Isoquinolin-3(4H)-il]ethyl]-urea (ensifentrin)

Abstract

The present invention relates to the field of processes for the preparation of active ingredients for pharmaceutical use, and in particular to a process for the synthesis of N-[2-[(2E)-6,7-dihydro-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]- 2H-pyrimido[6,1-a]isoquinolin-3(4H)-il]ethyl]-urea, also known by the common name Ensifentrine.

Description

[0001] DESCRIPTION of the industrial invention titled:

[0002] ISOQUINOLIN-3(4H)-IL]ETHYL]-UREA (ENSIFENTRIN)

[0003] ********************

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to the field of processes for the preparation of active ingredients for pharmaceutical use, and in particular to an industrially applicable process for the preparation of N-[2-[(2E)-6,7-dihydro-9,10-dimethoxy-4-oxo-2- [(2,4,6-trimethylphenyl)imino]-2 / - / -pyrimido[6,1 -a]isoquinolin-3(4 / - / )-il]ethyl]-urea, also known by the common name Ensifentrine (CAS Registry Number: 1884461 - 72-6), a compound of pharmaceutical interest.

[0006] Ensifentrine is the first representative of a class of drugs, i.e. selective dual inhibitors of phosphodiesterases 3 and 4 (PDE3 and 4), capable of combining bronchodilatory and anti-inflammatory properties; in this way, it is configured as a possible effective treatment for COPD and other respiratory diseases, such as asthma and cystic fibrosis.

[0007] Furthermore, Ensifentrine also activates the cystic fibrosis transmembrane conductance regulator (CFTR), which is useful for reducing mucous membrane viscosity and improving mucociliary clearance; for these reasons, it is also a potential candidate as a new therapeutic option for the treatment of this clinical condition.

[0008] STATE OF THE ART

[0009] Ensifentrine is described and patented in EP 1165558 B1. Example 1 on page 14 of EP 1165558 B1 describes the reaction for obtaining it from the compound obtained in preparation 4 on page 13. The reaction of preparation 4 is schematically shown in Figure 1 of EP 1165558 B1 , from compound (3) to compound (4). Both in the experimental description and in Figure 1 , the reaction from intermediate (3) to intermediate (4) is carried out in the presence of hydrazine and chloroform. Both the reagent used, hydrazine, and the solvent used, chloroform, are incompatible with the preparation of pharmaceutical active ingredients for human use due to their well- known toxicity and carcinogenicity; it is therefore necessary to find a synthetic alternative that avoids their use and is at the same time industrially applicable. SUMMARY OF THE INVENTION

[0010] This object is achieved with the present invention, relating to a process for the preparation of N-[2-[(2E)-6,7-dihydro-9,10-dimethoxy-4-oxo-2-[(2,4,6- trimethylphenyl)imino]-2H-pyrimido[6, 1 -a]isoquinolin-3(4 / - / )-il]ethyl]-urea, also known by the common name Ensifentrine (CAS Registry Number: 1884461 -72-6), which comprises the following steps: a) reduction of intermediate (3) with a boron hydride to give intermediate (4): b) transformation of intermediate (4) into Ensifentrine:

[0011] (4i Ensifentrine

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] - Figure 1 shows the UPLC chromatogram relating to the chromatographic peak in question, obtained by analyzing the reaction product of Example 2.

[0014] - Figure 2 shows the XRD diffractogram of the reaction product of Example 2. - Figure 3 shows the UPLC chromatogram relating to the chromatographic peak in question, obtained by analyzing the reaction product of Example 3.

[0015] - Figure 4 shows the XRD diffractogram of the reaction product of Example 3.

