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CITRULLINE

December 20, 2020 1:15 pm / Leave a comment

CITRULLINE

CAS 372-75-8

L-Citrulline
瓜氨酸

Used for nutritional supplementation, also for treating dietary shortage or imbalance.

L-Citrulline

Molecular FormulaC₆H₁₃N₃O₃
Average mass175.186 Da

SYN

Hua Bai, Peijie Yang, Zhengjie Chen, Chongyan Xu, Zhaorul Li, Zigang Zhao, Luyan Jiang, Zongyi Yang, Jiang Li, “PROCESSES FOR THE PRODUCTION OF L-CITRULLINE.” U.S. Patent US20090142813, issued June 04, 2009.

US20090142813(S)-2-Amino-5-ureidopentanoic acid1725416[Beilstein]206-759-6[EINECS]372-75-8[RN]a-Amino-d-ureidovaleric Acid

Product Ingredients

INGREDIENT	UNII	CAS	INCHI KEY
Citrulline malate	PAB4036KHO	70796-17-7	DROVUXYZTXCEBX-WCCKRBBISA-N

CitrullineCAS Registry Number: 372-75-8
CAS Name:N5-(Aminocarbonyl)-L-ornithine
Additional Names: d-ureidonorvaline; a-amino-d-ureidovaleric acid; Nd-carbamylornithine
Molecular Formula: C6H13N3O3Molecular Weight: 175.19
Percent Composition: C 41.13%, H 7.48%, N 23.99%, O 27.40%Line Formula: H2NCONH(CH2)3CH(NH2)COOH
Literature References: An amino acid, first isolated from the juice of watermelon, Citrullus vulgaris Schrad., Cucurbitaceae: Wada, Biochem. Z.224, 420 (1930); isoln from casein: Wada, ibid.257, 1 (1933). Synthesis from ornithine through copper complexes: Kurtz, J. Biol. Chem.122, 477 (1938); by alkaline hydrolysis of arginine: Fox, ibid.123, 687 (1938); from cyclopentanone oxime: Fox et al.,J. Org. Chem.6, 410 (1941). Crystallization: Matsuda et al.,JP71 174 (1971 to Ajinomoto), C.A.74, 126056u (1971). Crystal and molecular structure: Naganathan, Venkatesan, Acta Crystallogr.27B, 1079 (1971); Ashida et al.,ibid.28B, 1367 (1972). Use in asthenia and hepatic insufficiency: FR2198739 (1974 to Hublot & Vallet), C.A.82, 144952c (1975). Clinical trial in treatment of lysinuric protein intolerance: J. Rajantie et al.,J. Pediatr.97, 927 (1980); T. O. Carpenter et al.,N. Engl. J. Med.312, 290 (1985).Properties: Prisms from methanol + water, mp 222°. [a]D20 +3.7° (c = 2). pK1 2.43; pK2 9.41. Sol in water. Insol in methanol, ethanol.Melting point: mp 222°pKa: pK1 2.43; pK2 9.41Optical Rotation: [a]D20 +3.7° (c = 2) Derivative Type: HydrochlorideCAS Registry Number: 34312-10-2Molecular Formula: C6H13N3O3.HClMolecular Weight: 211.65Percent Composition: C 34.05%, H 6.67%, N 19.85%, O 22.68%, Cl 16.75%Properties: Crystals, dec 185°. [a]D22 +17.9° (c = 2).Optical Rotation: [a]D22 +17.9° (c = 2) Derivative Type: Malate (salt)CAS Registry Number: 54940-97-5Trademarks: Stimol (Biocodex)Molecular Formula: C6H13N3O3.C4H6O5Molecular Weight: 309.27Percent Composition: C 38.84%, H 6.19%, N 13.59%, O 41.39% Therap-Cat: Treatment of asthenia.

Asklepion is developing an iv formulation of citrulline, Citrupress, for the potential treatment of pulmonary hypertension and for the potential prevention of clinical sequelae of acute lung injury complicating congenital heart repair surgery in pediatric patients, and also investigating the drug for the potential treatment of acute sickle cell crisis. In August 2016, a phase III study was initiated for preventing clinical sequelae of acute lung injury?in pediatric patients undergoing cardiopulmonary bypass (CPB) for heart defects; in July 2019, results were expected in October 2019.

Citrulline is an amino acid. It is made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine as a by-product of the reaction catalyzed by NOS family. Its name is derived from citrullus, the Latin word for watermelon, from which it was first isolated.

The organic compound citrulline is an α-amino acid.^[2] Its name is derived from citrullus, the Latin word for watermelon. Although named and described by gastroenterologists since the late 19th century, it was first isolated from watermelon in 1914 by Japanese researchers Yotaro Koga and Ryo Odake^[3]^{[note 1]} and further codified by Mitsunori Wada of Tokyo Imperial University in 1930.^[4] It has the formula H₂NC(O)NH(CH₂)₃CH(NH₂)CO₂H. It is a key intermediate in the urea cycle, the pathway by which mammals excrete ammonia by converting it into urea. Citrulline is also produced as a byproduct of the enzymatic production of nitric oxide from the amino acid arginine, catalyzed by nitric oxide synthase.^[5]

Biosynthesis

Citrulline is made from ornithine and carbamoyl phosphate in one of the central reactions in the urea cycle. It is also produced from arginine as a byproduct of the reaction catalyzed by NOS family (NOS; EC 1.14.13.39).^[6] It is made from arginine by the enzyme trichohyalin at the inner root sheath and medulla of hair follicles.^[7] Arginine is first oxidized into N-hydroxyl-arginine, which is then further oxidized to citrulline concomitant with release of nitric oxide.

Citrulline is also made by enterocytes of the small intestine.^[2]^[8]

Function

Several proteins contain citrulline as a result of a posttranslational modification. These citrulline residues are generated by a family of enzymes called peptidylarginine deiminases (PADs), which convert arginine into citrulline in a process called citrullination or deimination with the help of calcium ion. Proteins that normally contain citrulline residues include myelin basic protein (MBP), filaggrin, and several histone proteins, whereas other proteins, such as fibrin and vimentin are susceptible to citrullination during cell death and tissue inflammation.

Circulating citrulline concentration is a biomarker of intestinal functionality.^[9]^[10

PAPER

Biochemistry, 53(41), 6511-6519; 2014

PAPER

Journal of the Chemical Society of Pakistan, 34(2), 451-454; 2012

PAPER

Journal of Agricultural and Food Chemistry, 66(33), 8841-8850; 2018

https://pubs.acs.org/doi/10.1021/acs.jafc.8b02858

l-Citrulline is a nonessential amino acid with a variety of physiological functions and can be enzymatically produced by arginine deiminase (ADI, EC 3.5.3.6). The enzymatic-production approach is of immense interest because of its mild conditions, high yield, low cost, and environmental benignity. However, the major hindrances of l-citrulline industrialization are the poor thermostability and enzyme activity of ADI. Hence, in this work, directed evolution and site-directed mutagenesis aided with in silico screening, including the use of b-factor values and HoTMuSiC, were applied to a previously identified ADI from Enterococcus faecalis SK23.001 (EfADI), and a triple-site variant R15K–F269Y–G292P was obtained. The triple-site variant displays a 2.5-fold higher specific enzyme activity (333 U mg^–1), a lower K_m value of 6.4 mM, and a 6.1-fold longer half-life (t_1/2,45°C = 86.7 min) than wild-type EfADI. This work provides a protein-engineering strategy to improve enzyme activity and thermostability, which might be transferrable to other ADIs and enzymes.

PAPER

ACS Sustainable Chemistry & Engineering, 7(9), 8522-8529; 2019

https://pubs.acs.org/doi/10.1021/acssuschemeng.9b00301

Biocatalytic transformation of carbamate formed readily from CO₂ and NH₃ provides attractive green routes for mitigation of these important environmental pollutants. Accordingly, a coupled-enzyme system was developed for the one-pot production of citrulline through carbamoylation of ornithine in aqueous solutions of CO₂ and NH₃. Hyperthermophilic ornithine carbamoyltransferases are produced recombinantly in E. coli with carbamate kinases known to have a propensity for carbamoyl phosphate synthesis. Importantly, in vitro biocatalysis is carried out by E. coli cell lysate prepared through coexpression of the required recombinant enzymes in a single bacterial culture, greatly reducing limitations normally associated with protein production and purification. Acetate kinase that is endogenous in the lysate also recycles the required ATP cofactor, which would otherwise have been required in costly stoichiometric amounts. Recombinant lysates catalyze the production of carbamoyl phosphate with substoichiometric ATP (>300 turnovers) as well as its in situ reaction with ornithine to give citrulline in high yield (>95%) and g L^–1 h^–1 titers. The system is active over a wide range of NH₃ concentrations (2.5 mM – 2 M), and >90% conversions of NH₃ may be reached within 1.5 h. Aqueous NH₃ used to sequester CO₂ gas (10% v/v) may be directly used as the biocatalyst feedstock. In preliminary studies, citrulline is found to be an effective organic nitrogen fertilizer of the wheat grass Brachypodium distachyon. Therefore, lysates described here constitute a cost-effective biocatalytic platform for one-pot production of a promising organic nitrogen fertilizer, under mild reaction conditions, from environmental pollutants as feedstock.

PATENT

WO 2015050276

https://patents.google.com/patent/WO2015050276A1/en

PATENT

WO2018125999 claiming method for maintaining the coupling of endothelial nitric oxide synthase.

PATENT

WO-2020247853

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020247853&tab=PCTDESCRIPTION&_cid=P11-KIWJPR-87833-1

Process for preparing citrulline from a transition metal complex of ornithine using cyanate useful to reduce the incidence or severity of cardiopulmonary bypass-induced pulmonary injury due to free radical formation in a patient during cardiopulmonary bypass.

Ornithine is an alpha amino acid with a terminal amino group opposite the alpha carbon.

Citrulline is an alpha amino acid with a terminal carbamido group in the same position as the terminal amino group of ornithine. Dr. A. Kurtz described synthesis of racemic citrulline from racemic ornithine in 1938 (J. Biol. Chem., 122:477-484), and that disclosure was followed up by synthesis of optically active /-citrulline from /-ornithine in 1949 (J. Biol. Chem., 180: 1253-1267). Optical activity was preserved by complexing the starting material (/-ornithine) in a transition metal complex via the alpha amino and carboxyl groups, then reacting the terminal amino group with urea to from a carbamido derivative (see Figure 1). Kurth 1949 describes numerous other syntheses, all depending on the transition metal complex to preserve the alpha amino acid character of the starting compound while derivatizing other parts of the molecule. An example of this synthesis is described in Example 1 below.

Details of various steps in the improved processes developed by the present inventors for producing pharmaceutical grade citrulline are discussed below.

Synthesis of Citrulline from Ornithine

[00014] The present inventors preserved the stereochemical structure around the alpha carbon of the alpha amino acid during reaction of amino groups elsewhere on the compound by complexing the alpha end of the molecule with a transition metal atom, as reported by

Kurth 1938 and 1949. The initial production of the /-ornithine-copper complex is carried out as described by Kurtz. Kurtz describes a variety of transition metals as the complexing metal in the 1949 paper, but the preferred metal is copper (II), based on the ease of forming stable complexes and the ease with which copper (II) may subsequently be removed from the product. The copper is typically supplied as cupric sulfate, although complex formation from copper (II) acetate, cupric carbonate, or cupric oxide have also been reported.

[00015] The present inventors have discovered an alternative method of derivatizing the terminal amino group of the complexed alpha amino acid using cyanate rather than the urea reaction reported by Kurth. An example of this improved synthesis is shown in Figure 2A and described in Example 3 below. Use of cyanate as the derivatizing agent has been found to produce fewer distinct product compounds, which simplifies purification of the desired citrulline product. Kurth carried out urea derivatization by refluxing the copper complex in the presence of excess urea. Cyanate derivatization may be carried out at lower temperatures (e.g. 55°C-65°C) which may contribute to higher yield of citrulline, based on the initial amount of ornithine. Cyanate is preferably provided in excess, and the reaction is driven by precipitation of the citrulline: copper complex. The precipitated complex is washed with water to remove unreacted copper (e.g., wash until no blue coloration persists in the filtrate). The precipitated copper complex of citrulline may be recovered and dried.

Enriching Citrulline as a Copper Complex

[00016] The inventors have discovered that the relative citrulline content of the reaction

product(s) can be enhanced by reprecipitation of the citrulline: copper complex. Precipitated copper complex of citrulline (produced, for example, by reaction of a ornithine: copper complex with urea or cyanate in water) may be dried. The

citrulline: copper complex may be redissolved by suspending the precipitate in water and acidifying the suspension until the complex dissolves. Acidification may be

accomplished by adding concentrated acid, preferably hydrogen chloride, to the suspension while stirring. Once the copper: citrulline complex solution is clear, base (typically sodium hydroxide) is added to bring the pH up to 7-10. Both the acidification and subsequent neutralization steps are actively cooled (temperature not more than 45°C) to protect the citrulline product from hydrolysis or reaction to produce side products. The precipitate is washed with water (e.g., until the filtrate is free of chloride by checking the filtrate for turbidity with silver nitrate), and then the precipitate is dried. Reprecipitation under these conditions is selective for citrulline: copper complex over ornithine: copper complex, because the ornithine complex is more soluble in water. If the dried complex contains higher than the desired level of ornithine contamination (e.g., greater than 10 mole% ornithine – as measured by NMR, for example), the complex may be redissolved and reprecipitated as necessary to further lower the relative amount of ornithine.

Recovering Citrulline from Its Copper Complex

[00017] Once the ornithine content in the copper: citrulline complex precipitate is sufficiently low

(preferably less than 10 mole% ornithine), the precipitate is resuspended in water and citrulline is freed from the complex by removing the copper as an inorganic precipitate, typically copper sulfide (See Figure 2B). Sulfide may be introduced in a variety of salt forms, but the inventors have found it preferable to use hydrogen sulfide gas as the sulfide source. In a preferred mode, the aqueous suspension is placed in a stirred, pressure vessel. The air is then pumped out of the reactor’s head space to form an under pressure. The reactor is then repressurized with hydrogen sulfide gas over the aqueous suspension (preferably at low temperature, e.g., 0°C-5°C, to maximize the solubility of hydrogen sulfide). Hydrogen sulfide is continuously added to the reactor to maintain parity with ambient pressure during consumption of this gas. Copper salts will precipitate, leaving citrulline in solution. As hydrogen sulfide is consumed, the pressure in the vessel decreases; the reaction is complete when the pressure stabilizes. Reaction of hydrogen sulfide with residual copper salts (for example chloride or sulfate) will lower the pH; typically the pH will be below 4, preferably pH~3. Copper salts typically include copper (II) sulfide, but may also include copper (I) sulfide and copper oxide. The solution temperature is elevated for filtration, typically to about 30°C, to promote solubility of the citrulline and drive off excess hydrogen sulfide gas, while precipitated copper salts are removed by filtration.

Purifying Citrulline

[00018] For pharmaceutical use, the active compound must be substantially free of contaminants, and further purification steps are necessary to produce a pharmaceutical grade product. For the purposes of this invention, substantially free of contaminants is considered to include: ornithine not more than (NMT) 0.8%, individual specified impurities NMT 0.15%, individual unspecified (unknown) impurities NMT 0.1%; total related substances NMT 1.3%, and Cu not more than lOppm. For citrulline manufactured from ornithine using copper complex to protect the alpha amino acid functions, the inventors have found that desired purification after citrulline is released from the copper complex can be achieved by activated carbon adsorption of contaminants and solvent/anti- solvent crystallization of the active pharmaceutical component.

[00019] The citrulline-containing aqueous solution remaining after removal of precipitated copper salts is neutralized to stabilize the citrulline against hydrolysis, to enhance adsorption of residual copper to activated carbon, and to facilitate solvent/anti-solvent precipitation of citrulline; pH is preferably adjusted to 5.9 ± 0.2, the isoelectric point of citrulline. The neutralized citrulline solution may be passed through a nano-filter to remove any bacteria and/or bacterial cell wall fragments that contaminate the solution. The nano-filtered solution may be held in a semi-sterile reservoir for staging purposes between the subsequent purification steps. The neutralized citrulline solution is treated with activated carbon, either by mixing with carbon dust or passing the solution through an activated carbon adsorber bed. The aqueous citrulline-containing effluent from the activated carbon is mixed with an anti-solvent to induce anti-solvent crystallization. Suitable anti solvents are miscible with water, including aliphatic alcohols, such as 2-propanol, ethanol or methanol, as well as acetone. A preferred antisolvent for citrulline is acetone, when mixed with approximately two volumes of water (e.g., 1 volume of water to 1.8 volumes of acetone). Acetone is preferably pre-cooled so that the resultant suspension is 0°C- 10°C. The cooled suspension may be collected in a reservoir or processed by filtration immediately to recover the citrulline precipitate.

Microbial control:

[00020] Because citrulline synthesis and purification occur in aqueous solution, there is increased risk of microbial contamination and endotoxin accumulation in the product. Washing the citrulline: copper precipitate, and addition of H2S to acid solution minimize any accumulation of microbes. From the exposure of the complex to FES until treatment with acetone the aqueous solutions of citrulline are preferably kept in sealed vessels to limit microbial contamination and growth. Enclosing the purification steps to minimize contact with the environment and use of sterile filters to capture potential microbial contamination allows the manufacturing to be performed in an ISO 8 cleanroom. Alternatively, the final purification steps can be carried out in a sterile GMP environment of the sort used for aseptic filling of sterile dosage products (e.g., ISO Class 5/6).

[00021] If examination of the solution prior to the anti-solvent precipitation shows the amounts of microbes or endotoxin levels exceed those aceptable for injectable therapeutic compositions (e.g., 50 EU/g API, more preferably 20 EU/g), the product may be subjected to nano-filtration to remove microbes and endotoxin, before being recovered by anti-solvent precipitation and drying. The citrulline and water molecules pass through the nano-filtration membrane, but the larger bacteria and bacterial cell wall fragments are retained by the filter.

Filter press

[00022] The reaction mixtures may be pumped through a filter press to collect / remove the

suspended solids. See the general picture in Figure 3, and the attached photograph in Figure 4. The press is composed of a series of plates 1 which are then hydraulically pressed together. The hydraulic pressure ensures that the system is sealed. The suspension is then pumped through a central tube 2 where it spreads-out across several chambers 3 between the plates. The walls of the plates have a filter sheet, which allows the filtrate to flow past and exit via an internal cavity 4.

[00023] The general advantage of a filter press is that it allows a high surface area for filtration.

This effect greatly accelerates the portion-wise collection and washing of the complex and API. This system may be used to collect the copper salts after exposure to hydrogen sulfide. In the latter case, the suspension is pumped from the reactor into the press, and the filtrate may then be passed through an in-line 5 pm filter to catch any residual particulate copper, then an in-line sterile 0.2 pm filter at the entry port of a semi-sterile container for holding.

The press may be used to collect:

• Crude citrulline copper complex

• The complex after the pH-driven re-precipitation

• Precipitated copper salts (where citrulline leaves as solution in the filtrate)

• Precipitated citrulline from anti-solvent precipitation prior to drying

Semi-sterile containers

[00024] A useful semi-sterile container is basically a closed vessel equipped with a stirrer and ports for the addition and removal of liquid, and a pH meter. The container should be sterilized (e.g., treated with isopropyl alcohol solution and rinsed with water) directly prior to use and not opened during use. A sterile, air filter attached to the lid allows air to flow into the container as the liquid is being pumped out. The pH adjustment may be performed in this container, before treatment with activated carbon. The container is not particularly suitable for the long-term storage of the solutions.

Activated carbon adsorber bed

[00025] The solution may be pumped from the semi sterile container through the activated carbon bed (a column packed with granulated activated carbon) pre-flushed with argon. The liquid is then returned to the semi-sterile container via an in-line 5 pm filter and the 0.2 pm sterile filter at the entry port. If the solution is pumped in a cyclic manner with the stirrer activated for not less than 6 hours, the sterile filter acts as a“microbial scrubber” continually collecting any microbes in the solution. The activated carbon primarily removes any organic impurities and will also remove any residual dissolved copper ions. The 5 pm filter catches any carbon particles which detach from the bed.

Sterile bags

[00026] After processing in the activated carbon adsorber bed, the solution may be passed into a single use sterile bag via another sterile filter. The solution may be stored longer in the bag than in the semi-sterile container. At this point, a test for the presence of microbes and/or bacterial endotoxins can be carried out. If endotoxins are observed, then the cut off (nano-filtration) membrane may be employed. If not, the citrulline is ready to be

recovered from the solution by anti-solvent precipitation. Collection of the solution in a sterile bag allows the citrulline solution to be processed batch-wise, where conveniently sized portions of citrulline are precipitated and recovered in the filter press.

Solvent/Anti-solvent Mixing

[00027] The aqueous citrulline solution is mixed with pre-cooled anti-solvent to precipitate the citrulline from solution. After mixing with anti-solvent, the threat posed by bacterial growth is not higher than that for other APIs. The addition of the organic solvent makes the resulting solution bacteriostatic at a minimum. This precipitation improves the purity of citrulline, reducing, in particular, the ornithine levels, and allows for the rapid extraction of citrulline from solution.

Final drying

[00028] The precipitate is dried to remove residual acetone and water. Drying may be carried-out in a conical dryer, firstly to drive off the acetone anti-solvent, then moisture and finally the water of crystallization. The conical dryer can also be used to homogenize the product. The final, dry product of anti-solvent precipitation may be stored, and ultimately dissolved in sterile aqueous diluent for therapeutic administration.

[00029] On dissolution in sterile aqueous media, citrulline prepared as described herein may be used to treat pulmonary hypertension (WO/2000/073322), bronchopulmonary dysplasia (WO/2009/099998), sickle cell crisis (WO/2018/157137), cardiac surgery patients (WO/2005/082042), cardiopulmonary bypass patients (WO/2018/125999), and vasospasm as a complication of subarachnoid hemorrhage (WO/2009/099999), by parenteral administration as described in these documents, incorporated herein by reference.

EXAMPLES

Example 1. Synthesis of citrulline from ornithine using urea.

[00030] L-Citrulline is synthesized from L-omithine and urea. A flow chart of the reaction is shown in Figure 1 A.

[00031] L-Citrulline is prepared synthetically starting from L-ornithine hydrochloride. Into a 120- L reactor containing approximately 50 liters of water, 10 kilograms of L-omithine hydrochloride is added and dissolved. The solution is neutralized with potassium hydroxide and then converted to its copper complex by the addition of 15kg copper sulfate (molar equivalent amount). The copper complex protects the 2-amino carboxylic acid functionality in the molecule while chemistry is performed on the terminal amino group. The L-ornithine copper complex is then exposed to an excess of urea at reflux, which promotes its conversion to the copper complex of L-citrulline. The resulting copper complex of L-citrulline then is precipitated and collected by filtration.

[00032] The isolated copper complex of L-citrulline is dried and testing is performed. The

appearance is verified, and an in-use performance test is done to determine suitability to proceed.

Example 2. Purification of citrulline from copper-citrulline complex.

[00033] L-Citrulline synthesized from L-ornithine and urea is purified by resin-based purification and recrystallization. A flow chart of the reaction is shown in Figure IB.

[00034] In a 120-L reactor, ~13 kilograms of the L-citrulline copper complex prepared in

Example 1 is added to a stirring solution of sodium sulfide (Na2S) in water

(approximately 8 kilograms Na2S in 50 liters of water), causing the precipitation of copper sulfide and the freeing of L-citrulline. The solution is filtered to remove the copper salts. The pH of the resulting aqueous solution containing the sodium salt of L- citrulline and residual sodium sulfide is lowered to 4 by the addition of an acidic ion exchange resin (such as Amberlite™). A constant stream of argon gas is passed through the solution to remove the residual sulfide as hydrogen disulfide. The pH of the solution is then raised to 5.9 ± 0.2 using sodium hydroxide to form isoelectric L-citrulline.

Activated carbon is then added to the reaction mixture to remove residual impurities, in particular residual copper ions. The solids (Amberlite™ and activated carbon) are then removed by filtration, and the filtrate is concentrated to approximately 50 liters (either by evaporation or reverse osmosis). L-citrulline is then precipitated from the aqueous solution by the addition of an equal part of acetone, and the mixture is cooled to near 0°C. The precipitate is collected by filtration and dried in a vacuum oven.

[00035] The non-sterile bulk powder is then reconstituted and processed for endotoxin reduction and sterile filtration steps followed by crystallization, drying and micronization in an aseptic environment. The sterile bulk powder is then used as the“raw material” for aseptic filling into glass vials to produce the finished drug product which may be reconstituted with a sterile diluent prior to use.

Example 3. Synthesis of citrulline from ornithine using cyanate

[00036] L-Citrulline was prepared synthetically starting from L-omithine hydrochloride. Into a reactor containing sodium hydroxide (11 kg) in water (170 kg), L-ornithine hydrochloride (44 kg) was added and dissolved. The temperature was maintained at no more than 40°C by active cooling. The ornithine was then converted to its copper complex by the addition of 0.5 molar equivalents of copper sulfate (33 kg) and stirring at ambient temperature for more than 15 minutes. The copper complex protects the 2-amino carboxylic acid functionality of the molecule while chemistry is performed on the terminal amino group. A molar excess of potassium cyanate (32 kg) is then added to the L-ornithine copper complex, and the solution is held at 55°C-65°C for 4.0-4.5 hours, which promotes its conversion to the copper complex of L-citrulline. The resulting copper complex of L-citrulline precipitates during the reaction, and it is collected by filtration.

Example 4. Purification of therapeutic grade citrulline.

[00037] The dry copper: citrulline complex produced in Example 3 is added to a reactor

containing water, which is stirred to resuspend the complex. Concentrated hydrogen chloride solution is added to convert the complex into a solution of copper (II) chloride and citrulline hydrochloride, while the temperature of the reactor is maintained at no more than 45°C by active cooling. Once the contents of the reactor are in solution, sodium hydroxide is added to raise the pH to 7-10, while the temperature is maintained at no more than 40°C. The copper complex of citrulline then precipitates. The precipitate is collected and washed with water until no blue coloration persists in the filtrate.

[00038] The washed precipitate is tested to determine the relative ornithine content. If ornithine is greater than 10 mole%, the precipitate is redissolved and resuspended as described above, until the ornithine content is lowered to not more than 10 mole%.

[00039] Once the precipitate achieves the desired ornithine content, it is resuspended in water in a stirred reactor, and hydrogen sulfide gas is introduced into the suspension to precipitate copper sulfide and dissolve citrulline. The solution is warmed to 30°C ± 2°C to ensure citrulline is fully solubilized, and precipitated copper salts are removed by filtration. The citrulline-containing filtrate is passed thorough micro- and sterile-filtrations and collected in a semi-sterile reactor.

[00040] Activated carbon is used to remove residual impurities, in particular an organic

component and residual copper ions. The pH of the resulting aqueous solution containing L-citrulline and residual copper is adjusted to 5.9 ± 0.2 with sodium hydroxide to form isoelectric citrulline solution. The isoelectric citrulline solution is treated with active carbon granules, preferably by passing the solution through an active carbon adsorber bed, and passed through micro and sterile filters after the active carbon treatment.

[00041] L-citrulline is then precipitated from the aqueous solution by the addition of acetone anti solvent, and the mixture is cooled to near 0°C. Addition of 1.5 to 2 volume equivalents of acetone produce dihydrate crystals of citrulline. The precipitate is collected by filtration. The crystals are dried in a vacuum in a conical dryer at temperature of no more than 45°C to remove acetone and water, resulting in an anhydrous crystalline solid. This solid citrulline corresponds to the orthorhombic d form anhydrous crystals reported by Allouchi, et al., 2014 ( Cryst . Growth Des., 14: 1279-1286).

[00042] Either the dihydrate crystals or the anhydrous crystals may be used therapeutically. The solid or an aqueous solution/suspension may be administered enterally, or the solid may be redissolved for parenteral administration. To produce a final therapeutic product, the non-sterile bulk powder was reconstituted and underwent endotoxin reduction and sterile filtration steps followed by crystallization, drying and micronization in an aseptic environment. The sterile bulk powder was then used as the“raw material” for aseptic filling into glass vials to produce the finished drug product which was reconstituted with a sterile diluent prior to use.

References

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^ “Nos2 – Nitric Oxide Synthase”. Uniprot.org. Uniprot Consortium. Retrieved 10 February 2015.
^ Cox M, Lehninger AL, Nelson DR (2000). Lehninger principles of biochemistry (3rd ed.). New York: Worth Publishers. p. 449. ISBN 978-1-57259-153-0. Retrieved 13 March 2020.
^ Rogers, G. E.; Rothnagel, J. A. (1983). “A sensitive assay for the enzyme activity in hair follicles and epidermis that catalyses the peptidyl-arginine-citrulline post-translational modification”. Current Problems in Dermatology. 11: 171–184. doi:10.1159/000408673. ISBN 978-3-8055-3752-0. PMID 6653155.
^ DeLegge, Mark H. (2019-01-01), Corrigan, Mandy L.; Roberts, Kristen; Steiger, Ezra (eds.), “Chapter 7 – Enteral Access and Enteral Nutrition in Patients With Short Bowel Syndrome”, Adult Short Bowel Syndrome, Academic Press, pp. 81–96, doi:10.1016/b978-0-12-814330-8.00007-x, ISBN 978-0-12-814330-8, retrieved 2020-11-10
^ Fragkos, Konstantinos C.; Forbes, Alastair (2017-10-12). “Citrulline as a marker of intestinal function and absorption in clinical settings: A systematic review and meta-analysis”. United European Gastroenterology Journal. 6 (2): 181–191. doi:10.1177/2050640617737632. PMC 5833233. PMID 29511548.
^ Crenn, P.; et al. (2000). “Post-absorptive plasma citrulline concentration is a marker of intestinal failure in short bowel syndrome patients”. Gastroenterology. 119 (6): 1496–505. doi:10.1053/gast.2000.20227. PMID 11

Names


IUPAC name2-Amino-5-(carbamoylamino)pentanoic acid^[1]
Identifiers
CAS Number	627-77-0^[SciFinder]13594-51-9 R^[SciFinder]372-75-8 S
3D model (JSmol)	Interactive image
3DMet	B01217
Beilstein Reference	1725417, 1725415 R, 1725416 S
ChEBI	CHEBI:18211
ChEMBL	ChEMBL444814
ChemSpider	810553200 R9367 S
DrugBank	DB00155
ECHA InfoCard	100.006.145
EC Number	211-012-2
Gmelin Reference	774677 S
IUPHAR/BPS	722
KEGG	D07706
MeSH	Citrulline
PubChem CID	833 637599 R9750 S
UNII	29VT07BGDA
CompTox Dashboard (EPA)	DTXSID80883373
InChI [show]
SMILES [show]
Properties
Chemical formula	C₆H₁₃N₃O₃
Molar mass	175.188 g·mol⁻¹
Appearance	White crystals
Odor	Odourless
log P	−1.373
Acidity (pK_a)	2.508
Basicity (pK_b)	11.489
Thermochemistry
Heat capacity (C)	232.80 J K⁻¹ mol⁻¹
Std molar entropy (S^o₂₉₈)	254.4 J K⁻¹ mol⁻¹
Related compounds
Related alkanoic acids	N-Acetylaspartic acid Aceglutamide N-Acetylglutamic acid Pivagabine
Related compounds	Bromisoval Carbromal
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).
verify (what is ?)
Infobox references

///////CITRULLINE, L-Citrulline, 瓜氨酸 ,

Melatonin

December 14, 2020 1:35 am / Leave a comment

Melatonin

メラトニン

Formula	C13H16N2O2
CAS73-31-4	73-31-4
Mol weight	232.2783

APPROVED, Melatobel, JAPAN 2020/3/25

200-659-6[EINECS]

200-797-7[EINECS]

205542[Beilstein]

73-31-4[RN]

Acetamide, N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-N-[2-(5-methoxy-1H-indol-3-yl)ethyl]-Acetamide

MelatoninCAS Registry Number: 73-31-4CAS Name:N-[2-(5-Methoxy-1H-indol-3-yl)ethyl]acetamide

Additional Names:N-acetyl-5-methoxytryptamine

Trademarks: Regulin (Young)

Molecular Formula: C13H16N2O2Molecular Weight: 232.28Percent Composition: C 67.22%, H 6.94%, N 12.06%, O 13.78%

Literature References: A hormone of the pineal gland, also produced by extra-pineal tissues, that lightens skin color in amphibians by reversing the darkening effect of MSH, q.v. Melatonin has been postulated as the mediator of photic-induced antigonadotrophic activity in photoperiodic mammals and has also been shown to be involved in thermoregulation in some ectotherms and in affecting locomotor activity rhythms in sparrows. Isoln from the pineal glands of beef cattle: Lerner et al.,J. Am. Chem. Soc.80, 2587 (1958); Wurtman et al.,Science141, 277 (1963). Structure: Lerner et al.,J. Am. Chem. Soc.81, 6084 (1959). Crystal and molecular structure: A. Wakahara, Chem. Lett.1972, 1139. Synthesis from 5-methoxyindole as starting material by two different routes: Szmuszkovicz et al.,J. Org. Chem.25, 857 (1960). Biochemical role of melatonin: Chem. Eng. News45, 40 (May 1, 1967). Pharmacological studies: Barchas et al.,Nature214, 919 (1967). Identification of antigonadal action sites in mouse brain: J. D. Glass, G. R. Lynch, Science214, 821 (1981). Binding studies in human hypothalamus: S. M. Reppert et al.,Science242, 78 (1988). Efficacy in control of estrus in red deer: G. W. Asher, Anim. Reprod. Sci.22, 145 (1990). Reviews: M. K. Vaughn, Int. J. Rev. Physiol.24, 41-95 (1981); D. C.Klein et al.,Life Sci.28, 1975-1986 (1981). Book: Advan. Biosci.vol. 29, N. Birau, W. Schlott, Eds. (Pergamon Press, New York, 1981) 420 pp. Review of etiological role in clinical disease: A. Miles, D. Philbrick, Crit. Rev. Clin. Lab. Sci.25, 231-253 (1987); in psychiatric disorders: eidem,Biol. Psychiatry23, 405-425 (1988).Properties: Pale yellow leaflets from benzene, mp 116-118°. uv max: 223, 278 nm (e 27550, 6300).Melting point: mp 116-118°Absorption maximum: uv max: 223, 278 nm (e 27550, 6300)Therap-Cat-Vet: Control of estrus.

