Through Ritter Reaction with Acetonitrile and Its Toxicity to

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Synthesis Organonitrogen Compounds from Patchouli Alcohol

1

Through Ritter Reaction with Acetonitrile and Its Toxicity to

2

Artemia salina

Leach.

3 4

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

6

Chemistry Department, Brawijaya University 7

Jln. Veteran, Malang 65145, Indonesia 8

email : nisachemist@gmail.com 9

10

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

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ABSTRACT

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Patchouli oil contains a compound with biological activities to human body called the 14

patchouli alcohol that can be further developed in medical field. This research aimed to 15

synthesize organonitrogen compound from patchouli alcohol through Ritter reaction with 16

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

17

alcohol from patchouli oil using fractional distillation under reduced pressure method. The 18

synthesis of organonitrogen compound is done at room temperature with the mol ratio of 19

patchouli alcohol: acetonitrile: sulfuric acid is 1:1,5:4 for 24 hours. The result showed that the 20

amount of patchouli alcohol produced from fractional distillation is 65,25%. The main 21

product yielded from the synthesis between patchouli alcohol and acetonitrile through Ritter 22

reaction is 36,93% of N-(4,8a,9,9-tetramethyl decahydro-1,6-metanonaftalen-1-il) acetamide. 23

Starting material used have LC50 of 77,39 ppm. The product of synthesis have higher toxicity 24

level than starting material, which have LC50 value is 10,39 ppm with the potential as medical 25

compound. 26

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

reaction, toxicity 28

29 30

INTRODUCTION 31

Indonesia is the largest producer of patchouli oil in the world, where 90 % of the world’s 32

patchouli oil needs are supplied from Indonesia [1]. Given the scale abundance of patchouli 33

oil is a challenge for people to explore it further into new products and to increase the resale 34

value. One is the use of patchouli oil in the development of therapeutic areas. 35

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

Patchouli oil is shown to have pharmacological activity as an agent inhibiting platelet 37

activating factor (PAF) [2], anti microbial, sedative agents, antiseptic [3], antiviral [4], and 38

antifungal [5]. Pharmacological activity is influenced by the content patchouli alcohol is the 39

mayor compound in patchouli oil [3]. Patchouli alcohol can inhibit influenza virus [4]. This 40

encourages the optimation efforts patchouli alcohol as a pro drug compounds through 41

molecular modification of the structure to be beneficial in the medicine world. 42

Research have been done to modify the structure of patchouli alcohol during the 43

formation ester compound is carried out using acetic acid and acid catalyst producing 44

patchouli acetate compound [6] and dehydration becomes patchoulene by using a strong acid, 45

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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]. 52

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

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 82

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

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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 Na2SO4 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. 99

The process is then performed by fractional distillation under reduced pressure, the residue 100

was obtained then analyzed with GC-MS and FT-IR 101

102

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

out of concentrated sulfuric acid, the mixture is left at room temperature while stirring with a 110

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 Na2SO4 115

anhydrous, then filtered. The solution was concentrated with nitrogen gas. The compounds 116

synthesized were analyzed functional groups with FT-IR and analyzed the content of its 117

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

test substances at varying concentrations and progressive were prepared in DMSO (dimethyl 124

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

under a microscope the number of death larvae in each solution used to evaluate the toxicity 127

of the solution. Tests were carried out in triplicate. 128

Percent of lethality shrimp Artemia salina calculated at each concentration by the 129

formula [13]: 130

131

where Nt is the number of shrimp larvae that died after incubation for 24 hours and No is the 132

total number of shrimp larvae are included. LC50 value is then determined by linear 133

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RESULT AND DISCUSSION 140

141

Isolation Patchouli Alcohol from Patchouli Oil 142

143

The yield of patchouli alcohol was obtained from fractional distillation under reduced 144

pressure 65,25 % at 100 mmHg. 145

146

Table 1. The data content of patchouli alcohol 147

Parameters Before Distillation

After Distillation

Colour of Oil Brownish

yellow

Brown

Content of GC-MS analysis 16,91 % 65,25% 148

149

Figure 2. The chromatogram of patchouli alcohol was obtained from fractional distillation 150