[0016] DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention relates to a process for the synthesis and purification of N-[2- [(2E)-6,7-dihydro-9,10-dimethoxy-4-oxo-2-[(2,4,6-trimethylphenyl)imino]-2 / - / - pyrimido-[6,1 -a]-isoquinolin-3(4 / - / )-il]ethyl]-urea (Ensifentrine). Through the present invention, it is possible to avoid the use of a reagent and solvent incompatible with the preparation of pharmaceutical active ingredients for human use, and it is also possible to simplify the purification procedure of the active ingredient itself. In EP 1165558 B1 , the transformation from (3) into (4) is obtained using hydrazine according to a procedure well known to organic chemists, called the Gabriel amine synthesis, in its Ing-Manske variant (Jie Jack Li, Name Reactions, SPRINGER, pages 273-274). The reaction, illustrated in the scheme below, produces phthalhydrazide as a byproduct.

[0018] (3) (4)

[0019] Intermediate (3) in Scheme 1 of EP 1165558 B1 , which is the starting compound of the present invention, is obtained following the indications of EP 1165558 B1 (preparation 3 on page 13). The process of the invention comprises steps a) and b) of Scheme 1 , shown below:

[0020] Ensifentrine

[0021] Scheme 1 Intermediates (3) and (4) of the process are the same as those described in EP 1165558 B1 , but in step a) intermediate (3) is transformed into intermediate (4) without using hydrazine and chloroform, and in step b) intermediate (4) is transformed into Ensifentrine without using sodium cyanate in water and hydrochloric acid.

[0022] With both these modifications, the purification of Ensifentrine is achieved by crystallization from solvents rather than by chromatographic purification, as reported in Example 1 of EP 1165558 B1. Furthermore, both these modifications avoid the formation of an impurity with m / z = 521 detectable in UPLC-MS, not identified in the cited patent, the removal of which involves a significant loss of Ensifentrine during purification by precipitation.

[0023] More specifically, in step a):

[0024] (3) (4) to carry out the transformation of intermediate (3) into intermediate (4), a boron hydride is used, which is selected from lithium borohydride (LiBF ) and sodium borohydride (NaBF ), preferably sodium borohydride (NaBF ), using C1-C3 alcohols, pure or in a mixture thereof, optionally in the presence of water, as a solvent.

[0025] Preferably, an aqueous alcoholic mixture is used, even more preferably isopropanol (I PA) and water.

[0026] The reaction temperature is between 25 and 75 °C, preferably between 35 and 65 °C, for a time between 5 and 16 hours.

[0027] The transformation of step a) of the invention proceeds as described below, following a completely different reaction route as compared to the synthesis of EP 1165558 B1.

[0028] to

[0029] An intermediate of this transformation, also detectable by UPLC-MS analysis, is compound (3’), which is not described in the literature.

[0030] Intermediate (3’) is transformed into compound (4) by the addition of an acid, which can be selected among CH3COOH, H2SO4, HCI, used pure, in aqueous solution, in a mixture or in sequence with each other.

[0031] A similar transformation, but on different substrates, structurally much simpler and not polyfunctionalized as in the case of the invention, is described in the article “An exceptionally mild deprotection of phthalimides”, J. 0. Osby et al., Tetrahedron Lett. 25(20) 2093-96 (1984). In the final recovery of the product, as described in the representative procedure on page 2095, a chromatographic purification is used by employing an ion exchange resin (Dowex 50 (H+) resin). This procedure is complex and industrially demanding. In the case of the invention, this operation is not required. Below, in step b) of the invention:

[0032] •4) Ensifentrine

[0033] Ensifentrine formation is obtained by reaction of carbonyldiimidazole and intermediate (4), followed by addition of hexamethyldisilazane and subsequently water, or by the addition of aqueous ammonia.

[0034] In the case of using hexamethyldisilazane, the reaction solvent is an ether, preferably selected among methyltetrahydrofuran (2Me-THF), diethyl ether, diisopropyl ether, and methyl te / Y-butyl ether and, more preferably, tetrahydrofuran (THF), at a temperature between 25 and 90 °C, preferably between 40 and 90 °C, for at least 1 hour. The reaction is preferably carried out in a closed reactor, observing an increase in internal pressure which, as the inventors have experimentally verified, is accompanied by a reduction in the formation of the impurity having mass [M++1] = 895 which was identified to be the structure shown in the figure below:

[0035] Subsequently, water is added and the mixture is stirred at a temperature between 25 and 75 °C for at least 1 hour.