Melatonin is a hormone primarily released by the pineal gland that regulates the sleep–wake cycle.^[3]^[4] As a dietary supplement, it is often used for the short-term treatment of insomnia, such as from jet lag or shift work, and is typically taken by mouth.^[5]^[6]^[7] Evidence of its benefit for this use, however, is not strong.^[8] A 2017 review found that sleep onset occurred six minutes faster with use, but found no change in total time asleep.^[6] The melatonin receptor agonist medication ramelteon may work as well as melatonin supplements,^[6] at greater cost but with different adverse effects, for some sleep conditions.^[9]

Side effects from melatonin supplements are minimal at low doses for short durations.^[3]^[10] They may include somnolence (sleepiness), headaches, nausea, diarrhea, abnormal dreams, irritability, nervousness, restlessness, insomnia, anxiety, migraine, lethargy, psychomotor hyperactivity, dizziness, hypertension, abdominal pain, heartburn, mouth ulcers, dry mouth, hyperbilirubinaemia, dermatitis, night sweats, pruritus, rash, dry skin, pain in the extremities, symptoms of menopause, chest pain, glycosuria (sugar in the urine), proteinuria (protein in the urine), abnormal liver function tests, increased weight, tiredness, mood swings, aggression and feeling hungover.^[11]^[12]^[10]^[13]^[14] Its use is not recommended during pregnancy or breastfeeding or for those with liver disease.^[7]^[14]

In animals (including humans), melatonin is involved in synchronizing the circadian rhythm, including sleep–wake timing, blood pressure regulation, and seasonal reproduction.^[15] Many of its effects are through activation of the melatonin receptors, while others are due to its role as an antioxidant.^[16]^[17]^[18] In plants, it functions to defend against oxidative stress.^[19] It is also present in various foods.^[10]

Melatonin was discovered in 1958.^[3] It is sold over the counter in Canada and the United States;^[10]^[13] in the United Kingdom, it is a prescription-only medication.^[7] It is not approved by the US Food and Drug Administration (FDA) for any medical use.^[10] In Australia and the European Union, it is indicated for difficulty sleeping in people over the age of 54.^[20]^[11] In the European Union, it is indicated for the treatment of insomnia in children and adolescents.^[12] It was approved for medical use in the European Union in 2007.^[11]

SYN

https://www.ch.ic.ac.uk/local/projects/s_thipayang/synth.html

SYNTHESIS

Chemical Synthesis of Melatonin
The methods for the chemical synthesis of melatonin are generally not so complicated and do not involve more than three steps of conversion. Three synthesis reactions of melatonin from primary literatures are shown below;

Reaction 1

In 1958 melatonin was first isolated and characterised by A.B.Lerner. It was know as one of a substituted 5-hydroxyindole derivative in the pineal gland that could lighten pigment cells. It had not been know to exist in biological tissue although it had been isolated as a urinary excretion product in rats after administration of 5-hydroxytryptamine.
Melatonin or N-acetyl-5-methoxytryptamine (40 mg) was prepared by reducing 100 mg of 5-methoxyindole-3-acetonitrile with 160 mg of sodium and 2 ml of ethanol. Then the product was acetylated with 4 ml of both glacial acetic acid and acetic anhydride at 100 ^oC for 1 minute. Purification was achieved by countercerrent distribution and silicic acid chromatography.

Reaction 2

5-Methoxytryptamine hydrochloride (1g, 4.75 mmole) was dissolved in pyridine (10 ml) and acetic anhydride (10 ml) and kept overnight at 20 ^oC. The solution was poured onto iced, neutralised with dilute hydrochloric acid and extracted with chloroform (2×25 ml). The combined extracts were washed with water, dried in MgSO₄ and evaporated to afford a liquid of N,N diacetyltryptamine derivative. The liquid was then poured into water (50 ml) and extracted with chlroform (2×25 ml). The combined organic layers were washed with water (25 ml), dried in MgSO₄ and evaporated to dryness. The residual solid crystallised from benzene to afford melatonin 819 mg, 80% yield.

Reaction 3

The more reactive indoles (1a-1d) were alkylated at the 3 position by reaction with nitroethene generated in situ by thermolysis of nitroethyl acetate. The nitroethyl acetate used for this purpose was prepared by acetylation of nitroethanol with acetic anhydride using NaOAc as a catalyst. These conditions constitute a substantial improvement of the overal yield of the reation. Reduction of the nitroethylated indoles (2a-d) by hydrogenation over PtO₂, followed by acetylation fo the resluting tryptamines with acetic anhydride-pyridine completed the synthesis of melatonin and its derivatives (4a-d).

Biological Synthesis and Metabolism of Melatonin

The biosynthesis of melatonin (Fig.1) is initiated by the uptake of the essential amino acid tryptophan into pineal parenchymal cells. Tryptophan is the least abundant of essential amino acids in normal diets. It is converted to another amino acid, 5-hydroxytryptophan, through the action of the enzyme tryptopahn hydroxylase and then to 5-hydroxytryptamine (serotonin) by the enzyme aromatic amino acid decarboxylase. Serotonin concentrations are higher in the pineal than in any other organ or in any brain region. They exhibit a striking diurnal rhythm remaining at a maximum level during the daylight hours and falling by more than 80% soon after the onset of darkness as the serotonin is converted to melatonin, 5-hydroxytryptophol and other methoxyindoles. Serotonin’s conversion to melatonin involves two enzymes that are characteristic of the pineal : SNAT (serotonin-N-acetyltransferase) which converts the serotonin to N-acetylserotonin, and HIOMT (hydroxyindole-O-methyltrasferase) which trasfers a methyl group from S-adenosylmethionine to the 5-hydroxyl of the N-acetylserotonin. The activities of both enzymes rise soon after the onset of darkness because of the enhanced release of norepinephrine from sympathetic neurons terminating on the pineal parenchymal cells.
Another portion of the serotonin liberated from pineal cells after the onset of darkness is deaminated by the enzyme monoamine oxidase (MAO) and then either oxidized to form 5-hydroxyindole acetic acid or reduced to form 5-hydroxytryptophol (Fig.1). Both of these compounds are also substrates for HIOMT and can thus be converted in the pineal to 5-methoxyindole acetic acid 5-methoxytryptophol (Fig.1). The level of this latter indole, like that of melatonin, rises markedly in the pineal with the onset of darkness. Since 5-methoxytryptophol synthesis does not require the acetylation of serotonin, the nocturnal increase in pineal SNAT activity cannot be the trigger that causes pineal methoxyindole levels to rise. More likely, a single unexplained process- the intraparenchymal release of stored pineal serotonin, which then becomes accessible to both SNAT and MAO. This process ultimately controls the rates at which all three major pineal methoxyindoles are synthesized and generates the nocturnal increases in pineal melatonin and 5-methoxytryptophol. The proportion of available serotonin acetylated at any particular time of day or night depends on the relative activities of pineal SNAT and MAO at that time. The rates of methylation of all three 5-hydroxyindoles formed from pinela serotonin depends on HIOMT activity.Fig.1 Biosynthesis of pineal methoxyindoles from serotonin

Serotonin may be either acetylated to form N-acetylserotonin through the action of the enzyme serotonin-N-acetyltransferase (SNAT), or oxidatively deaminated by monoamine oxidase (MAO) to yield an unstable aldehyde. This compound is then either oxidized to 5-hydroxyindole acetic acid by the enzyme aldehyde dehydrogenase (ADH), or reduced to from 5-hydroxytryptophol by aldehyde reductase (AR). Each of these 5-hydroxyindole derivatives of serotonin is a substrate for hydroxyindole-O-methyltrasferase (HIMOT). The enzymatic trasfer of a methyl group from S-adenosylmethionine to these hydroxyindoles yields melatonin (5-hydroxy-N-acetyltryptamine), 5-methoxyindole acetic acid and 5-methoxytryptophol respectively. Pineal serotonin is synthesized from the essential amino acid tryptophan by 5-hydroxylation folloed by decarboxylation. The first step in ths enzymic sequence is catalysed by tryptophan hydroxylase. The second step is catalysed by aromatic L-amino acid decarboxylase.

Medical uses

In the European Union it is indicated for the treatment of insomnia in children and adolescents aged 2–18 with autism spectrum disorder (ASD) and / or Smith–Magenis syndrome, where sleep hygiene measures have been insufficient^[12] and for monotherapy for the short-term treatment of primary insomnia characterized by poor quality of sleep in people who are aged 55 or over.^[11]

Sleep disorders

Positions on the benefits of melatonin for insomnia are mixed.^[8] An Agency for Healthcare Research and Quality (AHRQ) review from 2015 stated that evidence of benefit in the general population was unclear.^[8] A review from 2017, found a modest effect on time until onset of sleep.^[3] Another review from 2017 put this decrease at six minutes to sleep onset but found no difference in total sleep time.^[6] Melatonin may also be useful in delayed sleep phase syndrome.^[3] Melatonin appears to work as well as ramelteon but costs less.^[6]

Melatonin is a safer alternative than clonazepam in the treatment of REM sleep behavior disorder – a condition associated with the synucleinopathies like Parkinson’s disease and dementia with Lewy bodies.^[21]^[22]^[23] In Europe it is used for short-term treatment of insomnia in people who are 55 years old or older.^[24] It is deemed to be a first line agent in this group.^[6]

Melatonin reduces the time until onset of sleep and increases sleep duration in children with neurodevelopmental disorders.^[25]

Dementia

A 2020 Cochrane review found no evidence that melatonin helped sleep problems in people with moderate to severe dementia due to Alzheimer’s disease.^[26] A 2019 review found that while melatonin may improve sleep in minimal cognitive impairment, after the onset of Alzheimer’s it has little to no effect.^[27] Melatonin may, however, help with sundowning.^[28]

Jet lag and shift work

Melatonin is known to reduce jet lag, especially in eastward travel. If the time it is taken is not correct, however, it can instead delay adaption.^[29]

Melatonin appears to have limited use against the sleep problems of people who work shift work.^[30] Tentative evidence suggests that it increases the length of time people are able to sleep.^[30]

Adverse effects

Melatonin appears to cause very few side effects as tested in the short term, up to three months, at low doses.^{[clarification needed]} Two systematic reviews found no adverse effects of exogenous melatonin in several clinical trials and comparative trials found the adverse effects headaches, dizziness, nausea, and drowsiness were reported about equally for both melatonin and placebo.^[31]^[32] Prolonged-release melatonin is safe with long-term use of up to 12 months.^[33] Although not recommended for long term use beyond this, low-dose melatonin is generally safer, and a better alternative, than many prescription and over the counter sleep aids if a sleeping medication must be used for an extended period of time. Low-doses of melatonin are usually sufficient to produce a hypnotic effect in most people. Higher doses do not appear to result in a stronger effect, but instead appear to cause drowsiness for a longer period of time.^[34]

Melatonin can cause nausea, next-day grogginess, and irritability.^[35] In the elderly, it can cause reduced blood flow and hypothermia.^[36]^{[needs update]} In autoimmune disorders, evidence is conflicting whether melatonin supplementation may ameliorate or exacerbate symptoms due to immunomodulation.^[37]^[38]^{[needs update]}

Melatonin can lower follicle-stimulating hormone levels.^[39] Melatonin’s effects on human reproduction remain unclear.^[40]

In those taking warfarin, some evidence suggests there may exist a potentiating drug interaction, increasing the anticoagulant effect of warfarin and the risk of bleeding.^[41]

Functions

When eyes receive light from the sun, the pineal gland’s production of melatonin is inhibited and the hormones produced keep the human awake. When the eyes do not receive light, melatonin is produced in the pineal gland and the human becomes tired.

Circadian rhythm

In animals, melatonin plays an important role in the regulation of sleep–wake cycles.^[42] Human infants’ melatonin levels become regular in about the third month after birth, with the highest levels measured between midnight and 8:00 am.^[43] Human melatonin production decreases as a person ages.^[44] Also, as children become teenagers, the nightly schedule of melatonin release is delayed, leading to later sleeping and waking times.^[45]

Antioxidant

Melatonin was first reported as a potent antioxidant and free radical scavenger in 1993.^[46] In vitro, melatonin acts as a direct scavenger of oxygen radicals and reactive nitrogen species including OH^•, O₂⁻^•, and NO^•.^[47]^[48] In plants, melatonin works with other antioxidants to improve the overall effectiveness of each antioxidant.^[48] Melatonin has been proven to be twice as active as vitamin E, believed to be the most effective lipophilic antioxidant.^[49] Via signal transduction through melatonin receptors, melatonin promotes the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase.^[50]^[51]

Melatonin occurs at high concentrations within mitochondrial fluid which greatly exceed the plasma concentration of melatonin.^[52]^[53]^[54] Due to its capacity for free radical scavenging, indirect effects on the expression of antioxidant enzymes, and its significant concentrations within mitochondria, a number of authors have indicated that melatonin has an important physiological function as a mitochondrial antioxidant.^[50]^[52]^[53]^[54]^[55]

The melatonin metabolites produced via the reaction of melatonin with reactive oxygen species or reactive nitrogen species also react with and reduce free radicals.^[51]^[55] Melatonin metabolites generated from redox reactions include cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-methoxykynuramine (AMK).^[51]^[55]

Immune system

While it is known that melatonin interacts with the immune system,^[56]^[57] the details of those interactions are unclear. An antiinflammatory effect seems to be the most relevant. There have been few trials designed to judge the effectiveness of melatonin in disease treatment. Most existing data are based on small, incomplete trials. Any positive immunological effect is thought to be the result of melatonin acting on high-affinity receptors (MT1 and MT2) expressed in immunocompetent cells. In preclinical studies, melatonin may enhance cytokine production,^[58] and by doing this, counteract acquired immunodeficiences. Some studies also suggest that melatonin might be useful fighting infectious disease^[59] including viral, such as HIV, and bacterial infections, and potentially in the treatment of cancer.

Biosynthesis

Overview of melatonin biosynthesis

In animals, biosynthesis of melatonin occurs through hydroxylation, decarboxylation, acetylation and a methylation starting with L-tryptophan.^[60] L-tryptophan is produced in the shikimate pathway from chorismate or is acquired from protein catabolism. First L-tryptophan is hydroxylated on the indole ring by tryptophan hydroxylase to produce 5-hydroxytryptophan. This intermediate (5-HTP) is decarboxylated by pyridoxal phosphate and 5-hydroxytryptophan decarboxylase to produce serotonin.

Serotonin is itself an important neurotransmitter, but is also converted into N-acetylserotonin by serotonin N-acetyltransferase with acetyl-CoA.^[61] Hydroxyindole O-methyltransferase and S-adenosyl methionine convert N-acetylserotonin into melatonin through methylation of the hydroxyl group.^[61]

In bacteria, protists, fungi, and plants, melatonin is synthesized indirectly with tryptophan as an intermediate product of the shikimate pathway. In these cells, synthesis starts with D-erythrose 4-phosphate and phosphoenolpyruvate, and in photosynthetic cells with carbon dioxide. The rest of the synthesising reactions are similar, but with slight variations in the last two enzymes.^[62]^[63]

It has been hypothesized that melatonin is made in the mitochondria and chloroplasts.^[64]

Mechanism

Mechanism of melatonin biosynthesis

In order to hydroxylate L-tryptophan, the cofactor tetrahydrobiopterin (THB) must first react with oxygen and the active site iron of tryptophan hydroxylase. This mechanism is not well understood, but two mechanisms have been proposed:

1. A slow transfer of one electron from the THB to O₂ could produce a superoxide which could recombine with the THB radical to give 4a-peroxypterin. 4a-peroxypterin could then react with the active site iron (II) to form an iron-peroxypterin intermediate or directly transfer an oxygen atom to the iron.

2. O₂ could react with the active site iron (II) first, producing iron (III) superoxide which could then react with the THB to form an iron-peroxypterin intermediate.

Iron (IV) oxide from the iron-peroxypterin intermediate is selectively attacked by a double bond to give a carbocation at the C5 position of the indole ring. A 1,2-shift of the hydrogen and then a loss of one of the two hydrogen atoms on C5 reestablishes aromaticity to furnish 5-hydroxy-L-tryptophan.^[65]

A decarboxylase with cofactor pyridoxal phosphate (PLP) removes CO₂ from 5-hydroxy-L-tryptophan to produce 5-hydroxytryptamine.^[66] PLP forms an imine with the amino acid derivative. The amine on the pyridine is protonated and acts as an electron sink, enabling the breaking of the C-C bond and releasing CO₂. Protonation of the amine from tryptophan restores the aromaticity of the pyridine ring and then imine is hydrolyzed to produce 5-hydroxytryptamine and PLP.^[67]

It has been proposed that histidine residue His122 of serotonin N-acetyl transferase is the catalytic residue that deprotonates the primary amine of 5-hydroxytryptamine, which allows the lone pair on the amine to attack acetyl-CoA, forming a tetrahedral intermediate. The thiol from coenzyme A serves as a good leaving group when attacked by a general base to give N-acetylserotonin.^[68]

N-acetylserotonin is methylated at the hydroxyl position by S-adenosyl methionine (SAM) to produce S-adenosyl homocysteine (SAH) and melatonin.^[67]^[69]

Regulation

In vertebrates, melatonin secretion is regulated by activation of the beta-1 adrenergic receptor by norepinephrine.^[70] Norepinephrine elevates the intracellular cAMP concentration via beta-adrenergic receptors and activates the cAMP-dependent protein kinase A (PKA). PKA phosphorylates the penultimate enzyme, the arylalkylamine N-acetyltransferase (AANAT). On exposure to (day)light, noradrenergic stimulation stops and the protein is immediately destroyed by proteasomal proteolysis.^[71] Production of melatonin is again started in the evening at the point called the dim-light melatonin onset.

Blue light, principally around 460–480 nm, suppresses melatonin biosynthesis,^[72] proportional to the light intensity and length of exposure. Until recent history, humans in temperate climates were exposed to few hours of (blue) daylight in the winter; their fires gave predominantly yellow light.^{[citation needed]} The incandescent light bulb widely used in the 20th century produced relatively little blue light.^[73] Light containing only wavelengths greater than 530 nm does not suppress melatonin in bright-light conditions.^[74] Wearing glasses that block blue light in the hours before bedtime may decrease melatonin loss. Use of blue-blocking goggles the last hours before bedtime has also been advised for people who need to adjust to an earlier bedtime, as melatonin promotes sleepiness.^[75]

Pharmacology

Pharmacodynamics

In humans, melatonin is a full agonist of melatonin receptor 1 (picomolar binding affinity) and melatonin receptor 2 (nanomolar binding affinity), both of which belong to the class of G-protein coupled receptors (GPCRs).^[51]^[76] Melatonin receptors 1 and 2 are both G_i/o-coupled GPCRs, although melatonin receptor 1 is also G_q-coupled.^[51] Melatonin also acts as a high-capacity free radical scavenger within mitochondria which also promotes the expression of antioxidant enzymes such as superoxide dismutase, glutathione peroxidase, glutathione reductase, and catalase via signal transduction through melatonin receptors.^[50]^[51]^[52]^[53]^[54]^[55]

Pharmacokinetics

When used several hours before sleep according to the phase response curve for melatonin in humans, small amounts (0.3 mg^[77]) of melatonin shift the circadian clock earlier, thus promoting earlier sleep onset and morning awakening.^[78] Melatonin is rapidly absorbed and distributed, reaching peak plasma concentrations after 60 minutes of administration, and is then eliminated.^[61] Melatonin has a half life of 35–50 minutes.^[79] In humans, 90% of orally administered exogenous melatonin is cleared in a single passage through the liver, a small amount is excreted in urine, and a small amount is found in saliva.^[5] The bioavalibility of melatonin is between 10 and 50%.^[61]

Melatonin is metabolized in the liver by cytochrome P450 enzyme CYP1A2 to 6-hydroxymelatonin. Metabolites are conjugated with sulfuric acid or glucuronic acid for excretion in the urine. 5% of melatonin is excreted in the urine as the unchanged drug.^[61]

Some of the metabolites formed via the reaction of melatonin with a free radical include cyclic 3-hydroxymelatonin, N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), and N1-acetyl-5-methoxykynuramine (AMK).^[51]^[55]

The membrane transport proteins that move melatonin across a membrane include, but are not limited to, glucose transporters, including GLUT1, and the proton-driven oligopeptide transporters PEPT1 and PEPT2.^[51]^[55]

For research as well as clinical purposes, melatonin concentration in humans can be measured either from the saliva or blood plasma.^[80]

History

Melatonin was first discovered in connection to the mechanism by which some amphibians and reptiles change the color of their skin.^[81]^[82] As early as 1917, Carey Pratt McCord and Floyd P. Allen discovered that feeding extract of the pineal glands of cows lightened tadpole skin by contracting the dark epidermal melanophores.^[83]^[84]

In 1958, dermatology professor Aaron B. Lerner and colleagues at Yale University, in the hope that a substance from the pineal might be useful in treating skin diseases, isolated the hormone from bovine pineal gland extracts and named it melatonin.^[85] In the mid-70s Lynch et al. demonstrated that the production of melatonin exhibits a circadian rhythm in human pineal glands.^[86]

The discovery that melatonin is an antioxidant was made in 1993.^[87] The first patent for its use as a low-dose sleep aid was granted to Richard Wurtman at MIT in 1995.^[88] Around the same time, the hormone got a lot of press as a possible treatment for many illnesses.^[89] The New England Journal of Medicine editorialized in 2000: “With these recent careful and precise observations in blind persons, the true potential of melatonin is becoming evident, and the importance of the timing of treatment is becoming clear.”^[90]

It was approved for medical use in the European Union in 2007.^[11]

Other animals

In vertebrates, melatonin is produced in darkness, thus usually at night, by the pineal gland, a small endocrine gland^[91] located in the center of the brain but outside the blood–brain barrier. Light/dark information reaches the suprachiasmatic nuclei from retinal photosensitive ganglion cells of the eyes^[92]^[93] rather than the melatonin signal (as was once postulated). Known as “the hormone of darkness”, the onset of melatonin at dusk promotes activity in nocturnal (night-active) animals and sleep in diurnal ones including humans.

Many animals use the variation in duration of melatonin production each day as a seasonal clock.^[94] In animals including humans,^[95] the profile of melatonin synthesis and secretion is affected by the variable duration of night in summer as compared to winter. The change in duration of secretion thus serves as a biological signal for the organization of daylength-dependent (photoperiodic) seasonal functions such as reproduction, behavior, coat growth, and camouflage coloring in seasonal animals.^[95] In seasonal breeders that do not have long gestation periods and that mate during longer daylight hours, the melatonin signal controls the seasonal variation in their sexual physiology, and similar physiological effects can be induced by exogenous melatonin in animals including mynah birds^[96] and hamsters.^[97] Melatonin can suppress libido by inhibiting secretion of luteinizing hormone and follicle-stimulating hormone from the anterior pituitary gland, especially in mammals that have a breeding season when daylight hours are long. The reproduction of long-day breeders is repressed by melatonin and the reproduction of short-day breeders is stimulated by melatonin.

During the night, melatonin regulates leptin, lowering its levels.

Cetaceans have lost all the genes for melatonin synthesis as well as those for melatonin receptors.^[98] This is thought to be related to their unihemispheric sleep pattern (one brain hemisphere at a time). Similar trends have been found in sirenians.^[98]

Plants

Until its identification in plants in 1987, melatonin was for decades thought to be primarily an animal neurohormone. When melatonin was identified in coffee extracts in the 1970s, it was believed to be a byproduct of the extraction process. Subsequently, however, melatonin has been found in all plants that have been investigated. It is present in all the different parts of plants, including leaves, stems, roots, fruits, and seeds, in varying proportions.^[19]^[99] Melatonin concentrations differ not only among plant species, but also between varieties of the same species depending on the agronomic growing conditions, varying from picograms to several micrograms per gram.^[63]^[100] Notably high melatonin concentrations have been measured in popular beverages such as coffee, tea, wine, and beer, and crops including corn, rice, wheat, barley, and oats.^[19] In some common foods and beverages, including coffee^[19] and walnuts,^[101] the concentration of melatonin has been estimated or measured to be sufficiently high to raise the blood level of melatonin above daytime baseline values.

Although a role for melatonin as a plant hormone has not been clearly established, its involvement in processes such as growth and photosynthesis is well established. Only limited evidence of endogenous circadian rhythms in melatonin levels has been demonstrated in some plant species and no membrane-bound receptors analogous to those known in animals have been described. Rather, melatonin performs important roles in plants as a growth regulator, as well as environmental stress protector. It is synthesized in plants when they are exposed to both biological stresses, for example, fungal infection, and nonbiological stresses such as extremes of temperature, toxins, increased soil salinity, drought, etc.^[63]^[102]^[103]

Occurrence

Dietary supplement

Melatonin is categorized by the US Food and Drug Administration (FDA) as a dietary supplement, and is sold over-the-counter in both the US and Canada.^[5] FDA regulations applying to medications are not applicable to melatonin,^[15] though the FDA has found false claims that it cures cancer.^[104] As melatonin may cause harm in combination with certain medications or in the case of certain disorders, a doctor or pharmacist should be consulted before making a decision to take melatonin.^[29] In many countries, melatonin is recognized as a neurohormone and it cannot be sold over-the-counter.^[105]

Food products

Naturally-occurring melatonin has been reported in foods including tart cherries to about 0.17–13.46 ng/g,^[106] bananas and grapes, rice and cereals, herbs, plums,^[107] olive oil, wine^[108] and beer. When birds ingest melatonin-rich plant feed, such as rice, the melatonin binds to melatonin receptors in their brains.^[109] When humans consume foods rich in melatonin, such as banana, pineapple, and orange, the blood levels of melatonin increase significantly.^[110]

Beverages and snacks containing melatonin were being sold in grocery stores, convenience stores, and clubs in May 2011.^[111] The FDA considered whether these food products could continue to be sold with the label “dietary supplements”. On 13 January 2010, it issued a Warning Letter to Innovative Beverage, creators of several beverages marketed as drinks, stating that melatonin, while legal as a dietary supplement, was not approved as a food additive.^[112] A different company selling a melatonin-containing beverage received a warning letter in 2015.^[113]

Commercial availability

Immediate-release melatonin is not tightly regulated in countries where it is available as an over-the-counter medication. It is available in doses from less than half a milligram to 5 mg or more. Immediate-release formulations cause blood levels of melatonin to reach their peak in about an hour. The hormone may be administered orally, as capsules, gummies, tablets, or liquids. It is also available for use sublingually, or as transdermal patches.^{[medical citation needed]}

Formerly, melatonin was derived from animal pineal tissue, such as bovine. It is now synthetic, which limits the risk of contamination or the means of transmitting infectious material.^[15]^[114]

Melatonin is the most popular over-the-counter sleep remedy in the US, resulting in sales in excess of US$400 million during 2017.^[115]

Research

A bottle of melatonin tablets. Melatonin is available in timed-release and in liquid forms.

Various uses and effects of melatonin have been studied. A 2015 review of studies of melatonin in tinnitus found the quality of evidence low, but not entirely without promise.^[116]

Headaches

Tentative evidence shows melatonin may help reduce some types of headaches including cluster and hypnic headaches.^[117]^[118]

Cancer

A 2013 review by the National Cancer Institutes found evidence for use to be inconclusive.^[119] A 2005 review of unblinded clinical trials found a reduced rate of death, but that blinded and independently conducted randomized controlled trials are needed.^[120]

Protection from radiation

Both animal^[121] and human^[122]^[123]^[124] studies have shown melatonin to protect against radiation-induced cellular damage. Melatonin and its metabolites protect organisms from oxidative stress by scavenging reactive oxygen species which are generated during exposure.^[125] Nearly 70% of biological damage caused by ionizing radiation is estimated to be attributable to the creation of free radicals, especially the hydroxyl radical that attacks DNA, proteins, and cellular membranes. Melatonin has been described as a broadly protective, readily available, and orally self-administered antioxidant that is without known, major side effects.^[126]

Epilepsy

A 2016 review found no beneficial role of melatonin in reducing seizure frequency or improving quality of life in people with epilepsy.^[127]

Secondary dysmenorrhoea

A 2016 review suggested no strong evidence of melatonin compared to placebo for dysmenorrhoea secondary to endometriosis.^[128]

Delirium

A 2016 review suggested no clear evidence of melatonin to reduce the incidence of delirium.^[129]

Gastroesophageal reflux disease

A 2011 review said melatonin is effective in relieving epigastric pain and heartburn.^[130]

Psychiatry

Melatonin might improve sleep in people with autism.^[131] Children with autism have abnormal melatonin pathways and below-average physiological levels of melatonin.^[132]^[133] Melatonin supplementation has been shown to improve sleep duration, sleep onset latency, and night-time awakenings.^[132]^[134]^[135] However, many studies on melatonin and autism rely on self-reported levels of improvement and more rigorous research is needed.

While the packaging of melatonin often warns against use in people under 18 years of age, studies suggest that melatonin is an efficacious and safe treatment for insomnia in people with ADHD, including children. However, larger and longer studies are needed to establish long-term safety and optimal dosing.^[136]

Melatonin in comparison to placebo is effective for reducing preoperative anxiety in adults when given as premedication. It may be just as effective as standard treatment with midazolam in reducing preoperative anxiety. Melatonin may also reduce postoperative anxiety (measured 6 hours after surgery) when compared to placebo.^[137]

Some supplemental melatonin users report an increase in vivid dreaming. Extremely high doses of melatonin increased REM sleep time and dream activity in people both with and without narcolepsy.^[138] Some evidence supports an antidepressant effect.^[139]

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^ Burkhardt S, Tan DX, Manchester LC, Hardeland R, Reiter RJ (October 2001). “Detection and quantification of the antioxidant melatonin in Montmorency and Balaton tart cherries (Prunus cerasus)”. Journal of Agricultural and Food Chemistry. 49 (10): 4898–902. doi:10.1021/jf010321. PMID 11600041.
^ González-Flores D, Velardo B, Garrido M, González-Gómez D, Lozano M, Ayuso MC, Barriga C, Paredes SD, Rodríguez AB (2011). “Ingestion of Japanese plums (Prunus salicina Lindl. cv. Crimson Globe) increases the urinary 6-sulfatoxymelatonin and total antioxidant capacity levels in young, middle-aged and elderly humans: Nutritional and functional characterization of their content”. Journal of Food and Nutrition Research. 50(4): 229–36.
^ Lamont KT, Somers S, Lacerda L, Opie LH, Lecour S (May 2011). “Is red wine a SAFE sip away from cardioprotection? Mechanisms involved in resveratrol- and melatonin-induced cardioprotection”. Journal of Pineal Research. 50 (4): 374–80. doi:10.1111/j.1600-079X.2010.00853.x. PMID 21342247. S2CID 8034935.
^ Hattori A, Migitaka H, Iigo M, Itoh M, Yamamoto K, Ohtani-Kaneko R, et al. (March 1995). “Identification of melatonin in plants and its effects on plasma melatonin levels and binding to melatonin receptors in vertebrates”. Biochemistry and Molecular Biology International. 35(3): 627–34. PMID 7773197.
^ Sae-Teaw M, Johns J, Johns NP, Subongkot S (August 2013). “Serum melatonin levels and antioxidant capacities after consumption of pineapple, orange, or banana by healthy male volunteers”. Journal of Pineal Research. 55 (1): 58–64. doi:10.1111/jpi.12025. PMID 23137025. S2CID 979886.
^ Catherine Saint Louis (14 May 2011). “Dessert, Laid-Back and Legal”. The New York Times.
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^ Bebida Beverage Company U.S. Food and Drug Administration 4 March 2015 (Accessed 8 December 2017)
^ “Melatonin”. Drugs.com. Retrieved 17 August 2011.
^ Loria K. “Does Melatonin Really Help You Sleep?”. Consumer Reports. Retrieved 16 March 2019.
^ Miroddi M, Bruno R, Galletti F, Calapai F, Navarra M, Gangemi S, Calapai G (March 2015). “Clinical pharmacology of melatonin in the treatment of tinnitus: a review”. European Journal of Clinical Pharmacology. 71 (3): 263–70. doi:10.1007/s00228-015-1805-3. PMID 25597877. S2CID 16466238.
^ Peres MF, Masruha MR, Zukerman E, Moreira-Filho CA, Cavalheiro EA (April 2006). “Potential therapeutic use of melatonin in migraine and other headache disorders”. Expert Opinion on Investigational Drugs. 15 (4): 367–75. doi:10.1517/13543784.15.4.367. PMID 16548786. S2CID 28114683.
^ Evars S, Goadsby P (2005). “Review: Hypnic headache” (PDF). Practical Neurology. 5(3): 144–49. doi:10.1111/j.1474-7766.2005.00301.x. S2CID 1196313. Retrieved 12 January 2018.
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^ Mills E, Wu P, Seely D, Guyatt G (November 2005). “Melatonin in the treatment of cancer: a systematic review of randomized controlled trials and meta-analysis”. Journal of Pineal Research. 39 (4): 360–6. doi:10.1111/j.1600-079X.2005.00258.x. PMID 16207291. S2CID 22225091.
^ Meltz ML, Reiter RJ, Herman TS, Kumar KS (March 1999). “Melatonin and protection from whole-body irradiation: survival studies in mice”. Mutation Research. 425 (1): 21–7. doi:10.1016/S0027-5107(98)00246-2. PMID 10082913.
^ Reiter RJ, Herman TS, Meltz ML (December 1996). “Melatonin and radioprotection from genetic damage: in vivo/in vitro studies with human volunteers”. Mutation Research. 371 (3–4): 221–8. doi:10.1016/S0165-1218(96)90110-X. PMID 9008723.
^ Pattanittum P, Kunyanone N, Brown J, Sangkomkamhang US, Barnes J, Seyfoddin V, Marjoribanks J (March 2016). “Dietary supplements for dysmenorrhoea”. The Cochrane Database of Systematic Reviews. 3 (3): CD002124. doi:10.1002/14651858.cd002124.pub2. PMC 7387104. PMID 27000311.
^ Reiter RJ, Herman TS, Meltz ML (February 1998). “Melatonin reduces gamma radiation-induced primary DNA damage in human blood lymphocytes”. Mutation Research. 397 (2): 203–8. doi:10.1016/S0027-5107(97)00211-X. PMID 9541644.
^ Tan DX, Manchester LC, Terron MP, Flores LJ, Reiter RJ (January 2007). “One molecule, many derivatives: a never-ending interaction of melatonin with reactive oxygen and nitrogen species?”. Journal of Pineal Research. 42 (1): 28–42. doi:10.1111/j.1600-079X.2006.00407.x. PMID 17198536. S2CID 40005308.
^ Shirazi A, Ghobadi G, Ghazi-Khansari M (July 2007). “A radiobiological review on melatonin: a novel radioprotector”. Journal of Radiation Research. 48 (4): 263–72. Bibcode:2007JRadR..48..263S. doi:10.1269/jrr.06070. PMID 17641465.
^ Brigo F, Igwe SC, Del Felice A (August 2016). “Melatonin as add-on treatment for epilepsy”. The Cochrane Database of Systematic Reviews (8): CD006967. doi:10.1002/14651858.CD006967.pub4. PMC 7386917. PMID 27513702.
^ Pattanittum P, Kunyanone N, Brown J, Sangkomkamhang US, Barnes J, Seyfoddin V, Marjoribanks J (March 2016). “Dietary supplements for dysmenorrhoea”. The Cochrane Database of Systematic Reviews. 3: CD002124. doi:10.1002/14651858.CD002124.pub2. PMC 7387104. PMID 27000311.
^ Siddiqi N, Harrison JK, Clegg A, Teale EA, Young J, Taylor J, Simpkins SA (March 2016). “Interventions for preventing delirium in hospitalised non-ICU patients” (PDF). The Cochrane Database of Systematic Reviews. 3: CD005563. doi:10.1002/14651858.CD005563.pub3. PMID 26967259.
^ Kandil TS, Mousa AA, El-Gendy AA, Abbas AM (January 2010). “The potential therapeutic effect of melatonin in Gastro-Esophageal Reflux Disease”. BMC Gastroenterology. 10: 7. doi:10.1186/1471-230X-10-7. PMC 2821302. PMID 20082715.
^ Braam W, Smits MG, Didden R, Korzilius H, Van Geijlswijk IM, Curfs LM (May 2009). “Exogenous melatonin for sleep problems in individuals with intellectual disability: a meta-analysis”. Developmental Medicine and Child Neurology (Meta-analysis). 51 (5): 340–9. doi:10.1111/j.1469-8749.2008.03244.x. PMID 19379289. S2CID 35553148.
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^ Veatch OJ, Pendergast JS, Allen MJ, Leu RM, Johnson CH, Elsea SH, Malow BA (January 2015). “Genetic variation in melatonin pathway enzymes in children with autism spectrum disorder and comorbid sleep onset delay”. Journal of Autism and Developmental Disorders. 45 (1): 100–10. doi:10.1007/s10803-014-2197-4. PMC 4289108. PMID 25059483.
^ “Melatonin improves sleep quality and behavior in children with asperger disorder”. Truthly. Retrieved 15 February 2015.
^ Giannotti F, Cortesi F, Cerquiglini A, Bernabei P (August 2006). “An open-label study of controlled-release melatonin in treatment of sleep disorders in children with autism”. Journal of Autism and Developmental Disorders. 36 (6): 741–52. doi:10.1007/s10803-006-0116-z. PMID 16897403. S2CID 19724241.
^ Bendz LM, Scates AC (January 2010). “Melatonin treatment for insomnia in pediatric patients with attention-deficit/hyperactivity disorder”. The Annals of Pharmacotherapy. 44(1): 185–91. doi:10.1345/aph.1M365. PMID 20028959. S2CID 207263711.
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^ Valdés-Tovar M, Estrada-Reyes R, Solís-Chagoyán H, Argueta J, Dorantes-Barrón AM, Quero-Chávez D, et al. (August 2018). “Circadian modulation of neuroplasticity by melatonin: a target in the treatment of depression”. British Journal of Pharmacology. 175(16): 3200–3208. doi:10.1111/bph.14197. PMC 6057892. PMID 29512136.

