WO2017212059A1 - Synthesis of photoactivable caged cyclofen-oh and derivatives thereof - Google Patents

Synthesis of photoactivable caged cyclofen-oh and derivatives thereof Download PDF

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WO2017212059A1
WO2017212059A1 PCT/EP2017/064203 EP2017064203W WO2017212059A1 WO 2017212059 A1 WO2017212059 A1 WO 2017212059A1 EP 2017064203 W EP2017064203 W EP 2017064203W WO 2017212059 A1 WO2017212059 A1 WO 2017212059A1
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Ludovic Jullien
Isabelle AUJARD
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Centre National de la Recherche Scientifique CNRS
Universite Pierre et Marie Curie
Universite Paris Sciences et Lettres
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Universite Pierre et Marie Curie
Universite Paris Sciences et Lettres
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/18—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted otherwise than in position 3 or 7
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C201/00—Preparation of esters of nitric or nitrous acid or of compounds containing nitro or nitroso groups bound to a carbon skeleton
    • C07C201/06—Preparation of nitro compounds
    • C07C201/12—Preparation of nitro compounds by reactions not involving the formation of nitro groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C213/00—Preparation of compounds containing amino and hydroxy, amino and etherified hydroxy or amino and esterified hydroxy groups bound to the same carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C37/00—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring
    • C07C37/11—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by reactions increasing the number of carbon atoms
    • C07C37/20—Preparation of compounds having hydroxy or O-metal groups bound to a carbon atom of a six-membered aromatic ring by reactions increasing the number of carbon atoms using aldehydes or ketones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/12—Systems containing only non-condensed rings with a six-membered ring
    • C07C2601/14—The ring being saturated

Definitions

  • the present invention relates to a new process of manufacturing of photoactivable caged precursors, which are useful for understanding the spatiotemporal dynamics of biological cells. Especially, the invention relates to a process of manufacturing of caged eye I o fen - OH and derivatives thereof, more specifically of compounds of formula (I):
  • n R 1 , R 1' , R 2 , and R 3 are as defined below.
  • Cells respond to external signals by modifying their internal state and their environment.
  • cellular differentiation and intra-ce!lular signaling are essential for the coordinated development of the organism. Revealing and understanding the spatiotemporal dynamics of these complex interaction networks is a major goal in biology.
  • Photoactivation methods have proved particularly attractive for such control of protein' s activity restoration due to their fast spatio-temporal dynamics.
  • genetically encoded photoactivabie proteins were designed to intrinsically bear a non-photoactive site which can be activated by an inducer released upon photo-activation of a smal l lipophilic caged precursor.
  • This method has been successfully implemented to photo-control gene expression in eukaryotic systems with one- and two-photon excitation.
  • the Applicant has developed such a system to photocontrol protein activity (Sinha et al., ChemBioChem, 2010, 11, 653-663).
  • a steroid-related inducer was used since various proteins, such as for example Engrailed, Otx2, Gal4, p53, kinases such as Raf- 1, ( 're or Flp recombinases, fused to a steroid receptor were shown to be activated by binding of an appropriate inducer such as cyclofen-OH ( Figure 1).
  • an appropriate inducer such as cyclofen-OH
  • the steroid receptor in absence of this appropriate inducer (Ind) forms a cytoplasmic assembly with a chaperone complex (cc), inactivating the fusion protein (Prot).
  • the function of the protein is restored in the presence of the inducer which binds to the receptor and disrupts the complex.
  • the photocontrol of the activity of the targeted protein is performed by fusing it to the extensively used modified estrogen receptor iigand-binding domain (ER 1 ) specific for the non-endogenous 4-hydroxy-cyclofen inducer, also called cyclofen-OH.
  • the overall yields are respectively of 22%, 9.6% and 11% for cind, c'Ind and c"Ind. These low yields are not compatible with synthesis at large scales.
  • the caged precursor is obtained from the inducer (cyclofen-OH). A careful purification of the caged precursor is thus required so that the amount of remai ning inducer in the fi nal product be lower than 2%. Otherwise, the inducer present as an impurity will directly activate the protein, without control by photo-activation. In order to ensure such a low content of cyclofen-OH in the final product, a 1 1 PI X ' purification was needed. This is also not compatible with a scale-up process.
  • Patent application WO2008/030771 discloses modulators of estrogen receptor for therapeutic uses.
  • Compounds of WO2008/030771 are cyclic alkylidene derivatives whose general formula encompasses cyclofen-OH.
  • WO2008/030771 discloses three routes of synthesis, especially the route of synthesis of Scheme 2 below, leading to compound (8) via intermediate (7) (example 3 of WO2008/030771):
  • the overall yield for protected intermediate (7) is of 22%. As mentioned above with the other route of synthesis, such low yield is not suitable for scaling-up the process.
  • alkoxy refers to any group O-alkyl, wherein alky! is as herein defined. Suitable alkoxy groups include for example methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, t-butoxy, sec-butoxy, and n-pentoxy.
  • alkyl refers to a hydrocarbyl radical of formula CnH2n + i wherein n is a number greater than or equal to 1.
  • alkyl groups of this invention comprise from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. Suitable alkyl groups include methyl, ethyl, propyl (n-propyl, i-propyl, n-butyl), butyl (i-butyl, s-butyl and t-butyl), pentyl and its isomers (e.g. n-pentyl, iso-pentyl), and hexyl and its isomers (e.g. n-hexyl, iso-hexyl).
  • alkylaniino refers to a group of formula -NH(alkyl), wherein alkyl is as herein defined.
  • alky!oxyaryl refers to any group aryl-O-alkyl, wherein aryl and alkyl are as herein defined.
  • alkyloxycarbonylalkyloxy refers to any group -0-alkyl-(CO)-0-alkyl, wherein alky] is as herein defined.
  • amino refers to the Nl ⁇ i group.
  • aryl refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphtyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic.
  • the aromatic ring may optionally include one to two additional rin s (either cycloalkyl, heterocyciyl or heteroaryl) fused thereto.
  • Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein.
  • Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl.
  • arylethynyl refers to a group of formula ( ' ⁇ ( '-aryl, wherein aryl is as herein defined.
  • dialkylaniino refers to a group of formula N(alkyl K alkyl ), wherein alkyl is as herein defined.
  • halo refers to fluoro, chloro, bromo, or iodo.
  • haloalkoxy refers to any group alkoxy group substituted by one or more halo group.
  • An example haloalkoxy group is -OCF3.
  • - haloalkyl refers to any group alkyl group substituted by one or more halo group.
  • haloalkyl groups examples include CCb and CBr 3 .
  • salt refers to the acid addition and base salts thereof.
  • Suitable acid additi n salts are formed from acids which form preferably non-toxic salts.
  • Non-limiting examples include the acetate, trifluoroacetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, tetrafluoroborate, camsylate, citrate, cyciamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexaf!uorophosphate, hibenzate, hydrochloride/chioride, hydrobromide bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sul phate, naphthylate, 2-napsylate, nicotinate, nitrate, or
  • Suitable base salts are formed from bases which form preferably non-toxic salts.
  • Non-limiting examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, 2- (diethylamino)ethanol, ethanolamine, morpholine, 4- (2- hydroxyefhyi)morpholine and zinc salts.
  • Hemisalts of acids and bases may also be formed, for example, hemisuiphate and hemicalcium salts.
