DE102019129221B4 - Process for the synthesis of DOPA with a photolabile protecting group and intermediates for its production - Google Patents

Abstract

Process for the enantioselective synthesis of DOPA with a photolabile protecting group comprising the following steps- etherifying dihydroxybenzaldehyde (A) with a photolabile protecting group R according to the following first process stepwherein R is selected from the group consisting of:2-nitrobenzyl, 1-(2-nitrophenyl )ethyl, 7-nitroindolin-1-yl, (7-methoxycoumarin-4-yl)methyl, {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl, (6-bromo-7-hydroxycoumarin- 4-yl)methyl, deoxybenzoinyl, p-hydroxyphenacyl, (8-bromo-7-hydroxyquinolin-2-yl)methyl, [7-(diethylamino)coumarin-4-yl]methyl to an ether (B), as the first intermediate ,- reacting the ether (B) with N-acetylglycine (C) to form an azlactone (D) as a second intermediate according to the following, second process step- opening the azlactone (D) according to the following, third process step by reacting the azlactone (D) in alcoholic solution to an opened azlactone (E) as a third intermediate,- asymmetric hydrogenation of the opened azla ctons (E) according to the following, fourth process step by reacting the opened azlactone (E), which was obtained as an intermediate in the previous step, with S,S Et-Duphos (2S,5S)-1-[2-[(2S,5S )-2,5-diethylphospholan-1-yl]phenyl]-2,5-diethylphospholane or "DUPHOS") and bisoctadienyl rhodium tetrafluoroborate to a fourth intermediate (F)- deprotection of intermediate (F) resulting from opening of the azlactones (E) formed in the third process step, to give a protecting group R-substituted-DOPA (G) according to the following reaction scheme by reacting the fourth intermediate (F) with acid.

Description

The invention relates to methods for the synthesis of DOPA with a photolabile protecting group and intermediates for its production.

Various adhesives exist in human and veterinary medicine for wound treatment or hemostasis, while there are no adhesives available for the treatment of fractures, for example. However, the products available on the market have serious disadvantages. These include, in particular, the toxicity potential of the adhesives (e.g. cyanoacrylate), the sometimes insufficient control of the adhesive effect with the result of unwanted adhesion of tissue and instruments, high treatment costs and the risk of transmitting infectious agents from substances of human and animal origin (e.g. fibrin ). Mussels produce a biological peptide-based glue that allows them to stick to various surfaces in water - an environment in which most glues do not work - while resisting marine tidal forces. The high proportion of the non-canonical amino acid DOPA (L-3,4-dihydroxyphenylalanine) is decisive for the adhesive effect of the proteins. If DOPA is used with a photolabile ONB protecting group, as has already been used in numerous studies, it enables the production of photoactivatable proteins or, more precisely, photoactivatable protein glues, which are cleaved off by irradiation with light of wavelength 365 nm. This strategy has the following advantages: 1) The undesired oxidation of DOPA is prevented. 2) The adhesive effect can be activated by light irradiation, if required, by cleaving the photolabile ONB protecting group.

A synthesis for this component is described in the journal Eur. J. Org. Chem, 2018, 2053-2063, pp. 2053-2063. It includes 8 stages with an overall yield of 4%. The last step involves enzymatic resolution, which could pose a problem from a pharmacological point of view.

In the published US application US 2014/0023603 A1 new active ingredients with skin-lightening and antioxidant effects and an activity in relation to the peroxisome proliferator-activated receptor are described. On page 22 in Table 8 with reference numerals 86 and 87 (Z)-2-methoxy-4-((2-methyl-5-oxooxazol-4(5H)-ylidene)methyl)phenyl acetate and (Z)-2-ethoxy -4-((2-methyl-5-oxooxazol-4(5H)-ylidene)methyl)phenyl acetate. These substances are similar to intermediates that are formed in the process presented here, with the intermediates presented here carrying photolabile substituents that are compatible with the US application US 2014/0023603 A1 manufacturing processes presented are not compatible.

The technical article Biotechnology and Polymers, Plenum Press, New York, 1991, pp. 229-243 reports on L-3,4-dihydroxyphenylalanine (DOPA) and rearrangements to N-acetyl-α,β-dehydro-DOPA. A characterization of unsubstituted N-acetyl-α,β-dehydro-DOPAethyl ester begins on page 234 of this technical article.

