A crystalline dipeptide useful for the synthesis of elamipretide

Abstract

To develop new methods to synthesize elamipretide that allow the purification after coupling reactions.SOLUTION: Disclosed are crystalline forms of L-Lys(Boc)-Phe-NH2 and Boc-D-Arg-DMT.SELECTED DRAWING: None

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 651,430, filed April 2, 2018. [Background technology]

[0002] Elamipretide (MTP-131) is a mitochondrial targeted peptide compound with therapeutic potential to treat diseases associated with mitochondrial dysfunction. Elamipretide contains four amino acid residues and has been synthesized following typical linear and convergent solution-phase peptide synthesis methods. The synthetic routes to produce elamipretide used thus far require the preparation of various differentially protected peptides, such that certain protecting groups are selectively removed to expose the deprotected compound to peptide coupling, while leaving other protecting groups to prevent unwanted side reactions. Even with protecting groups, such coupling reactions and related steps generate impurities. Thus, there is a need to develop novel methods to purify elamipretide that allow for purification after the coupling reaction. Crystallization of the target reaction product is one method to achieve the necessary purification. Summary of the Invention [Means for solving the problem]

[0003] Crystalline forms of L-Lys(Boc)-Phe-NH2 and Boc-D-Arg-DMT are disclosed, where DMT is an abbreviation for dimethyltyrosine, an intermediate in the synthesis of elamipretide. [Brief description of the drawings]

[0004] [Figure 1] FIG. 1 is an XRPD pattern of crystalline L-Lys(Boc)-Phe-NH2. [Diagram 2] 1 is an XRPD pattern of crystalline Boc-D-Arg-DMT. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0005] Elamipretide has been shown to have various therapeutic effects in diseases related to mitochondrial dysfunction. Conventional synthetic routes to elamipretide present challenges in terms of scale-up, as they rely on chromatographic separation to increase the levels of the desired intermediates. Herein, we disclose crystalline forms of L-Lys(Boc)-Phe-NH2 and Boc-D-Arg-DMT that can be used as purified intermediates in the synthesis of elamipretide.

[0006] One aspect of the present invention relates to a crystalline form of Compound (I), which is also known as L-Lys(Boc)-Phe-NH2: [ka]

[0007] The crystalline form of compound (I) can be used in the synthesis of elamipretide.

[0008] In certain embodiments, the polymorph of the crystalline form is characterized by powder X-ray diffraction (XRD). θ represents the diffraction angle measured in degrees (°). In certain embodiments, the diffractometer used in XRD measures the diffraction angle as two times the diffraction angle θ. Thus, in certain embodiments, the diffraction patterns described herein refer to the X-ray intensity measured against the angle 2θ.

[0009] In certain embodiments, the crystalline form of Compound (I) is not solvated (e.g., the crystal lattice does not contain molecules of a solvent). In certain alternative embodiments, the crystalline form of Compound (I) is solvated. In some cases, the solvent is water.

[0010] In one aspect, the invention features a crystalline form of Compound (I) having characteristic peaks in the X-ray powder diffraction (XRPD) pattern shown in FIG.

[0011] In another aspect, the invention features a crystalline form of Compound (I) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at the values ​​of 2 theta (°2θ) set forth in Table 1.

[0012] In another aspect, the invention features a crystalline form of Compound (I) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at values ​​of two-theta (°2θ) of 4.7, 6.2, 12.4, 15.8, 16.5, 18.0, 18.2, 18.8, and 19.8.

[0013] In another aspect, the invention features a crystalline form of Compound (I) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at values ​​of two theta (°2θ) of 4.7, 6.2, 11.3, 12.4, 13.3, 15.0, 15.8, 16.5, 17.0, 17.7, 18.0, 18.2, 18.8, 19.8, 22.0, and 22.8.

[0014] The relative intensity and 2θ values ​​of each peak in Table 1 and Figure 1 may change or shift under certain conditions while the crystal form is the same. One skilled in the art would be able to readily determine whether a given crystal form is the same crystal form as that described in Table 1 and Figure 1 by comparing their XRPD data.

[0015] One aspect of the present invention relates to a crystalline form of compound (II), which is also called Boc-D-Arg-DMT and may also be depicted in the form of a zwitterion: [ka]

[0016] The crystalline form of compound (II) can be used in the synthesis of elamipretide.

[0017] In certain embodiments, the polymorph of the crystalline form is characterized by powder X-ray diffraction (XRD). θ represents the diffraction angle measured in degrees (°). In certain embodiments, the diffractometer used in XRD measures the diffraction angle as two times the diffraction angle θ. Thus, in certain embodiments, the diffraction patterns described herein refer to the X-ray intensity measured against the angle 2θ.

[0018] In certain embodiments, the crystalline form of Compound (II) is not solvated (e.g., the crystal lattice does not contain molecules of a solvent). In certain alternative embodiments, the crystalline form of Compound (II) is solvated. In some cases, the solvent is water.

[0019] In one aspect, the invention features a crystalline form of Compound (II) having characteristic peaks in the X-ray powder diffraction (XRPD) pattern shown in FIG.

[0020] In another aspect, the invention features a crystalline form of Compound (II) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at the values ​​of 2 theta (°2θ) set forth in Table 2.

[0021] In another aspect, the invention features a crystalline form of Compound (II) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at values ​​of two-theta (°2θ) of 9.3, 12.1, 16.6, 17.6, 18.0, 18.8, and 19.4.

