DE NOVO Synthesis of Cyclocreatine and Subsequent Conversion to Cyclocreatine Phosphate Via a Continuous Flow Reactor (CFR) System

Abstract

A highly efficient and safe N-cyanation and N-phosphorylation reagent for the de novo synthesis of pharmaceutically acceptable cyclocreatine and salts was achieved using trichloroacetonitrile in lieu of highly toxic cyanogen bromide (CNBr) for generating the required cyclocreatine (CCr) intermediates, followed by highly effective N-phosphorylation through pH control using phosphoryl chloride with high conversion to the corresponding cyclocreatine phosphate (CCrP) targets. A continuous flow reactor system was engineered to improve the efficiency of the process and deliver a product with improved yield, safety, and cost efficiency.

Claims

1. A process for synthesizing a compound of formula (IV) or a pharmaceutically acceptable salt thereof, wherein R5 is a mono-valent cation and R6 is H or a cation, using a continuous flow reactor (CFR) system including at least two flow cells, the method comprising: ##STR00017## providing an aqueous solution of cyclocreatine intermediate of formula (III), wherein R1 is H and R2 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Na or CH.sub.2CO.sub.2Y, the Y being a carboxyl protecting group; providing a first aqueous solution of a base; providing phosphoryl chloride; mixing the aqueous solution of cyclocreatine intermediate of formula (III), the first aqueous solution of the base, and the phosphoryl chloride in a first flow cell to generate an aqueous solution of cyclocreatine phosphate as a Zwitterion complex in a first stage; collecting the aqueous solution of cyclocreatine phosphate in a second flow cell; and injecting a second aqueous solution of the base to the second flow cell in a second stage so as to control pH value and to generate an aqueous solution of cyclocreatine phosphate disodium dihydrate (CCrPNa.sub.2).

2. The process of claim 1 further comprising mixing the aqueous solution of cyclocreatine intermediate of formula (III) and the first aqueous solution of the base to form a first feedstock, and mixing the first feedstock with the phosphoryl chloride in the first flow cell to generate the aqueous solution of cyclocreatine phosphate as a Zwitterion complex in the first stage.

3. The process of claim 1, wherein the pH value in the second flow cell is controlled in a range between 7.45 and 7.71.

4. The process of claim 1, wherein the base is selected from a group including NaOH, KOH, Na.sub.2CO.sub.3, K.sub.2CO.sub.3, NaHCO.sub.3, KHCO.sub.3, and a combination thereof.

5. The process of claim 1, each of R5 and R6 cations, independent of each other, is selected from a group including Na and K.

6. The process of claim 1, wherein the pharmaceutical acceptable salt of the compound of formula (IV) is in a hydrated form including cyclocreatine phosphate disodium dihydrate.

7. A method for synthesizing a compound of formula (IV) or a pharmaceutically acceptable salt thereof, ##STR00018## the method comprising: reacting a compound of formula (I): ##STR00019## with a compound of formula (II)
R.sub.4C?N (II) to generate an intermediate of formula (III): ##STR00020## or an acceptable salt thereof, and reacting the intermediate of formula (III) with phosphoryl chloride by N-phosphorylation to yield final product of formula (IV): ##STR00021## wherein: R1 is H, R2 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Y, wherein Y is Na, K, or a carboxyl protecting group, R3 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Y, wherein Y is Na, K, or a carboxyl protecting group, R4 is CCl.sub.3, R5 is a mono-valent cation, and R6 is H or a cation.

8. The method of claim 7, wherein each of R2 and R3, independent of each other, is CH.sub.2CO.sub.2CH.sub.2C.sub.6H.sub.5.

9. The method of claim 7, wherein each of R5 and R6 cations, independent of each other, is selected from a group including Na, K, and a combination thereof.

10. A method for synthesizing cyclocreatine of formula (III) or a pharmaceutically acceptable salt thereof, the method comprising: ##STR00022## reacting a compound of formula (I): ##STR00023## with a compound of formula (II)
R.sub.4C?N (II) to generate cyclocreatine of formula (III) or an acceptable salt thereof, ##STR00024## wherein: R1 is H, R2 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Y, wherein Y is Na, K, or a carboxyl protecting group, R3 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Y, wherein Y is Na, K, or a carboxyl protecting group, and R4 is CCl.sub.3.

11. The method of claim 10, wherein each of R2 and R3, independent of each other, is CH.sub.2CO.sub.2CH.sub.2C.sub.6H.sub.5.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 is a .sup.31P NMR spectrum of cyclocreatine phosphate disodium dihydrate (CCrPNa.sub.2) product prepared in a prior work;

[0032] FIG. 2 is a flow diagram of continuous flow reactor (CFR) system for synthesizing CCrPNa.sub.2 according to one embodiment of the present invention.

