetyl alcohol, and stearyl alcohol); fatty acid esters of fatty alcohols (polyalkylene glycols, such as polypropylene glycol and polyethylene glycol), sorbitan, sucrose, and cholesterol; amides such as dimethylacetamide (DMA), benzyl benzoate DMA, dimethylformamide, N-hydroxyethyl-lactamide, N,N-dimethylacetamide-amides, 2-pyrrolidinone, 1-methyl-2-pyrrolidinone, and polyvinylpyrrolidone; acetate esters, such as monoacetin, diacetin, and triacetin; aliphatic and aromatic esters, such as ethyl caprylate or octanoate, alkyl oleate, benzyl benzoate, or benzyl acetate; dimethylsulfoxide (DMSO); esters of glycerin (e.g., mono, di, and tri-glyceryl citrates and tartrates), ethyl benzoate, ethyl acetate, ethyl carbonate, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, glyceryl monostearate, glyceride esters (e.g., mono, di, or tri-glycerides), fatty acid esters (e.g., isopropyl myristate), fatty acid derived PEG esters (e.g., PEG-hydroxyoleate and PEG-hydroxystearate), N-methyl pyrrolidinone, pluronic 60, polyoxyethylene sorbitol oleic polyesters (e.g., Poly(ethoxylated)30-60 sorbitol poly(oleate)2-4, poly(oxyethylene)i5-20 monooleate, poly(oxyethylene)i5-20 mono 12-hydroxystearate, and poly(oxyethylene)i5-2o mono ricinoleate), polyoxyethylene sorbitan esters (e.g., polyoxyethylene- sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and POLYSORBATES 20, 40, 60, and 80, polyvinylpyrrolidone, alkyleneoxy modified fatty acid esters (e.g., polyoxyl 40 hydrogenated castor oil and polyoxyethylated castor oils, such as CREMOPHOR EL solution or CREMOPHOR RH 40 solution), saccharide fatty acid esters (i.e. , the condensation product of a monosaccharide (e.g., pentoses, such as, ribose, ribulose, arabinose, xylose, lyxose, and xylulose; hexoses, such as glucose, fructose, galactose, mannose, and sorbose; trioses; tetroses; heptoses; and octoses), disaccharide (e.g., sucrose, maltose, lactose, and trehalose), oligosaccharide, or a mixture thereof with one or more C4-C22 fatty acids (e.g., saturated fatty acids, such as caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, and stearic acid; and unsaturated fatty acids, such as palmitoleic acid, oleic acid, elaidic acid, erucic acid, and linoleic acid), and steroidal esters); ethers such as diethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethyl ether), and glycofurol (tetrahydrofurfuranyl alcohol polyethylene glycol ether); ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone; hydrocarbons such as benzene, cyclohexane, dichloromethane, dioxolanes, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylenesulfone, tetramethylenesulfoxide, toluene, dimethylsulfoxide (DMSO); and tetramethylene sulfoxide; oils such as mineral oils, vegetable oils, glycerides, animal oils, oleic oils, alkyl, alkenyl, or aryl halides, monoethanolamine; petroleum benzine; trolamine; omega-3 polyunsaturated fatty acids such as alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid, or docosahexaenoic acid; polyglycol ester of 12-hydroxystearic acid and polyethylene glycol (SOLUTOL HS-15, from BASF, Ludwigshafen, Germany); polyoxyethylene glycerol; sodium laurate; sodium oleate; and sorbitan monooleate. Other pharmaceutically acceptable solvents for use in the invention are well known to those of ordinary skill in the art.

[0212] Additional components can cryoprotective agents; agents for preventing reprecipitation of the dithienopyrrole compound or salt surface; active, wetting, or emulsifying agents (e.g., lecithin, polysorbate-80, TWEEN 80, pluronic 60, and polyoxyethylene stearate); preservatives (e.g., ethyl-p-hydroxybenzoate); microbial preservatives (e.g., benzyl alcohol, phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal, and paraben); agents for adjusting pH or buffering agents (e.g., acids, bases, sodium acetate, sorbitan monolaurate, etc.); agents for adjusting osmolarity (e.g., glycerin); and diluents (e.g., water, saline, electrolyte solutions, etc.).

[0213] One embodiment provides a composition comprising at least one fluorescent compound as described herein, such as a compound of Formula I, and dimethyl sulfoxide (DMSO).

[0214] Another embodiment provides a composition or formulation comprising an effective amount of a compound of Formula (I) and at least one pharmaceutically acceptable carrier. Examples of gel-based compositions or formulations include those in U.S. Patent Publications US 2021 / 0236657 A1 (U.S. Pat. Appln. No. 17 / 266,549) and US 2023 / 0098686 A1 (U.S. Pat. Appln. No. 17 / 797,819), the contents of which are incorporated in their entirety herein by reference.

[0215] Another embodiment provides a gel-based composition or formulation comprising an effective amount of a compound of Formula (I) and 5-10% sodium alginate and / or 18-26% PEO- PPO-PEO triblock copolymer.

[0216] Formulations comprising one or more of the compounds disclosed herein can be used to image nerves or nerve tissue. In particular embodiments, the formulations of the disclosure can be used to image nerves or nerve tissue in a subject. In particular embodiments, images of nerves can be obtained intraoperatively during FGS. In particular embodiments, the visualization of nerves during FGS allows surgery to be performed on tissue of interest while sparing nerves so as to reduce incidence of nerve injury during surgery. The area where surgery is performed or nearby regions can be surgically exposed. Surgery can be performed on organs, which include tissues such as nerve tissue, muscle tissue, and adipose tissue. The surgery can be laparoscopic, which is minimally invasive and includes the use of a thin, tubular device (laparoscope) that is inserted through a keyhole incision into a part of a subject’s body, such as the abdomen or pelvis. The surgery can be assisted by a robot. Robot-assisted surgery can offer more precision, flexibility, and control, and is often associated with minimally invasive surgery.

[0217] In particular embodiments, the fluorophore concentration (effective amount) in a formulation that is directly applied to nerve tissue includes a concentration range of 40 to 300 pg / mL. In particular embodiments, the fluorophore concentration in a formulation for direct application includes 40 pg / mL, 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, and 200 pg / mL. In particular embodiments, the fluorophore concentration in a formulation for direct application is 50 pg / mL. In particular embodiments, the fluorophore concentration in a formulation for direct application is 200 pg / mL.

[0218] A formulation of the disclosure can be systemically applied to a subject for imaging of nerves. In particular embodiments, systemic application of a formulation includes intravenous injection of the formulation into a subject.

[0219] A formulation that is directly applied to a tissue can be allowed to penetrate the tissue for a given amount of time after direct application. In particular embodiments, the formulation can be allowed to penetrate the tissue for 30 seconds to 15 minutes, for 1 to 10 minutes, for 1 to 5 minutes, for 1 minute, for 2 minutes, for 3 minutes, for 4 minutes, or for 5 minutes. In particular embodiments, the formulation can be allowed to penetrate the tissue for 1 to 2 minutes. A formulation that is systemically applied to a subject can be administered a sufficient time before imaging such that the formulation can reach the area to be imaged and is present in such area at the time of imaging. In particular embodiments, a formulation that is systemically applied to a subject can be administered a sufficient time prior to imaging to allow uptake of the formulation by tissue in the subject. In particular embodiments, the formulation may be administered up to or less than 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours before imaging. The amount of time required may depend on the nerve imaging application and the administration site. In particular embodiments, the formulation is administered no more than 30 minutes, 1 hour, 2 hours, 3 hours, or 4 hours before imaging. In particular embodiments, the formulation is administered no more than 2 hours before imaging.

[0220] Tissue stained by a formulation including a fluorophore by direct application can be washed with buffer prior to imaging of the stained tissue. Washing of tissue stained by a formulation including a fluorophore can include flushing the tissue with an appropriate buffer and removing the buffer. In particular embodiments, the stained tissue can be washed 1 to 18 times, 1 to 10 times, 1 to 6 times, 1 time, 2 times, 3 times, 4 times, 5 times, or 6 times, with wash buffer. In particular embodiments, the stained tissue can be washed 6 times. In particular embodiments, the wash buffer is phosphate-buffered saline (PBS). In particular embodiments, washing the stained tissue removes unbound fluorophore. In particular embodiments, washing the stained tissue increases the nerve signal intensity and / or the signal to background ratio (SBR) as compared to no washing of the stained tissue. In particular embodiments, washing the stained tissue resolubilizes the fluorophore and allows for further diffusion of the fluorophore into the nerve tissue.

[0221] Imaging a tissue stained by a formulation including a fluorophore includes applying light to tissue that has been stained with a formulation of the disclosure. The light can be at a wavelength sufficient to excite the fluorophore in the formulation to fluoresce. In particular embodiments, light to excite the fluorophore is at a wavelength in the near infrared spectra. In particular embodiments, the fluorophore of a formulation emits at a wavelength in the near infrared spectra. In particular embodiments, the near infrared spectra includes a wavelength of 700 to 900 nm.

