USOO9018226B2 (12) United States Patent (10) Patent No.: US 9,018,226 B2 Srivastava et al. (45) Date of Patent: Apr. 28, 2015 (54) ANTIPSYCHOTICAGENTS AND (56) References Cited STANDARDIZED ANTIPSYCHOTC PUBLICATIONS FRACTIONS FROM RAUVOLIFLA TETRAPHYLLA AND PROCESS OF THEIR Akinloye, B.A. et al.: Stem bark alkaloids of Rauwolfia volkensii. SOLATION Planta Medica, vol. 37, pp. 361-366, 1979.* * cited by examiner (75) Inventors: Santosh Kumar Srivastava, Lucknow Primary Examiner — Charanjit Aulakh (IN); Ashok Kumar Agrawal, Lucknow (74) Attorney, Agent, or Firm — Abelman, Frayne & (IN); Subhash Chandra Singh, Schwab Lucknow (IN); Vinay Kumar Khanna, (57) ABSTRACT Lucknow (IN); Janardan Singh, The present invention relates to bioactive extracts its fractions Lucknow (IN); Chandeshwar Nath, and isolation of compound from Rauwolfia tetraphylla. The Lucknow (IN); Madan Mohan Gupta, extracts and fractions are useful for the treatment of psychosis based on in-vivo Validation on animal model and proportional Lucknow (IN); Shikha Gupta, Lucknow binding affinities for dopaminergic-D, Cholinergic (musca (IN); Ram Kishor Verma, Lucknow rinic) and Serotonergic (5HT) receptors for antipsychotic (IN); Anirban Pal, Lucknow (IN); activity. The present invention relates to novel antipsychotic Dnyaneshwar Umrao Bawankule, activity in the leaf alkaloids of Formula 1 and 2 named tet rahydroalstonine, 10-methoxytetrahydroalstonine, isoreser Lucknow (IN); Dharmendra Saikia, piline, 10-demethoxyreserpiline, 11-demethoxyreserpiline, Lucknow (IN); Anil Kumar Gupta, reserpiline and C-yohimbine. The present invention also Lucknow (IN); Anupam Maurya, relates to processes for obtaining antipsychotic extracts as Lucknow (IN); Suman Preet Singh well as for the isolation of alkaloids of formula 1 and 2 from Khanuja, Lucknow (IN) the leaves of Rauwolfia tetraphylla. The present invention particularly relates to significant antipsychotic activity in the (73) Assignee: Council of Scientific & Industrial MeOH extract, ethylacetate and chloroform fractions of R. tetraphylla and in the isolated compounds C-yohimbine, res Research, New Delhi (IN) erpiline and in a mixture 10-demethoxyreserpiline and (*) Notice: Subject to any disclaimer, the term of this 11-demethoxyreserpiline in 1:1.5 ratios for treating psycho patent is extended or adjusted under 35 sis without any extra pyramidal symptoms (EPS). U.S.C. 154(b) by 583 days. Formula-1 (21) Appl. No.: 13/262,040 (22) PCT Filed: Mar. 31, 2010 (86). PCT No.: PCT/N2O1 O/OOO2O8 S371 (c)(1), (2), (4) Date: Mar. 1, 2012 H3COOC > 1. R = R2 = OMe R3 = C-H (Isoreserpiline) (87) PCT Pub. No.: WO2010/113180 H- 2. R = R2 = OMe R3 = B-H (Reserpiline) PCT Pub. Date: Oct. 7, 2010 He 3. R1 = OMe R2 = HR = B-H (11-Demethoxy reserpiline) (65) Prior Publication Data H- 4. R = HR2 = OMe R3 = B-H (10-Demethoxy reserpiline) He 5. R1 = R = H R3 = C-H (Tetrahydroalstonine) US 2012/O184576A1 Jul. 19, 2012 H 7. R1 = OMe R2 = H R3 = C-H (10-Methoxytetrahudroalstonine) Formula 2 (30) Foreign Application Priority Data Mar. 31, 2009 (IN) ............................. 658/DELA2009 (51) Int. Cl. A6 IK3I/4375 (2006.01) CO7D 49/22 (2006.01) A6 IK 36/24 (2006.01) (52) U.S. Cl. CPC ...................................... A61K 36/24 (2013.01) (6. C-Yohimbine) (58) Field of Classification Search USPC ............................................. 