Multi-Gram Scale Synthesis and Characterization of Mometasone Furoate EP Impurity C

Mometasone furoate is a synthetic corticosteroid used in the treatment of skin inflammatory conditions, hay fever and asthma. The industrial manufacturing routes to mometasone furoate are generally accompanied by the formation of numerous process impurities that need to be detected and quantified, as requested by regulatory authorities. The ready availability of such impurities in the required quantity and purity is therefore essential for toxicological studies, analytical method development and process validation. Herein, we report the multi-gram scale preparation of 21′-chloro-(16′α-methyl-3′,11′,20′-trioxo-pregna-1′,4′-dien-17′-yl)-furan-2-carboxylate (mometasone furoate EP impurity C), one of the known impurities of mometasone furoate. This study also includes the systematic investigation of the final acylation step, as well as the characterization of the difuroate enol ether intermediate and its conversion to the target impurity C.


Introduction
Mometasone furoate (MF, 1, Scheme 1) is a steroidal active pharmaceutical ingredient (API) employed as ointment, cream or lotion in the treatment of inflammatory skin disorders such as atopic dermatitis eczema and psoriasis [1].MF (1) is also used as metered spray to alleviate the symptoms of seasonal allergic rhinitis (e.g., hay fever) and to treat nasal polyps [2], as well as in aerosol and dry powder inhalers for the prevention of asthma attacks [3].MF (1) was patented by Schering Corp. in 1981 and launched on the market by Merck Sharp & Dohme Corp./Schering Corp. in 1987 under the brand name of Elocon ® [4,5].MF is mentioned on the World Health Organization's list of essential medicines and among the top 300 most prescribed drugs in the United States in 2020 [6,7].The processing methods of MF (1) are based on two approaches that share the same reaction steps but in a different order and starting material (Scheme 1) [4, 8,9].Although some minor changes have been proposed over the years to improve both the product yield and quality profile [10], the process to MF (1) generally leads to the formation of numerous impurities [11].According to the International Conference on Harmonization (ICH) guidelines, impurity profiling represents a mandatory duty within the production process of APIs and involves both toxicological assessments and analytical activities aimed at detecting, elucidating and quantifying the API-related, process-related or stability-related impurities [12][13][14][15][16][17].Moreover, impurity profiling plays a crucial role in the routine activities of modern API manufacturing including in-process control, method and process validation, definition of starting materials/reagents specifications and carry-over activities.In addition, the impurity profiling requires intensive efforts for drawing ad hoc synthetic routes of high-purity reference standards for impurities that are present at a very low level (e.g., 0.10%) in the final API and, therefore, are difficult to isolate [18].Pharmaceutical companies frequently outsource to contract development and manufacturing organizations (CDMOs) the management of impurity synthesis for supporting regulatory requirements with a subsequent increase in the cost for process development studies.
validation, definition of starting materials/reagents specifications and carry-over activities.In addition, the impurity profiling requires intensive efforts for drawing ad hoc synthetic routes of high-purity reference standards for impurities that are present at a very low level (e.g., 0.10%) in the final API and, therefore, are difficult to isolate [18].Pharmaceutical companies frequently outsource to contract development and manufacturing organizations (CDMOs) the management of impurity synthesis for supporting regulatory requirements with a subsequent increase in the cost for process development studies.Twenty known impurities, namely impurities A-T, and one unknown impurity (impurity U) are reported in the corresponding monograph of MF (1) in the European Pharmacopoeia [11].Although known MF impurities are commercially available from specialized suppliers [19], the procedures for the preparation of most of them are not available in the literature.Recently, Das and co-workers reported the preparation of some impurities of corticosteroids starting from tetraene acetate (3) and, among them, the synthesis of six impurities related to MF (impurities G, H, K, L, N and Q) [20].Although structurally similar to each other and related to the structure of MF (1), the preparation of these impurities requires the design and the development of ad hoc synthetic processes to provide the target impurity on-demand, in the desired amount and purity grade.
In this context, herein we report the synthesis of 21′-chloro-(16′α-methyl-3′,11′,20′trioxo-pregna-1′,4′-dien-17′-yl)-furan-2-carboxylate (mometasone furoate EP impurity C, 2) (Scheme 2).This impurity (CAS: 1305334-31-9) is commercially available as a reference standard (mg scale) from different specialized suppliers [21], but its preparation has never been reported so far, thus making the development of a reliable synthetic route for its preparation highly desirable for the steroidal community.Twenty known impurities, namely impurities A-T, and one unknown impurity (impurity U) are reported in the corresponding monograph of MF (1) in the European Pharmacopoeia [11].Although known MF impurities are commercially available from specialized suppliers [19], the procedures for the preparation of most of them are not available in the literature.Recently, Das and co-workers reported the preparation of some impurities of corticosteroids starting from tetraene acetate (3) and, among them, the synthesis of six impurities related to MF (impurities G, H, K, L, N and Q) [20].Although structurally similar to each other and related to the structure of MF (1), the preparation of these impurities requires the design and the development of ad hoc synthetic processes to provide the target impurity on-demand, in the desired amount and purity grade.

