Production of hydroxysteroids via reduction of ketosteroids in continuous flow

Description

"PRODUCTION OF HYDROXYSTEROIDS VIA REDUCTION OF KETOSTEROIDS IN CONTINUOUS FLOW" UTILITY MODEL FIELD

[001] The present utility model relates to the field of pharmacy. It concerns an innovative process for the production of hydroxysteroids via the reduction of ketosteroids in a continuous flow. BASIS OF THE UTILITY MODEL

[002] With the increasing demand for green and environmentally beneficial reactions, it is important to explore alternative chemical routes that address these issues.1 Within this context, steroids are a vast family of chemical compounds with 3 or 4 rings of 5 to 6 carbon atoms, which may contain a ketone or alcohol functional group in their structure. Modifications to the chain can affect the biological activity of these compounds, as most sex hormones and even anabolic steroids are steroids.

[003] Of the steroid hormones, the main ones that can be highlighted are the estrone derivatives. The main active biochemical molecule of estrogen is estradiol, responsible for controlling endometrial growth, breastfeeding, and sexual development. In addition to its derivatives, which also have female sexual functions and are used to treat dysregulations in the sexual organs.2

[004] Within the realm of sexual activity and hormones, estrogens are highly important in livestock activity. Combined with progesterone, the hormone is useful for stimulating estrus and synchronizing the fertile period without affecting pregnancy. The reproductive period of cattle can therefore be regulated by the combination of sex hormones.3

[005] They are also used in the pharmaceutical industry for hormonal regulation in transgender women and correction of sexual problems in women. Petition 870250008349, dated 01 / 31 / 2025, page 10 / 25 / 8 cisgender. As an example, promestriene (estradiol 3-propyl 17ε-methyl diether) is a medication used to treat vaginal atrophy in postmenopausal women.4

[006] The synthesis of these drugs can be done by transformations of other hormones, such as estrone, which can be obtained and purified from the urine of pregnant animals and humans or prepared by cyclopentadienes via Diels-Alder.5-7 Biotransformations in these compounds have several advantages in chemical routes, including chiral selectivity in the development of substances. The intention in reducing ketosteroids is the conversion to hydroxysteroids maintaining the carbon chain and only transforming the C=O group into C-OH, however parallel reactions can occur and perform reduction of C=C bonds.

[007] In addition to the mechanisms of biotransformation, the choice of a reducing agent is essential to the topic of green chemistry. Some reducing agents already known as metals in a gaseous hydrogen stream, metal hydrides, borohydrides and renewable catalysts are widely used for the reduction of interest.

[008] Double catalysts such as copper-cobalt in aqueous medium have already been used in green reductions and have obtained good results. Since the hydrogen donor comes from water and the catalyst is reusable, the catalytic reduction follows excellent yields for the subject matter of interest. However, such a reaction is only functional for aromatic aldehydes and ketones, proving inefficient for aliphatic systems.8

[009] In 1938, Russell Marker and Ewald Rohrmann wanted to study and understand the conformation of estradiols, which were made from the catalytic reduction of estrone.9 For this study, aluminum isopropylate and isopropanol were used. The reaction was carried out under reflux in a water bath for 5 hours, without reporting the yield obtained, but the formation of the two conformers (α, β) was obtained.

[0010] In 1999, Maureen Wilson and colleagues interested in pathogenic fungi of bananas decided to investigate the biotransformations caused by these pathogens.10 The fungi were previously prepared for the Petition 870250008349, dated 01 / 31 / 2025, page 11 / 25 3 / 8 catalytic use and applied to the steroid solubilized in organic solvent. After the reaction and collection of the corresponding alcohol, the yield performed was low at approximately (1-1.5%).

[0011] In 2006, Paula Kiuru proposed a fast and efficient synthetic route for the hydrogenation of estrone using microwaves and deuterium.11 The steroid was reduced with LÍBD4 irradiated by microwaves at room temperature over a long period of 2.5 hours. The final product obtained showed a yield of 95%.

[0012] In 2008, Animesh Aditya and colleagues, while offering undergraduate courses, decided to study the stereochemistry of organic reactions.12 For this, the target was estrone under a reduction using borohydrides, first dissolved in 95% ethanol, and reacted with NaBH4 under heating at 70°C for 30 minutes.

[0013] Two years later, Wang and colleagues, in an attempt to use aromatic solvents to obtain fluoridated arenes of diaylodon salts, tested the reduction of estrone via borohydrides. 13 Starting from estrone solubilized in methanol, the reaction was carried out in a basic medium with the addition of NaBH4 in methanol.

[0014] The use of borohydrides is an excellent way to reduce these ketone bodies due to favorable conditions such as temperature and reaction time, as well as the low cost of the reagent.