151

Synthesis Patchouli Acetamide Through Ritter Reaction 152

153

Based on the analysis using GC-MS using a column Rtx-wax, the chromatogram 154

obtained compounds synthesized as shown in Figure 3. Chromatogram compounds 155

synthesized showed a peak that has 14% area of more than 1% and 12 peaks that have M+ = 156

263 with a retention time sequence, shown in Table 2. Peak with M+ = 263 major expected 157

outcome is a product synthesis, namely N-(4,8a,9,9-tetramethyldecahydro-6,1-1-yl 158

methanonaphtalena) acetamide, or better known as patchouli acetamide. 159

160

161

Figure 3. The chromatogram compound synthesis 162

163

Table 2. Data Synthesis The Compound Allegations 164

Peak Retention Time

(minute)

% Area m/z

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5

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

Mass spectrum at peak 39 which has 5 area, 34,31% showed a peak of m/z = 30,41,43, 166

67, 79, 91, 107, 121, 133, 147, 161, 177, 189, 204, 220, 258, and 263 (shown in Figure 4). 167

Presence of the carbonyl group in patchouli acetamide compound results cleavage α, peakk 168

m/z = 263 release CH3 so that produce peak m/z = M+ -15 = 248, then the release CO to form 169

peak of m/z = 220. Cleavage α to the C carbonyl produces a peak m/z = M+ - 220 = 43. Peak 170

m/z = M+ - 59 = 204 is obtained from the release NH2COCH3, then the release CH3 to form 171

the peak m/z = M+ - 74 = 189. Peak m/z = 220 of the release of C3H7 (M+ - 43) then release 172

NH2COCH3 thus forming the peak m/z = 161. Peak m/z = 161 have thus generated the 173

release of C4H8 peak m/z = 91. Peak m/z = 91 release C2H2 respectively so as to produce the 174

peak m/z = 41. Suggested fragmentation pattern for compound patchouli acetamide shown in 175

Figure 6. 176

177

178

Figure 4. The Mass Spectrum Peak 39 with Retention Time 25,8 minutes. 179

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H N C

O CH2

e

H N C

O CH3 C

O CH3

m/z = 263

m/z = 43

e

H N C

O

CH3

H N C

O

m/z = 248 - CH3

H N CH3

m/z = 220 - CO

- NH2COCH3

m/z = 204

- CH3

H2C

m/z = 189

H N C

O

e

- C3H7 H N C

O

m/z = 220 - NH2COCH3

m/z = 161 - CH2

m/z = 147 - C4H8

m/z= 91

- C2H2

m/z = 67

m/z = 41

181

Figure 5. Suggested fragmentation for patchouli acetamide 182

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Figure 6. Plausible mechanism reaction the Formation patchouli acetamide 185

186

187 188

Figure 7. Stucture of patchouli acetamide 189

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Figure 8. Infrared spectrum of product synthesis 192

193 194

Table 3. Data functional group product synthesis 195

Wavenumber(cm-1) 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

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

groups of data in Table 3, show a strong absorption at wavenumber 3299 cm-1 which is the N-199

H stretching vibration of secondary amide. Uptake was supported by the absorption at 200

wavenumber 1647,10 cm-1 which is the vibration of the amide carbonyl stretching and at 201

wavenumber 1546,80 cm-1 which is an N-H bending vibration. This indicates that the 202

presence of secondary amide compounds contained in the compounds synthesized. Based on 203

the percent results between the mass of the synthesis results compared with the theoretical 204

mass, percent yield of product patchouli acetamide through Ritter reaction is 36,93%. 205

206

Toxicity Test to Artemia salina Leach. 207

208

Table 4. The calculation result data LC50 from BSLT test 209

No. Sampel Graph Equation LC50

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Product synthesis containing patchouli acetamide 36,93% have a higher toxic properties, 211

where the compounds are patchouli acetamide which has a lone pair of N atom and the 212

carbonyl group has two lone pair on O atom. Presence of three lone pairs on these 213

compounds (patchouli acetamide) its active side to form hydrogen bonds with DNA [...]. This 214

binding causes the protein synthesis process is hampered and toxic effect at low doses. 215

Based on research that has been done, it can be concluded that the main products of the 220

synthesis of patchouli alcohol with acetonitrile trough Ritter reaction is N-(4,81,9,9-221

tetramethyl decahydro-6-1-1-yl methanonaphtalene) acetamide or patchouli acetamide, 36,93 222

%. 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

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