Synthesis of Drugs Assignment

Synthesis of Atropine

Name : Sujia Nospiatdi Bp Number : 1011014024 International Class

FACULTY OF PHARMACY ANDALAS UNIVERSITY PADANG 2012

ATROPINE 1. Information Of Atropine SDBS No: 7817 CAS Registry No.: 51-55-8 Molecular Formula: C17H23NO3 Molecular Weight: 289.4 Compound Name: atropine (+-)-hyoscyamine endo-8-methyl-8-azabicyclo(3.2.1)octan-3-ol (+-)-tropate

2. Effects : Antimuscarinic M-cholinoblockers are competitive antagonists of acetylcholine as well as other Mcholinoblockers in terms of postsynaptic M-cholinoreceptors 3. Side Effect : a. Local iritation b. Foliculas konjungtivitis c. Contact dermatitis d. Eczematoid dermatitis e. Light sensitifity 4. Indication a. Glaucoma b. Hypersensitif

c. dilating pupils for severe inflammatory illnesses d. eye trauma e. Cholesystis 5. SAR (Structure Activity Relationship) Comparison of atropine with acetylcholine
Me

NMe3 CH2 CH2 O C O

N H CH2 OH O C O CH

CH3

Relative positions of ester and nitrogen similar in both molecules Nitrogen in atropine is ionised Amine and ester are important binding groups (ionic + H-bonds) Aromatic ring of atropine is an extra binding group (vdW) Atropine binds with a different induced fit - no activation Atropine binds more strongly than acetylcholine Others SAR of atropine are

The Analogues are fully ionised The Analogues unable to cross the blood brain barrier No CNS side effects Tertiary amine (ionised) or a quaternary nitrogen Aromatic ring Ester N-Alkyl groups (R) can be larger than methyl (unlike agonists) Large branched acyl group R = aromatic or heteroaromatic ring Branching of aromatic/heteroaromatic rings is important

6. Principe Of Synthesis Reaction Condensation Reduction Hidrogenation Esterefication 7. Reaction a) Condensation of maleyl aldehyde with methylamine and acetonedicarboxylic acid gives tropenone (14.1.1). which is the main starting material for the synthesis of both atropine and scopolamine

b) The carbonyl group of tropenone is reduced with reacted by LiAlH4, thus forming tropenol (14.1.2)

c) The double bond between C6 and C7 of the tropane ring is hydrogenated, giving tropine (14.1.3).

d) Esterification of the tropenol gives the desired atropine (14.1.4)

8. Isolastion and purification of product Actually so many ways to isolated atropine from plant whther Atropa Belladona or Datura Sp. There are: 1) Isolation Materials 1. Equipment: 1.1 Appropriate Glassware (large beakers, graduated cylinders) 1.2 Separation Funnel 1.3 Mortar and Pestle 1.4 Magnetic Stirrer/Hotplate 1.5 Stir Bar 1.6 Thermometer 1.7 Large Buchner Funnel 1.8 Whatman #5 Coarse Buchner Filters 1.9 1000mL Buchner Flask 1.10 pH Meter 1.11 Chiral AGP column 100 x 4.0mm from ChromTech 1.12 LC pump model A, HP series from Eldex 1.13 Peak Simple Data System, Single Channel 1.14 PC Computer with Window 1.15 VUV-24: Variable Single Wavelength UV/VIS Detector from ChromTech 2. Reagents: 2.1 NaOH pellets (Sigma Aldrich 2005-2006, CAS No. 1310-73-2, product S5881-500G) 2.2 Chloroform (Sigma Aldrich 2005-2006, CAS No. 67-66-3, product 496189-1L) 2.3 2-propanol (Sigma Aldrich 2005-2006, CAS No. 67-63-0, product 34965-1L) 2.4 Octanoic Acid (Sigma Aldrich 2005-2006, CAS No. 124-07-2, product O3907-1L) 2.5 HCl (Sigma Aldrich 2005-2006, CAS No. 7647-01-0, product 38281-1EA) 2.6 Sod.Ph.B. (sodium phosphate buffer) 3. Standards: 3.1 200, 400, 600, 800, 1000ppm atropine in distilled water Solubility: 2.5 g/mL stable for several days at 4C Poisonous. Keep away from sunlight. (Sigma Aldrich 2005-2006, CAS No. 5908-99-6, product A0257-25G)

