Synthesis and evaluate the degradation of isoamyl nitrite - Vu Ngoc Toan

Chemistry & Environment V.N. Toan, N.M. Tri, L.H. Minh, “Synthesis and evaluate the degradation of isoamyl nitrite.” 86 SYNTHESIS AND EVALUATE THE DEGRADATION OF ISOAMYL NITRITE Vu Ngoc Toan*, Nguyen Minh Tri, Le Hong Minh Abstract: Isoamyl nitrite (IAN) is one of antidotes for cyanide poisoning. IAN can be synthesized by many different ways. IAN is degraded easily in normal conditions, requiring the evaluation of degradation after synthesis. Physical - chemical properties as well as the results of synthesis and evaluation of degradation are presented in this paper. The results showed that the product stored in vacuum ampoule, with stabilizer K2CO3 (2 wt %), increased the shelf life more than 12 months, yield 80-85%. Keywords: Isoamyl nitrite, Antidote, Cyanide. 1. INTRODUCTION Inhaling isoamyl nitrite (IAN) has been first aid measure for cyanide-poisoned victims in many years [8, 9]. However, the effectiveness of IAN is still controversial, nowday. Some studies said that IAN could not produce more than 6% methemoglobin, while the necessary quantity antidote is 15% for LD50 dose of cyanide [9]. Other reports said that IAN is only support solution for treatment cyanide victims. Now the protective effect of IAN is attributed to its vasodilatory effect that can reverse the early cyanide induced vasoconstriction [2, 9]. Artificial ventilation with IAN broken into ampul bags has been reported as a life saving therapy in cyanide poisoned dogs, prior to induction of significant level of methemoglobinemia [6]. In addition, IAN is used for treating heart diseases and angina. Especially, IAN is also used as stimulant drug, combining with other drugs to create a feeling like using cocaine or methylenedioxyl methamphetamine [5]. Therefore, IAN was only synthesized in small amounts for medical purposes. IAN can be easily synthesized in laboratory, but it quickly decomposes into other products under normal conditions. In this paper, we present method for synthesis, determine structure of product and as the level of degradation when using stabilizer. 2. EXPERIMENTAL 2.1. Chemical, equipment and method 2.1.1. Chemical, equipment Isoamyl alcohol (Merck); Sodium nitrite (Merck); Con. sulfuric acid (Fluka); Anhydrous NaHCO3, K2CO3, CaCl2, Three neck round bottom flask (500 ml); Mechanical stirrer with glass or teflon stirrer; Pressure equalising dropping funnel capacity 100 ml; Research Journal of Military Science and Technology, Special Issue, No.54A, 05 - 2018 87 Na2SO4 (Acros); Deionized water. Measuring cylinder capacity 50 ml; Pipette 2, 5 and 10 ml; Fractional distillation equipment,.. 2.1.2. Research method a. Density: The density of IAN was determined on DMA 5000 M (USA) density meter at test temperature of 25 ± 0.1 oC . b. Refractive index: The product’s refractive index was measured on the Atago NAR-1T liquid measuring instrument at test temperature of 25 ± 0.1 oC, range of refractive index: 1.3000-1.7000, accuracy of ± 0.1 %. c. FTIR-spectra: The FTIR-spectra was recorded on Impact 410 FT-IR Spectrometer (Nicolet, USA) in form of KBr powder. d. NMR spectra: The 1H NMR and 13C NMR spectra were recorded on an Advance Spectrometer AV500 (Bruker, Germany) at 500.13 MHz and 125.77 MHz, respectively, using DMSO-d6 as solvent and TMS as an internal reference substance. e. Evaluation method of product degradation: NMR spectra is appropriate analytical method to evaluate the degradation of IAN [3]. Record the NMR spectra of the internal reference substance (using benzyl benzoate) and the sample containing the product to be quatifield over time. Integrate the triplet at 4.6 ppm (I -CH2- in IAN) and the signal from the internal substance, the singlet at 5.3 ppm (II -CH2 aryl), at least five times and determine the average integral. The amount of IAN can be calculated as follows: % Isoamyl nitrite= AI AII E.WI E.WII mg of II mg of sample X 100 Where: AI = integral value of the signal representing (-CH2-) isoamyl nitrite; AII = integral value of the signal representing (-CH2 aryl), benzyl benzoate; E.WI = formula weight of I/2 = 58.57; E.WII = formula weight of II/2 = 106.12; mg of sample = sample weight in milligrams corrected for the stabilizer added. 