Results and Discussion
The synthesis of the target product 2 started from the cheap and readily available 9β,11β-epoxy-17α,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-diene (6), namely 8-DM (Scheme 2) [22].Being 2 a process impurity structurally related to the parent API, we adopted a strategy similar to the synthetic sequence for the manufacturing of MF (1) shown in Scheme 1B.The synthesis consisted of the initial manipulation of ring C by converting the 11,12-epoxide moiety to the desired C11-oxo functionality, followed by the chlorination at C21-position and the final acylation reaction at the C17-position (Scheme 2).Initially, the route was attempted at mg scale.Thus, 8-DM (6) was treated with acetic anhydride and potassium acetate in N,N-dimethylacetamide (DMAC) at 25 °C affording the corresponding 21-acetoxy intermediate 9 (step a).The crude 9 obtained by aqueous extractive work-up (95% recovery) was then submitted to the epoxide ring-opening by treatment with hydrobromic acid in AcOH at 25 °C.After aqueous extractive work-up, the desired bromohydrin derivative 10 was isolated in 90% yield and good purity (step b).The crude bromohydrin 10 was then de-halogenated under Barton's conditions [23,24] by treatment with CrCl3•6H2O, zinc dust and thioglycolic acid in DMF and DMSO affording the desired 11β-hydroxy intermediate 11 in 82% isolated yield from 6 after chromatographic purification (step c).Oxidation of 11 by treatment with pyridinium chlorochromate (PCC) in CH2Cl2 afforded the corresponding 11-oxo derivative 12 in 74% yield after chromatographic purification (step d).Deprotection of the acetyl group at C21-position was achieved under mild alkaline conditions with 95% crude recovery after aqueous extractive work-up (step e).The C21 free hydroxyl group was activated by treatment with methanesulfonyl chloride (MsCl) and 4-dimethylaminopyridine (DMAP) in CH2Cl2 at 0 °C.The methanesulfonate intermediate thus obtained was converted into the corre-

