Process for the Production of Ferroportin Inhibitors
20230212159 · 2023-07-06
Inventors
- Stefan Reim (St. Gallen, CH)
- Erik PHILIPP (St. Gallen, CH)
- Maria WILHELM (St. Gallen, CH)
- Cyrill BLATTER (St. Gallen, CH)
Cpc classification
A61P7/00
HUMAN NECESSITIES
C07D413/12
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates to a new process for preparing compounds of the formula (I)
##STR00001##
and pharmaceutically acceptable salts thereof, which act as ferroportin inhibitors being suitable for the use as medicaments in the prophylaxis and/or treatment of diseases caused by a lack of hepcidin or of iron metabolism disorders leading to increased iron levels or increased iron absorption, including iron overload, thalassemia, sickle cell disease and hemochromatosis.
Claims
1-16. (canceled)
17. A process for preparing a compound of the formula (I), and pharmaceutically acceptable salts thereof, ##STR00075## comprising at least one step comprising reacting a compound of the formula (IM-3) with a compound of the formula (RM-3) ##STR00076## to provide the compound of the formula (I); wherein X.sup.1 is N, S or O; and X.sup.2 is N, S or O; with the proviso that one of X.sup.1 and X.sup.2 is N and that X.sup.1 and X.sup.2 are different; m is an integer of 1, 2, or 3; n is an integer of 1, 2, 3 or 4; o is an integer of 1, 2, 3 or 4; A represents a CH-group, a CH.sub.2-CH-group or a CH.sub.2-CH.sub.2-CH-group; R.sup.1 and R.sup.2 are independently selected from the group consisting of hydrogen and C.sub.1-C.sub.4-alkyl, which may be substituted with 1 or 2 substituents; R.sup.3 represents 1, 2 or 3 optional substituents, which may independently be selected from the group consisting of halogen, cyano, C.sub.1-C.sub.4-alkyl, C.sub.1-C.sub.3-halogenoalkyl; C.sub.1-C.sub.4-alkoxy, and a carboxyl group; R.sup.4 is selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.3-alkyl, and C.sub.1-C.sub.3-halogenoalkyl; R.sup.5 is selected from the group consisting of aryl which may carry 1 to 3 substituents, and mono- or bicyclic heteroaryl which may carry 1 to 3 substituents; and R.sup.6 is selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.4-alkyl, which may be substituted with 1 or 2 substituents; C.sub.1-C.sub.3-halogenoalkyl.
18. The process of claim 17, further comprising the step of reacting a compound of the formula (IM-2) with a compound of the formula (RM-2) to form the compound of the formula (IM-3): ##STR00077## .
19. The process of claim 18, further comprising a step of preparing the compound of the formula (IM-2) by converting a compound (RM-1) into the compound (IM-1) followed by ester cleavage: ##STR00078## wherein R.sub.y represents hydrogen or halogen.
20. The process of claim 19, wherein the compound of the formula (IM-1) is prepared according to one of the following reaction schemes: ##STR00079## ##STR00080## ##STR00081## .
21. The process of claim 19, wherein the preparation of the compound (IM-2) from the compound (RM-1) via the intermediate compound (IM-1) is carried out in one process step in a one-pot reaction.
22. The process according to claim 17, wherein R.sup.5 represents a monocyclic heteroaryl group, which may carry 1 to 3 substituents, which may independently be selected from C.sub.1-C.sub.3-alkyl, halogen and C.sub.1-C.sub.3-halogenoalkyl,.
23. The process of 17, comprising the following reaction steps: Step 1: ##STR00082## ##STR00083## ##STR00084## followed by Ester Cleavage: ##STR00085## Step 2: ##STR00086## Step 3: ##STR00087## ##STR00088## wherein R.sub.y is selected from C.sub.1-C.sub.3-alkyl, halogen and C.sub.1-C.sub.3-halogenoalkyl.
24. The process of claim 17, wherein at least one of the following conditions are fulfilled: A represents a CH-group; m represents 1; o represents 1; R.sup.1 and R.sup.2 each represent hydrogen; R.sup.4 represents hydrogen; R.sup.6 represents hydrogen; R.sup.3 represents hydrogen; X.sup.1 is N and X.sup.3 is O or S, forming a group ##STR00089## ##STR00090## or X.sup.1 is O or S and X.sup.2 is N, forming a group ##STR00091## ##STR00092## wherein in each case * indicates the binding position to the carbonyl-group and ** indicates the second binding position.
25. The process of claim 17 for preparing a compound of the formula (II), and pharmaceutically acceptable salts thereof ##STR00093## the process comprising the following process steps: Step 1-a: ##STR00094## ##STR00095## ##STR00096## followed by Ester Cleavage: ##STR00097## Step 2-a: ##STR00098## Step 3-a: ##STR00099## .
26. The process of claim 17 for preparing a compound of the formula (II′), and pharmaceutically acceptable salts thereof ##STR00100## the process comprising the following process steps: Step 1-a: ##STR00101## ##STR00102## ##STR00103## followed by Ester Cleavage: ##STR00104## Step 2-a: ##STR00105## Step 3-a: ##STR00106## .
27. The process of claim 17, further comprising the step of converting the compound of the formula (I) into a pharmaceutically acceptable salt or solvate thereof based on acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid.
28. The process of claim 25, further comprising the step of converting the compound of the formula (II) into a pharmaceutically acceptable salt or solvate thereof based on acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid.
29. The process of claim 26, further comprising the step of converting the compound of the formula (II′) into a pharmaceutically acceptable salt or solvate thereof based on acids selected from the group consisting of benzoic acid, citric acid, fumaric acid, hydrochloric acid, lactic acid, malic acid, maleic acid, methanesulfonic acid, phosphoric acid, succinic acid, sulfuric acid, tartaric acid and toluenesulfonic acid.
30. The process of claim 27, where the pharmaceutically acceptable salt of the compound of the formula (I) is a salt selected from the group consisting of mono-HCl salt (1HCl), triple-HCl salt (3HCl), H.sub.2SO.sub.4-salt, 0.5 H.sub.3PO.sub.4 salt and 1H.sub.3PO.sub.4 salt.
31. The process of claim 28, where the pharmaceutically acceptable salt of the compound of the formula (II) is a salt selected from the group consisting of mono-HCl salt (1HCl), triple-HCl salt (3HCl), H.sub.2SO.sub.4-salt, 0.5 H.sub.3PO.sub.4 salt and 1H.sub.3PO.sub.4 salt.
32. The process of claim 29, where the pharmaceutically acceptable salt of the compound of the formula (II′) is a salt selected from the group consisting of mono-HCl salt (1HCl), triple-HCl salt (3HCl), H.sub.2SO.sub.4-salt, 0.5 H.sub.3PO.sub.4 salt and 1H.sub.3PO.sub.4 salt.
33. The process of claim 17, wherein the at least one step is carried out in one telescoped one-pot reaction.
34. The process of claim 23, wherein the steps 1 and 2, respectively, are carried out in one telescoped one-pot reaction and if present, the step of converting the intermediate compound IM-3 into a salt of the compound (I) or (II) or (II′) is carried out in one telescoped one-pot reaction.
35. The process of claim 25, wherein the steps 1-a and 2-a, respectively, are carried out in one telescoped one-pot reaction and/or, if present, the step of converting the intermediate compound IM-3b into a salt of the compound (II′) is carried out in one telescoped one-pot reaction.
36. A compound of the formula (IM-3) ##STR00107## wherein X.sup.1 and X.sup.2 are different and independently represent O or S and X.sup.1 is N, S or O; and X.sup.2 is N, S or O; with the proviso that one of X.sup.1 and X.sup.2 is N and that X.sup.1 and X.sup.2 are different; o is an integer of 1, 2, 3 or 4; A represents a CH-group, a CH.sub.2-CH-group or a CH.sub.2-CH.sub.2-CH-group; R.sup.1 is hydrogen or C.sub.1-C.sub.4-alkyl, which may be substituted with 1 or 2 substituents; R.sup.4 is selected from the group consisting of hydrogen, halogen, C.sub.1-C.sub.3-alkyl, and C.sub.1-C.sub.3-halogenoalkyl; R.sup.5 is selected from the group consisting of aryl which may carry 1 to 3 substituents, and mono- or bicyclic heteroaryl which may carry 1 to 3 substituents; and or wherein the compound of the formula (IM-3) has the formula (IM-3-a) ##STR00108## wherein X.sup.1 and X.sup.2 are different and independently represent O or S and R.sub.y is selected from C.sub.1-C.sub.3-alkyl, halogen and C.sub.1-C.sub.3-halogenoalkyl; or wherein the compound of the formula (IM-3) has the formula (IM-3-b) ##STR00109## or wherein the compound of the formula (IM-3) has the formula (IM-3-b′) ##STR00110## .
37. A 3HCI salt of the compound according to the formula (II) or (II′) ##STR00111## ##STR00112## or a solvate, hydrate or polymorph thereof, which is characterized by at least one of the following purity criteria: (i) a total impurity content of less than 2.00% rel. area, (ii) a purity of ≥ 97.80% rel. area, ≥ 98.00% rel. area, ≥ 98.50% rel. area, or ≥ 99.00% rel. area; (iii) containing one or more of the impurities at relative retention times 0.59, 0.65, 0.83, and 1.37 in an amount of not more than 0.20% rel. area; or (iv) absence of impurities at relative retention times: 0.59, 0.65, 0.83, and 1.37; wherein the impurity content, the purity, the relative retention times and the rel. area values are determined by HPLC.
Description
DESCRIPTION OF THE FIGURES
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
EXAMPLES
[0243] TABLE-US-00001 Abbreviations DCM dichloromethane DSC differential scanning calorimetry IPC HPLC High Pressure Liquid Chromatography PXRD Powder X-ray diffraction THF Tetrahydrofuran Mw Molecular Weight
1A. Process for Preparing an Intermediate Compound (IM-2-a) With Crystallization from HCl
[0244] TABLE-US-00002 Chemicals Chemical Molar Mass Eq. Amount (th) Amount (is) g/mol mmol 1,2-dibromoethane 187.86 120.5 1.7 22.63 g Li-tert-butoxide 80.06 198.4 2.8 15.88 g THF - - - 70 ml 15 ml 15 ml ethyl 4-oxazolcarboxylate 141.12 70.9 1.0 10.00 g Pd(PPh.sub.3).sub.4 - - - 2.24 g Lithium hydroxide 23.95 106.3 1.5 2.55 g water - - - 35ml isopropyl acetate - - - 2x 70 ml hydrochloric acid 20% - - - x ml x ml
Reaction
[0245] ##STR00047##
Procedure
[0246] The vessel is purged with N.sub.2 for ≥ 15 min.
[0247] LitOBu is charged, 70 ml THF are dosed and stirred for ≥ 10 min at 20 - 25° C.
[0248] The mixture is heated to 55 - 60° C. and stirred ≥ 5 min.
[0249] A solution of 1,2-dibromoethane in 15 ml THF is dosed at 55 - 60° C.
[0250] The mixture is stirred for ≥ 30 min at this temperature.
[0251] The mixture is cooled to 45 - 50° C.
[0252] Pd(PPh.sub.3).sub.4 is added, purged with some ml THF and stirred at this temperature for ≥ 5 min.
[0253] The mixture is heated to 60 - 65° C. and a solution of ethyl 4-oxazolcarboxylate in 15 ml THF is dosed at 60 - 65° C. (exothermic).
[0254] The mixture is stirred at this temperature ≥ 30 min.
[0255] The mixture is cooled to 20 - 25° C.
[0256] A solution of LiOH in 35 ml water is dosed at 20 - 25° C. and stirred overnight (exotherm).