[0222] Imaging a tissue stained by a formulation including a fluorophore includes obtaining fluorescence images of the stained tissue by optical imaging systems such as ones described in the Examples.

[0223] In particular embodiments, imaging a tissue includes observing fluorescence images of the stained tissue. The fluorescence images can include still images (whether printed or on screen), or real-time images on a video monitor. In particular embodiments, the individual images of nerves obtained by staining of the nerves with the present formulations can be used for diagnostic purposes and for documentation of nerve location. By observing the fluorescence images the surgical team can determine the absence or presence of a nerve in the image. The surgical team can thus use information about the presence / absence or location of one or more nerves to determine how they will perform the surgical procedure. For example, based on information obtained through the disclosed methods, the surgical team may decide to perform a surgical cut at a point in the tissue where they are less likely to inadvertently cut or surgically contact a particular nerve based on the perceived absence of a nerve in an area of the tissue.

[0224] The information obtained from the obtained image can aid in grafting the ends of the nerves if they are transected. In the event of transection, nerve grafts can be applied directly to the ends to facilitate sprouting of regenerative neural fibers. In this case, the light visible from the fluorescence of the ends of transected nerves provides a target to guide the anastomosis of the nerves by the nerve graft.

[0225] Formulations of the present disclosure to detect nerve tissue can also be provided as kits. Kits for detecting nerve tissue can include, in different containers: (i) a water-based formulation comprising a fluorophore, and (ii) one or more wash buffers. Kits can also include a notice in the form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use, or sale for human administration. The notice may state that the provided active ingredients can be administered to a subject. The kits can include further instructions for using the kit, for example, instructions regarding: directly applying the formulations to a tissue; washing to remove excess formulation; systemically administering the formulations to a subject; applying light for visualization of the fluorophores; capturing fluorescent images of the tissue; proper disposal of related waste; and the like. The instructions can be in the form of printed instructions provided within the kit or the instructions can be printed on a portion of the kit itself. Instructions may be in the form of a sheet, pamphlet, brochure, CD-ROM, or computer-readable device, or can provide directions to instructions at a remote location, such as a website. In particular embodiments, kits can also include some or all of the necessary laboratory and / or medical supplies needed to use the kit effectively, such as syringes, ampules, tubing, gloves, tubes, buffers, and the like. Variations in contents of any of the kits described herein can be made.

[0226] General All reagents were purchased from Sigma Aldrich, Fisher Scientific, or TCI. Unless otherwise indicated, all commercially available starting materials were used directly without further purification. Analytical TLC was performed on Millipore ready-to-use plates with silica gel 60 (F254, 32-63 p.m). Purification was performed on a Biotage Isolera Flash System using prepacked silica gel cartridges or on a reverse phase preparative HPLC (Agilent 1250 Infinity HPLC).

[0227] LCMS characterization

[0228] Mass-to-charge ratio and purity of the Oxazine compounds were characterized on an Agilent 6244 time-of-flight LCMS with diode array detector VL+. Sample (10 uL) was injected into a C18 column (Poroshell 120, 4.6 x 50 mm, 2.7 micron), and eluted with a solvent system of A (H2O, 0.1% FA) and B (MeCN, 0.1% FA) at 0.4 mL / min, from A / B = 90 / 10 to 5 / 95 over 10 min, maintained at A / B = 5 / 95 for additional 5 min. Ions were detected in positive ion mode by setting the capillary voltage at 4 kV and gas temperature at 350° C.

[0229] UV-Vis absorption and fluorescence spectroscopy

[0230] UV-Vis and fluorescence spectra were collected on a SpectraMax M5 spectrometer with a Microplate reader (Molecular Devices, Sunnyvale, CA). All absorbance spectra were reference corrected. Extinction coefficient was calculated from Beer’s Law plots of absorbance versus concentration. Relative quantum yields are reported using HITCI as reference. Excitation emission matrices (EEMs) were collected on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies), using 5-nm step size. The band pass for excitation and emission was 10 nm.

[0231] Water solubility measurements.

[0232] Each screening candidate was dissolved in a 1 mL mixture of chloroform and methanol (equal volume) with final stock concentrations ranging from 10 to 50 mM. The solvent was then removed in vacuo before 200 .L of DI water was added. The test sample was then vortexed before sonicated in an ultrasonic bath for 30 minutes. The undissolved pellet was removed by centrifugation at 13,000 rpm for 5 minutes. The supernatant was sampled and diluted with water before measured for absorbance using a SpectraMax M5 spectrometer with a Microplate reader (Molecular Devices, Sunnyvale, CA). The water solubility of each screening candidate was then calculated using Beer’s Law plots of absorbance versus concentration. The water solubility concentration unit (mM) of each sample was then converted and reported as mg / mL. Experimental LogD measurements.

[0233] Each screening candidate was dissolved in DMSO at a concentration of 10 mM. The stock solution was sampled (2 pL) and added to a 1 mL mixture of 1 -octanol and PBS buffer (equal volume). The solution was then vortexed for 30 mins at room temperature before centrifuged at 13,000 rpm for 5 minutes. The PBS buffer and 1-octanol layers were separated and measured for absorbance using a SpectraMax M5 spectrometer with a Microplate reader (Molecular Devices, Sunnyvale, CA). Sample concentration in each phase was then calculated using Beer’s Law plots of absorbance versus concentration. The experimental LogD value for each screening candidates was calculated using the equation below.

[0234] Sample concentration in PBS buffer LogD = Log(- - : - - - r)

[0235] Sample concetration in 1 — octanol

[0236] Nerve-Specificity Screening using Direct / Topical Administration

[0237] Each compound was screened for its tissue-specificity using a previously published direct / topical administration strategy in murine brachial plexus and sciatic nerves. Each compound from the nerve dye library was formulated in phosphate buffered saline solution at 125 pM. 100 pL of the formulated Oxazine were incubated on the exposed brachial plexus or sciatic nerve for 5 minutes. The fluorophore containing solution was removed and the area was irrigated with saline 18 times to remove any unbound fluorophore. Co-registered fluorescence and color images were collected of each stained area 30 minutes after Oxazine direct / topical administration using a custom-built macroscopic imaging system with 620 / 60 nm excitation and 700 / 75 nm bandpass emission filters. Custom written MatLab code was used to analyze the tissue specific fluorescence where regions of interest were selected on the nerve, muscle and adipose tissue using the white light images. These regions of interest were then analyzed on the co-registered matched fluorescence images permitting assessment of the nerve to muscle and nerve to adipose ratios.

[0238] Nerve-Specificity Screening using Systemic Administration

[0239] Each compound was screened for its tissue-specificity using a previously published systemic administration strategy in murine brachial plexus and sciatic nerves. Each compound from the Oxazine library was formulated in phosphate buffered saline solution at 2 mM. 100 pL of the formulated Oxazine were administered intravenously 2 hours before exposing the brachial plexus and sciatic nerves. Co-registered fluorescence and color images were collected of each nerve site using a custom-built macroscopic imaging system with 620 / 60 nm excitation and 700 / 75 nm bandpass emission filters. Custom written MatLab code was used to analyze the tissue specific fluorescence where regions of interest were selected on the nerve, muscle and adipose tissue using the white light images. These regions of interest were then analyzed on the co-registered matched fluorescence images permitting assessment of the nerve to muscle and nerve to adipose ratios in blinded manner.

[0240] Chemical synthesis

[0241] Scheme 1 : Synthetic route to ARM25-04. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 3-methoxyazetidine, dioxane, 102 °C; b) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; c) 3-(diethylamino)phenol, PPSE, CF3CH2OH, 83 °C.

[0242] 3-methoxy-1-(3-methoxyphenyl)azetidine (2): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)3(97.92 mg, 106.93 pmol), 3-methoxyazetidine hydrochloride (145.36 mg, 1.18 mmol), Xphos (149.70 mg, 320.79 pmol), and CS2CO3 (1.08 g, 3.31 mmol). The vial was evacuated under vacuum and backfilled 5x with N2before the vial was sealed with crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (10 mL). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 1 (500 mg, 2.30 mmol), delivered by syringe through the septum cap. The reaction was heated to 102 °C and stirred vigorously for 4 h. The reaction was then cooled to rt, diluted with DCM (20 mL) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to provide compound 2 (194.5 mg, 94.12%) as a yellow oil. (E)-3-methoxy-1-(3-methoxy-4-((4-nitrophenyl)diazenyl)phenyl)azetidine (3): A solution of compound 2 (50 mg, 258.74 pmol) in MeOH (1 mL) was treated with HCI (2 M, 10 ml_) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (64.37 mg, 271.67 pmol) was added to the solution in 3 portions over 15 mins and stirred at 0 °C for 1 h. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 3 as a dark red solid, used in the next step without further purification.

