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

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:

E

////////////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-]

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

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

2d

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

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

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).

Further reading

• The non‐target impact of spinosyns on beneficial arthropods
• Spinosad toxicity to pollinators and associated risk

References

1. ^ 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.
2. ^ 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.
3. ^ 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.
4. ^ 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.
5. ^ 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.
6. ^ 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.
7. ^ 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.
8. ^ 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.
9. ^ “Spinosad international brands”. Drugs.com. 3 January 2020. Retrieved 30 January2020.
10. ^ “Spinosad US brands”. Drugs.com. 3 January 2020. Retrieved 30 January 2020.
11. ^ “Spinosad – brand name list from”. Drugs.com. Retrieved 2012-10-20.
12. ^ “UC Davis School of Vet Med”. Vetmed.ucdavis.edu. Retrieved 2012-10-20.
13. ^ “Safer Flea Control | Insects in the City”. Citybugs.tamu.edu. Retrieved 2012-10-20.
14. ^ “Codling Moth and Leafroller Control Using Chemicals” (PDF). Entomology.tfrec.wsu.edu. Retrieved 2012-10-20.
15. ^ 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.
16. ^ 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

//////////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) bothFigure 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

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.

“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

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

PATENT

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

PATENT

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

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

//////////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

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

See also: List of selective estrogen receptor modulators and Benzothiophene

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

1. Grese TA, Dodge JA. Selective Estrogen Receptor Modulators (SERMs). Curr Pharm Des. 1998;4:71-92.
2. Bryant HU, Dere WH. Selective estrogen receptor modulators: an alternative to hormone replacement therapy. Proc Soc Exp Biol Med. 1998;217:45-52.
3. 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.
4. 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.
5. 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

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/B978-0-12-805157-3.00002-8Piperidine-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].

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50. ^ 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.
51. ^ 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.
52. ^ 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.
53. ^ 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.
54. ^ 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.
55. ^ 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.
56. ^ 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.
57. ^ 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.
58. ^ 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.
59. ^ 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.
60. ^ 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.
61. ^ Nordt CA, DiVasta AD (2008). “Gynecomastia in adolescents”. Curr. Opin. Pediatr. 20 (4): 375–82. doi:10.1097/MOP.0b013e328306a07c. PMID 18622190. S2CID 205834072.
62. ^ 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.
63. ^ 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.
64. ^ 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.
65. ^ 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.

Further reading

• Barrett-Connor E (2001). “Raloxifene: risks and benefits”. Ann N Y Acad Sci. 949 (1): 295–303. Bibcode:2001NYASA.949..295B. doi:10.1111/j.1749-6632.2001.tb04036.x. PMID 11795366. S2CID 41412601.
• Heringa M (2003). “Review on raloxifene: profile of a selective estrogen receptor modulator”. Int J Clin Pharmacol Ther. 41 (8): 331–45. doi:10.5414/cpp41331. PMID 12940590.
• Sporn MB, Dowsett SA, Mershon J, Bryant HU (2004). “Role of raloxifene in breast cancer prevention in postmenopausal women: clinical evidence and potential mechanisms of action”. Clin Ther. 26 (6): 830–40. doi:10.1016/s0149-2918(04)90127-0. PMID 15262454.
• Vogel VG (2009). “The NSABP Study of Tamoxifen and Raloxifene (STAR) trial”. Expert Rev Anticancer Ther. 9 (1): 51–60. doi:10.1586/14737140.9.1.51. PMC 2785111. PMID 19105706.
• Wickerham DL, Costantino JP, Vogel VG, Cronin WM, Cecchini RS, Ford LG, Wolmark N (2009). “The use of tamoxifen and raloxifene for the prevention of breast cancer”. Recent Results Cancer Res. Recent Results in Cancer Research. 181: 113–9. doi:10.1007/978-3-540-69297-3_12. ISBN 978-3-540-69296-6. PMC 5110043. PMID 19213563.
• Vogel VG (2011). “Update on raloxifene: role in reducing the risk of invasive breast cancer in postmenopausal women”. Breast Cancer: Targets and Therapy. 3: 127–37. doi:10.2147/BCTT.S11288. PMC 3846694. PMID 24367182.
• Yang ZD, Yu J, Zhang Q (2013). “Effects of raloxifene on cognition, mental health, sleep and sexual function in menopausal women: a systematic review of randomized controlled trials”. Maturitas. 75 (4): 341–8. doi:10.1016/j.maturitas.2013.05.010. PMID 23764354.

