Through Ritter Reaction with Acetonitrile and Its Toxicity to

  1 Synthesis Organonitrogen Compounds from Patchouli Alcohol

  2 Through Ritter Reaction with Acetonitrile and Its Toxicity to

  3 Artemia salina Leach.

  4

  5 Khoirun Nisyak*, M. Farid Rahman, dan Sutrisno

  6

  7 Chemistry Department, Brawijaya University

  8 Jln. Veteran, Malang 65145, Indonesia 9 email : nisachemist@gmail.com

  10

  11 Received day moth year; Accepted day moth year (will be given)

  12 ABSTRACT

  13

  14 Patchouli oil contains a compound with biological activities to human body called the 15 patchouli alcohol that can be further developed in medical field. This research aimed to

  16

  synthesize organonitrogen compound from patchouli alcohol through Ritter reaction with

  17

  acetonitrile and discover its toxicity towards Artemia salina Leach. The isolation of patchouli

  18 alcohol from patchouli oil using fractional distillation under reduced pressure method. The 19 synthesis of organonitrogen compound is done at room temperature with the mol ratio of 20 patchouli alcohol: acetonitrile: sulfuric acid is 1:1,5:4 for 24 hours. The result showed that the 21 amount of patchouli alcohol produced from fractional distillation is 65,25%. The main

  22

  product yielded from the synthesis between patchouli alcohol and acetonitrile through Ritter 23 reaction is 36,93% of N-(4,8a,9,9-tetramethyl decahydro-1,6-metanonaftalen-1-il) acetamide.

  24 Starting material used have LC of 77,39 ppm. The product of synthesis have higher toxicity ₅₀ 25 level than starting material, which have LC value is 10,39 ppm with the potential as medical ₅₀ 26 compound.

  27 Key word: patchouli oil, patchouli alcohol, synthesis organonitrogen compound, Ritter 28 reaction, toxicity

  29

  30

  31 INTRODUCTION

  32 Indonesia is the largest producer of patchouli oil in the world, where 90 % of the world’s 33 patchouli oil needs are supplied from Indonesia [1]. Given the scale abundance of patchouli 34 oil is a challenge for people to explore it further into new products and to increase the resale 35 value. One is the use of patchouli oil in the development of therapeutic areas.

  36 Various studies have shown that the potential of patchouli oil as a base for medicine.

  37 Patchouli oil is shown to have pharmacological activity as an agent inhibiting platelet 38 activating factor (PAF) [2], anti microbial, sedative agents, antiseptic [3], antiviral [4], and 39 antifungal [5]. Pharmacological activity is influenced by the content patchouli alcohol is the 40 mayor compound in patchouli oil [3]. Patchouli alcohol can inhibit influenza virus [4]. This 41 encourages the optimation efforts patchouli alcohol as a pro drug compounds through 42 molecular modification of the structure to be beneficial in the medicine world.

  43 Research have been done to modify the structure of patchouli alcohol during the 44 formation ester compound is carried out using acetic acid and acid catalyst producing 45 patchouli acetate compound [6] and dehydration becomes patchoulene by using a strong acid,

  46 H SO [7]. Modify the structure of patchouli alcohol in this study was directed at the

  2

  4 formation of organonitrogen compounds, i.e hydrocarbons containing N atom in a variety of

  47 bonds. Organonitrogen compounds have properties related to biological and physiological 48 potential as a cure for diseases related to disorders of the central nervous system and its 49 ability to interact with receptors of the body [8]. Organonitrogen compounds have biological 50 activity as vasodilator, anti-inflammatory, antiviral, antimicrobial, analgesic, antidepressants, 51 antischistosoma, antitumor, and anticonsvulsan [9].

  (95-97%), diethyl eter,

  4

  anhydrous, CH

  3 CN, Na

  2 CO

  3

  , H

  2 SO

  4

  85 and DMSO, was purchased from MERCK, Artemia salina eggs was purchased from UIN

  84 General remarks: Na

  86 Malang, and patchouli alcohol (65,25%) was obtained from fractional distillation under

  87 reduced pressure towards patchouli oil that purchased from Blitar, East Java.

  88

  89 Instrumentation

  90

  91 FT-IR Shimadzu 8400S and Gas Chromatography-Mass Spectrometer GCMS-QP2010S

  92 Shimadzu.

  93 OH

  2 SO

  83 Materials

  52 Patchouli alcohol has a rigid structure and including the tertiary alcohol group, which has

  66

  53 activity related to the formation of a stable carbocation. Reaction that can be used in the 54 formation organonitrogen compound with alcohol compound as the starting material through 55 formation of carbocations is the Ritter reaction. Ritter reaction is a reaction to the formation

  56 N-alkyl carboxyamide from aliphatic or aromatic nitrile and carbocations in strong acid

  57 media [10]. In the Ritter reaction will be produced carbocations as intermediates. Formation 58 of a stable carbocation is due to the protonation of a strong acid, which carbocations are later 59 attacked by the nucleophilic nitrogen atom. Acetonitrile used in this study as a nitrile reagent 60 that attacks the carbocations.