[0022] In another aspect, the invention features a crystalline form of Compound (II) having characteristic peaks in an X-ray powder diffraction (XRPD) pattern at values ​​of two theta (°2θ) of 9.3, 12.1, 13.7, 16.3, 16.6, 17.6, 18.0, 18.8, 19.4, 21.3, 23.0, 24.2, and 25.1.

[0023] The relative intensity and 2-theta (2θ) values ​​of each peak in Table 2 and Figure 2 may change or shift under certain conditions while the crystal form is the same. One skilled in the art should be able to readily determine whether a given crystal form is the same crystal form as those set forth in Table 2 and Figure 2 by comparing their XRPD data. EXAMPLES

[0024] material and method TIFF0007663642000003.tif101160

[0025] Example 1. L-Lys(Boc)-Phe-NH 2 (Compound I) Exemplary Synthetic Routes [ka]

[0026] Synthesis of compound 3 TIFF0007663642000005.tif214170

[0027] Synthesis of compound 1 TIFF0007663642000006.tif95170TIFF0007663642000007.tif168170

[0028] Microscopy of compound 1 product slurry JPEG0007663642000008.jpg79103

[0029] Preliminary solubility of compound I in a single solvent TIFF0007663642000009.tif51115

[0030] Preliminary precipitation test of compound I (MeOH / water) TIFF0007663642000010.tif87170

[0031] In-process precipitation results for compound I (MeOH / water): TIFF0007663642000011.tif41169

[0032] Example 2. Boc-D-Arg-DMT-OH [ka]

[0033] Manufacturing process for producing compound 8 TIFF0007663642000013.tif139170

[0034] Process for producing the HCl salt of compound II TIFF0007663642000014.tif116170

[0035] Preparation of the zwitterionic form of compound II [ka] 1. Suspend the crude HCl salt in CHOH / H0 (1 / 1, v / v). 2. Heat the suspension to 45-50°C. 3. After a clear solution is formed, add an aqueous solution of Na2CO3 (1.2 eq.) During the addition, a solid begins to precipitate. 4. The suspension is stirred at 45° C. for 1 hour, then cooled to 15° C. and stirred for an additional hour. 5. The solid is isolated by filtration and dried to provide Compound II having high purity (99.4 area%) by HPLC. The calculated w / w assay correcting for residual solvent, water content, and residue on ignition for this demonstration run was 98.7%.

[0036] The formation of the zwitterionic compound resulted in a high purity material that was stable, easy to handle, and highly crystalline.

[0037] Example 3. XRPD peak list of crystalline L-Lys(Boc)-Phe-NH2 [Table 1-1] [Table 1-2]

[0038] Example 4. XRPD peak list for crystalline Boc-d-Arg-DMT [Table 2-1] [Table 2-2]

[0039] Incorporation by Reference All US patents and US and PCT published patent applications referred to in the above description are hereby incorporated by reference in their entirety.

[0040] equivalent Although the present invention has been described in some detail by way of examples and illustrations for clarity of understanding, it will be apparent to one skilled in the art that the same can be practiced by modifying or altering the present invention within a range of conditions, formulations and other parameters without affecting the scope of the invention or specific embodiments thereof, and that such modifications or alterations are intended to be included within the scope of the appended claims.

Claims

1. A compound of formula II in crystalline form or a pharma- ceutically acceptable salt thereof. 【Chemistry 1】

2. Formula II: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof, comprising: (a) Formula I: 【Chemistry 3】 and a HCl salt represented by CH ３ OH and H ２ 2. Combining the mixture of 0 with a suspension of 100% by weight ... A method comprising:

3. (b) heating the suspension, thereby forming a solution. The method of claim 2 , further comprising:

4. The method of claim 3, wherein the suspension is heated to about 45°C to about 50°C.

5. (c) Na ２ CO ３ to the solution formed in (b), thereby forming a second suspension. The method of claim 3 further comprising:

6. About 1.2 equivalents of Na relative to the amount of the compound of formula I ２ CO ３ The method according to claim 5, further comprising the addition of

7. (d) agitating the second suspension. The method of claim 5 further comprising:

8. 8. The method of claim 7, wherein the second suspension is heated to about 45° C. with stirring.

9. 9. The method of claim 8, wherein the second suspension is stirred at about 45° C. for about 1 hour.

10. (e) cooling the second suspension to about 15° C. The method of claim 8 further comprising:

11. 11. The method of claim 10, wherein the second suspension is stirred at about 15°C for about 1 hour.

12. (f) isolating the compound of Formula II in solid form from said second suspension. The method of claim 10 further comprising:

13. 13. The method of claim 12, wherein (f) comprises filtering the second suspension to recover the compound of formula II in solid form.

14. (g) drying the compound of formula II, thereby forming a dried compound of formula II. The method of claim 13 further comprising:

15. 15. The method of claim 14, wherein the dried compound of formula II has a purity of 99.4% as measured by HPLC.

16. 15. The method of claim 14, wherein the dried compound of formula II is crystalline.

17. Formula II: 【Chemistry 4】 or a pharma- ceutically acceptable salt thereof, comprising: (a) Na ２ CO ３ with a solution of a compound of formula I: 【Chemistry 5】 and HCl salts represented by CH ３ OH and H ２ and adding the mixture of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29, 32 A method comprising:

18. About 1.2 equivalents of Na relative to the amount of the compound of formula I ２ CO ３ The method of claim 17, further comprising adding

19. (b) isolating the compound of formula II in solid form from said suspension.

20. The method of claim 17, further comprising:

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