External links

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

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

Melatonin

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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139. ^ 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.

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

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

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

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

1. ^ “Citrulline – Compound Summary”. PubChem Compound. USA: National Center for Biotechnology Information. 16 September 2004. Identification. Retrieved 1 May 2012.
2. ^ Jump up to:^a b Banerjee, Aryamitra (2014-01-01), Gupta, Ramesh C. (ed.), “Chapter 15 – Gastrointestinal toxicity biomarkers”, Biomarkers in Toxicology, Boston: Academic Press, pp. 269–277, doi:10.1016/b978-0-12-404630-6.00015-4, ISBN 978-0-12-404630-6, retrieved 2020-11-10
3. ^ Fragkos, Konstantinos C.; Forbes, Alastair (September 2011). “Was citrulline first a laxative substance? The truth about modern citrulline and its isolation” (PDF). Nihon Ishigaku Zasshi. [Journal of Japanese History of Medicine]. 57 (3): 275–292. ISSN 0549-3323. PMID 22397107.
4. ^ Fearon, William Robert (1939). “The Carbamido Diacetyl Reaction: A Test For Citrulline”. Biochemical Journal. 33 (6): 902–907. doi:10.1042/bj0330902. PMC 1264464. PMID 16746990.
5. ^ “Nos2 – Nitric Oxide Synthase”. Uniprot.org. Uniprot Consortium. Retrieved 10 February 2015.
6. ^ 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.
7. ^ 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.
8. ^ 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
9. ^ 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.
10. ^ 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	810 553200 R 9367 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	833637599 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 (pKa)	2.508
Basicity (pKb)	11.489
Thermochemistry
Heat capacity (C)	232.80 J K−1 mol−1
Std molar entropy (So298)	254.4 J K−1 mol−1
Related compounds
Related alkanoic acids	N-Acetylaspartic acidAceglutamideN-Acetylglutamic acidPivagabine
Related compounds	BromisovalCarbromal
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  ,

Ansuvimab-zykl

FDA APPROVED, 12/21/2020, EBANGA

To treat ebola

https://www.fda.gov/drugs/drug-safety-and-availability/fda-approves-treatment-ebola-virus

The U.S. Food and Drug Administration approved Ebanga (Ansuvimab-zykl), a human monoclonal antibody, for the treatment for Zaire ebolavirus (Ebolavirus) infection in adults and children. Ebanga blocks binding of the virus to the cell receptor, preventing its entry into the cell.

Zaire ebolavirus is one of four Ebolavirus species that can cause a potentially fatal human disease. It is transmitted through blood, body fluids, and tissues of infected people or wild animals, and through surfaces and materials, such as bedding and clothing, contaminated with these fluids. Individuals who care for people with the disease, including health care workers who do not use correct infection control precautions, are at the highest risk for infection.

During an Ebola outbreak in the Democratic Republic of the Congo (DRC) in 2018-2019, Ebanga was evaluated in a clinical trial (the PALM trial). The PALM trial was led by the U.S. National Institutes of Health and the DRC’s Institut National de Recherche Biomédicale with contributions from several other international organizations and agencies.

In the PALM trial, the safety and efficacy of Ebanga was evaluated in a multi-center, open-label, randomized controlled trial. 174 participants (120 adults and 54 pediatric patients) with confirmed Ebolavirus infection received Ebanga intravenously as a single 50 mg/kg infusion and 168 participants (135 adults and 33 pediatric patients) received an investigational control. The primary efficacy endpoint was 28-day mortality. The primary analysis population was all patients who were randomized and concurrently eligible to receive either Ebanga or the investigational control during the same time period of the trial. Of the 174 patients who received Ebanga, 35.1% died after 28 days, compared to 49.4% of the 168 patients who received a control.

The most common symptoms experienced while receiving Ebanga include: fever, tachycardia (fast heart rate), diarrhea, vomiting, hypotension (low blood pressure), tachypnea (fast breathing) and chills; however, these are also common symptoms of Ebolavirus infection. Hypersensitivity, including infusion-related events, can occur in patients taking Ebanga, and treatment should be discontinued in the event of a hypersensitivity reaction.