[0036] In the case of using aqueous ammonia, the reaction time is between 30 minutes and 6 hours, preferably between 1 and 3 hours.

[0037] In the case of using ammonia, the reaction solvent is an ether, preferably selected among methyltetrahydrofuran (2Me-THF), diethyl ether, diisopropyl ether, and methyl te / Y-butyl ether, and more preferably tetrahydrofuran (THF), at the reflux temperature of the reaction mixture for at least 1 hour. Subsequently, water is added and the mixture is stirred at a temperature between 10 °C and 50 °C for at least 1 hour.

[0038] In EP 1165558 B1 , the transformation from (4) into Ensifentrine is obtained through the experimental description of Example 1 on page 14. Crude Ensifentrine is purified first by silica gel chromatography, then with a subsequent treatment with diethyl ether.

[0039] In the case of the invention, chromatographic purification is not required. The crude Ensifentrine obtained is purified by crystallization through solvent exchange. The crystallization solvents are selected among dichloromethane (DCM), acetone, methyl ethyl ketone (MEK), cyclohexanone, methyl isobutyl ketone (MIBK) and methanol (MeOH), pure or in mixtures thereof.

[0040] The invention will be further illustrated by the following examples.

[0041] INSTRUMENTS, METHODS AND EXPERIMENTAL CONDITIONS

[0042] NMR

[0043] Spectrometer: NMR JEOL 400 YH (400 MHz); Software: JEOL Delta v5.1.1 ;

[0044] Spectra were recorded in deuterated solvents such as: Chloroform-d, 99.8% D, containing 0.1 % (v / v) tetramethylsilane (TMS) as internal standard; and Chloroform- d, “100%”, 99.96% D, containing 0.03% (v / v) TMS, and DMSO-d6.

[0045] MS

[0046] Spectrometer: WATERS SYNAPT G2-Si QTof

[0047] Sample ionization is achieved via ESI (electrospray ionization).

[0048] HPLC

[0049] HPLC: Agilent 1260 Infinity II; Detector UV-DAD

[0050] Mobile phase A: Water / Methanol / TFA, 95:5:0.05 v / v / v

[0051] Mobile phase B: Acetonitrile / Methanol / TFA, 95:5:0.05 v / v / v

[0052] Chromatographic conditions:

[0053] Column: Luna Omega Polar 150 x 4.6 mm; 3.0 pm

[0054] Flow rate: 1 .0 mL / min

[0055] Detector: 220 and 364 nm

[0056] Injection volume: 5 pL

[0057] Sample solution: Dilution with Water / Acetonitrile, 50:50 v / v (first, add the required percentage of ACN, then bring to volume with water).

[0058] UPLC

[0059] Chromatographic system: Waters Acquity UPLC;

[0060] Detector: Acquity UPLC PDA diode array

[0061] Column: Acquity UPLC BEH C18 1 .7 pm (2.1 x 50 mm), T = 35 °C

[0062] Solvent A: acetonitrile containing 0.1 % v / v formic acid

[0063] Solvent B: water containing 0.1 % v / v formic acid

[0064] Gradient:

[0065] TLC

[0066] MERCK: TLC silica gel 60 F254 Aluminium sheets 20 x 20 cm, cod. 1 .0554.0001 .

[0067] HPTLC

[0068] MERCK: TLC silica gel 60 F254, cod. 1.3727.0001 with concentrating zone 10 x 2.5 cm.

[0069] TLC detectors

[0070] UV: 254 nm lamp

[0071] Cerium phosphomolybdate: 25 g of phosphomolybdic acid and 10 g of cerium(IV) sulfate are dissolved in 600 mL of H2O. 60 mL of concentrated sulfuric acid (98% H2SO4 by weight) are added and brought to a volume of 1 L with H2O. The TLC plate is impregnated with the solution and then heated until the products are detected.