[0015] In 2012, in a proposal to carry out aqueous reactions of sparingly soluble compounds with high melting points, Xiaoxue Cui and her team proposed a route using estrone. 14 The reduction to estradiol was carried out under mild conditions, such as 60 °C and 1.5 h in aqueous medium, a favorable route due to the conditions and reagents used, in addition to

[0016] In 2020, the study by Chinna Ayya Swamy P and team was published on the versatility of chiral imidazo[1,5-a]pyridine-oxazolines in the selectivity of ketone reduction.15 In an attempt to carry out the synthesis at room temperature (40 °C), a Rhodium complex as a corresponding imidazole salt was used as a catalyst for the reduction of estrone via hydrosilylation. Petition 870250008349, dated 01 / 31 / 2025, page 12 / 25 4 / 8 The synthetic route proved to be extremely effective for stereoselectivity and with yields above 99%.

[0017] In 2021, Mautschke studied the selective reduction of ketosteroids using Zirconium and MOF 808 complexes.16 By carrying out the reaction in alcoholic solvents at 120°C and varying the temperature between 8h and 24h, the study was conducted on the proportion of estradiol diastereoisomers formed, with yields reaching up to 99%.

[0018] Based on the results obtained, we see great advantages in exploring the production of hydroxysteroids by continuous flow reduction of ketosteroids using NaBFk. SUMMARY OF THE UTILITY MODEL

[0019] The purpose of this utility model is the scalable production of high-potency drugs, which increasingly raises concerns about occupational safety. Within the production chain, minimizing the number of operations involving the handling of bioactive compounds leads to greater safety in the process. In this context, the use of reactors under continuous flow conditions for the synthesis of organic compounds has proven to be an excellent synthetic tool in fine chemical industries due to the advantages inherent in the use of such systems.

[0020] Among these, we can mention: High control of reaction variables; Miniaturization of equipment for carrying out reactions under high temperatures and pressures; Possibility of generating and applying highly reactive chemical species; Reduction of downtime due to automation; Significant reduction of handling operations; High reproducibility; Integration of the chemical reaction and its purification under the same continuous flow conditions; Higher catalyst / substrate ratio in heterogeneously catalyzed reactions.

[0021] The use of borohydrides in the reduction of ketosteroids allows the production of only one diastereoisomer, 173-hydroxysteroids, due to steric hindrance by the methyl group at position 18. Therefore, the attack of Petition 870250008349, dated 01 / 31 / 2025, page 13 / 25 5 / 8 hydride is formed by the anterior face of the steroid (α face), producing a β-alcohol, which can be analyzed by proton NMR.

[0022] The synthetic route of interest for the production of 17β-hydroxysteroids was carried out under continuous flow conditions. For the flow system setup, the reactor (characterize in a general way) connected to the reactor pump (characterize in a general way) was used. The reaction was carried out by varying the temperature (range of 20-60°C) and the reaction time (5-120 minutes) to obtain the best conversion.

[0023] Under the tested conditions, the most efficient reduction was achieved using 15 minutes, 0.2 mol.L-1 of NaBH4 and 20°C with 100% conversion.

[0024] Similar evaluations were performed with the ketosteroid 3O-propyl Estrone. Tests were performed varying time (5-120 minutes), amount of Sodium Borohydride (0.1-0.5 mol / L-1) and temperature (20-60°C)

[0025] Under the tested conditions, the most efficient reduction was achieved using 30 minutes, 0.2 mol.L-1 of NaBH4 and 20°C as the process temperature with a conversion of 97%.

[0026] SYNTHESIS OF 3-O-PROPYL ESTRONE: The ketosteroid 3-O-propyl estrone was synthesized from the alkylation of the phenol group present in estrone. Estrone (2 mmol) was solubilized in 4 mL of dimethylformamide (DMF) with K2CO3 (6.5 mmol) under stirring and, after 10 minutes, bromopropane (3 mmol) was added and stirred for 20 h. At the end of the reaction, 10 mL of water and 25 mL of ethyl acetate were added. The organic phase was separated and washed with water (5 x 10 mL), dried with MgSO4 and after evaporation of the solvent a white solid was obtained with a yield of 82.1%.

[0027] ESTRONE REDUCTION: The ketosteroid estrone (0.1 mol.L-1, 0.8 mmol) was solubilized in 8 mL of THF and, separately, the reducing agent NaBH4 (0.2 mol.L-1, 1.6 mmol) was solubilized in 8 mL of ethanol. Syringes were filled with each solution and placed for injection under a flow rate of 0.0334 mL.min-1 (reaction time = 15 min, reactor volume = 1 mL). The reaction effluent was collected for 3 Petition 870250008349, dated 01 / 31 / 2025, page 14 / 25 The residence time (45 min) was 6 / 8 times that of the reactor filling and stabilization, and after this time, the product was collected in a concentrated 10 mL solution of NH4Cl. Then, the product (estradiol) was recovered by liquid-liquid extraction in ethyl acetate (3 x 10 mL), dried with MgSO4, and rotary evaporated. The conversion obtained was 100% with a yield of 98.5%.