Patients who receive Ebanga should avoid the concurrent administration of a live virus vaccine against Ebolavirus. There is the potential for Ebanga to inhibit replication of a live vaccine virus and possibly reduce the efficacy of this vaccine.

Ebanga was granted an Orphan Drug designation, which provides incentives to assist and encourage drug development for rare diseases. Additionally, the agency granted Ebanga a Breakthrough Therapy designation.

FDA granted the approval to Ridgeback Biotherapeutics, LP.

Ansuvimab, sold under the brand name Ebanga, is a monoclonal antibody medication for the treatment of Zaire ebolavirus (Ebolavirus) infection.^[1][2]

The most common symptoms include fever, tachycardia (fast heart rate), diarrhea, vomiting, hypotension (low blood pressure), tachypnea (fast breathing) and chills; however, these are also common symptoms of Ebolavirus infection.^[1]

Ansuvimab was approved for medical use in the United States in December 2020.^[1][2]

Chemistry

The drug is composed of a single monoclonal antibody (mAb) and was initially isolated from immortalized B-cells that were obtained from a survivor of the 1995 outbreak of Ebola virus disease in Kikwit, Democratic Republic of Congo.^[3] In work supported by the United States National Institutes of Health and the Defense Advanced Projects Agency, the heavy and light chain sequences of ansuvimab mAb was cloned into CHO cell lines and initial production runs were produced by Cook Phamica d.b.a. Catalent under contract of Medimmune.^[4][5]

Mechanism of action

Neutralization

Ansuvimab is a monoclonal antibody therapy that is infused intravenously into patients with Ebola virus disease. Ansuvimab is a neutralizing antibody,^[3] meaning it binds to a protein on the surface of Ebola virus that is required to infect cells. Specifically, ansuvimab neutralizes infection by binding to a region of the Ebola virus envelope glycoprotein that, in the absence of ansuvimab, would interact with virus’s cell receptor protein, Niemann-Pick C1 (NPC1).^[6][7][8] This “competition” by ansuvimab prevents Ebola virus from binding to NPC1 and “neutralizes” the virus’s ability to infect the targeted cell.^[6]

Effector function

Antibodies have antigen-binding fragment (Fab) regions and constant fragment (Fc) regions. The Neutralization of virus infection occurs when the Fab regions of antibodies binds to virus antigen(s) in a manner that blocks infection. Antibodies are also able to “kill” virus particles directly and/or kill infected cells using antibody-mediated “effector functions” such as opsonization, complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent phagocytosis. These effector functions are contained in the Fc region of antibodies, but is also dependent on binding of the Fab region to antigen. Effector functions also require the use of complement proteins in serum or Fc-receptor on cell membranes. Ansuvimab has been found to be capable of killing cells by antibody-dependent cell-mediated cytotoxicity.^[3] Other functional killing tests have not been performed.

History

Ansuvimab is a monoclonal antibody that is being evaluated as a treatment for Ebola virus disease.^[9] Its discovery was led by the laboratory of Nancy Sullivan at the United States National Institute of Health Vaccine Research Center and J. J. Muyembe-Tamfum from the Institut National pour la Recherche Biomedicale (INRB) in the Democratic Republic of Congo, working in collaboration with the Institute of Biomedical Research and the United States Army Medical Research Institute of Infectious Diseases.^[3][10] Ansuvimab was isolated from the blood of a survivor of the 1995 outbreak of Ebola virus disease in Kikwit, Democratic Republic of Congo roughly ten years later.^[3]

In 2018, a Phase 1 clinical trial of ansuvimab was conducted by Martin Gaudinski within the Vaccine Research Center Clinical Trials Program that is led by Julie E. Ledgerwood.^[5][4][11] Ansuvimab is also being evaluated during the 2018 North Kivu Ebola outbreak.^[12]

Ansuvimab has also shown success with lowering the mortality rate from ~70% to about 34%. In August 2019, Congolese health authorities, the World Health Organization, and the U.S. National Institutes of Health promoted the use of ansuvimab, alongside REGN-EB3, a similar Regeneron-produced monoclonal antibody treatment, over other treatments yielding higher mortality rates, after ending clinical trials during the outbreak.^[13][14]

