Benzo [ c ] phenoxazine fluorophores for near-infrared nerve retention
阅读说明：本技术 近红外保留神经用苯并[c]吩噁嗪荧光团 (Benzo [ c ] phenoxazine fluorophores for near-infrared nerve retention ) 是由 S·L·吉布斯 L·G·王 C·W·巴思 于 2019-09-11 设计创作，主要内容包括：提供了近红外保留神经用荧光化合物、包含其的组合物及其在医疗程序中的使用方法。(Fluorescent compounds for near-infrared nerve retention, compositions comprising the same, and methods of use thereof in medical procedures are provided.)
1. A compound of the formula I, wherein,
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2selected from hydrogen and C1-C3An alkyl group;
R3selected from the group: hydrogen, halogen and C1-C3An alkyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
2. The compound of claim 1, wherein R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, halogen and C1-C3An alkyl group; and R is4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
3. The compound of claim 1, wherein R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group; and R is4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
4. The compound of claim 1, wherein R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group; and R is4And R5Each independently is C1-C3An alkyl group.
5. The compound of claim 1, wherein R1Is hydrogen; r2Is C1-C2An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C2An alkyl group; and R is4And R5Each independently is C1-C2An alkyl group.
6. The compound of claim 1, wherein R1Selected from hydrogen and C1-C3An alkyl group; r2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
7. The compound of claim 1, wherein R1Selected from hydrogen and C1-C3An alkyl group; r2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C2Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C2An alkyl group.
8. The compound of claim 1, comprising formula II:
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2selected from hydrogen and C1-C3An alkyl group;
R3selected from the group: hydrogen, halogen and C1-C3An alkyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
9. The compound of claim 8, having formula II, wherein R1Is hydrogen; r2Is C1-C3An alkyl group; and R is3Selected from the group: hydrogen, halogen and C1-C3An alkyl group.
10. The compound of claim 8, having formula II, wherein R1Is hydrogen; r2Is C1-C3An alkyl group; and R is3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group.
11. The compound of claim 8, having formula II, wherein R1Is hydrogen; r2Is C1-C2An alkyl group; and R is3Selected from the group: hydrogen, F, Cl and C1-C2An alkyl group.
12. The compound of claim 8, having formula II, wherein R1Selected from hydrogen and C1-C3An alkyl group; and is
R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
13. The compound of claim 8, having formula (la)II, wherein R1Selected from hydrogen and C1-C2An alkyl group; and is
R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C2Alkyl substituents.
14. The compound of claim 1, comprising formula III:
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R4and R5Each independently selected from hydrogen and C1-C3An alkyl group;
R6、R7and R8Each independently is hydrogen or C1-C3An alkyl group;
x is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by C1-C3Alkyl substitution.
15. The compound of claim 14, having formula III, wherein R is1Selected from hydrogen and C1-C2An alkyl group;
R4and R5Each independently selected from hydrogen and C1-C2An alkyl group;
R6、R7and R8Each independently is hydrogen or C1-C2An alkyl group; and is
X is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by C1-C2Alkyl substitution.
16. The compound of claim 14, having formula III, wherein R is1Selected from hydrogen and-CH3；
R4And R5Each independently selected from hydrogen and C1-C2An alkyl group;
R6、R7and R8Each independently is hydrogen or-CH3(ii) a And is
X is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by-CH3And (4) substitution.
17. The compound of claim 1, having the formula:
wherein R is3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
18. The compound of claim 1, selected from the group of:
19. a composition comprising at least one compound of claim 1 and a pharmaceutically acceptable carrier or excipient.
20. A method of imaging a target region of a subject, the method comprising contacting the target region of the subject with a compound of claim 1 and detecting the compound in a target using fluorescence or near-infrared imaging.
Background
Accidental nerve transection or injury is a major disease associated with many surgical interventions, resulting in persistent post-operative numbness, chronic pain, and/or paralysis. Nerve retention can be a difficult task due to patient-to-patient variability and the difficulty of nerve visualization in the operating room. Currently, nerve detection in an operating room is mainly completed through electromyography monitoring and direct visualization under ultrasound or white light. Fluorescence-guided surgery facilitates real-time accurate visualization of important neural structures, and can greatly improve patient prognosis. However, there are no clinically approved neuro-specific contrast agents. Contrast agents falling within the Near Infrared (NIR) window (650-900nm) are particularly attractive for fluorescence guided surgery because absorption, scattering and autofluorescence are all at local minima, maximizing tissue light penetration in this range. To date, there are no NIR nerve-specific fluorophores, with oxazine 4 having the longest emission wavelength (635 nm maximum) among the nerve-specific fluorophores reported to highlight peripheral nerves. This is a particularly challenging problem because nerve-specific contrast agents must have a relatively low molecular weight to cross the blood-nerve barrier. Complicating this requirement is that the NIR fluorophore must have a sufficient degree of conjugation to reach the NIR wavelength, increasing its molecular weight by definition. There remains a need for NIR nerve-specific contrast agents that can improve nerve visualization for diagnostic procedures and be used during fluorescence-guided surgery. Here we report our work aimed at synthesizing key libraries of systematically modified NIR fluorophores to determine factors that modulate the neural specificity of the fluorophore.
Disclosure of Invention
There is provided a compound of formula I:
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2selected from hydrogen and C1-C3An alkyl group;
R3selected from the group: hydrogen, halogen and C1-C3An alkyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
Also provided are pharmaceutically acceptable compositions comprising an effective amount of a compound of formula I, and methods of using such formulations and compounds in imaging techniques.
Drawings
FIG. 1 shows the Excited Emission Matrix (EEM) of compound LGW05-25 in phosphate buffered saline solution containing 10% DMSO.
Figure 2 depicts a comparison represented by systemic administration and direct administration of compound LG 05-25.
Figure 3A provides a graph of the mean quantitative fluorescence intensity per second after direct administration to nerve, fat and excised muscle tissue.
Figure 3B provides a graph of calculated nerve-to-muscle, nerve-to-excised, and nerve-to-fat ratios determined after direct administration of the compounds.
Figures 4A, 4B, and 4C provide graphs of mean quantitative fluorescence intensity per second for nerve, muscle, and adipose tissue after systemic administration.
Figures 4E and 4F provide graphs representing calculated nerve-to-muscle and nerve-to-fat ratios after systemic administration.
Detailed Description
Also provided herein are embodiments comprising compounds of formula Ia, formula Ib, and formula Ic, or pharmaceutically acceptable salts thereof, as follows:
other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, halogen and C1-C3An alkyl group; and R is4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
Other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group; and R is4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
Other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is C1-C3An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group; and R is4And R5Each independently is C1-C3An alkyl group.
Other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is C1-C2An alkyl group; r3Selected from the group: hydrogen, F, Cl and C1-C2An alkyl group; and R is4And R5Each independently is C1-C2An alkyl group.
Other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is C1-C2An alkyl group; r3Is C1-C2An alkyl group; and R is4And R5Each independently is C1-C2An alkyl group.
Other separate embodiments provide compounds of formula I, formula Ia, formula Ib, and formula Ic, wherein, in each case, R1Is hydrogen; r2Is an ethyl group; r3Is C1-C2An alkyl group; and R is4And R5Each is ethyl.
Another embodiment comprises compounds of formula Ia
Wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2and R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C3An alkyl group.
Another embodiment comprises compounds of formula Ia
Wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2and R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C2Alkyl substituent group substitution; and is
R4And R5Each independently selected from hydrogen and C1-C2An alkyl group.
Another embodiment comprises a compound of formula II:
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R2selected from hydrogen and C1-C3An alkyl group;
R3selected from the group: hydrogen, halogen and C1-C3An alkyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
Also provided in separate embodiments are compounds selected from formula IIa, formula IIb, and formula IIc, wherein in each case R1、R2And R3As defined above for formula II:
other separate embodiments provide compounds of formula II, formula IIa, formula IIb and formula IIc, wherein, in each case, R1Is hydrogen; r2Is C1-C3An alkyl group; and R is3Selected from the group: hydrogen, halogen and C1-C3An alkyl group.
Other separate embodiments provide compounds of formula II, formula IIa, formula IIb and formula IIc, wherein, in each case, R1Is hydrogen; r2Is C1-C3An alkyl group; and R is3Selected from the group: hydrogen, F, Cl and C1-C3An alkyl group.