[0243] 3-(diethylamino)-7-(3-methoxyazetidin-1-yl)phenoxazin-5-ium (ARM25-04): ) 3-

[0244] (diethylamino)phenol (105 mg, 636.45 pmol) and compound 3 (246.79 mg, 635.45 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (106.52 pL, 826.09 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-12%, over 5 cv, isocratic hold at 12%, 10 cv) in CH2CI2containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM25-04 (107.8 mg, 50.13%) as a dark green film.

[0245]

[0246] Scheme 2: Synthetic route to ARM25-17. Reagents and conditions: : a) MOMCI, NaH, DMF, 0 °C; b) Pd2(dba)3, Xphos, CS2CO3, 2-oxa-6-azaspiro[3.3]heptane, dioxane, 102 °C; c) Etl, Na2CC>3, MeCN, 83 °C; d) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C e) 6, PPSE, CF3CH2OH, 83 °C.

[0247] 1-bromo-3-(methoxymethoxy)benzene (5): Under N2, a solution of compound 4 (20.00 g, 115.6 mmol) in anhydrous DMF (25 mL) was chilled in an ice bath for 30 mins prior to the addition of NaH (6.01 g, 150.28 mmol) portion-wise over 10 mins. The reaction mixture was then stirred for 1 h while the temperature was maintained at 0 °C. Methoxymethyl chloride (9.66 mL, 127.16 mmol) was then added to the solution dropwise and the resulting reaction mixture slowly warmed to rt overnight, or about 16 h. The reaction was then chilled in an ice bath, quenched by the careful addition of 1 N HCI (100 mL), and the crude product extracted with EtOAc (3 x 100 mL). The combined organic layers were rinsed with a 5% LiCI solution and brine, then dried over anhydrous Na2SC>4. Solvent was removed under reduced pressure and the residue was purified by flash column chromatography with silica gel, using Hexanes as eluent to give compound 5 (20.92 g, 83.36%) as a translucent, colorless oil. 6-(3-(methoxymethoxy)phenyl)-2-oxa-6-azaspiro[3.3]heptane (6): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)3(210.94 mg, 230.35 pmol), Xphos (322.48 mg, 691.05 pmol), and Cs2CO3(2.33 g, 7.14 mmol). The vial evacuated under vacuum and backfilled 5x with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (5 mL). To the suspension was added 2-oxa-6-azaspiro[3.3]heptane (228.35 pL, 2.53 mmol) via syringe through the septum cap and the mixture was stirred for 5 mins at rt prior to the addition of compound 5 (500 mg, 2.30 mmol), delivered by syringe through the septum cap. The reaction was heated to 102 °C and stirred vigorously for 6 h. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to afford compound 7 (340 mg, 62.47%) as an amber oil.

[0248] A / ,A / -diethyl-3-methoxyaniline (8): In a 250 mL recovery flask under N2, compound 7 (9.09 mL, 81.2 mmol, 1 .0 equiv.) and Na2CO3(17.21 g, 162.4 mmol) were suspended in 50 mL anhy. MeCN. To the mixture at rtwas added iodoethane (13.06 mL, 162.4 mmol) before the reaction was placed on a pre-heated oil bath (83 °C) and refluxed overnight. Once complete, MeCN was removed from the reaction under reduced pressure and the residue was resuspended in 50 mL DCM and filtered through a pre-packed Celite® funnel. Purification by column chromatography with a gradient of ethyl acetate (1-10%) in hexanes over 10 column volumes afforded compound 8 as a translucent, colorless oil (12.71 g, 87.31%).

[0249] (E)-N,N-diethyl-3-methoxy-4-((4-nitrophenyl)diazenyl)aniline (9): A solution of compound 8 (5.00 g, 27.89 mmol) in MeOH (10 mL) was treated with HCI (2 M, 50 mL) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (6.94 g, 29.29 mmol) was added to the solution in 3 portions over 15 mins and stirred at 0 °C for 1 h. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 9 as a bright red solid, used in the next step without further purification.

[0250] 3-(diethylamino)-7-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenoxazin-5-ium (ARM25-17):

[0251] Compounds 7 (200 mg, 850.04 pmol) and 3 (318.24 mg, 850.04 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (10 mL) containing trimethylsilyl polyphosphate (142.5 pL, 1.11 mmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-12%, over 5 cv, isocratic hold at 12%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM25-17 as a green film (55.1 mg, 18.5%)

[0252] Scheme 3: Synthetic route to ARM25-18. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 6-oxa-1-azaspiro[3.3]heptane hemioxalate, dioxane, 102 °C; b) 3, PPSE, CF3CH2OH, 83 °C.

[0253] 1-(3-(methoxymethoxy)phenyl)-6-oxa-1-azaspiro[3.3]heptane (10): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)s (295.31 mg, 322.49 pmol), Xphos (451.47 mg, 967.47 pmol), 6-oxa-1-azaspiro[3.3]heptane hemioxalate (1.02 g, 3.55 mmol), and CS2CO3 (3.26 g, 10.0 mmol). The vial was evacuated under vacuum and backfilled 5x with N2 before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1,4-dioxane (7 mL). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (700 mg, 3.22 mmol), delivered by syringe through the septum cap. The reaction heated to 102 °C and stirred vigorously for 4 h. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to provide compound 10 (470.2 mg, 61.97%) as a yellow oil.

[0254] 3-(diethylamino)-7-(6-oxa-1-azaspiro[3.3]heptan-1-yl)phenoxazin-5-ium (ARM25-18):

[0255] Compounds 10 (59.8 mg, 222.02 pmol) and 9 (76.01 mg, 222.02 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (37.22 pL, 288.63 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-12%, over 5 cv, isocratic hold at 12%, 10 cv) in CH2CI2containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM 25- 18 (7.9 mg, 4.35%) as a green film.

[0256] Scheme 4: Synthetic route to ARM25-70. Reagents and conditions: a) PPSE, CF3CH2OH, 83 °C.

[0257] 4-(3-(methoxymethoxy)phenyl)rnorpholine (11): A flame-dried microwave vial was charged with a magnetic stir bar, Pd2(dba)3(421.88 mg, 460.7 pmol), Xphos (644.95 mg, 1.38 mmol), and CS2CO3 (2.1 g, 6.45 mmol). The vial was evacuated under vacuum and backfilled 5 times with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (10 ml_). Morpholine (437.13 pL, 5.07 mmol) was added through the septum cap via syringe and the resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (1 g, 4.61 mmol), delivered by syringe through the septum cap. The reaction mixture was then heated to 102 °C and stirred vigorously for 4 hrs. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was then deposited directly onto silica gel and concentrated to dryness. Purification by silica gel flash chromatography using a mobile phase of DCM and Hexanes provided compound 26 (418.5 mg, 40.69%) as a translucent colorless oil.

[0258] 3-(diethylamino)-7-morpholinophenoxazin-5-ium (ARM25-70): Compounds 11 (85 mg, 380.7 pmol) and 9 (125.01 mg, 380.7 pmol) were dissolved in a solution of trifluoroethanol (5 mL) containing trimethylsilyl polyphosphate (63.82 pL, 494.91 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-70 (33.7 mg, 26.16%) as a dark green film.

[0259] Scheme 5: Synthetic route to ARM 19-38. Reagents and conditions: a) MOMCI, K2CO3, acetone, 0 °C; b) Fe, NH4CI, EtOH, H2O, 90 °C; c) 1-bromo-2-methoxyethane, Lil, K2CO3, MeCN, 83 °C; d) 6, PPSE, CF3CH2OH, 83 °C.

[0260] 1-(methoxymethoxy)-3-nitrobenzene (13): Under N2, compound 11 (12.5 g, 89.86 mmol) and K2CO3 (18.63 g, 134.79 mmol) were suspended in anhy. acetone (100 ml_), stirring for 30 mins before the solution was chilled in an ice bath for an additional 30 mins and methoxymethyl chloride (7.51 mL, 98.84 mmol) added to the chilled solution dropwise over 10 mins. The resulting mixture was left slowly warming to rt overnight. Once complete, the reaction was chilled in an ice bath and filtered through a pre-packed Celite® funnel. Solvent was removed under reduced pressure and the residue was purified by flash column chromatography with silica gel, using Hexanes as eluent to give compound 12 (15.21 g, 92.41%) as a colorless oil.

[0261] 3-(methoxymethoxy)aniline (14): In a 500 mL recovery flask, 12 (15 g, 81.89 mmol), iron dust (25.15 g, 450.42 mmol), and NH4CI ( 3.07 g, 57.33 mmol) were suspended in a mixture of EtOH (90 mL) and water (10 mL), placed on a pre-heated oil bath (90 °C) and refluxed for 3 hrs. The reaction was then cooled to rt and filtered through a pre-packed Celite® funnel. Filtered solids were washed with MeOH (10 mL) and the filtrate dried under reduced pressure. The residue was resuspended in water (50 mL) and the crude product extracted with DCM (3 x 50 mL). The combined organic layers were dried over anhy. Na2SO4, filtered, and dried under reduced pressure. Purification by column chromatography with silica gel and ethyl acetate in hexanes (E:H 1 :4) as eluent afforded 13 (9.28 g, 73.98%) as a colorless oil.