[0072] DRX

[0073] Powder X-ray diffraction (XRD) analysis was performed using a Bruker® D2 Phaser diffractometer (2nd ed.) operating in Bragg-Brentano geometry and equipped with a 6-position rotating multi-sample holder. The X-ray source is a copper anode X-ray tube, operated at 30 kV and 10 mA. The analytical wavelength used is the Ka of copper (A = 1.54184 A). K|3 is filtered using a nickel filter. The X-ray detector is a LYNXEYE® solid-state linear detector. The samples were deposited as a thin layer on “zero background” type silicon sample holders. The diffractogram was recorded in the angular range 4.0-40° 29 with 0.016° increments and a scanning rate of 1.0 s / increment. The sample was rotated at 60 rpm during analysis. The data were analyzed using the software DIFFRAC.EVA (Bruker®).

[0074] NOTES

[0075] The water used in the experimental descriptions is to be intended as distilled water, unless otherwise indicated.

[0076] The organic solvents used in the experimental descriptions are to be intended of a “technical” grade, unless otherwise indicated.

[0077] The reagents and catalysts used in the experimental descriptions are to be intended of commercial quality, unless otherwise indicated.

[0078] EXAMPLE 1

[0079] In a flask under an inert N2 atmosphere, intermediate (3) (solid, 4.88 g) is charged in isopropanol (140 mL) and water (25 mL). The mixture is heated to Tintemai = 36 °C and a first portion of sodium borohydride (1.3 g) is added. The mixture is stirred at Tintemai = 36 °C, after 7 hours a second portion of sodium borohydride (0.327 g) is added, and after 5.5 hours a third portion of sodium borohydride (0.327 g) is added. The reaction mixture is observed to be biphasic. The temperature is raised by 10 °C (46 °C) and the mixture is allowed to react for a further 16 hours. The internal temperature is lowered to 0 / 5 °C using an ice bath, and 1 M hydrochloric acid (100 mL) is added dropwise. The mixture is heated to Tintemai = 60 °C for 5.5 hours, and then to 25 °C for 16 hours. UPLC-MS analysis confirmed the completion of the reaction.

[0080] Isopropanol is distilled off under static vacuum (approximately 190 mL of solvent distilled), water (50 mL) is added, the temperature is lowered to 0 / 5 °C and 30% w / w sodium hydroxide (6 mL) is added dropwise until a final pH of 8.66 is reached. The mixture is stirred at 25 °C for 1 hour, then the solid is filtered and washed with water (25 mL) obtaining 4.15 g of intermediate (4) usable for the subsequent steps.

[0081] The weight obtained exceeds that expected from a quantitative transformation, but the inventors have experimentally verified that the by-products present, probably borates derived from the hydride, do not have negative effects on the subsequent reaction.

[0082] EXAMPLE 2 4) Ensifantrin*

[0083] In a flask under an inert N2 atmosphere, intermediate (4) (solid, 0.52 g) is charged in tetrahydrofuran (10.5 mL), carbonyldiimidazole (0.388 g) is added, the mixture is heated to Tintemai = 37 °C and allowed to react for 1 hour (total dissolution is observed after approximately 40 minutes), and the progress of the reaction is monitored by TLC.

[0084] A TLC check, using dichloromethane / methanol 95:5 v / v as eluent, shows that the reaction is complete.

[0085] Hexamethyldisilazane (1 mL) is added, and the mixture is heated to Tintemai = 62 °C; after 2 hours, a second portion of hexamethyldisilazane (0.25 mL) is added and the mixture is stirred at Tintemai = 62 °C for 1 hour. Water (10 mL) is added, stirring at Tintemai = 62 °C for 2 hours, and then at 25 °C for a further 16 hours.

[0086] A second portion of tetrahydrofuran (10 mL) is added, heating to Tintemai = 62 °C for 1 hour and monitoring by TLC.

[0087] A TLC check, using dichloromethane / methanol 95:5 v / v as eluent, shows that the reaction is complete.