514/280; 546/48 See application file for complete search history. 21 Claims, 8 Drawing Sheets U.S. Patent Apr. 28, 2015 Sheet 1 of 8 US 9,018,226 B2 OO SO so is 40 2O SS O 25 s s es as slas Dose mg/kg) year is extrati Creferrt fractice Fig 1 airbs 3i s: s 3. i st i t s i: r U.S. Patent Apr. 28, 2015 Sheet 2 of 8 US 9,018,226 B2 Big Too?age·11, Fig o 3 3) Ch. 22) t -----Y- w" ix. sm- ww.m-W-a-row..., "Xmes Fig.4 U.S. Patent Apr. 28, 2015 Sheet 3 of 8 US 9,018,226 B2 ---- - rs - OO 100g 50ag 25ug 12.5ig 100 g 50g 25ig i2.5ig K K{{4 Fig.6 U.S. Patent Apr. 28, 2015 Sheet 4 of 8 US 9,018,226 B2 Anti-psychotic activity evaluation of known drugs o too i to i ii" f : ggi 20 r O e 2 a. 0. Dose (mg/kg) Dose (mg/kg) Risperidone Olanzapine Values are mean of five animals in each group % inhibition calculated with respect to amphetamine induced hyperactivity Fig.7 U.S. Patent Apr. 28, 2015 Sheet 5 of 8 US 9,018,226 B2 Rauwolfiatetraphylla (powdered leaves, 190g) Extracted with MeOH overnight MeOH extract (1A, 42g) Dissolved in distill water l Aqueous extract Extracted with n-Hexane Aqueous solution Hexane fraction* (1B) Extracted with EtOAc Aqueous solution EtOAc fraction* (1C) Extracted with n-BuOH (saturated with water) Aqueous fraction BuOH fraction (1E) (1D) *Washed with water, dried over anhydrous Na2SO4 Solvent removed under vacuum Fig. 8: Flow chart 1 U.S. Patent Apr. 28, 2015 Sheet 6 of 8 US 9,018,226 B2 EtOAc extract (1C) of Example 2 (43gm) Vacuum liquid chromatography (hexane: EtOAc 1:1) Fr 18-26(5.2gm) Column chromatography (hexane: EtOAc 65:35) Fr(s). 170-179,182-183,186,207-217 were polled Fr 189-206 (800mg) Flash chromatography (Hexane: EtOAc 70:30) Fr (33-43)(416mg) ol- Yohimbine(K001) (15mg) Fast Centrifugal Partition Chromatography (FCPC) (teuMeether: ACN:water +.8mMTFA 4:1:5) Fr 19-20 o-Yohimbine(K001) (40.3mg) Fig:9 Flow chart 2 U.S. Patent Apr. 28, 2015 Sheet 7 of 8 US 9,018,226 B2 Dry leaves (190gm) uns with MeOH overnight X4 MeOH extract (42gm, 1A) Extracted with 2% HCl(20x150ml) Acidic, extract (3000ml) Extracted with Hexane Acidic extract Hexane fraction* Extracted with CHCl3 (2A) Acidic extract CHCl3 fraction* (2B) Basified with Na2CO3 in cold up to PH-9and extracted with CHCl3 Basic extract CHCls fraction* Basified with NaOH in cold up to PH-12 (2C) Extracted with CHCl3 Basic extract CHCl fraction* (2D) eutralized and concentrated Aqueous fraction (2E) (d) Washed with water, dried over anhydrous Na2SO4 Solvent removed under vacuum Fig 10; Flow chart 3 U.S. Patent Apr. 28, 2015 Sheet 8 of 8 US 9,018,226 B2 CHCl, extract (2C) of Example 3 Purified over flash, eluted with Fr (8-12) Hexane: EtOAc (3:1) Ttrahydroalstonin (K006) Purified by chromatography Fr (8-12) -D -0 Fr (13-15) F19-24) alstonin (K003) 10-Methoxytetrahydroalstonin (K003) Fr (17-21) Isoreserpiline (K005) 10-Demethoxyreserpiline (K004) Fr (66-81) Isomeric mixture 1 1-Demethoxyre serpiline (K004) Fr (98-129) o-Yohimbine (K001) (120 mg) Fr (137-168) Reserpiline (K002) (134.8mg) Fig. 11: Flow chart 4 US 9,018,226 B2 1. 