[0262] A / , / V-bis(2-methoxyethyl)-3-(methoxymethoxy)aniline (15): In a 250 mL recovery flask under N2, compound 13 (2.00 g, 13.06 mmol), lithium iodide (1.22 g, 9.14 mmol), and K2CO3 (4.51 g, 32.64 mmol) were suspended in 20 mL anhy. MeCN. To the mixture at rt was added 1-bromo-2- methoxyethane (3.99 mL, 42.43 mmol) before placing the reaction on a pre-heated oil bath (83 °C) to reflux overnight. Once complete, MeCN was removed from the reaction under reduced pressure and the residue was resuspended in 20 mL DCM and filtered through a pre-packed Celite® funnel. Purification by column chromatography with a gradient of ethyl acetate (1-10%) in hexanes over 10 column volumes afforded compound 15 as a translucent, bronze-colored oil (3.05 g, 86.68%).

[0263] 3-(bis(2-methoxyethyl)amino)-7-(3-methoxyazetidin-1-yl)phenoxazin-5-ium (ARM 19-38): Compounds 14 (59.8 mg, 222.02 pmol) and 2 (76.01 mg, 222.02 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (37.22 pL, 288.63 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-12%, over 5 cv, isocratic hold at 12%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM 19- 38 (56.9 mg, 64.31 %) as a blue film.

[0264] Scheme 6: Synthetic route to ARM 19-36. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 2-oxa-6-azaspiro[3.3]heptane, dioxane, 102 °C; b) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; c) 14, PPSE, CF3CH2OH, 83 °C.

[0265] 6-(3-methoxyphenyl)-2-oxa-6-azaspiro[3.3]heptane (16): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)s (48.96 mg, 53.47 pmol), 2-oxa-6-azaspiro[3.3]heptane oxalate (111.25 mg, 588.12 pmol), Xphos (74.85 mg, 160.40 pmol), and CS2CO3 (540.02 mg, 1.66 mmol). The vial was evacuated under vacuum and backfilled 5x with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (1 ml_). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 1 (67.70 pL, 534.66 pmol), delivered by syringe through the septum cap. The reaction was heated to 102 °C and stirred vigorously for 6 h. The reaction was then cooled to rt, diluted with DCM (20 mL) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to provide compound 16 (58.10 mg, 52.94%) as a yellow oil.

[0266] (E)-6-(3-methoxy-4-((4-nitrophenyl)diazenyl)phenyl)-2-oxa-6-azaspiro[3.3]heptane (17): A solution of compound 16 (50 mg, 243.60 pmol) in MeOH (1 mL) was treated with HCI (2 M, 10 mL) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (64.37 mg, 271.67 pmol) was added to the solution in 3 portions over 15 mins and stirred for 1 hr. at 0 °C. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 17 as a dark red solid, used in the next step without further purification.

[0267] 3-(bis(2-methoxyethyl)amino)-7-(2-oxa-6-azaspiro[3.3]heptan-6-yl)phenoxazin-5-ium

[0268] (ARM19-36): Compounds 15 (60 mg, 222.77 pmol) and 17 (78.94 mg, 222.77 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (37.34 pL, 289.60 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-10%, over 5 cv, isocratic hold at 10%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM 19-36 (10.80 mg, 11.81 %) as a dark blue film.

[0269]

[0270] Scheme 7: Synthetic route to ARM 19-37. Reagents and conditions: a) Pd2(dba)3, Xphos, Cs2CO3, 6-oxa-1-azaspiro[3.3]heptane hemioxalate, dioxane, 102 °C; b) 2M HCI, p- nitrobenzenediazonium tetrafluoroborate, 0 °C; c) 14, PPSE, CF3CH2OH, 83 °C.

[0271] 1-(3-methoxyphenyl)-6-oxa-1-azaspiro[3.3]heptane (18): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)3(269.28 mg, 294.06 pmol), 6-oxa-1- azaspiro[3.3]heptane hemioxalate (932.56 mg, 3.23 mmol), Xphos (411.67 mg, 882.18 pmol), and CS2CO3 (2.97 g, 9.12 mmol). The vial was evacuated under vacuum and backfilled 5x with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhydrous 1 ,4- dioxane (5 ml_). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 1 (371 .62 pL, 2.94 mmol), delivered by syringe through the septum cap. The reaction was heated to 102 °C and stirred vigorously for 4 h. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to provide compound 18 (516.90 mg, 85.64%) as an amber oil.

[0272] (E)-1-(3-methoxy-4-((4-nitrophenyl)diazenyl)phenyl)-6-oxa-1-azaspiro[3.3]heptane (19): A solution of compound 18 (100 mg, 487.19 pmol) in MeOH (1 mL) was treated with HCI (2 M, 10 mL) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (121.20 mg, 511.55 pmol) was added to the solution in 3 portions over 15 mins and stirred for 1 hr. at 0 °C. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 19 as a dark red solid, used in the next step without further purification.

[0273] 3-(bis(2-methoxyethyl)amino)-7-(6-oxa-1-azaspiro[3.3]heptan-1-yl)phenoxazin-5-ium

[0274] (ARM19-37): Compounds 14 (71.2 mg, 264.35 pmol) and 18 (93.68 mg, 264.35 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (44.31 pL, 343.65 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-10%, over 5 cv, isocratic hold at 10%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM 19-37 (34.6 mg, 31.9%) as a dark blue film.

[0275] Scheme 8: Synthetic route to ARM19-91. Reagents and conditions: a) ) Pd2(dba)3, Xphos, Cs2CO3, 3-methoxyazetidine, dioxane, 102 °C; b) 2-chloroacetic chloride, K2CO3, MeCN, 80 °C; c) BH3-THF, THF, 0 °C to rt; d) Mel, Na2CO3, MeCN, 80 °C; e) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; f) 20, PPSE, CF3CH2OH, 80 °C. 3-methoxy-1-(3-(methoxymethoxy)phenyl)azetidine (20): A flame-dried microwave vial was charged with a magnetic stir bar Pd2(dba)3(421.88 mg, 460.70 mmol), 3-methoxyazetidine hydrochloride (626.26 mg, 5.07 mmol), Xphos (644.95 mg, 1.38 mmol), and CS2CO3 (4.65 g, 14.28 mmol). The vial was evacuated under vacuum and backfilled 5x with N2before the vial was sealed with crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (10 ml_). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (1 g, 4.61 mmol), delivered by syringe through the septum cap. The reaction was heated to 102 °C and stirred vigorously for 4 h. The reaction was then cooled to rt, diluted with DCM (20 ml_) and filtered through a pre-packed Celite® funnel. The filtrate was deposited directly onto silica gel, concentrated to dryness, and purified by silica gel flash chromatography with a mobile phase of ethyl acetate and hexanes to provide compound 20 (722 mg, 70.19%) as a yellow oil.

[0276] 6-methoxy-2H-benzo[b][1,4]oxazin-3(4H)-one (21): Compound 21 was synthesized following a slightly modified protocol reported by Zhang et al.2Under N2atmosphere, compound 20 (2.00 g, 14.4 mmol) was dissolved in anhydrous MeCN (20 ml_), to which 2-chloroacetyl chloride (1.37 mL, 17.3 mmol) was added dropwise. Following the addition of K2COa (4.97 g, 25.9 mmol), the reaction mixture was heated to 80 °C and stirred overnight. It was then cooled to room temperature, diluted with DCM, and filtered through Celite. The solvent was removed in vacuo, and the residue was purified by silica gel flash chromatography using a mobile phase of EtOAc and Hexanes, provided compound 21 (2.21 g, 86%) as a brown solid.1H NMR (400 MHz, CDCI3) 5 9.16 (br s, 1 H), 6.90 (d, J= 8.9 Hz, 1 H), 6.52 (dd, J= 8.9, 2.8 Hz, 1 H), 6.42 (d, J= 2.8 Hz, 1 H), 4.57 (s, 2H), 3.76 (s, 3H).13C{1H} NMR (101 MHz, CDCh) 6 166.9, 155.5, 137.8, 127.0, 117.3, 109.0, 102.3, 67.5, 56.0. HRMS(ESI) [M+H]+ m / z found 180.0682, calcd for C9HioN03+180.0656.

[0277] 6-methoxy-3,4-dihydro-2H-benzo[b][1,4]oxazine (22): A solution of compound 21 (2.00 g, 11.2 mmol) in anhydrous THF (30ml_) was stirred in an ice bath under N2for 30 mins. Borane tetrahydrofuran complex solution (1 M, 30 mL) was added to the solution above dropwise over 30 mins using a syringe pump, while the temperature of the solution was maintained below 5 °C. The resulting reaction mixture was left to stir in the ice bath and slowly warm to rt. After 24 h, the solution was placed in an ice bath again and excess borane reagent was destroyed by carefully adding MeOH until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography with silica gel, using DCM / Hexane as eluent, providing compound 22 (1.62 g, 88%) as a light pink solid.1H NMR (400 MHz, CDCI3) 6 6.71 (d, J= 8.6 Hz, 1 H), 6.27-6.22 (m, 2H), 3.72 (s, 3H), 3.42-3.41 (m, 2H).13C{1H} NMR (101 MHz, CDCh) 6 154.5, 138.7, 133.2, 117.2, 104.5, 101.9, 65.0, 5.8, 41.2. HRMS(ESI) [M+H]+ m / z found 166.0836, calcd for CgH^NC 166.0863.