  61

  62

  63

  64

  65

  67

  82

  68 Figure 1. Structure of Patchouli Alcohol

  69

  70 Organonitrogen compounds have the ability to interact with receptors of the body

  71 because of the hetero group that has a certain affinity and polarity that resulted in the 72 occurrence of molecular interactions through biochemical mechanisms in the body [11].

  73 Further studies to determine the ability of the products synthesized as a drug compound to be

  74 done through the toxicity test by brine shrimp lethality test (BSLT). The results of toxicity 75 test the method BSLT reflects its potential as a drug compound. Wijdharti, et al [12] states 76 that BSLT method is a method that is simple, rapid, inexpensive, and reliable and usually 77 done at the preliminary stage of the screening materials that are thought to have anticancer 78 properties before stepping to the test in vitro against tumor cell sustainably.

  79

  80

  81 EXPERIMENTAL SECTION

  H H

  Procedure

  94

  95 Isolation Patchouli Alcohol from Patchouli Oil

  96 A number of 75 mL of patchouli oil that has been analyzed compounds with GC-MS,

  97 dried with Na

  4

  anhydrous and then inserted into the flask capacity 100 mL and put in a

  98 series distillation apparatus with condenser column of 60 cm and vigreux column of 30 cm.

2 SO

  Synthesis of Patchouli Acetamide Compound Through Ritter Reaction 103

  104 A number of 28,2 mL of (contain 0,1 mol patchouli alcohol) is inserted into the flask 105 100 mL three-neck equipped with a thermometer. A number of 7,8 mL of acetonitrile (0,15 106 mol) is added to the flask. The mixture was cooled to 0 °C, cold condition are maintained 107 with the addition of salt around the flask. A number of 22 mL of 97% sulfuric acid (0,4 mol) 108 was added slowly dropwise to the mixture while stirring with a magnetic stirrer. Having run 109 110 out of concentrated sulfuric acid, the mixture is left at room temperature while stirring with a magnetic stirrer for 24 hours. Synthesis mixture was poured into the erlenmeyer flask 111 containing 100 mL of cold distilled water. Then added 100 mL of diethyl eter, stirred, and 112 separated by a separating funnel. 113

  Obtained organic phase was neutralized with saturated sodium carbonate solution and 114 saturated sodium chloride solution was added. Neutral organic phase was dried with Na

  99 The process is then performed by fractional distillation under reduced pressure, the residue 100 101 was obtained then analyzed with GC-MS and FT-IR 102

  4

  115 anhydrous, then filtered. The solution was concentrated with nitrogen gas. The compounds 116 117 synthesized were analyzed functional groups with FT-IR and analyzed the content of its components by GC-MS. 118

  119 120

  Toxicity Test to Artemia salina Leach. 121

  122

  Artemia salina eggs are incubated in brine at pH 7-8 (48 h). Then, series of solutions of 123

  124 test substances at varying concentrations and progressive were prepared in DMSO (dimethyl sufoxide) and brine. A defined number of larvae introduced into each solution. All solution 125 sand control solutions containing no active substance were left stirring for 24 hours. Counting 126 127 under a microscope the number of death larvae in each solution used to evaluate the toxicity of the solution. Tests were carried out in triplicate. 128

  Percent of lethality shrimp Artemia salina calculated at each concentration by the 129 130 formula [13]: 131 132 where Nt is the number of shrimp larvae that died after incubation for 24 hours and No is the total number of shrimp larvae are included. LC

  50

  133 value is then determined by linear 134 regression on analysis of the similarities between the percent of deaths as the y-axis and the concentration as the x axis. 135

  136 137 138 139

  2 SO

  140 RESULT AND DISCUSSION 141 142 Isolation Patchouli Alcohol from Patchouli Oil 143 144 The yield of patchouli alcohol was obtained from fractional distillation under reduced 145 pressure 65,25 % at 100 mmHg. 146 147 Table 1. The data content of patchouli alcohol

  Parameters Before After Distillation Distillation

  Colour of Oil Brownish Brown yellow Content of GC-MS analysis 16,91 % 65,25%

  148 149 150 Figure 2. The chromatogram of patchouli alcohol was obtained from fractional distillation 151 152 Synthesis Patchouli Acetamide Through Ritter Reaction 153 154 Based on the analysis using GC-MS using a column Rtx-wax, the chromatogram 155 obtained compounds synthesized as shown in Figure 3. Chromatogram compounds