[0257] IPC via HPLC: ≥ 95 % conversion
[0258] The mixture is extracted three times with isopropyl acetate. The organic phases are discarded. The aqueous phase is cooled to 0 - 5° C. and the pH is measured.
[0259] The pH is adjusted with HCl 20 % to 0.9 - 1.1 by keeping the temperature ≤ 10° C.
[0260] The suspension is stirred at - 5 to 0° C. for ≥ 45 min.
[0261] The suspension is filtered, washed with 50 ml cooled (<_ 5° C.) HCl of pH 0, 20 ml of cooled (<_ 5° C.) water and is dried in vacuo at 45° C. to dryness.
TABLE-US-00003 Yield Theory Found Comments Yield Crude - Yield Final Product (pale brown solid) 9.9 g 6.5 g 66% uncorr. HPLC = 98% area q-NMR = 98% m/m
1B. Alternative Process Step for Preparing Intermediate Compound (IM-1-a)
[0262] In an alternative (but less preferred) process according to Example 1a above the process steps of stage 1 and stage 2 described therein can alternatively be carried out by starting with a compound RM-1 wherein R.sub.y is chlorine and R.sup.4 is hydrogen and which is indicated herein as RM-1-a′, leading to intermediate compound IM-1-a.
TABLE-US-00004 Chemicals Chemical Molar Mass Eq. Amount q/mol mmol ethyl 2-chlorooxazole-4-carboxylate 175.57 885.69 1.0 155.50 g tributyl(vinyl)tin 317.1,1 877.96 1.0 278.40 g Pd(PH.sub.3P).sub.2Cl.sub.2 701.9 43.60 - 30.60 g dioxane - - - 1500 ml
Reaction
[0263] ##STR00048##
Procedure
[0264] Ethyl 2-chlorooxazole-4-carboxylate, tributyl(vinyl)tin and Pd(Ph.sub.3P).sub.2Cl.sub.2 are charged in dioxane under nitrogen. The mixture is heated to reflux OT 100 - 110° C. for ≥ 4 h. The mixture is cooled to IT 20 - 25° C., filtrated over Celite and the filtercake washed with 200 ml of dioxane. The filtrate is evaporated in vacuo to dryness and the crude product purified by chromatography. [0265] Column: Kp-Sil 1500 g [0266] Eluent: EtOAc/Heptane 20:80 [0267] Method: Duration 7 CV, no gradient, threshold 20 mAU; [0268] Crude dissolved in EtOAc/Heptane 20:80, two columns used at this scale
TABLE-US-00005 Yield Theory Found Comments Yield Crude - 453.40 g Yield Final Product 146.02 g 112.34 g 76.93% NMR conform to structure
1C. Alternative Process Step for Preparing Intermediate Compound (IM-1-a)
[0269] In a further alternative (preferred) process according to Example 1a above the process steps of stage 1 and stage 2 described therein can alternatively be carried out by starting with a compound RM-1 wherein R.sub.y is chlorine and R.sup.4 is hydrogen and which is indicated herein as RM-1-a′, leading to intermediate compound IM-1-a.
Reaction
[0270] ##STR00049##
Procedure
[0271] A RBF was charged with ethyl 2-chlorooxazole-4-carboxylate (RM-1-a′ / 1.0 eq), 2-Me THF (9 V) and water (1 V) were added under nitrogen atmosphere at 25-30° C. To this mixture vinylboronic acid pinacol ester (1.2 eq) and potassium carbonate (2.5 eq) were added at 25-30° C. and the resultant mixture was degassed with nitrogen for 15 minutes. Pd(PPh.sub.3).sub.4 (0.05 eq) was added under a nitrogen atmosphere and the reaction mixture was warmed to 80° C. The reaction mixture was stirred for 8-12 h at 80-85° C. and reaction completion was monitored by TLC/HPLC. After completion of the reaction, the reaction mixture was cooled to 25-30° C. and diluted with water (5 V). The phases were separated and the aqueous phase was extracted with 2-Me THF (5 V). The combined organic phases were washed with water (5 V) followed by brine solution (5 V) and then dried over sodium sulfate. The organic phase was filtered and concentrated below 50° C. under vacuum to obtain the crude product as brown liquid. The crude product was purified using silica gel (60-120 mesh) column chromatography eluting with ethyl acetate and n-heptane to obtain the pure product.
[0272] NMR conform to structure.
Yield
[0273] 55-60%
Purity
[0274] Purity of the obtained material was in the range of 96-99%.
[0275] By further purification using n-heptane crystallization at lower temperature below 0° C. a purity of >98% was obtained. When testing this highly purified material for Palladium content using ICP-MS, an amount in the range of 10-250 ppm was found, which was further reduced by treatment with a Siliabond thiol scavenger (heterogeneous Pd scavenger treatment) to a content below 25 ppm.
2. Process for Preparing an Intermediate Compound (IM-2-a) With Crystallization From Water
[0276] TABLE-US-00006 Chemicals Chemical Molar Mass Eq. Mass th. Mass is g/mol mmol IM-1-a 167.16 598.2 1.0 100.00 g 100.14 g THF - - - 500 ml 500 ml water - - - 500 ml 500 ml lithium hydroxide 23.95 658.1 1.1 15.76 g 15.76 g HCl 20% - - - x ml x ml DCM - - - 2 x 500 ml 2 x 500 ml magnesium sulfate - - - x g xg
Reaction
[0277] The starting compound IM-1-a can be prepared according to process steps ,,stage 1″ and ,,stage 2″ of Example 1a or as described in Example 1b (less preferred).
##STR00050##
Procedure
[0278] IMa is charged into the reactor and dissolved in THF.
[0279] The solution is cooled to 3 - 7° C.
[0280] A solution of LiOH (15.76 g in 500 ml water) is added ≥ 15 min (slightly exotherm) at 3 - 7° C. The mixture is stirred ≥ 3 h at 3 - 7° C.
[0281] IPC via LC/MS ≥ 97% conversion.
[0282] The mixture is extracted twice with DCM. The DCM phase extractions and separations are done with the aq. phase at 5° C. but without active cooling using DCM having room temperature.
[0283] The phases are rested ≥ 30 min.
[0284] The organic layers are discarded.
[0285] The vessel is cleaned with HCl 20% and ethanol.
[0286] HCl 20% is dosed to the aq. phase at 3 - 7° C. till pH 0.5 - 1.0 (slightly exotherm, the HCl should not rinse at the vessel walls). Towards the end of HCl dosing during crystallization the stirrer speed is raised from 80 to 300 rpm for a good mixing of the suspension.
[0287] The suspension is stirred ≥ 30 min at 3 - 7° C.
[0288] The suspension is filtered and the reactor rinsed once with the mother liquor.
[0289] The wet cake is washed with water of 5 - 10° C. and dried at 45° C./vacuum < 50 mbar to dryness. The transfer of the fine suspension on the filter is leaving some residues in the vessel but these are easily removed by one rinse with the mother liquor.
TABLE-US-00007 Yield Theory Found Comments Yield Crude - Final Yield 83 g 77 g 93% uncorr. NMR Assay: 97% m/m Corr. Yield: 90%
3A. Process for Preparing an Intermediate Compound (IM-3-b) With Crystallization From Water
[0290] TABLE-US-00008 Chemicals Chemical Molar Mass Eq. amount th. amount is g/mol mmol IM-2-a 139.11 718.9 1.00 100.00 g DCM - - - 900 ml 4-methylmorpholine 101.15 2731.7 3.80 276.31 g ethylchloroformate 108.52 898.6 1.25 97.51 g RM-2-a 199.05 898.6 1.25 178.86 g NaCl-sol. 10 % - - - 2 x 900 ml HCl 10 % 3x 450 ml
Reaction
[0291] ##STR00051##
Procedure
[0292] The starting compound IM-2-a may be prepared as described in Example 1 or 2.
[0293] Under inert atmosphere compound IM-2-a is suspended in DCM and 4-methylmorpholine is added at -5 to 0° C.
[0294] Afterwards ethylchloroformate is added keeping the temperature ≤ 0° C. (exothermic addition). Compound RM-2-a (grinded) is added in portions keeping the temperature ≤ 0° C.
[0295] The suspension is stirred ≥ 2 h at -5 to 0° C.
[0296] IPC control by LC/MS conversion ≥ 93% area.
[0297] The mixture is heated to 15 - 25° C. and extracted two times with NaCl 10% solution.
[0298] The organic phase is extracted three times with 450 ml HCl 10% w/w.
[0299] The combined aqueous phases are adjusted to pH 2 with 30% NaOH and afterwards to pH 7 - 8 with 5% NaOH keeping the temperature ≤ 10° C.
[0300] The suspension is filtered, washed two times with 400 ml of water and dried in vacuum at 45° C. to dryness.
TABLE-US-00009 Yield Theory Found Comments Yield Crude - Final Yield 178 g 162 g 91% uncorr NMR assay: 99% Corr. Yield: 90%
3B. Process for Preparing Intermediate Compound (IM-3-b) - Telescoped Synthesis via IM-2-a
Reaction
[0301] ##STR00052##
Process Variant 1
[0302] TABLE-US-00010 Chemicals Chemical Molar Mass Eq. Amount (th) Amount (is) q/mol mmol IM-1-a 167.16 59.8 1.0 10.00 g THF - - - 50 ml Water - - - 50 ml LiOH 23.95 71.8 1.2 1.72 g DCM - - - 3 x 50 ml 3 x HCl 20% - - - x ml x ml DCM - - - x ml 4-methylmorpholine 101.15 227.3 3.80 22.99 g ethylchloroformate 108.52 74.8 1.25 8.11 g RM-2-a 199.05 74.8 1.25 14.88 g NaCl 10 % - - - 2 x 150 ml HCl 10% 3 x 75 ml
Procedure
[0303] The starting compound IM-1-a may be prepared as described in Example 1 or 2.
[0304] The starting compound IM-1-a is dissolved in THF. A solution of 1.72 g LiOH in 20 ml water is added at IT 0 - 5° C. The mixture is stirred at 3 - 7° C. for ≥ 3 h.
[0305] IPC via HPLC-MS ≥ 97 % conversion.
[0306] The mixture is adjusted to IT 15 - 20° C. and the pH set to 0.8 - 1.2 by addition of HCl 20 % at this temperature range. The mixture is extracted three times with DCM. The combined organic phases are dried via azeotropic solvent removal by distilling off 50 ml of volume at OT 30° C. / 600 mbar and addition of 50 ml DCM afterwards. This procedure is repeated till water content is 0.13%, determined by Karl Fischer.
[0307] The solution is filled up to a volume of 160 ml with DCM and cooled to IT -5 - 0° C. 4-methylmorpholine is added in this temperature range. Ethylchloroformate is added at this temperature range (exothermic). RM-2-a (grinded) is added in portions at IT ≤ 0° C. The mixture is stirred at -5 - 0° C. for ≥ 3 h.
[0308] IPC via HPLC-MS ≥ 93% conversion.
[0309] The mixture is extracted two times with NaCl 10 % solution. The water phases are discarded. The organic phase is extracted three times with 75 ml of HCl 10 %. The organic phase is discarded. The combined water phases are adjusted at IT ≤ 10° C. to pH 2 with NaOH 30% first and afterwards with NaOH 5% to pH 7 - 8. After stirring ≥ 15 min at ≤ 10° C. the suspension is filtered and washed two times with 60 ml water. The filter cake is dried at OT 45° C. / < 100 mbar to dryness.