[0278] 6-methoxy-4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazine (22): To a suspension of compound 21 (1.00 g, 6.05 mmol) and Na2COs (1.28 g, 12.1 mmol) in anhydrous MeCN (10 ml_) under N2was added Mel (0.400 ml_, 6. 36 mmol) at rt. The reaction mixture was heated to 80 °C and stirred overnight. It was then cooled to room temperature, diluted with DCM, and filtered through Celite. The solvent was removed in vacuo, and the residue was purified by silica gel flash chromatography with silica gel, using DCM / Hexane as eluent to give compound 22 (814 mg, 75%) as a burgundy oil.1H NMR (400 MHz, CDCh) 6 6.69 (d, J= 8.5 Hz, 1 H), 6.27 (d, J= 2.8 Hz, 1 H), 6.20 (dd, = 8.6, 2.8 Hz, 1 H), 4.25-4.23 (m, 2H), 3.75 (s, 3H), 3.28-3.25 (m, J= Hz, 2H), 2.88 (s, 3H).13C{1H} NMR (101 MHz, CDCh) 0 154.8, 138.7, 137.2, 115.8, 101.6, 99.8, 64.7, 55.7, 49.3, 38.8. HRMS(ESI) [M+H]+ m / z found 180.1013, calcd for CIOHI4N02+180.1020.

[0279] (E)-6-methoxy-4-methyl-7-((4-nitrophenyl)diazenyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine (23): A solution of compound 22 (400 mg, 2.23 mmol) in MeOH (1 mL) was treated with HCI (2 M, 10 mL) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (555.22 mg, 2.34 mmol) was added to the solution in 3 portions over 15 mins and stirred for 1 hr. at 0 °C. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 23 as a dark red solid, used in the next step without further purification.. HRMS(ESI) [M+H]+ m / z found 329.1292, calcd for C16H17N4CV 329.1245.

[0280] 8-(3-methoxyazetidin-1-yl)-4-methyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM19-91): Compounds 20 (100 mg, 447.88 pmol) and 25 (147.05 mg, 447.88 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (75.08 L, 582.25 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-10%, over 5 cv, isocratic hold at 10%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM 19-91 (31.30 mg, 20.65%) as a dark green film.

[0281] Scheme 9: Synthetic route to ARM25-19. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0282] 4-methyl-8-(2-oxa-6-azaspiro[3.3]heptan-6-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM25-19): Compounds 6 (100 mg, 425.02 pmol) and 25 (139.55 mg, 425.02 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (6 ml_) containing trimethylsilyl polyphosphate (71.25 pL, 552.53 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-10%, over 5 cv, isocratic hold at 10%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM25-19 (13.5 mg, 9.06%) as a blue-green film.

[0283] Scheme 10: Synthetic route to ARM25-20. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C. 4-methyl-8-(6-oxa-1-azaspiro[3.3]heptan-1-yl)-3,4-dihydro-2H-[1 ,4]oxazino[2,3- b]phenoxazin-6-ium (ARM25-20): Compounds 10 (122 mg, 518.52 pmol) and 25 (170.25 mg, 518.52 pmol) were dissolved in a solution of 2,2,2-trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (86.92 pL, 674.08 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. The residue was initially purified by silica gel flash chromatography using a mobile phase of MeOH (0-20%, over 10 cv, isocratic hold at 20%, 10 cv) in acetone containing 0.5% formic acid, followed by a second silica gel column with a mobile phase of MeOH (gradient, 0-10%, over 5 cv, isocratic hold at 10%, 10 cv) in CH2CI2 containing 0.5% formic acid. The fractions containing product were pooled and evaporated to afford ARM25-20 (17.4 mg, 9.58%) as a blue-green film.

[0284] Scheme 11 : Synthetic route to ARM 19-42. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 3-methoxypyrrolidine, dioxane, 102 °C; b) 25, PPSE, CF3CH2OH, 83 °C.

[0285] 3-methoxy-1-(3-(methoxymethoxy)phenyl)pyrrolidine (26) A flame-dried microwave vial was charged with a magnetic stir bar, Pd2(dba)3(828.99 mg, 905.27 pmol), Xphos (1.27 g, 2.72 mmol), and Cs2CO3(4.13 g, 12.67 mmol). The vial was evacuated under vacuum and backfilled 5 times with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4- dioxane (20 mL). 3-methoxypyrrolidine (1.07 mL, 9.96 mmol) was added through the septum cap via syringe and the resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (1.97 g, 9.05 mmol), delivered by syringe through the septum cap. The reaction mixture was then heated to 102 °C and stirred vigorously for 4 hrs. The reaction was then cooled to rt, diluted with DCM (200 mL) and filtered through a pre-packed Celite® funnel. The filtrate was then deposited directly onto silica gel and concentrated to dryness. Purification by silica gel flash chromatography using a mobile phase of DCM and Hexanes provided compound 25 (1.80 g, 83.44%) as a light-yellow oil.

[0286] 8-(3-methoxypyrrolidin-1-yl)-4-methyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6- ium (ARM19-42): Compounds 26 (97.4 mg, 410.45 pmol) and 25 (131.69 mg, 401.11 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (67.24 pL, 521.44 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-42 (99.8 mg, 68.99%) as a brilliant green film.

[0287] Scheme 12: Synthetic route to LG 19-48. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 2-oxa-6-azaspiro[3.4]octane, dioxane, 102 °C; b) 25, PPSE, CF3CH2OH, 83 °C.

[0288] 6-(3-(methoxymethoxy)phenyl)-2-oxa-6-azaspiro[3.4]octane (27): A flame-dried microwave vial was charged with a magnetic stir bar, Pd2(dba)3(274.22 mg, 460.7 pmol), Xphos (419.22 mg, 898.36 pmol), and CS2CO3 (3.02 g, 9.28 mmol). The vial was evacuated under vacuum and backfilled 5 times with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (6 mL). 2-oxa-6-azaspiro[3.4]octane (345.14 pL, 3.29 mmol) was added through the septum cap via syringe and the resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (650 mg, 2.99 mmol), delivered by syringe through the septum cap. The reaction mixture was then heated to 102 °C and stirred vigorously for 4 hrs. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was then deposited directly onto silica gel and concentrated to dryness. Purification by silica gel flash chromatography using a mobile phase of DCM and Hexanes provided compound 27 (455.2 mg, 60.73%) as a brown oil.

[0289] 4-methyl-8-(2-oxa-6-azaspiro[3.4]octan-6-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM 19-48): Compounds 27 (100 mg, 401.11 pmol) and 25 (134.76 mg, 410.45 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (68.81 pL, 533.59 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-48 (41.3 mg, 28.25%) as a blue-green film.

[0290] Scheme 13: Synthetic route to ARM 19-49 Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, 2-oxa-5-azaspiro[3.4]octane hemioxalate, dioxane, 102 °C; b) 25, PPSE, CF3CH2OH, 83 °C.

[0291] 5-(3-(methoxymethoxy)phenyl)-2-oxa-5-azaspiro[3.4]octane (28): A flame-dried microwave vial was charged with a magnetic stir bar, Pd2(dba)s (274.22 mg, 299.45 pmol), Xphos (419.22 mg, 898.36 pmol), 2-oxa-5-azaspiro[3.4]octane hemioxalate (1.04 g, 3.29 mmol) and CS2CO3 (3.02 g, 9.28 mmol). The vial was evacuated under vacuum and backfilled 5 times with N2 before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4-dioxane (6 mL). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (650 mg, 2.99 mmol), delivered by syringe through the septum cap. The reaction mixture was then heated to 102 °C and stirred vigorously for 4 hrs. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was then deposited directly onto silica gel and concentrated to dryness. Purification by silica gel flash chromatography using a mobile phase of DCM and Hexanes provided compound 28 (570 mg, 76.35%) as a yellow oil.

[0292] 4-methyl-8-(2-oxa-5-azaspiro[3.4]octan-5-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM19-49): Compounds 28 (65.4 mg, 262.32 pmol) and 25 (86.13 mg, 262.32 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (43.97 pL, 341.02 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-49 (26.3 mg, 27.51%) as a blue-green film.

[0293] Scheme 14: Synthetic route to ARM25-71. Reagents and conditions: a) PPSE, CF3CH2OH, 83 °C.

[0294] 4-methyl-8-morpholino-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM25-71): Compounds 11 (105 mg, 470.28 pmol) and 25 (154.41 mg, 470.28 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (78.83 pL, 611 .36 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-71 (3.4 mg, 2.14%) as a dark green film.