In the international PCT application WO02/00245 A1 discloses neurotoxic oligomers for use in the treatment of Alzheimer's disease. One of these oligomers, presented there in 7B , is called IsoDiatee and occurs in diastereomeric form, with the configuration diversity doubling with each additional monomer.. These substances are similar to intermediates formed in the process presented here, but the intermediates presented here carry different and photolabile substituents.

The object of the invention is to provide a process for the enantioselective production of DOPA with a photolabile protective group.

The object of the invention is achieved by a method according to claim 1.

The method according to claim 1 is presented below in its best possible form, namely a fully synthetic, enantioselective synthesis which comprises 5 stages and produces the product in an overall yield of 5-7%. The key step is an asymmetric hydrogenation with a chiral rhodium species.

wobei R ausgesucht ist aus der Gruppe bestehend aus:

wobei die Cumarin-Abkömmlinge für R den besonderen Vorteil haben, dass diese beim photoinduzierten Austritt beim Einsatz am Patienten keine toxischen Nebenprodukte bilden.General synthetic route:

where R is selected from the group consisting of:

where the coumarin derivatives for R have the particular advantage that they do not form any toxic by-products in the event of photo-induced escape when used on patients.

Preferred synthesis route:

Description of the individual steps

1. Introduction of the photosensitive leaving group R

Preferred introduction of the nitrobenzyl group as a light-sensitive leaving group

30.9 g of sodium hydride (774 mmol; 2.03 eq. 60% in mineral oil) are washed three times with 100 ml of hexane each time and dried under fine vacuum. It is suspended in 500 ml DMF (abs.) and cooled in an ice bath. 52.6 g of dihydroxybenzaldehyde (381 mmol) are added in portions over the course of 15 minutes and the mixture is stirred in an ice bath for 30 minutes. 82.3 g o-nitrobenzyl bromide (381 mmol; 1 eq.) are dissolved in 160 ml DMF (abs.) and added dropwise within 25 minutes.

After 20 minutes, a mixture of 1.95 ml of water and 250 ml of 2M hydrochloric acid is poured in, and 700 ml of ethyl acetate are added. The mixture is subsequently stirred for a few minutes and then filtered through a G3 frit. The phases are separated and the aqueous phase is extracted three more times with 700 ml of ethyl acetate each time. The combined organic phases are washed three times with 700 ml of water each time and dried over sodium sulfate.

After filtration and concentration, 99.95 g of yellow-orange oil are obtained.

This oil is dissolved in 300 mL of warm methanol. 500 ml of water are added and the mixture is heated to 80-85° C. for 15 minutes. The resulting chewing-gum-like mass is separated off by filtration through a G3 frit, dried at 40° C. on a rotary evaporator and smoked off with methanol. 79.83 g of a resinous mass are isolated.

150 ml of dichloromethane are added and the mixture is heated to boiling. It is allowed to crystallize at 4°C. It is filtered through a G3 frit and washed five times with 20 ml of cold dichloromethane each time.

23.26 g of product (85 mmol, 22%, 09-19-11-08 P8571) are obtained as a white solid.
¹ H NMR data for R equals nitrobenzyl
1H ⁽ DMSO): s 10.38 (1H, OH); s 9.76 (1H, CHO); dd 8.15(8.2/1.1Hz, 1H, CH _ar NO ₂ benzyl); d 7.92 (7.4 Hz, 1H, CH _ar NO ₂ benzyl), dt 7.82 (7.6-7.7/1-1.2 Hz, 1H, CH _ar NO 2 benzyl), m 7, 61-7.66 (1H, CH _ar NO ₂ benzyl); m 7.45-7.49, 2H, 2xCH _ar ); m 7.01-7.05 (1H, CH _ar ); s 5.53 (2H, _CH2 benzyl)