Results and Discussion
The synthesis of the target product 2 started from the cheap and readily available 9β,11β-epoxy-17α,21-dihydroxy-16α-methyl-3,20-dioxo-pregna-1,4-diene (6), namely 8-DM (Scheme 2) [22].Being 2 a process impurity structurally related to the parent API, we adopted a strategy similar to the synthetic sequence for the manufacturing of MF (1) shown in Scheme 1B.The synthesis consisted of the initial manipulation of ring C by converting the 11,12-epoxide moiety to the desired C11-oxo functionality, followed by the chlorination at C21-position and the final acylation reaction at the C17-position (Scheme 2).Initially, the route was attempted at mg scale.Thus, 8-DM (6) was treated with acetic anhydride and potassium acetate in N,N-dimethylacetamide (DMAC) at 25 • C affording the corresponding 21-acetoxy intermediate 9 (step a).The crude 9 obtained by aqueous extractive work-up (95% recovery) was then submitted to the epoxide ring-opening by treatment with hydrobromic acid in AcOH at 25 • C.After aqueous extractive work-up, the desired bromohydrin derivative 10 was isolated in 90% yield and good purity (step b).The crude bromohydrin 10 was then de-halogenated under Barton's conditions [23,24] by treatment with CrCl 3 •6H 2 O, zinc dust and thioglycolic acid in DMF and DMSO affording the desired 11β-hydroxy intermediate 11 in 82% isolated yield from 6 after chromatographic purification (step c).Oxidation of 11 by treatment with pyridinium chlorochromate (PCC) in CH 2 Cl 2 afforded the corresponding 11-oxo derivative 12 in 74% yield after chromatographic purification (step d).Deprotection of the acetyl group at C21-position was achieved under mild alkaline conditions with 95% crude recovery after aqueous extractive work-up (step e).The C21 free hydroxyl group was activated by treatment with methanesulfonyl chloride (MsCl) and 4-dimethylaminopyridine (DMAP) in CH 2 Cl 2 at 0 • C. The methanesulfonate intermediate thus obtained was converted into the corresponding 21-chloro derivative 14 by heating the crude mixture (Scheme 2, step f ).The crude 14 was finally submitted to the furoylation reaction at the C-17 tertiary alcohol group (step g).
The reaction was initially performed using pyridine (2 equiv.)as the base and 2-furoyl chloride (1.2 equiv.) in CH 2 Cl 2 at 25 • C.Under these conditions, a partial conversion of 14 was observed affording the target product 2 in 27% isolated yield (Table 1, entry 1).The use of Et 3 N (2 equiv.)instead of pyridine in the presence of DMAP (0.1 equiv.)as the acyl transfer reagent resulted in the formation of the target product 2 (46% yield) along with unreacted 14 after 24 h at 25 • C (entry 2).However, when the reaction was scaled-up at 1 g, a longer reaction time (up to 48 h) and higher temperature (reflux) were needed (entry 3).Interestingly, the use of higher amounts of Et 3 N (4 equiv.),DMAP (0.25 equiv.)and 2-furoyl chloride (2 equiv.)resulted in the almost quantitative consumption of 14 and in the formation of a different reaction product (76% yield) with a TLC retention factor similar to that of the target product 2 (Table 1, entry 4).High resolution mass spectrometry (HRMS) analysis showed a mass of m/z 579.1785 that corresponds to the formula C 32 H 31 ClO 8 in positive ion mode.Nuclear magnetic resonance (NMR) analysis confirmed the presence of a second 2-furoyl group at the side chain.In particular, the comparison of the 1 H-and 13 C-NMR data with that of compound 2 clearly evidenced the loss of 21-methylene protons at around 4 ppm and the formation of a double bond associated with a single vinylic proton (singlet at 6.31 ppm and CH at 111.5 ppm) of the chloro vinyl ether moiety bearing the second furoyl group.Moreover, key spatial correlations in the NOESY experiment confirmed the Z-geometry at the double bond of the chloro vinyl ether group (see Supplementary Materials).Based on the analytical and spectroscopic data and supported by the literature analysis [25], we unambiguously assigned the structure of the side product as the difuroate enol ether 15.It is worth noting that although 15 is not recognized among the known process impurities of MF (1), it is commercially available from some specialized suppliers of API impurities [26].We therefore focused our efforts at finding mild hydrolytic conditions to convert difuroate enol ether 15 to the desired mometasone furoate EP impurity C (2) (Table 2).Heggie et al. reported the conversion of traces of difuroate side products like 15 to MF (1) by treatment with concentrated HCl and AcOH at 15-25 • C [10].However, under these conditions 15 may undergo an acid-catalysed migration of the angular 18-CH 3 group to the electron deficient C-17 carbon atom via a carbonium ion by either a stepwise or concerted mechanism (Scheme 3) [25].
In our case, the use of 12 N HCl resulted in the formation of unidentified side product(s) along with the almost quantitative consumption of 15 (Table 2, entries 1-3).Also, the use of 3 N HCl in AcOH at 0 • C and p-toluenesulfonic acid (pTSA) in H 2 O/acetone gave traces of the desired target product 2 along with unreacted 14 (entries 4 and 5).Interestingly, the use of HClO 4 in CH 2 Cl 2 at 0 • C afforded the target product 2 in 46% isolated yield (entry 6).
Lower temperatures (−10 • C) and diluted conditions gave a better outcome (72% yield) (Table 2, entry 7).In our case, the use of 12 N HCl resulted in the formation of unidentified side product(s) along with the almost quantitative consumption of 15 (Table 2, entries 1-3).Also, the use of 3 N HCl in AcOH at 0 °C and p-toluenesulfonic acid (pTSA) in H2O/acetone gave traces of the desired target product 2 along with unreacted 14 (entries 4 and 5).Interestingly, the use of HClO4 in CH2Cl2 at 0 °C afforded the target product 2 in 46% isolated yield (entry 6).Lower temperatures (−10 °C) and diluted conditions gave a better outcome (72% yield) (Table 2, entry 7).Accordingly, the target product 2 was obtained in 33% yield from 8-DM (6) over eight steps at mg scale (Scheme 2 and Table 3).Having optimized the conversion of the difuroate enol ether 15 to the target mometasone furoate EP impurity C (2) (step h) and evaluated the feasibility of the synthetic strategy shown in Scheme 2, we finally aimed at scaling the process at the multi-gram scale (27 g of 6).At this point, in order to make the synthetic route easily applicable in industrial settings, we focused on improving work-up and isolation procedures by avoiding, when possible, tedious chromatographic purifications and aqueous extractive work-ups (Table 3).In particular, intermediates 9 and 10 were obtained by simple precipitation from water, the chromatographic purification of 11 was replaced by crystallization and intermediate 15 was purified by simple filtration on silica gel.Flash chromatography followed by crystallization were required to obtain 2 in 30% overall yield and ≥96% purity at multi-gram scale with solvent-saving isolation and purification procedures with respect to initial small-scale synthesis (Table 3).Target product 2 was fully characterized by mono-and bidimensional NMR, HRMS and IR analyses, while its purity (≥96%) was assessed by quantitative normal phase (NP) and reverse phase (RP) C18-thin layer chromatography, quantitative 1 H-NMR in the presence of dimethyl sulfone (DMS) as the internal standard and high-performance liquid chromatography (HPLC) analysis (see Supplementary Materials).HRMS analysis in positive ion mode showed a m/z 485.1728, which corresponds to the formula C 27 H 29 ClO 6 , and a m/z 486.1759, which corresponds to the p + 1 (abund.27.29%), conforming the presence of a chlorine atom.The comparison of 1 H-and 13 C-NMR spectra of 2 with those of the starting material 8-DM (6) confirmed the presence of the conjugated dienone at the A ring of the steroid backbone (signals at δ 6.11, 6.23 and 7.68 ppm in the proton spectra and carbon nuclei at δ 186.2, 165.9, 154.8, 127.8 and 124.3 ppm).The presence of two additional carbonyl groups at C11 and C20 position was proved by the signals at δ 207.5 and 196.1, respectively, in the carbon spectrum.The presence of a methylene group bearing the chlorine atom at the side chain was confirmed by the presence of a multiplet at 4.03-4.11ppm, which corresponds to the signal at 44.6 ppm in the 13 C-NMR spectrum.Finally, the signals at δ 6.57, 7.29 and 7.64 in the 1 H-NMR spectrum and the carbon nuclei at δ 112.5, 120.2, 142.8, 147.5 and 158.0 ppm are related to the presence of the 2-furoyl group at the C17 position (carbon at 96.8 ppm).Scalar and spatial correlation in homonuclear 1 H-1 H COSY and NOESY experiments confirmed the aforementioned atom arrangement (see Supplementary Materials).