External links

"Melatonin". Drug Information Portal. U.S. National Library of Medicine.

Clinical data


Pronunciation	/ˌmɛləˈtoʊnɪn/ (listen)
Trade names	Circadin, Slenyto, others^[1]
Other names	N-acetyl-5-methoxy tryptamine^[2]
AHFS/Drugs.com	Consumer Drug Information
License data	EU EMA: by INNUS DailyMed: Melatonin
Routes of administration	By mouth, sublingual, transdermal
ATC code	N05CH01 (WHO)
Physiological data
Source tissues	pineal gland
Target tissues	wide spread, including brain, retina, and circulatory system
Receptors	melatonin receptor
Precursor	N-acetylserotonin
Metabolism	Liver via CYP1A2 mediated 6-hydroxylation
Legal status
Legal status	AU: OTC / Rx-onlyCA: OTCUK: POM (Prescription only)EU: Rx-onlyIn general: ℞ (Prescription only)
Pharmacokinetic data
Bioavailability	30–50%
Metabolism	Liver via CYP1A2 mediated 6-hydroxylation
Metabolites	6-hydroxymelatonin, N-acetyl-5lhydroxytryptamine, 5-methoxytryptamine
Elimination half-life	30–50 minutes^[3]
Excretion	Kidney
Identifiers
IUPAC name [show]
CAS Number	73-31-4
PubChem CID	896
IUPHAR/BPS	224
DrugBank	DB01065
ChemSpider	872
UNII	JL5DK93RCL
KEGG	D08170
ChEBI	CHEBI:16796
ChEMBL	ChEMBL45
CompTox Dashboard (EPA)	DTXSID1022421
ECHA InfoCard	100.000.725
Chemical and physical data
Formula	C₁₃H₁₆N₂O₂
Molar mass	232.283 g·mol⁻¹
3D model (JSmol)	Interactive image
Melting point	117 °C (243 °F)
SMILES [hide]COC1=CC2=C(NC=C2CCNC(C)=O)C=C1
InChI [hide]InChI=1S/C13H16N2O2/c1-9(16)14-6-5-10-8-15-13-4-3-11(17-2)7-12(10)13/h3-4,7-8,15H,5-6H2,1-2H3,(H,14,16)Key:DRLFMBDRBRZALE-UHFFFAOYSA-N

//////////Melatonin, Melatobel, メラトニン , JAPAN 2020, 2020 APPROVALS

RALOXIFENE

December 4, 2020 2:16 am / Leave a comment

Keoxifene hydrochloride, Raloxifene hydrochloride, LY-139481(free base), LY-156758, Optruma, Loxifen, EvistaTitle: RaloxifeneCAS Registry Number: 84449-90-1CAS Name: [6-Hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl][4-[2-(1-piperidinyl)ethoxy]phenyl]methanoneAdditional Names: keoxifeneManufacturers’ Codes: LY-139481Molecular Formula: C28H27NO4SMolecular Weight: 473.58Percent Composition: C 71.01%, H 5.75%, N 2.96%, O 13.51%, S 6.77%Literature References: Nonsteroidal, selective estrogen receptor modulator (SERM). Prepn: C. D. Jones, EP62503; idem,US4418068 (1982, 1983 both to Lilly); idemet al.,J. Med. Chem.27, 1057 (1984). Review of pharmacology and toxicology: J. Buelke-Sam et al.,Reprod. Toxicol.12, 217-221 (1998); of clinical pharmacology and pharmacokinetics: D. Hochner-Celnikier, Eur. J. Obstet. Gynecol. Reprod. Biol.85, 23-29 (1999); of clinical efficacy in osteoporosis: D. Agnusdei, ibid. 43-46. Clinical effect on risk of breast cancer: S. R. Cummings et al.,J. Am. Med. Assoc.281, 2189 (1999); on reduction of fracture risk: B. Ettinger et al.,ibid.282, 637 (1999).Properties: Crystals from acetone, mp 143-147°. uv max (ethanol): 290 nm (e 34000).Melting point: mp 143-147°Absorption maximum: uv max (ethanol): 290 nm (e 34000)
Derivative Type: HydrochlorideCAS Registry Number: 82640-04-8Manufacturers’ Codes: LY-156758Trademarks: Evista (Lilly)Molecular Formula: C28H27NO4S.HClMolecular Weight: 510.04Percent Composition: C 65.94%, H 5.53%, N 2.75%, O 12.55%, S 6.29%, Cl 6.95%Properties: Crystals from methanol/water, mp 258°. uv max (ethanol): 286 nm (e 32800).Melting point: mp 258°Absorption maximum: uv max (ethanol): 286 nm (e 32800)
Therap-Cat: Antiosteoporotic.Keywords: Antiosteoporotic; Selective Estrogen Receptor Modulator (SERM).

Raloxifene, sold under the brand name Evista among others, is a medication used to prevent and treat osteoporosis in postmenopausal women and those on glucocorticoids.^[4] For osteoporosis it is less preferred than bisphosphonates.^[4] It is also used to reduce the risk of breast cancer in those at high risk.^[4] It is taken by mouth.^[4]

Common side effects include hot flashes, leg cramps, swelling, and joint pain.^[4] Severe side effects may include blood clots and stroke.^[4] Use during pregnancy may harm the baby.^[4] The medication may worsen menstrual symptoms.^[5] Raloxifene is a selective estrogen receptor modulator (SERM) and therefore a mixed agonist–antagonist of the estrogen receptor (ER).^[4] It has estrogenic effects in bone and antiestrogenic effects in the breasts and uterus.^[4]

Raloxifene was approved for medical use in the United States in 1997.^[4] It is available as a generic medication.^[4]^[6] A month supply in the United Kingdom costs the NHS about 3.50 £ as of 2019.^[6] In the United States the wholesale cost of this amount is about $16.^[7] In 2017, it was the 330th most commonly prescribed medication in the United States, with more than 900 thousand prescriptions.^[8

Medical uses

Raloxifene is used for the treatment and prevention of osteoporosis in postmenopausal women.^[9] It is used at a dosage of 60 mg/day for both the prevention and treatment of osteoporosis.^[10] In the case of either osteoporosis prevention or treatment, supplemental calcium and vitamin D should be added to the diet if daily intake is inadequate.^[11]

Raloxifene is used to reduce the risk of breast cancer in postmenopausal women. It is used at a dosage of 60 mg/day for this indication.^[10] In the Multiple Outcomes of Raloxifene (MORE) clinical trial, raloxifene decreased the risk of all types of breast cancer by 62%, of invasive breast cancer by 72%, and of invasive estrogen receptor-positive breast cancer by 84%.^[12] Conversely, it does not reduce the risk of estrogen receptor-negative breast cancer.^[12] There were no obvious differences in effectiveness of raloxifene in the MORE trial for prevention of breast cancer at a dosage of 60 mg/m²/day relative to 120 mg/m²/day.^[12] In the Study of Tamoxifen and Raloxifene (STAR) trial, 60 mg/day raloxifene was 78% as effective as 20 mg/day tamoxifen in preventing non-invasive breast cancer.^[13] Women with undetectable levels of estradiol (<2.7 pg/mL) have a naturally low risk of breast cancer and, in contrast to women with detectable levels of estradiol, do not experience significant benefit from raloxifene in terms of reduction of breast cancer risk.^[12]

Contraindications

Raloxifene is contraindicated in lactating women or women who are or who may become pregnant.^[14] It also may be of concern to women with active or past history of venous thromboembolic events, including deep vein thrombosis, pulmonary embolism, and retinal vein thrombosis.^[15]

Side effects

Common side effects of raloxifene include hot flashes (25–28% vs. 18–21% for placebo),^[12] vaginal dryness, and leg cramps (generally mild; 5.5% vs. 1.9% for placebo).^[14]^[1]^[16] Raloxifene does not cause breast tenderness, endometrial hyperplasia, menstrual bleeding, or endometrial cancer.^[17] It does not appear to affect cognition or memory.^[15]^[12] Raloxifene is a teratogen; i.e., it can cause developmental abnormalities such as birth defects.

Raloxifene may infrequently cause serious blood clots to form in the legs, lungs, or eyes.^[1] Other reactions experienced include leg swelling/pain, trouble breathing, chest pain, and vision changes. Black box warnings were added to the label of raloxifene in 2007 warning of increased risk of death due to stroke for postmenopausal women with documented coronary heart disease or at increased risk for major coronary events, as well as increased risk for deep vein thrombosis and pulmonary embolism.^[14] The risk of venous thromboembolism with raloxifene is increased by several-fold in postmenopausal women (RR = 3.1).^[18]^[12] Raloxifene has a lower risk of thromboembolism than tamoxifen.^[13] In the MORE trial, raloxifene caused a 40% decrease in risk of cardiovascular events in women who were at increased risk for coronary artery disease, although there was no decrease in cardiovascular events for the group as a whole.^[12]

A report in September 2009 from Health and Human Services’ Agency for Healthcare Research and Quality suggests that tamoxifen and raloxifene, used to treat breast cancer, significantly reduce invasive breast cancer in midlife and older women, but also increase the risk of adverse side effects.^[19]

A recent human case report in July 2016 suggests that raloxifene may in fact, at some point, also stimulate breast cancer growth leading to a reduction of advanced breast cancer disease upon the withdrawal of the drug.^[20]

Unlike other SERMs, such as tamoxifen, raloxifene has no risk of uterine hyperplasia or endometrial cancer (RR = 0.8).^[1]^[18]^[13]

Raloxifene does not increase the incidence of breast pain or tenderness in postmenopausal women.^[16]^[21]

Overdose

Raloxifene has been studied in clinical trials across a dosage range of 30 to 600 mg/day, and was well-tolerated at all dosages.^[16]

Pharmacology

Pharmacodynamics

Mechanism of action

Raloxifene is a selective estrogen receptor modulator (SERM) and hence is a mixed agonist and antagonist of the estrogen receptor (ER) in different tissues.^[4] It has estrogenic activity in some tissues, such as bone and the liver, and antiestrogenic activity in other tissues, such as the breasts and uterus.^[4] Its affinity (K_d) for the ERα is approximately 50 pM, which is similar to that of estradiol.^[16] Relative to estradiol, raloxifene has been reported to possess about 8 to 34% of the affinity for the ERα and 0.5 to 76% of the affinity for the ERβ.^[22]^[23] Raloxifene acts as a partial agonist of the ERα and as a pure antagonist of the ERβ.^[24]^[25] In contrast to the classical ERs, raloxifene is an agonist of the G protein-coupled estrogen receptor (GPER) (EC₅₀ = 10–100 nM), a membrane estrogen receptor.^[26]^[27]

Clinical effects

Raloxifene has antiestrogenic effects in the mammary glands in preclinical studies.^[16] In accordance, raloxifene reduces breast density in postmenopausal women, a known risk factor for breast cancer.^[28] It does not stimulate the uterus in postmenopausal women, and results in no increase in risk of endometrial thickening, vaginal bleeding, endometrial hyperplasia, or endometrial cancer.^[29]^[16]^[21] At the same time, raloxifene has minimal antiestrogenic effect in the uterus in premenopausal women.^[29] This may possibly be due to inadequate tissue exposure of the uterus to raloxifene in these estrogen-rich individuals.^[29]

In premenopausal women, raloxifene increases levels of follicle-stimulating hormone (FSH) and estradiol.^[12] Conversely, in postmenopausal women, raloxifene has been found to reduce levels of the gonadotropins, luteinizing hormone (LH) and FSH, while not affecting levels of estradiol.^[12]^[29] Raloxifene also decreases prolactin levels in postmenopausal women.^[29] In men, raloxifene has been found to disinhibit the hypothalamic–pituitary–gonadal axis (HPG axis) and thereby increase total testosterone levels.^[30]^[31]^[32]^[33] Due to the simultaneous increase in sex hormone-binding globulin (SHBG) levels however, free testosterone levels often remain unchanged in men during therapy with raloxifene.^[30]

Raloxifene has estrogenic effects on liver protein synthesis.^[12] It increases SHBG levels in both pre- and postmenopausal women as well as in men.^[12]^[30] The medication decreases levels of total and low-density lipoprotein (LDL) cholesterol, C-reactive protein, apolipoprotein B, and homocysteine.^[12]^[29] Conversely, it has little effect on levels of triglycerides and high-density lipoprotein (HDL).^[12] Raloxifene has been shown to inhibit the oxidation of LDL cholesterol in vitro.^[16] The medication has been found to decrease insulin-like growth factor 1 (IGF-1) levels in pre- and postmenopausal women as well as in men.^[31] It has also been found to increase insulin-like growth factor binding protein 3 (IGFBP-3) levels in pre- and postmenopausal women.^[12] Due to activation of estrogen receptors in the liver, raloxifene has procoagulatory effects, such as decreasing levels of fibrinogen and influencing levels of other coagulation factors.^[12]^[29]^[16] For these reasons, raloxifene increases the risk of thrombosis.^[12]^[29]

Raloxifene increases bone mineral density in postmenopausal women but decreases it in premenopausal women.^[12] In the MORE trial, the risk of vertebral fractures was decreased by 30%, and bone mineral density was increased in the spine (by 2.1% at 60 mg, 2.4% at 120 mg) and femoral neck (2.6% at 60 mg, 2.7% at 120 mg).^[18] It has been found to possess estrogenic effects in adipose tissue in postmenopausal women, promoting a shift from an android fat distribution to a gynoid fat distribution.^[34]^[35] The medication has been found to increase levels of leptin, an adipokine.^[12]

AbsorptionPharmacokinetics

The absorption of raloxifene is approximately 60%.^[1]^[2] However, due to extensive first-pass metabolism, the absolute bioavailability of raloxifene is only 2.0%.^[1]^[2] Raloxifene is rapidly absorbed from the intestines upon oral administration.^[1] Peak plasma levels of raloxifene occur 0.5 to 6 hours after an oral dose.^[1]^[2]

Distribution

Raloxifene is widely distributed throughout the body.^[1] There is extensive distribution of raloxifene into the liver, serum, lungs, and kidneys.^[1] The volume of distribution of raloxifene with a single 30 to 150 mg oral dose is approximately 2348 L.^[1]^[36] Both raloxifene and its metabolites show high plasma protein binding (>95%), including to both albumin and α₁ acid glycoprotein, but not to sex hormone-binding globulin.^[1]^[2]

Metabolism

Raloxifene is metabolized in the liver and undergoes enterohepatic recycling.^[2] It is metabolized exclusively by glucuronidation and is not metabolized by the cytochrome P450 system.^[1]^[2] Less than 1% of radiolabeled material in plasma comprises unconjugated raloxifene.^[2] The metabolites of raloxifene include several glucuronides.^[1] The elimination half-life of raloxifene after a single dose is 27.7 hours (1.2 days), whereas its half-life at steady state at a dosage of 60 mg/day is 15.8 to 86.6 hours (0.7–3.6 days), with an average of 32.5 hours (1.4 days).^[1]^[2] The extended half-life of raloxifene is attributed to enterohepatic recirculation and its high plasma protein binding.^[1] Raloxifene and its glucuronide conjugates are interconverted by reversible metabolism and enterohepatic recycling, which prolongs the elimination half-life of raloxifene with oral administration.^[2] The medication is deconjugated into its active form in a variety of tissues, including liver, lungs, spleen, bone, uterus, and kidneys.^[1]

Elimination

Raloxifene is mainly excreted in bile and is eliminated in feces.^[1]^[2] Less than 0.2% of a dose is excreted unchanged in urine and less than 6% of a dose is excreted in urine as glucuronide conjugates.^[2]

Chemistry

Raloxifene hydrochloride has the empirical formula C₂₈H₂₇NO₄S•HCl, which corresponds to a molecular weight of 510.05 g/mol. Raloxifene hydrochloride is an off-white to pale-yellow solid that is slightly soluble in water.^[14]

Raloxifene is a benzothiophene derivative and is structurally distinct from the triphenylethylene SERMs like tamoxifen, clomifene, and toremifene.^[37] It is the only benzothiophene SERM to have been marketed.^[37] A benzothiophene SERM that was not marketed is arzoxifene (LY-353381).^[38] Bazedoxifene (Duavee, Viviant) and pipendoxifene (ERA-923) are structurally related to raloxifene but are technically not benzothiophenes and instead are indoles.^[38]

History

Raloxifene was approved in the United States for the prevention of postmenopausal osteoporosis in 1997, the treatment of postmenopausal osteoporosis in 1999, and to prevent or reduce the risk of breast cancer in certain postmenopausal women in 2007.^[39]^[40]^[41]^[42] It received orphan designation in 2005.^[39]

Society and culture

A bottle of raloxifene.

Names

Raloxifene is the generic name of the drug and its INN and BAN, while raloxifène is its DCF and raloxifene hydrochloride is its USAN, BANM, and JAN.^[43]^[44]^[45]^[46] It has also been known by the name keoxifene.^[43]^[44]^[46]

Raloxifene is sold mainly under the brand name Evista and to a lesser extent the brand name Optruma.^[46]^[44] It is also sold under a variety of other brand names in various countries.^[46]

Availability

Raloxifene is available widely throughout the world, including in the United States, Canada, the United Kingdom, Ireland, elsewhere throughout Europe, Australia, New Zealand, South Africa, Latin America, Southern, Eastern, and Southeastern Asia, and elsewhere in the world such as in Israel and Egypt.^[46]^[44]

Raloxifene is provided in the form of 60 mg oral tablets.^[10]

Controversy

An editorial in Lancet Oncology criticized the way that research about the medication for breast cancer prevention was released.^[47]

Research

Clinical studies of raloxifene for metastatic breast cancer in women have been conducted but found little effectiveness at 60 mg/day in those previously treated with tamoxifen, though modest effectiveness has been observed at higher doses.^[12]^[48] In contrast to tamoxifen, raloxifene is not approved for the treatment of breast cancer.^[49]

Raloxifene has been studied in men for a variety of uses, such as for treatment of schizophrenia, prostate cancer, and osteoporosis.^[50]^[51]^[52]^[53]^[54]^[33]^[32]^[55]^[56]^[57]^[58] It has been studied in combination with castration and bicalutamide, a nonsteroidal antiandrogen, for the treatment of prostate cancer.^[58]^[55]

Raloxifene has been studied as an adjunct in the treatment of schizophrenia in postmenopausal women.^[59] A 2017 meta-analysis concluded that it was safe and effective for this indication, although further studies with larger sample sizes are needed for confirmation.^[59] It may be effective in women with less severe symptoms.^[59]

A tissue-selective estrogen-receptor complex (TSEC) of estradiol and raloxifene has been studied in postmenopausal women.^[60]

Raloxifene (60 mg/day) was reported to be effective in the treatment of pubertal gynecomastia in adolescent boys in a small retrospective chart review.^[61]^[62]^[63] Other SERMs are also known to be effective in the treatment of gynecomastia.^[64]

Raloxifene has been reported to augment the antidepressant effects of selective serotonin reuptake inhibitors (SSRIs).^[65]

June 18th 2020, Exscalate4CoV, the private-public consortium supported by the EU’s Horizon 2020 programme for research and innovation, led by Dompé farmaceutici and currently representing 18 partners (including Fraunhofer Institute, CINECA, Chelonia Applied Science, Swiss Institute of Bioinformatics and others) has requested access to clinical trials for the use of Raloxifene in Covid 19 patients. Raloxifene, already proven effective against Mers and Sars in precliinical tests, has been indicated as effective against Sars-Cov2 by the “in-silico” research conducted by the consortium which has shown efficacy in countering the replication of the virus in cells. The IP for its use against Sars-Cov2 has already been protected on May 6 2020 in the name Dompé farmaceutici, Fraunhofer Institute and KU Leuven, to facilitate the largest possible access. Raloxifene would be used in mildly symptomatic Covid19 patients to halt the spread of infection. This result emerged from the first virtual (in silico) screening conducted on the Consortium’s supercomputers of more than 400.000 molecules (safe-in-man drugs and natural products) made available by Dompé farmaceutici and the partner Fraunhofer (IME) to the Consortium. The molecules were prioritized if in clinical stage or already on the market. 7.000 molecules with certain promising characteristics were tested.

SYN

Jones, Charles D.; Jevnikar, Mary G.; Pike, Andrew J.; Peters, Mary K.; Black, Larry J.; Thompson, Allen R.; Falcone, Julie F.; Clemens, James A. (1984). “Antiestrogens. 2. Structure-activity studies in a series of 3-aroyl-2-arylbenzo[b]thiophene derivatives leading to [6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl]-[4-[2-(1-piperidinyl)ethoxy]phenyl]methanone hydrochloride (LY 156758), a remarkably effective estrogen antagonist with only minimal intrinsic estrogenicity”. Journal of Medicinal Chemistry 27 (8): 1057–66.doi:10.1021/jm00374a021. PMID 6431104.

syn 1

EP 0062053; GB 2097788

Keoxifene has been synthesized using the following process: A portion of 6-methanesulfonyloxy-2-(4-methanesulfonyloxyphenyl)-3-[4-(2-pipendinoethoxy)benzoyl]benzo[b]thiophene hydrochloride (I) was combined with denatured alcohol and 5N sodium hydroxide, and stirred under a nitrogen atmosphere. The reaction mixture was evaporated to dryness under vacuum, and the residue dissolved in water and washed with diethyl ether. The water layer was degassed under vacuum, and then nitrogen was bubbled through it to remove all traces of ether. The mixture was then acidified with 1N hydrochloric acid, and then made basic with excess sodium bicarbonate The precipitate was collected by filtration and washed with cold water to obtain crude product, which was purified on a column of silica gel. The column was eluted first with 700 ml of 5% methanol in chloroform, followed by 1l of 10% methanol in chloroform. The impurities came off first, and the product-containing fractions were combined and evaporated under vacuum to obtain a yellow oil. The oil was dissolved in acetone seeded and chilled in a freezer to obtain the purified product.

syn2

J Label Compd Radiopharm 1995,36(1),43

The synthesis of radiolabeled raloxifene has been reported: The esterification of 3,5-dibromo-4-hydroxybenzoic acid (I) with methanol/HCl gives the corresponding methyl ester (II), which is condensed with 1-(2-chloroethyl)piperidine (III) by means of K2CO3 in DMF yielding 3,5-dibromo-4-[2-(1-piperidyl)ethoxy]benzoic acid methyl ester (IV). The hydrolysis of (IV) with NaOH in methanol affords the corresponding free acid (V), which by treatment of SOCl2 in toluene is converted to the acyl chloride (VI). The Friedel-Crafts condensation of (VI) with 6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene (VII) by means of AlCl3 in dichloromethane gives [3,5-dibromo-4-[2-(1-piperidinyl)ethoxy]phenyl]-[6-methoxy-2-(4-methoxy phenyl)benzo[b]thien-3-yl]methanone (VIII), which is demethylated with AlCl3 and ethylmercaptane to dibromoraloxifene (IX). Finally, this compound is submitted to hydrogenolysis with tritium over Pd/C in methanol.

syn 3

Bioorg Med Chem Lett 1997,7(8),993

The two major metabolites of raloxifene, the glucuronide conjugates (VI) and (VIII) are synthesized as follows: The partial silylation of raloxifene (I) with tert-butyldimethylsilyl chloride (TBDMS-Cl) by means of dimethylaminopyridine (DMAP) in THF/DMF gives a mixture of the monosilylated compounds (II) and (III), which are separated by chromatography. Compounds (II) and (III) are independently condensed with methyl 1,2,3,4-tetra-O-acetyl-D-glucuronate (IV) by means of BF3.OEt2 in dichloromethane yielding protected glucuronides (V) and (VII), respectively. Finally, both compounds are deprotected by a treatment first with LiOH in dioxane to hydrolyzed the ester groups, and then with tetrabutylammonium fluoride in THF to eliminate the silyl groups, thus obtaining the desired metabolites (VI) and (VIII), respectively.

syn 4

Tetrahedron Lett 1999,40(28),5155

Two related new syntheses of raloxifene have been described: 1) The acylation of N-(6-methoxy-1-benzothiophen-2-yl)-N,N-dimethylamine (I) with 4-fluorobenzoyl chloride (II) by heating at 100 C in chlorobenzene gives the 3-acyl derivative (III), which is condensed with 4-methoxyphenylmagnesium bromide (IV) in THF yielding 3-(4-fluorobenzoyl)-6-methoxy-2-(4-methoxyphenyl)-1-benzothiophene (V). The condensation of (V) with 1-(2-hydroxyethyl)piperidine (VI) by means of NaH in DMF affords the ether (VII), which is finally demethylated with AlCl3 and ethanethiol. 2) The intermediate (III) can also be condensed first with 1-(2-hydroxyethyl)piperidine (VI) by means of NaH as before giving the piperidinoethyl ether (VIII), which is then condensed with the Grignard reagent (IV) affording the previously reported ether (VII).

syn

Org Chem Ind J, Volume: 14( 3)

https://www.tsijournals.com/articles/industrially-viable-demethylation-reaction-in-synthesis-of-raloxifene-hydrochloride-13848.html

A GREEN PROCESS FOR DEMETHYLATION REACTION IN SYNTHESIS OF RALOXIFENE HYDROCHLORIDEAuthors : Ramadas Chavakula *, Chakradhar Saladi J S, Narayana Rao Mutyalaa , Vijaya Raju Maddalaa and Raghu Babu Kb

A green process for demethylation reaction in synthesis of raloxifene hydrochloride by using aluminium chloride and odorless decanethiol as demethylation agent instead of aluminium chloride and ethanethiol (foul smell) under normal conditions is described.

Raloxifene hydrochloride [1], is an estrogen agonist/antagonist, commonly referred to as a Selective Estrogen Receptor Modulator (SERM) [1,2] that belongs to the benzothiophene class of compounds. Raloxifene decreases the resorption of bone and reduces the biochemical markers of bone turnover to the premenopausal range [3–5]. Raloxifene hydrochloride may also lower the chance of developing a certain type of breast cancer (invasive breast cancer) in post-menopausal women [6,7]. It can be synthesized [3] directly from aroylation of 6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene [2] by the acid chloride(4) of 4-[2-(1-piperidinyl)ethoxy]benzoic acid hydrochloride [3] in the presence of AlCl3 followed by addition of ethanethiol (FIG. 1).

Experimental Section

4-[2-(1-Piperidinyl)ethoxy]benzoic acid hydrochloride [3] and 6-methoxy-2-(4-methoxyphenyl) benzo[b] thiophene [2] were prepared by procedures reported previously [3]. Decanethiol was from commercial source. All melting points are uncorrected and were determined in capillary tubes on an Electothermal melting point apparatus. ¹H NMR spectra were recorded on a Brucker ADVANCE 400 MHz spectrometer, using DMSO-d6 as solvent and TMS as internal standard. Electrospray ionization mass spectroscopy was performed using an ion trap mass spectrometer (Model 6310 Agilent). All reactions were monitored and checked by Thin Layer Chromatography (TLC) using methanol and spots examined by a UV lamp.

Preparation of [6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophen-3-yl][4-[2-(1-piperidyl)ethoxy]phenyl] methanone hydrochloride (Raloxifene hydrochloride) [1]

To a solution of 4-[2-(1-piperidinyl)ethoxy]benzoic acid hydrochloride (3) (14.3 g, 0.05 mol) in methylene dichloride (400 mL) and pyridine (0.5 mL) at 25ºC to 35ºC, thionyl chloride (23.8 g, 0.20 mol) was added dropwise under argon for 15-30 minute. The reaction mixture was stirred for 2 hr. at 40ºC to 45ºC. Excess thionyl chloride and solvent were removed in vacuum at 40◦C to afford 15.0 g of the crude acid chloride hydrochloride salt [4]. The crude solid acid chloride hydrochloride [4] was dissolved in methylene dichloride (150 mL), cooled to 0ºC to 10ºC, 6-methoxy-2-(4-methoxyphenyl)benzo[b] thiophene [2] (10.8 g, 0.04 mol) was added. Then, anhydrous aluminium chloride (37.0 g, 0.28 mol) was added portion wise over a period of 30 min and then the mixture was allowed to warm to 30ºC and stirred for 2 hr at 25-35ºC. Then decanethiol (28.0 g, 0.16 mol) was added and stirred for 2 hr. at 25-35ºC. The reaction mixture was quenched with mixture of methanol (100 mL), ice (200 g) and Conc. HCl (15 mL) and stirred for 1 hr. at 25-35ºC. The precipitated solid was collected, washed with water (100 mL X 2) and dried at 65ºC for 4 h to afford 20.0 g of crude compound 1, which was crystallized from methanol/water (23/1, vol/vol) to yield 13.6 g of compound 1 (53.3 %yield) as a white solid, MP 258-260°C, liter 3, 258°C ; ¹H NMR: δ 1.34, 1.72 [2H, m, (CH₂CH₂)₂CH₂], 1.76 [4H, m, N(CH₂CH₂)₂], 2.96 (2H, m, N-CH2), 3.43 [4H, m, N(CH₂CH₂)₂], 4.44 (2H, m, O-CH₂), 6.67 (2H, d, Ar), 6.85 (1H, d, Ar), 6.95 (2H, d, Ar), 7.18 (2H, d, Ar), 7.25 (1H, d, Ar), 7.35 (1H, s, Ar), 7.70 (2H, d, Ar), 9.77 (1H, s, OH), 9.82 (1H, s, OH), 10.16 (1H, brs, NH), MS (ESI): m/z 474.6 (M +H). “This procedure has been scaled up using 250g of compound 1.”

Results and Discussion

Commonly used thiols like ethanethiol and benzyl mercaptan in demethylation reactions have a foul smell making them difficult and unpleasant to use in the laboratory without fume hoods. The problem becomes even worse in industry on a large scale. Odorless substitutes are therefore always required. Few papers [8,9] discuss the use of long chain thiols to minimize odor, so we used this work as a basis for choosing a long chain thiol for our demethylation reaction. We now report a new, highly active demethylation reagent, an aluminum chloride and decanethiol, characterized by rapid action under mild conditions, easy workup of the reaction product, and high yield (FIG. 2.).

Figure 2: Synthesis of Raloxifene hydrochloride.

Conclusion

In conclusion, we have found that decanethiol is odorless thiol compared to ethanethiol. We believe that removing the foul-smelling thiols and use of these odorless thiols will greatly improve the greenchemistry.

References

Grese TA, Dodge JA. Selective Estrogen Receptor Modulators (SERMs). Curr Pharm Des. 1998;4:71-92.
Bryant HU, Dere WH. Selective estrogen receptor modulators: an alternative to hormone replacement therapy. Proc Soc Exp Biol Med. 1998;217:45-52.
Jones CD, Jevnikar MG, Pike AJ, et al. Antiestrogens. 2. Structure-activity studies in a series of 3-aroyl-2-arylbenzo [b] thiophene derivatives leading to [6-hydroxy-2-(4-hydroxyphenyl) benzo [b] thien-3-yl]-[4-[2-(1-piperidinyl) ethoxy] phenyl] methanone hydrochloride (LY 156758), a remarkably effective estrogen antagonist with only minimal intrinsic estrogenicity. J Med Chem. 1984;27:1057-66.
Sato M, Grese TA, Dodge JA, et al. Emerging therapies for the prevention or treatment of postmenopausal osteoporosis. J Med Chem. 1999;42:1-24.
Draper MW, Flowers DE, Huster WJ, et al. A controlled trial of raloxifene (LY139481) HCl: impact on bone turnover and serum lipid profile in healthy postmenopausal women. J Bone Miner Res. 1996;11:835-42.

paper

https://www.sciencedirect.com/science/article/abs/pii/S0223523412001122

Recent advances in the synthesis of raloxifene: A selective estrogen receptor modulator - ScienceDirect

syn

https://www.tandfonline.com/doi/abs/10.1080/00397911.2014.943348?journalCode=lsyc20

Piperidine Nucleophilic Substitution Without Solvent: An Efficient Synthesis of RaloxifeneYewei Yang,Tao Zhang,Wenhai Huang &Zhenrong Shen Pages 3271-3276 |

https://doi.org/10.1080/00397911.2014.943348

Mild and high-yielding synthesis is described for raloxifene via piperdine nucleophilic substitution of a new raloxifene intermediate 3-aroyl-2-aryl-substituted benzo[b]thiophenes, which is obtained by acylation of para-substituted benzoyl chlorides and 2-arylbenzo[b]thiophenes. The key step is solvent free and offers valuable advantages, such as low cost, and is suitable for industrial production.

Keywords: Friedel–Crafts acylation, green chemistry, nucleophiles, raloxifene, SERM

The improved synthesis of raloxifene 1 was accomplished as shown in Scheme 2. Methyl p-hydroxybenzoate 2, 1-bromo-2-chloroethane, and K2CO3 were refluxed in acetone, yielding compound 3 in 94% yield. Without prior purification, 3 was hydrolyzed to the corresponding p-substituted benzoyl acids 4 in 100% yield. The application of general reaction conditions of methanol as solvent and hydrochloric as acid would afford the substitution impurity 4-(2-methoxyethoxy)-benzoic acid. To control this impurity during reaction, various solvents such as alcohol, ethyl acetate, acetone, and tetrahydrofuran (THF) were screened, and THF gave the best result from the view of impurity formation and yield. Compound 4 is a solid and was easily isolated from THF by adding water. Then 4 was transferred to acid chlorides 5 and substantially reacted with benzothiophene 6 using AlCl3 in dichloromethane at 50 C to afford aroylated benzothiophene 7 in two steps, with yield of 95% (79% from method A[8] and 65.5% from method B[3]). With the requisite 7 in hand, we next examined piperidine nucleophilic substitution to produce the desired beno[b]thien-3-yl ketones 8. In general using reaction conditions A (acetone, NaI, K2CO3, reflux, 70%) and B (acetonitrile, NaI, K2CO3, reflux, 85%), impurity formation was observed from the beginning of the reaction. We screened various conditions and were delighted to found that using excess piperidine at reflux temperature gave negligible impurity formation. Piperidine was not only reagent but also solvent. The isolated product 8 was stable and was converted into the desired raloxifene 1 as reported. In conclusion, we have developed a viable alternative route for the synthesis of raloxifene. The new synthesis would have been better able to support the increase in bulk demand for this drug for the chemoprevention of breast cancer and novel formulations. Our synthetic route has several advantages: the use of difunctionalized coumpunds 5 as key intermediate makes Friedel–Crafts acylation and nucleophilic substitution highly efficient. The using of piperine as reagent and solvent avoids the large waste streams derived from neutralization reaction of sodium hydride. The cost of the new route is less than the current route of manufacture.