TABLE-US-00011 Yield Theory Found Comments Yield Crude - Final Yield off-white powder 14.78 g 10.35 g 70.03 % HPLC Purity = 99.8% Water = 0.04%
[0310] In experiments with a DCM water content of 0.04% by azeotroping the yield was even up to 85%.
Process Variant 2
[0311] TABLE-US-00012 Chemicals Chemical Molar Mass Eq. Amount (th) g/mol mmol IM-1-a 167.16 2392.9 1.00 400.00 g THF - - 5.00 2000.0 ml aq. LiOH 34.4 g/l 23.95 2871.5 1.2 1999.8 ml HCl 20% - - - x ml DCM - - 3x 5.0 3x 2000.0 ml 2.0 2.0 800.0 ml 800.0 ml 3x 10.0 3x 4000.0 ml DMF - - 7.0 1.5 2800.0 ml 600.0 ml 4-methylmorpholine 101.15 9332.4 3.90 943.97 g ethylchloroformate 108.52 3230.4 1.35 350.57 g RM-2-a 199.05 2991.1 1.25 595.39 g Water - - 11.0 2.5 2.5 4400.0 ml 1000.0 ml 1000.0 ml
Procedure
[0312] The starting compound IM-1-a may be prepared as described in Example 1 or 2.
[0313] The starting compound IM-1-a is charged in a reactor and filled with THF. The solution is cooled to 0 - 5° C. and a LiOH solution is dosed at IT ≤ 5° C. The solution is stirred at IT 0 - 5° C. ≥ 60 min.
IPC via HPLC
[0314] If IPC IM-1-a is < 0.2% a/a, the pH is adjusted to pH 0.5 - 1.0 at 15 - 20° C. with HCl 20%.
[0315] The mixture is extracted 3 x with DCM. The combined organic phases are dried over MgSO.sub.4, filtrated and the filter cake washed with DCM. DCM is evaporated at OT 32 - 37° C. / 400 mbar. 8.5 eq. of DMF are added. THF is evaporated at 32 - 37° C. / to 35 mbar. (End points of distillation when no further solvent is condensing.)
IPC via KF ≤ 0.2% Water
[0316] If IPC is out of spec, 10eq. of DCM are added and distilled off at 32 - 37° C. again followed by IPC KF.
[0317] 4-Methylmorpholine is added to the DMF solution at IT -5 to 0° C. Ethylchloroformate is added at IT = -5° C. to 0° C. RM-2-a (milled) is added at IT -5° C. to 0° C. The mixture is stirred for ≥ 5 h at IT -5 to 0° C.
IPC via HPLC
[0318] If IPC IM-3-a > 85% a/a, water is added slowly at IT < 10° C. The pH of the reaction mixture is adjusted to pH 6 - 8 with NaOH 30% if necessary. The product suspension is stirred ≥ 60 min at IT 0 - 5° C., filtered, washed twice with water and dried at 45° C. in vacuo.
Yield
[0319] Yield = 448.42 g = 75.6% [0320] HPLC Assay = 98.2% [0321] HPLC Purity = 99.7%
3C. Process for Preparing Intermediate Compound (IM-3-b) - Telescoped Synthesis via IM-2-a
Reaction
[0322] ##STR00053##
Process Variant 1
[0323] TABLE-US-00013 Chemicals Chemical Molar Mass Eq. Amount (th) Amount (is) q/mol mmol 1,2-dibromoethane 187.86 120.5 1.70 22.63 g lithium tert-butoxid 80.06 198.4 2.80 15.88 g THF - - - 70 ml 15 ml 15 ml ethyl 4-oxazolcarboxylate 141.12 70.9 1.00 10.00 g Pd(PPh.sub.3).sub.4 - - 2.7 % 2.24 g LiOH 23.95 106.3 1.50 2.55 g MTBE - - - 2x 70 ml HCl 20% - - - x ml x ml THF - - - 50 ml dichlormethane - - - 3 x 50 ml 3 x 4-methylmorpholine 101.15 269.3 3.80 27.24 g ethylchloroformate 108.52 88.6 1.25 9.61 g RM-2-a 199.05 88.6 1.25 17.63 g NaCl soln. 10% - - - 2x 100 ml 2 x
Procedure
[0324] The vessel is purged ≥ 15 min with N.sub.2. LitOBu is charged to the vessel, 70 ml THF are added and the mixture is stirred at IT 20 - 25° C. for ≥ 10 min.
[0325] The mixture is heated up to IT 53 - 57° C. and stirred for ≥ 5 min.
[0326] A solution of 1,2-dibromoethane in 15 ml THF is added at IT 55 - 60° C. (exothermic), the mixture is stirred for ≥ 30 min at IT 58 - 62° C.
[0327] The mixture is cooled to IT 43 - 47° C.
[0328] Pd(PPh.sub.3).sub.4 is added and stirred for ≥ 5 min.
[0329] The mixture is heated to IT 58 - 62° C., a solution of (ethyl 4-oxazolcarboxylate in 15 ml THF is added at IT 60 - 65° C.
[0330] The mixture is stirred for ≥ 30 min at IT 58 - 62° C.
IPC via HPLC for Information
[0331] no Ethyl 4-oxazolcarboxylate [0332] ethylester product 72% [0333] tert-butylester product 23% [0334] Intermediate product IM-1-a 5%
[0335] The mixture is cooled to IT 20 - 25° C.
[0336] A solution of LiOH in 35 ml water is added at IT 20 - 27° C. and the mixture is stirred at IT 23-27° C. for ≥ 16 h.
[0337] IPC via HPLC ≥ 93%
[0338] 35 ml water are added and the mixture extracted two times with 70 ml TBME.
[0339] 50 ml THF are added and the pH is adjusted at IT 15 - 20° C. to 0.5 - 1.0 with HCl 20%.
[0340] The mixture is extracted three times with 50 ml of DCM
[0341] The combined organic phases are dried over Na.sub.2SO.sub.4 (15 - 20 g), filtered and the filter cake washed with 10 ml DCM.
[0342] DCM phase = 0.51% water via Karl Fischer.
[0343] OT is set to -20° C.
[0344] 4-Methylmorpholine is added at IT -5 to 0° C.
[0345] Ethylchloroformate is added at IT -5 to 0° C.
[0346] RMa (grinded) is added in portions at IT ≤ 0° C. and the mixture is stirred at IT -5 to 0° C. for > 3 h.
[0347] IPC via HPLC ≥ 90%.
[0348] The mixture is extracted two times with 100 ml NaCl 10%.
[0349] The intermediate layer is kept with the organic phase.
[0350] The organic phase is extracted three times with 80 ml HCl 10%.
[0351] The aq. solution is filtered and the pH adjusted first to 2 - 5 with NaOH 30% and afterwards with NaOH 5% to pH 7 - 8. IT is kept ≤ 10° C. during pH addition.
[0352] The suspension is filtered and washed two times with 60 ml of water.
[0353] The product is dried at 45° C. / < 100 mbar to dryness.
Appearance
[0354] HPLC Purity: 99.0 %
TABLE-US-00014 Yield Theory Found Comments Yield Crude - Final Yield beige powder 17.52 g 12.36 g 70.60 % HPLC Purity = 99.8% Water = 0.04%
[0355] In experiments with a DCM water content of 0.04% by azeotroping the yield was even up to 85%.
Process Variant 2
TABLE-US-00015 Chemicals Chemical Molar Mass Eq. Theory g/mol mmol lithium-t-butoxid (fBuOLi) 80.06 99.2 2.80 7.94 g THF - - 7.00 35.0 ml 1,2-dibromomethane (DBE) 187.86 60.2 1.70 11.32 g THF - - 1.50 7.5 ml Pd(PPh.sub.3).sub.4 (catalyst) 1155.56 1.0 0.027 1.11 g ethyl 4-oxazolcarboxylate 141.12 35.4 1.00 5.00 g THF - - 1.50 7.5 ml LiOH 23.95 42.5 1.20 1.02 g water - - 3.5 17.5 ml 17.5 87.5 ml MTBE - - 2x 7.00 2x 35.0ml THF - - 10.00 50.0ml HCl 20% - - - x ml dichloromethane (DCM) - - 14.0 70.0 ml 7.0 35.0 ml 7.0 35.0 ml 3.0 15.0 ml 3.0 15.0 ml x 10.0 x 50.0 ml DMF - - 8.5 42.5 ml 1.5 7.5 ml 4-methylmorpholine 101.15 138.2 3.90 13.98 g ethylchloroformate (ECF) 108.52 47.8 1.35 5.19 g RM-2-a 199.05 44.3 1.25 8.82 g THF - - 1.95 9.8 ml water - - 13.0 65.0 ml 3.0 15.0 ml 3.0 15.0 ml
Procedure
[0356] A reactor is purged with nitrogen for 15 min, the condensator is cooled to -30C°.
[0357] tBuOLi is charged to the reactor and THF is added. The mixture is heated to Tl = 55° C. (TM = 58° C.) and stirred for 5 min. A solution of 1,2-dibromomethane in THF is added at Tl ≤ 60° C. The mixture is stirred at TI = 60° C. (TM = 63° C.) for 60 min. The mixture is cooled to Tl = 45° C.
[0358] Pd(PPh3)4 is added. The mixture is heated to TI = 60° C. (TM = 63° C.).
[0359] A solution of ethyl 4-oxazolcarboxylate in THF is added at Tl ≤ 65° C. The mixture is stirred for 30 min at Tl = 60° C. (TM = 63° C.). The mixture is cooled to Tl = 20 - 25° C. (TM = 20° C.).
[0360] IPC via HPLC-MS.
[0361] A solution of LiOH in water is added at Tl ≤ 25° C. The mixture is stirred at Tl = 25° C. for ≥ 16 h.
[0362] IPC via HPLC-MS
[0363] Water is added and the mixture is extracted 2x with MTBE. The MTBE phases are discarded. THF is added and the pH adjusted to 0.5 - 1.0 at 15° C.-20° C. with HCl 20%. The mixture is extracted 3x with DCM. The combined organic extracts are dried over MgSO.sub.4, filtered and the filter cake washed with DCM. DCM is evaporated at 35° C./400 mbar. 8.5 EQ DMF are added and THF evaporated at 35° C./ to 35 mbar. End points of distillation are when no further solvent is condensing.
[0364] IPC via KF ≤ 0.2% water, otherwise additional azeotroping.
[0365] For further azeotroping, 10 Eq DCM are added and evaporated at 35° C./400 mbar. IPC via KF is repeated.
[0366] This procedure is repeated till IPC via KF is in spec. 4-Methylmorpholine is added to DMF solution at Tl = -5° C. - 0° C. Ethylchloroformate is added at Tl = -5° C. - 0° C. RM-2-a (milled) is added at Tl = -5° C. - 0° C. The mixture is stirred ≥ 5 h bei Tl = -3° C.
[0367] IPC via HPLC-MS
[0368] If IPC IM2 > 85% a/a, water is added at IT < 10° C.
[0369] The pH of reaction mixture is adjusted to pH 6 - 8 with NaOH 30% if necessary. The product suspension is stirred ≥ 60 min at IT 0 - 5° C., filtered, washed twice with water and dried at 45° C. in vacuo.