  • pharmaceutically acceptable salts include hydrochloride/chioride, hydrobromide/bromide, bisulphate/sulphate, nitrate, citrate, and acetate.
  • sulfonate refers to group of formula -OSO2-R, wherein R represents alky], haloalkyl, optionally substituted aryl. Examples of preferred sulfonate groups are mesylate, in late and tosylate.
  • R 1 represents Ar 1 , -CO-Ph, -CH 2 -(o-COR)Ph, -CH 2 -(o-N0 2 )Ph or -CO-CO-NR 2 , wherein
  • Ar 1 represents a phenyl or coumarin group, optionally substituted by one or more group selected from nitro, alkoxy, halo, hydroxy 1, amino, alkyiami.no, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano;
  • Ph represents a phenyl group, optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haloalkyl, haloalkoxy, amino, alkylamino, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano; and
  • R represents aryl, alkyl or vinylcarboxy; wherein the aryl group is optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haloalkyl, haloalkoxy, amino, alkylamino, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano;
  • R 1 ' represents H, alkyl, haloalkyl, cyano or aryl
  • R 2 and R 3 represent each independently hydrogen, alkyl, carbomimidoyl; or R 2 and R 3 form together with the nitrogen atom to which they are attached an heterocyclic ring preferably selected from pyrolidinyl, piperidinyl, N-methyi-piperazinyl, morpholinyl and pyrrolyl; and n is an integer ranging from 1 to 6, preferably n is 1, 2, 3 or 4.
  • R 1 represents a moiety selected from:
  • R ⁇ R ⁇ R 6 , R 7 , R 8 , R 9 , R 10 , R", R 12 and R 13 represent each independently II, nitro, aikoxy, halo, hydroxy I, amino, alky!amino, dialkylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyi or cyano;
  • R", R 15 , R 16 , R 1 and R 18 represent each independently 11, aikoxy, halo, hydroxyl, alky I, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyi and cyano.
  • R 1 represents H or methyl, preferably R 1 represents H. According to one embodiment, R 1 represents haloalkyl, preferably -CCI3 or -C'Bn. According to one embodiment, R 1 represents aryl, preferably phenyl.
  • 11 is equal to 1 or 2.
  • R 2 and R 3 represent both methyl or R 2 and R 3 represent both ethyl. According to one embodiment, R 2 represents 11 and R 3 represents a carbomimidoyl group.
  • R 2 and R 3 form together with the nitrogen atom to which they are attached a heterocyclic ring selected from pyrolidinyl, piperidinyl, N-methyl- pipera/inyl, morpholinyl and pyrrolyl.
  • compounds of formula (I) are of formula
  • R 4 , R ⁇ R 6 , R 7 and R 8 represent each independently H, nitro, alkoxy, halo, hydroxyl, amino, alkylami no, dialkylam ino. alkyioxycarbonylaikyloxy, alkyloxyaryl, aryl, ai lvinylyl, arylethynyl or cyano.
  • R 4 is nitro. According to a specific embodiment, R 4 is nitro and R 8 is I I . According to another specific embodiment, R 4 and R 8 are both nitro.
  • R 5 is I I .
  • R 6 is an alkoxy, preferably methoxy. According to another specific embodiment, R 6 is H.
  • R is alkoxy, alkyioxycarbonylaikyloxy or alkyloxyaryl, preferably R is methoxy, ethoxy, ethyloxycarbonylmethoxy or methoxyphenyi.
  • compounds of formula (I) are of formula (lb) I I
  • R 9 , R 10 , R", R 12 and R 13 represent each independently 1 1. nitro, alkoxy, halo, hydroxy!, amino, alkylamino, dialkylamino, alkyioxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl or cyano.
  • R 9 is H.
  • R 10 is H.
  • R 13 is H.
  • R", R 10 and R " are H.
  • R" is 1 1, alkoxy or halo, preferably R " is 1 1, methoxy or bromo.
  • R 12 is alkoxy, hydroxy 1 or dialkylamino, preferably R 12 is methoxy, hydroxy! or dimethylamino.
  • the process of the invention comprises a fi st step of McMurry coupling on 4,4'- dihydroxybenzophenone, enabling to introduce the cyclohexyl ring.
  • the second step introduces the caging arylalkyl moiety by phenol mono substitution.
  • the second phenol group is then alkylated.
  • the process of the invention is a process of manufacturing a compound of formula (I) as defined above, comprising the following steps: a) performing a McMurry coupling between 4,4'-dihydroxybenzophenone and cyclohexanone to form intermediate (A):
  • R 1 and R 1 are as defined in formula (I),
  • step b) is performed in the dark, in a solvent in which intermediate (A) and caging halide (B) are soluble and in which intermediate (C) is not soluble; and c) alkylating, in the dark, the remaining phenol of intermediate (C) with derivative (D):
  • n, R 2 and R 3 are as defined in formula (I) and X 2 represents halo or sulfonate, preferably X 2 represents CI, Br, I, mesylate, triflate or tosyiate, more preferably X 2 represents CI; to afford compound of formula (I).
  • cyclohexanone is used at a proportion ranging from 1 to 10 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 3 to 6 equivalents, more preferably at a proportion of about 4.2 equivalents.
  • step a) is performed in presence of TiCl i and /inc.
  • zinc is used under the form of powder.
  • zinc is activated zinc.
  • T1CI4 is used at a proportion ranging from 2 to 15 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 4 to 10 equivalents, more preferably at a proportion of about 6.2 equivalents.
  • zinc is used at a proportion ranging from 2 to 30 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 6 to 20 equivalents, more preferably at a proportion of about 14 equivalents.
  • the solvent used in step a) is selected from tetrahydrofuran ( 1 1 II ⁇ ). 1,2-dimethoxyethane (DME), dioxane and tetrahydropyran; preferably the solvent is THF, more preferably dry 1 1 11 ⁇ .
  • the McMurry coupling of step a) is performed at a temperature ranging from 20 to 100°C, preferably at reflux of the solvent.
  • the McMurry coupling of step a) is performed for a duration ranging from 0.5 to 24 hours, preferably from 1 to 12 hours, more preferably for 2 hours.
  • Intermediate (A) obtained in step a) may be purified by flash chromatography or by precipitation.
  • intermediate (A) is purified by flash chromatography on silica gel, using preferably a mixture cyclohexane/ethyl acetate as solvent.
  • (A) is purified by precipitation and the solvent of precipitation is selected from dichloromethane, cyclohexane and a mixture ethyl acetate/cyclohexane.
  • caging halide (B) is used at a proportion ranging from 0.5 to 2 equivalents compared to intermediate (A), preferably at a proportion of 0.8 to 1 . 1 equivalents, more preferably at a proportion of about 0.95 equivalents.
  • the caging halide (B) is a bromide, i.e. X 1 represents Br.
  • caging halide (B) is a chloride, i.e. X 1 represents CI.
  • step b) is performed in presence of a base, preferably a base selected from K2CO3 and CS2CO3, more preferably the base is K2CO3.
  • K2CO3 is used at a proportion ranging from 0.5 to 2 equivalents compared to intermediate (A), preferably at a proportion of 0.8 to 1.1 equivalents, more preferably at a proportion of about 0.95 equivalents.
  • Nal or Kl may be added to perform step b) i the conversion rate is too low.
  • the solvent used in step b) is a solvent in which starting products are soluble and resulting compound is not soluble.