[0295]

[0296] Scheme 15: Synthetic route to ARM25-36. Reagents and conditions: a) AC2O, H2O, 50 °C to rt; b) BH3-THF, THF, 0 °C to rt; c) 1-bromo-2-methoxyethane, Lil, K2CO3, MeCN, 83 °C; d) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; e) BBr3, DCM, 0 °C; f) PPSE, CF3CH2OH, 83 °C.

[0297] A / -(3-methoxyphenyl)acetamide (30): Compound 7 (2.00 g, 16.2 mmol) was suspended in 20 mL DI water, to which acetic anhydride (4.61 mL, 48.7 mmol) was added dropwise. The reaction mixture was placed in an ultrasonication bath for 1 min, then stirred in a heated water bath (50 °C) for 10 min. The resulting solution was stirred overnight at rt. The solid product was collected via vacuum filtration and washed with small portions of DI water. The product was left in the funnel, air dried overnight, provided compound 30 (2.37 g, 88%) as an off-white solid, and used for the next step without further purification.

[0298] A / -ethyl-3-methoxyaniline (31): A solution of compound 30 (2.00 g, 12.1 mmol) in anhydrous THF (30mL) was stirred in an ice bath under N2 for 30 mins. Borane tetrahydrofuran complex solution (1 M, 30 mL) was added to the solution above dropwise over 30 mins using a syringe pump, while the temperature of the solution was maintained below 5 °C. The resulting reaction mixture was left to stir in the ice bath and slowly warm to rt. After 24 h, the solution was placed in an ice bath again and excess borane reagent was destroyed by carefully adding MeOH until no gas evolved. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography with silica gel, using DCM / Hexane as eluent, providing compound 31 (1.47 g, 80%) as an oil.

[0299] A / -ethyl-3-methoxy-A / -methylaniline (32): In a 250 mL recovery flask under N2, compound 31 (1 g, 6.61 mmol), lithium iodide (919.2 mg, 6.61 mmol), and Na2CO3(1.4 g, 13.23 mmol) were suspended in 20 mL anhy. MeCN. To the mixture at rt was added 1-bromo-2-methoxyethane (621.08 pL, 6.61 mmol) before placing the reaction on a pre-heated oil bath (83 °C) to reflux overnight. Once complete, MeCN was removed from the reaction under reduced pressure and the residue was resuspended in 20 mL DCM and filtered through a pre-packed Celite® funnel. Purification by column chromatography with a gradient of ethyl acetate (1-10%) in hexanes over 10 column volumes afforded compound 32 as a translucent oil (1 g, 72.25%).

[0300] (E)-N-ethyl-3-methoxy-N-methyl-4-((4-nitrophenyl)diazenyl)aniline (33): Compound 32 (400 mg, 2.42 mmol) was dissolved in MeOH (1 mL). The solution was chilled in an ice bath, then treated with HCI (2 M, 10 mL). After 15 mins, p-nitrobenzenediazonium tetrafluoroborate (602 mg, 2.54 mmol) was added to the solution in 3 portions over an additional 15 mins, then stirred at 0 °C for 1 h. During this time, the color of the reaction mixture changed from orange to dark red. After two hours, the solution was carefully neutralized with solid K2CO3until the pH value of the solution had risen above 7. The precipitate was collected via vacuum filtration and washed with small portions of DI water. The product was left in the funnel, air dried overnight, afforded compound 33 (714 mg, 94%) as a dark red solid, used for the next step without further purification.

[0301] 4-methyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ol (34): Compound 24 (100 mg, 0.558 mmol) was dissolved in anhydrous DCM (5 mL) under N2, and chilled in an ice bath. To the solution above, was added BBr3(1 M in DCM, 2 mL, 1.95 mmol) dropwise. The reaction mixture was slowly warmed up to rt and stirred overnight. The reaction flask was placed in an ice bath, after sufficient amount of time for cooling, water was carefully added to the reaction mixture to destroy excess BBr3and neutralized to pH ~ 7 with Na2CO3. The aqueous phase was then extracted with EtOAc (4 x 10 mL). The combined organics were dried over anhydrous Na2SC>4, filtered, and concentrated in vacuo. Flash chromatography on silica gel yielded compound 34 (61 mg, 66%) as a burgundy oil.

[0302] 8-(ethyl(2-methoxyethyl)amino)-4-methyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6- ium (ARM25-36): Compounds 34 (50 mg, 300.84 pmol) and 33 (107.82 mg, 300.84 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (50.43 pL, 391.1 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-36 (44 mg, 41.3%) as a dark blue film.

[0303] Scheme 16: Synthetic route to ARM25-34. Reagents and conditions: a) PPSE, CF3CH2OH, 83 °C.

[0304] 8-(bis(2-methoxyethyl)amino)-4-methyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6- ium (ARM25-34): Compounds 25 (100 mg, 371.28 pmol) and 15 (121.9 mg, 371.28 pmol) were dissolved in a solution of trifluoroethanol (6 ml_) containing trimethylsilyl polyphosphate (62.64 pL, 482.66 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-34 (28.4 mg, 19.9%) as a dark blue film.

[0305] Scheme 17: Synthetic route to ARM25-11. Reagents and conditions: a) Pd2(dba)3, Xphos, CS2CO3, c / s-3,4-dimethoxypyrrolidine hydrochloride, dioxane, 102 °C; b) PPSE, CF3CH2OH, 83 °C.

[0306] (+) / (-) 3,4-dimethoxy-1-(3-(methoxyrnethoxy)phenyl)pyrrolidine (29): A flame-dried microwave vial was charged with a magnetic stir bar, Pd2(dba)3(421.88 mg, 460.7 pmol), Xphos (644.95 mg, 1.38 mmol), c / s-3,4-dimethoxypyrrolidine hydrochloride (849.51 mg, 5.07 mmol) and CS2CO3 (4.65 g, 14.28 mmol). The vial was evacuated under vacuum and backfilled 5 times with N2before the vial was sealed with a crimp cap and the solid mixture suspended in anhy. 1 ,4- dioxane (10 ml_). The resulting mixture was stirred for 5 mins at rt prior to the addition of compound 5 (1 g, 4.61 mmol), delivered by syringe through the septum cap. The reaction mixture was then heated to 102 °C and stirred vigorously for 4 hrs. The reaction was then cooled to rt, diluted with DCM (50 mL) and filtered through a pre-packed Celite® funnel. The filtrate was then deposited directly onto silica gel and concentrated to dryness. Purification by silica gel flash chromatography using a mobile phase of DCM and Hexanes provided a racemic mixture of 29 (1.17 g, 94.93%) as an orange oil that was used in the next step without additional purification.

[0307] (+) / (-) 8-(3,4-dimethoxypyrrolidin-1-yl)-4-methyl-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM25-11): Compounds 29 (100 mg, 374.08 pmol) and 25 (122.82 mg, 374.08 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (62.71 pL, 486.3 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-11 (24 mg, 16.78%) as a dark green film.

[0308]

[0309] Scheme 18: Synthetic route to LGW19-44. Reagents and conditions: a) Etl, Na2CO3, MeCN, 80 °C; b) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; c) 20, PPSE, CF3CH2OH, 80 °C.

[0310] 4-ethyl-6-methoxy-3,4-dihydro-2H-benzo[b][1,4]oxazine (35): To a suspension of compound 23 (1.00 g, 6.05 mmol) and Na2CC>3 (1.28 g, 12.1 mmol) in anhydrous MeCN (10 mL) under N2was added Etl (0.501 mL, 6.17 mmol) at rt. The reaction mixture was heated to 80 °C and stirred overnight. It was then cooled to room temperature, diluted with DCM, and filtered through Celite. The solvent was removed in vacuo, and the residue was purified by silica gel flash chromatography with silica gel, using DCM / Hexane as eluent to give compound 35 (1.01 g, 86%) as a light brown oil.

[0311] (E)-4-ethyl-6-methoxy-7-((4-nitrophenyl)diazenyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine

[0312] (36): A solution of compound 36 (400 mg, 2.07 mmol) in MeOH (1 mL) was treated with HCI (2 M, 10 mL) and chilled in an ice bath. Once chilled (0 °C), p-nitrobenzenediazonium tetrafluoroborate (515 mg, 2.17 mmol) was added to the solution in 3 portions over 15 mins and stirred for 1 hr. at 0 °C. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 36 as a bright red solid, used in the next step without further purification.

[0313] 4-ethyl-8-(3-methoxyazetidin-1-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium

[0314] (ARM19-44): Compounds 20 (100 mg, 447.88 pmol) and 36 (153.34 mg, 447.88 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (75.08 pL, 582.25 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-44 (62.8 mg, 39.79%) as a dark green film.

[0315] Scheme19: Synthetic route to ARM 19-55. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0316] 4-ethyl-8-(2-oxa-6-azaspiro[3.3]heptan-6-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM19-55): Compounds 6 (100 mg, 425.02 pmol) and 36 (145.51 mg, 425.02 pmol) were dissolved in a solution of trifluoroethanol (6 ml_) containing trimethylsilyl polyphosphate (71.25 pL, 552.53 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-55 (20.4 mg, 13.17%) as a green film.