[0088] Tetrahydrofuran is distilled under vacuum, and 1 M hydrochloric acid (7 mL) is added. The mixture is extracted with dichloromethane (2x25 mL), and the combined organic phases are washed with water (1x10 mL). The organic phase is concentrated by evaporating approximately 30 mL of solvent at atmospheric pressure with a Dean-Stark apparatus. 15 mL of acetone are added, and another 15 mL of solvent are evaporated at atmospheric pressure. Precipitation is observed. The mixture is cooled to 25 °C and stirred for 30 minutes. It is filtered, washing with acetone (3 mL). It is dried under vacuum in a rotary evaporator at 45 °C for 1 hour, and a 230 mg sample of Ensifentrine is recovered. An additional 40 mg of Ensifentrine is recovered by concentrating the mother liquors. The two samples, after UPLC-MS analysis, are combined for a total of 270 mg as they are of comparable quality.

[0089] MS: m / z 478 = MH+;1H-NMR in accordance with the description in EP 1165558 B1 , Example 1 , page 14.

[0090] Figure 1 shows, in the upper part, the UPLC chromatogram relating to the chromatographic peak in question.

[0091] Figure 2 shows the XRD diffractogram of the product obtained in the example.

[0092] EXAMPLE 3 Under a nitrogen atmosphere, intermediate (4) (solid, 390 mg) is suspended in THF (8 mL) and carbonyldiimidazole (0.29 mg) is added. The temperature is raised to 33-37 °C and stirred for 5 hours.

[0093] UPLC-MS check showed the almost complete disappearance of intermediate (4). After cooling to 5 °C, 30% by weight aqueous ammonium hydroxide (5 mL) is added. The mixture is brought to reflux and stirred for 1 hour. UPLC check (acetonitrile / water) confirmed the completion of the reaction.

[0094] THF is evaporated under vacuum at 45 °C using a rotary evaporator, and precipitation is observed. Water (3 mL) is added, and the mixture is stirred at 25 °C for 1 hour and filtered, washing with water (2 mL). The wet crude product is suspended in 1 N HCI (5 mL) and ethyl acetate (5 mL) is added. The phases are separated, and the aqueous phase is extracted twice with dichloromethane (10 mL). The organic phases are combined and washed with water.

[0095] The solvent is concentrated, under vacuum at 45 °C using a rotary evaporator, to approximately one-third of the initial volume, and MEK (10 mL) is added. Precipitation is observed. The mixture is stirred at 25 °C for 1 hour and filtered, washing with MEK (3 mL). The solid is dried with a rotary evaporator at 45 °C for 1 hour under vacuum, and 237 mg of solid are recovered.

[0096] The solid from MEK is suspended in DCM (10 mL) and stirred at reflux until completely dissolved. Acetone (15 mL) is added. DCM is evaporated at atmospheric pressure to a volume of approximately 15 mL. Precipitation is observed. The mixture is stirred at 25 °C for 16 hours and filtered, washing with acetone. The solid is dried with rotary evaporator under vacuum for 30 minutes, and the Ensifentrine sample (194 mg) is recovered, which, upon UPLC analysis, shows a purity of 99.89%.

[0097] MS: m / z 478 = MH+;1H-NMR in accordance with the description in EP 1165558 B1 , Example 1 , page 14.

[0098] Figure 3 shows, in the upper part, the UPLC chromatogram relating to the chromatographic peak in question

[0099] Figure 4 shows the XRD diffractogram of the product obtained in the example.

[0100] EXAMPLE 4

[0101] (4; Ensifentrine

[0102] In a pressure-tight reactor under a nitrogen atmosphere, intermediate (4) (solid, 20 g) is suspended in THF (0.6 L), and carbonyldiimidazole (29.8 g) is added. The temperature is brought to 40 °C, and the mixture is stirred for 2 hours (closed reactor, internal overpressure).

[0103] HPTLC check (eluent: dichloromethane / methanol, 9:1 v / v): the reaction is complete, intermediate (4) is not detectable.

[0104] The internal pressure is returned to atmospheric pressure, and 111 g of hexamethyldisilazane are added.

[0105] With the reactor closed, the mixture is heated to Tintemai = 70-75 °C and stirred at this temperature for 16 hours (internal overpressure).