Other separate embodiments provide compounds of formula II, formula IIa, formula IIb and formula IIc, wherein, in each case, R1Is hydrogen; r2Is C1-C2An alkyl group; and R is3Is selected fromThe group of: hydrogen, F, Cl and C1-C2An alkyl group.
Another embodiment comprises compounds of formula IIa, wherein R1Selected from hydrogen and C1-C3An alkyl group; and is
R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
Yet another embodiment comprises compounds of formula IIa, wherein R1Selected from hydrogen and C1-C2An alkyl group; and is
R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C2Alkyl substituents.
Another embodiment provides a compound of formula III:
wherein R is1Selected from hydrogen and C1-C3An alkyl group;
R4and R5Each independently selected from hydrogen and C1-C3An alkyl group;
R6、R7and R8Each independently is hydrogen or C1-C3An alkyl group;
x is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by C1-C3Alkyl substitution.
Also provided are compounds of formula III, wherein R1Selected from hydrogen and C1-C2An alkyl group;
R4and R5Each independently selected from hydrogen and C1-C2An alkyl group;
R6、R7and R8Each independently is hydrogen or C1-C2An alkyl group; and is
X is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by C1-C2Alkyl substitution.
Further provided are compounds of formula III, wherein R1Selected from hydrogen and-CH3；
R4And R5Each independently selected from hydrogen and C1-C2An alkyl group;
R6、R7and R8Each independently is hydrogen or-CH3(ii) a And is
X is selected from oxygen and carbon, wherein the carbon atom is optionally substituted by-CH3And (4) substitution.
Within each embodiment herein for compounds of formula III, there is another embodiment wherein R1、R6、R7、R8And X is as defined for the examples in question and R4And R5Each is ethyl.
Another embodiment provides a compound of formula IV:
wherein R is3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-or six-membered fused ring, optionally containing an epoxy heteroatom, and which is interrupted by 0,1, 2 or 3C1-C3Alkyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a five-membered thickRings with 0,1, 2 or 3C atoms1-C3Alkyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Form a five-membered fused ring, which is substituted by 0,1, 2 or 3C atoms, together with the carbon atom to which it is bonded1-C2Alkyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atoms to which they are bonded, form a five-membered fused ring, which is substituted with 0,1, 2 or 3 methyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a six-membered fused ring, which is substituted by 0,1, 2 or 3C1-C3Alkyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3Together with the carbon atom to which they are bonded form a six-membered fused ring, which is substituted by 0,1, 2 or 3C1-C2Alkyl substituents.
Another embodiment includes compounds of formula IV, wherein R3Is methyl and R2Is an ethyl group;
or, when R is3And 10H-benzo [ c ]]Bonding of the 9-carbon of the phenoxazine core, R2And R3To which it is bondedThe carbon atoms together form a six membered fused ring, which is substituted with 0,1, 2 or 3 methyl substituents.
Examples of compounds within the groups described herein include those selected from the group consisting of:
3- (diethylamino) -10H-benzo [ c ] phenoxazin-10-iminium;
3- (diethylamino) -9-methyl-10H-benzo [ c ] phenoxazin-10-iminium;
(E) -N- (3- (diethylamino) -10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium;
(Z) -N- (3- (diethylamino) -9-methyl-10H-benzo [ c ] phenoxazin-10-ylidene) ethanaminium;
(E) -N- (3- (diethylamino) -11-methyl-10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium;
(Z) -N- (9-chloro-3- (diethylamino) -10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium;
(Z) -N- (3- (diethylamino) -9-fluoro-10H-benzo [ c ] phenoxazin-10-ylidene) ethanaminium;
3- (diethylamino) -10, 11-dihydro-9H-benzo [ H ] pyrido [3,2-b ] phenoxazin-12-ium;
3- (diethylamino) -10, 11-dihydrobenzo [ h ] [1, 4] oxazino [2,3-b ] phenoxazin-12-ium;
(E) -N- (3- (diethylamino) -8-methyl-10H-benzo [ c ] phenoxazin-10-ylidene) ethanaminium;
3- (diethylamino) -9, 10-dihydrobenzo [ h ] pyrrolo [3,2-b ] phenoxazin-11-ium;
3- (diethylamino) -9,11, 11-trimethyl-11H-benzo [ H ] pyrido [3,2-b ] phenoxazin-12-ium;
n- (3- (diethylamino) -10H-benzo [ c ] phenoxazin-10-ylidene) -N-methylmethanamine; and
n- (3- (diethylamino) -10H-benzo [ c ] phenoxazin-10-ylidene) -N-ethylethylamine.
Also provided herein are pharmaceutical or medical compositions comprising one or more fluorophore compounds described herein and a pharmaceutically or medically acceptable carrier or excipient. In some embodiments, the composition is intended for direct/topical application. Direct administration refers to the administration of the compound or composition directly to the target tissue or organ, such as by irrigation, nebulization, spraying, rubbing, smearing, brushing, or other direct administration. Systemic administration refers to the administration of a compound or composition such that the entire target system, organ or tissue receives a dispersion of the compound sufficient for imaging or other medical purposes. Systemic administration includes parenteral administration, such as intravenous, intramuscular, or subcutaneous administration by injection, infusion, or other means.
Suitable pharmaceutically acceptable non-aqueous solvents that may be used as carriers or excipients for the compounds of the invention include the following (and mixtures thereof): alcohols (these include, for example, sigma-glycerol formal, beta-glycerol formal, 1, 3-butanediol; aliphatic or aromatic alcohols such as methanol, ethanol, propanol, isopropanol, butanol, tert-butanol, hexanol, octanol, pentene hydrate, benzyl alcohol, glycerol (glycerol), ethylene glycol, hexylene glycol, tetrahydrofurfuryl 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 O-lactamide (N-hydroxyyethyo-lactamide), N-dimethylacetamide-amide, 2-pyrrolidone, 1-methyl-2-pyrrolidone, and polyvinylpyrrolidone; acetates such as glycerol monoacetate, glycerol diacetate, and glycerol triacetate; aliphatic and aromatic esters such as ethyl octanoate (caprylate) or ethyl octanoate (octanoate), alkyl oleate, benzyl benzoate or benzyl acetate; dimethylsulfoxide (DMSO); esters of glycerol (e.g., mono-, di-, and triglycerol citrates and tartrates), ethyl benzoate, ethyl acetate, ethyl carbonateEsters, ethyl lactate, ethyl oleate, fatty acid esters of sorbitan, glycerol monostearate, glycerol esters (e.g., mono-, di-and triglycerides), fatty acid esters (e.g., isopropyl myristate), fatty acid-derived PEG esters (e.g., PEG-hydroxyoleate and PEG-hydroxystearate), N-methylpyrrolidone, pluronic 60, polyoxyethylene sorbitol oleic polyesters (e.g., poly (ethoxylated)30-60Sorbitol poly (oleate)2-4Poly (oxyethylene)15-20Monooleate, poly (oxyethylene)15-20Mono 12-hydroxystearate and poly (oxyethylene)15-20Monoricinoleate esters), polyoxyethylene sorbitan esters (e.g., polyoxyethylene-sorbitan monooleate, polyoxyethylene-sorbitan monopalmitate, polyoxyethylene-sorbitan monolaurate, polyoxyethylene-sorbitan monostearate, and POLYSORBATE 20, 40, 60, and 80, polyvinylpyrrolidone, alkyleneoxy-modified fatty acid esters (e.g., polyoxyethylene 40 hydrogenated castor oil and polyoxyethylated castor oil, such as CREMOPHOR EL solution or CREMOPHOR RH 40 solution), saccharide fatty acid esters (i.e., condensation products of monosaccharides (e.g., pentoses, such as ribose, ribulose, arabinose, xylose, lyxose, and xylulose; hexoses, such as glucose, fructose, galactose, mannose, and sorbose; triose; tetrose; heptose, and octose), disaccharides (e.g., sucrose, maltose, lactose and trehalose), oligosaccharides, or a mixture thereof with one or more C4-C22Fatty acids (e.g., mixtures of 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 steroid esters); ethers such as diethyl ether, tetrahydrofuran, dimethyl isosorbide, diethylene glycol monoethyl ether and tetraethylene glycol (tetrahydrofurfuryl alcohol polyglycol ether); ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone; hydrocarbons such as benzene, cyclohexane, dichloromethane, dioxolane, hexane, n-decane, n-dodecane, n-hexane, sulfolane, tetramethylene sulfone, tetramethylene sulfoxide, toluene, dimethyl sulfoxide (DMSO); and tetramethylene sulfoxide;oils such as mineral oil, vegetable oil, glyceride, animal oil, oleic oil, alkyl, alkenyl or aryl halides, monoethanolamine; petroleum ether; triethanolamine; omega-3 polyunsaturated fatty acids, such as alpha-linolenic acid, eicosapentaenoic acid, docosapentaenoic acid or docosahexaenoic acid; polyethylene glycol esters of 12-hydroxystearic acid and polyethylene glycol (SOLUTOL HS-15 from BASF, Lodviesha, Germany); polyoxyethylene glycerin; sodium laurate; sodium oleate; and sorbitan monooleate. Other pharmaceutically acceptable solvents for use in the present invention are well known to those of ordinary skill in the art.