Preparation of [4-(2-Chloro-ethoxy)-phenyl]-[6-methoxy-2- (4-methoxy-phenyl)-benzo[b]thiophen-3-yl]-methanone (7) Under an N2 atmosphere, 5 was added to a mixture of 6 (20.25 g, 75 mmol) and AlCl3 (13.30 g, 100 mmol) in DCM (2 mL), and the mixture was stirred for 12 h. The reaction was monitored by TLC (n-hexane/EtOAc, 4:1). After the reaction was judged complete, the reaction mixture was allowed to cool. The crude mixture was poured into H2O and extracted with EtOAc. The organic layer was separated and concentrated. The residue was crystallized from EtOAc to give the product 7 (32.26 g, 95%): yellow solid crystals; mp 119–120 C; IR (KBr) nmax: 2960, 2835, 1647, 1599, 1472, 1251, 1169, 1032, 830 cm1 ; 1 H NMR (400 MHz, CDCl3) d 7.76 (d, J ¼ 8.8 Hz, 2H), 7.53 (d, J ¼ 8.8 Hz, 1H), 7.32 (d, J ¼ 8.4 Hz, 2H), 7.31 (s, 1H), 6.95 (dd, J ¼ 8.4, 2.4 Hz, 1H), 6.75 (dd, J ¼ 9.2, 7.2 Hz, 4H), 4.20 (t, J ¼ 4.0 Hz,2H), 3.87 (s, 3H), 3.78 (t, J ¼ 6.0 Hz, 2H), 3.74 (s, 3H); 13C NMR (100 MHz, CDCl3) d 193.1, 162.1, 159.7, 157.6, 142.7, 139.9, 133.8, 132.3, 130.9, 130.2, 130.1, 125.9, 123.9, 114.8, 114.1, 113.9, 104.4, 67.8, 55.6, 55.2, 41.5; MS (EI) m/z (%):452 (Mþ, 100.0), 437 (13.0), 297 (25.0), 183 (39.0), 121 (44.0). HRMS m/z (EI) calcd. for C25H22ClO4S: (MþH) þ: 453.0927; found: 453.0933.
Preparation of [6-Methoxy-2-(4-methoxy-phenyl)-benzo[b] thiophen-3-yl]-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-methanone (8) Under an N2 atmosphere, a mixture of 7 (8.50 g, 19 mmol) and piperdine (30 ml) was stirred under reflux for 12 h. The reaction was monitored by TLC (n-hexane/EtOAc, 4:1). After the reaction was judged complete, the reaction mixture was allowed to cool. The mixture was concentrated for recovery of piperidine. EtOAc was added and the residue was washed with saturated NaHCO3 aqueous solution. The organic layer was separated and concentrated to give the product 8 (8.80 g, 94%): yellow viscous oil; IR (KBr) nmax: cm1 2933, 1645, 1597, 1535, 1501, 1470, 1249, 1164, 1030, 827; 1 H NMR (400 MHz, CDCl3) d7.76 (d, J ¼ 8.8 Hz, 2H), 7.52 (d, J ¼ 8.8 Hz, 1H), 7.33 (d, J ¼ 8.8 Hz, 2H), 7.30 (d, J ¼ 2.4 Hz, 1H), 6.94 (dd, J ¼ 8.8, 2.0 Hz, 1H), 6.75 (dd, J ¼ 7.2, 5.2 Hz, 4H), 4.08 (t, J ¼ 6.0 Hz, 2H), 3.86 (s, 3H), 3.73 (s, 3H), 2.71 (t, J ¼ 6.0 Hz, 2H), 2.46 (s, 4H), 1.60–1.54 (m, 4H), 1.43–1.41 (m, 2H).13C NMR (100 MHz, CDCl3) d 193.2, 163.0, 159.7, 157.6, 142.4, 140.1, 133.9, 132.3, 130.6, 130.4, 130.2, 126.0, 124.0, 114.8, 114.2, 114.1, 104.5, 66.3, 57.7, 55.6, 55.2, 55.1, 25.9, 24.1. MS (EI) m/z (%): 501 (Mþ, 100.0), 452 (12.0), 402 (21.0), 297 (24.0), 98 (100.0). HRMS m/z (EI) calcd. for C30H32NO4S: (MþH) þ: 502.2052; found: 502.2055.REFERENCES 1. Clemett, D.; Spencer, C. M. Drugs 2000, 60 (2), 379–411. 2. Land, S. R. JAMA 2007, 298 (9), 973–973. 3. Dadiboyena, S. Eur. J. Med. Chem. 2012, 51, 17–34. 4. Schmid, C. R.; Sluka, J. P.; Duke, K. M. Tetrahedron Lett. 1999, 40 (4), 675–678. 5. Bradley, D. A.; Godfrey, A. G.; Schmid, C. R. Tetrahedron Lett. 1999, 40 (28), 5155–5159. 6. Shinde, P. S.; Shinde, S. S.; Renge, A. S.; Patil, G. H.; Rode, A. B.; Pawar, R. R. Lett. Org. Chem. 2009, 6 (1), 8–10.7. Sach, N. W.; Richter, D. T.; Cripps, S.; Tran-Dube, M.; Zhu, H. C.; Huang, B. W.; Cui, J.; Sutton, S. C. Org. Lett. 2012, 14 (15), 3886–889. 8. Jones, C. D.; Jevnikar, M. G.; Pike, A. J.; Peters, M. K.; Black, L. J.; Thompson, A. R.; Falcone, J. F.; Clemens, J. A. J. Med. Chem. 1984, 27 (8), 1057–1066. 9. Grese, T. A.; Cho, S.; Finley, D. R.; Godfrey, A. G.; Jones, C. D.; Lugar, C. W.; Martin, M. J.; Matsumoto, K.; Pennington, L. D.; Winter, M. A.; Adrian, M. D.; Cole, H. W.; Magee, D. E.; Phillips, D. L.; Rowley, E. R.; Short, L. L.; Glasebrook, A. L.; Bryant, H. U. J. Med. Chem. 1997, 40 (2), 146–167.
synChapter 2 – 1-Substituted PiperidinesAuthor links open overlay panelRubenVardanyan
https://doi.org/10.1016/B978-0-12-805157-3.00002-8 Piperidine-Based Drug DiscoveryHeterocyclic Drug Discovery2017, Pages 83-1011-Substituted Piperidines

Ruben Vardanyan, in Piperidine-Based Drug Discovery, 2017

Raloxifene (7685)

Raloxifene (Evista) (1.3.4) is a second-generation selective estrogen receptor modulator that functions as an estrogen antagonist on breast and uterine tissues, and an estrogen agonist on bone. Raloxifene is an antiresorptive agent, a new representative of a class of drugs that prevent the loss of bone mass, i.e., used to treat osteoporosis and similar diseases in postmenopausal women and those postmenopausal women at increased risk of invasive breast cancer [41–53].

It was shown that raloxifene can have some affect on cognition, mental health, sleep, and sexual function in menopausal women [54]. Raloxifene was used also as an adjuvant treatment in postmenopausal women with schizophrenia [55].

The first reported synthesis of the raloxifene scaffold consists in Friedel-Crafts aroylation in 1,2-dichloroethane and using AlCl₃ as a catalyst by coupling of 4-(2-(piperidin-1-yl)ethoxy)benzoyl chloride (2.3.15) with benzothiophene derivative (2.3.16) followed by alkaline hydrolysis of mesyl groups, which give the desired raloxifene (2.3.4) [56–58] (Scheme 2.9).

The key intermediate – 6-methoxy-2-(4-methoxyphenyl)benzo[b]thiophene (2.3.16) – was prepared by the cyclization-rearrangement of 1-(4-methoxyphenyl)-2-((3-methoxyphenyl)thio)ethan-1-one (2.3.20) induced by polyphosphoric acid (PPA). This rearrangement (Kost rearrangement [59]) is general for 3-(R-substituted)indoles, -benzofurans, and -benzothiophenes, which are converted to the corresponding 2-isomers by heating with PPA.

The synthesis started from thiophenol (2.3.18) and bromoketone (2.3.19), which were coupled in presence of KOH in ethanol/water solution. Obtained (2.3.20) was heated with PPA to give a mixture that is easily separable by crystallization isomeric 2-phenylbenzo[b]thiophenes (2.3.21) and (2.3.22), where preferable, isomer (2.3.22) predominates. Cleavage of the methoxy groups in (2.3.22) was done conveniently with pyridine hydrochloride to give (2.3.23), which was easily converted to mesylate (2.3.16) with methanesulfonyl chloride in pyridine and 4-dimethylaminopyridine as a catalyst (Scheme 2.10).

The second reagent—4-(2-(piperidin-1-yl)ethoxy)benzoyl chloride (2.3.15)—was prepared starting with 4-hydroxybenzoate (2.3.24), which with 1-(2-chloroethyl)piperidine (2.3.25) in anhydrous DMF, and K₂CO₃ or sodium hydride, gave methyl 4-(2-(piperidin-1-yl)ethoxy)benzoate (2.3.26) hydrolyzed in MeOH/water NaOH solution. The acid (2.3.26) was converted to its chloride (2.3.15) with SOCl₂ in 1,2-dichloroethane and a catalytic amount of DMF (Scheme 2.11).

Another novel convenient synthesis of raloxifene (2.3.4) have been proposed [60]. According to this method anisaldehyde (2.3.28) was transformed to corresponding cyanohydrin (2.3.29) using a mixture of sodium cyanide ethanol containing triethylamine through which HCl gas was passed over 30 minutes at 5–10°C.

Gaseous HCl was added to the solution of prepared cyanohydrin (2.3.29) in ethanol at room temperature over 30 minutes in order to give p-methoxybenzaldehyde cyanohydrin iminoether hydrochloride (2.3.30). Then, hydrogen sulfide was bubbled into a solution of the methyl imidate (2.3.30) and triethylamine in methanol at 0°C to give α-(4-methoxy phenyl)-α-hydroxy-N,N dimethylthioacetamide (2.3.31).

To the obtained α-hydroxythioamide (2.3.31) dissolved-in-methylene chloride methanesulfonic acid was slowly added, which transformed the starting material to 2-N,N-dimethylamino-6-methoxy benzo[β]thiophene (2.3.32).

The obtained 2-dimethylaminobenzothiophene (2.3.32) and known 4-(2-piperidinoethoxy)-benzoyl chloride (2.3.15) were partially dissolved in chlorobenzene and the mixture was warmed in a 100–105°C to give 2-(4-methoxyphenyl)-6-methoxy-3-[4-(piperidinoethoxy)benzoyl]-benzo[β]thiophene (2.3.33). 4-Methoxyphenylmagnesium bromide (2.3.34) in THF was added to chilled to 0°C prepared compound (2.3.33) in THF, which gave 2-(4-methoxyphenyl)-6-methoxy-3-[4-(piperidinoethoxy)benzoyl] benzo[β] thiophene (2.3.35). To the prepared benzothiophene (2.3.35) suspended in chlorobenzene was added AlCl₃, followed by the addition of n-propanethiol, and the mixture was heated at 35°C. After the workup with aqueous HCl, the desired raloxifene (2.3.4) was separated [60] (Scheme 2.12).

There exist plenty of modifications for these two approaches, as reviewed in [61,62].

Clinical data


Trade names	Evista, Optruma, others
Other names	Keoxifene; Pharoxifene; LY-139481; LY-156758; CCRIS-7129
AHFS/Drugs.com	Monograph
MedlinePlus	a698007
License data	EUEMA: by INNUSDailyMed: RaloxifeneUSFDA: Evista
Pregnancy category	AU: X (High risk)US: X (Contraindicated)
Routes of administration	By mouth
Drug class	Selective estrogen receptor modulator
ATC code	G03XC01 (WHO)
Legal status
Legal status	In general: ℞ (Prescription only)
Pharmacokinetic data
Bioavailability	2%^[1]^[2]
Protein binding	>95%^[1]^[2]
Metabolism	Liver, intestines (glucuro- nidation);^[1]^[2]^[3]CYP450 system not involved^[1]^[2]
Elimination half-life	Single-dose: 28 hours^[1]^[2] Multi-dose: 33 hours^[1]
Excretion	Feces^[2]
Identifiers
IUPAC name [show]
CAS Number	84449-90-1 82640-04-8 (hydrochloride)
PubChemCID	5035
IUPHAR/BPS	2820
DrugBank	DB00481
ChemSpider	4859
UNII	YX9162EO3I
ChEBI	CHEBI:8772
ChEMBL	ChEMBL81
PDB ligand	RAL (PDBe, RCSB PDB)
CompTox Dashboard (EPA)	DTXSID3023550
ECHA InfoCard	100.212.655
Chemical and physical data
Formula	C₂₈H₂₇NO₄S
Molar mass	473.584 g·mol⁻¹
3D model (JSmol)	Interactive image
SMILES [hide]O=C(c1c3ccc(O)cc3sc1c2ccc(O)cc2)c5ccc(OCCN4CCCCC4)cc5
InChI [hide]InChI=1S/C28H27NO4S/c30-21-8-4-20(5-9-21)28-26(24-13-10-22(31)18-25(24)34-28)27(32)19-6-11-23(12-7-19)33-17-16-29-14-2-1-3-15-29/h4-13,18,30-31H,1-3,14-17H2Key:GZUITABIAKMVPG-UHFFFAOYSA-N

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^ James F. Holland; Raphael E. Pollock (2010). Holland-Frei Cancer Medicine 8. PMPH-USA. pp. 743–. ISBN 978-1-60795-014-1.
^ Blum A, Hathaway L, Mincemoyer R, Schenke WH, Csako G, Waclawiw MA, Panza JA, Cannon RO (2000). “Hormonal, lipoprotein, and vascular effects of the selective estrogen receptor modulator raloxifene in hypercholesterolemic men”. Am. J. Cardiol. 85 (12): 1491–4, A7. doi:10.1016/s0002-9149(00)00802-x. PMID 10856400.
^ Doran PM, Riggs BL, Atkinson EJ, Khosla S (2001). “Effects of raloxifene, a selective estrogen receptor modulator, on bone turnover markers and serum sex steroid and lipid levels in elderly men”. J. Bone Miner. Res. 16 (11): 2118–25. doi:10.1359/jbmr.2001.16.11.2118. PMID 11697809. S2CID 28216610.
^ Dimaraki EV, Symons KV, Barkan AL (2004). “Raloxifene decreases serum IGF-I in male patients with active acromegaly”. Eur. J. Endocrinol. 150 (4): 481–7. doi:10.1530/eje.0.1500481. PMID 15080777.
^ Duschek EJ, Gooren LJ, Netelenbos C (2004). “Effects of raloxifene on gonadotrophins, sex hormones, bone turnover and lipids in healthy elderly men” (PDF). Eur. J. Endocrinol. 150 (4): 539–46. doi:10.1530/eje.0.1500539. PMID 15080785.
^ Smith MR, Fallon MA, Lee H, Finkelstein JS (2004). “Raloxifene to prevent gonadotropin-releasing hormone agonist-induced bone loss in men with prostate cancer: a randomized controlled trial”. J. Clin. Endocrinol. Metab. 89 (8): 3841–6. doi:10.1210/jc.2003-032058. PMID 15292315.
^ Jump up to:^a ^b Ho TH, Nunez-Nateras R, Hou YX, Bryce AH, Northfelt DW, Dueck AC, Wong B, Stanton ML, Joseph RW, Castle EP (2017). “A Study of Combination Bicalutamide and Raloxifene for Patients With Castration-Resistant Prostate Cancer”. Clin Genitourin Cancer. 15 (2): 196–202.e1. doi:10.1016/j.clgc.2016.08.026. PMID 27771244. S2CID 19043552.
^ Khodaie-Ardakani MR, Khosravi M, Zarinfard R, Nejati S, Mohsenian A, Tabrizi M, Akhondzadeh S (2015). “A Placebo-Controlled Study of Raloxifene Added to Risperidone in Men with Chronic Schizophrenia”. Acta Med Iran. 53 (6): 337–45. PMID 26069170.
^ Weickert TW, Weinberg D, Lenroot R, Catts SV, Wells R, Vercammen A, O’Donnell M, Galletly C, Liu D, Balzan R, Short B, Pellen D, Curtis J, Carr VJ, Kulkarni J, Schofield PR, Weickert CS (2015). “Adjunctive raloxifene treatment improves attention and memory in men and women with schizophrenia”. Mol. Psychiatry. 20 (6): 685–94. doi:10.1038/mp.2015.11. PMC 4444978. PMID 25980345.
^ Jump up to:^a ^b Fujimura, Tetsuya; Takayama, Kenichi; Takahashi, Satoru; Inoue, Satoshi (2018). “Estrogen and Androgen Blockade for Advanced Prostate Cancer in the Era of Precision Medicine”. Cancers. 10 (2): 29. doi:10.3390/cancers10020029. ISSN 2072-6694. PMC 5836061. PMID 29360794.
^ Jump up to:^a ^b ^c Wang Q, Dong X, Wang Y, Li X (2017). “Raloxifene as an adjunctive treatment for postmenopausal women with schizophrenia: a meta-analysis of randomized controlled trials”. Arch Womens Ment Health. 21 (1): 31–41. doi:10.1007/s00737-017-0773-2. PMID 28849318. S2CID 4524617.
^ Carneiro AL, de Cassia de Maio Dardes R, Haidar MA (July 2012). “Estrogens plus raloxifene on endometrial safety and menopausal symptoms–semisystematic review”. Menopause. 19 (7): 830–4. doi:10.1097/gme.0b013e31824a74ce. PMID 22549172. S2CID 196380398.
^ Nordt CA, DiVasta AD (2008). “Gynecomastia in adolescents”. Curr. Opin. Pediatr. 20 (4): 375–82. doi:10.1097/MOP.0b013e328306a07c. PMID 18622190. S2CID 205834072.
^ Leung KC, Leung AC (2017). “Gynecomastia in Infants, Children, and Adolescents”. Recent Pat Endocr Metab Immune Drug Discov. 10 (2): 127–137. doi:10.2174/1872214811666170301124033. PMID 28260521.
^ Lawrence SE, Faught KA, Vethamuthu J, Lawson ML (July 2004). “Beneficial effects of raloxifene and tamoxifen in the treatment of pubertal gynecomastia”. J. Pediatr. 145 (1): 71–6. doi:10.1016/j.jpeds.2004.03.057. PMID 15238910.
^ Kanakis, G. A.; Nordkap, L.; Bang, A. K.; Calogero, A. E.; Bártfai, G.; Corona, G.; Forti, G.; Toppari, J.; Goulis, D. G.; Jørgensen, N. (2019). “EAA clinical practice guidelines—gynecomastia evaluation and management”. Andrology. 7 (6): 778–793. doi:10.1111/andr.12636. ISSN 2047-2919. PMID 31099174.
^ Sugiyama, Nobuhiro; Barros, Rodrigo P.A.; Warner, Margaret; Gustafsson, Jan-Åke (2010). “ERβ: recent understanding of estrogen signaling”. Trends in Endocrinology & Metabolism. 21 (9): 545–552. doi:10.1016/j.tem.2010.05.001. ISSN 1043-2760. PMID 20646931. S2CID 43001363.

External links

“Raloxifene”. Drug Information Portal. U.S. National Library of Medicine.

///////Keoxifene hydrochloride, Raloxifene hydrochloride, LY-139481, LY 156758, Optruma, Loxifen, Evista

REPROXALAP

November 13, 2020 8:40 am / Leave a comment

2-(3-Amino-6-chloroquinolin-2-yl)propan-2-ol.png

REPROXALAP

レプロキサラップ;

ADX-102

2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol

C12H13ClN2O, 236.7 g/mol

CAS 916056-79-6

UNII-F0GIZ22IJH

2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol

Phase 3 Clinical

Aldeyra Therapeutics is developing reproxalap, which binds and traps free aldehydes, formulated using Captisol technology licensed from Ligand Pharmaceuticals as an eye drop formulation, for treating acute noninfectious anterior uveitis, allergic conjunctivitis and dry eye syndrome.

PATENT

product case, WO2006127945 ,

EU states until 2026

expire US in 2029 with US154 extension.

PATENTS

WO2018170476

https://patentscope.wipo.int/search/es/detail.jsf;jsessionid=E1AD23F43DBB0FFDC006418CE99FCC69.wapp2nB?docId=WO2018170476&recNum=12607&office=&queryString=&prevFilter=&sortOption=Fecha+de+publicaci%C3%B3n%2C+orden+descendente&maxRec=70962897

United States patent application serial number US 13/709,802, filed December 10, 2012 and published as US 2013/0190500 on July 25, 2013 (“the ‘500 publication,” the entirety of which is hereby incorporated herein by reference), describes certain aldehyde scavenging compounds. Such compounds include com ound A:

[0036] Compound A, (6-chloro-3-amino-2-(2-hydroxypropyl)-l-azanaphthalene), is designated as compound A in the ‘500 publication and the synthesis of compound A is described in detail at Example 5 of the ‘500 publication, and is reproduced herein for ease of reference.

Example A – General Preparation of Compound A

Compound A

[00436] The title compound was prepared according to the steps and intermediates (e.g., Scheme 1) described below and in the ‘500 publication, the entirety of which is incorporated herein by reference.

Step 1: Synthesis of Intermediate A- 1

[00437] To a 2 L round bottom flask was charged ethanol (220 mL), and pyridine (31 g, 392 mmol) and the resulting solution stirred at a moderate rate of agitation under nitrogen. To this solution was added ethyl bromopyruvate (76.6 g, 354 mmol) in a slow, steady stream. The reaction mixture was allowed to stir at 65±5° C. for 2 hours.

Step 2: Synthesis of Intermediate A-2

[00438] Upon completion of the 2-hour stir time in example 1, the reaction mixture was slowly cooled to 18-22° C. The flask was vacuum-purged three times at which time 2-amino-5-chloro-benzaldehyde (ACB) (50.0 g, 321 mmol) was added directly to the reaction flask as a solid using a long plastic funnel. Pyridine (64.0 g, 809 mmol) was added followed by an EtOH rinse (10 mL) and the reaction mixture was heated at 80±3° C. under nitrogen for about 16 hours (overnight) at which time HPLC analysis indicated that the reaction was effectively complete.

Step 3: Synthesis of Intermediate A-3

[00439] The reaction mixture from example 2 was cooled to about 70° C. and morpholine (76.0 g, 873 mmol)) was added to the 2 L reaction flask using an addition funnel. The reaction mixture was heated at 80±2° C. for about 2.5 hours at which time the reaction was considered complete by HPLC analysis (area % of A-3 stops increasing). The reaction mixture was cooled to 10-15° C. for the quench, work up, and isolation.

Step 4: Isolation of Intermediate A-3

[00440] To the 2 L reaction flask was charged water (600 g) using the addition funnel over 30-60 minutes, keeping the temperature below 15° C. by adjusting the rate of addition and using a cooling bath. The reaction mixture was stirred for an additional 45 minutes at 10-15° C. then the crude A-3 isolated by filtration using a Buchner funnel. The cake was washed with water (100 mLx4) each time allowing the water to percolate through the cake before applying a vacuum. The cake was air dried to provide crude A-3 as a nearly dry brown solid. The cake was returned to the 2 L reaction flask and heptane (350 mL) and EtOH (170 mL) were added and the mixture heated to 70±3° C. for 30-60 minutes. The slurry was cooled to 0-5° C. and isolated by filtration under vacuum. The A-3 was dried in a vacuum drying oven under vacuum and 35±3° C. overnight (16-18 hours) to provide A-3 as a dark green solid.

Step 5: Synthesis of Compound A

[00441] To a 2 L round bottom flask was charged methylmagnesium chloride (200 mL of 3.0 M solution in THF, 600 mmol). The solution was cooled to 0-5° C. using an ice bath.

[00442] A 500 mL flask (magnetic stirring) was charged with 22.8 grams A-3 from example 4 and THF (365 mL), stirred to dissolve then transferred to an addition funnel on the 2 L Reaction Flask. The A-3 solution was added drop-wise to the reaction flask over 5.75 hours, keeping the temperature of the reaction flask between 0-5° C throughout the addition. At the end of the addition the contents of the flask were stirred for an additional 15 minutes at 0-5° C. then the cooling bath was removed and the reaction was allowed to stir overnight at ambient temperature.

[00443] The flask was cooled in an ice bath and the reaction mixture was carefully quenched by adding EtOH (39.5 g, 857 mmol) drop-wise to the reaction mixture, keeping the temperature of the reaction mixture below 15° C. during the course of the addition. An aqueous solution of H4C1 (84.7 g H4C1 in 415 mL water) was then carefully added and the mixture stirred under moderate agitation for about 30 minutes then transferred to a separately funnel to allow the layers to separate. Solids were present in the aqueous phase so HO Ac (12.5 g) was added and the contents swirled gently to obtain a nearly homogeneous lower aqueous phase. The lower aqueous layer was transferred back to the 2 L reaction flask and stirred under moderate agitation with 2-methylTHF (50 mL) for about 15 minutes. The original upper organic layer was reduced in volume to approximately 40 mL using a rotary evaporator at≤40° C. and vacuum as needed. The phases in the separatory funnel were separated and the upper 2-MeTHF phase combined with the product residue, transferred to a 500 mL flask and vacuum distilled to an approximate volume of 25 mL. To this residue was added 2-MeTHF (50 mL) and distilled to an approximate volume of 50 mL. The crude compound A solution was diluted with 2-MeTHF (125 mL), cooled to 5-10° C. and 2M H2S04 (aq) (250 mL) was slowly added and the mixture stirred for 30 minutes as the temperature was allowed to return to ambient. Heptane (40 mL) was charged and the reaction mixture stirred for an additional 15 minutes then transferred to a separatory funnel and the layers were allowed to separate. The lower aqueous product layer was extracted with additional heptane (35 mL) then the lower aqueous phase was transferred to a 1 L reaction flask equipped with a mechanical stirrer and the mixture was cooled to 5-10° C. The combined organic layers were discarded. A solution of 25% NaOH(aq) was prepared (NaOH, 47 g, water, 200 mL) and slowly added to the 1 L reaction flask to bring the pH to a range of 6.5-8.5.

[00444] EtOAc (250 mL) was added and the mixture was stirred overnight. The mixture was transferred to a separatory funnel and the lower phase discarded. The upper organic layer was washed with brine (25 mL) then the upper organic product layer was reduced in volume on a rotary evaporator to obtain the crude compound A as a dark oil that solidified within a few minutes. The crude compound A was dissolved in EtOAc (20 mL) and filtered through a plug of silica gel (23 g) eluting with 3/1 heptane/EtOAc until all compound A was eluted (approximately 420 mL required) to remove most of the dark color of compound A. The solvent was removed in vacuo to provide 14.7 g of compound A as a tan solid. Compound A was taken up in EtOAc (25 mL) and eluted through a column of silica gel (72 g) using a mobile phase gradient of 7/1 heptane/EtOAc to 3/lheptane/EtOAc (1400 mL total). The solvent fractions containing compound A were stripped, compound A diluted with EtOAc (120 mL) and stirred in a flask with Darco G-60 decolorizing carbon (4.0 g) for about 1 hour. The mixture was filtered through celite using a fitted funnel, rinsing the cake with EtOAc (3 x 15 mL). The combined filtrates were stripped on a rotary evaporator and compound A dissolved in heptane (160 mL)/EtOAc(16 mL) at 76° C. The

homogeneous solution was slowly cooled to 0-5° C, held for 2 hours then compound A was isolated by filtration. After drying in a vacuum oven for 5 hours at 35° C. under best vacuum, compound A was obtained as a white solid. HPLC purity: 100% (AUC).

Example 1 – Preparation of Free Base Forms A and B of Compound A

Compound A

[00445] Compound A is prepared according to the method described in detail in Examples 1-5 of the ‘500 publication, the entirety of which is hereby incorporated herein by reference.

PATENT

example 5 [WO2018039197A1]

https://patents.google.com/patent/WO2018039197A1/en

Exam le 5: Synthesis of NS2

NS2

[00190] 2-(3-amino-6-chloroquinolin-2-yl)propan-2-ol. To a 2 L round bottom flask was charged methylmagnesium chloride (200 mL of 3.0 M solution in THF, 600 mmol). The solution was cooled to 0-5 °C using an ice bath.

[00191] A 500 mL flask (magnetic stirring) was charged with 22.8 grams A-3a from Example 4 and THF (365 mL), stirred to dissolve, and then transferred to an addition funnel on the 2 L reaction flask. The A-3a solution was added drop-wise to the reaction flask over 5.75 hours, keeping the temperature of the reaction flask between 0-5 °C throughout the addition. At the end of the addition the contents of the flask were stirred for an additional 15 minutes at 0-5 °C, then the cooling bath was removed and the reaction was allowed to stir overnight at ambient temperature.

[00192] The flask was cooled in an ice bath and the reaction mixture was carefully quenched by adding EtOH (39.5 g, 857 mmol) drop-wise to the reaction mixture, keeping the temperature of the reaction mixture below 15 °C during the course of the addition. An aqueous solution of H4CI (84.7 g H4CI in 415 mL water) was then carefully added and the mixture stirred under moderate agitation for about 30 minutes then transferred to a separatory funnel to allow the layers to separate. Solids were present in the aqueous phase so HOAc (12.5 g) was added and the contents swirled gently to obtain a nearly homogeneous lower aqueous phase. The lower aqueous layer was transferred back to the 2 L reaction flask and stirred under moderate agitation with 2-methyl-tetrahydrofuran (2-MeTHF) (50 mL) for about 15 minutes. The original upper organic layer was reduced in volume to approximately 40 mL using a rotary evaporator at < 40 °C under vacuum as needed. The phases in the separatory funnel were separated and the upper 2-MeTHF phase combined with the product residue was transferred to a 500 mL flask and vacuum distilled to an approximate volume of 25 mL. To this residue was added 2-MeTHF (50 mL) and the mixture again distilled to an approximate volume of 50 mL. The crude compound NS2 solution was diluted with 2-MeTHF (125 mL), cooled to 5-10 °C, and 2 M H₂S0₄ (aq) (250 mL) was slowly added and the mixture stirred for 30 minutes as the temperature was allowed to return to ambient. Heptane (40 mL) was charged and the reaction mixture stirred for an additional 15 minutes then transferred to a separatory funnel, and the layers were allowed to separate. The lower aqueous product layer was extracted with additional heptane (35 mL), then the lower aqueous phase was transferred to a 1 L reaction flask equipped with a mechanical stirrer, and the mixture was cooled to 5-10 °C. The combined organic layers were discarded. A solution of 25% NaOH (aq) was prepared (NaOH, 47 g, water, 200 mL) and slowly added to the 1 L reaction flask to bring the pH to a range of 6.5 – 8.5.

[00193] EtOAc (250 mL) was added and the mixture was stirred overnight. The mixture was transferred to a separatory funnel and the lower phase discarded. The upper organic layer was washed with brine (25 mL), then the upper organic product layer was reduced in volume on a rotary evaporator to obtain a obtain the crude compound NS2 as a dark oil that solidified within a few minutes. The crude compound NS2 was dissolved in EtOAc (20 mL) and filtered through a plug of silica gel (23 g) eluting with 3/1 heptane/EtOAc until all compound NS2 was eluted (approximately 420 mL required) to remove most of the dark color of compound NS2. The solvent was removed in vacuo to provide 14.7 g of compound NS2 as a tan solid. Compound NS2 was taken up in EtOAc (25 mL) and eluted through a column of silica gel (72g) using a mobile phase gradient of 7/1 heptane/EtOAc to 3/1 heptane/EtOAc (1400 mL total). The solvent fractions containing compound NS2 were evaporated. Compound NS2 was diluted with EtOAc (120 mL) and stirred in a flask with Darco G-60 decolorizing carbon (4.0 g) for about 1 hour. The mixture was filtered through celite using a firtted funnel, rinsing the cake with EtOAc (3 x 15 mL). The combined filtrates were evaporated on a rotary evaporator and compound NS2 dissolved in heptane (160 mL)/EtOAc (16 mL) at 76 °C. The homogeneous solution was slowly cooled to 0-5 °C, held for 2 hours, then compound NS2 was isolated by filtration. After drying in a vacuum oven for 5 hours at 35 °C under best vacuum, compound NS2 was obtained as a white solid. HPLC purity: 100% (AUC); HPLC (using standard conditions): A-2: 7.2 minutes; A-3 : 11.6 minutes.

Preparation of ACB

[00194] After a N₂ atmosphere had been established and a slight stream of N₂ was flowing through the vessel, platinum, sulfided, 5 wt. % on carbon, reduced, dry (9.04 g, 3.0 wt. % vs the nitro substrate) was added to a 5 L heavy walled pressure vessel equipped with a large magnetic stir-bar and a thermocouple. MeOH (1.50 L), 5-chloro-2-nitrobenzaldehyde (302.1 g, 1.63 mol), further MeOH (1.50 L) and Na₂C0₃ (2.42 g, 22.8 mmol, 0.014 equiv) were added. The flask was sealed and stirring was initiated at 450 rpm. The solution was evacuated and repressurized with N₂ (35 psi), 2x. The flask was evacuated and repressurized with H₂ to 35 psi. The temperature of the solution reached 30 °C w/in 20 min. The solution was then cooled with a water bath. Ice was added to the water bath to maintain a temperature below 35 °C. Every 2h, the reaction was monitored by evacuating and repressurizing with N₂ (5 psi), 2x prior to opening. The progress of the reaction could be followed by TLC: 5-Chloro-2-nitrobenzaldehyde (Rf = 0.60, CH2CI2, UV) and the intermediates (R_f = 0.51, CH2CI2, UV and R_f = 0.14, CH2CI2, UV) were consumed to give ACB (R_f = 0.43, CH2CI2, UV). At 5 h, the reaction had gone to 98% completion (GC), and was considered complete. To a 3 L medium fritted funnel was added celite (ca. 80 g). This was settled with MeOH (ca. 200 mL) and pulled dry with vacuum. The reduced solution was transferred via cannula into the funnel while gentle vacuum was used to pull the solution through the celite plug. This was chased with MeOH (4 x 150 mL). The solution was transferred to a 5 L three-necked round-bottom flask. At 30 °C on a rotavap, solvent (ca. 2 L) was removed under reduced pressure. An N2 blanket was applied. The solution was transferred to a 5L four-necked round-bottomed flask equipped with mechanical stirring and an addition funnel. Water (2.5 L) was added dropwise into the vigorously stirring solution over 4 h. The slurry was filtered with a minimal amount of vacuum. The collected solid was washed with water (2 x 1.5 L), 2-propanol (160 mL) then hexanes (2 x 450 mL). The collected solid (a canary yellow, granular solid) was transferred to a 150 x 75 recrystallizing dish. The solid was then dried under reduced pressure (26-28 in Hg) at 40°C overnight in a vacuum-oven. ACB (> 99% by HPLC) was stored under a N2 atmosphere at 5°C.