Yield
[0370] Yield = 5.5 g = 62.4% [0371] HPLC Assay = 99.6% m/m [0372] HPLC Purity = 99.7% a/a
4.Process for Preparing the Compound (II) in the Form of a 3HCl-Salt - Telescoped Synthesis with Solvent Exchange
Process Variant 1 (Solvent Extraction)
[0373] TABLE-US-00016 Chemicals Chemical Molar Mass Eq. Mass th. Mass is g/mol mmol RM-3-a 161.20 486.4 1.2 78.4 g 78.4 g water - - - 6500 ml 6500 ml NaOH 30% 39.99 40.5 0.1 5.4 g 5.4 g IM-3-b 247.23 404.5 1.0 100.00 g 100 g DCM 4x 3200 ml 4x 3200 ml EtOAc 3x 3200 ml 3x 3200 ml ethanol 1x 3200 ml 2x 3200 ml 1x5600 ml 1x4600 ml HCl 32% 36.46 100.4 (2.5) 114.4 g 114.4 g
Reaction
[0374] ##STR00054##
Procedure
[0375] The intermediate compound IM-3-b may be prepared as described in Example 3.
[0376] Compound RM-3-a (grinded) is charged to the reactor and suspended in water at 20 - 25° C. NaOH 30% is dosed and the suspension stirred at 20 - 25° C. until a solution is formed (approx 15 min).
[0377] Intermediate compound IM-3-b (grinded) is added and the mixture heated to 60 - 65° C. for 72 h. IPC via LC/MS conversion ≥ 93% area.
[0378] The mixture is cooled to 20 - 25° C. and DCM is added.
[0379] The pH is adjusted to 3.9 - 4.1 with HCl 10%.
[0380] The mixture is stirred for 5 min and the phases are rested for 1h. The lower phase is discarded. The DCM extraction is repeated three times.
[0381] The aq. phase is adjusted to pH 9.9 - 10.1 with NaOH 15% and EtOAc is added.
[0382] The mixture is stirred for 5 min and the phases are rested for 1 h. The lower phase is discarded.
[0383] The EtOAc extraction is repeated two times.
[0384] The organic phases are combined (volume = 8.5 I) and are concentrated under vacuum / OT 40° C. to a volume of 1.7 l (⅕ of volume).
[0385] 3.2 l EtOH is added and the solution is concentrated under vacuum / OT 40° C. to a volume of 1.7 l (½ of volume).
[0386] 3.2 l EtOH is added and the solution is concentrated under vacuum / OT 40° C. to a volume of 1.05 | (⅓ of volume).
[0387] 4.55 | EtOH (5.6 l - 1.05 l) is added and the solution is filtered and heated to 55 - 60° C.
[0388] HCl 32% is dosed within ≥ 20 min at 55 - 60° C. and the suspension is cooled slowly to 0 - 5° C. within ≥ 3 h.
[0389] The suspension is stirred at 0 - 5° C. ≥ 1 h and filtered.
[0390] The filtered cake is washed with 0.8 l EtOH and dried at 45° C. / vacuum < 50 mbar to dryness.
TABLE-US-00017 Yield Theory Found Comments Yield Crude - Yield final Product (white to off-white powder) 209.4 g 142.3 g 68% uncorr / 65 % corr HPLC Assay: 75.8% m/m free base, 96.2% m/m salt HPLC Purity: 99.2% area The HPLC purity profile is shown in
Process Variant 2 (Oil Separation)
Reaction
[0391] ##STR00055##
TABLE-US-00018 Chemicals Chemical Molar Mass Eq. Mass th. g/mol mmol RM-3-a 161.20 40.4 1.0 6.52 g water - - - 100 ml NaOH 30% 39.99 0.4 0.01 54 mg IM-3-b 247.23 40.4 1.0 10.00 g EtOH 99% - - 560 ml HCl 32% 36.46 100.0 (2.5) 11.51 g
Procedure
[0392] RMa is charged to a reactor and suspended in water. NaOH 30% is added. IM-3-b is added and the mixture heated to IT 58 - 62° C. for ≥ 72 h. IPC via LC/MS product ≥ 75% area (all compounds integrated).
[0393] The mixture is cooled to 5 - 25° C. and allowed to rest for ≥ 16 h. The bottom oil phase is separated by drain off the reactor, decanting or suction off the water phase by vacuum. The separated oil phase is dissolved in 560 ml EtOH. The solution is filtered and heated to IT 60 -65° C. 0.7 mg seeding crystals are added. HCl 32% is dosed within 20 min at IT 60 - 65° C. with a stirrer speed of 100 rpm. After HCl addition the stirrer speed is lowered to 60 rpm and the suspension is cooled slowly to IT 0 - 5° C. within ≥ 4 h. The suspension is stirred at 0 - 5° C. ≥ 1 h and filtered. The filter cake is washed with 105 ml EtOH and dried at 45° C./vacuum < 50 mbar to dryness.
TABLE-US-00019 Yield Theory Found Comments Yield Crude - Yield final Product 20.93 g 13.7 g 65.40% HPLC Assay: 77.4% m/m free base, 98.3% m/m salt Corr. Yield = 64.3% HPLC Purity: 99.7% area The HPLC purity profile is shown in
Preparation of Polymorph PM2 of the Compound (II) in the Form of a 3HCl-Salt
[0394] The polymorph form PM2 is obtained by hot recrystallization of a solution of the compound (II) as the 3HCl salt in the form of polymorph PM1 as obtained in Example 4 described above.
[0395] The hot recrystallization was carried out in a solvent mixture of toluene : methanol in a ratio of 1:1.
[0396] The .sup.1H NMR spectrum of polymorph PM2 is essentially unchanged compared to that of polymorph PM1 but shows a sharp singlet at ~δ3.16 ppm assigned to methanol that suggests a methanol content of ~2 mole percent.
[0397] Elemental analysis of polymorph PM2 to determine the amount of chloride provided an average value of 20.0% (m/m), which is in good accordance with the expected value of 20.5% for a 3:1 HCl:free base salt. [0398] DSC: 226° C. [0399] PXRD analysis: polymorph form PM2 (PXRD pattern according to
Preparation of Polymorph PM3 of the Compound (II) in the Form of a 3HCl-Salt
[0400] The polymorph form PM3 is obtained by preparing a saturated solution of the compound (II) as the 3HCl salt in the form of polymorph PM1 as obtained in Example 4 described above in a solvent mixture of acetone : water in a ratio of 9:1 (v/v) at 50° C., cooling to 5° C., then precipitating a white solid by adding acetone.
[0401] The .sup.1H NMR spectrum of polymorph PM3 exhibits only minor differences compared to that of polymorph PM1 with a slight shift in the broad resonances.
[0402] Elemental analysis of polymorph PM3 to determine the amount of chloride provided an average value of 19.3% (m/m), which is in good accordance with the expected value of 20.5% for a 3:1 HCl:free base salt. [0403] DSC: 169° C. [0404] PXRD analysis: polymorph form PM3 (PXRD pattern according to
5. Process for Preparing the Compound (II) in the Form of the HCl Salt (Mono-Salt) -Telescoped Synthesis
[0405] TABLE-US-00020 Chemicals Chemical Molar Mass Eq. amount th. amount is g/mol mmol RM-3-a 161.20 97.3 1.2 15.68 g water - - - 1300 ml NaOH 30% 39.99 8.2 0.1 1.08 g IM-3-b 247.23 81.0 1.0 20.00 g HCl 20 % x ml DCM - - 650 ml ethanol 160 ml
Reaction
##STR00056##
Procedure
[0406] The intermediate compound IM-3-b may be prepared as described in Example 3.
[0407] Compound RM-3-a (grinded) is suspended in water at 20 - 25° C. and NaOH 30% is added.
[0408] The suspension is stirred for ≥ 15 min at 20 - 25° C. to form a solution.
[0409] Intermediate compound IM-3-b (grinded) is added and the suspension heated to 60 - 65° C. for ≥ 72 h.
[0410] IPC control via HPLC ≥ 93% conversion.
[0411] The emulsion is cooled to 20 - 25° C. and 650 ml DCM are added.
[0412] The mixture is stirred for ≥ 10 min and the phases are rested for ≥ 1 h. The water phase is discarded.
[0413] 650 ml water are added to the DCM phase and the pH is adjusted to 5.4 - 5.6 with HCl 20%. The mixture is stirred vigorous for ≥ 10 min and the phases are rested for ≥ 1 h. The DCM phase is discarded.
[0414] The water phase is stirred for ≥ 16 h and the resulting suspension is filtered.
[0415] The wet cake is washed with 80 ml of EtOH.
[0416] The filtered cake is dried at 50° C. / < 100 mbar to dryness.
TABLE-US-00021 Yield Theory Found Comments Yield Crude Product - Yield final Product 36.0 g 21.0 g 59.00% HPLC Assay: 88.7% m/m free base, 96.6% m/m Corr. Yield = 57% HPLC Purity = 98.4% area DSC = 173° C.
6. Process for Preparing the Compound (II) in the Form of the H.SUB.2.SO.SUB.4 Salt - Telescoped Synthesis
[0417] TABLE-US-00022 Chemicals Chemical Molar Mass Eq. amount th. amount is g/mol mmol RM-3-a 161.20 97.3 1.2 15.68 g water - - - 1300 ml NaOH 30% 39.99 8.2 0.1 1.08 g IM-3-b 247.23 81.0 1.0 20.00 g H.sub.2SO.sub.4 20% x ml DCM - - 640 ml ethanol 160 ml
Reaction
[0418] ##STR00057##
Procedure
[0419] The intermediate compound IM-3-b may be prepared as described in Example 3.
[0420] Compound RM-3-a (grinded) is suspended in water at 20 - 25° C. 30% NaOH is added and the mixture stirred ≥ 15 min at 20 - 25° C.
[0421] Intermediate compound IM-3-b (grinded) is added and the mixture heated to 60 - 65° C. for ≥ 72 h.
[0422] IPC conversion via HPLC ≥ 93%.
[0423] The mixture is cooled to 20 - 25° C.
[0424] 640 ml DCM are added, stirred and the phases are rested for ≥ 1 h. The water phase is discarded.
[0425] 640 ml water are added to the DCM phase and the pH adjusted to 3.4 - 3.6 with 20% H.sub.2SO.sub.4. The phases are rested for ≥ 1 h and the DCM phase is discarded.
[0426] The water phase is stirred and cooled to 0 - 5° C. within ≥ 2 h.
[0427] The suspension is heated to 40° C. and stirred for ≥ 16 h at this temperature. The suspension is cooled to 0 - 5° C. within ≥ 4 h and stirred at 0 - 5° C. for ≥ 1 h.
[0428] The suspension is filtered and the filtered cake washed with 160 ml ethanol.
[0429] The filtered cake is dried at 50° C./< 100 mbar vacuum to dryness.
TABLE-US-00023 Yield Theory Found Comments Yield Crude - Yield final Product (white to off-white powder) 24.6 g 60 % uncorr. HPLC Assay: 76.4% m/m free base, 94.8% m/m 1:1 salt Corr. Yield = 57% HPLC Purity: 98.3% area H.sub.2O/KF = 2.3% DSC: 176° C.
7. Process for Preparing the Compound (II) in the Form of the 0.5 H.SUB.3.PO.SUB.4 Salt -Telescoped Synthesis
[0430] TABLE-US-00024 Chemicals Chemical Molmasse Eq. Amount (th) Amount (is) g/mol mmol RM-3-a 161.2 48.64 1.2 7.84 g water - - - 650 ml NaOH 30% 39.99 4.1 0.1 0.54 g IM-3-b 247.23 40.5 1.0 10.00 g DCM 4x320 ml ethylacetate 3x320 ml ethanol 320, 110, 40 ml H.sub.3PO.sub.4 30% 5 x 6.5 ml
Reaction
##STR00058##
Procedure
[0431] The intermediate compound IM-3-b may be prepared as described in Example 3.
[0432] Compound RM-3-a (grinded) is suspended in water at 20 - 25° C. and NaOH 30% is added.