The additional component may be a cryoprotectant; an agent for preventing the surface of a dithienopyrrole compound or salt from reprecipitation; active agents, wetting or emulsifying agents (e.g., lecithin, polysorbate-80, tween 80, pluronic 60, and polyoxyethylene stearate); preservatives (e.g., ethyl paraben); microbial preservatives (e.g., benzyl alcohol, phenol, m-cresol, chlorobutanol, sorbic acid, thimerosal, and parabens); reagents or buffers for adjusting pH (e.g., acids, bases, sodium acetate, sorbitan monolaurate, etc.); agents for adjusting osmolarity (e.g., glycerol); and diluents (e.g., water, saline, electrolyte solutions, etc.).
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).
Definition of
As used herein, the term "administering" or the like refers to a method for enabling the delivery of an agent or composition disclosed herein to a 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 topical injection). In some embodiments, the administration is topical. Administration techniques optionally used with the agents and methods described herein include, for example, as discussed in: goodman (Goodman) and Gilman (Gilman), "Pharmacological Basis of Therapeutics (The Pharmacological Basis of Therapeutics)", current edition; pegoman (Pergamon); and Remington (Pharmaceutical Sciences), Current edition, Mark Publishing Co (Mack Publishing Co.), Iston, Pa.
The term "effective amount" or "medically effective amount" or similar terms refer to an amount of a compound or composition as described herein sufficient to cover a target area to accomplish binding to one or more nerves such that they can be identified by a related imaging technique, particularly near infrared imaging techniques.
The term "imaging" herein refers to the use of fluorescent compounds in conventional medical imaging techniques including, but not limited to, those associated with fluorescence image guided surgery (including minimally invasive laparoscopic or endoscopic techniques), computer assisted surgery or surgical navigation, radiosurgery or radiotherapy, interventional radiology, fluorescence microscopy and laser confocal microscopy. These techniques may include near infrared wavelengths from about 650nm to 900 nm.
The term "tag" refers to a molecule that facilitates visualization and/or detection of the targeting molecules disclosed herein. In some embodiments, the label is a fluorescent moiety.
As used herein, the term "neuron" refers to an electrically excitable cell that processes and transmits information by electrical and chemical signaling. Neurons have a cell body (i.e., a somatic cell (soma)), dendrites, and axons. Neurons are electrically excitable, maintaining a voltage gradient across their membrane by an ion pump that combines with ion channels embedded in the membrane to produce intracellular-extracellular concentration differences of ions (e.g., sodium, potassium, chloride, and calcium). Neurons may or may not include myelin sheaths. The term "neuron" is intended to include any tissue (e.g., the sinoatrial node or the atrioventricular node) or structure associated therewith (e.g., the neuromuscular junction).
The term "nerve" means a bundle of nerve axons. Within the nerve, each axon is surrounded by a layer of connective tissue called the endoneurium. The axons are bundled together to form bundles, and each bundle is wrapped in a layer of connective tissue, called the perineurium. The entire nerve is encapsulated in a layer of connective tissue called the epineurium. The term "nerve" is intended to include any tissue (e.g., the sinoatrial node or the atrioventricular node) or structure associated therewith (e.g., the neuromuscular junction).
The terms "patient," "individual," and "subject" are used interchangeably. As used herein, they refer to any mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is non-human, including domestic animals (cattle, pigs, horses, goats, sheep, etc.), pets (dogs, cats, etc.), and research animals (mice and rats).
As used herein, the term "pharmaceutically acceptable" refers to a material that does not abrogate the biological activity or properties of an agent described herein (such as binding to a desired target) and is relatively non-toxic (i.e., the toxicity of the material significantly outweighs the benefit of the material). In some cases, pharmaceutically acceptable materials can be administered to an individual without causing significant undesirable biological effects or interacting significantly in a deleterious manner with any component of the composition in which they are contained.
As used herein, the terms "robotic surgery," "robotic-assisted surgery," or "computer-assisted surgery" refer to a surgical technique involving a robotic system that controls movement of a medical instrument to perform a surgical procedure of precise, flexible, and/or minimally invasive actions designed to limit surgical trauma, blood loss, pain, scarring, and the amount of post-operative patient recovery time and/or complications, such as infection of a surgical area. Examples of robotic surgery include those performed using the da Vinci Surgical System (Intuitive surgery, Sunnyvale, CA, USA) approved by the united states food and Drug Administration in 2000.
As used herein, the term "surgery" or "surgical method" refers to any method for manipulating, altering or causing an effect by physical intervention. These methods include, but are not limited to, open surgery, endoscopic surgery, laparoscopic surgery, minimally invasive surgery, robotic surgery, any procedure that may affect any neuron or nerve, such as placement of a retractor during spinal surgery, electrically conductive cardiac tissue or nerve ablation, epidural injection, intrathecal injection, neuron or nerve block, implant devices (such as neuron or nerve stimulators), and implant pumps. These methods may also include biopsy or other invasive techniques for collecting 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 neural cell or an organ or tissue associated with one or more neural cells or neural 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., neural tissue, nervous system tissue, nerve bundles, etc.). It is understood that neural and neural-related targets include targets related to the brain and spinal cord of the Central Nervous System (CNS) and nerves of the Peripheral Nervous System (PNS).
Also provided herein are methods of imaging neural tissue tumors (neoplasms) including gliomas, such as brain glioma (bliimatosis cerb), oligodendroastrocytoma, choroid plexus papilloma, ependymoma, astrocytoma (fibroastrocytoma and glioblastoma multiforme), dysplastic neuroepithelial tumors, oligodendroglioma, medulloblastoma, and primitive neuroectodermal tumors; neuroepithelial tumors such as ganglioneuroma, neuroblastoma, atypical teratoid rhabdoid tumor, retinoblastoma, and olfactory neuroblastoma; and sphingomyma tumors, such as neurofibroma (neurofibrosarcoma and neurofibromatosis), schwannomas (Schannomas), schwannomas, acoustic neuroma and neuroma.
A method of imaging a region of interest in a subject is provided, the method comprising contacting a region of interest in a subject with a compound selected from those described herein, and detecting the compound in the target using fluorescence or near-infrared imaging.
Also provided is a method of imaging one or more nerves in a region of interest of a subject, the method comprising contacting the region of interest of the subject with a compound selected from those described herein, and detecting the compound in the target using fluorescence imaging.
Also provided is a method of imaging one or more nerves in a region of interest of a subject, the method comprising contacting the region of interest of the subject with a compound selected from those described herein and detecting the compound in the target using near infrared imaging.
Also provided is a method of minimizing nerve damage in a target region of a subject during a medical procedure, the method comprising the steps of:
a) contacting a target area of a subject with a compound selected from those herein;
b) detecting one or more nerves in the target region to which the compound binds using fluorescence imaging; and
c) actions of the medical procedure that may damage one or more detected nerves are minimized.
The above methods can be used to identify and minimize damage to nerves that may result from medical procedures, including traumatic, thermal, and radiological damage, or from the application of therapeutic agents, anesthetics, or anesthetics to the target area.
In some embodiments, the medical procedure referred to in the above methods is a surgical procedure. In other embodiments, the medical procedure is a biopsy procedure, a radiology procedure, or administration of an anesthetic or anesthesia to the subject. In further embodiments, the medical procedure in the above methods is the insertion or implantation of a medical device (including a medical pump, stent, pacemaker, port, artificial joint, valve, screw, pin, plate, rod, cosmetic implant, neurostimulator, etc.).
Also provided is the use of any of the compounds disclosed herein in the preparation of a composition for imaging one or more nerves in a subject using near infrared imaging.
Also provided is a kit comprising a container having a composition comprising a medically useful amount of a compound described herein, and a set of instructions for using the composition in a neuroimaging procedure.