2 ANTIPSYCHOTICAGENTS AND They have been reported to possess various biological activi STANDARDIZED ANTIPSYCHOTC ties such as antitumor, antimicrobial, antihypertensive (Ver FRACTIONS FROM RAUVOLIFLA poorte, R. In Alkaloids: Biochemistry, Ecology and Medici TETRAPHYLLA AND PROCESS OF THEIR nal Applications; Roberts, M. F. & Wink, M., (Eds.); Plenum SOLATION Press: New York, 1998, pp.397-433), antileishmanial activity, antibacterial activity. RELATED APPLICATIONS The genus Rauwolfia belongs to the family Apocynaceae and five species of this genus are native to India. R. tetra This application is a S371 of PCT/IN2010/000208 filed phylla L. Syn. R. canescens L. (Compend. Indian Med. Plants, Mar. 31, 2010, and claims priority from Indian Patent Appli 10 Vol. 1, Rastogi&Mehrotra, PID, New Delhi, 1990, p. 340) is cation No. 65.8/DEL/2009 filed Mar. 31, 2009, both incorpo an economically important plant, which is cultivated on com rated by reference in their entirety. mercial scale in India. The plant is important because of the presence of nearly 30 alkaloids in its roots: ajmalicine, reser FIELD OF INVENTION pine, Sapagine, deserpidine, rescinnamine, Serpentine, ajma 15 lidine, alloyohimbine, chandrine, corynathine, iscajmaline, The present invention relates to novel antipsychotic activ neo ajmaline, papaverine, raunatine, raunoline, rauwolscine ity in the leaf extracts of Rauwolfia tetraphylla and their or (C-yohimbine), reserpiline, reserpinine, reserpoxidine, useful standardized herbal formulations for the treatment of serpinine, serpentinine, thambine, ajmaline and yohimbine psychosis based on proportional binding affinities for dopam (Farooqi and Sreeramu, 2001, Cultivation of Medicinal and inergic-D and Serotonergic (5HT) receptors in vitro and Aromatic Crops. University Press Ltd., India, pp: 210-211), J. amphitamine induced hyperactive mouse model in-vivo for Amer. Chem. Soc. 79(5):1217-1222. antipsychotic activity. The present invention further relates to From the leaves of R. tetraphylla N(O)-demethylaccidine, a process for obtaining antipsychotic extracts from the leaves tetraphylline, tetraphyllicine (Phytochemistry, 28(3): 1989, of R. tetraphylla, which are non toxic and devoid of extra 961-962), J. Amer. Chem. Soc. 79(5): 1217-1222), aricine, pyramidal side effects. The present invention also relates to 25 isoreserpiline, tetrahydroalstonine, a yohimbine isomer (Rev. novel antipsychotic activity in the leafalkaloids of Formula 1 Cubana Farm. 1982, 16,28; Chem. Abstr. 1982,97, 107095 q: and 2 named tetrahydroalstonine, 10-methoxytetrahydroal Rev. Cubana Farm. 1982, 16, 316; Chem. Abstr. 1983, 98, stonine, isoreserpiline, 10-demethoxyreserpiline, 176193 c); O.-yohimbine and reserpiline have been isolated 11-demethoxyreserpiline, reserpiline and C.-yohimbine. The (Rev. Cubana Farm. 1982, 16, 251; Chem. Abstr. 1983, 98, present invention further relates to processes for obtaining 30 157873 h), (Compendium of Indian Medicinal Plants: Vol. 3 antipsychotic extracts as well as for the isolation of alkaloids 1980-1984 by Ram P. Rastogi, B. N. Mehrotra). of formula 1 and 2 from the leaves of R. tetraphylla. The Yohimbine has been used to facilitate recall of traumatic present invention particularly relates to significant antipsy memories in the treatment of posttraumatic stress disorder chotic activity in the MeOH extract, ethylacetate and chloro (PTSD). Yohimbine is used in arteriosclerosis and angina form fractions of R.