2. Formation of the azlactone

Preferred formation of the azlactone with R equal to nitrobenzyl

23.26 g product of the precursor (85 mmol), 9.08 g sodium acetate (111 mmol, 1.3 eq.) and 11.98 g N-acetylglycine (102 mmol, 1.2 eq.) are premixed dry and in 40 ml acetic anhydride (426 mmol, 5 eq.) suspended. The mixture is heated at 120° C. for 4 hours. An easily stirrable mixture forms first, then a yellow suspension. After cooling, 70 ml of water are added and crushed mechanically to achieve stirrability. It is filtered through a G3 frit and washed six times with 23 ml each time of a 1:1 mixture of water and ethanol and dried at 40.degree.
31.4 g of a yellow solid (79 mmol, 93%, 09-19-II-15 P8579) are isolated as the product.
¹ H NMR data for R equals nitrobenzyl
¹ H (DMSO): dd 8.18 (0.7/8.2 Hz, 1H, CH _ar NO ₂ benzyl); d 8.03 (1.7Hz, 1H, CH _ar ); dd 7.83 (1.7/8.3 Hz, 1H, CH _ar ); dt 7.80 (1/7.5-7.7 Hz, 1H, CH _ar NO ₂ benzyl); d 7.73 (6.9Hz, 1H, CH _ar NO ₂ benzyl); m 7.6-7.66 (presumably dt, 1H, CH _ar NO ₂ benzyl); d 7.28 (8.3Hz, 1H, CH _ar ); s 7.18 (1H; _{C H olef} ); s 5.57 (2H, _CH2 benzyl); s 2.37 (3H; _CH3 ); s 2.31 (3H; CH ₃ COO-)

3. Opening of the azlactone

Preferred opening of the azlactone with R equal to nitrobenzyl

31.4 g product of the precursor are suspended in 184 ml ethanol and treated with 56 ml triethylamine and heated under reflux for 28 h. Reaction control is possible after dilution with DCM with the mixture EE/PE 2:1. It is taken up in 1.6 l of ethyl acetate and 1 l of water and 1 l of sodium chloride solution are added. The pH is brought to 1 with 56 ml of hydrochloric acid (25%) and the phases are separated. The aqueous phase is extracted four more times with 800 ml of ethyl acetate each time. The combined organic phases are washed with 600 ml of saline solution and dried over sodium sulfate. After filtration and concentration, 32.15 g of crude product are isolated as a red-yellow solid. It is dissolved in 200 ml of ethanol while it is hot, allowed to cool briefly and 3.57 g of activated charcoal are added. It is boiled and filtered warm through Celite. Rinse with 250 ml of warm ethanol. The ethanol is distilled off until a thick suspension has formed (approx. 60 ml) and is allowed to crystallize at 8.degree. The precipitated solid is filtered off, washed with a total of 70 ml of cold ethanol and dried at 40.degree. 18.08 g of product (58%) are obtained as a pale yellow solid (09-19-11-19 P8598).

4.26 g of product (09-19-I-47 P8459, 95%) are isolated as a red-brown solid.
¹ H NMR data for R equals nitrobenzyl
1H (DMSO): s 9.73 (1H; NH ¹ ); s 9.41 (1H, OH2); dd 8.17 (1-2/8.2 Hz, 1H, CH _ar NO ₂ benzyl 3); d 7.91 (7.4Hz, 1H, CH _ar NO2 benzyl ₄ ); dt 7.82 (1.2-1.4/7.4-7.6 Hz, CH _ar NO ₂ benzyl 5); m 7.60-7.66 (presumably dt, 1H, CH _ar NO ₂ benzyl 6); d 7.37 (1.9Hz, 1H, CH _ar 7); dd 7.14 (1.8-1.9/8.3Hz, 1H, CH _ar 8); s 7.10 (1H, _{C H olef} 9); d 6.89 (8.3Hz, 1H, CH _ar 10); s 5.50 (2H, CH ₂ benzyl 11); q 4.14 (7.1Hz, CH 2-O 12); s 1.93 (3H, CH3 _COO13 ); t 1.22 (7.11Hz, 3H, CH3 _{-CH2 O14} )

4. Asymmetric Hydrogenation

Preferred asymmetric hydrogenation with R equal to nitrobenzyl

220 ml of methanol are degassed by bubbling nitrogen through them. 23 mg S,S Et-Duphos (0.15 mol%) are degassed twice in a heated flask and dissolved in 14 ml degassed methanol, and degassed twice again. 23.1 mg of bisoctadienyl rhodium tetrafluoroborate (0.55 mol %) are added and the mixture is degassed twice. The solution is stirred at room temperature for one hour. 4.53 g of the product from the precursor are suspended in 205 ml of degassed methanol, the catalyst solution is added and the suspension is sealed in the autoclave. It is flushed three times with nitrogen and three times with hydrogen and finally stirred under hydrogen at 5 bar at room temperature. After 17 hours the autoclave is opened.