General purpose
All reagents were purchased from Sigma Aldrich (Sigma Aldrich), siemer femtole (Fisher Scientific), tokyo chemical co ltd (TCI), or Ark Pharm. All commercially available starting materials were used without further purification unless otherwise indicated. Analytical Thin Layer Chromatography (TLC) was performed on Millipore (Millipore) ready-to-use plates with silica gel 60(F254, 32-63 μm). Flash chromatography was performed on adsorbent technology (Sorbent Technologies) silica gel for column chromatography or on a Biotage Isolera Flash System (Flash System) using SNAP Ultra columns. High Resolution Mass Spectra (HRMS) were measured on an Agilent (Agilent)6244 time-of-flight LCMS with diode array detector VL +.
LCMS and purity characterization
Characterization of benzo [ c ] on Agilent 6244 time-of-flight tandem Liquid Chromatography Mass Spectrometry (LCMS) with diode array detector VL +)]Mass to charge ratio and purity of the phenoxazine compound. Sample (10. mu.L) was injected onto a C18 column (Poroshell 120, 4.6X 50mm, 2.7 microns) and applied with A (H) at 0.4 mL/min2O, 0.1% FA) and B (MeCN, 01% FA), eluting from a/B-90/10 to 5/95 in 10 minutes and holding at a/B-5/95 for an additional 5 minutes. Ions were detected in positive ion mode by setting the capillary voltage to 4kV and the gas temperature to 350 ℃. Purity was calculated by area under curve analysis of absorbance at 254 nm.
UV-Vis absorption and fluorescence Spectroscopy
UV-Vis and fluorescence spectra were collected on a SpectraMax M5 spectrometer with a microplate reader (Molecular Devices, Santo, Calif.). All absorption spectra were reference corrected. The extinction coefficient was calculated from the Beer's Law plot of absorbance versus concentration (Beer's Law plot). Relative quantum yields were reported using HITCI as a reference. Excitation Emission Matrices (EEMs) were collected using 5nm steps in a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). The bandpass of excitation and emission was 10 nm.
In FIG. 1, the stimulated emission matrix (EEM) of LGW05-25 in phosphate buffered saline solution containing 10% DMSO. The fluorophore concentration was 10. mu.M. The color scale of EEM (C-J) is normalized to the maximum of the graph.
Calculation of physicochemical Properties
The physicochemical properties (https:// www.chemaxon.com) were calculated using Marvin (17.21.0 Chemaxon).
Neural specific screening using direct/topical application
Each compound was tissue-specific screened in the murine brachial plexus and sciatic nerve using a previously published direct/topical strategy.1Will be derived from benzo [ c]Each compound of the phenoxazine library was formulated at 500 μ M in a previously used co-solvent formulation (10% DMSO, 5% Kolliphor, 65% serum, and 20% phosphate buffered saline). 100 μ L of formulated oxazine was incubated on exposed brachial plexus or sciatic nerve for 5 minutes. The fluorophore-containing solution was removed and the area was washed 18 times with saline to remove any unbound fluorophores. In benzo [ c)]Co-registered fluorescence and color images of each stained area were collected 30 minutes after direct/topical application of phenoxazine using a custom built macroscopic imaging system with 710/75nm excitation and 810nm bandpass emission filters. Tissue specific fluorescence was analyzed using custom-written MatLab code, where white light images were used to select target regions on nerves, muscle, excised muscle, and adipose tissue. These target regions are then analyzed on co-registered matched fluorescence images to blindly assess the nerve-muscle ratio, nerve-excised muscle ratio, and nerve-fat ratio.
Neural specific screening using systemic administration
Each compound was tissue-specific screened in the murine brachial plexus and sciatic nerve using previously published systemic administration strategies.1Will be derived from benzo [ c]Each compound of the phenoxazine library was formulated at 2.5mM in the previously used co-solvent formulation (10% DMSO, 5% Kolliphor, 65% serum, and 20% phosphate buffered saline). Before exposing the brachial plexus and sciatic nerve, 200 μ L of formulated fluorophore was administered intravenously. Co-registered fluorescence and color images of each nerve site were collected using a custom-constructed macroscopic imaging system with 710/75nm excitation and 810nm bandpass emission filters. Tissue specific fluorescence was analyzed using custom-written MatLab code, where white light images were used to select target regions on nerve, muscle and adipose tissue. These target regions are then analyzed on co-registered matched fluorescence images to blindly assess the nerve-muscle ratio and nerve-fat ratio.
Resonance structure
It is to be understood that the fluorescent compounds described herein may exist in any possible resonance form. For example, a compound depicted by formula I:
the following structure may be equivalently referred to as used:
wherein, in each case, the general expression "- - - - -" denotes a single or double bond required to satisfy the valence requirements.
Chemical synthesis
Scheme 1
Scheme 1: synthetic route to LGW 05-25. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) ac of2O，H2O, from 50 ℃ to room temperature; d) BH3-THF, 0 ℃ to room temperature; e) ac of2O，H2O, from 50 ℃ to room temperature; f) BH3-THF, 0 ℃ to room temperature; g) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; h) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；i)TfOH，100℃。
N- (5-hydroxynaphthalen-2-yl) acetamide (2)
Compound 1(4g, 25.23mmol) was suspended in 40mL of deionized water, and acetic anhydride (9.50mL, 100.51mmol) was added dropwise thereto. The reaction mixture was placed in an ultrasonic bath for 1 minute and then stirred in a water bath (50 ℃) for 10 minutes. The resulting solution was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 2(4.32g, 85%) as a pale grey solid which was used in the next step without further purification.
6- (ethylamino) naphthalene-1-ol (3)
A solution of 2(4g, 19.88mmol) in anhydrous THF (60mL) in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 60mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 3(3.23g, 87%) as a dark solid.
N-Ethyl-N- (5-hydroxynaphthalen-2-yl) acetamide (4)
Compound 3(3g, 16.02mmol) was suspended in 30mL of deionized water, and acetic anhydride (6.06mL, 64.09mmol) was added dropwise thereto. The reaction mixture was placed in an ultrasonic bath for 1 minute and then stirred in a water bath (50 ℃) for 10 minutes. The resulting solution was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 4(3.51g, 96%) as a pale grey solid which was used in the next step without further purification.
6- (diethylamino) naphthalene-1-ol (5)
A solution of 4(3.5g, 15.27mmol) in anhydrous THF (45mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 45mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 5(2.67g, 81%) as a dark oil.
N- (5-iodo-2-methylphenyl) acetamide (7)
Compound 6(2g, 8.58mmol) was dissolved in 2mL of DMSO, and acetic anhydride (2.43mL, 25.75mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for an additional 2 hours (h). 18mL of deionized water was added to the reaction mixture and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 7(2.09g, 89%) as a light grey solid which was used in the next step without further purification.
N-Ethyl-5-iodo-2-methylaniline (8)
A solution of 7(2.5g, 9.09mmol) in anhydrous THF (27mL) was placed in an ice bath under N2Stirred for 30 minutes. The solution was added to the solution using a syringe pump within 30 minutesTo the solution was added borane tetrahydrofuran complex solution (1M, 27mL) while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 8(1.99g, 84%) as a clear oil.
N, N-diethyl-5- (3- (ethylamino) -4-methylphenoxy) naphthalen-2-amine (9)
Compound 9 was synthesized using a slightly modified protocol published by Maiti (Maiti) and Buchwald (Buchwald).2An oven or flame dried microwave glass tube was charged with a magnetic stir bar, Compound 8(260mg, 0.996mmol), 5(195mg, 0.905mmol) and CuI (17mg, 0.091mmol), 2-picolinic acid (22mg, 0.181mmol) and anhydrous K3PO4(384mg, 1.81 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 9(258mg, 82%) as a colourless oil.
(E) -N, N-diethyl-5- (5- (ethylamino) -4-methyl-2- ((4-nitrophenyl) diazenyl) phenoxy) Naphthalene-2-amines(10)
Compound 9(0.175g, 0.5mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.13g, 0.55mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 10(0.223g, 90%) which was used in the next step without further purification.
(Z) -N- (3- (diethylamino) -9-methyl-10H-benzo [ c)]Phenoxazin-10-ylidene) ethylamine (LGW05-25)
Compound 10(0.1g, 0.201mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (1mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW05-25(45mg, 62%) as a dark green solid.