[0106] It is cooled to 20-25 °C and checked by HPLC.

[0107] The reactor is changed, 10 °C pre-cooled water (400 mL) is added, and the mixture is stirred for 1 hour at 70-75 °C.

[0108] It is brought back to 20-25 °C; a second portion of tetrahydrofuran (200 mL) and water (400 mL) is added, and tetrahydrofuran is distilled off at 50 °C under reduced pressure.

[0109] It is brought back to 25 °C and stirred at this temperature for 1 hour. The resulting solid is filtered and washed with water while on the filter (24.9 g wet solid weight).

[0110] The solid is dried under reduced pressure and T = 50 °C for 17 hours, obtaining 18.6 g of Ensifentrine.

[0111] HPLC check: Ensifentrine peak %area = 98.95%.

[0112] The solid is purified by crystallization from DCM / MeOH first, and then from MEK, obtaining, after drying under reduced pressure at T = 50 °C for 18 hours, Ensifentrine (yellow solid, 17.66 g) of pharmaceutical grade in which all the impurities present have a %area by HPLC lower than 0.10. LIPLC and X-ray diffractometry analyses performed on the product obtained in this example provided a chromatogram and a diffractogram similar to those in Figures 1 and 2, respectively.

Claims

CLAIMS1. Process for the synthesis of N-[2-[(2E)-6,7-dihydro-9,10-dimethoxy-4-oxo-2- [(2,4,6-trimethylphenyl)imino]-2 / - / -pyrimido[6,1-a]isoquinolin-3(4 / - / )-yl]ethyl]- urea, known by the common name Ensifentrine, comprising the steps of: a) reduction of intermediate (3) with a boron hydride selected between sodium borohydride (NaBH4) and lithium borohydride (LiBH4) to give intermediate (4):b) transformation of intermediate (4) into Ensifentrine:

2. Process according to claim 1 , wherein the boron hydride used in step a) is sodium borohydride.

3. Process according to any one of claims 1 or 2, wherein step a) is carried out in a solvent selected from C1 -C3 alcohols, pure or in mixtures with each other, or aqueous mixtures thereof.

4. Process according to claim 3, wherein said solvent is a mixture of isopropanol (I PA) and water.

5. Process according to any one of the preceding claims, wherein step a) iscarried out using an acid selected among CH3COOH, H2SO4 and HCI.

6. Process according to any one of the preceding claims, wherein step a) is carried out at a temperature between 25 and 75 °C for a time between 5 and 16 hours.

7. Process according to any one of the preceding claims, wherein step b) is carried out by adding to intermediate 4, in this order, carbonyldiimidazole (CDI), hexamethyldisilazane, and water.

8. Process according to claim 7, wherein the solvent used is an ether selected among diethyl ether, diisopropyl ether, methyl-te / Y-butyl ether, tetrahydrofuran (THF), and methyltetrahydrofuran (2MeTHF), and the reaction is carried out at a temperature between 25 and 90 °C with an overall reaction time of at least 2 hours.

9. Process according to claim 8, wherein the reaction is carried out in a closed reactor.

10. Process according to any one of claims 1 to 6, wherein step b) is carried out by adding to intermediate 4, in this order, carbonyldiimidazole (CDI) and aqueous ammonia.11 . Process according to claim 10, wherein the solvent used is an ether selected among diethyl ether, diisopropyl ether, methyl-te / Y-butyl ether, tetrahydrofuran (THF), and methyltetrahydrofuran (2MeTHF), and the reaction is carried out at the reflux temperature of the mixture with a reaction time of at least 1 hour, after which water is added and the system is kept under stirring at a temperature between 10 and 50 °C for at least 1 hour.

12. Process according to any one of the preceding claims, wherein the crude Ensifentrine obtained is purified by crystallization via solvent exchange with a solvent selected among dichloromethane (DCM), acetone, methyl ethyl ketone (MEK), cyclohexanone, methyl isobutyl ketone (MIBK) and methanol, either pure or in mixture with each other.

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