a
Scheme 3. (A) Proposed mechanism for the formation and acid-promoted hydrolysis of difuroate enol ether 15 from intermediate 14; (B) proposed mechanism for the formation of rearranged side product 16 by acid−catalysed migration of the angular 18−CH3 by stepwise pathway from target product 2, or (C) by concerted mechanism from enol ether 15 [25].

Scheme 3 .
Scheme 3. (A) Proposed mechanism for the formation and acid-promoted hydrolysis of difuroate enol ether 15 from intermediate 14; (B) proposed mechanism for the formation of rearranged side product 16 by acid−catalysed migration of the angular 18−CH 3 by stepwise pathway from target product 2, or (C) by concerted mechanism from enol ether 15 [25].

Table 1 .
Screening for the furoylation reaction of intermediate 14.
a Reaction performed at 100 mg scale.b Reaction performed at 1 g scale.c Yield refers to difuroate enol ether 15.

Table 2 .
Screening for the conversion of difuroate enol ether 15 to target 2.

Table 2 .
Screening for the conversion of difuroate enol ether 15 to target 2.

Table 3 .
Comparison of the results obtained for the preparation of mometasone furoate EP impurity C (2) at mg and multi-gram scale.
a Starting from 500 mg of 6. b Starting from 27 g of 6. c Esteemed by 1 H-NMR analysis.d Crude recovery.e Isolated yield.f Determined by quantitative NP-and RP 18 -TLC, HPLC-DAD analysis and quantitative 1 H-NMR in the presence of dimethyl sulfone (standard for quantitative NMR, TraceCERT ® ) as the standard (see Supplementary Data).