PATENT

WO-2020223717

Process for preparing reproxalap as acetaldehyde dehydrogenase inhibitor useful for treating ocular diseases and cancer.

PATENT

WO-2020223685

Novel crystalline forms of reproxalap (compound 1; designated as Forms A and B) as acetaldehyde dehydrogenase inhibitor useful for treating ocular diseases and cancer.

PATENT

WO 2020123730

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020123730&tab=PCTDESCRIPTION&_cid=P21-KHG0DI-30040-1

//////////REPROXALAP, レプロキサラップ , ADX-102, Phase 3 Clinical

CC(C)(C1=C(C=C2C=C(C=CC2=N1)Cl)N)O

Lercanidipine

November 1, 2020 2:19 am / Leave a comment

LercanidipineCAS Registry Number: 100427-26-7CAS Name: 1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid 2-[(3,3-diphenylpropyl)methylamino]-1,1-dimethylethyl methyl esterAdditional Names: methyl 1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate; methyl 1,1-dimethyl-2-[N-(3,3-diphenylpropyl)-N-methylamino]ethyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate; masnidipineMolecular Formula: C36H41N3O6Molecular Weight: 611.73Percent Composition: C 70.68%, H 6.76%, N 6.87%, O 15.69%Literature References: Dihydropyridine calcium channel blocker. Prepn: D. Nardi et al.,EP153016; eidem,US4705797 (1985, 1987 both to Recordati). Pharmacology: G. Bianchi et al.,Pharmacol. Res.21, 193 (1989). Clinical evaluation in hypertension: E. Rimoldi et al.,Acta Ther.20, 23 (1994).
Derivative Type: HydrochlorideCAS Registry Number: 132866-11-6Manufacturers’ Codes: Rec-15-2375; R-75Trademarks: Lerdip (Recordati); Zanidip (Napp)Molecular Formula: C36H41N3O6.HClMolecular Weight: 648.19Percent Composition: C 66.71%, H 6.53%, N 6.48%, O 14.81%, Cl 5.47%Properties: Prepd as the hemihydrate, mp 119-123°. LD50 in mice (mg/kg): 83 i.p.; 657 orally (Nardi).Melting point: mp 119-123°Toxicity data: LD50 in mice (mg/kg): 83 i.p.; 657 orally (Nardi)
Therap-Cat: Antihypertensive.Keywords: Antihypertensive; Dihydropyridine Derivatives; Calcium Channel Blocker; Dihydropyridine Derivatives.

Masnidipine hydrochloride, Lercanidipine hydrochloride, TJN-324, Rec-15/2375, Lercan, Cardiovasc, Lerzam, Zanidip, Lerdip, Lercadip, Zanedip

Syn 1

EP 0153016; JP 60199874; US 4772621; US 4968832

Two new related ways for the synthesis of lercanidipine have been reported: 1) The condensation of diketene (I) with the aminoalcohol (II) gives the corresponding acetoacetate ester (III), which is allowed to react with 3-nitrobenzaldehyde (IV) by means of HCl in chloroform yielding the expected benzylidene derivative (V). Finally, this compound is cyclized with methyl 3-aminocrotonate (VI) in refluxing isopropanol. 2) By esterification of 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid monomethyl ester (VIII) with alcohol (II) by means of SOCl2 in DMF/dichloromethane.

PATENT

https://patents.google.com/patent/WO2007054969A2/en

WO2007054969A2 - Process for the preparation of lercanidipine and ...

PATENT

https://patents.google.com/patent/EP1860102A1/en

PATENT

SPINOSAD

November 1, 2020 2:17 am / Leave a comment

Spinosad

Spinosyn A: The chemical name is: 1H-as-Indaceno[3,2- d]oxacyclododecin-7,a5-dione, 2-[(6-deoxy-2,3,4-tri-O-methyl-alphaL-mannopyranosyl)oxy]-13-[[2R,5S,6R)-5-(dimethylamino) tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-14-metyl-, (2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-

Spinosyn D: The chemical name is: 1H-as-Indaceno[3,2- d]oxacyclododecin-7,15-dione, 2-[(6-deoxy-2,3,4-tri-O-methyl-alphaL-mannopyranosyl)oxy]-13-[[2R,5S,6R)-5-(dimethylamino) tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-4,14-dimetyl-, (2S,3aSR,5aS,5bS,9S,13S,14R,16aS,16bS)-

168316-95-8

Molecular FormulaC₈₃H₁₃₂N₂O₂₀
Average mass1477.938 Da
Comfortis
Conserve
EC 434-300-1
Natroba
NaturaLyte
Spinosad
Tracer
Tracer Naturalyte
UNII-XPA88EAP6V
XDE 105

Natroba (Spinosad) Suspension 0.9% ParaPro Pharma

New Drug Application (NDA): 022408 appr 01/18/2011

Click to access 022408Orig1s000ChemR.pdf

spinosad, is a new molecular entity, and a fermentation product produced by the actinomycete, Saccharopolyspora spinosa. Spinosad contains two components, spinosyn A and D. T

Figure 1. Structure of spinosyn A and DTitle: SpinosynsCAS Registry Number: 131929-60-7Literature References: Class of fermentation derived 12 membered macrocyclic lactones in a unique tetracyclic ring. At least 20 spinosyns have been isolated from Saccharopolyspora spinosa; variations in the two sugars account for most of the structural and insecticidal activity differences. Isolation and biological activity: L. D. Boeck et al.,EP375316 (1990 to Lilly); eidem,US5496931 (1996 to DowElanco); and structure determn: H. A. Kirst et al.,Tetrahedron Lett.32, 4839 (1991). Soil degradation: K. A. Hale, D. E. Portwood, J. Environ. Sci. HealthB31, 477 (1996). HPLC determn in vegetables: L.-T. Yeh et al.,J. Agric. Food Chem.45, 1746 (1997); in soil and water: S. D. West, ibid. 3107. Uptake and metabolism in larvae: T. C. Sparks et al.,Proc. Beltwide Cotton Conf.2, 1259 (1997). Mode of action study: V. L. Salgado et al.,Pestic. Biochem. Physiol.60, 103 (1998). Review of physical and biological properties: C. V. DeAmicis et al.,ACS Symp. Ser.658, 144-154 (1997). Review: G. D. Crouse, T. C. Sparks, Rev. Toxicol.2, 133-146 (1998).
Derivative Type: Spinosyn Acas 131929-60-7CAS Name: (2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-2-[(6-Deoxy-2,3,4-tri-O-methyl-a-L-mannopyranosyl)oxy]-13-[[(2R,5S,6R)-5-(dimethylamino)tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl-2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-14-methyl-1H-as-indaceno[3,2-d]oxacyclododecin-7,15-dioneAdditional Names: lepicidin AManufacturers’ Codes: A-83543A; LY-232105Molecular Formula: C41H65NO10Molecular Weight: 731.96Percent Composition: C 67.28%, H 8.95%, N 1.91%, O 21.86%Literature References: Total synthesis: L. A. Paquette et al.,J. Am. Chem. Soc.120, 2553 (1998).Properties: White, odorless crystalline solid, mp 118°. pKa 8.1. uv max (methanol): 243 nm (e 11000). [a]27436 -262.7° (methanol). Vapor pressure: 2.4 ´ 10-10. Soly in water (ppm): 290 (pH 5), 235 (pH 7), 16 (pH 9), distilled 20. Soly (w/v%): methanol 19, acetone 17, dichloromethane >50, hexane 0.45%. LD50 in rats (mg/kg): 3783-5000 orally (Crouse).Melting point: mp 118°pKa: pKa 8.1Optical Rotation: [a]27436 -262.7° (methanol)Absorption maximum: uv max (methanol): 243 nm (e 11000)Toxicity data: LD50 in rats (mg/kg): 3783-5000 orally (Crouse)
Derivative Type: Spinosyn DCAS Registry Number: 131929-63-0Manufacturers’ Codes: A-83543DMolecular Formula: C42H67NO10Molecular Weight: 745.98Percent Composition: C 67.62%, H 9.05%, N 1.88%, O 21.45%Properties: Odorless, white crystalline solid. mp 169°. pKa 7.8. uv max (methanol): 243 nm (e 11000). [a]27436 -297.5° (methanol). Vapor pressure: 2.0 ´ 10-10. Soly in water (ppm): 28 (pH 5), 0.329 (pH 7), 0.04 (pH 9), distilled 1.3. Soly (w/v%): methanol 0.25, acetone 1.0, dichloromethane 45, hexane 0.07%.Melting point: mp 169°pKa: pKa 7.8Optical Rotation: [a]27436 -297.5° (methanol)Absorption maximum: uv max (methanol): 243 nm (e 11000)
Derivative Type: SpinosadCAS Registry Number: 168316-95-8Manufacturers’ Codes: XDE-105; DE-105Trademarks: Conserve (Dow AgroSci.); Justice (Dow AgroSci.); Naturalyte (Dow AgroSci.); SpinTor (Dow AgroSci.); Success (Dow AgroSci.); Tracer (Dow AgroSci.)Literature References: Mixture of spinosyns A and D. Effect on beneficial insects: D. Murray, R. Lloyd, Australian Cottongrower18, 62 (1997).Properties: Light grey to white crystals (tech). LD50 in rats, mallard ducks, quail (mg/kg): >3600, >2000, >2000 orally (Crouse).Toxicity data: LD50 in rats, mallard ducks, quail (mg/kg): >3600, >2000, >2000 orally (Crouse)
Use: Insecticide.(2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-13-{[(2R,5S,6R)-5-(Dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl]oxy}-9-ethyl-14-methyl-7,15-dioxo-2,3,3a,5a,5b,6,7,9,10,11,12,13,14,15,16a,16b-hexadecahydro-1H ;-as-indaceno[3,2-d]oxacyclododecin-2-yl 6-deoxy-2,3,4-tri-O-methyl-α-L-mannopyranoside – (2S,3aR,5aS,5bS,9S,13S,14R,16aS,16bS)-13-{[(2R,5S,6R)-5-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl]ox y}-9-ethyl-4,14-dimethyl-7,15-dioxo-2,3,3a,5
1H-as-Indaceno[3,2-d]oxacyclododecin-7,15-dione, 2-[(6-deoxy-2,3,4-tri-O-methyl-α-L-mannopyranosyl)oxy]-13-[[(2R,5S,6R)-5-(dimethylamino)tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl-2,3,3a,5a,5b ,6,9,10,11,12,13,14,16a,16b-tetradecahydro-14-methyl-, (2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-, compd. with (2S,3aR,5aS,5bS,9S,13S,14R,16aS,16bS)-2-[(6-deoxy-2,3,4-tri-O-methyl-α-L-mannopyranosyl)o xy]-13-[[(2R,5S,6R)-5-(dimethylamino)tetrahySpinosad[USAN] [Wiki]168316-95-8 [RN]1H-as-Indaceno[3,2-d]oxacyclododecin-7,15-dione, 2-[(6-deoxy-2,3,4-tri-O-methyl-α-L-mannopyranosyl)oxy]-13-[[2R,5S,6R)-5-(dimethylamino) tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl-2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-4,14-dimetyl-,(2S,3aSR,5aS,5bS,9S,13S,14R,16aS,16bS)-1H-as-Indaceno[3,2-d]oxacyclododecin-7,a5-dione, 2-[(6-deoxy-2,3,4-tri-O-methyl-α-L-mannopyranosyl)oxy]-13-[[2R,5S,6R)-5-(dimethylamino) tetrahydro-6-methyl-2H-pyran-2-yl]oxy]-9-ethyl-2,3,3a,5a,5b,6,9,10,11,12,13,14,16a,16b-tetradecahydro-14-metyl-, (2R,3aS,5aR,5bS,9S,13S,14R,16aS,16bR)-NAF-144Spinosad|spinosyn A and D (mixture)spinosyn A and D (mixture)

Spinosad is an insecticide based on chemical compounds found in the bacterial species Saccharopolyspora spinosa. The genus Saccharopolyspora was discovered in 1985 in isolates from crushed sugarcane. The bacteria produce yellowish-pink aerial hyphae, with bead-like chains of spores enclosed in a characteristic hairy sheath.^[1] This genus is defined as aerobic, Gram-positive, nonacid-fast actinomycetes with fragmenting substrate mycelium. S. spinosa was isolated from soil collected inside a nonoperational sugar mill rum still in the Virgin Islands. Spinosad is a mixture of chemical compounds in the spinosyn family that has a generalized structure consisting of a unique tetracyclic ring system attached to an amino sugar (D-forosamine) and a neutral sugar (tri-Ο-methyl-L-rhamnose).^[2] Spinosad is relatively nonpolar and not easily dissolved in water.^[3]

Spinosad is a novel mode-of-action insecticide derived from a family of natural products obtained by fermentation of S. spinosa. Spinosyns occur in over 20 natural forms, and over 200 synthetic forms (spinosoids) have been produced in the lab.^[4] Spinosad contains a mix of two spinosoids, spinosyn A, the major component, and spinosyn D (the minor component), in a roughly 17:3 ratio.^[1

Mode of action

Spinosad is highly active, by both contact and ingestion, in numerous insect species.^[5] Its overall protective effect varies with insect species and life stage. It affects certain species only in the adult stage, but can affect other species at more than one life stage. The species subject to very high rates of mortality as larvae, but not as adults, may gradually be controlled through sustained larval mortality.^[5] The mode of action of spinosoid insecticides is by a neural mechanism.^[6] The spinosyns and spinosoids have a novel mode of action, primarily targeting binding sites on nicotinic acetylcholine receptors (nAChRs) of the insect nervous system that are distinct from those at which other insecticides have their activity. Spinosoid binding leads to disruption of acetylcholine neurotransmission.^[2] Spinosad also has secondary effects as a γ-amino-butyric acid (GABA) neurotransmitter agonist.^[2] It kills insects by hyperexcitation of the insect nervous system.^[2] Spinosad so far has proven not to cause cross-resistance to any other known insecticide.^[7]

Use

Spinosad has been used around the world for the control of a variety of insect pests, including Lepidoptera, Diptera, Thysanoptera, Coleoptera, Orthoptera, and Hymenoptera, and many others.^[8] It was first registered as a pesticide in the United States for use on crops in 1997.^[8] Its labeled use rate is set at 1 ppm (1 mg a.i./kg of grain) and its maximum residue limit (MRL) or tolerance is set at 1.5 ppm. Spinosad’s widespread commercial launch was deferred, awaiting final MRL or tolerance approvals in a few remaining grain-importing countries. It is considered a natural product, thus is approved for use in organic agriculture by numerous nations.^[5] Two other uses for spinosad are for pets and humans. Spinosad has recently been used in oral preparations (as Comfortis) to treat C. felis, the cat flea, in canines and felines; the optimal dose set for canines is reported to be 30 mg/kg.^[2]

Spinosad is sold under the trade names, Comfortis, Trifexis, and Natroba.^[9]^[10] Trifexis also includes milbemycin oxime. Comfortis and Trifexis brands treat adult fleas on pets; the latter also prevents heartworm disease. Natroba is sold for treatment of human head lice. Spinosad is also commonly used to kill thrips.^[11]^[12]^[13]

Spinosyn A

Spinosyn A does not appear to interact directly with known insecticidal-relevant target sites, but rather acts via a novel mechanism.^[6] Spinosyn A resembles a GABA antagonist and is comparable to the effect of avermectin on insect neurons.^[4] Spinosyn A is highly active against neonate larvae of the tobacco budworm, Heliothis virescens, and is slightly more biologically active than spinosyn D. In general, spinosyns possessing a methyl group at C6 (spinosyn D-related analogs) tend to be more active and less affected by changes in the rest of the molecule.^[7] Spinosyn A is slow to penetrate to the internal fluids of larvae; it is also poorly metabolized once it enters the insect.^[7] The apparent lack of spinosyn A metabolism may contribute to its high level of activity, and may compensate for the slow rate of penetration.^[7]

Safety and ecotoxicology

Spinosad has high efficacy, a broad insect pest spectrum, low mammalian toxicity, and a good environmental profile, a unique feature of the insecticide compared to others currently used for the protection of grain products.^[5] It is regarded as natural product-based, and approved for use in organic agriculture by numerous national and international certifications.^[8] Spinosad residues are highly stable on grains stored in bins, with protection ranging from 6 months to 2 years.^[5]^{[clarification needed]} Ecotoxicology parameters have been reported for spinosad, and are:^[14]

in rat (Rattus norvegicus Bergenhout, 1769), acute oral: LD₅₀ >5000 mg/kg (nontoxic)
in rat (R. norvegicus), acute dermal: LD₅₀ >2000 mg/kg (nontoxic)
in California quail (Callipepla californica Shaw, 1798), oral toxicity: LD₅₀ >2000 mg/kg (nontoxic)
in duck (Anas platyrhynchos domestica Linnaeus, 1758), dietary toxicity: LC₅₀ >5000 mg/kg (nontoxic)
in rainbow trout (Oncorhynchus mykiss Walbaum, 1792), LC_50-96h = 30.0 mg/l (slightly toxic)
in Honeybee (Apis mellifera Linnaeus, 1758), LD₅₀ = 0.0025 mg/bee (highly toxic if directly sprayed on and of dried residues).

Chronic exposure studies failed to induce tumor formation in rats and mice; mice given up to 51 mg/kg/day for 18 months resulted in no tumor formation.^[15] Similarly, administration of 25 mg/kg/day to rats for 24 months did not result in tumor formation.^[16]

syn

EP 0375,316 (1994, to DowElanco)

US 5496931 (1996 to DowElanco)

PATENT

CN 102190694

https://patents.google.com/patent/CN102190694A/en

Pleocidin compounds (spinosyns) is soil actinomycete thorn many armfuls of bacterium Saccharopolysporaspinosa of sugar secondary metabolites behind aerobic fermentation under developing medium.Pleocidin belongs to macrolides compound, it comprises one a plurality of chiral carbon tetracyclic ring systems (Macrolide tetracycle), big ring is gone up the 9-hydroxyl and is being linked two different hexa-atomic sugar respectively with the 17-hydroxyl, wherein that 17 connections is an aminosugar (Forosamine sugar), and that connect on the 9-position is a rhamnosyl (Rhamnose sugar).Tetracyclic ring system is by one 5,, 6,5-is suitable-and anti–anti–three-loop system condenses one 12 membered macrolide to be formed, and wherein contains an alpha, beta-unsaturated ketone and an independently two key.When 6 on ring is pleocidin A when being substituted by hydrogen, in mixture, account for 85-90%, when ring 6 bit substituents when connecing methyl, be pleocidin D then, in mixture, account for about 10-15%.Up to the present B, C, D, E, F, G, K, L, M, N, O, P, Q, R, S, T, U, more than 20 derivative such as V, W etc. have been found and have isolated it to comprise Spinosyn A.

The commercialization kind has pleocidin Spinosyns (mixture of pleocidin A and pleocidin D) at present, the s-generation pleocidin insecticides Spinetoram. latter is got through semisynthesis by the thick product pleocidin L of biological method preparation and the mixture of J, promptly by 5 of pleocidin J, 6 two key selective reductions, reach 3 ‘ O-ethylization of rhamnosyl and obtain its major ingredient, ethylizing by 3 ‘ O-of pleocidin L rhamnosyl obtains its minor consistuent.

The pleocidin compound can be controlled lepidopteran, Diptera and Thysanoptera insect effectively.It can prevent and treat the pest species of some blade of eating in a large number in Coleoptera and the Orthoptera well.Pleocidin has very high activity to lepidopterous larvaes such as Heliothis virescens, bollworm, beet armyworm, prodenia litura, cabbage looper, small cabbage moth and rice-stem borers, and they are suitable environmental protection, have interesting toxicology character.

U.S. Patent No. 5362634 discloses the derivative that natural pleocidin is replaced by methyl or ethyl on C-21, U.S. Patent application No.60/153513 has disclosed the natural butenyl pleocidin derivative that the 3-4 carbochain replaces on C-21.Pleocidin derivative (John Daeuble, ThomasC.Sparks, Peter Johnson, Paul R.Graupner, the Bioorganic ﹠amp that can prepare C-21 position different substituents by replacement(metathesis)reaction; Medicinal Chemistry17 (2009) 4197-4205).U.S. Patent No. 6001981A, WO 9700265A have openly opened the chemosynthesis of pleocidin compound and have modified, and comprise aminosugar and rhamnosyl and the big chemically modified that encircles in the structure.

PATENT

https://patents.google.com/patent/WO2002077005A1/en

Spinosyns (A83543) are produced by derivatives of Saccharopolyspora spinosa NRRL18395 including strains NRRL 18537, 18538, 18539, 18719, 18720, 18743 and 18823 and derivatives thereof. A more preferred nomenclature for spinosyns is to refer to the pseudoaglycones as spinosyn A 17-Psa, spinosyn D 17-Psa, etc., and to the reverse pseudoaglycones as spinosyn A 9-Psa, spinosyn D 9-Psa, etc. (see Kirst et al., 1991). The known members of this family have been referred to as factors or components, and each has been given an identifying letter designation. These compounds are hereinafter referred to as spinosyn A, B, etc. The spinosyn compounds are useful for the control of arachnids, nematodes and insects, in particular Lepidoptera and Diptera species, and they are quite environmentally friendly and have an appealing toxicological profile. [0004] U.S. Patent No. 5,362,634 and corresponding European Patent Application No. 375316 Al disclose spinosyns A, B, C, D, E, F, G, H, and J. WO 93/09126 discloses spinosyns L, M, N, Q, R, S, and T. WO 94/20518 and US 5,6704,486 disclose spinosyns K,

O, P, U, V, W, and Y, and derivatives thereof. A large number of synthetic modifications to spinosyn compounds have been made, as disclosed in U.S. Patent No. 6,001,981 and WO

97/00265.

PAPER

J. Am. Chem. Soc. 120, 2553 (1998).

References

^ Jump up to:^a ^b Mertz, Frederick; Raymond C. Yao (Jan 1990). “Saccharopolyspora spinosa sp. nov. Isolated from soil Collected in a Sugar Mill Rum Still”. International Journal of Systematic Bacteriology. 40 (1): 34–39. doi:10.1099/00207713-40-1-34.
^ Jump up to:^a ^b ^c ^d ^e Qiao, Meihua; Daniel E. Snyder; Jeffery Meyer; Alan G. Zimmerman; Meihau Qiao; Sonya J. Gissendanner; Larry R. Cruthers; Robyn L. Slone; Davide R. Young (12 September 2007). “Preliminary Studies on the effectiveness of the novel pulicide, spinosad, for the treatment and control of fleas on dogs”. Veterinary Parasitology. 150 (4): 345–351. doi:10.1016/j.vetpar.2007.09.011. PMID 17980490.
^ Crouse, Gary; Thomas C Sparks; Joseph Schoonover; James Gifford; James Dripps; Tim Brue; Larry L Larson; Joseph Garlich; Chris Hatton; Rober L Hill; Thomas V Worden; Jacek G Martynow (27 September 2000). “Recent advances in the chemistry of spinosyns”. Pest Manag Sci. 57 (2): 177–185. doi:10.1002/1526-4998(200102)57:2<177::AID-PS281>3.0.CO;2-Z. PMID 11455648.
^ Jump up to:^a ^b Watson, Gerald (31 May 2001). “Actions of Insecticidal Spinosyns on gama-Aminobutyric Acid Responses for Small-Diameter Cockroach Neurons”. Pesticide Biochemistry and Physiology. 71: 20–28. doi:10.1006/pest.2001.2559.
^ Jump up to:^a ^b ^c ^d ^e Hertlein, Mark; Gary D. Thompson; Bhadriraju Subramanyam; Christos G. Athanassiou (12 January 2011). “Spinosad: A new natural product for stored grain protection”. Stored Products. 47 (3): 131–146. doi:10.1016/j.jspr.2011.01.004. Retrieved 3 May 2012.
^ Jump up to:^a ^b Orr, Nailah; Andrew J. Shaffner; Kimberly Richey; Gary D. Crouse (30 April 2009). “Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites”. Pesticide Biochemistry and Physiology. 95: 1–5. doi:10.1016/j.pestbp.2009.04.009.
^ Jump up to:^a ^b ^c ^d Sparks, Thomas; Gary D crouse; Gregory Durst (30 March 2001). “Natural products as insecticides: the biology, biochemistry and quantitative structure-activity relationships of spinosyns and spinosoids”. Pest Manag Sci. 57 (10): 896–905. doi:10.1002/ps.358. PMID 11695182.
^ Jump up to:^a ^b ^c Sparks, Thomas; James E. Dripps; Gerald B Watson; Doris Paroonagian (6 November 2012). “Resistance and cross-resistance to the spinosyns- A review and analysis”. Pesticide Biochemistry and Physiology. 102: 1–10. doi:10.1016/j.pestbp.2011.11.004. Retrieved 17 November 2011.
^ “Spinosad international brands”. Drugs.com. 3 January 2020. Retrieved 30 January2020.
^ “Spinosad US brands”. Drugs.com. 3 January 2020. Retrieved 30 January 2020.
^ “Spinosad – brand name list from”. Drugs.com. Retrieved 2012-10-20.
^ “UC Davis School of Vet Med”. Vetmed.ucdavis.edu. Retrieved 2012-10-20.
^ “Safer Flea Control | Insects in the City”. Citybugs.tamu.edu. Retrieved 2012-10-20.
^ “Codling Moth and Leafroller Control Using Chemicals” (PDF). Entomology.tfrec.wsu.edu. Retrieved 2012-10-20.
^ Stebbins, K. E. (2002). “Spinosad Insecticide: Subchronic and Chronic Toxicity and Lack of Carcinogenicity in CD-1 Mice”. Toxicological Sciences. 65 (2): 276–287. doi:10.1093/toxsci/65.2.276. PMID 11812932. Retrieved 2015-03-08.
^ Yano, B. L. (2002). “Spinosad Insecticide: Subchronic and Chronic Toxicity and Lack of Carcinogenicity in Fischer 344 Rats”. Toxicological Sciences. 65 (2): 288–298. doi:10.1093/toxsci/65.2.288. PMID 11812933. Retrieved 2015-03-08.

External links

“Spinosad”. Drug Information Portal. U.S. National Library of Medicine.
Monograph

Identifiers
Spinosyn A
Spinosyn D
CAS Number	168316-95-8 (A)131929-60-7 (D)
ChEBI	CHEBI:9230 (A)CHEBI:9232 (D)
ChEMBL	ChEMBL1615373
ChemSpider	16736513
ECHA InfoCard	100.103.254
PubChem CID	183094 (A)443059 (D)
CompTox Dashboard (EPA)	DTXSID7032478
InChI [show]
Properties
Chemical formula	C₄₁H₆₅NO₁₀ (A) C₄₂H₆₇NO₁₀ (D)
Pharmacology
ATCvet code	QP53BX03 (WHO)
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).

//////////Spinosad

Dr. Darrin Lew https://www.drdarrinlew.us/insect-control/production-of-spinosad.html

Production of Spinosad

Last Updated on Tue, 29 Oct 2019 | Insect Control

Spinosad is produced directly from the fermentation of a strain of Saccharo-polyspora spinosa. Production strains of S. spinosa have been selected for increased titers of spinosyns A and D, however, no genetic engineering techniques have been used in this process and no genetically-modified organisms are used in the production process. After fermentation, the spinosyn A and D mixture is extracted from the fermentation broth, precipitated and dried to create technical spinosad, which is then formulated into end-use products. Spinosad technical material is also produced under pharmaceutical manufacturing guidelines to be used as a flea control agent in companion animals.

5.9.2 Production of Spinetoram

Production of spinetoram begins with the fermentation of a mutant strain of Saccharopolyspora spinosa that produces primarily spinosyns J and L, rather than spinosyns A and D. This strain was generated through mutagenesis of S. spinosa. However, like the spinosad-producing strains, no genetic engineering techniques were used in this process and no genetically-modified organisms are used in the production process. After fermentation, the spinosyn J and L mixture is extracted from the fermentation broth and precipitated in preparation for the two chemical synthesis steps required to produce spinetoram. The solvents used in extracting and precipitating the spinosyn J and L mixture are recycled.

Spinosyns J and L, unlike spinosyns A and D, have a free hydroxyl group at the 30-position on the rhamnose sugar, which allows for chemical manipulation of this site (see Figure 5.10). In the first synthetic step, the free hydroxyl at the 30-position in spinosyn J and spinosyn L is ethylated to yield a mixture of 30-O-ethyl spinosyn J and 30-O-ethyl spinosyn L. This material is then hydrogenated to yield a mixture of spinetoram-J (30-O-ethyl-5,6-dihydro spinosyn J; see Figure 5.2, structure 5.5) and spinetoram-L (30-O-ethyl spinosyn L; see Figure 5.2, structure 5.6). The hydrogenation conditions are selective and reduce only the disubstituted double bond between C5 and C6 in the 30-O-ethyl spinosyn J intermediate, leaving the 30-O-ethyl spinosyn L unchanged. The material is crystallized from the reaction mixture and dried to create technical spinetoram, which is then formulated into end-use products.

5.9.3 Formulation Attributes of the Spinosyns

To meet a variety of market needs, spinosad and spinetoram products span a very wide range of formulation types (see Table 5.8).

The range of possible formulations for any pesticide is determined by the physical and chemical properties of the active ingredient. Three primary properties determine the formulation characteristics of the spinosyns: (1) both Spinosyn Insecticide Figure 5.10 Chemical synthesis steps in spinetoram manufacturing.

Table 5.8 Spinosyn product formulation types and associated uses.

Formulation type

Use pattern

Suspension concentrate

Emulsifiable concentrate Wettable granule Wettable powder Dustable powder Sprayable bait Granular bait Bait stations Granules Tablets

Chewable tablets Gel, paste Creme rinse

Crops, ornamentals, forestry, stored grain, animal health, public health, turf, home and garden Public health Crops

Crops, ornamentals, seed treatment

Stored grain, crops

Crops

Crops, animal health, urban pests

Urban pests

Public health

Animal health

Urban pests

Public health are fermentation-derived mixtures; (2) both are weak bases; and (3) both have significant solubility in organic solvents.

As fermentation-derived products, spinosad and spinetoram are mixtures composed primarily of two similar, but not identical molecules. In terms of physical properties, a significant difference between the major and minor components of both spinosad and spinetoram is the presence or absence of a methyl group at C6 on the tetracycle (see Table 5.9). With regard to components of spinosad, spinosyn D (methyl group at C6) has a melting point 71 °C higher than that of spinosyn A (hydrogen at C6), and the water solubility of spinosyn D (at pH 7) is almost 1000-fold lower than that of spinosyn A. With regard to the components of spinetoram, spinetoram-L (methyl group at C6) has a melting point 72 °C lower than that of spinetoram-J (hydrogen at C6), and the water solubility of spinetoram-L (at pH 7) is four-fold higher than that of spinetoram-J. The melting points and water solubilities of the mixtures that constitute technical spinosad and technical spinetoram are determined by the relative ratios of the major and minor components.

The predominant components of both spinosad and spinetoram all have pKa values of about 8 (see Table 5.9). As a weak base, the solubility of spinosyns in water increases as the pH is reduced. From a formulation perspective, at pH level above 5, the spinosyns behave like high-melting solids with little water solubility, which results in the predominant agricultural formulations being suspension concentrates and wettable granule formulations composed of milled crystalline particles. Acid salts of spinosyns can be produced and are used in animal health formulations. The basic nature of the spinosyns is also a consideration when combining multiple active ingredients into the same formulation.

The spinosyns have significant solubility in organic solvents (see Table 5.9). This property is relatively rare in high-melting solids with limited water solubility, and has proven to be useful in a number of formulations for

Table 5.9 Selected physical properties of spinosyn A, spinosyn D, spinetoram-

J, and spinetoram-L.

Table 5.9 Selected physical properties of spinosyn A, spinosyn D, spinetoram-

J, and spinetoram-L.

Property	Spinosyn A133	Spinosyn D133	Spinetoram-J134	Spinetoram-L134
Melting point, °C	84-99.5a	161.6-170a	143.4b	70.8b
Water solubility,	235	0.332	11.3	46.7
mg/lc’d’e
pKaf	8.10e	7.87e	7.86g	7.59g
Solubility in organic solvents, mg/Lc
Acetone	168 000	10100	>250000	>250000
Ethyl acetate	194000	19 000	>250000	>250000
w-Heptane	12 400	300	23 900	>250000
Methanol	190000	2520	163 000	>250000
Xylene	> 250 000	64000	>250000	>250000

“Visual determination. bDiffential scanning calorimetry. cShake flask. ^Buffered to pH 7. eAt 20 °C.

fCapillary zone electrophoresis. gAt 25 °C.

“Visual determination. bDiffential scanning calorimetry. cShake flask. ^Buffered to pH 7. eAt 20 °C.

fCapillary zone electrophoresis. gAt 25 °C.

non-agricultural markets, such as mosquito control and animal health. It is also a consideration when combining the spinosyns with other active ingredients.

////////

https://aem.asm.org/content/82/18/5603

MILVEXIAN

October 24, 2020 2:20 am / 1 Comment on MILVEXIAN

MILVEXIAN

ミルベクシアン;

Molecular Formula,C28-H23-Cl2-F2-N9-O2

Molecular Weight, 626.4441

BMS-986177, JNJ-70033093; JNJ-3093, WHO 11401

CAS 1802425-99-5

(5R,9S)-9-(4-(5-Chloro-2-(4-chloro-1H-1,2,3-triazol-1-yl)phenyl)-6-oxopyrimidin-1(6H)-yl)-21-(difluoromethyl)-5-methyl-21H-3-aza-1(4,2)-pyridina-2(5,4)-pyrazolacyclonaphan-4-one

Prevention and Treatment of Thromboembolic Disorders

Milvexian, also known as BMS-986177, is a blood coagulation factor XIa inhibitor.Bristol-Myers Squibb , in collaboration with Janssen , is developing milvexian (BMS-986177, JNJ-70033093; JNJ-3093), an antithrombotic factor XIa (FXIa) inhibitor, for the oral prevention and treatment of thrombosis.

PATENT

WO-2020210629

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020210629&tab=PCTDESCRIPTION&_cid=P11-KGN1SB-37057-1

Process for preparing milvexian as FXIa and/or plasma kallikrein inhibitors useful for treating deep vein thrombosis, stroke, and atherosclerosis.

(9i?,13ri)-13-{4-[5-chloro-2-(4-chloro- 1 //- 1 2.3-triazol- 1 -yl)phenyl |-6-o\o- 1 6-dihydropyri midin- 1 -yl }-3-(difluoromethyl)-9-methyl-3,4,7,15-tetraazatricyclo[12.3.1.0^{2 (5}]octadeca-l(18),2(6),4,14,16-pentaen-8-one, has the structure of Formula (I):

PATENT

WO2020210613

PATENT

WO2016053455

PATENT

product case WO2016053455 novel macrocyclic compounds are FXIa and/or plasma kallikrein inhibitors.