[0433] The suspension is stirred for ≥ 15 min at 20 - 25° C. to form a solution. Intermediate compound IM-3-b (grinded) is added and the suspension heated to 60 - 65° C. for ≥ 72 h.
[0434] IPC control via HPLC ≥ 93% conversion.
[0435] The emulsion is cooled to 20 - 25° C. and 320ml DCM are added.
[0436] The pH is adjusted to 3.9 - 4.1 with HCl 20%.
[0437] The mixture is stirred vigorous for ≥ 10 min and the phases rested for ≥ 1 h. The organic phase is discarded.
[0438] This DCM extraction is repeated further 3 times.
[0439] The water phase is adjusted to pH 9.9 - 10.1 with NaOH 30%.
[0440] 320 ml EtOAc are added and the mixture is stirred vigorous for ≥ 10 min. The phases are rested for ≥ 1 h. The water phase is discarded.
[0441] This EtOAc is extraction is repeated further 2 times.
[0442] The combined organic phases are concentrated in vacuum at 40° C. to 13 ml.
[0443] 320 ml ethanol are added and the solution is concentrated to a volume of 18 ml.
[0444] 110 ml of ethanol are added and 5 x 6.5 ml H.sub.3PO.sub.4 30% are added at 20 - 25° C. The mixture is stirred ≥ 24 h at 28 - 32° C. The suspension is cooled to 20 - 25° C. within ≥ 1 h and filtered. The filtered cake is washed with 40 ml of EtOH.
[0445] The filtered cake is dried at 45° C. / < 100 mbar to dryness.
TABLE-US-00025 Yield Theory Found Comments Yield Crude - Yield Final Product 18.5 g 8.8 g 48.00 % HPLC Assay: 85.2% m/m free base, 95.4% m/m 2:1 salt Corr. Yield = 45.8% HPLC Purity = 99.5% area P = 3.3% DSC = 253° C.
8. Process for Preparing the Compound (II) in the Form of the H.SUB.3.PO.SUB.4 Salt -Transformation to the 1:1 Salt
[0446] TABLE-US-00026 Chemicals Chemical Molmasse Eq. Amount (th) Amount (is) g/mol mmol Compound (II) 0.5 H.sub.3PO.sub.4 salt 914.85 13.1 12 g ethanol - - - 180 ml
Reaction
##STR00059##
Procedure
[0447] The 0.5 H.sub.3PO.sub.4 salt of compound (II) can be prepared as described in Example 7.
[0448] The compound (II) 0.5 H.sub.3PO.sub.4 salt is suspended in ethanol at 20 - 25° C. and stirred for 4 days at this temperature.
[0449] The suspension is filtered and the wet cake dried at 45° C. / < 100 mbar to dryness.
TABLE-US-00027 Yield Theory Found Comments Yield Crude Product - Yield Final Product 13.3 g 6.6 g 50.00% Yield: 6.6 g = 50% with regard to free base HPLC Assay: 78.3% m/m free base, 97% m/m 1:1 salt Corr. Yield = 48.5% HPLC Purity: 98.7% area P = 5.7% DSC: 182° C.
9A. Process for Preparing an Intermediate Compound (IM-3-b′)
[0450] TABLE-US-00028 Chemicals Chemical Molar Mass Eq. Quantiy (th.) Quantiy (eff.) q/mol mmol IM-2-a (2-Vinyloxazole-4 carboxylic acid) 139.11 15.4 1.00 2.14 g 2.15 g Dichlormethane - - - 40 ml 40 ml 4-Methylmorpholine 101.15 58.5 3.80 5.91 g 5.89 g Ethylchloroformate 108.52 19.2 1.25 2.09 g 2.08 g RM-2-a′ (2-(Aminomethyl)-3-bromopyridine) 259.96 19.2 1.25 5.00 g 5.03 g NaCl liquid 10% - - - 3 x 40 ml 3 x 40 ml
Reaction
[0451] ##STR00060##
Procedure
[0452] The reaction is carried out under an N.sub.2 flow.
[0453] 2-vinyloxazole-4-carboxylate (IM-2-a) is suspended in dichloromethane and cooled to -5° C. 4-Methylmorpholine is added dropwise in such a way that the temperature did not exceed 0° C.
[0454] (exothermic).
[0455] Ethyl chloroformate is added dropwise in such a way that the temperature did not exceed 0° C.
[0456] (exothermic).
[0457] After a stirring time of 20 min, 2-(aminomethyl)-3-bromopyridine (RM-2-a′) is added in such a way that the temperature did not exceed 0° C.
[0458] The mixture is stirred overnight at 0° C. - 5° C.
[0459] The mixture is washed three times with 10% NaCl solution.
[0460] The organic phases are concentrated at 45° C. on a rotavap.
[0461] EtOAc / Heptane 20:80 (1.7 ml / g) is added to the crude product, stirred at RT for 4 h, filtered off, washed twice with EtOAc/Hepatne and dried at 50° C. to constant weight.
TABLE-US-00029 Yield Theory Found Comments Yield 4.76 g 4.50 g 94.54 % 4.5 g of beige solid observed = 94.5% yield of th. .sup.1H-NMR corresponds to structure observed = 94.5% yield of th.
9B. Process for Preparing the Compound (II′)
[0462] TABLE-US-00030 Chemicals Chemical Molar Mass Eq. Quantiy (th.) Quantiy (eff.) g/mol mmol RM-3-a (Benzimidazolethylamine) 161.20 16.7 1.2 2.70 g 2.72 g Water - - - 225.00 225 ml NaOH solution 30% 39.99 1.4 0.1 0.19 g 0.25 g IM-3-b′ 308.14 14.0 1.0 4.30 g 4.31 g Dichlormethane - - - 4x 110 ml 4 x 110 ml Ethylacetate - - - 3x 110 ml 3 x 110 ml
Reaction
[0463] ##STR00061##
Procedure
[0464] Benzimidazole-ethylamine (RM-3) is grounded in a mortar and suspended in water.
[0465] 30% sodium hydroxide solution are added dropwise and stirred for 10 min at RT.
[0466] The intermediate Compound IM-3-b′ is added to the mixture and stirred at an internal temperature of 63° C. for 72 hours.
[0467] The mixture is cooled to RT.
[0468] 110ml DCM are added and stirred for 10 minutes.
[0469] The pH value is set to 4 with HCl 20%.
[0470] The water phase is extracted 4 times with 110 ml DCM and rested for 1 hour for phase separation, DCM phases are discarded.
[0471] The water phase is adjusted to pH 10 with NaOH 30%, extracted 3 times with EtOAc and rested for 1 hour for phase separation, the water phases are discarded.
[0472] The EtOAc phases are dried with magnesium sulfate, filtered and concentrated on a Rotavap to dryness.
TABLE-US-00031 Yield Theory Found Comments Yield 6.56 g 4.18 g 63.72% 4.18 g of an brown oil are observed = 63.7% yield of th. .sup.1H-NMR corresponds to structure
9C. Process for Preparing the 3HCl-Salt of Compound (II′)
[0473] TABLE-US-00032 Chemicals Chemical Molar Mass Eq. Quantiy (th.) Quantiy (eff.) g/mol mmol Compound (ll′) 469.34 8.9 1.0 4.16 g 4.16 g Ethanol - - - 168 ml 170 ml HCl 32% 36.46 27.9 3.15 3.18 g 3.23 g Ethanol - - - 40 ml 40 ml
Reaction
[0474] ##STR00062##
##STR00063##
Procedure
[0475] Compound (II′) is dissolved in ethanol and heated to an internal temperature of 40° C.
[0476] HCl 32% is added dropwise.
[0477] The suspension is cooled to 0° C. slowly.
[0478] The suspension is stirred at 0° C. for 1 h.
[0479] The suspension is filtered, the wet cake washed with ethanol and dried at 40° C. to dryness.
TABLE-US-00033 Yield Theory Found Comments Yield 5.13 g 4.02 g 78.36 % 4.02 g of a white solid observed = 78.4% yield of th. .sup.1H-NMR corresponds to structure
10. .SUP.1.H-NMR, PXRD and DSC Analysis of Intermediate and Example Compounds
10.1 NMR-Analysis
[0480] NMR analysis of Intermediate Compounds IM-1-a, IM-2-a and IM-3-b, which have been prepared with the process as described in Examples 1b, 1a and 3a, respectively, have been carried out providing the following NMR-data.
TABLE-US-00034 IM-1-a according to Example 1b .sup.1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.24 (s, 1 H) 6.64 (dd, J=17.6, 11.3 Hz, 1 H) 6.29 (dd, J=17.7, 0.8 Hz, 1 H) 5.76 (dd, J=11.4, 0.8 Hz, 1 H) 4.40 (q, J=7.3 Hz, 2 H) 1.39 (t, J=7.2 Hz, 3 H) IM-1-a according to Example 1c .sup.1H NMR (400 MHz, CDCl3): δ 8.14 (s, 1H), 6.63 - 6.56 (m, 1H), 6.27 - 6.22 (m, 1H), 5.71 - 5.68 (m, 1H), 4.36 (q, J = 6.8 Hz, 2H), 1.35 (t, J = 7.20 Hz, 3H) IM-2-a .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.73 (s, 2 H) 8.46 (s, 1 H, internal standard=1,2,4,5-Tetrachloro-3-nitrobenzene) 6.71 (dd, J=17.5, 11.2 Hz, 1 H) 6.22 (dd, J=17.7, 1.0 Hz, 2 H) 5.82 (dd, J=11.2, 0.8 Hz, 2 H) IM-3-b .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.57 - 8.67 (m, 2 H) 8.45 (s, 1 H) 8.41 (dt, J=4.7, 1.3 Hz, 1 H) 7.71 (ddd, J=10.2, 8.6, 1.3 Hz, 1 H) 7.42 (dt, J=8.5, 4.4 Hz, 1 H) 6.72 (dd, J=17.6, 11.3 Hz, 1 H) 6.24 (dd, J=17.7, 0.8 Hz, 1 H) 5.83 (dd, J=11.4, 0.8 Hz, 1 H) 4.67 (dd, J=5.6, 1.5 Hz, 2 H)
[0481] NMR analysis of Example Compounds (II) in the form of the 3HCl salt (polymorph form PM1, PM2 and PM2), 1HCl salt, H.sub.2SO.sub.4 salt, 0.5H.sub.3PO.sub.4 salt and 1H.sub.3PO.sub.4 salt, which have been prepared with the process as described in Examples 4, 5, 6, 7 and 8, respectively, have been carried out providing the following NMR-data.