[0317] Scheme 20: Synthetic route to ARM19-56. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0318] 4-ethyl-8-(6-oxa-1-azaspiro[3.3]heptan-1-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM19-56): Compounds 10 (100 mg, 425.02 pmol) and 36 (145.51 mg, 425.02 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (71.25 pL, 552.53 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-56 (9.6 mg, 6.2%) as a blue-green film.

[0319] Scheme 21 : Synthetic route to ARM19-66. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0320] 4-ethyl-8-(3-methoxypyrrolidin-1-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM19-66): Compounds 26 (100 mg, 421.41 pmol) and 36 (144.27 mg, 421.41 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (70.64 pL, 547.83 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-66 (29 mg, 18.78%) as a brilliant green film.

[0321]

[0322] Scheme 22: Synthetic route to ARM19-54. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0323] 4-ethyl-8-(2-oxa-6-azaspiro[3.4]octan-6-yl)-3,4-dihydro-2H-[1 ,4]oxazino[2,3-fe]phenoxazin- 6-ium (ARM19-54): Compounds 27 (103 mg, 413.14 pmol) and 36 (141.44 mg, 413.14 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (69.26 pL, 537.08 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-54 (78.7 mg, 50.33%) as a dark green film.

[0324] Scheme 23: Synthetic route to ARM25-13. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0325] 4-ethyl-8-(2-oxa-5-azaspiro[3.4]octan-5-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin- 6-ium (ARM25-13): Compounds 28 (147.5 mg, 589.25 pmol) and 36 (201.73 mg, 589.25 pmol) were dissolved in a solution of trifluoroethanol (7 mL) containing trimethylsilyl polyphosphate (98.78 pL, 766.03 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-13 (64.6 mg, 28.97%) as a dark green film.

[0326] Scheme 24: Synthetic route to ARM25-72. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0327] 4-ethyl-8-morpholino-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM25-72):

[0328] Compounds 11 (106 mg, 474.76 pmol) and 36 (162.54 mg, 474.76 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (79.59 pL, 617.18 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-72 (8 mg, 4.78%) as a dark green film.

[0329] Scheme 25: Synthetic route to ARM25-37. Reagents and conditions: a) BBr3, DCM, 0 °C; b) PPSE, CF3CH2OH, 80 °C.

[0330] 4-ethyl-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-ol (37): A solution of 35 (900 mg, 10 mmol) in anhydrous DCM (14 mL) was stirred in an ice bath under N2for 30 mins. Boron tribromide solution (1 M, 14 mL) was added to the solution using a syringe pump over 30 mins while maintaining the temperature of the solution below 5° C. The resulting reaction mixture was left in the ice bath for an additional 90 minutes, then removed from the ice bath and allowed to slowly warm to rt. After 2 h, the solution was again placed in an ice bath and any excess reagent was quenched by the careful addition of brine that was then extracted 4x with DCM. The organic layers were pooled together and the solvent was evaporated under reduced pressure to give a residue that was purified by flash column chromatography with silica gel using DCM / Hexane as eluent to give 37 (756 mg, 91 %) as brown oil.

[0331] 4-ethyl-8-(ethyl(2-methoxyethyl)amino)-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6- ium (ARM25-37): Compounds 37 (30 mg, 167.41 pmol) and 36 (60 mg, 167.41 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (28.06 pL, 217.64 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-37 (24.6 mg, 39.88%) as a dark blue film.

[0332] Scheme 26: Synthetic route to ARM25-35. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0333] 8-(bis(2-methoxyethyl)amino)-4-ethyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM25-35): Compounds 15 (100 mg, 371.28 pmol) and 36 (144.19 mg, 371.28 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (62.24 pL, 482.66 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-35 (44 mg, 29.74%) as a dark blue film.

[0334] Scheme 27: Synthetic route to ARM25-12. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0335] 8-(bis(2-methoxyethyl)amino)-4-ethyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6- ium (ARM25-12): Compounds 29 (100 mg, 374.08 pmol) and 36 (128.07 mg, 374.08 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (62.71 pL, 486.3 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-12 (10 mg, 6.74%) as a dark green film.

[0336] Scheme 28: Synthetic route to ARM 19-39. Reagents and conditions: a) 1-bromo-2- m ethoxy ethane, Lil, K2CO3, MeCN, 83 °C; b) 2M HCI, p-nitrobenzenediazonium tetrafluoroborate, 0 °C; c) 20, PPSE, CF3CH2OH, 80 °C.

[0337] 6-methoxy-4-(2-methoxyethyl)-3,4-dihydro-2H-benzo[b][1,4]oxazine (38): In a 100 mL recovery flask under N2, compound 22 (2.00 g, 12.11 mmol), lithium iodide (1.13 g, 8.47 mmol), and Na2CC>3 (2.57 g, 24.21 mmol) were suspended in 20 mL anhy. MeCN. To the mixture at rt was added 1-bromo-2-methoxyethane (1.25 mL, 13.32 mmol) before placing the reaction on a pre-heated oil bath (83 °C) to reflux overnight. Once complete, MeCN was removed from the reaction under reduced pressure and the residue was resuspended in 20 mL DCM and filtered through a pre-packed Celite® funnel. Purification by column chromatography with a gradient of ethyl acetate in hexanes afforded the target, 38, as a colorless translucent oil (2.15 g, 79.42%).

[0338] (E)-6-methoxy-4-(2-methoxyethyl)-7-((4-nitrophenyl)diazenyl)-3,4-dihydro-2H- benzo[b][1,4]oxazine (39): A solution of compound 38 (499.2 mg, 2.23 mmol) in MeOH (1 mL) was treated with HCI (2 M, 10 mL) and chilled in an ice bath. Once chilled (0 °C), p- nitrobenzenediazonium tetrafluoroborate (553.7 mg, 2.34 mmol) was added to the solution in 3 portions over 15 mins and stirred for 1 hr. at 0 °C. The appearance of the reaction mixture changed rapidly from a light yellow to deep red, forming a visible precipitate that was collected in a Buchner funnel, washed with DI water, and left to air-dry overnight, affording compound 31 as a dark red solid, used in the next step without further purification.

[0339] 8-(3-methoxyazetidin-1-yl)-4-(2-methoxyethyl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM 19-39): Compounds 20 (147.5 mg, 589.25 pmol) and 39 (201.73 mg, 589.25 pmol) were dissolved in a solution of trifluoroethanol (7 mL) containing trimethylsilyl polyphosphate (98.78 pL, 766.03 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-39 (14.1 mg, 8.23%) as a dark blue film.

[0340]

[0341] Scheme 29: Synthetic route to ARM19-33. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0342] 4-(2-methoxyethyl)-8-(2-oxa-6-azaspiro[3.3]heptan-6-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM19-33): Compounds 6 (104.2 mg, 442.87 pmol) and 39 (164.92 mg, 442.87 pmol) were dissolved in a solution of trifluoroethanol (6 ml_) containing trimethylsilyl polyphosphate (74.24 pL, 575.73 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-33 (10.2 mg, 5.84%) as a dark blue film.

[0343] Scheme 30: Synthetic route to ARM19-34. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0344] 4-(2-methoxyethyl)-8-(6-oxa-1-azaspiro[3.3]heptan-1-yl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM19-34): Compounds 10 (88.3 mg, 375.29 pmol) and 39 (139.75 mg, 375.39 pmol) were dissolved in a solution of trifluoroethanol (6 ml_) containing trimethylsilyl polyphosphate (62.91 pL, 487.88 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-34 (72.2 mg, 48.77%) as a dark blue film.

[0345] Scheme 31 : Synthetic route to ARM25-33. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0346] 8-(diethylamino)-4-(2-methoxyethyl)-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM25-33): 3-diethylaminophenol (100 mg, 605.19 pmol) and 39 (253.21 mg, 605.2 pmol) were dissolved in a solution of trifluoroethanol (6 mL) containing trimethylsilyl polyphosphate (101.45 pL, 786.75 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM25-33 (155 mg, 69.5%) as a dark blue film.

[0347]

[0348] Scheme 32: Synthetic route to ARM25-35. Reagents and conditions: a) Ac2O, H2O, 50 °C to rt; b) BH3-THF, THF, 0 °C to rt; c) 2-methoxyacetyl chloride, H2O, 50 °C to rt; d) BH3-THF, THF, 0 °C to rt; f) PPSE, CF3CH2OH, 83 °C.

[0349] A / -(3-(methoxymethoxy)phenyl)acetamide (40): Compound 14 (200 mg, 1.3 mmol) was suspended in 20 mL acetonitrile, to which acetic anhydride (370.26 pL, 3.92 mmol) was added dropwise. The reaction mixture was placed in an ultrasonication bath for 1 min, then stirred in a heated water bath (50 °C) for 10 min. The resulting solution was stirred overnight at rt. The solid product was collected via vacuum filtration and washed with small portions of DI water. The product was left in the funnel, air dried overnight, provided compound 40 as an off-white solid, used for the next step without further purification.