Scheme 2: synthetic route to LGW 06-65. Reagents and conditions: a) CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; b) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；c)TfOH，100℃。
5- (3-Aminophenoxy) -N, N-diethylnaphthalen-2-amine (12)
Compound 12 was synthesized using a slightly modified protocol published by motto and branharder.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 11(190mg, 0.868mmol), 5(170mg, 0.789mmol)CuI (15mg, 0.079mmol), 2-pyridinecarboxylic acid (19.5mg, 0.158mmol) and anhydrous K3PO4(335mg, 1.58 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 12(178mg, 74%) as a colourless oil.
(E) -5- (5-amino-2- ((4-nitrophenyl) diazenyl) phenoxy) -N, N-diethylnaphthalen-2-amine (13)
Compound 12(0.1g, 0.326mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, p-nitrophenyltetrafluoroborate diazonium salt (0.085g, 0.359mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 13(0.125g, 81%) which was used in the next step without further purification.
3- (diethylamino) -10H-benzo [ c]Phenoxazine-10-iminium (LGW06-65)
Compound 13(0.05g, 0.11mmol) was charged to a round bottom flask and stirred under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and washed with waterAnhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW06-65(9mg, 26%) as a dark green solid.
Scheme 3: synthetic route to LGW 06-83. Reagents and conditions: a) CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; b) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；c)TfOH，100℃。
5- (3-amino-4-methylphenoxy) -N, N-diethylnaphthalen-2-amine (14)
Compound 14 was synthesized using a slightly modified protocol published by motto and branharder.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 6(218mg, 0.935mmol), 5(183mg, 0.85mmol), CuI (16mg, 0.085mmol), 2-picolinic acid (21mg, 0.17mmol), and anhydrous K3PO4(361mg, 1.7 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 14(248mg, 91%) as a colourless oil.
(E) -5- (5-amino-4-methyl-2- ((4-nitrophenyl) diazenyl)]Phenoxy) -N, N-diethylnaphthalene-2- Amine (15)
Compound 14(0.14g, 0.437mmol) was dissolved in 1mL of methanol. Will be provided withThe solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.109g, 0.459mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 15(0.184g, 90%) which was used in the next step without further purification.
3- (diethylamino) -9-methyl-10H-benzo [ c]Phenoxazine-10-iminium (LGW06-83)
Compound 15(0.05g, 0.106mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW06-83(11mg, 31%) as a dark green solid.
Scheme 4: synthetic route to LGW 06-84. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; d) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；e)TfOH，100℃。
N- (3-iodophenyl) acetamide (17)
Compound 16(1.5g, 6.85mmol) was dissolved in 2mL of DMSO, and acetic anhydride (2.59mL, 27.39mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for a further 2 hours. 18mL of deionized water was added to the reaction mixture and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 17(1.57g, 88%) as a solid which was used in the next step without further purification.
N-ethyl-3-iodoaniline (18)
A solution of 17(1.5g, 5.75mmol) in anhydrous THF (17mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 17mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 18(1.08g, 76%) as a clear oil.
N, N-diethyl-5- (3- (ethylamino) phenoxy) naphthalen-2-amine (19)
Compound 19 was synthesized using a slightly modified protocol published by motto and branharder.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 18(225mg, 0.991mmol), 5(178mg, 0.828mmol), CuI (16mg, 0.083mmol), 2-picolinic acid (21mg, 0.166mmol) anhydrous K3PO4(351mg, 1.66 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was deionized with 10mLDiluted with water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 19(173mg, 62%) as a colourless oil.
(E) -N, N-diethyl-5- (5- (ethylamino) -2- ((4-nitrophenyl) diazenyl) phenoxy) naphthalen-2-amine (20)
Compound 19(0.150g, 0.448mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyl tetrafluoroborate diazonium salt (0.112g, 0.471mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 20(0.189g, 87%) which was used in the next step without further purification.
(E) -N- (3- (diethylaminoisobutyl) -10H-benzo [ c ]]Phenoxazin-10-ylidene) ethylamine (LGW06-84)
Compound 20(0.05g, 0.103mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature (rt) and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM).
The product containing fractions were collected and evaporated to give LGW06-84(15mg, 42%) as a dark green solid.
Scheme 5: synthetic route to LGW 07-14. Reagents and conditions: a) CuI, LiI, DMEDA, dioxane, 110 ℃; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
7-iodo-1, 2,3, 4-tetrahydroquinoline (22)Compound 22 was synthesized using a slightly modified protocol published by krappars (Klapars) and buchward.3An oven or flame dried microwave glass tube was charged with a magnetic stir bar, compound 21(600mg, 2.83mmol), CuI (54mg, 0.283mmol), and LiI (757mg, 5.66 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (77 μ Ι) was quickly added to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (3mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 22(701mg, 96%).
N, N-diethyl-5- ((1,2,3, 4-tetrahydroquinolin-7-yl) oxy) naphthalen-2-amine (23)
Compound 23 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 22(290mg, 1.12mmol), 5(219mg, 1.02mmol), CuI (19mg, 0.102mmol), 2-picolinic acid (25mg, 0.204mmol), and anhydrous K3PO4(432mg, 2.04 mmol). The glass tube was evacuated under vacuum and N was used2Backfill 5Next, the tube was then immediately sealed with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 23(292mg, 83%) as a colorless oil.
(E) -N, N-diethyl-5- ((6- ((4-nitrophenyl) diazenyl) -1,2,3, 4-tetrahydroquinolin-7-yl) oxy Yl) naphthalen-2-amines(24) Compound 23(0.160g, 0.462mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyl tetrafluoroborate diazonium salt (0.115g, 0.485mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 24(0.196g, 86%) which was used in the next step without further purification.
3- (diethylamino) -10, 11-dihydro-9H-benzo [ H ]]Pyrido [3,2-b]Phenoxazin-12-ium (LGW07-14)
Compound 24(0.05g, 0.101mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera rapid system using a SNAP Ultra column, with mobile phaseDCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-14(23mg, 64%) as a dark green solid.
Scheme 6: synthetic route to LGW 07-16. a) Yb (OTf)3Acetone, room temperature; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
7-iodo-2, 2, 4-trimethyl-1, 2-dihydroquinoline (25)Compound 25 was synthesized using a modified protocol published by Belov frazimmil (Belov Vladimir) et al.4In N2Next, compound 16(0.550mL, 4.57mmol) was diluted in acetone (20mL) and ytterbium (III) trifluoromethanesulfonate (0.283g, 0.457mmol) was added to the above solution. The resulting solution was stirred at room temperature for 72 hours. After that, the reaction mixture was concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with water, brine and over anhydrous Na2SO4And (5) drying. The organic solvent was removed using a rotary evaporator. The crude product was purified by flash column chromatography using DCM/hexane as eluent to give compound 25(0.65g, 48%).
N, N-diethyl-5- ((2,2, 4-trimethyl-1, 2-dihydroquinolin-7-yl) oxy) naphthalen-2-amine (26)
Compound 26 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 25(330mg, 1.10mmol), 5(216mg, 1.0mmol), CuI (19mg, 0.10mmol), 2-picolinic acid (25mg, 0.20mmol), and anhydrous K3PO4(426mg, 2.01 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hThen (c) is performed. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 26(277mg, 71%) as a colourless oil.
(E) -N, N-diethyl-5- ((2,2, 4-trimethyl-6- ((4-nitrophenyl) diazenyl) -1, 2-dihydroquine Lin-7-yl) oxy) naphthalen-2-amine (27)Compound 26(0.193g, 0.499mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyl tetrafluoroborate diazonium salt (0.124g, 0.524mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 27(0.219g, 82%) which was used in the next step without further purification.
3- (diethylamino) -9,11, 11-trimethyl-11H-benzo [ H ]]Pyrido [3,2-b]Phenoxazin-12-ium salts (LGW07-16)
Compound 27(0.05g, 0.093mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). Collecting the product-containing fractionPartitioned and evaporated to give LGW07-16(17mg, 46%) as a dark green solid.
Scheme 7: synthetic route to LGW 07-17. Reagents and conditions: a) CuI, LiI, DMEDA, dioxane, 110 ℃; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
6-iodo-3, 4-dihydro-2H-benzo [ b ]][1,4]Oxazine (29)Compound 29 was synthesized using a slightly modified protocol published by krappa and buchward.3A magnetic stir bar, Compound 28(500mg, 2.34mmol), CuI (49mg, 0.257mmol), and LiI (688mg, 5.14mmol) were loaded into an oven or flame dried microwave glass tube. The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (55 μ L) was quickly added to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (2.5mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 29(554mg, 91%).