TABLE-US-00035 Example 4 PM1 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.86 (t, J=5.7 Hz, 1 H) 8.61 (s, 1 H) 8.45 (d, J=4.8 Hz, 1 H) 7.86 (ddd, J=9.8, 8.6, 1.1 Hz, 1 H) 7.75 - 7.82 (m, 2 H) 7.44 - 7.61 (m, 3 H) 4.66 (br d, J=5.1 Hz, 2 H) 3.77 (s, 4 H) 3.49 (br t, J=6.7 Hz, 2 H) 3.38 (br t, J=7.1 Hz, 2 H) 3.01 (s, 35 H; internal standard=dimethylsulfone) Example 5 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.66 (t, J=5.7 Hz, 1 H) 8.63 (s, 1 H) 8.37 (dt, J=4.7, 1.3 Hz, 1 H) 7.70 (ddd, J=10.2, 8.6, 1.3 Hz, 1 H) 7.46 - 7.62 (m, 2 H) 7.40 (dt, J=8.6, 4.3 Hz, 1 H) 7.01 - 7.28 (m, 2 H) 4.62 (dd, J=5.8, 1.3 Hz, 2 H) 3.08 - 3.67 (m, 12 H) 1.06 (t, J=7.1 Hz, 1 H) Example 6 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 10.00 (br s, 1 H) 8.64 (s, 1 H) 8.58 (t, J=5.7 Hz, 1 H) 8.36 (dt, J=4.6, 1.5 Hz, 1 H) 7.70 (ddd, J=10.2, 8.6, 1.3 Hz, 1 H) 7.46 - 7.62 (m, 2 H) 7.41 (dt, J=8.5, 4.4 Hz, 1 H) 7.06 - 7.24 (m, 2 H) 4.62 (dd, J=5.7, 1.4 Hz, 2 H) 3.39 - 3.70 (m, 4 H) 3.27 (dt, J=10.3, 7.0 Hz, 4 H) Example 7 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.58 (t, J=5.7 Hz, 1 H) 8.53 (s, 1 H) 8.37 (dt, J=4.8, 1.4 Hz, 1 H) 7.70 (ddd, J=10.0, 8.5, 1.3 Hz, 1 H) 7.43 - 7.56 (m, 2 H) 7.33 - 7.43 (m, 1 H) 7.05 - 7.18 (m, 2 H) 4.61 (dd, J=5.6, 1.3 Hz, 2 H) 2.98 - 3.25 (m, 8 H) Example 8 .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ ppm 8.59 (br t, J=5.7 Hz, 1 H) 8.55 (s, 1 H) 8.37 (dt, J=4.7, 1.3 Hz, 1 H) 7.69 (ddd, J=10.0, 8.5, 1.3 Hz, 1 H) 7.43 - 7.56 (m, 2 H) 7.40 (dt, J=8.5, 4.4 Hz, 1 H) 7.00 - 7.20 (m, 2 H) 4.61 (dd, J=5.6, 1.0 Hz, 2 H) 3.05 - 3.35 (m, 8 H)
[0482] NMR analysis of Intermediate Compound IM-3-b′, Compound (II′) and the 3HCl salt of Compound (II′), which have been prepared with the process as described in Examples 9a, 9b and 9c, respectively, have been carried out providing the following NMR-data.
TABLE-US-00036 IM-3-b′ according to Example 9a .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ = 8.66 (s, 1H), 8.63 - 8.48 (m, 2H), 8.11 (dd, J = 1.5, 8.1 Hz, 1H), 7.31 (dd, J = 4.7, 8.0 Hz, 1H), 6.74 (dd, J = 11.3, 17.6 Hz, 1H), 6.24 (dd, J = 0.8, 17.7 Hz, 1H), 5.84 (dd, J = 0.8, 11.2 Hz, 1H), 4.63 (d, J = 5.3 Hz, 2H) (II′) according to Example 9b .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ = 8.56 (dd, J= 1.4, 4.7 Hz, 2H), 8.54 (s, 2H), 8.51 (t, J = 5.5 Hz, 2H), 8.45 (s, 3H), 8.09 (dd, J = 1.4, 8.0 Hz, 2H), 7.50 - 7.41 (m, 4H), 7.30 (dd, J = 4.7, 8.0 Hz, 2H), 7.14 - 7.06 (m, 4H), 4.62 (d, J = 5.3 Hz, 4H), 3.07 - 2.92 (m, 16H) (II′) 3HCl according to Example 9c .sup.1H NMR (400 MHz, DMSO-d.sub.6) δ = 16.16 - 14.70 (m, 1H), 10.56 - 9.46 (m, 2H), 8.71 (t, J = 5.7 Hz, 1H), 8.64 (s, 1H), 8.56 (dd, J = 1.4, 4.7 Hz, 1H), 8.12 (dd, J = 1.3, 8.1 Hz, 1H), 7.82 - 7.76 (m, 2H), 7.58 - 7.51 (m, 2H), 7.32 (dd, J = 4.7, 8.0 Hz, 1H), 4.60 (d, J = 5.6 Hz, 2H), 3.54 - 3.44 (m, 2H), 3.44 - 3.35 (m, 2H)
10.2 PXRD-Analysis
[0483] Further PXRD analysis of Example Compounds (II) in the form of the 3HCl salt (PM1, PM2 and PM3), H.sub.2SO.sub.4 salt and 1 H.sub.3PO.sub.4 salt, which have been prepared with the process as described in Examples 4, 6 and 8, respectively, have been carried out providing the PXRD spectra as shown in
Method
[0484] Sample preparation and measurement: [0485] Sample preparation: 10 to 20 mg sample was placed between two acetate foils in Stoe transmission sample holder; the sample was rotated during the measurement [0486] Stoe Stadi P (G.52.SYS.S072); Mythen1K Detector; Cu-Ka1 radiation; standard measurement conditions: transmission; 40 kV and 40 mA tube power; curved Ge monochromator; [0487] - 0.02° 2Theta step size, 48 s step time, 1.5-50.5° 2Theta scanning range; detector mode: step scan; 1° 2Theta detector step;
Data Evaluation
[0488] the d-value Analysis was performed with software EVA from Bruker, version 14, 0, 0, 0 [0489] background was subtracted [0490] only lines up to 35° 2theta were listed [0491] calculation of relative intensity with formula in Excel
TABLE-US-00037 PXRD Conditions -Compound (II) 3HCl salt (PM1, PM2 and PM3 of
TABLE-US-00038 Polymorph PM1 Angle 2-Theta ° d value Angstrom Intensity Relative intensity % 3.89 22.70 s 36.5 7.96 11.10 m 24.4 10.01 8.83 w 12 12.00 7.37 w 12.6 14.32 6.18 w 9.8 16.53 5.36 s 32.5 19.09 4.65 m 15 22.20 4.00 w 12 24.18 3.68 m 20.1 26.10 3.41 m 15.4 27.69 3.22 w 13.6
TABLE-US-00039 Polymorph PM2 Angle 2-Theta ° d value Angstrom Intensity Relative intensity % 16.96 5.23 vs 100 25.28 3.52 vs 96.5 11.73 7.54 vs 83.2 28.32 3.15 s 65.9 25.48 3.49 s 63.6 20.10 4.41 s 62 26.20 3.40 s 57.4 17.63 5.03 s 54 24.07 3.69 s 52.9 17.18 5.16 s 50.8 19.03 4.66 s 50.5 22.52 3.94 s 50.1 19.73 4.50 s 47.6 23.19 3.83 s 45.5 21.25 4.18 s 44.8 23.72 3.75 s 44.3 18.28 4.85 s 44 21.55 4.12 s 37.5 20.74 4.28 s 36.1 28.89 3.09 s 33.2 24.74 3.60 s 31.3 30.32 2.95 m 27.8 29.21 3.05 m 27 26.65 3.34 m 25.5 29.68 3.01 m 25.1 27.39 3.25 m 24.7 35.16 2.55 m 24.4 35.70 2.51 m 22.9 34.82 2.57 m 21.5 36.64 2.45 m 18.5 37.16 2.42 m 18.1 37.68 2.39 m 16.8 39.01 2.31 m 16.6 33.56 2.67 m 15.6 41.22 2.19 w 13.5 39.99 2.25 w 12.8
TABLE-US-00040 Polymorph PM3 Angle 2-Theta ° d value Angstrom Intensity Relative intensity % 14.75 6.00 vs 100 10.28 8.60 vs 93.9 16.97 5.22 vs 90.1 26.55 3.35 vs 75.2 18.05 4.91 vs 74.5 22.10 4.02 s 69.2 27.13 3.28 s 68.1 26.11 3.41 s 60.2 28.87 3.09 s 60.1 19.71 4.50 s 57.4 20.74 4.28 s 55.2 5.13 17.21 s 53.9 26.28 3.39 s 53.9 21.68 4.10 s 52.9 27.71 3.22 s 51.3 25.14 3.54 s 50.8 17.84 4.97 s 50.2 22.55 3.94 s 50.2 23.01 3.86 s 49.4 19.29 4.60 s 49 28.22 3.16 s 47.3 6.39 13.82 s 44.3 15.49 5.72 s 42.9 24.54 3.62 s 37.9 25.50 3.49 s 37.3 10.96 8.07 s 36.3 18.94 4.68 s 36.2 31.54 2.83 s 34.8 23.24 3.83 s 34.4 12.81 6.91 s 33.3 31.95 2.80 s 32.8 29.75 3.00 s 31.7 24.79 3.59 m 28.5 28.60 3.12 m 25.2 35.62 2.52 m 24.9 25.81 3.45 m 24.5 34.68 2.58 m 20.4 38.94 2.31 m 19.4 36.58 2.45 m 19.2 33.86 2.64 m 18.8 38.71 2.32 m 18.8 30.19 2.96 m 17.7 30.94 2.89 m 16.9 31.24 2.86 m 16.9 33.28 2.69 m 16.8 38.43 2.34 m 15.9 33.47 2.67 m 15.3 40.87 2.21 m 15.2 39.51 2.28 w 14.7 30.52 2.93 w 14.3 35.39 2.53 w 13.9 32.66 2.74 w 12.9 38.07 2.36 w 12.9 40.53 2.22 w 11.5 40.04 2.25 w 10.7
TABLE-US-00041 PXRD Conditions -Compound (II) H.sub.2SO.sub.4 salt (
TABLE-US-00042 PXRD Conditions -Compound (II) H.sub.3PO.sub.4 salt (
TABLE-US-00043 Angle / 2-Theta ° d value / Angstrom Intensity Relative intensity [%] 4.87 18.14 m 28.1 11.26 7.85 m 21.9 12.01 7.37 s 31 13.16 6.72 s 42.5 14.72 6.01 vs 74.3 15.49 5.72 vs 74.7 16.02 5.53 m 23.4 16.47 5.38 vs 83.7 16.97 5.22 s 43.7 17.44 5.08 s 66.4 18.42 4.81 vs 82.7 19.65 4.51 s 35.2 20.04 4.43 m 18.9 20.45 4.34 s 58.7 21.65 4.10 m 26.7 22.09 4.02 s 35.5 22.62 3.93 m 20.8 23.31 3.81 m 16 24.10 3.69 m 21.7 25.06 3.55 s 37.7 25.41 3.50 s 52.5 26.13 3.41 vs 100 26.89 3.31 s 41.6 27.42 3.25 w 14 27.89 3.20 w 11.1 28.66 3.11 m 25.9 29.09 3.07 m 19.6 29.66 3.01 w 9 30.62 2.92 w 8.1 30.95 2.89 w 9.9 31.28 2.86 w 8.8 31.57 2.83 w 10.7 33.32 2.69 w 11 33.77 2.65 w 8.4 34.35 2.61 w 7.8 35.00 2.56 w 12 35.52 2.53 w 13.5 36.81 2.44 w 9.7 37.32 2.41 w 6 38.79 2.32 w 6.2 39.14 2.30 w 6.4 40.89 2.21 w 7.2
10.3 DSC Measurement
[0492] Differential scanning calorimetry is performed in a sealed gold pan with a heating rate of 10° C./min.
10.4 HPLC Determination of Identity, Impurity Profiles and Purity Degree
[0493] Identity and assay of the compounds of the present invention base and its impurities are determined by a qualitative and quantitative analysis by high performance liquid chromatography (HPLC) with diode array detection using an in-house method based on Ph. Eur. 2.2.29.
[0494] Identity of the synthesized compounds is confirmed by comparison to the retention times and spectra of the corresponding reference substances. Assay is calculated based on an external calibration curve of the tested compound in the form of the base for both the salt form and its impurities.