2.2. Process for the preparation of IAN In a 500 ml three neck round bottom flask, 120 ml (97.2 g) of isoamyl alcohol was placed and 100 g of pure sodium nitrite was added. The mixture was cooled to 10° C and while stirring, 32.5 ml of concentrated sulfuric acid was droped very slowly. During this procedure, the nitrite should be kept from settling to the bottom, a process which was easily accomplished by having the stirring so vigorous that a clear layer of alcohol is never visible above the nitrite. During this addition of sulfuric acid, the temperature was an important consideration and should be kept below 25° C. Chemistry & Environment V.N. Toan, N.M. Tri, L.H. Minh, “Synthesis and evaluate the degradation of isoamyl nitrite.” 88 After all of the sulfuric acid added (about 60 minutes), the stirring is continued more 1h30 and water gradually added. The obtained solution was poured in a large separatory funnel where IAN rises to the surface, then added water to salt mixture, stirred, allowed to settle, and the nitrite again poured off together with most of the water. The extraction was continued until no more IAN rises to the surface. It was not necessary to dissolve all of the salt but the small lumps must be broken up and the remaining salt should be perfectly white. The yield volume of crude material was about 130-135 ml. IAN was washed once with clean water to remove any acid, dried for a few minutes over anhydrous CaCl2 or Na2SO4 and distilled. The fraction which boils from 99-100 oC was collected yielding 125-130 ml (110.0- 114.5 g) of IAN. 2.3. Evaluation of the degradation of IAN Main degradation products of IAN are aldehyde, alcohol and NO gas. The degradation of product was carried out in four conditions: photochemical environment, sealed glass jars, under vacuum ampoule with and without stabilizer (K2CO3). Solvent: carbon tetrachloride, internal reference substance: benzyl benzoate (Sigma Aldrich, 99.7 % purity). Procedure: Transfer 4 to 5 mEq of internal reference substance, accurately weighed, to a semimicro sampling tube, added 2 to 3 mL of carbon tetrachloride, apply a sampling valve and septum, thereby sealing the tube, and determine the weight of the sealed assembly. Open the valve, introduce about 500 µL of IAN with a micro syringe, close the valve, and determine the weight of the sealed assembly when it has attained constant weight. Shake the sampling tube and valve assembly, and transfer about 500 µL of the solution to a precision NMR tube as directed for absolute method of quantitation under nuclear magnetic resonance. With no spinning, or with the spinning adjusted so that the spinning side bands of neither the substance under assay nor the internal reference substance interfere with the regions to be integrated, record as AI the average area of the reference standard singlet appearing at about 5.3 ppm, representing the methylene protons of benzyl benzoate, and record as AII the average area of the multiplet with a band center at about 4.8 ppm, representing the alpha methylene protons of isoamyl nitrite, with reference to the tetramethylsilane singlet at 0 ppm. Calculate the quantity of C5H11NO2 in the IANtaken, using 58.57 as the equivalent weight of IAN( EWI ) and 106.12 as that of benzyl benzoate ( EWII ). Repeat the NMR measurements after 15, 30, 60, 90 and 120 minutes. Conducted similarly to condition 2, 3 and 4 samples after 1, 2, 3 and 4 weeks. 3. RESULTS AND DISCUSSION 3.1. Synthesis of IAN Research Journal of Military Science and Technology, Special Issue, No.54A, 05 - 2018 89 There are many influence factor in reaction, including: the rate of reagent, temperature, reaction time,therein, temperature holds the most important role. While testing various synthetic methods, sodium nitrite was dissolved in water and then poured into isoamyl alcohol, drop concentrated sulfuric acid with stirring. It was found that the temperatute changes very fast, the surface was reddish brown (or dark yellow), solution was dark yellow, indicating the formation of NO2 gas. Thus, we stirred a mixture of sodium nitrite with isoamyl alcohol without dissolve in water. Concentrated sulfuric acid was drop slowly, the reaction solution was light yellow and no reddish-brown gas appears on the solution surface. The product evaporated vigorously, and formed sodium sulfate so it required strong stirring to push the IAN to the bottom of the