5- ((3, 4-dihydro-2H-benzo [ b)][1,4]Oxazin-6-yl) oxy) -N, N-diethylnaphthalen-2-amine (30)
Compound 30 was synthesized using a slightly modified protocol published by motto and branharder.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 29(290mg, 1.11mmol), 5(217mg, 1.01mmol), CuI (19mg, 0.101mmol), 2-picolinic acid (25mg, 0.202mmol), and anhydrous K3PO4(429mg, 2.02 mmol). Putting a glass tubeEvacuating under vacuum and applying N2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 30(253mg, 72%) as a colourless oil.
(E) -N, N-diethyl-5- ((7- ((4-nitrophenyl) diazenyl) -3, 4-dihydro-2H-benzo [ b)][1,4] Oxazin-6-yl) oxy) naphthalen-2-amine (31)Compound 30(0.130g, 0.373mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.093g, 0.392mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 31(0.175g, 94%) which was used in the next step without further purification.
3- (diethylamino) -10, 11-dihydrobenzo [ h][1,4]Oxazino [2,3-b]Phenoxazin-12-ium (LGW07- 17)
Compound 31(0.05g, 0.101mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. Using SNAP Ultra column, the residue was purified on Biotage Isolera flash system with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-17(19mg, 53%) as a dark solid.
Scheme 8: synthetic routes to LGW 07-48. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) CuI, LiI, DMEDA, dioxane, 110 ℃; d) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; e) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；f)TfOH，100℃。
N- (5-bromo-2-chlorophenyl) acetamide (33)Compound 32(2g, 9.69mmol) was dissolved in 2mL of DMSO, and acetic anhydride (3.66mL, 38.75mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for a further 2 hours. 18mL of deionized water was added to the reaction mixture and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 33(2.13g, 88%) as a solid which was used in the next step without further purification.
5-bromo-2-chloro-N-ethylaniline (34)A solution of 33(2.0g, 8.05mmol) in anhydrous THF (24mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 24mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure using DCM/hexane as eluent and the residue was purified by flash column chromatography over silica gel,yield 34(1.77g, 94%).
2-chloro-N-ethyl-5-iodoaniline (35)Compound 35 was synthesized using a slightly modified protocol published by krappa and buchward.3A magnetic stir bar, Compound 34(1.20g, 5.12mmol), CuI (98mg, 0.512mmol), and LiI (1.37g, 10.23mmol) were loaded into an oven or flame dried microwave glass tube. The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (121 μ L) was quickly added to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (5mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 35(1.25g, 87%).
5- (4-chloro-3- (ethylamino) phenoxy) -N, N-diethylnaphthalen-2-amine (36)
Compound 36 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 35(315mg, 1.12mmol), 5(219mg, 1.02mmol), CuI (19mg, 0.102mmol), 2-picolinic acid (25mg, 0.203mmol), and anhydrous K3PO4(432mg, 2.03 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 36(316mg, 84%) as a colourless oil.
(E) -5- (4-chloro-5- (ethylamino) -2- ((4-nitro)Phenyl) diazenyl]Phenoxy) -N, N-diethylnaphthalene- 2-amine (37)Compound 36(0.150g, 0.407mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.101g, 0.427mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 37(0.187g, 89%), which was used in the next step without further purification.
(Z) -N- (9-chloro-3- (diethylamino) -10H-benzo [ c)]Phenoxazin-10-ylidene) ethylamine (LGW07-48)Compound 37(0.05g, 0.097mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-48(15mg, 41%) as a dark green solid.
Scheme 9: synthetic route to LGW 07-50. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) CuI, LiI, DMEDA, dioxane, 110 ℃; d) compound 5, CuI, 2-picolinic acid,K3PO4DMSO, 85 ℃; e) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；f)TfOH，100℃。
N- (5-bromo-2-fluorophenyl) acetamide (39)Compound 38(2g, 10.53mmol) was dissolved in 2mL of DMSO, and acetic anhydride (3.97mL, 42.1mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for a further 2 hours. 18mL of deionized water was added to the reaction mixture and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 39(1.99g, 81%) as a solid which was used in the next step without further purification.
5-bromo-N-ethyl-2-fluoroaniline (40)A solution of 39(1.5g, 6.46mmol) in anhydrous THF (19mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 19mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give 40(1.28g, 91%).
N-Ethyl-2-fluoro-5-iodoaniline (41)Compound 41 was synthesized using a slightly modified protocol published by krappa and buchward.3A magnetic stir bar, Compound 40(1.0g, 4.59mmol), CuI (87mg, 0.459mmol), and LiI (1.23g, 9.17mmol) were loaded into an oven or flame dried microwave glass tube. The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (109 μ Ι) was added quickly to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (4mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4Diluted with Cl solution and then with DCM (4 as a crude product)25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 41(1.02g, 84%).
N, N-diethyl-5- (3- (ethylamino) -4-fluorophenoxy) naphthalen-2-amine (42)
Compound 42 was synthesized using a slightly modified protocol published by motto and branharder.2An oven or flame dried microwave glass tube was charged with a magnetic stir bar, Compound 41(180mg, 0.679mmol), 5(133mg, 0.617mmol), CuI (12mg, 0.062mmol), 2-picolinic acid (15mg, 0.124mmol), and anhydrous K3PO4(262mg, 1.23 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 42(174mg, 80%) as a colourless oil.
(E) -N, N-diethyl-5- (5- (ethylamino) -4-fluoro-2- ((4-nitrophenyl) diazenyl) phenoxy) naphthalene- 2-amine (43)
Compound 42(0.150g, 0.426mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyl tetrafluoroborate diazonium salt (0.106g, 0.447mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight,compound 43(0.171g, 80% yield) was obtained and used in the next step without further purification.
(Z) -N- (3- (diethylamino) -9-fluoro-10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium (LGW07-50)
Compound 43(0.05g, 0.100mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-50(12mg, 33%) as a dark green solid.
Scheme 10: synthetic route to LGW 07-92. Reagents and conditions: a) MeI, K2CO3MeCN, 80 ℃; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
3-iodo-N, N-dimethylaniline (44)
At room temperature, in N2Down Compound 11(1g, 4.57mmol) and K2CO3(0.757g, 5.48mmol) to a suspension in anhydrous MeCN (10mL) MeI (0.853mL, 13.7mmol) was added. The reaction mixture was then heated to 80 ℃ and stirred for an additional 12 hours. The solution was cooled to room temperature and concentrated under reduced pressure. The crude product was diluted with deionized water and the resulting suspension was extracted with DCM (3 × 50 mL). The combined organic layers were saltedWashed with water and anhydrous Na2SO4And (5) drying. The solvent was removed using a rotary evaporator and the residue was purified by flash column chromatography on silica gel using EtOAc/hexane as eluent to give compound 44(0.87g, 77%).
5- (3- (dimethylamino) phenoxy) -N, N-diethylnaphthalen-2-amine (45)
Compound 45 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, compound 44(200mg, 0.810mmol), 5(158mg, 0.736mmol), CuI (14mg, 0.074mmol), 2-picolinic acid (18mg, 0.147mmol), and anhydrous K3PO4(312mg, 1.47 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 45(202mg, 82%) as a colorless oil.
(E) -5- (5- (dimethylamino) -2- ((4-nitrophenyl) diazenyl) phenoxy) -N, N-diethylnaphthalene-2- Amine (46)
Compound 45(0.100g, 0.299mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.074g, 0.314mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 46 (0)105g, 72%) which was used in the next step without further purification.
N- (3- (diethylamino) -10H-benzo [ c)]Phenoxazin-10-ylidene) -N-methylmethanamin (LGW07-92)Compound 46(0.05g, 0.103mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-92(8mg, 22%) as a dark green solid.
Scheme 11: synthetic route to LGW 07-98. Reagents and conditions: a) EtI, K2CO3MeCN, 80 ℃; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
N, N-diethyl-3-iodoaniline (47)In N2Next, compound 11(1g, 4.57mmol) and K are added2CO3(0.757g, 5.48mmol) to a suspension in anhydrous MeCN (10mL) EtI (1.1mL, 13.7mmol) was added. The reaction mixture was then heated to 80 ℃ and stirred for an additional 12 hours. The solution was cooled to room temperature and concentrated under reduced pressure. The crude product was diluted with deionized water and the resulting suspension was extracted with DCM (3 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4And (5) drying. The solvent was removed using a rotary evaporator and the residue was taken up using EtOAc/hexane as eluentThe product was purified by flash column chromatography on silica gel to give compound 47(0.94g, 75%).