Discovery

A 2016 paper describes the efforts of how ansuvimab was originally developed as part of research efforts lead by Dr. Nancy Sullivan at the United States National Institute of Health Vaccine Research Center and Dr. J. J. Muyembe-Tamfum from the Institut National de Recherche Biomedicale (INRB) in the Democratic Republic of Congo.^[3][10] This collaborative effort also involved researchers from Institute of Biomedical Research and the United States Army Medical Research Institute of Infectious Diseases.^[3][10] A survivor from the 1995 outbreak of Ebola virus disease in Kikwit, Democratic Republic of Congo donated blood to the project that began roughly ten years after they had recovered.^[3] Memory B cells isolated from the survivor’s blood were immortalized, cultured and screened for their ability to produce monoclonal antibodies that reacted with the glycoprotein of Ebola virus. Ansuvimab was identified from one of these cultures and the antibody heavy and light chain gene sequences were sequenced from the cells.^[3] These sequences were then cloned into recombinant DNA plasmids and purified antibody protein for initial studies was produced in cells derived from HEK 293 cells.^[3]

Ansuvimab and mAb100 combination

In an experiment described in the 2016 paper, rhesus macaques were infected with Ebola virus and treated with a combination of ansuvimab and another antibody isolated from the same subject, mAb100. Three doses of the combination were given once a day starting 1 day after the animals were infected. The control animal died and the treated animals all survived.^[3]

Ansuvimab monotherapy

In a second experiment described in the 2016 paper, rhesus macaques were infected with Ebola virus and only treated with ansuvimab. Three doses of ansuvimab were given once a day starting 1 day or 5 days after the animals were infected. The control animals died and the treated animals all survived.^[3] Unpublished data referred to in a publication of the 2018 Phase I clinical trial results of ansuvimab, reported that a single infusion of ansuvimab provided full protection of rhesus macaques and was the basis of the dosing used for human studies.^[5][4]

Development

Ansuvimab was developed by the Vaccine Research Center with support of the United States National Institutes of Health and the Defense Advanced Projects Agency. The heavy and light chain sequences of ansuvimab mAb were cloned into CHO cell lines to enable large-scale production of antibody product for use in humans.^[4][5]

Human safety testing

In early 2018,^[9] a Phase 1 clinical trial of ansuvimab’s safety, tolerability and pharmacokinetics was conducted by Dr. Martin Gaudinski within the Vaccine Research Center Clinical Trials Program that is led by Dr. Julie E. Ledgerwood.^[5][4][11] The study was performed in the United States at the NIH Clinical Center and tested single dose infusions of ansuvimab infused over 30 minutes. The study showed that ansuvimab was safe, had minimal side effects and had a half-life of 24 days.^[5][4]

Ridgeback Biotherapeutics

A license for ansuvimab was obtained by Ridgeback Biotherapeutics in 2018, from the National Institutes of Health–National Institute of Allergy and Infectious Diseases.^[15] Ansuvimab was given orphan drug status in May 2019 and March 2020.^[16][17][18]

Experimental use in the Democratic Republic of Congo

During the 2018 Équateur province Ebola outbreak, ansuvimab was requested by the Democratic Republic of Congo (DRC) Ministry of Public Health. Ansuvimab was approved for compassionate use by the World Health Organization MEURI ethical protocol and at DRC ethics board. Ansuvimab was sent along with other therapeutic agents to the outbreak sites.^[19][20][11] However, the outbreak came to a conclusion before any therapeutic agents were given to patients.^[11]