4. Isolation Procedure Obtaining the target is done by this simplified objective: Manipulating the solubility of the alkaloid by switching it from organic base to inorganic salt and then retrieving the base by using a powerful solvent. Measurements are based on the assumption one plant contains 800 - 1,000ppm atropine. 1 Because atropine is in all plant tissues, liquefy the entire plant into a paste. Freezing and thawing the material is an optional tactic to rupture cells, however this can denature the alkaloids. 2 Let the paste dry. Do not expose to excessive heat or air currents. 3 Add the dried material to 500mL distilled acidified water (pH 6.0) and heat solution in a double boiler (using water) to 80C. Heat the solution for 30-45 minutes. 4 Filter out insoluble material using vacuum suction and a coarse filter. Save the filtered solution. 5 Adjust pH of the solution to 9.9 with a strong solution of NaOH. Keep heated at 50C for 30 minutes while stirring. 6 Let solution cool to room temperature while stirring. 7 Use 2mL aliquots of chloroform until all the inorganic salt that has precipitated out dissolves. Administer as little solvent as possible and continue stirring for efficient use of the solvent. 8 Pour the contents into a separating funnel. The bottom layer is your target. 9 Collect the target into a suitable container (i.e. no degradable rubbers or plastics and an air tight seal). 10 Purify the extract by running it through a chromatograph using CHIRALAGP 100 x 4.0 mm column with a 2% 2-propanol and 5mM octanoic acid in 0.01M sod.ph.b (pH 7.0) mobile phase. Set detection to 265nm. Atropine has ~ 8.1min retention time. If overlapping of peaks occurs, see section (7.0). For storage keep in a suitable container away from moisture, light, and heat. 11 To determine relative concentration (ppm) in the original plant, dilute atropine in 1L of distilled water and run sample against 5 standards in the same column (with fitting attenuation).

5. Marker Compound The marker compound scopolamine has similar characteristics to that of the target. It can only be found in the seed of the plant but is very inconsistent in concentration: 53 - 3,050ppm (http://www.swsbm.com/Constituents/Datura_stramonium.txt). Although scopolamine is expected to precipitate at a pH of 7.6-7.8, most of it may re-dissolve when the pH is elevated (6.4). Scopolamine has a retention time of ~ 6.0min and is very similar in structure (+1 oxygen) to that of atropine. Because of this, size exclusion chromatography will not separate adequately and therefore will not prevent overlapping of peaks. An extra procedure is to use a reverse phase column: Column: Eluent: Flow Rate: Detector: Column Temp.: Shodex Asahipak ODP-50 4D (4.6mmID*150mm) 50mM Phosphate buffer (pH10**)/CH3CN=70/30 0.6mL/min UV (210nm) 30deg-C

(http://www.shodex.com/english/dc040211.html) This column can be integrated with the equipment listed (5.1) in the extraction procedure. Its intended use is to separate particularly large amounts of scopolamine that are present in the atropine extract. Because the concentration of scopolamine varies extensively, quantitative analysis of the marker compound on a batch-by-batch basis may be necessary. From usage of the column in the extraction procedure (6.9), overlapping of peaks or joined peaks will give an indication that more separation is required. **This pH is referenced from its website listed. The chromatogram (8.5) demonstrates separation, but other data suggests some precipitation of atropine could occur.

6. Conceptual Diagrams 1 Typical readout of atropine extract

2 Buchner Filtration Apparatus

3 Typical Liquid Chromatography System

4 Datura Stramonium Plant

5 Scopolamine and Atropine Readout

6 Separation Funnel

2) Isolation Of Atropine From Datura Stramonium (1) Dry 500 gms. seeds of Datura stramonium (2) Boil the seeds in water for 1-1.5 hours in abeaker (3) Strain the extract through a fine pored mess (4) Add 4 ml of conc. Sulphuric acid into the liquid (5) Leave for half an hour (6) Take out the clear layer of liquor with the help of a pipette,