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2016053455

Scheme 1

4M HCI or TFA

1c 1a

Scheme 2

Scheme 3

EXAMPLES

Example 1. Preparation of (9i?,135)-13-{4-[5-chloro-2-(4-chloro-lH-l,2,3-triazol-l-yl)phenyl]-6-oxo- 1 ,6-dihydropyrimidin- 1 -yl} -3-(difluoromethyl)-9-methyl-3,4,7, 15-tetraazatricyclo[ 12.3.1.0²‘⁶] -8-one trifluoroacetate

1A. Preparation of l-(difluoromethyl)-4-nitro-lH-pyrazole

CS₂CO₃ (14.41 g, 44.2 mmol) was suspended in a solution of 4-nitro-lH-pyrazole (5.00 g, 44.2 mmol) and DMF (40 mL). After heating to 120 °C for 5 min, solid sodium 2-chloro-2,2-difluoroacetate (13.48 g, 88 mmol) was added in 10 equal portions over 20 min. The reaction was complete after 10 min of additional heating. The mixture was added to a separatory funnel containing 100 mL water and extracted with Et₂0 (2 x 50 mL). The combined organic layers were concentrated. Purification by normal-phase chromatography eluting with a gradient of hexanes/EtOAc yielded l-(difluoromethyl)-4-nitro-lH-pyrazole (6.99 g, 42.9 mmol, 97% yield) as a clear, colorless oil. 1H NMR (500MHz, CDCI₃) δ 8.58 (s, 1H), 8.22 (s, 1H), 7.39 – 7.05 (t, J= 60 Hz, 1H).

IB. Preparation of (S)-tert-butyl (l-(4-(l-(difluoromethyl)-4-nitro-lH-pyrazol-5-yl)pyridin-2-yl)but-3 -en- 1 -yl)carbamate

To a N₂ flushed, 500 mL RBF was added {S)-tert-bvXy\ (l-(4-chloropyridin-2-yl)but-3-en-l-yl)carbamate, prepared as described in Example 3, (10 g, 35.4 mmol), 1-(difluoromethyl)-4-nitro-lH-pyrazol (6.34 g, 38.9 mmol) and dioxane (100 mL). The solution was bubbled with N₂ for 5 min. Then Pd(OAc)₂ (0.40 g, 1.7 mmol),

di(adamantan-l-yl)(butyl)phosphine (1.27 g, 3.5 mmol), K₂CO₃ (14.7 g, 106 mmol) and PvOH (1.08 g, 10.61 mmol) were added. The reaction mixture was bubbled with N₂ for 5 min then the reaction mixture was heated to 100 °C for 3 h. After this time, the solution was cooled to rt and water (200 mL) was added. The reaction mixture was then extracted with EtOAc (2 x 200 mL). The combined organic extracts were washed with water (200 mL), brine (200 mL), dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by normal phase chromatography eluting with a gradient of hexanes/EtOAc afforded (S)-tert-butyl ( 1 -(4-( 1 -(difluoromethyl)-4-nitro- lH-pyrazol-5 -yl)pyridin-2-yl)but-3 -en- 1 -yl)carbamate (12.91 g, 31.5 mmol, 89% yield) as a slightly yellow oil. MS(ESI) m/z: 410.4 [M+H]⁺. 1H NMR (400MHz, CDC1₃) δ 8.80 (dd, J=5.1, 0.7 Hz, 1H), 8.36 (s, 1H), 7.34 (s, 1H), 7.31 (dd, J=5.1, 1.5 Hz, 1H), 7.27 – 6.91 (t, J=58 Hz, 1H), 5.79 – 5.63 (m, 1H), 5.16 – 5.03 (m, 2H), 4.92 (d, J=5.9 Hz, 1H), 2.67 (t, J=6.4 Hz, 2H), 1.46 (br. s., 9H).

1C. Preparation of
(l-(4-(4-amino-l -(difluoromethyl)- lH-pyrazol-5-yl)pyridin-2-yl)but-3 -en- 1 -yl)carbamate

To a 100 mL, 3-necked RBF was added a solution of (S)-tert-butyl (l-(4-(l-(difluoromethyl)-4-nitro-lH-pyrazol-5-yl)pyridin-2-yl)but-3-en-l-yl)carbamate (0.78 g, 1.90 mmol) in MeOH (12 mL) and a solution of NH₄C1 (1.02 g, 19 mmol) in water (3 mL). To the solution was added Fe (0.53 g, 9.49 mmol). The reaction mixture was heated to 65 °C for 3 h. Water (50 mL) was added. After cooling to rt, the mixture was filtered through a CELITE® pad and rinsed with MeOH (200 mL). The filtrate was concentrated in vacuo. The residue was partitioned between EtOAC (100 mL) and water (100 mL). The organic phase was separated, washed with water (100 mL), brine (100 mL), dried over Na₂S0₄, filtered and concentrated in vacuo. Purification by normal phase chromatography eluting with a gradient of DCM/MeOH yielded (S)-tert-butyl (l-(4-(4-amino- 1 -(difluoromethyl)- lH-pyrazol-5 -yl)pyridin-2-yl)but-3 -en- 1 -yl)carbamate (0.585 g, 1.54 mmol, 81% yield) as an oil. MS(ESI) m/z: 380.1 [M+H]⁺. 1H NMR (400MHz,

CDC1₃) δ 8.70 (dd, J=5.0, 0.7 Hz, 1H), 7.43 (s, 1H), 7.36 (s, 1H), 7.32 (dd, J=5.1, 1.5 Hz, 1H), 7.28 – 6.97 (t, J=58 Hz, 1H), 5.80 – 5.66 (m, 1H), 5.65 – 5.53 (m, 1H), 5.13 – 5.03 (m, 2H), 4.87 (br. s., 1H), 3.22 (br. s., 2H), 2.65 (t, J=6.5 Hz, 2H), 1.52 – 1.37 (m, 9H).

ID. Preparation of tert-butyl ((5)-l-(4-(l-(difiuoromethyl)-4-((i?)-2-methylbut-3-enamido)- lH-pyrazol-5-yl)pyridin-2-yl)but-3-en- 1 -yl)carbamate

To a N₂ flushed, 3 -necked, 250 mL RBF was added a solution of {S)-tert-bvXy\ (1-(4-(4-amino-l-(difluoromethyl)-lH-pyrazol-5-yl)pyridin-2-yl)but-3-en-l-yl)carbamate (5 g, 13.18 mmol) and EtOAc (50 ml). The solution was cooled to -10 °C and (R)-2-methylbut-3-enoic acid, as prepared in Example 2, (1.72 g, 17.13 mmol), pyridine (4.26 ml, 52.7 mmol). and T3P® (23.54 ml, 39.5 mmol) were added. The cooling bath was removed and the solution was allowed to warm to rt and then stir over a period of 20 h. Water (30 mL) and EtOAc (30 mL) were added and the mixture was stirred for 30 min. The organic phase was separated and the aqueous layer was extracted with EtOAc (30 mL). The combined organic extracts were washed with brine (50 mL), dried over

Na₂SC”4, filtered and concentrated in vacuo. Purification by normal phase

chromatography eluting with a gradient of hexanes/EtOAc gave tert-butyl ((5)-l-(4-(l-(difluoromethyl)-4-((i?)-2-methylbut-3-enamido)-lH-pyrazol-5-yl)pyridin-2-yl)but-3-en-l-yl)carbamate (5.69 g, 12.33 mmol, 94% yield). MS(ESI) m/z: 462.2 [M+H]⁺. 1H NMR (400MHz, CDC1₃) δ 8.75 (dd, J=5.0, 0.6 Hz, 1H), 8.37 (s, 1H), 7.32 (t, J=59 Hz, 1H), 7.28 (br. s., 1H), 7.20 (s, 1H), 5.97 – 5.85 (m, 1H), 5.78 – 5.65 (m, 1H), 5.56 – 5.44 (m, 1H), 5.28 – 5.19 (m, 2H), 5.12 (d, J=2.0 Hz, 2H), 4.91 – 4.82 (m, 1H), 3.20 – 3.11 (m, 1H), 2.72 – 2.62 (m, 2H), 1.48 – 1.43 (s, 9H), 1.33 (d, J=6.8 Hz, 3H).

IE. Preparation of tert-butyl N-[(9i?,10E,135)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,10,14,16-hexaen-13-yl] carbamate

To a N₂ flushed, 2 L, 3 -necked, RBF was added a solution of tert-butyl ((S)-l-(4-(1 -(difluoromethyl)-4-((i?)-2-methylbut-3 -enamido)- lH-pyrazol-5 -yl)pyridin-2-yl)but-3 -en-l-yl)carbamate (3 g, 6.50 mmol) in EtOAc (1300 ml). The solution was sparged with argon for 15 min. Grubbs II (1.38 g, 1.63 mmol) was added in one portion. The reaction mixture was heated to reflux for 24 h. After cooling to rt, the solvent was removed and the residue was purified by normal phase chromatography eluting with a gradient of DCM/MeOH to yield tert-butyl N-[(9R, 10E, 135)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,10,14,16-hexaen-13-yl]carbamate (2.13 g, 4.91 mmol, 76% yield) as a tan solid. MS(ESI) m/z: 434.4 [M+H]⁺. 1H NMR (400MHz, CDC1₃) δ 8.71 (d, J=5.1 Hz, 1H), 7.78 (s, 1H), 7.44 – 7.40 (m, 1H), 7.36 (br. s., 1H), 7.27 (t, J=58 Hz, 1H), 6.87 (s, 1H), 6.49 – 6.39 (m, 1H), 5.78 (s, 1H), 4.80 (br. s., 2H), 3.18 – 3.08 (m, 1H), 3.08 – 2.98 (m, 1H), 2.06 – 1.93 (m, 1H), 1.51 (s, 9H), 1.19 (d, J=6.6 Hz, 3H).

IF. Preparation of tert-butyl N-[(9i?,135)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,14,16-pentaen-13-yl]carbamate

Pd/C (0.60 g, 0.570 mmol) was added to a 250 mL Parr hydrogenation flask containing a solution of tert-butyl N-[(9i?,10E,135)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,10,14,16-hexaen-13-yljcarbamate (2.46 g, 5.68 mmol) in EtOH (100 mL). The flask was purged with N₂ and pressurized to 55 psi of H₂ allowed to stir for 18 h. The reaction was filtered through CELITE® and concentrated to yield tert-butyl N-[(9i?,135)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,14,16-pentaen-13-yl]carbamate (2.17 g, 88% yield) as a tan solid. MS(ESI) m/z: 436.3 [M+H]⁺. 1H NMR (400MHz, DMSO-d₆) δ 9.32 (s, 1H), 8.71 (d, J=5.0 Hz, 1H), 7.96 (t, J=58 Hz, 1H), 7.43 (s, 1H), 7.32 (d, J=4.8 Hz, 1H), 7.22 (d, J=7.3 Hz, 1H), 4.66 (d, J=8.3 Hz, 1H), 2.62 (br. s., 1H), 1.88 (d, J=12.8 Hz, 1H), 1.77 – 1.59 (m, 2H), 1.42 – 1.28 (m, 9H), 1.15 (d, J=18.2 Hz, 2H), 0.83 (d, J=7.0 Hz, 3H).

I G. Preparation of (9R, 13S)-l 3-amino-3-(difiuoromethyl)-9-methyl-3,4,7, 15-tetraazatricyclo[ 12.3.1.0²‘⁶]octadeca- 1(18),2(6),4, 14,16-pentaen-8-one

4 N HC1 in dioxane (3.88 mL, 15.5 mmol) was added to a solution of tert-butyl N-[(9R, 13S)-3-(difluoromethyl)-9-methyl-8-oxo-3,4,7, 15-tetraazatricyclo[12.3.1.0²‘⁶] octadeca-l(18),2(6),4,14,16-pentaen-13-yl]carbamate (2.25 g, 5.2 mmol) in MeOH (10 mL). The reaction was allowed to stir at rt for 2 h. The reaction was cooled in an ice bath, and 7 N NH₃ in MeOH (13.3 mL, 93.0 mmol) was added. After 5 min, the reaction was diluted with CH₂C1₂ (80 mL) and the solid that formed was filtered. The filtrate was concentrated to yield (9i?,135)-13-amino-3-(difluoromethyl)-9-methyl-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,14,16-pentaen-8-one (1.3 g, 3.88 mmol, 75% yield). MS(ESI) m/z: 336.3 [M+H]⁺. 1H NMR (400MHz, DMSO-d₆) δ 9.33 (s, 1H), 8.71 (d, J=5.0 Hz, 1H), 7.94 (t, J=58 Hz, 1H), 7.85 (s, 1H), 7.40 (s, 1H), 7.32 (d, J=5.0 Hz, 1H), 4.01 (dd, J=10.2, 5.1 Hz, 1H), 2.63 – 2.53 (m, 1H), 1.90 – 1.69 (m, 2H), 1.53 -1.36 (m, 2H), 1.16 – 1.00 (m, 1H), 0.85 (d, J=7.0 Hz, 3H).

1H. Preparation of (9i?,135)-13-{4-[5-chloro-2-(4-chloro-lH-l,2,3-triazol-l-yl)phenyl]-6-oxo- 1 ,6-dihydropyrimidin- 1 -yl} -3-(difluoromethyl)-9-methyl-3 ,4,7, 15-tetraazatricyclo [12.3.1.0²‘⁶]octadeca- 1 ( 18),2(6),4, 14,16-pentaen-8-one.

To a 100 mL flask containing a white suspension of 6-(5-chloro-2-(4-chloro-lH-l,2,3-triazol-l-yl)phenyl)pyrimidin-4-ol (0.83 g, 2.7 mmol), as prepared in Example 4 in ACN (36 mL) was added HATU (1.12 g, 3.0 mmol) and DBU (0.53 mL, 3.5 mmol). The resulting clear, yellow solution was stirred at rt. After 5 min, (9i?,135)-13-amino-3-(difluoromethyl)-9-methyl-3,4,7,15-tetraazatricyclo[12.3.1.0²‘⁶]octadeca-l(18),2(6),4,14,16-pentaen-8-one (0.9 g, 2.68 mmol) was added and the resulting suspension was stirred at rt for 3 h. The reaction was then concentrated and purified by normal phase silica gel chromatography, eluting with a gradient of 0% to 100% EtOAc in hexanes to yield (9i?,135)-13-{4-[5-chloro-2-(4-chloro-lH-l,2,3-triazol-l-yl)phenyl]-6-oxo- 1 ,6-dihydropyrimidin- 1 -yl} -3-(difluoromethyl)-9-methyl-3 ,4,7, 15-tetraazatricyclo [12.3.1.0²‘⁶]octadeca-l(18),2(6),4,14,16-pentaen-8-one (0.87 g, 50% yield) as a white solid. MS(ESI) m/z: 626.2 [M+H]⁺. 1H NMR (500MHz, CD₃OD) δ 8.91 – 8.83 (m, 1H), 8.78 – 8.71 (m, 1H), 8.33 (s, 1H), 7.88 (d, J=2.5 Hz, 1H), 7.74 (s, 2H), 7.69 – 7.67 (m, 1H), 7.65 (s, 1H), 7.63 (t, J=58 Hz, 1H), 7.52 – 7.50 (m, 1H), 6.36 (d, J=0.8 Hz, 1H),

6.06 – 5.95 (m, 1H), 2.76 – 2.65 (m, 1H), 2.36 – 2.21 (m, 1H), 2.08 – 1.93 (m, 2H), 1.63 -1.53 (m, 1H), 1.53 – 1.42 (m, 1H), 0.99 (d, J=6.9 Hz, 3H). Analytical HPLC (Method A): RT = 8.87 min, purity = 99.7%.

///////////MILVEXIAN, BMS 986177, JNJ 70033093, JNJ 3093, WHO 11401, ミルベクシアン ,

C[C@@H]1CCC[C@H](N2C=NC(=CC2=O)c3cc(Cl)ccc3n4cc(Cl)nn4)c5cc(ccn5)c6c(NC1=O)cnn6C(F)F

Sofpironium bromide

October 1, 2020 2:54 am / 1 Comment on Sofpironium bromide

Sofpironium bromide.png

File:Sofpironium bromide.jpg

Sofpironium bromide

ソフピロニウム臭化物

BBI 4000

[(3R)-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidin-1-ium-3-yl] (2R)-2-cyclopentyl-2-hydroxy-2-phenylacetate;bromide

Formula	C22H32NO5. Br
CAS	1628106-94-4 BASE 1628251-49-9
Mol weight	470.3972

PMDA APPROVED JAPAN 2020/9/25, Ecclock

Anhidrotic

Sofpironium Bromide

1-ambo-(3R)-3-{[(R)-(Cyclopentyl)hydroxy(phenyl)acetyl]oxy}-1-(2-ethoxy-2-oxoethyl)-1-methylpyrrolidinium bromide

C₂₂H₃₂BrNO₅ : 470.4
[1628106-94-4]

SYN

PATENT

WO 2018026869

https://patents.google.com/patent/WO2018026869A1/en

Certain glycopyrronium salts and related compounds, as well as processes for making and methods of using these glycopyrronium salts and related compounds, are known. See, for example, US Patent No. 8,558,008, which issued to assignee Dermira, Inc. See also, for example, US Patent No. 2,956,062, which issued to assignee Robins Co Inc. A H. See also, for example, International Patent Application Publication Nos. WO 98/00132 Al and WO 2009/00109A1, both of which list applicant Sepracor, Inc., as well as US Patent Nos. 6,063,808 and 6,204,285, both of which issued to assignee Sepracor, Inc. Certain methods of treating hyperhidrosis using glycopyrronium salts and related compounds are known. See, for example GB 1,080,960. Certain forms of applying glycopyrrolate compounds to a subject are known. See, for example US Patent Nos. 6,433,003 and 8,618,160, both of which issued to assignee Rose U; also US Patent Nos. 7,060,289; 8,252,316; and 8,679,524, which issued to PurePharm, Inc.

[0004] One glycopyrronium salt which is useful in certain medical applications is the following compound:

[0005] As illustrated above, the absolute configuration at the three asymmetric chiral positions is 2R3’R1’RS. This means that the carbon indicated with the number, 2, has the stereochemical R configuration. The carbon indicated with the number, 3′, also has the stereochemical R configuration. The quatemary ammonium nitrogen atom, indicated with a positive charge, may have either the R or the S stereochemical configuration. As drawn, the compound above is a mixture of two diastereoisomers.

[0006] Certain processes for making glycopyrronium salts are known. However, these processes are not as safe, efficient, stereospecific, or stereoselective as the new processes disclosed herein, for example with respect to large-scale manufacturing processes. Certain publications show that higher anticholinergic activity is attributed to the 2R3’R configuration. However, to date, processes for making the 2R3’R isomers, as well as the 2R3’R1’R isomers are low yielding, involve too many reaction steps to be economically feasible, use toxic materials, and/or are not sufficiently stereospecific or stereoselective with respect to the products formed.

EXAMPLE 2

[0179] The below synthetic description refers to the numbered compounds illustrated in FIG. 2. Numbers which refer to these compounds in FIG. 2 are bolded and underlined in this Example.

[0180] Synthesis of R(-)-Cyclopentylmandelic acid (4)

[0181] R(-)-cyclopentylmandelic acid (compound 4) can be synthesized starting with

R(-)-mandelic acid (compound 1) according to Example 1.

[0182] Step 1 : Making Compound 2.

[0183] R(-)-mandelic acid (1) was suspended in hexane and mixed with pivaldehyde and a catalytic amount of trifluoromethanesulfonic acid at room temperature to form a mixture. The mixture was warmed to 36 °C and then allowed to react for about 5 hours. The mixture was then cooled to room temperature and treated with 8% aqueous sodium bicarbonate. The aqueous layer was removed and the organic layer dried over anhydrous sodium sulfate. After filtration and removal of the solvent under vacuum, the crude product was recrystallized to give (5R)-2-(tert-butyl)-5-phenyl-l,3-dioxolan-4-one (compound 2) in 88% yield (per S-enantiomer yield).

[0184] Step 2: Making Compound 3.

[0185] Compound 2 was reacted with lithium hexamethyl disilazide (LiHMDS) in hexane at -78 °C under stirring for one hour. Next, cyclopentyl bromide was added to the reaction mixture including compound 2 and LiHMDS . The reaction was kept cool for about four (4) hours and then slowly warmed to room temperature and allowed to react for at least twelve (12) more hours. The resulting mixture was then treated with 10% aqueous ammonium chloride. The aqueous layer was discarded and the organic layer dried over anhydrous sodium sulfate. The solvent was removed under vacuum and the residue recrystallized from hexane to give pure product (5R)-2-(tert-butyl)-5-cyclopentyl-5-phenyl- l,3-dioxolan-4-one (3) in 63% yield (per S-enantiomer yield).

[0186] Step 3: Making Compound 4.

[0187] R(-)-cyclopentylmandelic acid (compound 4) was prepared by providing compound 3 in aqueous methanolic potassium hydroxide at 65 °C for four hours. After cooling this mixture to room temperature and removing the methanol under vacuum, the aqueous solution was acidified with aqueous hydrochloric acid. The aqueous solution was then extracted twice with ethyl acetate and the organic phase dried with anhydrous sodium sulfate. After removing the solvent and performing a recrystallization, pure R(-)- cyclopentylmandelic acid (compound 4) was obtained in 62% yield (based on S-enantiomer yield).

[0188] Next, a racemic mixture of l -methyl-3-pyrridinol (20) was provided:

[0189] Synthesis of 2R3 ‘R-glycopyrrolate base (8)

[0190] Step 4: Making Compound 8.

[0191] Enantiomerically pure R(-)-cyclopentylmandelic acid (4) was coupled to racemic l-methyl-3-pyrridinol (20) using 1, 1 -carbonyldiimideazole (CDI) activated esterification to make an enantiomerically pure mixture of the following erythro- and threo- glycopyrrolate bases (compounds 8 and 21, respectively):

[0192] The 2R3’R-glycopyrrolate base (compound 8) was then resolved using the 5- nitroisophthalate salt procedure in Finnish Patent 49713, to provide enantiomerically pure 2R3 Έ. {erythro) as well as pure 2R3 ‘S {threo). In this example, the 2R3 ‘S {threo) was discarded. The 2R3 Έ. {erythro) was separated as stereomerically pure compound 8.

[0193] Step 6: Making Compound 9.

[0194] The glycopyrrolate base, compound 8, was treated in dry acetonitrile with methyl bromoacetate at room temperature under stirring for three (3) hours. The crude product was dissolved in a small volume of methylene chloride and poured into dry ethyl ether to obtain a precipitate. This procedure was repeated three times to provide (3R)-3-((R)- 2-cyclopentyl-2-hydroxy-2-phenylacetoxy)-l -(2-ethoxy-2-oxoethyl)-l-methylpyrrolidin-l – ium bromide, also known as 3′(R)-[R-Cyclopentylphenylhydroxyacetoy]- -ethyl- l ‘methoxycarbonylpyrrolidinium bromide (compound 9) in 89% yield. Compound 9 included the following stereoisomers:

Synthesis of 9a, 9b, 13a, and 13b.

Publication Number	Title	Priority Date	Grant Date
US-2019161443-A1	Processes for making, and methods of using, glycopyrronium compounds	2016-08-02

ClinicalTrials.gov

CTID	Title	Phase	Status	Date
NCT02058264	A Safety, Tolerability and Preliminary Efficacy Study of BBI-4000 in Subjects With Axillary Hyperhidrosis	Phase 1	Completed	2014-09-11

NIPH Clinical Trials Search of Japan

CTID	Title	Status	Date
JapicCTI-184249	A repeatedly applied study of BBI-4000 in patients with primary hyperhidrosis	complete	2018-12-13
JapicCTI-184003	A long term safety study of BBI-4000 gel in patients with primary axillary hyperhidrosis	complete	2018-06-15
JapicCTI-183948	A confirmatory study of BBI-4000 gel in patients with primary axillary hyperhidrosis	complete	2018-05-07
UMIN000020546	A skin irritation study of BBI-4000 in healthy adult males (phase 1)	Complete: follow-up complete	2016-01-18

////////////Sofpironium bromide, Ecclock, 2020 APPROVALS, JAPAN 2020, Anhidrotic, ソフピロニウム臭化物 , BBI 4000

CCOC(=O)C[N+]1(CCC(C1)OC(=O)C(C2CCCC2)(C3=CC=CC=C3)O)C.[Br-]

Tetrahydrobiopterin,

September 29, 2020 5:58 am / Leave a comment

Kuvan (Saproterin Dihydrochloride Tablets): Uses, Dosage, Side Effects, Interactions, Warning

Sapropterin

Sapropterin dihydrochloride, Dapropterin dihydrochloride, R-THBP, 6R-BH4, SUN-0588, Phenoptin, Biopten, Biobuden, Bipten

Approval:US: Dec’07, EU: Dec’08

IUPAC Name

(6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-3,4,5,6,7,8-hexahydropteridin-4-one

SMILES

[H][C@@]1(CNC2=C(N1)C(=O)NC(N)=N2)[C@@H](O)[C@H](C)O

сапроптерин [Russian] [INN]

سابروبتيرين [INN]

沙丙蝶呤 [Chinese] [INN]

17528-72-2
27070-47-9
Sun 0588

6R-BH4
R-THBP
Sapropterin
Sapropterina
sapropterinum
Tetrahydrobiopterin

Title: Sapropterin

CAS Registry Number: 62989-33-7

CAS Name: (6R)-2-Amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-4(1H)-pteridinone

Additional Names: (6R)-L-erythro-tetrahydrobiopterin; dapropterin; R-THBP; 6R-BH4

Molecular Formula: C9H15N5O3

Molecular Weight: 241.25

Percent Composition: C 44.81%, H 6.27%, N 29.03%, O 19.90%

Literature References: Natural cofactor of the aromatic amino acid hydroxylases required for catecholamine and serotonin biosynthesis. Identification of cofactor activity: S. Kaufman, Proc. Natl. Acad. Sci. USA 50, 1085 (1963). Prepn of (6R,S)-BH4: B. Schircks et al., Helv. Chim. Acta 61, 2731 (1978). Chromatographic separation of diastereoisomers: S. W. Bailey, J. E. Ayling, J. Biol. Chem. 253, 1598 (1978). Absolute configuration of natural isomer: W. L. F. Armarego et al., Aust. J. Chem. 35, 785 (1982). Stereospecific synthesis: S. Matsuura et al., Heterocycles 23, 3115 (1985); H. Sakai, T. Kanai, EP 191335; eidem, US 4713454 (1986, 1987 both to Shiratori; Suntory). Bioavailability: G. Kapatos, S. Kaufman, Science 212, 955 (1981). Effect on neurotransmitter monoamine biosynthesis: S. Miwa et al., Arch. Biochem. Biophys. 239, 234 (1985). LC determn in biological samples: Y. Tani, T. Ishihara, Life Sci. 46, 373 (1990). Therapeutic potential in hyperphenylalaninemia: S. Kaufman, J. Nutr. Sci. Vitaminol, Suppl., 601 (1992).

Properties: pK¢ 5.05. uv max (0.1 N HCl): 265 nm (e 14000).

pKa: pK¢ 5.05

Absorption maximum: uv max (0.1 N HCl): 265 nm (e 14000)

Derivative Type: Dihydrochloride

CAS Registry Number: 69056-38-8

Manufacturers’ Codes: SUN-0588

Trademarks: Biopten (Maruho)

Molecular Formula: C9H15N5O3.2HCl

Molecular Weight: 314.17

Percent Composition: C 34.41%, H 5.45%, N 22.29%, O 15.28%, Cl 22.57%

Properties: Crystals from HCl, mp 245-246° (dec). [a]D25 -6.81° (c = 0.665 in 0.1 M HCl). uv max (2 M HCl): 264 nm (e 16770).

Melting point: mp 245-246° (dec)

Optical Rotation: [a]D25 -6.81° (c = 0.665 in 0.1 M HCl)

Absorption maximum: uv max (2 M HCl): 264 nm (e 16770)

Therap-Cat: In treatment of hyperphenylalaninemia.

Keywords: Enzyme Cofactor

INGREDIENT	UNII	CAS	INCHI KEY
Sapropterin dihydrochloride	RG277LF5B3	69056-38-8	RKSUYBCOVNCALL-NTVURLEBSA-N

NAME	DOSAGE	STRENGTH	ROUTE	LABELLER	MARKETING START	MARKETING END
Sapropterin Dihydrochloride	Tablet	100 mg/1	Oral	Par Pharmaceutical, Inc.	2017-02-02	2019-05-05

Experimental Properties

PROPERTY	VALUE	SOURCE
melting point (°C)	250-255 °C (hydrochloride salt)	Not Available
water solubility	>20 mg/mL (dichloride salt)	Not Available
logP	-1.7	Not Available

Tetrahydrobiopterin (BH₄, THB), also known as sapropterin (INN),^[2]^[3] is a cofactor of the three aromatic amino acid hydroxylase enzymes,^[4] used in the degradation of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotonin (5-hydroxytryptamine, 5-HT), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and is a cofactor for the production of nitric oxide (NO) by the nitric oxide syntheses.^[5] Chemically, its structure is that of a (dihydropteridine reductase) reduced pteridine derivative (Quinonoid dihydrobiopterin).^[6]

Medical use

Tetrahydrobiopterin is available as a tablet for oral administration in the form of sapropterin dihydrochloride (BH4*2HCL).^[7]^[8]^[9] It was approved for use in the United States as a tablet in December 2007^[10]^[11] and as a powder in December 2013.^[12]^[11] It was approved for use in the European Union in December 2008,^[9] Canada in April 2010,^[11] and Japan in July 2008.^[11] It is sold under the brand names Kuvan and Biopten.^[9]^[8]^[11] The typical cost of treating a patient with Kuvan is US$100,000 per year.^[13] BioMarin holds the patent for Kuvan until at least 2024, but Par Pharmaceutical has a right to produce a generic version by 2020.^[14]

Sapropterin is indicated in tetrahydrobiopterin deficiency caused by GTP cyclohydrolase I (GTPCH) deficiency, or 6-pyruvoyltetrahydropterin synthase (PTPS) deficiency.^[15] Also, BH4*2HCL is FDA approved for use in phenylketonuria (PKU), along with dietary measures.^[16] However, most people with PKU have little or no benefit from BH4*2HCL.^[17]

Sapropterin (tetrahydrobiopterin or BH4) is a cofactor in the synthesis of nitric oxide. It is also essential in the conversion of phenylalanine to tyrosine by the enzyme phenylalanine-4-hydroxylase; the conversion of tyrosine to L-dopa by the enzyme tyrosine hydroxylase; and conversion of tryptophan to 5-hydroxytryptophan via tryptophan hydroxylase.

Sapropterin commonly known as tetrahydrobiopterin (THB or BH4) developed by BioMarin and marketed as Sapropterin dihydrochloride under the brand name of KUVAN®. It is indicated for the treatment of phenylketonuria (PKU) and tetrahydrobiopterin deficiencies. Sapropterin dihydrochloride is chemically known as (6R)-2-amino-6-[(lR, 2S)-1, 2- dihydroxypropyl]-5,6,7,8-tetrahydro-4(lH)-pteridinone dihydrochloride and structurally represented as below.

Sapropterin dihydrochloride

Due to its vital role in the conversion of L-tyrosine into L-DOPA, which is the precursor for dopamine, a deficiency in tetrahydrobiopterin can cause severe neurological disorders unrelated to toxic build-up of L-phenylalanine; dopamine is a crucial neurotransmitter, and is the precursor of norepinephrine and epinephrine. Thus, a deficiency of tetrahydrobiopterin can result in phenylketonuria (PKU) from L-phenylalanine concentrations or hyperphenylalaninemia (HP A), as well as monoamine and nitric oxide neurotransmitter deficiency or chemical imbalance. The chronic presence of PKU can result in severe brain damage, including symptoms of mental retardation, speech impediments like stuttering, slurring, seizures or convulsions and behavioural abnormalities.

In an article published in Bio Chem J 347 (1): 1-16, tetrahydrobiopterin is reported to be biosynthesized from guanosine triphosphate (GTP) by three chemical reactions mediated by the enzymes GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SR).

Preparation of Sapropterin is reported with a mixture of R & S isomers in Helv. Chim. Acta, 60, 1977, 211-214, by catalytic reduction of L-biopterin of formula (2). Similar process with slight modifications is also published in Hel. Chim. Acta, 61, 1978, 2731- 2738.

(2)

In another publication reported in Helv. Chim. Acta, 62, 1979, 2577-2580, separation of the diastereomers (6R) and (6S)-5,6,7,8-tetrahydro-L-biopterin is reported by fractional crystallization of corresponding tetraacetyl derivative followed by hydrolysis using aq. HC1.

In another process published in Heterocycles, 23(12), 1985, 3115-3120, Sapropterin dihydrochloride of formula (1) is prepared by catalytic hydrogenation of L- biopterin of formula (2) in the presence of Pt02 under latm hydrogen pressure in 0.1 M potassium phosphate buffer at pH 11.8 for 18hr followed by filtration and recrystallization from 8M HC1. With slight modifications in the above reaction conditions like using platinum black, aq. base solutions like tetraethylammonium hydroxide or triethylamine etc. under 100 Kg/cm2 hydrogen pressure / 0° C / pH 12.0 / 1000 rpm / 20h/3N HCl-EtOH with 85% yield is disclosed in US4713454. In another process disclosed in US4595752, L-biopterin of formula (2) is catalytically reduced in the presence of platinum oxide in aq. base / acid solutions like (10% aq. potassium carbonate, aq. sodium carbonate, aq. potassium acetate and 0.1 N aq. HCl) under bubbling of hydrogen gas for 5-30hr at room temperature followed by filtration and isolated as HCl salt of formula (1) using aq. HCl and ethanol to obtain Sapropterin dihydrochloride.

In another approach disclosed in WO2005049614, racemic isomers of Sapropterin dihydrochloride are prepared from L-neopterin.

In another process disclosed in WO2009088979, the diacetyl biopterin is hydrolysed in the presence of aq. diethyl amine-n-butanol mixture at 40°C for 16hr at pH >11.5 followed by hydrogenation in the presence of platinum black using 50 bar hydrogen pressure at 25 °C. Product of formula (1) isolated as HCl salt from ethanol or butanol.

In another process disclosed in US20130197222, Sapropterin dihydrochloride of formula (1) is prepared starting from condensation of crotonoic acid.

The process for preparation of key intermediate, L-biopterin of formula (2) is cited in the following references.

In an article published in J. Am. Chem. Soc, 1955, 77, 3167-3168, L-biopterin of formula (2) is reported to be first isolated from human urine. The melting point reported to be 250-280°C. In another article published in J. Am. Chem. Soc, 1956, 78, 5868-5871, L-biopterin of formula (2) is prepared starting from L-rhamnose. A slight modification in the reaction conditions mentioned above is disclosed in US3505329.

In the article published in Helv. Chim. Acta, 1969, 52, 1225-1228, L-biopterin of formula (2) along with 7-biopterin is synthesized by condensing 2, 4, 5-triamino-6-oxo-l, 6-dihydropyrimidine dihydrochloride with (1 -benzyl- l-phenyl-hydrazino)-5-desoxy-L- ribulose followed by oxidation of the tetrahydro derivative.

Later in the year 1974, in an article, J. Am. Chem. Soc, 1974, 96, 6781-6782, L-biopterin is reported to be prepared starting from L-rhamnose. In another approach published in Bull. Chem. Soc. Jpn., 1975, 48(12), 3767-3768, L- biopterin of formula (2) is prepared from 2, 4, 5-triamino-6-hydroxypyrimidine dihydrochloride is reacted with hydrazone derivative in aq. methanol at reflux temperature.

In another process disclosed in US5043446 (1989), L-biopterin process is claimed to be synthesized starting from D-ribose. Similar approach with slight variations in the process, later published in Liebigs Ann. Chem., 1989, 1267-1269.