Procedure
Apparatus
[0495] HPLC equipped with a pump, autosampler, column oven and diode array detector, and analytical balance (Category 1), pipettes (e.g. Gilson, Rainin), volumetric flasks (10, 25 and 50 ml), membrane filter (0.2 .Math.m)
Reagents
[0496] methanol (HPLC grade), Sigma (or equivalent) [0497] trifluoroacetic acid (TFA) for HPLC 100%, Sigma (or equivalent)
Mobile Phase
[0498] A: water / methanol (95+5 V/V; 0.1% TFA) [0499] B: methanol / water (95+5 V/V; 0.1% TFA)
Mobile Phase A Water / Methanol (95+5 V/V; 0.1% TFA)
[0500] Dilute 100 ml methanol to 2 liters with water R. Add 2.0 ml TFA then, mix thoroughly and degas in the ultrasonic bath.
Mobile Phase B Methanol / Water (95+5 V/V; 0.1 % TFA)
[0501] Dilute 50 ml water R to 1 liter with methanol. Add 1.0 ml TFA then, mix thoroughly and degas in the ultrasonic bath.
[0502] Stock solutions A and B (1000 mg/l): Prepare two stock solutions each of 1,000 mg/L of the compound to be tested, by dissolving 50 mg in 50 mL of mobile phase A.
[0503] Standard solutions A and B (40 mg/l): Dilute each of the stock solutions 1:25 with mobile phase A. Filter the samples through a 0.2 .Math.m filter into the HPLC glass vial.
SST Stock Solution
[0504] Weigh 10 mg of reference impurities, e.g. the intermediate compounds to be expected from the process, into a 10 ml volumetric flask, add 5 ml of methanol and dilute with water.
Impurity Solution C (1 Mg/l)
[0505] Pipette 100 .Math.l of stock solution A into a 100 ml volumetric flask and dilute with mobile phase A. Filter the samples through a 0.2 .Math.m filter into the HPLC glass vial.
Impurity Solution D/ SST Standard Solution (10 Mg/l)
[0506] Pipette 1000 .Math.l of stock solution B and 1000 .Math.l of SST stock solution into a 100 ml volumetric flask and dilute with mobile phase A. Filter the samples through a 0.2 .Math.m filter into the HPLC glass vial.
Chromatographic System
[0507] The liquid chromatograph is equipped with a column compartment maintained at a controlled temperature of 40° C. ± 5° C., a diode array detector (200 - 400 nm) and a 4.6 mm x 150 mm C18 column (e.g. Ascentis Express C18, 2.7 .Math.m). The flow rate is at 1.0 ml/min.
TABLE-US-00044 LC gradient time [min] A [%] B [%] -3.000 100 0 0.000 100 0 1.000 100 0 8.000 65 35 20.000 65 35 20.100 0 100 25.000 0 100
TABLE-US-00045 Run time: 20.0 min UV wavelength: 254 nm injection volume: 10 .Math.l for samples, variable volumes for standard solutions
System Suitability Testing (SST) and Quality Control Check (QC)
[0508] Adequate injections of SST standard solution prove the suitability of the chromatographic system if the RSD and retention times of the tested compound peak areas is ≤ 2%, respectively, resolution of the tested compound is ≥ 1.5 and the tailing factor of the tested compound peak is in the range 0.8 - 1.5.
[0509] Inject standard solution A six times at the beginning of the sequence (SST) and twice at the end of sequence (QC). The sequence may only be used if the requirements above are fulfilled.
Standard Curve
[0510] The standard curve for the tested compound (assay) may be obtained by e.g., injecting 5.0 .Math.L, 7.5 .Math.L and 10.0 .Math.L of standard solution A and 12.5 .Math.L and 15.0 .Math.L of standard solution B.
[0511] The standard curve for the tested compound impurities may be obtained by e.g., injecting 5.0 .Math.L and 10.0 .Math.L of impurity solution C and 5.0 .Math.l, 10.0 .Math.L and 15.0 .Math.L of impurity solution D.
[0512] The regression coefficient for both standard curves should be ≥ 0.9990.
Sample Preparation
Procedure - Impurities
[0513] Weigh accurately approximately 50 mg of the compound to be tested in its salt form into a 50-ml-flask and make up to the mark with mobile phase A. Prepare the sample solution twice. Filter the solution through a 0.2 .Math.m membrane filter and inject 10 .Math.l.
Procedure - Assay
[0514] Use both solutions prepared for ‘Impurities’ and dilute 1:25 with mobile phase A. Filter the solution through a 0.2 .Math.m membrane filter and inject 10 .Math.l.
Calculation
Qualitative Determination
[0515] The retention time of the tested compound should not deviate more than 0.3 min from the peak of standard A solution. The spectrum obtained for the tested compound in the sample correlates with the reference spectrum of the tested compound in the standard A solution. Determine the library match factor. The latter should be ≥ 990.
[0516] The retention time of any impurity of the tested compound should not deviate more than 0.1 min from the corresponding reference substance. The spectrum obtained for the impurity in the sample correlates with the reference spectrum of the corresponding impurity in the SST solution. Determine the library match factor. The latter should be ≥ 990.
Quantitative Determination - Assay
[0517] Plot the concentrations of the standard solutions expressed in amount of the tested compound in the form of its base (.Math.g) versus the peak area and extract the intercept (a) and the slope (b). The correlation coefficient obtained should not be less than 0.999. Calculate the amount of the tested compound base using the below formula:
TABLE-US-00046 y peak area (tested compound) of the sample a intercept of the standard curve b slope of the standard curve (area/.Math.g) V.sub.1 dilution (e.g., 50 mL) V.sub.2 dilution factor volume of the sample (e.g., 1.0 ml/25 ml = 25) V.sub.3 injection volume (e.g. 10 .Math.l) E amount of sample weighed (mg) 100 factor to calculate % [m/m]
Quantitative Determination - Impurities
Quantitative Determination of Impurities as %area
[0518] All peaks in the sample chromatogram at 254 nm are integrated. Peaks related to the blank are not considered. The percentage of total impurities and each single impurity ≥ 0.05% area is determined and reported.
Quantitative Determination of Impurities as % M/m
[0519] The concentrations of the standard solutions expressed in amount of the tested compound in the form of its base (.Math.g) are plotted versus the peak area and the intercept (a) and the slope (b) are extracted. The correlation coefficient obtained should not be less than 0.999. The amount of each impurity is calculated taking into consideration the response factor using the below formula:
TABLE-US-00047 y peak area (tested compound) of the sample a intercept of the standard curve b slope of the standard curve (area/.Math.g) V.sub.1 dilution (e.g., 50 mL) V.sub.2 injection volume (e.g. 10 .Math.l) E amount of sample weighed (mg) 100 factor to calculate % [m/m] RF Response Factor of each impurity vs. the tested compound
[0520] The above method is used to determine identity and assay the Compound (II) in the form a 3HCI salt (polymorph PM1) prepared with the process according to Example 4 (Process Variant 1 and 2).
TABLE-US-00048 Abbreviation Name Sum formula/ Molecular weight structure RM-2-a = RM2 = SP6 (3-fluoropyridin-2-yl)methanamine C.sub.6H.sub.7FN.sub.2 Mw:126.13 g/mol
TABLE-US-00049 Reference standards Information Storage Compound (II) 3HCI (VIT-2763) Content is based on free base with a content of 75.3% RT RM-2-a = RM2 = SP6 see response factor, 6.4 +2 - +8° C. RM-3-a = RM3 see response factor, 6.4 +2 - +8° C. IM-3-b =IM2 - RT SP1 see response factor, 6.4 RT SP3 see response factor, 6.4 RT SP4 see response factor, 6.4 RT SP5 see response factor, 6.4 RT SP7 see response factor, 6.4 RT
TABLE-US-00050 Impurity Response Factor RM-2-a = RM2 = SP6 1.140 RM-3-a = RM3 1.269 SP1 1.300 SP3 0.916 SP4 0.884 SP5 0.889 SP7 0.748
TABLE-US-00051 Results Substance Retention Time [min] RM-2-a = RM2 = SP6 2.30 RM-3-a = RM3 3.71 SP5 4.05 SP7 5.34 SP1 5.81 Compound (II) 3HC (VIT-2763) 8.33 IM-3-b =IM2 10.84 SP3 12.08 SP4 13.97
[0521]
TABLE-US-00052 No. Rel. RT [VIT-2763] Peak Name Height mAU Area mAU*min Name in Library Match in Library rel. Area % 1 0.48 RM3 0.593 0.05964 SP5 946.91 0.06 2 1.00 VIT-2763 950.122 104.71023 n.a. n.a. 99.75 3 1.27 unknown 0.593 0.08638 VIT-2763 809.42 0.08 4 1.48 SP3 0.640 0.11859 SP3 852.54 0.11
[0522]
TABLE-US-00053 No. Rel. RT [VIT-2763] Peak Name Height mAU Area mAU*min Name in Library Match in Library rel. Area % 1 0.70 SP1 1.287 0.06175 SP1 956.88 0.058 2 1.00 VIT-2763 1101.520 107.10545 n.a. n.a. 99.861 3 1.27 SP2 1.575 0.08782 VIT-2763 949.55 0.082
Comparative Example - Example Compound 127 According to WO2017068090A1
[0523]
TABLE-US-00054 No. Rel. RT [VIT-2763] Peak Name Height mAU Area mAU*min Name in Library Match in Library rel. Area % 1 0.27 RM2 0.649 0.05274 n.a. n.a. 0.05 2 0.48 RM3 0.714 0.06292 SP5 963.18 0.06 3 0.52 SP5 1.029 0.08730 SP5 984.98 0.08 4 0.59 unknown 8.853 0.65997 VIT-2763 948.21 0.63 5 0.65 SP7 3.709 0.29642 SP7 993.82 0.28 6 0.83 OH 7.957 0.59178 VIT-2763 942.23 0.57 7 0.94 unknown 1.012 0.07481 VIT-2763 914.21 0.07 8 1.00 VIT-2763 949.837 102.08108 VIT-2763 837.49 97.52 9 1.19 unknown 0.375 0.07308 n.a. n.a. 0.07 10 1.37 SP9 4.466 0.63416 SP9 0.50% 944.14 0.61 11 1.49 SP3 0.376 0.06125 n.a. n.a. 0.06
Conversion From Compound (II) Base to Compound (II) in 3HCI
[0524] VlT3HCl [% (m/m)] = VIT-2763 base [% (m/m)] x 517.81 [g/mol] / 408.44 [g/mol]
[0525] This corresponds to a conversion factor of 1.27.
11. Pharmacological Assays for Evaluation of the Activity of Compounds (II) and (II′)
[0526] The following table summarizes the activity of the compounds (II) and (II′) as ferroportin inhibitors compared to hepcidin:
TABLE-US-00055 Assay (II′) IC50 (nM) (II) IC50 (nM) Hepcidin IC50 (nM) n Hepcidin internalization (J774) 5.9 7.3 10.7 2 Biophysical Ferroportin-Hepcidin Binding (FP) 28 28 - 2 Iron response (HEK354 Blazer) 168 156 39 3 Fpn internalization and degradation (HEK354 FACS) 161 119 6 2
[0527] Compounds (II) and (II′) were tested in the form of their 3HCl-salts.
11.1 Hepcidin Internalization Assay (J774)
[0528] This cellular assay allows quantification of the binding of hepcidin to ferroportin (Fpn) through microscopic detection of internalization of a fluorescently labeled hepcidin into J774 cells. J774 is a mouse macrophage cell line which was shown to express Fpn endogenously upon incubation with iron (Knutson et al, 2005). Binding of hepcidin to Fpn triggers internalization and degradation of both hepcidin and Fpn. However, the TMR (6-carboxytetramethylrhodamine) fluorophore attached to hepcidin remains associated with the cell after degradation of the hepcidin peptide backbone. Therefore, microscopic detection of cell-associated TMR fluorescence is a measure of hepcidin binding to Fpn and internalization of hepcidin and Fpn. If TMR-hepcidin is prevented from binding to Fpn, cellular TMR fluorescence remains low (Dürrenberger et al, 2013). The effect of small molecular weight Fpn inhibitor compounds in this assay was evaluated in vitro as described below.