• 156 synthesized showed a peak that has 14% area of more than 1% and 12 peaks that have M =
• 157 263 with a retention time sequence, shown in Table 2. Peak with M = 263 major expected 158 outcome is a product synthesis, namely N-(4,8a,9,9-tetramethyldecahydro-6,1-1-yl 159 methanonaphtalena) acetamide, or better known as patchouli acetamide. 160 161 162 Figure 3. The chromatogram compound synthesis 163 164 Table 2. Data Synthesis The Compound Allegations

  Peak Retention Time % Area m/z (minute)

  28 22,150 1,37% 30, 41, 43, 67, 79, 91, 110, 121, 139, 152, 161, 181, 189, 207, 248, 263 29 22,400 0,55% 30, 41, 43, 60, 70, 91, 107, 121, 135,

  149, 163, 189, 206, 263 30 22,733 0,49% 30, 41, 43, 60, 79, 91, 98, 120, 137, 147, 163, 191, 206, 265 31 22,900 1,61% 30, 41, 43, 56, 81, 91, 107, 122, 136, 148, 163, 175, 189, 204, 263 32 23,325 1,71% 41, 43, 67, 91, 107, 121, 136, 148, 161, 175, 189, 204, 248, 263 33 23,625 0,81% 30, 41, 43, 67, 79, 91, 107, 120, 134, 149, 161, 189, 204, 263 34 23,867 1,38% 30, 41, 43, 67, 81, 91, 105, 124, 132, 152, 161, 178, 190, 206, 220, 248, 263 35 24,308 9,32% 30, 41, 43, 67, 79, 91, 107, 121, 133, 147, 161, 177, 189, 204, 220, 263 36 24,692 0,38% 30, 41, 43, 67, 84, 107, 122, 136, 148, 161, 189, 204, 263 37 24,917 0,99% 30, 41, 43, 67, 79, 91, 107, 120, 133, 148, 163, 177, 189, 204, 263 38 25,567 7,98% 30, 41, 43, 60, 81, 95, 114, 129, 151, 163, 177, 191, 206, 222, 250, 265 39 25,800 34,31% 30, 41, 43, 67, 79, 91, 107, 121, 133, 147, 161, 177, 189, 204, 220, 248, 263

  165 166 Mass spectrum at peak 39 which has 5 area, 34,31% showed a peak of m/z = 30,41,43, 167 67, 79, 91, 107, 121, 133, 147, 161, 177, 189, 204, 220, 258, and 263 (shown in Figure 4). 168 Presence of the carbonyl group in patchouli acetamide compound results cleavage

  α, peakk

• 169 m/z = 263 release CH so that produce peak m/z = M -15 = 248, then the release CO to form

  3

• 170 peak of m/z = 220. Cleavage
• 220 = 43. Peak

  α to the C carbonyl produces a peak m/z = M

• 171 m/z = M - 59 = 204 is obtained from the release NH COCH , then the release CH to form

  2

  3

  3

  172 the peak m/z = M - 74 = 189. Peak m/z = 220 of the release of C H (M - 43) then release

  3

  7

  173 NH COCH thus forming the peak m/z = 161. Peak m/z = 161 have thus generated the

  2

  3

  174 release of C H peak m/z = 91. Peak m/z = 91 release C H respectively so as to produce the

  4

  8

  2

  2

  175 peak m/z = 41. Suggested fragmentation pattern for compound patchouli acetamide shown in 176 Figure 6. 177 178 179 Figure 4. The Mass Spectrum Peak 39 with Retention Time 25,8 minutes. 180

  _{H - NH COCH e O m/z = 263 N C H e CH} O

₂

_e

_{H N C O CH O 3 C m/z = 43 CH O - C H 3 4 8 - CH 3 N C H N C CH O 3 m/z = 204}

  2

³

_{H N C m/z= 91 - C H 2 2 m/z = 248 - CO N CH H 3} - CH _{3 - C H 3 - NH COCH m/z = 41 7 N C H - CH 2 O 3 m/z = 147 2 m/z = 67 m/z = 220 m/z = 189} H C _{2 m/z = 220 m/z = 161}

  181 182 Figure 5. Suggested fragmentation for patchouli acetamide 183 184 185 Figure 6. Plausible mechanism reaction the Formation patchouli acetamide 186 187 188 189 Figure 7. Stucture of patchouli acetamide 190

  _{%T 75}

  90 _{2729 .0 9}

  60 _{45 63 34 44. 307 6. 25 15 116 11 07 88. 00 3. 111 6. 103 71 5. 70 41 989. 3 4. 4 96 84 1 9 0. 83 7 07. 40 2. 69 82 5 14. 96 24 15}