[0350] A / -ethyl-3-(methoxymethoxy)aniline (41): A solution of compound 40 (254.9 mg, 1.31 mmol) in anhydrous THF (10mL) was stirred in an ice bath under N2for 30 mins. Borane tetrahydrofuran complex solution (1 M, 4 mL) was added to the solution above dropwise over 30 mins using a syringe pump, while the temperature of the solution was maintained below 5 °C. The resulting reaction mixture was left to stir in the ice bath and slowly warm to rt. After 24 h, the solution was placed in an ice bath again and excess borane reagent was destroyed by carefully adding MeOH until no gas evolved. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography with silica gel, using DCM / Hexane as eluent, providing compound 41 (158 mg, 66.77%) as an oil.

[0351] A / -ethyl-A / -(3-hydroxyphenyl)-2-methoxyacetamide (42): Compound 41 (158 mg, 871.8 pmol) was suspended in 20 mL acetonitrile, to which 2-methoxyacetyl chloride (95.4 pL, 1.05 mmol) was added dropwise. The reaction mixture was placed in an ultrasonication bath for 1 min, then stirred in a heated water bath (50 °C) for 10 min. The resulting solution was stirred overnight at rt. The solid product was collected via vacuum filtration and washed with small portions of DI water. The product was left in the funnel, air dried overnight, provided compound 42 as a white solid, used for the next step without further purification.

[0352] M-ethyl-3-(methoxymethoxy)aniline (43): A solution of compound 42 (220 mg, 871.8 pmol) in anhydrous THF (10mL) was stirred in an ice bath under N2for 30 mins. Borane tetrahydrofuran complex solution (1 M, 4 mL) was added to the solution above dropwise over 30 mins using a syringe pump, while the temperature of the solution was maintained below 5 °C. The resulting reaction mixture was left to stir in the ice bath and slowly warm to rt. After 24 h, the solution was placed in an ice bath again and excess borane reagent was destroyed by carefully adding MeOH until no gas evolved. The solvent was evaporated under reduced pressure, and the residue was purified by flash column chromatography with silica gel, using DCM / Hexane as eluent, providing compound 43 (129.4 mg, 75.63%) as an oil.

[0353] 8-(bis(2-methoxyethyl)amino)-4-ethyl-3,4-dihydro-2H-[1,4]oxazino[2,3-b]phenoxazin-6-ium (ARM26-12): Compounds 39 (25 mg, 67.14 pmol) and 43 (17.04 mg, 87.28 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (11.25 pL, 87.28 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1% formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM26-12 (7.2 mg, 26.91 %) as a dark blue film.

[0354]

[0355] Scheme 20: Synthetic route to ARM19-35. Reagents and conditions: a) PPSE, CF3CH2OH, 80 °C.

[0356] 8-(bis(2-methoxyethyl)amino)-4-(2-methoxyethyl)-3,4-dihydro-2H-[1,4]oxazino[2,3- b]phenoxazin-6-ium (ARM 19-35): Compounds 15 (71.3 mg, 264.72 pmol) and 39 (98.58 mg, 264.72 pmol) were dissolved in a solution of trifluoroethanol (3 mL) containing trimethylsilyl polyphosphate (44.38 pL, 344.14 pmol). The reaction mixture was heated to 83 °C and stirred overnight. Once complete, the reaction was cooled to rt, the crude product was deposited directly onto silica gel, and the solvent removed under reduced pressure. Initial purification was done via silica gel flash chromatography using a mobile phase of MeOH and Acetone (gradient, 1-20% MeOH in Acetone). The semi-pure residue was purified again by silica gel flash chromatography using a mobile phase of CH2CI2 and MeOH containing 1 % formic acid (gradient, 1-10% of MeOH in CH2CI2). The fractions containing product were pooled and evaporated, affording ARM19-35 (41.4 mg, 36.5%) as a dark blue film.

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Claims

What is claimed:

1. A compound of Formula (I):wherein:X is selected from the group of a single bond and a double bond;Y is selected from the group of a single bond and a double bond; with the proviso that at least one of X and Y is a single bond;Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle of the formula:n3 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n4 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n3 and n4 is not less than 2 and not greater than 4; n5 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; n6 in each instance is an integer independently selected from the group of 0, 1 , 2, and 3; with the proviso that the sum of n5 and n6 is not less than 2 and not greater than 4;R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;88R4 is Ci-Ce alkyl when R3 is Ci-Ce alkyl; and R4 is -(CH2)ni-O-(CH2)n2-CH3 when R3 is - (CH2)ni-O-(CH2)n2-CH3; with the proviso that the integers of n1 and n2 in the R4-(CH2)ni-O-(CH2)n2-CH3 moiety may be the same or different from the integers of n1 and n2 in the R3-(CH2)ni-O-(CH2)n2-CH3moiety;R5is H; or R5is an oxygen atom that joins with R4to form a 2,3,4,5a,6a,11a-hexahydro- [1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II):R6is a ring heteroatom selected from the group of O and S; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

2. The compound of Claim 1 of Formula (II):wherein:X is selected from the group of a single bond and a double bond;Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;R2 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or R1 and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by R1 and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy;89or Ri and R2together with the nitrogen atom to which they are bound form a heterocyclic spirocycle selected from the group of:the wavy linein the Ri and R2heterocyclic spirocycle formulas represents the optional single bond or double bond through which Ri is bound to the adjacent 3-position carbon atom; andR3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

3. The compound of Claim 1 of Formula (III):wherein:X is selected from the group of a single bond and a double bond;Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy;90or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of:R3 is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; andR4is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

4. The compound of Claim 1 of Formula (III):wherein:X is selected from the group of a single bond and a double bond;Ri is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3;R2is a moiety of the formula -(CH2)ni-O-(CH2)n2-CH3; n1 in each instance is an integer independently selected from the group of 1 , 2, and 3; n2 in each instance is an integer independently selected from the group of 0, 1 , and 2; or Ri and R2together form a heterocyclic ring selected from the group of azetidinyl, pyrrolidinyl, and piperidinyl, wherein the heterocyclic ring formed by Ri and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or Ri and R2together with the nitrogen atom to which they are bound form a spirocyclic heterocycle selected from the group of:91R3is C1-C6 alkyl; andR4is C1-C6 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

5. The compound of Claim 1 , wherein R1 and R2 together form a heterocyclic ring selected from the group of azetidinyl and pyrrolidinyl, , wherein the heterocyclic ring formed by R1 and R2is substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

6. The compound of Claim 1 , wherein R1 and R2together form an azetidinyl ring substituted by 1 substituent selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

7. The compound of Claim 1 , wherein R1 and R2together form an azetidinyl ring substituted by 1 methoxy substituent; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

8. The compound of Claim 1 , wherein R1 and R2together form a pyrrolidinyl ring substituted by 1 or 2 substituents selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

9. The compound of Claim 1 , wherein R1 and R2together form a pyrrolidinyl ring substituted by 1 substituent selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical92isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

10. The compound of Claim 1 , wherein Ri and R2together form a pyrrolidinyl ring substituted by 1 methoxy substituent; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

11. The compound of Claim 1 , wherein Ri and R2together form a pyrrolidinyl ring substituted by 2 methoxy substituents; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

12. The compound of Claim 1 , wherein Ri and R2together with the nitrogen atom to which they are bound form a heterocyclic spirocycle selected from the group of:or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

13. The compound of Claim 1 , Rs is an oxygen atom that joins with R4 to form a 2,3,4,5a,6a,11a-hexahydro-[1 ,4]oxazino[2,3-b]phenoxazine compound of Formula (II):R3 is C1-C6 alkyl;93or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

14. The compound of Claim 1 of Formula (Hid):wherein:R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3; andR? is selected from the group of methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

15. The compound of Claim 14, wherein R3 is C1-C3 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

16. The compound of Claim 1 of Formula (Hie):wherein:R3is selected from the group of Ci-Ce alkyl and a moiety of the formula -(CH2)ni-O- (CH2)n2-CH3;R7 is selected from the group of methoxy and ethoxy; and Rs is selected from the group of H, methoxy and ethoxy; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.9417. The compound of Claim 16, wherein R3 is C1-C3 alkyl; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

18. The compound of Claim 1 , selected from the group of:or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

19. A composition comprising a pharmaceutically acceptable carrier or excipient and an effective amount of a compound of Claim 1 ; or a pharmaceutically acceptable salt, co-crystal, ester, solvate, hydrate, isomer (including optical isomers, racemates, or other mixtures thereof), tautomer, isotope, polymorph, or pharmaceutically acceptable prodrug thereof.

20. A method of detecting nerves in a tissue or organ, the method comprising: a) administering an effective amount of a composition comprising a compound of Claims 1 to the tissue or organ to form a stained tissue or a stained organ; and b) imaging the stained tissue or stained organ, thereby detecting nerves intraoperatively in the stained tissue or stained organ.

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