5- (3- (diethylamino) phenoxy) -N, N-diethylnaphthalen-2-amine (48)
Compound 48 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 47(225mg, 0.818mmol), 5(160mg, 0.743mmol), CuI (14mg, 0.074mmol), 2-picolinic acid (18mg, 0.147mmol), and anhydrous K3PO4(316mg, 1.49 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 48(198mg, 73%) as a colourless oil.
(E) -5- (5- (diethylamino) -2- ((4-nitrophenyl) diazenyl) phenoxy) -N, N-diethylnaphthalen-2-amine (49)Compound 48(0.100g, 0.276mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.069g, 0.290mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 49(0.025g, 18%) which was used in the next step without further purification.
N- (3- (diethylamino) -10H-benzo [ c)]Phenoxazin-10-ylidene) -N-ethylethylammonium (LGW07-98)
Compound 46(0.025g, 0.049mmol) was charged to a round bottom flask and stirred under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW07-98(6mg, 33%) as a dark green solid.
Scheme 12: synthetic route to LGW 08-06. Reagents and conditions: a) CuI, LiI, DMEDA, dioxane, 110 ℃; b) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; c) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；d)TfOH，100℃。
6-iodoindoline (51)Compound 51 was synthesized using a slightly modified protocol published by krappa and buchward.3An oven or flame dried microwave glass tube was charged with a magnetic stir bar, Compound 50(1.0g, 5.05mmol), CuI (106mg, 0.555mmol), and LiI (1.49g, 11.11 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (120 μ Ι) was quickly added to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (5mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4DryingAnd then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 51(1.03g, 83%).
N, N-diethyl-5- (indolin-6-yloxy) naphthalen-2-amine (52)
Compound 52 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 51(240mg, 0.98mmol), 5(201mg, 0.934mmol), CuI (18mg, 0.093mmol), 2-picolinic acid (23mg, 0.187mmol), and anhydrous K3PO4(396mg, 1.87 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 52(265mg, 85%) as a colourless oil.
(E) -N, N-diethyl-5- ((5- ((4-nitrophenyl) diazenyl) indolin-6-yl) oxy) naphthalen-2-amine (53)
Compound 52(0.100g, 0.301mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.078g, 0.331mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 53(0.113g, 78%) which was used in the next step without further purification.
3- (diethylamino) -9, 10-dihydrobenzo [ h]Pyrrolo [3,2-b]Phenoxazin-11-ium (LGW08-06)
Compound 53(0.05g, 0.104mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW08-06(17mg, 48%) as a dark solid.
Scheme 13: synthetic route to LGW 08-35. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) CuI, LiI, DMEDA, dioxane, 110 ℃; d) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; e) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；f)TfOH，100℃。
N- (3-bromo-2-methylphenyl) acetamide (55)Compound 54(4g, 21.5mmol) was dissolved in 4mL of DMSO, and acetic anhydride (8.11mL, 86.0mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for a further 2 hours. To the reaction mixture was added 36mL of deionized water, and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 55(4.48g, 91%) as a solid which was used without further purificationAnd (4) carrying out the next step.
3-bromo-N-ethyl-2-methylaniline (56)A solution of 55(2.0g, 8.77mmol) in anhydrous THF (26mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 26mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 56(1.67g, 89%).
N-Ethyl-3-iodo-2-methylaniline (57)Compound 57 was synthesized using a slightly modified protocol published by krappa and buchward.3An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, compound 56(1.0g, 4.67mmol), CuI (98mg, 0.514mmol), and LiI (1.38g, 10.28 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (111 μ Ι) was added quickly to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (4mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 57(1.05g, 86%).
N, N-diethyl-5- (3- (ethylamino) -2-methylphenoxy) naphthalen-2-amine (58)Compound 58 was synthesized using a slightly modified protocol published by motto and brahward.2A magnetic stir bar, compound 57(174mg, 0.664mmol), 5(130mg, 0.604mmol), CuI (12mg, 0.06mmol), 2-picolinic acid (15mg, 0.121mmol) and anhydrous K were charged into an oven or flame dried microwave glass tube3PO4(256mg, 1.21 mmol). Putting the glass tube in a vacuumEvacuating under the air and using N2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 58(189mg, 90%) as a colourless oil.
(E) -N, N-diethyl-5- (3- (ethylamino) -2-methyl-6- ((4-nitrophenyl) diazenyl) phenoxy) Naphthalene-2-amine (59)Compound 58(0.200g, 0.574mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.143g, 0.603mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 59(0.266g, 93%) which was used in the next step without further purification.
(E) -N- (3- (diethylamino) -11-methyl-10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium (LGW08-35)Compound 59(0.05g, 0.101mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera rapid system using a SNAP Ultra column, with DCM and DCM containing mobile phase0.5% formic acid in methanol (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW08-35(18mg, 50%) as a dark green solid.
Scheme 14: synthetic route to LGW 08-46. Reagents and conditions: a) ac of2O，H2O, from 50 ℃ to room temperature; b) BH3-THF, 0 ℃ to room temperature; c) CuI, LiI, DMEDA, dioxane, 110 ℃; d) compound 5, CuI, 2-pyridinecarboxylic acid, K3PO4DMSO, 85 ℃; e) i)2M HCl, p-nitrophenyltetrafluoroborate diazonium salt, 0 ℃; II) K2CO3，0℃；f)TfOH，100℃。
N- (3-bromo-5-methylphenyl) acetamide (61)Compound 60(4g, 21.5mmol) was dissolved in 4mL of DMSO, and acetic anhydride (8.11mL, 86.0mmol) was added dropwise thereto. The reaction mixture was stirred in a water bath (50 ℃) for 10 minutes and then at room temperature for a further 2 hours. To the reaction mixture was added 36mL of deionized water, and the resulting suspension was stirred at room temperature overnight. Thereafter, the solid was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried to give compound 61(4.26g, 87%) as a solid which was used in the next step without further purification.
3-bromo-N-ethyl-5-methylaniline (62)A solution of 61(2.0g, 8.77mmol) in anhydrous THF (26mL) was placed in an ice bath under N2Stirred for 30 minutes. To the above solution was added borane tetrahydrofuran complex solution (1M, 26mL) over 30 minutes using a syringe pump while maintaining the solution temperature below 5 ℃. The resulting reaction mixture was left in an ice bath and slowly warmed to room temperature. After 24 hours, the solution was placed in the ice bath again and excess borane reagent was destroyed by careful addition of methanol until no gas evolved. The solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give 62(1.72g, 92%).
N-ethyl-3-iodo-5-carbaldehydePhenylamine (63)Compound 63 was synthesized using a slightly modified protocol published by krappa and buchward.3An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, compound 62(1.0g, 4.67mmol), CuI (98mg, 0.514mmol), and LiI (1.38g, 10.28 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling was done 5 times and DMEDA (111 μ Ι) was added quickly to the reaction vessel before sealing the tube with a teflon cap. Anhydrous 1, 4-dioxane (4mL) was delivered by syringe. The reaction was then heated to 110 ℃ and stirred for 24 hours. After cooling to room temperature, the reaction mixture was quenched with 10mL of saturated NH4The Cl solution was diluted and then extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography on silica gel using DCM/hexane as eluent to give compound 63(0.99g, 81%).
N, N-diethyl-5- (3- (ethylamino) -5-methylphenoxy) naphthalen-2-amine (64)
Compound 64 was synthesized using a slightly modified protocol published by motto and brahward.2An oven or flame-dried microwave glass tube was charged with a magnetic stir bar, Compound 63(334mg, 1.28mmol), 5(250mg, 1.16mmol), CuI (22mg, 0.116mmol), 2-pyridinecarboxylic acid (29mg, 0.232mmol), and anhydrous K3PO4(493mg, 2.32 mmol). The glass tube was evacuated under vacuum and N was used2Backfilling 5 times, and immediately sealing the tube with a teflon cap. Anhydrous DMSO (2mL) was delivered by syringe. The reaction was then heated to 85 ℃ and stirred for 18 hours. After cooling to room temperature, the reaction mixture was diluted with 10mL of deionized water and extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified by flash column chromatography over silica gel using DCM/hexane as eluent to give compound 64(322mg, 79%) as a colorless oil.