In another approach published in Agric. Biol. Chem., 1989, 53, 2095-2100, L-biopterin is synthesized starting from (S)-ethyl lactate. Prior to this publication the methodology is claimed by the same authors in JP01-221380 (1989).

In another approach disclosed in US5037981 (1990), L-biopterin is synthesized from 2- methylfuran.

In the article, Synthesis, 1992, 303-308, L-biopterin is synthesized from (4S)-4(3P- Acetoxy-5-androsten-17P-ylcarbonyloxy)-2-pentynol.

In the approach published in J. Org. Chem., 1996, 61, 8698-8700, L-biopterin is synthesized from L-tartaric acid.

In the patent US7361759 (2005), L-biopterin of formula (2) is made from L-rhamnose diethyl mercaptal.

US 20120157671 application discloses the preparation of compound of formula (4a) is by reacting D-ribose of formula (3) with acetone in the presence of sulphuric acid at room temperature followed by neutralization with sodium carbonate and concentrated under vacuum.

Sapropterin | Nature Reviews Drug Discovery

Pharmaceutics 12 00323 g004 550

https://www.mdpi.com/1999-4923/12/4/323/htm

PATENT NUMBER	PEDIATRIC EXTENSION	APPROVED	EXPIRES (ESTIMATED)
US9216178	No	2015-12-22	2032-11-01
US9433624	No	2016-09-06	2024-11-17

PATENT NUMBER	PEDIATRIC EXTENSION	APPROVED	EXPIRES (ESTIMATED)
CA2545968	No	2010-03-09	2024-11-17
US7566714	Yes	2009-07-28	2025-05-17
US7612073	Yes	2009-11-03	2025-05-17
US8067416	Yes	2011-11-29	2025-05-17
USRE43797	Yes	2012-11-06	2025-05-17
US7947681	Yes	2011-05-24	2025-05-17
US7566462	Yes	2009-07-28	2026-05-16
US8318745	Yes	2012-11-27	2025-05-17
US8003126	Yes	2011-08-23	2026-05-16
US7727987	Yes	2010-06-01	2025-05-17

Synthesis Reference

Steven S. Gross, “Blocking utilization of tetrahydrobiopterin to block induction of nitric oxide synthesis.” U.S. Patent US5502050, issued October, 1984.

US5502050

SYN

Synthetic Reference

Hong, Hao; Gage, James; Chen, Chaoyong; Lu, Jiangping; Zhou, Yan; Liu, Shuangyong. Method for synthesizing sapropterin dihydrochloride. Assignee Asymchem Laboratories (Tianjin) Co., Ltd., Peop. Rep. China; Asymchem Life Science (Tianjin) Co., Ltd.; Tianjin Asymchem Pharmaceutical Co., Ltd.; Asymchem Laboratories (Fuxin) Co., Ltd.; Jilin Asymchem Laboratories Co., Ltd. WO 2013152609. (2013).

syn 1

EP 0191335. Aust J Chem 1984,37(2),355-66, Chem Lett 1984,5(5),735-8

Helv Chim Acta 1979,62(8),2577-80

This compound can be prepared in two related ways: 1) The catalytic hydrogenation of biopterin (I) with H2 over PtO2 aqueous K2HPO4 at pH 11.4 or aq. (Et)4NOH at pH 12 yields a solution which is acidified with HCl. After evaporation, the residue is crystallized in ethanol – HCl. 2) The acetylation of biopterin (I) with refluxing acetic anhydride gives the triacetyl derivative (II), which is hydrogenated with H2 over PtO2 in trifluoroacetic acid, yielding the (6RS)-mixture of triacetyl derivatives (III). Acetylation of (III) with refluxing acetic anhydride affords the tetracetyl (6RS)-derivative (IV), which by fractional crystallization or column chromatography of the dihydrochloride in methanol gives the desired compound as pure (6R)-isomer.

PATENT

https://patents.google.com/patent/WO2016189542A1/en

formula 1).

The present invention is shown in below scheme- 1

Experimental Section: Example-1: Preparation of (6R)-2-amino-6-[(lR, 2S)-1, 2-dihydroxypropyl]-5,6,7,8- tetrahydro-4(lH)-pteridinone dihydrochloride of formula (1):

Step (i): Preparation of 2, 3-O-isopropylidene-D-ribose of formula (4a)

Into a 5L, 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a condenser, were charged acetone (3.0 L), D-ribose (300.0 gm, 2.0 mole) and p-toluene sulfonic acid (11.5 gm). The solution was stirred and maintained at 20-25°C for 2.5-3.0hrs. After completion of reaction, the reaction mixture was neutralized with aq. base solution and filtered. The filtrate was evaporated to dryness to get 375.0 gm (98.8% by theory) of 2, 3-O-isopropylidene-D-ribose of formula (4a) as light brown colour oily residue. Purity: >95% by GC. Step (ii): Preparation of l-deoxy-3, 4-O-isopropylidene-D-allitol of formula (5a)

Into a 5L, 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a condenser, was charged, 2.0L, 3M methyl magnesium chloride and cooled to 10° C. To this stirred solution, a solution of 200gm of 2,3-0- isopropylidene-D-ribose of formula (4a) dissolved in 200 mL tetrahydrofuran was added. After completion of reaction, the reaction mixture was quenched with ammonium chloride, extracted with ethyl acetate and separated. The solvent was evaporated to dryness under vacuum to get 185gm of l-deoxy-3, 4-O-isopropylidene-D-allitol of formula (5a) as dark brown colour oily residue. The crude product was purified by crystallization from ethyl acetate/hexane mixture to get 130g (60% by theory) as white crystalline solid. Purity: >98% by GC.

JR (λ Cm-1, KBr disc): 3317.64, 2993.69-2976.90, 2926.08, 2873.26 (m) -CH3, 1074.35; 1 HNMR (400 MHz, DMSO-d6, EDl®j&¾ : (H2¾H3, J=6.8Hz, 3H),

1.148 (s, CH3, 3H), 1.290 (s,CH3), 3.415-3.357 (m, CH, 1H), 3.652-3.571 (m, CH2, 2H), 3.812-3.803 (d, 2 X CH, 2H), 4.00-3.969 (q, CH, 1H), 4.504-4.476 (t, ΟΗ, ΙΗ), 4.504- 4.476 (d, OH, 1H), 5.381-5.371 (d, OH, 1H): 13 CNMR (100 MHz, DMSO-d6, □ (ppm): 20.59, 25.35, 27.73, 63.18, 64.61 , 69.77, 76.82, 81.40, 107.31 ; Mass: 206.42 [M], 205.41 [M-l]. DSC (° C): 77.58° C Step (iii): Preparation of 5-deoxy-2, 3-O-isopropylidene-D-ribose of formula (6a)

Into a 5L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a condenser, were charged, 1.6 L of water and 270 gm of sodium meta periodate. The solution was cooled to 10-20°C. To the stirred solution, a solution of 200 gm of l-deoxy-3, 4-O-isopropylidene-D-allitol of formula (5a) dissolved in 1.4 L of isopropyl ether at 25°C. After addition, the reaction mixture was maintained at 25-30° C for l-2h. After completion of reaction, the layers were separated and the organic layer was washed with water, aq. sodium bicarbonate and separated. The excess solvent was removed by distillation under vacuum to get 145 gm (85.4% by theory) of 5- deoxy-2, 3-O-isopropylidene-D-ribose of formula (6a) as yellow oil. Purity: >98% by GC.

Step (iv & v): Preparation of 5-deoxy-L-ribose phenyl hydrazone of formula (8) a) Step (iv): Preparation of 5-deoxy-L-ribose of formula (7)

Into a 2L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a condenser, were charged 600ml of water and 200gm of 5- deoxy-2, 3-O-isopropylidene-D-ribose of formula (6a). To the stirred reaction mixture, 180gm of resin was charged and stirred for 8-10 h at 10-15° C. After completion of reaction, the resin was recovered and the filtrate was clarified by activated charcoal and filtered. The filtrate was distilled off under vacuum and the resulting 5-deoxy-L-ribose of formula (7) present water was directly used in the next step without further isolation and purification. The purity of 5-deoxy-L-ribose of formula (7) present in water was above 95% by TLC.

b) Step (v): Preparation of 5-deoxy-L-ribose phenyl hydrazone of formula (8)

Into a 2L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a condenser, were charged the above aq. solution of 5-deoxy-L- ribose of formula (7), 5.0 mL of acetic acid. To the stirred solution, 125g of phenyl hydrazine was charged and stirred the reaction mixture for l-2h at 25-35° C. After completion of reaction, the reaction product was filtered and washed with isopropyl ether. The wet product was dried to get 190g (73.9% by theory) of 5-deoxy-L-ribose phenyl hydrazone of formula (8) as yellow colour crystalline powder. Purity: >99.0% by HPLC. Step (VI toX): Preparation of L-erythro-biopterin of formula (2)

a) Step (vi): Preparation of triacetoxy-5-deoxy-L-ribose phenylhydrazone of formula (9)

Into a 10L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a guard tube, were charged 5L of ethyl acetate, 500g of 5- deoxy-L-ribose phenyl hydrazone of formula (8) and 54gm of 4-dimethylaminopyridine. The reaction mixture was cooled to 25-30° C and was added 730gm of acetic anhydride drop wise. The reaction mixture was maintained under stirring for 2-3h. After completion of reaction, the reaction mixture was washed with water, aq. sodium carbonate and water, and separated. The organic layer was used in the next stage without further isolation and purification.

b) Step (vii): Preparation of 1,2-diacetyl-biopterin of formula (10)

Into a 20L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, addition funnel, and a condenser, were charged, the above organic layer containing triacetoxy-5-deoxy-phenyl hydrazone of formula (9) obtained in step (vi), 3.0 L methanol and 4-hydroxy-2,5,6-triaminopyrimidine base (generated from 600 gm of corresponding sulphate salt) and salt (generated from 350 gm of tetra butyl ammonium bromide and 154g of 70% perchloric acid) and 5.3L water under stirring and heated and maintained at 35-40°C for 6-8h. The reaction mixture was then cooled to 20- 25°C and added 1.0 Kg 35% aq. hydrogen peroxide drop wise. The reaction mixture was maintained for 36-40h under stirring at 25-30°C and resulting product was filtered under suction. The wet product was washed with water and utilized in the next step without further purification.

c) Step (viii): Preparation oi -erythro biopterin of formula (2)

Into a 10L 4-necked round-bottomed flask equipped with a mechanical stirrer, condenser, thermometer socket, and addition funnel, were charged 1.35 L of aq. potassium hydroxide and the above wet product obtained from step (vii). The reaction mixture was heated to 45-50° C and maintained form 2-3h and filtered. The pH of the filtrate was adjusted to neutral and the resulting product was filtered and dried to get 205 g of crude L-erythro-biopterin of formula (2) as dark brown solid. Purity: >90% by HPLC

d) Step (ix): Preparation of potassium salt oi -erythro biopterin of formula (11a) Into a 10L 4 necked round-bottomed flask equipped with a mechanical stirrer, thermometer socket, and a glass stopper, were charged 650 mL water followed by HOg of potassium hydroxide and dissolved under stirring. The potassium hydroxide solution was cooled to 25-30° C and the above crude L-erythro-biopterin of formula (2) was charged under stirring. The resulting solution was then clarified using activated carbon and filtered. The potassium salt was regenerated from the solution by the addition of 8.5L of isopropyl alcohol. The resulting salt was filtered and washed with isopropyl alcohol. The wet product of formula (11a) was utilized in the next step without further purification.

e) Step (x): Preparation of pure L-er thro biopterin of formula (2) from potassium salt of L-erythro biopterin of formula (2)

Into a 5L 4 necked round-bottomed flask equipped with mechanical stirrer, thermometer socket, and addition funnel, were charged 3.2 L of water and the above wet potassium salt of formula (11a). The reaction mixture was stirred to dissolve completely. The resulting solution was clarified using activated carbon and filtered. The pH of the filtrate was adjusted to 6.0-7.0 to get pure L-erythro-biopterin of formula (2). The product was filtered and washed with water followed by isopropyl alcohol followed by isopropyl ether to get 130g of highly pure L-erythro biopterin of formula (2) with > 98% HPLC purity Appearance: pale brown coloured solid.

1H NMR (3N DC1) 5(ppm): 1.569-1.585(d, 3H), 4.596-4.657(p, 1H), 5.325-5.337(d, 1H), 9.355(s, 1H); Mass: 238.29(M+1), 239.22(M+2).

Step (xi): Preparation of Sapropterin dihydrochloride of formula (1)

Into a 5L 4 necked round-bottomed flask equipped with mechanical stirrer, and thermometer socket, were charged 1.8L of water, 250g of L-erythro-biopterin of formula (2) followed by 800mL of 20% aq. potassium carbonate solution under stirring. The solution was then added 90g of platinum oxide catalyst. The reaction mixture was then transferred into an autoclave and pressurized with 40 bar hydrogen gas and hydrogenated at room temperature for 24-30h under stirring. After completion of reaction, the catalyst was filtered off and the pH of the filtrate was acidified with concentrated hydrochloric acid. The water was evaporated under vacuum and the resulting crude Sapropterin dihydrochloride of formula (1) was isolated as pale yellow colour solid by addition of isopropanol/l-pentanol mixture. The product was dried in a vacuum oven to get 250g of crude Sapropterin dihydrochloride of formula (1). Step (xii): Purification of Sapropterin dihydrochloride of formula (1)

Into a 2L 4 necked round-bottomed flask equipped with a mechanical stirrer, thermometer socket, and reflux condenser, were charged 1L water and 250g of Sapropterin dihydrochloride of formula (1). The contents were stirred to dissolve completely. The clear solution was treated with activated charcoal and filtered. The filtrate was distilled off completely under vacuum to afford pale yellow solid. The product was isolated from isopropanol/l-pentanol mixture to get 225.0 g (90%) pure Sapropterin dihydrochloride of formula (1) as pale yellow to off-white solid. HPLC purity is >99.9%.

Example 2: Preparation of triacetoxy-5-deoxy-L-ribose phenylhydrazone of formula

(9)

Into a 10L 4 necked round-bottomed flask equipped with a mechanical stirrer, a thermometer socket, and a guard tube, were charged 50mL of ethyl acetate, 5.0g of 5- deoxy-L-ribose phenyl hydrazone of formula (8) and 0.54g of N, N-dimethylamino pyridine. The reaction mixture was cooled to 15-20°C and was added 7.2gm of acetic anhydride drop wise. The reaction mixture was maintained under stirring for 6-8h. After completion of reaction, the reaction mixture was washed with water, aq. sodium carbonate and water, and separated. The organic layer was distilled under reduced pressure and product was isolated from n-hexane to get 6.2g of triacetoxy-5 -deoxy-L- ribose phenylhydrazone of formula (9) 79.4% yield.

Appearance: Orange coloured solid.

Melting point: 70-75 °C.

1HNMR (CDC13): 1.275-1.29 l(d, 3H), 2.039(s, 3H), 2.085-2.095(d, 6H), 5.083-5.144(m, 1H), 5.390-5.416(t, 1H), 5.589-5.619(t, 1H), 6.849-6.886(t, 1H), 6.922-6.937(t, 1H), 6.966-6.987(d, 2H), 7.221-7.242(d, 2H), 7.563(s, 1H(D20 exchangeable).

13CNMR (CDC13): 15.325, 20.816-21.053, 68.482, 71.717, 73.043, 112.759, 120.510, 129.212, 132.105, 144.049, 169.496, 169.948. Example 3: Preparation of potassium salt of L-erythro biopterin of formula (11)

Into a 1.0L 4 necked round-bottomed flask equipped with a mechanical stirrer, thermometer socket, and a glass stopper, were charged 75 mL water followed by 3.7g of potassium hydroxide and dissolved under stirring. The potassium hydroxide solution was cooled to 25-30° C and 15.0g of crude L-erythro-biopterin of formula (2) was charged under stirring. The resulting solution was then clarified using activated carbon and filtered. The potassium salt was regenerated from the solution by the addition of 500mL of ethanol. The resulting salt was filtered and washed with ethanol and dried to get 9.1g of potassium salt of L-erythro biopterin of formula (11) with 52.3% yield. HPLC <98% Appearance: Brown coloured solid.

1H NMR (D20): 1.187-1.203(d, 3H), 4.158-4.220(p, 1H), 4.731-4.745(d, 1H), 8.623(s, 1H).

13C NMR (D20): 18.198, 70.645, 76.703, 128.811, 147.875, 149.410, 156.504, 164.774, 173.731.

Mass: 276.23(M+1), 277.21(M+2), 238.29(M-K+1); DSC (° C): 313.12°

Example 4: Preparation of Sapropterin dihydrochloride of formula (1)

Into a 5L 4 necked round-bottomed flask equipped with mechanical stirrer, and thermometer socket, were charged 1.8L of water, 250g of L-erythro-biopterin of formula (2) followed by 800ml of 20% aq. potassium hydroxide solution under stirring. The solution was then added 90gm of platinum oxide catalyst. The reaction mixture was then transferred into an autoclave and pressurized with 50 bar hydrogen gas and hydrogenated at room temperature for 24-30h under stirring. After completion of reaction, the catalyst was recovered by filtration and the filtrate was acidified with concentrated hydrochloric acid. The water was evaporated under vacuum and the resulting crude Sapropterin dihydrochloride of formula (1) was isolated as pale yellow colour solid by addition of ethanol- 1 -pentanol mixture. The product was dried in a vacuum oven to get 250g of crude Sapropterin dihydrochloride of formula (1). Example 5: Purification of Sapropterin dihydrochloride of formula (1)

Into a 2L 4 necked round-bottomed flask equipped with a mechanical stirrer, thermometer socket, and reflux condenser, were charged 1L water and 250g of Sapropterin dihydrochloride of formula (1). The contents were stirred to dissolve completely and the clear solution was treated with activated charcoal and filtered. The filtrate was distilled off completely under vacuum to afford pale yellow solid. The product 225.0 g (90%) was isolated ethanol- 1 -pentanol mixture as pure Sapropterin dihydrochloride of formula (1) as pale yellow to off-white solid. HPLC purity is >99.9%.

syn

PATENT

https://patents.google.com/patent/WO2016101211A1/en

was developed by Merck and was launched in the United States and the European Union in 2007 and 2008 under the trade name Kuvan. This product can be used to treat hyperphenylalaninemia (HPA) caused by tetrahydrobiopterin (BH4) deficiency. The structure is as follows:

The chemical name is: (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-4(1H)-fluorenone Dihydrochloride.

The oxaprozin hydrochloride can be obtained by hydrogenation of L-erythrobiopterin. The literature Liebigs Ann. Chem. 1989, 1267-1269 reports the preparation of L-erythrobiopterin starting from L-ribose. The preparation route is as follows:

Although the method is simple and easy to perform, it is a better preparation route, but the disadvantage is that the starting material L-ribose price is higher, thus causing the cost of sapropium hydrochloride to be high.

The literature for the preparation of L-erythrobiopteris is reported by the documents Helv. Chim. Acta, 1985, 1639-1643, US2011218339A, etc. The product of the acetylation reaction of the steroid compound 6 with 2,4,5-triaminopyrimidinone Cyclization in a methanol/water/pyridine system followed by aromatization with an iodine reagent to give an acetylated L- Red-type biopterin, followed by hydrolysis and deacetylation to obtain L-erythrobiopterin. The reaction equation is as follows:

Among them, compound 6 is used as a key intermediate, and many methods for its preparation are reported. The method reported in J. Am. Chem. Soc. 1974, 6781-6782, J. Am. Chem. Soc. 1976, 2301-2307, etc., uses L-rhamnose as a raw material, and reacts with ethanethiol to form a corresponding shrinkage. Sulfuraldehyde, oxidizing thiol to sulfone with an oxidizing agent, removing a carbon under alkaline conditions to obtain 5-deoxy-L-arabinose, and reacting 5-deoxy-L-arabinose with phenylhydrazine to obtain a key intermediate formula 6 . The synthetic route is as follows:

Although this method has been improved and improved many times, the ethanethiol used has a special malodor and requires the use of a deodorizing device, and its lower boiling point also causes inconvenience to the production.

Document J. Org. Chem. 1996, 8699-8700 reports that L-tartaric acid is used as a starting material, which is protected by hydroxyl group, carboxyl group, reduction, addition, deprotection to obtain 5-deoxy-L-ribose, 5-deoxy- The condensation of L-arabinose with phenylhydrazine gives key intermediates. The synthetic route is as follows:

The reducing agent used in the route of the acid chloride to reduce the aldehyde is bis(triphenylphosphine) copper borohydride (I), which has a high price and is not favorable for the control of industrialization cost. The reaction temperature of the format reagent with carbonyl addition and lactone reduction is -78 ° C, and the energy consumption in industrial production is high. In addition, the post-treatment of the multi-step reaction uses silica gel column color The spectrum is purified and it is difficult to achieve industrialization. Therefore, this route has great disadvantages in terms of cost and operability in industrial production.

Document CN201010151443.2 reports the use of L-arabinose as a starting material to obtain L-erythrobioptery through a multi-step reaction. The preparation route is as follows:

In reproducing the preparation method, we have found that the intermediate 2 is directly subjected to reduction and desulfonation reaction to prepare the intermediate 2, which has the disadvantages of low yield, low product purity, and difficulty in purification of the product. Therefore, it is necessary to find a simple, feasible and low-cost preparation route.

scheme synthetic route includes the following steps:

Example 1: Preparation of Product 1

To the reaction flask was added 10 L of anhydrous methanol, and 1.5 kg of the starting material L-arabinose was added under mechanical stirring. 250 g of concentrated sulfuric acid was added dropwise under a water bath, and the reaction was stirred for 20-24 hours. The reaction was monitored by TLC, and 350 g of sodium carbonate was added to the reaction system. Stir until pH = 7-8 and filter. The filtrate was concentrated under reduced pressure at 35 ° C to 40 ° C to dryness to yield 1.64 kg of oil, yield -100%.

Example 2: Preparation of product 2

The product 1, 4 L of pyridine and 5 L of acetonitrile were added to the reaction flask and dissolved by mechanical stirring. The mixture was cooled by stirring, and a solution of 1.8 kg of p-toluenesulfonyl 5 L acetonitrile was added dropwise at a temperature of 0 to 5 ° C. After completion of the dropwise addition, the reaction was stirred at room temperature 20-25 ° C for 4 hours. The TLC monitors the reaction.

After concentration, 12 L of ethyl acetate and 5 L of water were added to the concentrated residue, and the layers were stirred. The organic layer was washed with 1 mol/L hydrochloric acid, saturated sodium hydrogen carbonate and saturated brine and dried. Filtration and concentration of the filtrate gave 1.7 kg of pale yellow oil, yield 56.3%.

Example 3: Preparation of product 3

1.2 kg of product 2 was added to a 10 L reaction flask, dissolved with 6 L of methyl ethyl ketone, and 840 g of sodium iodide was added with stirring. After the addition, the temperature was refluxed for 12 hours, and the reaction was completed by TLC. The mixture was cooled to room temperature, filtered, and the filtrate was evaporated. It was dissolved in ethyl acetate, washed with water, and the aqueous layer was evaporated. The combined organic layers were washed with EtOAc EtOAc m.

Example 4: Preparation of product 4

To a 20 L reaction flask was added 900 g of product 3, 332 g of triethylamine dissolved in 9 L of methanol, 45 g of 10% Pd/C, vacuumed, hydrogenated twice, and hydrogenated at a constant temperature of 25-30 ° C for 16 hours. The reaction was completed by TLC, filtered, and the filtrate was concentrated under reduced pressure to give a residue. 4 L of ethyl acetate was added to the residue to precipitate a white solid. The mixture was stirred at 0 ° C for 30 min, and filtered. The filtrate was added to 2 L of a 0.4 mol/L sulfuric acid solution and the layers were separated. The aqueous layer was washed once with 50 mL of ethyl acetate to give an aqueous solution of product 4 (approximately 250 g).

Example 5: Preparation of product 5

The aqueous solution of product 4 was added to the reaction flask, and the reaction was heated at 75 ° C for 3 hours, and the reaction was completed by TLC (DCM: MeOH = 10:1). After cooling to room temperature, it was washed with 100 mL of ethyl acetate, and the aqueous layer was separated to give the product 5, i.e., about 213 g of aqueous solution of 5-deoxy-L-arabinose, which was directly reacted in the next step.

Example 6: Preparation of product 6

To the reaction flask, 2.5 L of ethyl acetate and 170 g of phenylhydrazine were added under nitrogen, and an aqueous solution of the product 5 was added dropwise with stirring at a temperature of 5 to 10 ° C (protected from light). The reaction was kept for 1 hour, and then the temperature was raised to 20-25 ° C for 30 min. The reaction was completed by TLC and the layers were separated. The aqueous layer was extracted with ethyl acetate and organic layers were combined. The organic layer was dried over anhydrous sodium sulfate and filtered.

The ethyl acetate solution of product 6 was added to the reaction flask under nitrogen, and 8 L of petroleum ether was slowly added with stirring. After the addition was completed, the mixture was cooled to -5 – 10 ° C and stirred for 1 hour, and filtered to give a beige solid. Drying under reduced pressure at 30-35 ° C gave a dry product of about 250 g, yield 71.4%.

Example 7: Preparation of product 7

To the reaction flask was added 2.5 L of ethyl acetate and 250 g of product 6. 30 g of DMAP was added with stirring. 400 ml of acetic anhydride was added dropwise at a temperature of 15 ° C, and the reaction was stirred at a temperature of 20-25 ° C for 3 hours. The reaction was monitored by TLC, and a hydrochloric acid solution was added at a temperature of 15 ° C to separate the layers. The organic layer was washed with saturated hydrochloric acid and saturated sodium hydrogen sulfate. The organic phase was separated, dried and filtered to give 371 g, m.

Example 8: Preparation of product 9

To the reaction flask was added 220 g of product 8, 2.2 L of purified water. Under stirring, 500 g of a product 7 in 5 L of methanol and 150 g of anhydrous lithium perchlorate dissolved in 1.5 L of water were added. After the addition was completed, the reaction was stirred at a temperature of 30 to 32 ° C for 20 hours. The reaction is completed and filtered. The filtrate was temperature-controlled at 15 ° C to 20 ° C, and 1 L of 30% hydrogen peroxide was added dropwise. After the addition, the reaction was kept at 20 ° C for 6 hours, and the solid was precipitated, filtered, and dried by blasting at 35-40 ° C to obtain 215 g of a brownish yellow product 9 in a yield of 47%.

Example 9: Preparation of product 10

To the reaction flask, 80 g of product 9, 400 ml of purified water, 300 ml of n-butanol, and 80 ml of diethylamine were added, and the mixture was stirred and heated to 45-50 ° C for 16 hours. After the TLC reaction is completed, the layers are separated, and the aqueous layer is separated to obtain an aqueous solution of the product 10, which is directly reacted in the next step.

Example 10: Preparation of Product I

An aqueous solution of product 10 was added to the autoclave, and 50 ml of triethylamine and 2 g of platinum dioxide were added thereto with stirring. The pressure was evacuated, the hydrogen was replaced three times, the pressure was controlled to 1.5 MPa, and the reaction was stirred at 35 ° C for 20 hours. After filtration, the filtrate was added to 30 ml of n-butanol for 5 min, and the mixture was allowed to stand to give an aqueous solution of product I. 200 ml of concentrated hydrochloric acid was added dropwise at a temperature of 10 ° C, and the aqueous solution was concentrated under reduced pressure to dryness. 500 ml of 95% ethanol was added to the crude product, and the mixture was heated to 55-60 ° C for 1 hour, then cooled to 35 ° C for 2 hours, filtered, and the filter cake was dried to give the product I35 g.

Example 11: Preparation of product 9′

To the reaction flask was added 1.25 L of ethyl acetate and 125 g of product 9. 15 g of DMAP was added with stirring. 200 ml of acetic anhydride was added dropwise at a temperature of 15 ° C, and the reaction was stirred at a temperature of 20-25 ° C for 3 hours. The reaction was monitored by TLC, and a hydrochloric acid solution was added at a temperature of 15 ° C to separate the layers. The organic layer was washed with saturated hydrochloric acid and saturated sodium hydrogen sulfate. The organic phase was separated, dried and concentrated to give 12,5 g of oil.

Example 12: Preparation of product 10

The product 9′ prepared in Example 11 was added to the reaction flask, 600 ml of purified water, 450 ml of n-butanol, and 120 ml of diethylamine were added, and the mixture was stirred and heated to 45-50 ° C for 16 hours. After the TLC reaction is completed, the layers are separated, and the aqueous layer is separated to obtain an aqueous solution of the product 10, which is directly reacted in the next step.

Example 13: Preparation of Product I

An aqueous solution of the product 10 prepared in Example 12 was added to the hydrogenation vessel, and 80 ml of triethylamine, 3 g of platinum dioxide was added thereto with stirring, and vacuum was applied thereto, and the pressure was controlled to 1.5 MPa, and the reaction was stirred at 35 ° C for 20 hours. After filtration, the filtrate was added to 45 ml of n-butanol for 5 min, and the mixture was allowed to stand to give an aqueous solution of product I. After cooling at 10 ° C, 300 ml of concentrated hydrochloric acid was added dropwise, and the aqueous solution was concentrated under reduced pressure to dryness. 750 ml of 95% ethanol was added to the crude product, and the mixture was heated to 55-60 ° C for 1 hour, then cooled to 35 ° C for 2 hours, filtered, and the filter cake was dried to give the product I 48.9 g.

///////////

https://patents.google.com/patent/US9365573B2/en

Sapropterin dihydrochloride, chemical name (6R)-2-amino-6-[(1R,2S)-1,2-dihydroxypropyl]-5,6,7,8-tetrahydro-4(1H)-pteridinone dihydrochloride, molecular formula C₉H₁₅N₅O₃.2HCl, and CAS registry number 69056-38-28, is a synthetic product of tetrahydrobiopterin (BH₄) dihydrochloride. BH₄is a cofactor of Phenylalanine Hydroxylase (PAH). Tyrosine is acquired from Phenylalanine (Phe) through hydroxylation under the action of PAH which is low in activity or even inactive in PKU patients, while BH₄is able to activate PAH, promote normal oxidative metabolism of Phe in the bodies of the patients, and reduce the Phe levels in the bodies of some patients. On Dec. 16, 2007, the sapropterin dihydrochloride tablets produced by BioMarin Pharmaceutical Inc. in USA were approved by the Food and Drug Administration (FDA) for marketing for treatment of PKU. Because of the effective activity of sapropterin dihydrochloride, it is extremely necessary to select a route applicable to industrial production with high product purity.

At present, BH₄is mainly synthesized by the following methods reported in literatures:

1. Preparation using 4-hydroxy-2,5,6-triaminopyrimidine (TAP) and 5-deoxy-L-arabinose as raw materials, please see literature E. L. Patterson et al., J. Am. Chem. Soc. 78, 5868(1956).

2. Preparation using TAP and 5-deoxy-L-arabinose phenylhydrazone as raw materials, please see literature Matsuura et al., Bull. Chem. Soc. Jpn., 48,3767 (1975);

3. Preparation by reaction of raw materials hydroxyl-protected TAP and 4-acetyl-2,3-epoxypentanal through oxidation of iodine and a dehydroxylation protecting group, please see literature Matsuura et al., Chemistry of Organic Synthesis, MI/g. 46. No. 6, P570(1988).

These traditional methods for preparing BH4 have the following major disadvantages: raw materials are expensive, arabinose which can be hardly acquired is used as a carbon atom radical for asymmetric synthesis; there are multiple steps in reactions with low yield, and low product purity, 5-deoxy-L-arabinose is easily degraded in a reaction solution, and products of the synthesis routes above have low stereoselectivity. To sum up, the traditional synthesis methods are not applicable to mass industrial production. Therefore, a synthesis route, which is applicable to industrial production with high product purity, high yield and high stereoselectivity, needs to be searched urgently.

tep 10: add 0.7 kg (0.05 g/g) of palladium 5% on carbon in the presence of the methanol solution containing 1.5 kg of acetylamino-7,8-dihydropteridine

obtained in Step 9, introduce hydrogen until the pressure of the reaction kettle is 0.8±0.05 MPa, control the temperature of the system at 25±5° C. and the pressure at 0.8±0.05 MPa, react for 82 hours, after reacting thoroughly, perform quenching in 31.9 kg (9 eq) of dilute hydrochloric acid having a concentration of 15%, and perform suction filtration and drying to the system to obtain a target product, i.e. a crude product of sapropterin dihydrochloride

recrystallize and purify the crude product by 29 L (20 ml/g) of methanol at 35±5° C. to obtain 0.8 kg of a pure product, with a yield of 45%, a purity of 98.3% and an enantiomeric excess of 99.1%.