  • the solvent used in step b) is a solvent in which intermediate (A) and caging halide (B) are soluble and in which intermediate (C) is not soluble.
  • Such solvent enables to easily recover intermediate (C) as a precipitate by filtration.
  • the solvent used in step b) is a mixture of water and of a miscibie organic solvent. Examples of organic solvents miscibie in water are acetonitrile, acetone, dioxane, I I 11 % alcohol (such as for example methanol, ethanol or butanol).
  • the solvent used in step b) is selected from water, acetonitrile, acetone, dioxane, I I II ⁇ .
  • alcohol such as for example methanol, ethanol or butanol
  • the solvent is a mixture of acetonitrile and water, more preferably acetonitriie/water 2/1 (v/v).
  • step b) is performed at a temperature ranging from 0 to 100°C, preferably at room temperature.
  • step b) is performed for a duration ranging from 1 to 72 hours, preferably from 4 to 48 hours, more preferably for 16 to 24 hours.
  • Step b) is performed in the dark in order to avoid degradation of resulting intermediate (C) which is photosensitive.
  • Intermediate (C) obtained in step b) forms a precipitate in the conditions of the reaction.
  • intermediate (C) does not need to be purified to be used in step c).
  • derivative (D) is used at a proportion ranging from I to 10 equivalents compared to intermediate (C), preferably at a proportion of 1 to
  • step c) is performed in presence of a base, preferably a base selected from CS2CO3 and 2 C03, more preferably the base is CS2CO3.
  • CS2CO3 is used at a proportion ranging from 2 to I 1 equivalents compared to intermediate (C), preferably at a proportion of 2 to
  • the solvent used in step c) is selected from acetone, acetonitrile and di methyl ormamide; preferably the solvent is acetone.
  • step c) is performed at a temperature ranging from 20 to 155°C, preferably at reflux of the solvent.
  • step c) is performed for a duration ranging from 1 to 48 hours, preferably from 6 to 24 hours, more preferably for 18 hours.
  • Step c) is performed in the dark in order to avoid degradation of final compound of formula (I) which is photosensitive.
  • Final compound (I) obtained in step c) is purified by flash chromatography.
  • compound (I) is purified by flash chromatography on silica gel, using preferably a mixture dichloromethane and methanol as solvent.
  • the process of the invention presents the advantage to have an overall yield of more than 30%, preferably of more than 40%, even more preferably of more than 45%. This overall yield is twofold compared to process disclosed in the prior art.
  • Figure 1 is a scheme representing the strategy to photo-activate properly engineered proteins: a protein (Prot) fused to the ER T2 receptor (ER T2 ) is inactivated by the assembly formed with a chaperone complex (cc). Upon photoactivation of a caged precursor (clnd), a non-endogenous inducer (Ind) is released, which binds to the LR 1 2 receptor and sets the protein fusion free from its assembly with the chaperone complex.
  • the inducer (Ind) may be 4-hydroxy-cycIofen (also called cyclofen-OH).
  • ACN acetonitrile
  • Mass spectra (chemical ionization and high resolution with NH3 or C ' i I i ) were performed by the Service de Spectrometrie de Masse de Chimie ParisTech or by the Service de Spectrometrie de Masse de 1'ICOA (Orleans).
  • Column chromatography was performed on silica gel 60 (0.040-0.063 nm; Merck).
  • Analytical thin-layer chromatography (TLC) was conducted on Merck silica gel 60 F254 precoated plates.
  • Titanium chloride (6.2 mL, 56 mmol) was added dropwise under argon to a stirred suspension of activated zinc powder (8.20 g, 126 mmol) in dry tetrahydrofuran (80 mL) at -10°C. When the addition was complete, the mixture was warmed to room temperature and then re fluxed for 2 h. A solution of 4,4'-dihydroxybenzophenone (2.0 g, 9 mmol ) and cyclohexanone (4 mL, 38 mmol) in dry tetrahydrofuran (120 ml . ) was added to the cooled suspension of the titanium reagent at 0-5 C and the mixture was re flu ed for 2 h.

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Abstract

The present invention relates to a new process of manufacturing of caged cyclofen-OH and derivatives thereof, of formula (I) or salts thereof, wherein n, R1, R1 ', R2, and R3 are as defined in the claims.

Description

SYNTHESIS OF PHOTOACTIVABLE CAGED CYCLOFEN-OH AND
DERIVATIV ES THEREOF
FIELD OF INVENTION The present invention relates to a new process of manufacturing of photoactivable caged precursors, which are useful for understanding the spatiotemporal dynamics of biological cells. Especially, the invention relates to a process of manufacturing of caged eye I o fen - OH and derivatives thereof, more specifically of compounds of formula (I):
Figure imgf000002_0001
or salts thereof, wherein n, R1, R1', R2, and R3 are as defined below.
BACKGROUND OF INVENTION
Cells respond to external signals by modifying their internal state and their environment. In multicellular organisms in particular, cellular differentiation and intra-ce!lular signaling are essential for the coordinated development of the organism. Revealing and understanding the spatiotemporal dynamics of these complex interaction networks is a major goal in biology.
While some of the most important players of these networks have been identified, much less is known of the quantitative rules that govern their interactions with one another and with other cellular components. Investigating these interactions requires the development of means to control or interfere spatially and temporally with these processes.
To address these issues, various approaches have been introduced to control proteins activity. One of these strategies directly acts at the protein level: the protein of interest is first inactivated and the activity of the protein is then restored with an appropriate stimulus in a controlled manner.
Photoactivation methods have proved particularly attractive for such control of protein' s activity restoration due to their fast spatio-temporal dynamics. For that purpose, genetically encoded photoactivabie proteins were designed to intrinsically bear a non-photoactive site which can be activated by an inducer released upon photo-activation of a smal l lipophilic caged precursor. This method has been successfully implemented to photo-control gene expression in eukaryotic systems with one- and two-photon excitation. Especially, the Applicant has developed such a system to photocontrol protein activity (Sinha et al., ChemBioChem, 2010, 11, 653-663). In this system, a steroid-related inducer was used since various proteins, such as for example Engrailed, Otx2, Gal4, p53, kinases such as Raf- 1, ( 're or Flp recombinases, fused to a steroid receptor were shown to be activated by binding of an appropriate inducer such as cyclofen-OH (Figure 1). As represented on Figure 1, in absence of this appropriate inducer (Ind), the steroid receptor ( ER 1 2) forms a cytoplasmic assembly with a chaperone complex (cc), inactivating the fusion protein (Prot). The function of the protein is restored in the presence of the inducer which binds to the receptor and disrupts the complex. The photocontrol of the activity of the targeted protein is performed by fusing it to the extensively used modified estrogen receptor iigand-binding domain (ER1 ) specific for the non-endogenous 4-hydroxy-cyclofen inducer, also called cyclofen-OH.
The Applicant evidenced that photo-releasing cyclofen-OH from a caged precursor (clnd) is an efficient strategy to restore the function of a protein fused to the ERT2 receptor. The dynamical effects of this system were investigated in cultured cells and in live zebrafish embryos. This method is compatible with a wide variety of proteins and may open up opportunities for the local spatio-temporal investigation of developmental pathways, the identification of stem cells and the study of cancer in a live organism. In above-referred study (Sinha et al., ChemBioChem, 2010, 11, 653-663), the Applicant used 3 caged precursors (cind, c'Ind and c"Ind) obtained through the following route of synthesis (Scheme 1):
Figure imgf000004_0001
cind c'Ind c'Ind Scheme 1 . Synthesis of caged precursors cind, c' Ind and c"Ind: a) cyclohexanone, TiC1.4, Zn, ΤΗ1 ·, reflux, 2 h; b) C1(CH2)2N(CH3)2- HC1, K2CO3, acetone/I I2O, reflux, 18 h; c) caging alcohol, P(Ph)3, diisopropylazo dicarboxylate, THF, sonication, 20 min.