(E) -N, N-diethyl-5- (5- (ethylamino) -3-methyl-2- ((4-nitrophenyl) diazenyl) phenoxy) Naphthalene-2-amines(65)
Compound 64(0.200g, 0.574mmol) was dissolved in 1mL of methanol. The solution was cooled in an ice bath and then treated with HCl (2M, 10 mL). After 15 minutes, the p-nitrophenyltetrafluoroborate diazonium salt (0.143g, 0.603mmol) was added in 5 portions to the above solution over 15 minutes, followed by stirring at 0 ℃ for an additional 1 hour. During this time, the color of the reaction mixture changed from orange to dark red. After that, carefully use solid K2CO3The solution was basified until the pH of the solution rose above 8. The dark red precipitate was collected by vacuum filtration and washed with a small amount of deionized water. The product was left in the funnel and air dried overnight to give compound 65(0.253g, 89%), which was used in the next step without further purification.
(E) -N- (3- (diethylamino) -8-methyl-10H-benzo [ c)]Phenoxazin-10-ylidene) ethylammonium (LGW08-46)
Compound 65(0.05g, 0.101mmol) was charged to a round bottom flask and heated under N2And (5) blowing down. TfOH (0.5mL) was added quickly to the reaction flask and the resulting solution was heated to 100 ℃. After 2 hours, the reaction mixture was cooled to room temperature and then poured into 25mL of ice-cold water. To the above solution was added dropwise sodium hydroxide solution (2M) until the pH of the solution rose to 4-5. The aqueous solution was extracted with DCM (4X 25 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4Dried and then concentrated in vacuo. The residue was purified on a Biotage Isolera flash system using a SNAP Ultra column with mobile phase DCM and methanol with 0.5% formic acid (gradient, 2-15% methanol in DCM). The product containing fractions were collected and evaporated to give LGW08-46(23mg, 64%) as a dark green solid.
Reference to the literature
1. Bartt, C.W. (Barth, C.W.); gibbs, s.l. (Gibbs, s.l.), "Direct Administration of a Nerve-Specific Contrast agent to Improve Radical Prostatectomy with Nerve retention (Direct Administration of Nerve-Specific Contrast to immediate Nerve Sparing radiation protection)", Theranostics (therapeutics) 2017,7(3),573 + 593.
2. Maidi, d.; buhward, S.L., "Orthogonal catalyst systems for O-and N-based catalysis of aminophenols based on Cu and Pd (Orthogonal Cu-and Pd-based catalysis systems)," J Am Chem Soc 2009,131(47),17423-9.
3. Krapa, a.; broowald, s.l., "copper-catalyzed halogen exchange in aryl halides: aromatic Finlstan Reaction (compressor-Catalyzed Halogen Exchange in Aryl Halides: An Aromatic Finkelstein Reaction). Journal of the American Chemical Society 2002,124(50),14844-14845.
4. Alloff frakemlier, n.; bociclib Mariano, L. (Bossi Mariano, L.); furin, J. (J.); boseski witelm, P. (boyarsky Vadim, P.); heir Stefan, W. (Hell Stefan, W.), "Rhodamine Spiroamides for Multicolor Single-Molecule switch Fluorescent nano-copies" (Rhodamine Spiroamides for Multicolor fluorescence Single-Molecule Switching nanoscoppy), Chemistry-European Journal (Chemistry-a European Journal)2009,15(41), 10762-.
In vivo neural specific screening of benzo [ c ] phenoxazine derivative libraries. Each compound was screened for its tissue specificity using a direct administration strategy (bart & gibbs, theranostics 7,573-593(2017)), in which nerve contrast was examined in the mouse brachial plexus and sciatic nerves. Benzo [ c ] phenoxazine derivatives were dissolved in the previously described co-solvent formulations for in vivo use (Gibbs-schtelus (Gibbs-Strauss) et al, Molecular imaging 10,91-101 (2011)). A previously optimized staining procedure was used (Gibbs) et al, american public library of science (PloS one)8, e73493(2013)), a brief description of which is as follows. By removing the overlying adipose and muscle tissue, the brachial plexus and sciatic nerve were surgically exposed. The benzo [ c ] phenoxazine compound was formulated at 500 μ M in a co-solvent formulation and 100 μ L was incubated on the exposed brachial plexus or sciatic nerve for 5 minutes. The fluorophore-containing solution was removed and the area was washed nine times with saline, then incubated with the blank for five minutes, and then washed nine more times with saline to remove any unbound fluorophores. Images were taken 30 minutes after staining was complete. Unstained nerve sites were used for all control images to quantify autofluorescence. Each benzo [ c ] phenoxazine derivative was screened in the presence of n-3 mice or 6 neural sites/fluorophore. The quantitative tissue fluorescence intensity after direct fluorophore administration was used to calculate the nerve-to-muscle, nerve-to-excised muscle, and nerve-to-fat ratios as measures of nerve contrast. Five lead candidates, LGW05-25, LGW07-14, LGW07-92, LGW08-35, and LGW08-46, were selected for further neurospecific screening studies by systemic administration. The dose and pharmacokinetic studies of the benzo [ c ] phenoxazine fluorophore library are briefly described below. Each lead candidate selected above was formulated in a co-solvent formulation, in which (IV) 500nmol of each compound in 200 μ L solution was administered intravenously and mice were sacrificed at 0.5, 1,2 and 4 hour time points prior to imaging. The non-injected animals were used for all control images to quantify autofluorescence. Similar to the quantitative protocol of direct administration, the quantitative tissue fluorescence intensity after systemic administration of the fluorophore was used to calculate the nerve-muscle ratio and nerve-fat ratio as a measure of nerve contrast. Each candidate was screened in the case of n-3 mice or 6 nerve sites/fluorophore, where two brachial plexus nerves and two sciatic nerve sites were averaged together, in total repeated twice for each animal, one for each nerve type.
An intraoperative fluorescence imaging system. In vivo rodent images are acquired using a custom small animal imaging system capable of real-time color and fluorescence imaging (hakman et al, Molecular pharmaceuticals (2015)). Briefly, the imaging system consisted of a QImaging EXi Blue monochrome camera for fluorescence detection (Surrey, British Columbia, CA) with a removable bayer filter for collecting co-registered color and fluorescence images. The PhotoFluor II light source was focused through a liquid light guide onto the surgical area and used unfiltered for white light illumination. For fluorescence excitation, PhotoFluor II was filtered with a 710 + -37.5 nm band pass emission filter. The resulting fluorescence was collected with a 810nm bandpass emission filter. All filters are available from Chroma Technology (Chroma Technology) (bellos Falls, VT), bellos, budd). The camera and light source positions were unchanged throughout all imaging studies, allowing quantitative comparisons of in vivo fluorescence intensities. The camera exposure time for fluorescence image collection is 5-5000 ms.
In vivo neural specific screening by direct administration of benzo [ c ] phenoxazine derivative libraries. As shown in FIGS. 3A and 3B, the fluorescence intensity was quantified on the mean per second for nerves, muscle, fat and excised muscle (3A). Calculated nerve-to-muscle ratio, nerve-to-excised muscle ratio, nerve-to-fat ratio (3B). Five lead candidates were selected for further neuro-specific screening studies by systemic administration, which were LGW05-25, LGW07-14, LGW07-92, LGW08-35 and LGW08-46 (indicated with blue arrows).
In vivo neural specific screening of selected benzo [ c ] phenoxazine candidates was performed by systemic administration. Mice were injected with 500nmol of each screening candidate in the cosolvent formulation and mice were sacrificed at 0.5 hour, 1 hour, 2 hours, and 4 hour time points. Mean quantitative fluorescence intensity per second for nerve, muscle and adipose tissue (4A, 4B and 4C). Calculated nerve-muscle and nerve-fat ratios (4D and 4E). Following systemic administration, four compounds were selected as lead benzo [ c ] phenoxazine nerve-specific fluorophores, which were LGW05-25, LGW07-14, LGW08-35, and LGW 08-46.
Note: mice were injected with 500nmol of each of the candidate screening co-solvent formulations and sacrificed at the 0.5 hour, 1 hour, 2 hour, and 4 hour time points. Lead candidates were selected after systemic administration and were LGW05-25, LGW07-14, LGW08-35 and LGW 08-46.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：凹凸修补用外用剂