[0028] REDUCTION OF 3-O-PROPYL ESTRONE: The ketosteroid 3-O-propyl estrone (0.1 mol.L-1, 0.8 mmol) was solubilized in 8 mL of THF and, separately, the reducing agent NaBH4 (0.2 mol.L-1, 1.6 mmol) was solubilized in 8 mL of ethanol. Syringes were filled with each solution and placed for injection at a flow rate of 0.0334 mL.min-1 (reaction time = 15 min, reactor volume = 1 mL). The reaction effluent was collected for 3 times the residence time (45 min) to fill and stabilize the reactor and, after this time, the product was collected in a concentrated solution of 10 mL of NH4Cl. Then, the product (estradiol 3-propyl ether) was recovered by liquid-liquid extraction on ethyl acetate (3 x 10 mL), dried with MgSO4 and rotary evaporated. The conversion obtained was 97.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 shows Scheme 1: Reduction of estrone using isopropanol and isopropylate.

[0030] Figure 2 presents Scheme 2: Estrone reduction via biotransformation with Fusarium oxysporum var. cubense and Colletotrichum musae.

[0031] Figure 3 shows Scheme 3: Microwave-assisted reduction of estrone via LIBD4.

[0032] Figure 4 shows Scheme 4: Reduction of estrone with sodium borohydride in ethanol.

[0033] Figure 5 shows Scheme 5: Reduction of estrone with sodium borohydride in basic medium and methanol.

[0034] Figure 6 shows Scheme 6: Estrone reduction in aqueous medium. Petition 870250008349, dated 01 / 31 / 2025, page 15 / 25 / 8

[0035] Figure 7 shows Scheme 7: Reduction with Rhodium catalyst via hydrosilylation.

[0036] Figure 8 shows Scheme 8: Rhodium catalyst.

[0037] Figure 9 presents Scheme 9: Stereospecific attack of borohydride on the carbonyl carbon of estrone.

[0038] Figure 10 shows Scheme 10: Estrone reduction reaction under continuous flow.

[0039] Figure 11 shows the reaction conditions for the reduction of estrone under continuous flow.

[0040] Figure 12 presents the Exploration of the reaction time and reduction of other ketosteroids under continuous flow. References 1. Ley SV Chem Rec. 2012;12(4):378-390. 2. Randall B. Barnes, Seth G. Levrant, Treatment of Postmenopausal Women (Third Edition), 2007, 767777 3. Vogg G., Souza CJH, Jaume CM & Moraes JCF 2004. Acta Scientiae Veterinariae. 32:41- 46 4. ]Del Pup, Linoa; Di Francia, Raffaeled; Cavaliere, Carlae; Facchini, Gaetanoe; Giorda, Giorgioa; De Paoli, Paoloc; Berretta, Massimilianob. Anticancer Drugs 2013, 24(10):p 989-998. 5. Kenneth J. Ryan; 1. Cancer Res August 1, 1982; 42 (8_Supplement): 3342s-3344s. 6. Edward A Doisy, Sydney A Thayer, Clement D Veler. US. Patent no. 1967350, 1934 7. Seiichi Takano, Kunio Ogasawara, US Patent No. 5424462, 1995 8. A. Call, C. Casadevall, F. Acuna-Parés, A. Casitas and J. Lloret-Fillol, Chem. Sci., 2017, 8, 4739-4749 9. Marker, ER, Rohrmann, EJ Am. Chem. Soc. 1938, 60, 12, 2927-2928 10. Maureen R. Wilson1, Winklet A. Gallimore, Paul B. Reese, Steroids. 64 , 1999 , 834 - 843 11. Paula S. Kiuru, Kristiina Wãhãlã, Steroids, Volume 71, Issue 1,2006, Pages 54-60 12. Animesh Aditya, David E. Nichols, and G. Marc Loudon J. Chem. Educ. 2008, 85, 11, 1535 Petition 870250008349, of 31 / 01 / 2025, p. 16 / 25 / 8 13. Bijia Wang, Linlin Qin, Kiel D. Neumann, ShriHarsha Uppaluri, Ronald L. Cerny, and Stephen G. DiMagno, Org. Lett. 2010, 12, 15, 3352-3355 14. Xiaoxue Cui, Bo Li, Tianzhen Liu and Chunbao Li, Green Chem., 2012,14, 668-672 15. Chinna Ayya Swamy P, Andrei Varenikov, and Graham de Ruiter, Organometallics 2020, 39, 2, 247-257 16. Philip C. Andrews, Tobias Beck, Benjamin H. Fraser, Peter C. Junk, Massimiliano Massi, Boujemaa Moubaraki, Keith S. Murray, Morry Silberstein, Polyhedron, 2009, 28, 2123-2130

Claims

1. An innovative process to minimize the number of handling operations of bioactive compounds within the production network, CHARACTERIZED by the production of hydroxysteroids via continuous flow reduction of ketosteroids, due to the fact that the system comprises: (a) production of hydroxysteroids by continuous flow reduction of ketosteroids using NaBH4 for the synthesis of organic compounds.

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