(7) Filter to obtain a transparent sherry-coloured solution (8) Add ammonium carbonate to the solution till saturation (9) Find colour change of the solution to black from sherry (10) Leave to obtain crystals of atropine in 15-30 minutes (11) Take out the upper supernatant with the help of a Pasteur pipette, (12) Dry the crystals on a filter paper (13) Dissolve dried crystals into boiling Alcohol (14) Evaporate alcohol and obtain pure crystals of atropine. 9. Identification of product Dilute solution of tropine when treated with concentrated nitric acid anf the mixture evaporated to dryness on the steam bath,produces a pale yellow residues. The residues gives a violet colouration when drop of freshly prepared of potassium hydoxide is added named vitali-marin. Thin Layer Chromatography Of Atropine One percent solution of atropine dissolved in 2 N acetic acid is spotted over silica gelG and eluted in the solvent system of strong ammonian solution,methanol (1,5:100). TLC plate is spread with an acidified iodoplatinate solution atropine gives the Rf value 0,18. Likewise atrophine sulfate shows the Rf 0,70 in the solvent system acetone: 0,5 M sodium chloride and spraying with Dragendorffs reagent.

Spectrum Analysis of Atropine SDBS-1H NMRSDBS No. 7817HSP-48-366 C17 H23 N O3 Atropine 399.65 MHz 0.040 g : 0.5 ml CDCl3

Assign. A B C D E F G J K L M N P Q R S T U V

Shift(ppm) 7.330 7.28 7.27 5.002 4.17 3.78 3.77 3.72 3.017 2.900 2.176 2.083 2.004 1.85 1.77 1.70 1.68 1.467 1.223

*1 *1

*2 *2 *3 *3 *4 *4 *4 *5 *5 *4

SDBS-13C NMRSDBS No. 7817CDS-10-167 C17 H23 N O3 Atropine

15.09 MHz 0.174 g : 0.8 ml CDCl3

ppm Int. Assign. 172.07 136.04 128.74 128.14 127.58 67.87 63.93 59.59 59.53 54.80 40.17 36.20 36.03 25.38 24.95 423 484 890 1000 445 544 396 643 637 440 385 390 396 363 308 1 2 3 4 5 6 7 8* 9* 10 11 12 # 13 # 14 *1 15 *1

SDBS-Mass MS-NW-6064 atropine C17H23NO3

SDBS NO. 7817 (Mass of molecular ion: 289)

Source Temperature: 160 C Sample Temperature: 120 C DIRECT, 75 eV

IR-KBR

10. Chemical Safety & Handling: a) Fire Potential: SLIGHT. [Sax, N.I. Dangerous Properties of Industrial Materials. 4th ed. New York: Van Nostrand Reinhold, 1975., p. 425] b) Hazardous Decomposition: When heated to decomposition it emits toxic fumes of /nitrogen oxides/. [Lewis, R.J. Sax's Dangerous Properties of Industrial Materials. 9th ed. Volumes 1-3. New York, NY: Van Nostrand Reinhold, 1996., p. 289]

c) Stability/Shelf Life: SENSITIVE TO LIGHT. [Sunshine, I. (ed.). CRC Handbook of Analytical Toxicology. Cleveland: The Chemical Rubber Co., 1969., p. C-13] d) SLOWLY AFFECTED BY LIGHT. /ATROPINE SULFATE/ [Osol, A. and J.E. Hoover, et al. (eds.). Remington's Pharmaceutical Sciences. 15th ed. Easton, Pennsylvania: Mack Publishing Co., 1975., p. 840] e) Atropine sulfate should be stored below 40 deg C, preferably at room temp. Freezing should be avoided. Minimum hydrolysis occurs at pH 3.5. Atropine sulfate 1 mg/ml was found to retain potency for 3 months at room temp when 0.5 or 1 ml of soln was packaged in Tubex. /Atropine sulfate/ [Trissel, L.A. Handbook on Injectable Drugs. 9th ed. Bethesda, MD. American Society of Health-System Pharmacists' Product Development. 1996., p. 109] f) Disposal Methods: SRP: At the time of review, criteria for land treatment or burial (sanitary landfill) disposal practices are subject to significant revision. Prior to implementing land disposal of waste residue (including waste sludge), consult with environmental regulatory agencies for guidance on acceptable disposal practices. Api Potensi kebakaran: Sedikit. [Sax, N.I. Berbahaya Sifat Bahan Industri. 4th ed. New York: Van Nostrand Reinhold, 1975, hlm.. 425]