  30 _{299. 3 98 3 .0 873 .74 2 .1 1647 .8 1546 14 56. 16 13 15 75. 294. 1} ^{599. 453.} _{4000 3500 3000 2500 2000 1750 1500 1250 1000 750 500 PATCHOULI ACETAMIDE} 2947 _1/cm

  191 192 Figure 8. Infrared spectrum of product synthesis 193 194 195 Table 3. Data functional group product synthesis

• -1

  

Wavenumber(cm ) Type of vibration

  3299, 98 Stretching N-H 2947,03 Stretching C-H 1647,10 Stretching C=O in amide 1546,80 Bending N-H 1456,16 Bending C-H in alkene

  196 197 198 Based on FT-IR spectrum of compounds synthesized in Figure 7 and the functional

• 1

  199 groups of data in Table 3, show a strong absorption at wavenumber 3299 cm which is the N- 200 H stretching vibration of secondary amide. Uptake was supported by the absorption at

• 1

  201 wavenumber 1647,10 cm which is the vibration of the amide carbonyl stretching and at

• 1

  202 wavenumber 1546,80 cm which is an N-H bending vibration. This indicates that the 203 presence of secondary amide compounds contained in the compounds synthesized. Based on 204 the percent results between the mass of the synthesis results compared with the theoretical 205 mass, percent yield of product patchouli acetamide through Ritter reaction is 36,93%. 206 207 Toxicity Test to Artemia salina Leach. 208 209 Table 4. The calculation result data LC from BSLT test

  50 No. Sampel Graph Equation LC

  50

  1 Starting material Y = 0,6462x 77,38 ppm

  2 R = 0,9809

  2 Product Synthesis Y = 4,575x 10,93 ppm

  2 R = 0,983

  210

  Product synthesis containing patchouli acetamide 36,93% have a higher toxic properties, 211 212 where the compounds are patchouli acetamide which has a lone pair of N atom and the carbonyl group has two lone pair on O atom. Presence of three lone pairs on these 213 214 compounds (patchouli acetamide) its active side to form hydrogen bonds with DNA [...] . This binding causes the protein synthesis process is hampered and toxic effect at low doses. 215

  , Thesis, 249 Department of Chemistry, Faculty of Mathematics and Natural Sciences Brawijaya 250 University, Malang, 2008. 251

  8. Hapsari, K.W., Reaksi Esterifikasi Patchouli Alkohol dalam Minyak Nilam (Patchouli oil) 248 dengan Asam Asetat Anhidrid Menggunakan Katalis H

  2 SO

  4

  dan H

  3 PO

  4

  9. Novitasari, R.D., Dehidrasi Patchouli Alkohol dalam Minyak Nilam (Patchouli Oil) 252 Menggunakan Asam Sulfat (H

  6. Ma’mun and Maryadhi, 2008, Jurnal Bul. Littro, 19(1), 95 – 99. 244

  4

  ) dan Asam Fosfat (H

  3 PO

  4

  ) dengan Variasi Pelarut, 253

  Thesis , Department of Chemistry, Faculty of Mathematics and Natural Sciences Brawijaya 254

  University, Malang, 2008. 255

  7. Aisyah, Y., Hastuti, P., Sastrohamidjojo, H., and Hidayat, C., 2008, Komposisi Kimia dan 245 Sifat Antibakteri Minyak Nilam (Pogostemon cablin), Majalah Farmasi Indonesia, 19(3), 246 151-156. 247

  5. Bulan, R., Esterifikasi Patchouli Alkohol Hasil Isolasi dari Minyak Nilam (Patchouli Oil), 241 Jurusan Kimia, Fakultas Matematika dan Ilmu Pengetahuan Alam Universitas Sumatera 242 Utara, Medan, 2004. 243

  216 217

  REFERENCES 231

  CONCLUSION 218

  219 Based on research that has been done, it can be concluded that the main products of the 220 221 synthesis of patchouli alcohol with acetonitrile trough Ritter reaction is N-(4,81,9,9-

  222 tetramethyl decahydro-6-1-1-yl methanonaphtalene) acetamide or patchouli acetamide, 36,93 %. Synthesized compounds have a toxicity level greater than starting material, which is 10,93 223 ppm, so the potential for drug compounds. 224

  225

  ACKNOWLEDGMENT 226

  227 The authors gratefully acknowledge DIKTI for financial support through the Program 228

  Kreativitas Mahasiswa 2011 program. 229 230

  232

  4. Sundaresan, Sigh, S.P., Mishra, A.N., Shasany, A.K., Darokar. M.P., Kalra, A., and Naqvi, 239 A.A., 2009, Journal of Essential Oil Research, 21(220). 240

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2 SO

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