[0529] J774 cells, harvested from ca. 80% confluent cultures, were plated at 8x10.sup.5 cells/ml in complete medium (DMEM, 10% FBS, 1% Penicillin-Streptomycin) containing 200 .Math.M Fe(III)NTA (nitrilotriacetic acid), 100 .Math.l per well of 96 well MicroClear plates (Greiner; Cat. 655090) and grown at 37° C. with 5% CO.sub.2. After overnight incubation, cells were washed 3 times with prewarmed DMEM w/o phenol red, 30 .Math.l/well of DMEM w/o phenol red was added after the final wash and 10 .Math.l/well of dilution series of test compounds were added in triplicates. J774 cells were pre-incubated with test compounds at 37° C. with 5% CO.sub.2 for 15 min. before TMR-hepcidin was added at 25 nM final concentration. Cells were incubated in a total volume of 50 .Math.l at 37° C. with 5% CO.sub.2 for 2 hours, then Hoechst 33342 dye was added to a final concentration of 0.5 .Math.g/ml to stain nuclei and further incubated for 10 min. at 37° C. with 5% CO.sub.2. Cells were washed 3 times with PBS and fixed in 100 .Math.l of 4% paraformaldehyde in PBS for 15 min. at room temperature. After removal of the paraformaldehyde solution, cells were washed 3 times with PBS leaving 100 .Math.l per well and the plates were sealed with foil plate seal. TMR (530-550 nm excitation / 575-625 nm emission / 400 ms exposure time) and Hoechst 33342 (360-370 nm excitation / 420-460 nm emission / 10 ms exposure time) fluorescence images were acquired using a ScanR plate imager (Olympus) with a 20x high NA objective. Four pictures were acquired per well and fluorescence channel covering ca. 1500 cells per well. The acquired image data was analysed with the ScanR image analysis software. Image analysis included detection of nuclei (Hoechst 33342 fluorescence), identification of cell-associated regions, application of a virtual channel and thresholding for rolling-ball-type background reduction, followed by application of the Sum(Mean) algorithm to measure the TMR fluorescence associated with cells as a quantitative measure for internalized TMR- hepcidin. IC.sub.50 values were calculated with the Sum(Mean) raw data using “log(inhibitor) vs. response” curve fitting of Prism 5 software (GraphPad Software Inc., version 5.02). For each data set the fit of the “log(inhibitor) vs. response (three parameters)″ model was compared to the fit of the “log(inhibitor) vs. response - Variable slope (four parameters)″ model and the IC.sub.50 data of the preferred model was used. IC.sub.50 data of the Compounds according to formula (II) and (II′), that were tested in the hepcidin internalization assay are shown in Table 1. The IC.sub.50 of unlabeled hepcidin in this assay is 0.015 ± 0.011 .Math.M.
TABLE-US-00056 Average (AVE) IC.sub.50 data of Compounds (II) and (II′) tested in the hepcidin internalization assay is shown for multiple measurements Exp. Comp. J774 IC50 (uM) Compound (II′) 0.006 Compound (II) 0.007
11.2 Biophysical Ferroportin-Hepcidin Binding Assay
[0530] This biophysical assay was developed to confirm inhibition of hepcidin binding to ferroportin (Fpn) more directly. Incubation of TMR-hepcidin with purified human Fpn isolated from Pichia pastoris yeast cells expressing human Fpn with a C-terminal FLAG affinity tag (Bonaccorsi di Patti, 2014) leads to increased fluorescence polarization (FP) of the TMR-hepcidin ligand. Compounds (II) and (II′) were tested for inhibition of binding of TMR-hepcidin to Fpn, as detected by dose-dependent decrease of the TMR FP signal, as described in detail below.
[0531] A mixture of 1.3 mM human Fpn and 30 nM TMR-hepcidin in FP assay buffer containing 50 mM Tris-HCI pH 7.3, 200 mM NaCI, 0.02% DDM, 0.1% BSA was plated into a 384 well black low volume round bottom plate (Corning, Cat. 3677) at 16 ml per well. 8 ml of serial dilutions of test compounds were added in duplicates to reach final Fpn and TMR-hepcidin concentrations of 1 mM and 20 nM, respectively. Plates were incubated for 90 minutes at room temperature and parallel (S) and perpendicular (P) fluorescence was measured in a Synergy H1 fluorescence reader (BioTek). FP values were calculated in mP according to the following formula.
[0532] IC.sub.50 values were determined with the calculated mP values as described for the hepcidin internalization assay and are listed in Table 2. The IC.sub.50 of unlabeled hepcidin in this assay is 0.37 ± 0.067 .Math.M.
TABLE-US-00057 Average (AVE) IC.sub.50 data of Compounds (II) and (II′) tested in the biophysical hepcidin-ferroportin binding assay is shown for multiple measurements. Exp. Comp. FP IC50 (uM) Compound (II′) 0.028 Compound (II) 0.028
11.3 Inhibition of Ferroportin Mediated Iron Export Activity in an Iron Response Assay
[0533] Intracellular iron levels are indirectly measured in this assay by monitoring the activity of a beta-lactamase (BLA) reporter gene fused to the human ferritin promoter and the associated iron regulatory element (IRE) contained within the 5′ untranslated region of the ferritin mRNA. Expression of ferroportin (Fpn) in such a cell line leads to iron efflux and lower iron levels as reflected by lower activity of the reporter gene. On the other hand, inhibition of Fpn-mediated iron efflux results in elevated cellular iron levels which is detected as increased reporter gene activity. Small molecular weight Fpn inhibitor compounds were tested for dose-dependent effects in this in vitro iron response assay as described below.
[0534] The HEK-293 cell line #354 was generated by stable integration of (i) a human Fpn-GFP fusion construct inserted in a derivative of the doxycycline-inducible pTRE-Tight-BI plasmid (Clontech, Cat. 631068) and (ii) a human ferritin promoter-BLA reporter gene into a derivative of the HEK-293 Tet-ON Advanced cell line (Clontech). To generate the ferritin-BLA reporter gene construct, a 1.4 kb fragment of the human ferritin H promoter was amplified by PCR from human genomic DNA (forward primer 5′-CAGGTTTGTGAGCATCCTGAA-3′; reverse primer 5′-GGCGGCGACTAAGGAGAGG-3′) and inserted in front of the BLA gene present in the pcDNA™6.2/cGeneBLAzer™-DEST plasmid (Invitrogen, Cat. 12578-043) thereby replacing the original CMV promoter and placing the IRE that regulates translation of the ferritin gene ca. 170 bp upstream of the start codon of the reporter gene. #354 cells were harvested from ca. 80% confluent cultures, seeded at 1.8x10.sup.5 cells/ml in DMEM/F12 GlutaMAX™ medium (Invitrogen, Cat. 31331-028) containing 10% FBS (Clontech, Cat. 631106), 1% Penicillin-Streptomycin, 200 .Math.g/ml Hygromycin B (Invitrogen, Cat. 10687-010), Blasticidin 5 .Math.g/ml, (Invitrogen, Cat. R210-01), 4 .Math.g/ml doxycycline (Clontech, Cat. 631311), 50 .Math.l per well of 384 well PDL-coated plates and grown at 37° C. with 5% CO.sub.2. After overnight incubation, 10 .Math.l/well of dilution series of the test compounds were added in quadruplicates and plates were further incubated overnight at 37° C. with 5% CO.sub.2. Cells were washed 3 times with HBSS leaving 25 .Math.l per well. BLA activity was detected by adding 5 .Math.l/well of the GeneBlazer reagent CCF4-AM (Invitrogen, Cat. K1085) to the cells. After incubation of the plates in the dark at 18° C. for 60 min., blue and green fluorescence signals were measured in a Safire2 fluorescence plate reader (Tecan) with excitation at 410 nm and emissions at 458 nm (blue) and 522 nm (green). The ratio of blue/green fluorescence as a measure for BLA activity was calculated and EC.sub.50 values were determined with the calculated blue/green fluorescence ratios as described for the hepcidin internalization assay. The EC.sub.50 data of the tested Compounds (II) and (II′) are listed in Table 3. The EC.sub.50 of hepcidin in this assay is 0.096 ± 0.063 .Math.M (n=37).
TABLE-US-00058 Average (AVE) EC.sub.50 data of Compounds (II) and (II′) tested in the iron response assay is shown for multiple measurements Exp. Comp. BLAzer EC50 (uM) Compound (II′) 0.168 Compound (II) 0.156
11.4 Ferroportin Internalization and Degradation Assay
[0535] HEK-293 cell line #354 (described in 11.3) was used to measure the capacity of the compounds to induce internalization and degradation of ferroportin (Fpn) by fluorescence activated cell sorting (FACS). Growing HEK-293 #354 cells in doxycycline containing media induced expression of human Fpn-GFP fusion protein on the cell surface. Data from 10 independent experiments showed that cultivation of HEK#354 cells for 48 h in the presence of 4 .Math.g/ml doxycycline induced in average 42.6% ± 6.4% Fpn-GFP-positive cells. Small molecular weight Fpn inhibitor compounds were tested for dose-dependent effects on the Fpn-GFP mean fluorescence intensity (MFI) on HEK-293 cell line #354, as described below.
[0536] HEK#354 cells were harvested from ca. 80% confluent cultures, seeded at 0.6x10.sup.6 cells/ml in DMEM/F12 GlutaMAX™ medium (Invitrogen, Cat. 31331-028) containing 10% FBS (Clontech, Cat. 631106), 1% Penicillin-Streptomycin (Invitrogen, Cat. 15140-122), 200 .Math.g/ml Hygromycin B (Invitrogen, Cat. 10687-010), Blasticidin 5 .Math.g/ml, (Invitrogen, Cat. R210-01), 4 .Math.g/ml doxycycline (Clontech, Cat. 631311), 50 .Math.l per well of 384 well plates (Greiner; Cat. 781091) and grown at 37° C. with 5% CO.sub.2. After overnight incubation, 10 .Math.l/well of dilution series of the test compounds were added in quadruplicates and plates were further incubated overnight at 37° C. with 5% CO.sub.2. Cells were washed once with FACS buffer (PBS containing 1% FBS, 2 mM EDTA and 0.05% NaN.sub.3), harvested in FACS buffer with 0.5 .Math.g/ml propidium iodide (Sigma, Cat. P4864) and analyzed in a flow cytometer (CANTO.sup.tm II, BD Biosciences) equipped with high throughput sampler. Live HEK#354 cells were gated as propidium iodide negative population and analyzed for expression of Fpn-GFP. MFI of Fpn-GFP of > 2000 live cells for each compound dilution was calculated using FlowJo (Tree Star’s, Oregon) and the potency of the Fpn-inhibitors to induce internalization and degradation of Fpn-GFP was calculated as described for the hepcidin internalization assay. EC.sub.50 data of the Compounds (II) and (II′) tested in the ferroportin internalization and degradation assay by FACS are listed in Table 4. The average EC.sub.50 value of hepcidin in this assay is 0.004 ± 0.002 .Math.M.
TABLE-US-00059 Average (AVE) EC.sub.50 data of Compounds (II) and (II′) tested in the ferroportin internalization and degradation assay is shown for multiple measurements Exp. Comp. EC50 (uM) Compound (II′) 0.161 Compound (II) 0.119