DETAILED PATENT DESCRIPTION

[0033] A highly efficient and sustainable method for synthesizing cyclocreatine phosphate (CCrP) from cyclocreatine (CCr) precursor has been developed using a Continuous Flow Reactor (CFR) system. Referring to FIG. 2, there illustrated is a flow diagram of CFR system for CCrPNa.sub.2 synthesis. This system specifically is a two-stage CFR system which leads to an aqueous solution of CCrPNa.sub.2 as an output feedstock. The subsequent crystallization of the CCrPNa.sub.2 yields a final product isolated as a pure powder.

[0034] The present invention further describes synthesis of CCrP as a Zwitterion complex in the first stage of the process by reacting cyclocreatine-based precursors and phosphoryl chloride in an aqueous media, followed by the second stage where such intermediate of the Zwitterion complex is converted to a hydrated salt by a reaction with sodium hydroxide to a defined pH range to generate the targeted cyclocreatine phosphate ionic salt.

[0035] As shown in FIG. 2, the continuous flow reaction (CFR) system according to an embodiment of the present invention comprises two flow reaction cells, in which two liquid feedstocks (A and B) are combined in the first reaction flow cell 1 to generate a homogeneous solution of the Zwitterion intermediate C. The Zwitterion intermediate C is transported to the second flow cell 2, where the reaction solution is controlled with pH adjustment using an auto titrator D, to generate an aqueous solution of final product E.

[0036] The present invention is directed at an improved synthetic method for commercial synthesis of CCrP and salts of formula (IV) in greater overall yield without using the extreme hazards associated with cyanogen bromide (2a) by incorporating an alternative de novo reagent to generate the required cyanamide intermediate, which leads to the formation of the CCr precursor compound of formula (III) in lieu of cyanogen bromide as outlined in Scheme 6.

##STR00008##

[0037] In scheme 6, the trichloroacetonitrile is used to replace cyanogen bromide (2a) used in Scheme 2 to generate CCr intermediates. Specifically, a compound of formula (I):

##STR00009##

undergoes N-cyanation using a compound of formula (II):

R.sub.4C?N (II)

to generate a cyclocreatine intermediate of formula (III):

##STR00010##

or an acceptable salt thereof, followed by N-phosphorylation using POCl.sub.3 to yield final target of formula (IV):

##STR00011##

where: R.sub.1 is H; R.sub.2 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Na or CH.sub.2CO.sub.2Y (where Y is a carboxyl protecting group); R.sub.3 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Na or CH.sub.2CO.sub.2Y (where Y is a carboxyl protecting group); R.sub.4 is CCl.sub.3; R.sub.5 is a mono-valent salt (e.g., Na, K, or equivalent); R.sub.6 is H or an acceptable salt.

##STR00012##

[0038] In Scheme 7, an embodiment of the present invention describes a method for the preparation of CCrP through directed N-cyanation of the secondary amine using trichloroacetonitrile in the presence of a base to generate the required CCr intermediate and chloroform after undergoing intramolecular cycloaddition. The crude CCr is then subjected to N-phosphorylation conditions to yield the CCrP (phosphagen) product under pH-controlled mild conditions. In this synthetic method, the CCr intermediate isolated is converted directly to CCrP without having to undergo stringent purification, lending a process which is amendable to large-scale commercial manufacturing with improved safety and efficiency.

[0039] As outlined in Scheme 7, according to the embodiment of the present invention, the CCr intermediate is prepared by the following procedure. A reaction vessel equipped with a stirrer is charged with the compound of formula (I) (1 eq.), acetonitrile ([amine]=1 mol mL), trichloroacetonitrile (1.1 eq.), and imidazole (10 mol %). The system is allowed to stir at 75-85? C. until the reaction is completed. The mixture is then concentrated to remove all of the acetonitrile and excess trichloroacetonitrile. The crude intermediate is then dissolved in dimethoxyethane (DME) to form a 0.2 M solution before charging sodium tert-pentoxide (NaOt-Am, 2 eq.) in one portion and stirring for 15-20 minutes at 20? C. Aqueous NaHCO.sub.3 is added, and the product extracted out using ethyl acetate. The organics are then dried over sodium sulfate and concentrated. The crude CCr collected is then dissolved in boiling water, and allowed to cool and chilled to fully precipitate product. Product is then filtered, washed with cold water and dried.

[0040] In the case where CCr contains unwanted impurities, CCr is dissolved in a boiling water, filtered, and chilled to precipitate CCr, followed by triturating with cold water and drying to generate pure CCr.

[0041] In another embodiment according to the present invention, detailed is a process for the preparation of a compound of formula (IV):

##STR00013##

by reacting a compound of formula (I):

##STR00014##

with a compound of formula (II)

R.sub.4C?N (II)

to generate an intermediate of formula (III):

##STR00015##

or an acceptable salt thereof, followed by N-phosphorylation to yield final target of formula (IV):

##STR00016##

where: R1 is H; R2 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Na or CH.sub.2CO.sub.2Y (where Y is a carboxyl protecting group); R3 is CH.sub.2CO.sub.2H or CH.sub.2CO.sub.2Na or CH.sub.2CO.sub.2Y (where Y is a carboxyl protecting group); R4 is CCl.sub.3; R5 is a mono-valent salt, such as Na, K, or equivalent; R6 is H or a cation forming an acceptable salt with CCrP of formula (IV).