Reaction control is possible with the mixture EE/PE 10:1. It is filtered through about 32 g of silica gel and the silica gel is rinsed with methanol. Fractions containing product are combined and concentrated. 4.68 g of crude product are obtained.

A total of 18.36 g of product is isolated in the 4 runs.

These are combined, treated with 3.61 g of activated charcoal and briefly heated to boiling. It is filtered while hot through Cellite and rinsed with 150 to 200 warm methanol. The solution is concentrated at 40°C. 17.79 g of light-colored solid are isolated (09-19-II-38 P8623, 98%)
¹ H NMR data for R equals nitrobenzyl
1H (DMSO): s 8.17 (1H, NH ¹ ); dd 8.15 (1.2/8.2Hz, 1H, CH _ar NO ₂ benzyl 2); dd 7.93 (0.7-0.8/7.7Hz, 1H, CH _ar NO ₂ benzyl 3); dt 7.80 (1.2/7.5-7.7 Hz, 1H, CH _ar NO ₂ benzyl 4); m 7.59-7.66 (1H, probably actually dt, CH _ar NO ₂ benzyl 5); d 6.85 (1.9Hz; 1H, CH _ar 6); d 6.75 (8Hz, 1H, CH _ar 7); dd 6.65 (1.9/8.1 Hz, 1H, CH _ar 8); s 5.43 (2H, _CH2 benzyl 9); m 4.30-4.37 (1H; CH-N 10); q 4.02 (7.1Hz, 2H, CH2 _-O11 ); dd 2.85 (5.8/13.8Hz, 1H, CH2 _-Ar12 ); dd 2.74 (8.8/13.8Hz, 1H, CH2 _-Ar13 ); s 1.76 (3H; CH3 _CO14 ); t 1.12 ( _7.1Hz , 3H, CH3 _CH2 15)

5. Deprotection

Preference is given to splitting off the protective groups where R is nitrobenzyl

17.79 g of precursor product (44.2 mmol) are suspended in 95 ml of dioxane and heated to obtain a solution. 95 ml of 32% hydrochloric acid are added and the mixture is heated under reflux for 5 h. It is concentrated at 60° C. and dissolved in 140 ml of 1M hydrochloric acid. It is extracted with 270 ml of ethyl acetate and twice with 230 ml of ethyl acetate. 19.81 g sodium bicarbonate are slowly added until pH 7 is reached. A greenish suspension forms which is cooled to 4°C. After 2 hours it is filtered, washed four times with 60 ml of cold water and dried at 50.degree. 9.33 g of yellow solid are isolated (28 mmol, 64%, 09-19-11-42 P8625) slightly impure.

8.10 g are suspended in 320 ml of methanol and stirred at room temperature for 1 hour 40 minutes. It is filtered through a G4 frit and washed twice with 80 ml of methanol each time and dried at 40.degree. 5.56 g of product (09-19-II-58 P8656, 69%) are isolated. The enantiomeric excess (ee) is 98%.
¹ H NMR data for R equals nitrobenzyl
1H (DMSO): s 8.17 (1H, NH ¹ ); dd 8.15 (1.2/8.2Hz, 1H, CH _ar NO ₂ benzyl 2); dd 7.93 (0.7-0.8/7.7Hz, 1H, CH _ar NO ₂ benzyl 3); dt 7.80 (1.2/7.5-7.7 Hz, 1H, CH _ar NO ₂ benzyl 4); m 7.59-7.66 (1H, probably actually dt, CH _ar NO ₂ benzyl 5); d 6.85 (1.9Hz; 1H, CH _ar 6); d 6.75 (8Hz, 1H, CH _ar 7); dd 6.65 (1.9/8.1 Hz, 1H, CH _ar 8); s 5.43 (2H, _CH2 benzyl 9); m 4.30-4.37 (1H; CH-N 10); q 4.02 (7.1Hz, 2H, CH2 _-O11 ); dd 2.85 (5.8/13.8Hz, 1H, CH2 _-Ar12 ); dd 2.74 (8.8/13.8Hz, 1H, CH2 _-Ar13 ); s 1.76 (3H; CH3 _CO14 ); t 1.12 ( _7.1Hz , 3H, CH3 _CH2 15)

The compounds D, E and F mentioned above have not yet been described in the literature.