In this route of synthesis, a fi st step of McMurry coupling enables to introduce the cyclohexyl ring on 4,4'-dihydroxybenzophenone. Cyclofen-OH is then obtained by phenol mono substitution. Caging of cyclofen-OH is then performed by a M i tsunobu reaction enabling to introduce the caging moiety.
Above route of synthesis presents the drawback to be not very reproducible.
Besides, the overall yields are respectively of 22%, 9.6% and 11% for cind, c'Ind and c"Ind. These low yields are not compatible with synthesis at large scales. Moreover, the caged precursor is obtained from the inducer (cyclofen-OH). A careful purification of the caged precursor is thus required so that the amount of remai ning inducer in the fi nal product be lower than 2%. Otherwise, the inducer present as an impurity will directly activate the protein, without control by photo-activation. In order to ensure such a low content of cyclofen-OH in the final product, a 1 1 PI X ' purification was needed. This is also not compatible with a scale-up process.
Above route of synthesis is thus not suitable for providing large amounts of caged precursors, which are required when dealing with demanded appl ications in animal models such as mice.
There is thus a need to provide a new process of synthesis of caged precursors of cyclofen- OH and derivatives thereof.
Patent application WO2008/030771 discloses modulators of estrogen receptor for therapeutic uses. Compounds of WO2008/030771 are cyclic alkylidene derivatives whose general formula encompasses cyclofen-OH. WO2008/030771 discloses three routes of synthesis, especially the route of synthesis of Scheme 2 below, leading to compound (8) via intermediate (7) (example 3 of WO2008/030771):
Figure imgf000005_0001
Scheme 2. Synthesis of estrogen modulator (8): a) ben/yl bromide. 2CO3, DMF, 60°C, 48 h; b) cyclooctanone, TiCU, Zn, Ti l l . 65°C, overnight; c) CKCH. hNlCH O. I K 'K
Csi( '( )·.. DMF, 70°C, overnight; d) H2, Pd/C, EtOAc, 2 h. In this route of synthesis, a fi st step of phenol monoprotection is followed by a McMurry coupling to introduce the cycloalkyl ring. The second phenol is then substituted by alkylation and the final compound is released after deprotection.
With this route of synthesis, the overall yield for protected intermediate (7) is of 22%. As mentioned above with the other route of synthesis, such low yield is not suitable for scaling-up the process.
There is thus a need to provide an improved process of manufacturing of caged precursors of cyclofen-OH and derivatives thereof, being reproducible, providing an overall higher yield, not requiring sensible purification steps and being suitable for scale-up. The process of the present invention has such properties, as evidenced in the example part.
DEFINITIONS
In the present invention, the following terms have the following meanings:
"alkoxy" refers to any group O-alkyl, wherein alky! is as herein defined. Suitable alkoxy groups include for example methoxy, ethoxy, n-propoxy, isopropoxy, n- butoxy, t-butoxy, sec-butoxy, and n-pentoxy.
"alkyl" refers to a hydrocarbyl radical of formula CnH2n+i wherein n is a number greater than or equal to 1. Generally, alkyl groups of this invention comprise from 1 to 12 carbon atoms, preferably from 1 to 6 carbon atoms. Alkyl groups may be linear or branched and may be substituted as indicated herein. Suitable alkyl groups include methyl, ethyl, propyl (n-propyl, i-propyl, n-butyl), butyl (i-butyl, s-butyl and t-butyl), pentyl and its isomers (e.g. n-pentyl, iso-pentyl), and hexyl and its isomers (e.g. n-hexyl, iso-hexyl).
"alkylaniino" refers to a group of formula -NH(alkyl), wherein alkyl is as herein defined.
"alky!oxyaryl" refers to any group aryl-O-alkyl, wherein aryl and alkyl are as herein defined. "alkyloxycarbonylalkyloxy" refers to any group -0-alkyl-(CO)-0-alkyl, wherein alky] is as herein defined.
"amino" refers to the Nl \i group.
"aryl" refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e. phenyl) or multiple aromatic rings fused together (e.g. naphtyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic. The aromatic ring may optionally include one to two additional rin s (either cycloalkyl, heterocyciyl or heteroaryl) fused thereto. Aryl is also intended to include the partially hydrogenated derivatives of the carbocyclic systems enumerated herein. Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl.
5- or 6- tetralinyl, naphthalen-1- or -2-yl, 4-, 5-, 6 or 7-indenyl, I - 2-, 3-, 4- or
5- acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, I - or 2-pentaienyi, 4- or 5-indanyl, 5-,
6- , 7- or 8-tetrahydronaphthyl, 1,2,3,4-tetrahydronaphthyl, 1,4-dihydronaphthyl, I -. 2-, 3-, 4- or 5-pyrenyl. - "arylethynyl" refers to a group of formula ( '≡( '-aryl, wherein aryl is as herein defined.
"aryivinylyl" refers to a group of formula -CH=CH-aryl, wherein aryl is as herein defined.
"carbaniiinidoyl*' refers to the moiety
Figure imgf000007_0001
- "dialkylaniino"' refers to a group of formula N(alkyl K alkyl ), wherein alkyl is as herein defined.
"halo" refers to fluoro, chloro, bromo, or iodo.
"haloalkoxy" refers to any group alkoxy group substituted by one or more halo group. An example haloalkoxy group is -OCF3. - "haloalkyl" refers to any group alkyl group substituted by one or more halo group.
Examples of preferred haloalkyl groups are CCb and CBr3.
"salt" refers to the acid addition and base salts thereof. Suitable acid additi n salts are formed from acids which form preferably non-toxic salts. Non-limiting examples include the acetate, trifluoroacetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, tetrafluoroborate, camsylate, citrate, cyciamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexaf!uorophosphate, hibenzate, hydrochloride/chioride, hydrobromide bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sul phate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form preferably non-toxic salts. Non-limiting examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, 2- (diethylamino)ethanol, ethanolamine, morpholine, 4- (2- hydroxyefhyi)morpholine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisuiphate and hemicalcium salts. Preferred, pharmaceutically acceptable salts include hydrochloride/chioride, hydrobromide/bromide, bisulphate/sulphate, nitrate, citrate, and acetate.
"sulfonate" refers to group of formula -OSO2-R, wherein R represents alky], haloalkyl, optionally substituted aryl. Examples of preferred sulfonate groups are mesylate, in late and tosylate.
"viny!carboxy" refers to a group of formula ( '( )-( i 1=( Ί h.