[0042] It should be understood that, throughout this patent application, the term a carboxyl protecting group refers to a functional group that protects the carboxyl group from being changed, removed, or eliminated from the molecule during preparation of CCr or CCrP. The carboxyl protecting group is a suitable chemical group which may be attached to the carboxyl functionality of a molecule, then removed at a later stage to reveal the intact functional group and molecule. Examples of suitable protecting groups for various functional groups are described in Theodora W. Greene, Peter G. M. Wuts: Protective Groups in Organic Synthesis, 3.sup.rd ed. Wiley Interscience, 1999; L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); L. Paquette, ed. Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995); each of which is incorporated by reference in its entirety.

EXAMPLES

Synthesis of Cyclocreatine Phosphate Disodium Dihydrate (CCrPNa.SUB.2.)

[0043] The synthesis of CCrPNa.sub.2 was carried out by steps of preparations of Feedstocks 1 and 2, N-Phosphorylation reaction to convert CCrNa to CCrPNa.sub.2, and isolation and purification of CCrPNa.sub.2.

[0044] 1.1 Preparation of Feedstock 1: De-ionized water (0.3 L, 222 mol) and sodium hydroxide flakes (38.6 g, 0.96 mol, 17 equivalent) were combined and allowed to stir for 5-10 minutes at room temperature, or until a clear solution resulted, at which point 2-imino-1-imidazolidineacetic acid cyclocreatine (8.0 g, 56 mmol was charged to the solution all at once and allowed to stir until a clear solution resulted (ca. 10-15 minutes). The concentration of the cyclocreatine sodium salt was 0.91 molar. The feedstock 1 was a clear and colorless solution.

[0045] 1.2 Preparation of Feedstock 2: Phosphoryl chloride (42 g, 25.6 mL, 273 mmol, 4.9 equivalent) was measured by mass under an inert atmosphere. The concentration of POCl.sub.3 was 10.7 molar as a neat liquid. The feedstock 2 was a clear and colorless liquid.

[0046] 1.3 N-Phosphorylation reaction to convert CCrNa to CCrPNa.sub.2: in reaction flow cell 1, the temperature was set to 35? C. The initiated additions of both feedstocks 1 and 2 to the reaction flow cell 1 were set at the following rates: feedstock 1 feeding rate was 50 mL/min (46 mmol/min) and feedstock 2 feeding rate was 5 mL/min (55 mmol/min) with a resonance time of 10-12 minutes under an inert nitrogen atmosphere. A clear solution was formed as the effluent (output) from the reaction flow cell 1 and collected in the reaction flow cell 2 under an inert atmosphere. The reaction flow cell 2 was assembled with an agitator and an auto titrator filled with 5 N NaOH aqueous solution using DI water. The pH of the effluent was typically between 2.4 and 2.6.

[0047] 1.4 Isolation and purification of CCrPNa.sub.2: using the auto titrator, 5 N NaOH solution was added to the reaction flow cell 2 with mild agitation to a targeted pH of 7.58 (7.45 to 7.71) while keeping the reaction temperature between 25 to 38? C. for over 10 minutes. While warm, the mixture remained a clear and colorless solution. However, once it was cooled to room temperature (19-20? C.) a cloudy white mixture would form. After adjusting the pH, the temperature was lowered to 5-7? C. and maintained with mild stirring for 30 minutes. The reaction mixture was then filtered through fluted filter paper to remove insoluble. A clear and colorless filtrate resulted. Upon concentrating to dryness (1-2 mmHg, 50-60? C.), a white free flowing powder resulted. DI water (250 mL) was charged to the white solid at room temperature and allowed to stir for 10-15 minutes (longer times are acceptable). Approximately 90-95% of the solid dissolved, leaving white insoluble material which was filtered away using fluted filter paper. The filtrate was concentrated back down to a white solid using vacuum (1-2 mmHg) and 50? C. DI water (250 mL) was charged to the solid and allowed to stir at room temperature (20-25? C.) for 10-15 minutes, followed by filtration to yield CCrPNa.sub.2 as a clear solution in water.

[0048] Hysol (denatured ethanol (EtOH) w/ethyl acetate (EtOAc)) was charged (10? volume, ca. 1.5 L) to the clear and colorless CCrP-water solution with moderate agitation at room temperature (20? C.) over 20 minutes. Agitation was then slowed, and the temperature was adjusted to 5-7? C. and maintained for 16 hours to allow full precipitation of CCrPNa.sub.2. Product was filtered off easily as a white crystalline solid with a clear and colorless filtrate. The product cake was then triturated with a cold (10-15? C.) 11% ethanol-water solution to remove residual water and impurities. Product was allowed to dry in a 40? C. oven overnight under 1-mmHg vacuum, yielding a white free flowing crystalline powder. (snow white in appearance). Material was extremely soluble in D.sub.2O for .sup.1H NMR analysis purposes. The product (disodium salt) was very soluble in water. Theoretical yield was 16.94 gram and the actual yield was 12.8 gram.