Reference List

1

Introduction of the nitrobenzyl group

2

formation of the azlactone

3

Opening of the azlactone

4

asymmetric hydrogenation

5

cleavage of the protecting groups

Claims (4)

Process for the enantioselective synthesis of DOPA with a photolabile protective group, comprising the following steps - etherification of dihydroxybenzaldehyde (A) with a photolabile protective group R according to the following first process step

where R is selected from the group consisting of: 2-nitrobenzyl, 1-(2-nitrophenyl)ethyl, 7-nitroindolin-1-yl, (7-methoxycoumarin-4-yl)methyl, {7-[bis(carboxymethyl) amino]coumarin-4-yl}methyl, (6-bromo-7-hydroxycoumarin-4-yl)methyl, deoxybenzoinyl, p-hydroxyphenacyl, (8-bromo-7-hydroxyquinolin-2-yl)methyl, [7- (Diethylamino)coumarin-4-yl]methyl to an ether (B) as a first intermediate, - reacting the ether (B) with N-acetylglycine (C) to an azlactone (D) as a second intermediate according to the following second process step

- Opening of the azlactone (D) according to the following, third process step

by reacting the azlactone (D) in an alcoholic solution to form an opened azlactone (E) as a third intermediate, asymmetric hydrogenation of the opened azlactone (E) according to the following fourth process step

by reacting the opened azlactone (E), obtained as an intermediate in the previous step, with S,S Et-Duphos (2S,5S)-1-[2-[(2S,5S)-2,5-diethylphospholane- 1-yl]phenyl]-2,5-diethylphospholane or "DUPHOS") and bisoctadienyl rhodium tetrafluoroborate to a fourth intermediate (F) - deprotecting intermediate (F) resulting from opening of the azlactone (E) in the third process step are, to a protecting group R substituted-DOPA (G) according to the following reaction scheme

by reacting the fourth intermediate (F) with acid. connection with the following structure

as an intermediate for the preparation of DOPA having a photolabile protecting group, namely R-(L-3,4-dihydroxyphenylalanine), where the radical R is selected from the group consisting of 2-nitrobenzyl, 1-(2-nitrophenyl)ethyl, 7 -nitroindolin-1-yl, (7-methoxycoumarin-4-yl)methyl, {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl, (6-bromo-7-hydroxycoumarin-4-yl) methyl, deoxybenzoinyl, p-hydroxyphenacyl, (8-bromo-7-hydroxyquinolin-2-yl)methyl, [7-(diethylamino)coumarin-4-yl]methyl. connection with the following structure

as an intermediate for the preparation of DOPA having a photolabile protecting group, namely R-(L-3,4-dihydroxyphenylalanine), where the radical R is selected from the group consisting of 2-nitrobenzyl, 1-(2-nitrophenyl)ethyl, 7 -nitroindolin-1-yl, (7-methoxycoumarin-4-yl)methyl, {7-[bis(carboxymethyl)amino]coumarin-4-yl}methyl, (6-bromo-7-hydroxycoumarin-4-yl) methyl, deoxybenzoinyl, p-hydroxyphenacyl, (8-bromo-7-hydroxyquinolin-2-yl)methyl, [7-(diethylamino)coumarin-4-yl]methyl. connection with the following structure

as an intermediate for the production of DOPA with a photolabile protecting group, namely R-(L-3,4-dihydroxyphenylalanine), wherein the radical R is selected from the group consisting of 2-nitrobenzyl, 1-(2-nitrophenyl)ethyl, 7-nitroindolin-1-yl, (7-methoxycoumarin-4-yl)methyl, {7-[bis( carboxymethyl)amino]coumarin-4-yl}methyl, (6-bromo-7-hydroxycoumarin-4-yl)methyl, deoxybenzoinyl, p-hydroxyphenacyl, (8-bromo-7-hydroxyquinolin-2-yl)methyl, [ 7-(diethylamino)coumarin-4-yl]methyl.

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