DETAILED DESCRIPTION Compounds This invention relates to a process of manufacturing of caged cyclofen-OH and derivatives thereof, more specifically of compounds of formula (I):
Figure imgf000009_0001
or salts thereof, wherein
R1 represents Ar1, -CO-Ph, -CH2-(o-COR)Ph, -CH2-(o-N02)Ph or -CO-CO-NR2, wherein
Ar1 represents a phenyl or coumarin group, optionally substituted by one or more group selected from nitro, alkoxy, halo, hydroxy 1, amino, alkyiami.no, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano;
Ph represents a phenyl group, optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haloalkyl, haloalkoxy, amino, alkylamino, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano; and
R represents aryl, alkyl or vinylcarboxy; wherein the aryl group is optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haloalkyl, haloalkoxy, amino, alkylamino, diaikylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl and cyano;
R1 ' represents H, alkyl, haloalkyl, cyano or aryl;
R2 and R3 represent each independently hydrogen, alkyl, carbomimidoyl; or R2 and R3 form together with the nitrogen atom to which they are attached an heterocyclic ring preferably selected from pyrolidinyl, piperidinyl, N-methyi-piperazinyl, morpholinyl and pyrrolyl; and n is an integer ranging from 1 to 6, preferably n is 1, 2, 3 or 4. According to one embodiment, R1 represents a moiety selected from:
Figure imgf000010_0001
wherein
the dotted line represents the point of attachment to the rest of the molecule, R is as defined in formula (I); and
R\ R\ R6, R7, R8, R9, R10, R", R12 and R13 represent each independently II, nitro, aikoxy, halo, hydroxy I, amino, alky!amino, dialkylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyi or cyano; and
R", R15, R16, R1 and R18 represent each independently 11, aikoxy, halo, hydroxyl, alky I, haloalkyl, haloalkoxy, amino, alkylamino, dialkylamino, alkyloxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyi and cyano.
According to one embodiment, R1 represents H or methyl, preferably R1 represents H. According to one embodiment, R1 represents haloalkyl, preferably -CCI3 or -C'Bn. According to one embodiment, R1 represents aryl, preferably phenyl.
According to one embodiment, 11 is equal to 1 or 2.
According to one embodiment, R2 and R3 represent both methyl or R2 and R3 represent both ethyl. According to one embodiment, R2 represents 11 and R3 represents a carbomimidoyl group.
According to one embodiment, R2 and R3 form together with the nitrogen atom to which they are attached a heterocyclic ring selected from pyrolidinyl, piperidinyl, N-methyl- pipera/inyl, morpholinyl and pyrrolyl. According to one embodiment, compounds of formula (I) are of formula
Figure imgf000011_0001
or salts thereof, wherein n, R1 ', R2 and R3 are as defined in formula (I); and
R4, R\ R6, R7 and R8 represent each independently H, nitro, alkoxy, halo, hydroxyl, amino, alkylami no, dialkylam ino. alkyioxycarbonylaikyloxy, alkyloxyaryl, aryl, ai lvinylyl, arylethynyl or cyano.
According to a specific embodiment, R4 is nitro. According to a specific embodiment, R4 is nitro and R8 is I I . According to another specific embodiment, R4 and R8 are both nitro.
According to a specific embodiment, R5 is I I .
According to a specific embodiment, R6 is an alkoxy, preferably methoxy. According to another specific embodiment, R6 is H.
According to a specific embodiment, R is alkoxy, alkyioxycarbonylaikyloxy or alkyloxyaryl, preferably R is methoxy, ethoxy, ethyloxycarbonylmethoxy or methoxyphenyi.
According to one embodiment, compounds of formula (I) are of formula (lb) I I
Figure imgf000012_0001
or salts thereof, wherein n, R1 ', R2 and R3 are as defined in formula (I); and
R9, R10, R", R12 and R13 represent each independently 1 1. nitro, alkoxy, halo, hydroxy!, amino, alkylamino, dialkylamino, alkyioxycarbonylalkyloxy, alkyloxyaryl, aryl, arylvinylyl, arylethynyl or cyano.
According to a specific embodiment, R9 is H. According to a specific embodiment, R10 is H. According to a specific embodiment, R13 is H. According to a specific embodiment, R", R 10 and R " are H. According to a specific embodiment, R" is 1 1, alkoxy or halo, preferably R " is 1 1, methoxy or bromo.
According to a specific embodiment, R12 is alkoxy, hydroxy 1 or dialkylamino, preferably R12 is methoxy, hydroxy! or dimethylamino.
In the present appl ication, when it is referred to compounds of formula (I), it includes references to all sub formulae thereof and to specific embodiments relative to substituents. Process
The process of the invention comprises a fi st step of McMurry coupling on 4,4'- dihydroxybenzophenone, enabling to introduce the cyclohexyl ring. The second step introduces the caging arylalkyl moiety by phenol mono substitution. The second phenol group is then alkylated. This route of synthesis is summarized in Scheme 3 below:
Figure imgf000013_0001
Scheme 3. Route of synthesis of the present invention.
According to one embodiment, the process of the invention is a process of manufacturing a compound of formula (I) as defined above, comprising the following steps: a) performing a McMurry coupling between 4,4'-dihydroxybenzophenone and cyclohexanone to form intermediate (A):
Figure imgf000013_0002
b) performing a phenol monosubstitution on intermediate (A) with caging halide (B):
Figure imgf000013_0003
(B) wherein R ' and R 1 are as defined in formula (I) and X ' represents halo, preferably X1 represents Br or CI, more preferably X1 represents Br, to form intermediate
Figure imgf000014_0001
wherein R1 and R 1 are as defined in formula (I),
wherein step b) is performed in the dark, in a solvent in which intermediate (A) and caging halide (B) are soluble and in which intermediate (C) is not soluble; and c) alkylating, in the dark, the remaining phenol of intermediate (C) with derivative (D):
Figure imgf000014_0002
wherein n, R2 and R3 are as defined in formula (I) and X2 represents halo or sulfonate, preferably X2 represents CI, Br, I, mesylate, triflate or tosyiate, more preferably X2 represents CI; to afford compound of formula (I).
Step a) According to one embodiment, cyclohexanone is used at a proportion ranging from 1 to 10 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 3 to 6 equivalents, more preferably at a proportion of about 4.2 equivalents.
According to one embodiment, step a) is performed in presence of TiCl i and /inc.
Advantageously, zinc is used under the form of powder. Advantageously, zinc is activated zinc. According to one embodiment, T1CI4 is used at a proportion ranging from 2 to 15 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 4 to 10 equivalents, more preferably at a proportion of about 6.2 equivalents.
According to one embodiment, zinc is used at a proportion ranging from 2 to 30 equivalents compared to 4,4'-dihydroxybenzophenone, preferably at a proportion of 6 to 20 equivalents, more preferably at a proportion of about 14 equivalents.
According to one embodiment, the solvent used in step a) is selected from tetrahydrofuran ( 1 1 II · ). 1,2-dimethoxyethane (DME), dioxane and tetrahydropyran; preferably the solvent is THF, more preferably dry 1 1 11 · . According to one embodiment, the McMurry coupling of step a) is performed at a temperature ranging from 20 to 100°C, preferably at reflux of the solvent.
According to one embodiment, the McMurry coupling of step a) is performed for a duration ranging from 0.5 to 24 hours, preferably from 1 to 12 hours, more preferably for 2 hours. Intermediate (A) obtained in step a) may be purified by flash chromatography or by precipitation. According to one embodiment, intermediate (A) is purified by flash chromatography on silica gel, using preferably a mixture cyclohexane/ethyl acetate as solvent. According to another embodiment, (A) is purified by precipitation and the solvent of precipitation is selected from dichloromethane, cyclohexane and a mixture ethyl acetate/cyclohexane.