Embodiment 5: main raw material:

and X═O

Step 1: add 836 kg (0.3 eq) of a tetrahydrofuran solution contaning a samarium catalyst having a concentration of 4%, 29.2 kg (0.3 eq) of (R)-(+)-1,1′-bi-2-naphthol, 28.4 kg (0.3 eq) of triphenylphosphine oxide, and 600 kg (10 kg/kg) of a 4 A molecular sieve to a 3000 L reaction kettle, after stirring uniformly, control the system temperature at 20±5° C., add 117.4 kg (2 eq) of meta-chloroperoxybenzoic acid, add 60 kg (1 eq) of benzyl crotonate

to the system after adding meta-chloroperoxybenzoic acid, react for 32 hours while preserving the temperature, add 19.6 kg (0.3 eq) of citric acid to the system to stop the reaction, and perform centrifugation, concentration and rectification to the system to obtain 40.5 kg of (2S,3R)-2,3-epoxy-benzyl butyrate

with a yield of 62%;

Step 2: add 36.8 kg (3 eq) of acetone, and 5.4 kg (0.6 eq) of lithium chloride to a 500 L enamel vessel, control the temperature at 15±5° C., add 40.5 kg (1 eq) of (2S,3R)-2,3-epoxy-benzyl butyrate

react for 7 hours while preserving the temperature, add 422 kg (2 eq) of a potassium bicarbonate aqueous solution having a concentration of 10%, and perform liquid separation, extraction and concentration to the system to obtain 44 kg of (4S,5S)-2,2,5-trimethyl-acetonide-benzyl butyrate

with a yield of 82%;

Step 3: add 352 L (8 ml/g) of ethanol, and 44 kg (1 eq) of (4S,5S)-2,2,5-trimethyl-2,3-acetonide-benzyl butyrate

to a 1000 L reaction kettle, increase the temperature to 37±5° C., add 4.8 kg (1.5 eq) of pure water and 53.2 kg (1.5 eq) of a sodium hydroxide aqueous solution having a concentration of 20%, react for 6 hours while preserving the temperature, perform centrifugation, dissolve a filter cake in 352 L (8 ml/g) of ethanol, add 71.0 kg (3 eq) of L-α-amphetamine, preserve the temperature at 22±5° C. for 4 hours, and perform centrifugation and drying to obtain 32.4 kg of (4S,5S)-2,2,5-trimethyl-2,3-acetonide-phenylacetylamino butyrate

with a yield of 62%;

Step 4: add 48 L (6 ml/g) of 1,4-dioxane, 8 kg (1 eq) of (4S,5S)-2,2,5-trimethyl-2,3-acetonide-phenylacetylamino butyrate

to a 72 L reaction bottle, then add a dilute sulphuric acid aqueous solution having a concentration of 10% to the system to regulate the pH at 2.5±0.5, control the temperature at −5±5° C., react for 1 hour, perform liquid separation to obtain an organic phase, add 7.0 kg of (2.0 eq) N,N-diisopropylethylamine to the organic phase, and concentrate the system to obtain 4.1 kg of (4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-methanoic acid

with a yield of 93.5%;

Step 5: add 49 L (12 ml/g) of 2-methyltetrahydrofuran, 4.1 kg of 1,3-dioxolan-4-methanoic acid

and 13.1 kg (4 eq) of N,N-diisopropylethylamine to a 100 L reaction bottle, reduce the temperature to −22±5° C., add 5.5 kg (2.0 eq) of ethyl chloroformate, react for 1.8 hours while preserving the temperature, introduce a diazomethane gas for 1.8 hours, add 18.5 kg (4.5 eq) of a hydrochloride ethanol solution having a concentration of 20%, react for 1.8 hours, add potassium bicarbonate to regulate the pH value to 8.5±0.5, and perform extraction, liquid separation and concentration to obtain 4.1 kg of (4S,5S)-2,2,5-trimethyl-5-chloroacetyl-1,3-dioxolane

with a yield of 83.7%;

Step 6: add 49 L (12 ml/g) of acetone, 4.1 kg of (4S,5S)-2,2,5-trimethyl-5-chloroacetyl-1,3-dioxolane

3.4 kg (2.5 eq) of sodium azide, and 1.8 kg (0.5 eq) of potassium iodide to a 72 L bottle, react the system for 26 hours while preserving the temperature at 34±5° C., perform filtering and concentration to obtain an acetone solution containing 3.9 kg of (4S,5S)-2,2,5-trimethyl-5-(2-azidoacetyl)-1,3-dioxolane

with a yield of 91.5%;

Step 7: add 46.4 L (12 ml/g) of methyl tert-butyl ether and 1.2 kg (0.3 g/g) of Raney nickel to a 100 L reaction kettle, introduce hydrogen until the system pressure is 0.8±0.1 MPa, regulate the pH of the system to 3±0.5 with concentrated sulfuric acid, add an acetonitrile solution containing 3.9 kg (1 eq) of (4S,5S)-2,2,5-trimethyl-5-(2-azidoacetyl)-1,3-dioxolane

react at 27±5° C. for 8.5 hours, perform suction filtration and concentration to obtain 2.3 kg of (3S,4S)-1-amino-3,4-dihydroxy-2-pentanone

with a yield of 89%;

Step 8: add 23 L (10 ml/g) of propanol, 6.9 L (3 ml/g) of pure water, 0.9 kg of (0.3 eq) of potassium iodide, 4.8 kg (1.2 eq) of compound A (2-amino-6-chloro-5-nitro-3H-pyrimidin-4-one), 2.3 kg (1 eq) of (3S,4S)-1-amino-3,4-dihydroxy-2-pentanone

and 10.5 kg (6 eq) of diisopropylamine to a 50 L reaction bottle, react the system for 7 hours while preserving the temperature at 72±5° C., then add a potassium dihydrogen phosphate-dipotassium phosphate aqueous solution to regulate the pH of the system to 7.5±0.5; and filter the system to obtain 2.5 kg of 2-acetylamino-5-nitro-6-((3S,4S)-3,3-dihydroxy-2-oxo-pentylamino)-pyrimidin-4-one

with a yield of 44%;

Step 9: add 1.25 kg (1 eq) of 2-acetylamino-5-nitro-6((3S,4S)-3,3-dihydroxy-2-oxo-pentylamino)-pyrimidin-4-one

50 L (40 ml/g) of ethanol and 0.5 kg (0.4 g/g) of 10% palladium on carbon to a 100 L autoclave, introduce hydrogen until the reaction system pressure is 0.8±0.05 MPa, control the temperature of the system at 27±5° C. and the pressure at 0.8±0.05 MPa, react for 24 hours, filter the system, and regulate the pH to 11±0.5 to obtain an ethanol solution containing 1.1 kg of acetylamino-7,8-dihydropteridine

which is used directly in the next step;

Step 10: add 0.44 kg (0.4 g/g) of palladium 10% on carbon in the presence of the ethanol solution containing 1.1 kg of acetylamino-7,8-dihydropteridine

obtained in Step 9, introduce hydrogen until the pressure of the reaction kettle is 0.8±0.05 MPa, control the temperature of the system at 25±5° C. and the pressure at 0.8±0.05 MPa, react for 80 hours, after reacting thoroughly, perform quenching in 20 kg (8 eq) of dilute hydrochloric acid having a concentration of 15%, and perform suction filtration and drying to the system to obtain a target product, i.e. a crude product of sapropterin dihydrochloride

recrystallize and purify the crude product by 21.4 L (20 ml/g) of ethanol at 35±5° C. to obtain 0.4 kg of a pure product, with a yield of 46.2%, a purity of 98.5% and an enantiomeric excess of 99.2%.

Embodiment 6: main raw material:

and X═N

Step 1: add 522 kg (0.05 eq) of a tetrahydrofuran solution containing a samarium catalyst having a concentration of 2%, 9.1 kg (0.05 eq) of (R)-(+)-1,1′-bi-2-naphthol, 8.9 kg (0.05 eq) of triphenylphosphine oxide, and 567 kg (7 kg/kg) of a 4 A molecular sieve to a 3000 L reaction kettle, after stirring uniformly, control the system temperature at 8±5° C., add 57.4 kg (0.eq) of a tert-butyl hydroperoxide toluene solution having a concentration of 50%, add 81.1 kg (1 eq) of (E)-N-isopropylbut-2-enamide

to the system after adding the tert-butyl hydroperoxide toluene solution, react for 34 hours while preserving the temperature, add 6.1 kg (0.05 eq) of citric acid to the system to stop the reaction, and perform centrifugation, concentration and rectification to the system to obtain 56.1 kg of (2S,3R)-2,3-epoxy-diisopropylamido butyrate

with a yield of 61.5%;

Step 2: add 11.4 kg (0.5 eq) of acetone, and 8.8 kg (0.1 eq) of zinc bromide to a 500 L enamel vessel, control the temperature at 20±5° C., add 56.1 kg (1 eq) of (2S,3R)-2,3-epoxy-diisopropylamido butyrate

react for 8.5 hours while preserving the temperature, add 329 kg (2 eq) of a sodium bicarbonate aqueous solution having a concentration of 10%, and perform liquid separation, extraction and concentration to the system to obtain 64.7 kg of (4S,5S)-2,2,5-trimethyl-2,3-acetonide-diisopropylamido butyrate

with a yield of 82%;

Step 3: add 259 L (4 ml/g) of tetrahydrofuran, and 64.7 kg (1 eq) of (4S,5S)-2,2,5-trimethyl-2,3-acetonide-diisopropylamido butyrate

to a 1000 L reaction kettle, increase the temperature to 27±5° C., add 2.9 kg (0.5 eq) of pure water and 29.9 kg (0.5 eq) of a methanol solution of sodium methoxide having a concentration of 29.9%, react for 4 hours while preserving the temperature, perform centrifugation, dissolve a filter cake in 194 L (3 ml/g) of tetrahydrofuran, add 39 kg (1 eq) of L-α-phenylethylamine, preserve the temperature at 18±5° C. for 3.5 hours, and perform centrifugation and drying to obtain 54.3 kg of 1-phenyltehanamine (4S,5S)-2,2,5-trimethyl-1,3-dioxolane-4-carboxylate

with a yield of 60%;

Step 4: add 30 L (3 ml/g) of 2-methyltetrahydrofuran, 10 kg (1 eq) of 1-phenyltehanamine (4S,5S)-2,2,5-trimethyl-1,3-dioxolane-4-carboxylate

to a 72 L reaction bottle, then add a dilute phosphoric acid aqueous solution having a concentration of 10% to the system to regulate the pH at 1.5±0.5, control the temperature at −5±5° C., react for 1 hour, perform liquid separation to obtain an organic phase, add 3.7 kg of (0.8 eq) N,N-diisopropylethylamine to the organic phase, and concentrate the system to obtain 5.3 kg of (4S,5S)-2,2,5-trimethyl-1,3-dioxolan-4-methanoic acid

with a yield of 92.5%;

Step 5: add 42 L (8 ml/g) of 1,4-dioxane, 5.3 kg of 1,3-dioxolan-4-methanoic acid

and 8.5 kg (2 eq) of N,N-diisopropylethylamine to a 100 L reaction bottle, reduce the temperature to −10±5° C., add 4 kg (21.0 eq) of propyl chloroformate, react for 2 hours while preserving the temperature, introduce a diazomethane gas for 2 hours, add 12 kg (2 eq) of a hydrochloride ethanol solution having a concentration of 20%, react for 2 hours, add sodium hydroxide to regulate the pH value to 7.5±0.5, and perform extraction, liquid separation and concentration to obtain 5.1 kg of (4S,5S)-2,2,5-trimethyl-5-chloroacetyl-1,3-dioxolane

with a yield of 81%; Step 6: add 41 L (8 ml/g) of tetrahydrofuran, 5.1 kg of (4S,5S)-2,2,5-trimethyl-5-chloroacetyl-1,3-dioxolane

3.1 kg (1 eq) of azidotrimethylsilane, and 0.5 kg (0.1 eq) of sodium iodide to a 72 L bottle, react the system for 30 hours while preserving the temperature at 12±5° C., perform filtering and concentration to obtain an acetone solution containing 4.6 kg of (4S,5S)-2,2,5-trimethyl-5-(2-azidoacetyl)-1,3-dioxolane

with a yield of 87.5%;

Step 7: add 28 L (6 ml/g) of 1,4-dioxane and 0.23 kg (0.05 g/g) of palladium 10% on carbon to a 50 L reaction kettle, introduce hydrogen until the system pressure is 0.8±0.1 MPa, regulate the pH of the system to 3±0.5 with acetic acid, add an acetonitrile solution containing 4.6 kg (1 eq) of (4S,5S)-2,2,5-trimethyl-5-(2-azidoacetyl)-1,3-dioxolane

react at 27±5° C. for 8.5 hours, react for 8.5 hours, perform suction filtration and concentration to obtain 2.7 kg of (3S,4S)-1-amino-3,4-dihydroxy-2-pentanone

with a yield of 87.7%;

Step 8: add 16.3 L (6 ml/g) of isopropanol, 2.7 L (1 g/g) of pure water, 0.4 kg of (0.1 eq) of sodium iodide, 4.8 kg (1.0 eq) of compound A (2-amino-6-chloro-5-nitro-3H-pyrimidin-4-one), 2.7 kg (1 eq) of (3S,4S)-1-amino-3,4-dihydroxy-2-pentanone

and 8.7 kg (4 eq) of sodium carbonate to a 50 L reaction bottle, react the system for 7 hours while preserving the temperature at 45±5° C., then add an ammonium formate-ammonia aqueous solution to regulate the pH of the system to 6.5±0.5; and filter the system to obtain 2.85 kg of 2-acetylamino-5-nitro-6((3S,4S)-3,3-dihydroxy-2-oxo-pentylamino)-pyrimidin-4-one

with a yield of 42.5%;

Step 9: add 2 kg (1 eq) of 2-acetylamino-5-nitro-6-((3S,4S)-3,3-dihydroxy-2-oxo-pentylamino)-pyrimidin-4-one

60 L (30 ml/g) of ethanol and 0.2 kg (0.1 g/g) of platinum dioxide to a 100 L autoclave, introduce hydrogen until the reaction system pressure is 0.6±0.05 MPa, control the temperature of the system at 20±5° C. and the pressure at 0.6±0.05 MPa, react for 20 hours, filter the system, and regulate the pH to 11±0.5 to obtain an ethanol solution containing 1.7 kg of acetylamino-7,8-dihydropteridine

which is used directly in the next step;

Step 10: add 0.2 kg (0.1 g/g) of platinum dioxide in the presence of the ethanol solution containing 1.7 kg of acetylamino-7,8-dihydropteridine

obtained in Step 9, introduce hydrogen until the pressure of the reaction kettle is 0.6±0.05 MPa, control the temperature of the system at 15±5° C. and the pressure at 0.6±0.05 MPa, react for 75 hours, after reacting thoroughly, perform quenching in 30 kg (5 eq) of dilute hydrochloric acid having a concentration of 10%, and perform suction filtration and drying to the system to obtain a target product, i.e. a crude product of sapropterin dihydrochloride

recrystallize and purify the crude product by 17 L (10 ml/g) of butanone at 15±5° C. to obtain 0.6 kg of a pure product, with a yield of 43%, a purity of 98.4% and an enantiomeric excess of 98.9%.

Thus, it can be seen that synthesis of a sapropterin dihydrochloride compound and an intermediate thereof disclosed in a method of the present disclosure can obtain a target product with a high purity, a high enantiomeric excess, and a high yield. The synthesis method uses readily-available raw materials, significantly reduces a synthesis route of sapropterin dihydrochloride. The technological conditions are stable, and there is less pollution in the whole operation process, hence providing an effective scheme for mass industrial production of sapropterin dihydrochloride.

The above are only preferred embodiments of the present disclosure and should not be used to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent replacements, improvements and the like within the spirit and principle of the present disclosure shall fall within the scope of protection of the present disclosure.

///////

USA

Patent No.	Patent Type	Assignee	Patent Expiry (Pediatric exclusivity)	Estimated Expiry	Status
US 4,713,454	Process	Shiratori Pharmaceutical Co., Ltd. (Narashino, JP) Suntory Limited (Osaka, JP)	NA	23-JAN-06	Expired

References

^ Jump up to:^a ^b “Sapropterin (Kuvan) Use During Pregnancy”. Drugs.com. 17 May 2019. Retrieved 4 March 2020.
^ “Sapropterin”. Drugs.com. 28 February 2020. Retrieved 4 March 2020.
^ “International Non-proprietary Names for Pharmaceutical Substances (INN)”. Fimea. Retrieved 4 March 2020.
^ Jump up to:^a ^b Kappock TJ, Caradonna JP (November 1996). “Pterin-Dependent Amino Acid Hydroxylases”. Chemical Reviews. 96 (7): 2659–2756. doi:10.1021/CR9402034. PMID 11848840.
^ Całka J (2006). “The role of nitric oxide in the hypothalamic control of LHRH and oxytocin release, sexual behavior and aging of the LHRH and oxytocin neurons”. Folia Histochemica et Cytobiologica. 44 (1): 3–12. PMID 16584085.
^ Bhagavan, N. V. (2015). Essentials of Medical Biochemistry With Clinical Cases, 2nd Edition. USA: Elsevier. p. 256. ISBN 978-0-12-416687-5.
^ Schaub J, Däumling S, Curtius HC, Niederwieser A, Bartholomé K, Viscontini M, et al. (August 1978). “Tetrahydrobiopterin therapy of atypical phenylketonuria due to defective dihydrobiopterin biosynthesis”. Archives of Disease in Childhood. 53 (8): 674–6. doi:10.1136/adc.53.8.674. PMC 1545051. PMID 708106.
^ Jump up to:^a ^b “Kuvan- sapropterin dihydrochloride tablet Kuvan- sapropterin dihydrochloride powder, for solution Kuvan- sapropterin dihydrochloride powder, for solution”. DailyMed. 13 December 2019. Retrieved 4 March 2020.
^ Jump up to:^a ^b ^c “Kuvan EPAR”. European Medicines Agency (EMA). 4 March 2020. Retrieved 4 March 2020.
^ “Drug Approval Package: Kuvan (Sapropterin Dihydrochloride) NDA #022181”. U.S. Food and Drug Administration (FDA). 24 March 2008. Retrieved 4 March 2020. Lay summary (PDF).
^ Jump up to:^a ^b ^c ^d ^e “Kuvan (sapropterin dihydrochloride) Tablets and Powder for Oral Solution for PKU”. BioMarin. Retrieved 4 March 2020.
^ “Drug Approval Package: Kuvan Powder for Oral Solution (Sapropterin Dihydrochloride) NDA #205065”. U.S. Food and Drug Administration (FDA). 28 February 2014. Retrieved 4 March 2020. Lay summary (PDF).
^ Herper M (28 July 2016). “How Focusing On Obscure Diseases Made BioMarin A $15 Billion Company”. Forbes. Retrieved 9 October 2017.
^ “BioMarin Announces Kuvan (sapropterin dihydrochloride) Patent Challenge Settlement”. BioMarin Pharmaceutical Inc. 13 April 2017. Retrieved 9 October 2017 – via PR Newswire.
^ “Tetrahydrobiopterin Deficiency”. National Organization for Rare Disorders (NORD). Retrieved 9 October 2017.
^ “What are common treatments for phenylketonuria (PKU)?”. NICHD. 23 August 2013. Retrieved 12 September 2016.
^ Camp KM, Parisi MA, Acosta PB, Berry GT, Bilder DA, Blau N, et al. (June 2014). “Phenylketonuria Scientific Review Conference: state of the science and future research needs”. Molecular Genetics and Metabolism. 112 (2): 87–122. doi:10.1016/j.ymgme.2014.02.013. PMID 24667081.
^ Jump up to:^a ^b Haberfeld, H, ed. (1 March 2017). Austria-Codex (in German). Vienna: Österreichischer Apothekerverlag. Kuvan 100 mg-Tabletten.
^ “Genetics Home Reference: GCH1”. National Institutes of Health.
^ Wu G, Meininger CJ (2009). “Nitric oxide and vascular insulin resistance”. BioFactors. 35(1): 21–7. doi:10.1002/biof.3. PMID 19319842. S2CID 29828656.
^ Kaufman S (February 1958). “A new cofactor required for the enzymatic conversion of phenylalanine to tyrosine”. The Journal of Biological Chemistry. 230 (2): 931–9. PMID 13525410.
^ Thöny B, Auerbach G, Blau N (April 2000). “Tetrahydrobiopterin biosynthesis, regeneration and functions”. The Biochemical Journal. 347 Pt 1: 1–16. doi:10.1042/0264-6021:3470001. PMC 1220924. PMID 10727395.
^ Kuzkaya N, Weissmann N, Harrison DG, Dikalov S (June 2003). “Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase”. The Journal of Biological Chemistry. 278 (25): 22546–54. doi:10.1074/jbc.M302227200. PMID 12692136.
^ Muller-Delp JM (November 2009). “Ascorbic acid and tetrahydrobiopterin: looking beyond nitric oxide bioavailability”. Cardiovascular Research. 84 (2): 178–9. doi:10.1093/cvr/cvp307. PMID 19744948.
^ Gori T, Burstein JM, Ahmed S, Miner SE, Al-Hesayen A, Kelly S, Parker JD (September 2001). “Folic acid prevents nitroglycerin-induced nitric oxide synthase dysfunction and nitrate tolerance: a human in vivo study”. Circulation. 104 (10): 1119–23. doi:10.1161/hc3501.095358. PMID 11535566.
^ “Search results for Kuvan”. ClinicalTrials.gov. U.S. National Library of Medicine.
^ “BioMarin Initiates Phase 3b Study to Evaluate the Effects of Kuvan on Neurophychiatric Symptoms in Subjects with PKU”. BioMarin Pharmaceutical Inc. 17 August 2010.
^ Burton B, Grant M, Feigenbaum A, Singh R, Hendren R, Siriwardena K, et al. (March 2015). “A randomized, placebo-controlled, double-blind study of sapropterin to treat ADHD symptoms and executive function impairment in children and adults with sapropterin-responsive phenylketonuria”. Molecular Genetics and Metabolism. 114 (3): 415–24. doi:10.1016/j.ymgme.2014.11.011. PMID 25533024.
^ Fernell E, Watanabe Y, Adolfsson I, Tani Y, Bergström M, Hartvig P, et al. (May 1997). “Possible effects of tetrahydrobiopterin treatment in six children with autism–clinical and positron emission tomography data: a pilot study”. Developmental Medicine and Child Neurology. 39 (5): 313–8. doi:10.1111/j.1469-8749.1997.tb07437.x. PMID 9236697. S2CID 12761124.
^ Frye RE, Huffman LC, Elliott GR (July 2010). “Tetrahydrobiopterin as a novel therapeutic intervention for autism”. Neurotherapeutics. 7 (3): 241–9. doi:10.1016/j.nurt.2010.05.004. PMC 2908599. PMID 20643376.
^ Channon KM (November 2004). “Tetrahydrobiopterin: regulator of endothelial nitric oxide synthase in vascular disease”. Trends in Cardiovascular Medicine. 14 (8): 323–7. doi:10.1016/j.tcm.2004.10.003. PMID 15596110.
^ Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, et al. (September 2003). “Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression”. The Journal of Clinical Investigation. 112 (5): 725–35. doi:10.1172/JCI17786. PMC 182196. PMID 12952921.
^ Khoo JP, Zhao L, Alp NJ, Bendall JK, Nicoli T, Rockett K, et al. (April 2005). “Pivotal role for endothelial tetrahydrobiopterin in pulmonary hypertension”. Circulation. 111 (16): 2126–33. doi:10.1161/01.CIR.0000162470.26840.89. PMID 15824200.
^ Cunnington C, Van Assche T, Shirodaria C, Kylintireas I, Lindsay AC, Lee JM, et al. (March 2012). “Systemic and vascular oxidation limits the efficacy of oral tetrahydrobiopterin treatment in patients with coronary artery disease”. Circulation. 125 (11): 1356–66. doi:10.1161/CIRCULATIONAHA.111.038919. PMC 5238935. PMID 22315282.
^ Romanowicz J, Leonetti C, Dhari Z, Korotcova L, Ramachandra SD, Saric N, et al. (August 2019). “Treatment With Tetrahydrobiopterin Improves White Matter Maturation in a Mouse Model for Prenatal Hypoxia in Congenital Heart Disease”. Journal of the American Heart Association. 8 (15): e012711. doi:10.1161/JAHA.119.012711. PMC 6761654. PMID 31331224.
^ Kraft VA, Bezjian CT, Pfeiffer S, Ringelstetter L, Müller C, Zandkarimi F, et al. (January 2020). “GTP Cyclohydrolase 1/Tetrahydrobiopterin Counteract Ferroptosis through Lipid Remodeling”. ACS Central Science. 6 (1): 41–53. doi:10.1021/acscentsci.9b01063. PMC 6978838. PMID 31989025.

External links

“Sapropterin”. Drug Information Portal. U.S. National Library of Medicine.

Tetrahydrobiopterin
INN: sapropterin
Clinical data

Trade names	Kuvan, Biopten
Other names	Sapropterin hydrochloride (JAN JP), Sapropterin dihydrochloride (USAN US)
AHFS/Drugs.com	Monograph
MedlinePlus	a608020
License data	EU EMA: by INN US DailyMed: Sapropterin US FDA: Sapropterin
Pregnancy category	AU: B1^[1] US: C (Risk not ruled out)^[1]
Routes of administration	By mouth
ATC code	A16AX07 (WHO)
Legal status
Legal status	AU: S4 (Prescription only) CA: ℞-only US: ℞-only In general: ℞ (Prescription only)
Pharmacokinetic data
Elimination half-life	4 hours (healthy adults) 6–7 hours (PKU patients)
Identifiers
IUPAC name [show]
CAS Number	62989-33-7
PubChem CID	135398654
IUPHAR/BPS	5276
DrugBank	DB00360
ChemSpider	40270
UNII	EGX657432I
KEGG	D08505 C00272
ChEBI	CHEBI:59560
ChEMBL	ChEMBL1201774
PDB ligand	H4B (PDBe, RCSB PDB)
CompTox Dashboard (EPA)	DTXSID1041138
ECHA InfoCard	100.164.121
Chemical and physical data
Formula	C₉H₁₅N₅O₃
Molar mass	241.251 g·mol⁻¹
3D model (JSmol)	Interactive image
SMILES [hide] CC(C(C1CNC2=C(N1)C(=O)N=C(N2)N)O)O
InChI [hide] InChI=1S/C9H15N5O3/c1-3(15)6(16)4-2-11-7-5(12-4)8(17)14-9(10)13-7/h3-4,6,12,15-16H,2H2,1H3,(H4,10,11,13,14,17)/t3-,4+,6-/m0/s1 Key:FNKQXYHWGSIFBK-RPDRRWSUSA-N

////////Sapropterin, сапроптерин , سابروبتيرين , 沙丙蝶呤 , Tetrahydrobiopterin,

Sapropterin Tablets - FDA prescribing information, side effects and uses

CILOFEXOR

September 5, 2020 4:53 am / Leave a comment

Cilofexor Chemical Structure

CILOFEXOR

C₂₈H₂₂Cl₃N₃O₅ ,

586.8 g/mol

1418274-28-8

GS-9674, Cilofexor (GS(c)\9674)

UNII-YUN2306954

YUN2306954

2-[3-[2-chloro-4-[[5-cyclopropyl-3-(2,6-dichlorophenyl)-1,2-oxazol-4-yl]methoxy]phenyl]-3-hydroxyazetidin-1-yl]pyridine-4-carboxylic acid

Cilofexor is under investigation in clinical trial NCT02943447 (Safety, Tolerability, and Efficacy of Cilofexor in Adults With Primary Biliary Cholangitis Without Cirrhosis).

Cilofexor (GS-9674) is a potent, selective and orally active nonsteroidal FXR agonist with an EC₅₀ of 43 nM. Cilofexor has anti-inflammatory and antifibrotic effects. Cilofexor has the potential for primary sclerosing cholangitis (PSC) and nonalcoholic steatohepatitis (NASH) research.

Gilead , following a drug acquisition from Phenex , is developing cilofexor tromethamine (formerly GS-9674), the lead from a program of farnesoid X receptor (FXR; bile acid receptor) agonists, for the potential oral treatment of non-alcoholic steatohepatitis (NASH), primary biliary cholangitis/cirrhosis (PBC) and primary sclerosing cholangitis. In March 2019, a phase III trial was initiated for PSC; at that time, the trial was expected to complete in August 2022.

PATENT

Product case WO2013007387 , expiry EU in 2032 and in the US in 2034.

https://patents.google.com/patent/WO2013007387A1/en

Figure imgf000039_0001

PATENT

WO2020150136 claiming 2,6-dichloro-4-fluorophenyl compounds.

PATENT

https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020172075&tab=PCTDESCRIPTION&_cid=P20-KEP1ZU-65392-1

WO-2020172075

Novel crystalline forms of cilofexor as FXR agonists useful for treating nonalcoholic steatohepatitis. Gilead , following a drug acquisition from Phenex , is developing cilofexor tromethamine (formerly GS-9674), the lead from a program of farnesoid X receptor (FXR; bile acid receptor) agonists, for the potential oral treatment of non-alcoholic steatohepatitis (NASH), primary biliary cholangitis/cirrhosis (PBC) and primary sclerosing cholangitis. In March 2019, a phase III trial was initiated for PSC; at that time, the trial was expected to complete in August 2022. Family members of the cilofexor product case WO2013007387 , expire in the EU in 2032 and in the US in 2034.

solid forms of compounds that bind to the NR1H4 receptor (FXR) and act as agonists or modulators of FXR. The disclosure further relates to the use of the solid forms of such compounds for the treatment and/or prophylaxis of diseases and/or conditions through binding of said nuclear receptor by said compounds.

[0004] Compounds that bind to the NR1H4 receptor (FXR) can act as agonists or modulators of FXR. FXR agonists are useful for the treatment and/or prophylaxis of diseases and conditions through binding of the NR1H4 receptor. One such FXR agonist is the compound of Formula I:

[0005] Although numerous FXR agonists are known, what is desired in the art are physically stable forms of the compound of Formula I, or pharmaceutically acceptable salt thereof, with desired properties such as good physical and chemical stability, good aqueous solubility and good bioavailability. For example, pharmaceutical compositions are desired that address

challenges of stability, variable pharmacodynamics responses, drug-drug interactions, pH effect, food effects, and oral bioavailability.

[0006] Accordingly, there is a need for stable forms of the compound of Formula I with suitable chemical and physical stability for the formulation, therapeutic use, manufacturing, and storage of the compound.

[0007] Moreover, it is desirable to develop a solid form of Formula I that may be useful in the synthesis of Formula I. A solid form, such as a crystalline form of a compound of Formula I may be an intermediate to the synthesis of Formula F A solid form may have properties such as bioavailability, stability, purity, and/or manufacturability at certain conditions that may be suitable for medical or pharmaceutical uses.

Description

IC₅₀ & Target

EC50: 43 nM (FXR)^[1]

In Vivo

Cilofexor (GS-9674; 30 mg/kg; oral gavage; once daily; for 10 weeks; male Wistar rats) treatment significantly increases Fgf15 expression in the ileum and decreased Cyp7a1 in the liver in nonalcoholic steatohepatitis (NASH) rats. Liver fibrosis and hepatic collagen expression are significantly reduced. Cilofexor also significantly reduces hepatic stellate cell (HSC) activation and significantly decreases portal pressure, without affecting systemic hemodynamics^[3].

Animal Model:	Male Wistar rats received a choline-deficient high fat diet (CDHFD)^[3]
Dosage:	30 mg/kg
Administration:	Oral gavage; once daily; for 10 weeks
Result:	Significantly increased Fgf15 expression in the ileum and decreased Cyp7a1 in the liver. Liver fibrosis and hepatic collagen expression were significantly reduced.

Clinical Trial

NCT Number	Sponsor	Condition	Start Date	Phase
NCT02943460	Gilead Sciences	Primary Sclerosing Cholangitis	November 29, 2016	Phase 2
NCT02808312	Gilead Sciences	Nonalcoholic Steatohepatitis (NASH)	July 13, 2016	Phase 1
NCT02781584	Gilead Sciences	Nonalcoholic Steatohepatitis (NASH)\|Nonalcoholic Fatty Liver Disease (NAFLD)	July 13, 2016	Phase 2
NCT02943447	Gilead Sciences	Primary Biliary Cholangitis	December 1, 2016	Phase 2
NCT03987074	Gilead Sciences\|Novo Nordisk A+S	Nonalcoholic Steatohepatitis	July 29, 2019	Phase 2
NCT03890120	Gilead Sciences	Primary Sclerosing Cholangitis	March 27, 2019	Phase 3
NCT02854605	Gilead Sciences	Nonalcoholic Steatohepatitis (NASH)	October 26, 2016	Phase 2
NCT03449446	Gilead Sciences	Nonalcoholic Steatohepatitis	March 21, 2018	Phase 2
NCT02654002	Gilead Sciences	Nonalcoholic Steatohepatitis (NASH)	January 2016	Phase 1

Patent ID	Title	Submitted Date
US2019142814	Novel FXR (NR1H4) binding and activity modulating compounds	2019-01-15
US2019055273	ACYCLIC ANTIVIRALS	2017-03-09
US10220027	FXR (NR1H4) binding and activity modulating compounds	2017-10-13
US10071108	Methods and pharmaceutical compositions for the treatment of hepatitis b virus infection	2018-02-19
US2018000768	INTESTINAL FXR AGONISM ENHANCES GLP-1 SIGNALING TO RESTORE PANCREATIC BETA CELL FUNCTIONS	2017-09-06

Patent ID	Title	Submitted Date	Granted Date
US9820979	NOVEL FXR (NR1H4) BINDING AND ACTIVITY MODULATING COMPOUNDS	2016-12-05
US9539244	NOVEL FXR (NR1H4) BINDING AND ACTIVITY MODULATING COMPOUNDS	2015-08-12	2015-12-03
US9895380	METHODS AND PHARMACEUTICAL COMPOSITIONS FOR THE TREATMENT OF HEPATITIS B VIRUS INFECTION	2014-09-10	2016-08-04
US2017355693	FXR (NR1H4) MODULATING COMPOUNDS	2017-06-12
US2016376279	FXR AGONISTS AND METHODS FOR MAKING AND USING	2016-09-12

Patent ID	Title	Submitted Date	Granted Date
US9139539	NOVEL FXR (NR1H4) BINDING AND ACTIVITY MODULATING COMPOUNDS	2012-07-12	2014-08-07
US2018133203	METHODS OF TREATING LIVER DISEASE	2017-10-27

ClinicalTrials.gov

CTID	Title	Phase	Status	Date
NCT03890120	Safety, Tolerability, and Efficacy of Cilofexor in Non-Cirrhotic Adults With Primary Sclerosing Cholangitis	Phase 3	Recruiting	2020-08-31
NCT02781584	Safety, Tolerability, and Efficacy of Selonsertib, Firsocostat, and Cilofexor in Adults With Nonalcoholic Steatohepatitis (NASH)	Phase 2	Recruiting	2020-08-13
NCT03987074	Safety, Tolerability, and Efficacy of Monotherapy and Combination Regimens in Adults With Nonalcoholic Steatohepatitis (NASH)	Phase 2	Completed	2020-07-29
NCT02943460	Safety, Tolerability, and Efficacy of Cilofexor in Adults With Primary Sclerosing Cholangitis Without Cirrhosis	Phase 2	Completed	2020-06-09
NCT02943447	Safety, Tolerability, and Efficacy of Cilofexor in Adults With Primary Biliary Cholangitis Without Cirrhosis	Phase 2	Completed	2020-02-11

ClinicalTrials.gov

CTID	Title	Phase	Status	Date
NCT03449446	Safety and Efficacy of Selonsertib, Firsocostat, Cilofexor, and Combinations in Participants With Bridging Fibrosis or Compensated Cirrhosis Due to Nonalcoholic Steatohepatitis (NASH)	Phase 2	Completed	2019-12-24
NCT02854605	Evaluating the Safety, Tolerability, and Efficacy of GS-9674 in Participants With Nonalcoholic Steatohepatitis (NASH)	Phase 2	Completed	2019-01-29
NCT02808312	Pharmacokinetics and Pharmacodynamics of GS-9674 in Adults With Normal and Impaired Hepatic Function	Phase 1	Completed	2018-10-30
NCT02654002	Study in Healthy Volunteers to Evaluate the Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of GS-9674, and the Effect of Food on GS-9674 Pharmacokinetics and Pharmacodynamics	Phase 1	Completed	2016-07-27

EU Clinical Trials Register

EudraCT	Title	Phase	Status	Date
2019-000204-14	A Phase 3, Randomized, Double-Blind, Placebo-Controlled Study Evaluating the Safety, Tolerability, and Efficacy of Cilofexor in Non-Cirrhotic Subjects with Primary Sclerosing Cholangitis	Phase 3	Restarted, Ongoing	2019-09-11
2016-002496-10	A Phase 2, Randomized, Double-Blind, Placebo-Controlled Study Evaluating the Safety, Tolerability, and Efficacy of GS-9674 in Subjects with Nonalcoholic Steatohepatitis (NASH)	Phase 2	Completed	2017-02-21
2016-002443-42	A Phase 2, Randomized, Double-Blind, Placebo Controlled Study Evaluating the Safety, Tolerability, and Efficacy of GS-9674 in Subjects with Primary Biliary Cholangitis Without Cirrhosis	Phase 2	Completed	2017-01-09
2016-002442-23	A Phase 2, Randomized, Double-Blind, Placebo Controlled Study Evaluating the Safety, Tolerability, and Efficacy of GS-9674 in Subjects with Primary Sclerosing Cholangitis Without Cirrhosis	Phase 2	Completed	2017-01-09

///////////CILOFEXOR, Cilofexor (GS(c)\9674), GS-9674, phase 3

C1CC1C2=C(C(=NO2)C3=C(C=CC=C3Cl)Cl)COC4=CC(=C(C=C4)C5(CN(C5)C6=NC=CC(=C6)C(=O)O)O)Cl

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