Approximately one month following the conclusion of the Équateur province outbreak, a distinct outbreak was noted in Kivu in the DRC (2018–20 Kivu Ebola outbreak). Once again, ansuvimab received approval for compassionate use by WHO MEURI and DRC ethic boards and has been given to many patients under these protocols.^[11] In November 2018, the Pamoja Tulinde Maisha (PALM [together save lives]) open-label randomized clinical control trial was begun at multiple treatment units testing ansuvimab, REGN-EB3 and remdesivir to ZMapp. Despite the difficulty of running a clinical trial in a conflict zone, investigators have enrolled 681 patients towards their goal of 725. An interim analysis by the Data Safety and Monitoring Board (DSMB) of the first 499 patient found that ansuvimab and REGN-EB3 were superior to the comparator ZMapp. Overall mortality of patients in the ZMapp and remdesivir groups were 49% and 53% compared to 34% and 29% for ansuvimab and REGN-EB3. When looking at patients who arrived early after disease symptoms appeared, survival was 89% for ansuvimab and 94% for REGN-EB3. While the study was not powered to determine whether there is any difference between REGN-EB3 and ansuvimab, the survival difference between those two therapies and ZMapp was significant. This led to the DSMB halting the study and PALM investigators dropping the remdesivir and ZMapp arms from the clinical trial. All patients in the outbreak who elect to participate in the trial will now be given either ansuvimab or REGN-EB3.^{[21][22][13][12]}

In October 2020, the U.S. Food and Drug Administration (FDA) approved atoltivimab/maftivimab/odesivimab (Inmazeb, formerly REGN-EB3) with an indication for the treatment of infection caused by Zaire ebolavirus.^[23]

FDA approves ansuvimab-zykl for Ebola virus infection

DECEMBER 21, 2020 BY JANICE REICHERThttps://www.antibodysociety.org/antibody-therapeutic/fda-approves-ansuvimab-zykl-for-ebola-virus-infection/embed/#?secret=zWW0Sr0BdW

On December 21, 2020, the US Food and Drug Administration approved Ebanga (ansuvimab-zykl) for the treatment for Zaire ebolavirus (Ebolavirus) infection in adults and children. Ebanga had been granted US Orphan Drug designation and Breakthrough Therapy designations. Ansuvimab is a human IgG1 monoclonal antibody that binds and neutralizes the virus.

The safety and efficacy of Ebanga were evaluated in the multi-center, open-label, randomized controlled PALM trial. In this study, 174 participants (120 adults and 54 pediatric patients) with confirmed Ebolavirus infection received Ebanga intravenously as a single 50 mg/kg infusion and 168 participants (135 adults and 33 pediatric patients) received an investigational control. The primary efficacy endpoint was 28-day mortality. Of the 174 patients who received Ebanga, 35.1% died after 28 days, compared to 49.4% of the 168 patients who received a control.

Ebanga is the 12th antibody therapeutic to be granted a first approval in the US or EU during 2020.

The Antibody Society maintains a comprehensive table of approved monoclonal antibody therapeutics and those in regulatory review in the EU or US. The table, which is located in the Web Resources section of the Society’s website, can be downloaded in Excel format.