Step b)
According to one embodiment, caging halide (B) is used at a proportion ranging from 0.5 to 2 equivalents compared to intermediate (A), preferably at a proportion of 0.8 to 1 . 1 equivalents, more preferably at a proportion of about 0.95 equivalents. According to one embodiment, the caging halide (B) is a bromide, i.e. X1 represents Br. According to another embodiment, caging halide (B) is a chloride, i.e. X 1 represents CI. According to one embodiment, step b) is performed in presence of a base, preferably a base selected from K2CO3 and CS2CO3, more preferably the base is K2CO3.
According to one embodiment, K2CO3 is used at a proportion ranging from 0.5 to 2 equivalents compared to intermediate (A), preferably at a proportion of 0.8 to 1.1 equivalents, more preferably at a proportion of about 0.95 equivalents.
According to one embodiment, Nal or Kl may be added to perform step b) i the conversion rate is too low.
According to one embodiment, the solvent used in step b) is a solvent in which starting products are soluble and resulting compound is not soluble. In other words, the solvent used in step b) is a solvent in which intermediate (A) and caging halide (B) are soluble and in which intermediate (C) is not soluble. Such solvent enables to easily recover intermediate (C) as a precipitate by filtration. According to a preferred embodiment, the solvent used in step b) is a mixture of water and of a miscibie organic solvent. Examples of organic solvents miscibie in water are acetonitrile, acetone, dioxane, I I 11 % alcohol (such as for example methanol, ethanol or butanol). According to one embodiment, the solvent used in step b) is selected from water, acetonitrile, acetone, dioxane, I I II ·. alcohol (such as for example methanol, ethanol or butanol) or a mixture thereof; preferably the solvent is a mixture of acetonitrile and water, more preferably acetonitriie/water 2/1 (v/v).
According to one embodiment, step b) is performed at a temperature ranging from 0 to 100°C, preferably at room temperature.
According to one embodiment, step b) is performed for a duration ranging from 1 to 72 hours, preferably from 4 to 48 hours, more preferably for 16 to 24 hours.
Step b) is performed in the dark in order to avoid degradation of resulting intermediate (C) which is photosensitive. Intermediate (C) obtained in step b) forms a precipitate in the conditions of the reaction. Advantageously, intermediate (C) does not need to be purified to be used in step c). Step c)
According to one embodiment, derivative (D) is used at a proportion ranging from I to 10 equivalents compared to intermediate (C), preferably at a proportion of 1 to
3 equivalents, more preferably at a proportion of about 2 equivalents.
According to one embodiment, step c) is performed in presence of a base, preferably a base selected from CS2CO3 and 2C03, more preferably the base is CS2CO3.
According to one embodiment, CS2CO3 is used at a proportion ranging from 2 to I 1 equivalents compared to intermediate (C), preferably at a proportion of 2 to
4 equivalents, more preferably at a proportion of about 3 equivalents.
According to one embodiment, the solvent used in step c) is selected from acetone, acetonitrile and di methyl ormamide; preferably the solvent is acetone.
According to one embodiment, step c) is performed at a temperature ranging from 20 to 155°C, preferably at reflux of the solvent.
According to one embodiment, step c) is performed for a duration ranging from 1 to 48 hours, preferably from 6 to 24 hours, more preferably for 18 hours.
Step c) is performed in the dark in order to avoid degradation of final compound of formula (I) which is photosensitive.
Final compound (I) obtained in step c) is purified by flash chromatography. According to one embodiment, compound (I) is purified by flash chromatography on silica gel, using preferably a mixture dichloromethane and methanol as solvent.
The process of the invention presents the advantage to have an overall yield of more than 30%, preferably of more than 40%, even more preferably of more than 45%. This overall yield is twofold compared to process disclosed in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a scheme representing the strategy to photo-activate properly engineered proteins: a protein (Prot) fused to the ERT2 receptor (ERT2) is inactivated by the assembly formed with a chaperone complex (cc). Upon photoactivation of a caged precursor (clnd), a non-endogenous inducer (Ind) is released, which binds to the LR 1 2 receptor and sets the protein fusion free from its assembly with the chaperone complex. The inducer (Ind) may be 4-hydroxy-cycIofen (also called cyclofen-OH).
EXAMPLES The present invention is further illustrated by the following examples. Abbreviations
ACN: acetonitrile;
DMF: dimethylformamide;
DMSO: dimethylsulfoxide;
EtOAc: ethyl acetate;
HPLC: high performance liquid chromatography;
HRMS: high resolution mass spectroscopy;
MS: mass spectroscopy;
NMR: nuclear magnetic resonance;
'i l l 1: tetrahydrofurane.
Material
Commercially available reagents were used as obtained. Microanalyses were performed by the Service de Microanalyses de Gif sur Yvette. The ' I I and 13C NMR spectra were recorded at room temperature on Bruker AM 250, 300 or 400 spectrometers; chemical shifts are reported in ppm with protonated solvent as internal reference ( ' I I, Ci K b in CDCb 7.26 ppm, CHD2SOCD3 in CD3SOCD3 2.49 ppm, CHD2COCD3 in CD3COCD3 2.05 ppm; 13C, 13CDCb in CDCb 77.0, 13CD3SOCD3 in CD3SOCD3 39.7 ppm, 1 ΤΊ )ι( '(Χ Ί )ι in CD3COCD3 29.9 ppm); coupling constants (J) are given in Hz. Mass spectra (chemical ionization and high resolution with NH3 or C 'i I i ) were performed by the Service de Spectrometrie de Masse de Chimie ParisTech or by the Service de Spectrometrie de Masse de 1'ICOA (Orleans). Column chromatography was performed on silica gel 60 (0.040-0.063 nm; Merck). Analytical thin-layer chromatography (TLC) was conducted on Merck silica gel 60 F254 precoated plates.
Example 1 : Synthesis of intermediate (A)
Step a): synthesis oi' 4-(CyclohexyIidene(4-hydroxyphenyl)methyl )phenol (A )
Figure imgf000019_0001
(A) Titanium chloride (6.2 mL, 56 mmol) was added dropwise under argon to a stirred suspension of activated zinc powder (8.20 g, 126 mmol) in dry tetrahydrofuran (80 mL) at -10°C. When the addition was complete, the mixture was warmed to room temperature and then re fluxed for 2 h. A solution of 4,4'-dihydroxybenzophenone (2.0 g, 9 mmol ) and cyclohexanone (4 mL, 38 mmol) in dry tetrahydrofuran (120 ml . ) was added to the cooled suspension of the titanium reagent at 0-5 C and the mixture was re flu ed for 2 h. After being cooled to room temperature, the reaction mixture was quenched with 10% (w/v) aqueous potassium carbonate (30 mL), filtered and extracted with ethyl acetate. The organic layer was washed with brine, dried over MgSC¼, and concentrated in vacuo. Flash column chromatography (cyclohexane/ethyl acetate, 3: 1 . v/v) or precipitation in dichloromethane afforded (A) as a white powder respectively with (2.15 g, 85%) and (2.1 g, 81%); precipitation can also be carried out in cyclohexane or in a EtOAc/cyclohexane mixture. Ή NMR (250 MHz, DMSO-de, δ): 9.28 (s, 2H), 6.82 id, 4 I I. J=8 Hz), 6.65 (d, 41 1. .1=8 Hz), 2. 14 (m, 4 I I ). 1 .52 (m, 61 1 ): 13C NMR (100 MHz, DMSO-de, δ): 155.5 (2C), 136.2, 1 33.8, 133.6 (2C), 130.3 (4C), 1 14.6 (4C), 31.9 (2C), 28.1(2C), 26.3. Example 2: Synthesis of compound (1-1)
Step b): synthesis of intermediate 4-((4-(4,5-diinethoxy-2-nitrobenzyloxy)phenyl) (cyclohexylideiie)methyDphenol (C- 1)
Figure imgf000020_0001
To a mixture of (A) (1 g, 3.5 mmol) and K2CO3 (470 mg, 3.4 mmol, 0.95 eq) in acetonitriie (A('N)/Ih() (60 rriL, 2/1, v/v) was added portionwise 4,5-dimethoxy-2- nitrobenzyl bromide (B-1 ) (940 mg, 3.4 mmol, 0.95 eq) over a period of 1 h. The resulting mixture was stirred in the dark for 16 h. The precipitate was filtered, washed with ACN/H2O and then dried in vacuo over P2O5. A white powder was obtained (1.4 g, (A)/(C-1 Vdisubstituted by-product: ε/96/4) and used without purification in the next step. 'II NMR (ppm, 300 Mil/, CDCI3, δ): 7.77 (s, III), 7.3 (s, III), 7.04 (ΑΑ'ΧΧ', 211, .!= 9 II/), 6.97 (ΛΛ'ΧΧ', 211, J= 9 II/), 6.91 (ΛΛ'ΧΧ', 211..1= 9 II/), 6.73 (ΛΛ'ΧΧ', 211, .1= 9 II/).5.48 (s, 211), 4.68 (s, III).3.97 (s, 311), 3.95 (s, 311), 2.23 (m, 411). 1.57 (m, 611).13C NMR (75 Mil/, DMSO-de, δ): 156.4.155.9.153.3.148.0, 139.9.137.1, 136.2, 133.7, 133.5, 130.7 (2C), 130.5 (2C), 122.5, I 14.9 (2C), 114.5 (2C), I I 1.5.108.4, 66.7. 56.3.56.2.32.1 (2C), 28.3 (2C), 26.4; MS (CI, NH3): m/z 493.30 (calculated average mass for [CisHigNOe + NH4]+: 493.23; HRMS (TOF MS ES+): m/z 476.2077 (calculated mass for [CisHigNOe + II|+: 476.2073. Step c): synthesis of 2-(4-((4-(4,5-Diniethoxy-2-iiitrobenzyloxy)phenyl) cycIohexylidene)inethyDphenoxy)- ,N-diinethylethaiianiine (I- 1)
Figure imgf000021_0001
A solution of (C-1) (800 mg, 1.68 mmol) in acetone (50 mL) was treated with Cs2C03 (1.64 g, 5 mmol, 3 eq.). 2-(Dimethylamino)ethylchlorid hydrochloride (D-l ) (484 mg, 3.3 mmol, 2 eq.) was added portionwise over a period of 30 min and the resulting suspension was stirred at reflux temperature in the dark for 18 h. After being cooled to room temperature, the reaction mixture was filtered. The precipitate was washed with dichloromethane and the solvent was evaporated. Flash chromatography (CHiCh/MeOH: 9/1) afforded (1-1) (640 mg, 70% yield). Ή NMR (400 MHz, CDCb, δ): 7.76 (s, i l l ). 7.35 (s, 1 I I ), 7.04 (ΑΑ'ΧΧ', 21 1, J=9 Hz), 7.00 (ΑΑ'ΧΧ' , 21 1. .1=9 I I/ ), 6.90 (ΑΑ'ΧΧ' , 21 1, .1=9 I I/ ). 6.82 (ΑΑ'ΧΧ', 21 1. .1=9 I I/ ). 5.47 (s, 2H), 4.03 (t, 21 1. .1= 6 I I/ ). 3.96 (s, 3 H), 3.94 (s, 3 H), 2.70 (t, 21 1, .1= 6 Hz), 2.32 (s, 6 H), 2.23 (m, 41 1 ). 1.58 (m, 6H); 13C NMR (100 MHz, CDCb, δ): 157.0. 156.2. 153.8, 147.7, 138.9. 138.5. 136.8, 137.7. 133.2. 131.0 (2C), 130.7 (2C), 129.6, 114.3 (2C), 113.8 (2C), 109.4, 107.9, 67.0, 65.8, 58.3, 56.3. 56.3. 45.8 (2C), 32.4 (2C), 28.6 (2C), 26.8.
The overall yield for the synthesis of compound (1-1) from 4,4'-dihydroxybenzophenone is of 47%. Example 3: Synthesis of compound (1-2)
Step b): synthesis of intermediate 4-((4-(6,7-dimethoxycoumarin)phenyl) (cyclohexylidene)inethyDphenol (C-2)
Figure imgf000022_0001
To a mixture of (A) (100 mg, 0.35 mmol) and K2CO3 (47 mg, 0.34 mmol, 0.95 eq) in ACN/H2O (7 ml., 2/1 , v/v) was added 4-bromomethyl-6,7-dimethoxycoumarin (B-2) (97 mg, 0.34 mmol, 0.95 eq). The resulting mixture was stirred in the dark for 24 h. The precipitate was filtered, washed with ACN/H2O and then dried in vacuo over P2O5. A white powder was obtained (120 mg, (A)/(C-2)/disubstituted by-product: ε/90/ΙΟ) and used without purification in the next step. Ί NMR (ppm, 300 MHz, acetone-d6, δ): 7.30 (s, 111), 7.08 (m, 411), 6.99 (s, 111).6.93 (ΑΛ'ΧΧ', 211, .1= 9 Hz), 6.73 (ΑΑ'ΧΧ', 211..1= 9 Hz), 6.4 (s, 1H), 5.41 (s, 211), 3.97 (s, 311), 3.987 (s, 311), 2.24 (m, 411), 1.59 (m, 611).13C NMR (75 MHz, CDC13, δ): 160.3, 156.1.155.9.152.7, 151.6, 149.0, 145.8, 136.9.136.4.133.5, 132.9, 130.5 (2C), 130.3 (2C), 114.8 (2C), I 14.5 (2C), 109.1, 108.7, 105.7, 100.3, 65.4. 56.1. 3 ! .9 (2C), 28.1 (2C), 26.2; MS (ESI): ml/, 499.5 (calculated mass for [C31H30O6 + 11|+: 499.2, m/z 521.5 (calculated mass for [C31H30O6 + Naf: 521.1 ). Step c): synthesis of 2-(4-((4-(4,5-Diniethoxy-2-iiitrobenzyloxy)phenyl) cycIohexylidene)inethyDphenoxy)- ,N-diniethylethaiianiine (1-2)
Figure imgf000023_0001
To a mixture of (C-2) (80 mg, 0.16 mmol) and CS2CO3 (157 mg, 0.48 mmol, 3 eq.) in acetone (5 ml.) was added 2-(dimethylamino)ethyichlorid hydrochloride (D-l ) (46 mg, 0.32 mmol.2 eq.). The resulting suspension was stirred at reflux temperature in the dark for 18 h. After being cooled to room temperature, the reaction mixture was filtered. The precipitate was washed with dichloromethane and the solvent was evaporated. Flash chromatography (CHiCh/MeOH: 9/1 v/v) afforded (1-2) (60 mg, 66% yield). Ί NMR (300 MHz, CDCb, δ): 7.26 (s, 1H), 7.07 (m, 4 H), 7.01 (ΑΑ'ΧΧ', 2FI, .1=9 Hz), 6.95 (s, 1H), 6.85 (ΑΑ'ΧΧ', 211, .1=9 Hz), 6.44 (s, III).5.38 (s, 2H), 4.09 (t, 211, .1=6 Hz), 3.93 (s, 3 II), 3.86 (s, 3 II).2.73 (t, 2 H, .!= 6 Hz), 2.30 (s, 6 II).2.23 (m, 411). 1.59 (m, 611); 13C NMR (75 MHz, CDCb, δ): 161.1, 158.3, 157.5.154.3.152.0.150.7. 147.4, 138.9. 137.7.136.6, 134.7.131.7 (2C), 131.6 (2C), I 15.4 (2C), I 14.8 (2C), 110.4, 110.4, 106.5. 101.1, 66.9.66.7.58.9, 56.8, 56.6.46.1 (2C), 33.1 (2C), 29.4 (2C), 27.6. MS (ESI): ml/, 570.5 (calculated mass for [C35H39O6 + H]+: 570.2.