References

1. ^ Jump up to:^a b c d “FDA Approves Treatment for Ebola Virus”. U.S. Food and Drug Administration. 21 December 2020. Retrieved 23 December 2020.  This article incorporates text from this source, which is in the public domain.
2. ^ Jump up to:^a b “Ridgeback Biotherapeutics LP Announces the Approval of Ebanga for Ebola” (Press release). Ridgeback Biotherapeutics LP. 22 December 2020. Retrieved 23 December 2020– via Business Wire.
3. ^ Jump up to:^{a b c d e f g h i j k l} Corti D, Misasi J, Mulangu S, Stanley DA, Kanekiyo M, Wollen S, et al. (March 2016). “Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody”. Science. 351 (6279): 1339–42. Bibcode:2016Sci…351.1339C. doi:10.1126/science.aad5224. PMID 26917593.
4. ^ Jump up to:^{a b c d e f} Clinical trial number NCT03478891 for “Safety and Pharmacokinetics of a Human Monoclonal Antibody, VRC-EBOMAB092-00-AB (MAb114), Administered Intravenously to Healthy Adults” at ClinicalTrials.gov
5. ^ Jump up to:^{a b c d e f} Gaudinski MR, Coates EE, Novik L, Widge A, Houser KV, Burch E, et al. (March 2019). “Safety, tolerability, pharmacokinetics, and immunogenicity of the therapeutic monoclonal antibody ansuvimab targeting Ebola virus glycoprotein (VRC 608): an open-label phase 1 study”. Lancet. 393 (10174): 889–898. doi:10.1016/S0140-6736(19)30036-4. PMC 6436835. PMID 30686586.
6. ^ Jump up to:^a b Misasi J, Gilman MS, Kanekiyo M, Gui M, Cagigi A, Mulangu S, et al. (March 2016). “Structural and molecular basis for Ebola virus neutralization by protective human antibodies”. Science. 351 (6279): 1343–6. Bibcode:2016Sci…351.1343M. doi:10.1126/science.aad6117. PMC 5241105. PMID 26917592.
7. ^ Côté M, Misasi J, Ren T, Bruchez A, Lee K, Filone CM, et al. (August 2011). “Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection”. Nature. 477 (7364): 344–8. Bibcode:2011Natur.477..344C. doi:10.1038/nature10380. PMC 3230319. PMID 21866101.
8. ^ Carette JE, Raaben M, Wong AC, Herbert AS, Obernosterer G, Mulherkar N, et al. (August 2011). “Ebola virus entry requires the cholesterol transporter Niemann-Pick C1”. Nature. 477 (7364): 340–3. Bibcode:2011Natur.477..340C. doi:10.1038/nature10348. PMC 3175325. PMID 21866103.
9. ^ Jump up to:^a b “NIH begins testing Ebola treatment in early-stage trial”. National Institutes of Health (NIH). 2018-05-23. Retrieved 2018-10-15.
10. ^ Jump up to:^a b c Hayden EC (2016-02-26). “Ebola survivor’s blood holds promise of new treatment”. Nature. doi:10.1038/nature.2016.19440. ISSN 1476-4687.
11. ^ Jump up to:^{a b c d e} “NIH VideoCast – CC Grand Rounds: Response to an Outbreak: Ebola Virus Monoclonal Antibody (mAb114) Rapid Clinical Development”. videocast.nih.gov. Retrieved 2019-08-09.
12. ^ Jump up to:^a b Kingsley-Hall A. “Congo’s experimental mAb114 Ebola treatment appears successful: authorities | Central Africa”. http://www.theafricareport.com. Retrieved 2018-10-15.
13. ^ Jump up to:^a b McNeil DG (12 August 2019). “A Cure for Ebola? Two New Treatments Prove Highly Effective in Congo”. The New York Times. Retrieved 13 August 2019.
14. ^ Molteni M (12 August 2019). “Ebola is Now Curable. Here’s How The New Treatments Work”. Wired. Retrieved 13 August 2019.
15. ^ “Ridgeback Biotherapeutics LP announces licensing of mAb114, an experimental Ebola treatment, from the National Institute of Allergy and Infectious Diseases” (Press release). Ridgeback Biotherapeutics LP. Retrieved 2019-08-17 – via PR Newswire.
16. ^ “Ansuvimab Orphan Drug Designations and Approvals”. accessdata.fda.gov. 8 May 2019. Retrieved 24 December 2020.
17. ^ “Ansuvimab Orphan Drug Designations and Approvals”. accessdata.fda.gov. 30 March 2020. Retrieved 24 December 2020.
18. ^ “Ridgeback Biotherapeutics LP Announces Orphan Drug Designation for mAb114”(Press release). Ridgeback Biotherapeutics LP. Retrieved 2019-08-17 – via PR Newswire.
19. ^ Check Hayden, Erika (May 2018). “Experimental drugs poised for use in Ebola outbreak”. Nature. 557 (7706): 475–476. Bibcode:2018Natur.557..475C. doi:10.1038/d41586-018-05205-x. ISSN 0028-0836. PMID 29789732.
20. ^ WHO: Consultation on Monitored Emergency Use of Unregistered and Investigational Interventions for Ebola virus Disease. https://www.who.int/emergencies/ebola/MEURI-Ebola.pdf
21. ^ Mole B (2019-08-13). “Two Ebola drugs boost survival rates, according to early trial data”. Ars Technica. Retrieved 2019-08-17.
22. ^ “Independent monitoring board recommends early termination of Ebola therapeutics trial in DRC because of favorable results with two of four candidates”. National Institutes of Health (NIH). 2019-08-12. Retrieved 2019-08-17.
23. ^ “FDA Approves First Treatment for Ebola Virus”. U.S. Food and Drug Administration(FDA) (Press release). 14 October 2020. Retrieved 14 October 2020.  This article incorporates text from this source, which is in the public domain.