The overall yield for the synthesis of compound (1-1) from 4,4'-dihydroxybenzophenone is of 42%.

Claims

1. A process of manufacturing of compounds of formula (I)
Figure imgf000024_0001
salts thereof, wherein
R1 represents Ar1, -CO-Ph, -CH2-(o-COR)Ph, -CH2-(o-N02)Ph or -CO-CO-NR2, wherein
Ar1 represents phenyl or coumarin groups, optionally substituted by a group selected from nitro, alkoxy, halo, hydroxyl, amino, alkylamino, dialkylamino, alkyloxycarbonylaikyloxy, alkyioxyaryl, aryi, arylvinylyl, arylethynyl, cyano;
Ph represents a phenyl group, optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haioalkyl, haloaikoxy, amino, alkylamino, dialkylamino, alkyloxycarbonylaikyloxy, alkyioxyaryl, aryl, arylvinylyl, arylethynyl and cyano; and
R represents aryl, alkyl or vinylcarboxy; wherein the aryl group is optionally substituted by one or more group selected from alkoxy, halo, hydroxyl, alkyl, haioalkyl, haloaikoxy, amino, alkylamino, dialkylamino, alkyloxycarbonylaikyloxy, alkyioxyaryl, aryl, arylvinylyl, arylethynyl and cyano;
R1' represents 11, alkyl, haioalkyl, cyano, aryl;
R2 and R3 represent each independently hydrogen, alkyl, carboimmidoyl; or R2 and R3 form together with the nitrogen atom to which they are attached an heterocyclic ring preferably selected from pyrolidinyl, piperidinyl, N-methyl- piperazinyl, morpholinyl, pyrrolyl; n is an integer ranging from 1 to 6, preferably n is 1, 2, 3 or 4, more preferably n is 1 or 2; comprising the following steps: a) performing a McMurry coupling between 4,4'-dihydroxybenzophenone and cyclohexanone to form intermediate (A):
Figure imgf000025_0001
b) performing a phenol monosubstitution on intermediate (A) with caging halide (B):
Figure imgf000025_0002
wherein K 1 and R1 ' are as defined in formula (I) and X1 represents halo, preferably X1 represents Br or CI, more preferably X1 represents Br, to form intermediate (C)
Figure imgf000025_0003
wherein R1 and R 1 are as defined in formula (I), wherein step b) is performed in the dark, in a solvent in which intermediate (A) and caging halide (B) are soluble and in which intermediate (C) is not soluble; and c) alkylating, in the dark the remaining phenol of intermediate (C) with derivative (D): f
(D) wherein n, R2 and R3 are as defined in formula (I) and X2 represents halo or sulfonate, preferably X2 represents CI, Br, I, mesylate, in late or tosylate, more preferably X2 represents CI, to afford compound of formula (I).
2. The process according to claim 1, wherein step a) is performed in presence of TiCU and zinc.
3. The process according to claim 1 or claim 2, wherein cyclohexanone is used at a proportion ranging from 1 to 10 equivalents compared to 4,4'- dihydroxybenzophenone, preferably at a proportion of 3 to 6 equivalents, more preferably at a proportion of about 4.2 equivalents.
4. The process according to anyone of claims 1 to 3, wherein the solvent used in step a) is selected from tetrahydrofuran, 1,2-dimethoxyethane, dioxane and tetrahydropyran; preferably the solvent is tetrahydrofuran, more preferably dry tetrahydrofuran .
5. The process according to anyone of claims 1 to 4, wherein intermediate (A) is purified by precipitation and the solvent of precipitation is selected from dichloromethane, cyclohexane and a mixture ethyl acetate/cyclohexane.
6. The process according to anyone of claims 1 to 5, wherein step b) is performed in presence of a base, preferably a base selected from K2CO3 and CS2CO3, more preferably the base is 2CO3.
7. The process according to anyone of clai ms 1 to 6, wherein caging halide (B) is used at a proportion ranging from 0.5 to 2 equivalents compared to intermediate (A), preferably at a proportion of 0.8 to 1.1 equivalents, more preferably at a proportion of about 0.95 equivalents. The process according to anyone of claims 1 to 7, wherein the solvent used in step b) is selected from acetonitrile, water, acetone, dioxane, tetrahydrofuran, alcohol or a mixture thereof; preferably the solvent is a mixture of acetonitrile and water, more preferably acetonitrile/water 2/1 (v/v).
The process according to anyone of claims 1 to 8, wherein step c) is performed in presence of a base, preferably a base selected from Cs _( '()¾ and K2CO3, more preferably the base is CS2CO3.
The process according to anyone of claims 1 to 9, wherein derivative (D) is used at a proportion ranging from 1 to 10 equivalents compared to intermediate (C), preferably at a proportion of 1 to 3 equivalents, more preferably at a proportion of about 2 equivalents.
The process according to anyone of claims 1 to 10, wherein the solvent used in step c) is selected from acetone, acetonitrile and di methyl ormamide; preferably the solvent is acetone. 12. The process according to anyone of claims 1 to 11, wherein compound (I) is purified by flash chromatography.
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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030771A1 (en) * 2006-09-08 2008-03-13 Smithkline Beecham Corporation Cyclic alkylidene compounds as selective estrogen receptor modulators

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008030771A1 (en) * 2006-09-08 2008-03-13 Smithkline Beecham Corporation Cyclic alkylidene compounds as selective estrogen receptor modulators

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEEPAK KUMAR SINHA ET AL: "Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One- and Two-Photon Illumination", CHEMBIOCHEM - A EUROPEAN JOURNAL OF CHEMICAL BIOLOGY., vol. 11, no. 5, 24 February 2010 (2010-02-24), DE, pages 653 - 663, XP055313167, ISSN: 1439-4227, DOI: 10.1002/cbic.201000008 *
LUDOVIC FOURNIER ET AL: "A Blue-Absorbing Photolabile Protecting Group for in Vivo Chromatically Orthogonal Photoactivation", ACS CHEMICAL BIOLOGY, vol. 8, no. 7, 19 July 2013 (2013-07-19), pages 1528 - 1536, XP055212890, ISSN: 1554-8929, DOI: 10.1021/cb400178m *

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