flask (using mechanical stirrers, glass stirrer or teflon). Water added must break most of salt and white mixen. Fast or slow processing affected the yield significantly, while the reaction time did not. Temperature was the crucial factor in success of the process. IAN is strongly degradable compound, so it was important to maintain the temperature below 25 oC during reaction. Product should be analyzed at least two weeks without stabilizer. There were many stabilizers can be used: epoxol 9-5 (epoxidized linseed oil) 2 % (w/v), Na3PO4 (2 %), K2CO3 (2 %), diphenylamine, [9]. The reaction occured through two stages: First, the reaction of sodium nitrite with sulfuric acid (or hydrochloric acid) produced nitrous acid, followed by reaction of nitrous acid with isoamyl alcohol. The process described in section 2.2 was optimized the factors, such as molar ratio, temperature, 3.2. Evaluation of product characteristic 3.2.1. Determine the physical properties Physical properties of IAN are shown in Table 1 below. Table 1. Physical properties of IAN. Properties Synthetic product Reference standard (Sigma- Aldrich) Color (eye) Light yellow Light yellow Density (25 oC, g/cm3) 0.8754 0.870-0.880 Boiling point (oC) 99-100 99-100 Refractive index (20 oC) 1.3855 1.3850-1.3860 3.2.2. Determine the structure of product a. FT-IR spectra: IR spectra of IAN appeared the absorption band of the functional groups present in the molecule. The characteristic IR spectra regions were 2963 cm-1 and 2878 cm -1 (CH), 1466 cm -1 and 1369 cm-1 (δCH2, δCH3, respectively). Especially, absorption band of the group –NO2 at 1648 cm -1. Chemistry & Environment V.N. Toan, N.M. Tri, L.H. Minh, “Synthesis and evaluate the degradation of isoamyl nitrite.” 90 b. NMR spectra: 1H NMR spectra (at the frequency of 500.13 MHz, measured in carbon tetrachloride solvent) of IAN showed signal regions, characteristic of each of the protons present in the compound . Resonance signals at δ=4,74 ppm (m, 2H) for proton in -CH2 (A). Resonance signals at δ=1,74-1,70 ppm (m, 1H) for proton –CH (C), at δ=1,66-1,62 ppm (m, 2H) for proton –CH2 (B). Resonance signals appeared at δ=0,96 ppm (d, 6H, J 6,50 Hz) for proton –CH3 (D). CH CH2 CH3 CH3 CH2 O N O (A)(B)(C) (D) (D) The 13C NMR spectra of the IAN indicated the chemical shift of the quivalent carbon atoms in the molecule. Resonance signals appeared at δ=66,8 ppm for carbon A, at δ=37,6 ppm for carbon B, at δ=25,9 ppm for carbon C and at δ=22,3 ppm for carbon D (the equivalent carbon atoms). c. Mass spectra: The molecular ion (M+ = 117) peak did not appear on the spectrum. Some major peak: m/z 71 (25 %), 60 (55 %), 57 (67 %), 43 (35 %), 41 (100 %, base peak), 30 (32 %), 29 (43 %). 3.3. Evaluation of the degradation of IAN 3.3.1. Evaluation of the degradation under photochemical environment The conversion of IAN into isoamyl alcohol occured rapidly in several other solvents, so all experiments were performed in carbon tetrachloride. This was also the solvent used to measure the NMR. The 1H NMR spectra of IAN appeared resonant signals at positions such as 3.2.2.b. Resonant signals at positions B and C were overlapping so that it was difficult to quantify by calculating on this group. Resonant signal at position A (t, CH2) and position D (singlet, 2CH3) were clearly separated, so using this to calculate was appropriate. The results of calculating the content of IAN in the product over time are presented in Table 2. Table 2. % IAN under photochemical environment. Time (min.) Content (isoamyl nitrite) (%) Content (benzyl benzoate) (%) 0 97.4 99.7 15 95.7 99.7 30 91.3 99.7 60 88.5 99.7 90 82.1 99.7 120 79.5 99.7 Research Journal of Military Science and Technology, Special Issue, No.54A, 05 - 2018 91 The content of IAN required for human (USP 38) is 80.0-105.0 %. After 120 minutes, under photochemical environment, the product has decomposed. 3.3.2. Evaluation of the degradation under sealed glass jars Quantitative IAN results are presented in Table 3. Table 3. % IANstoraged under sealed glass jars. Time (weeks) Content (isoamyl nitrite) (%) Content (benzyl benzoate) (%) 0 97.4 99.7 1 92.0 99.7 2 88.3 99.7 3 80.6 99.7 4 70.2 99.7 After about 3 weeks, the product was degraded . 3.3.3. Evaluation of the degradation under vacuum ampoule and under vacuum ampoule /K2CO3 IAN was packed in glass ampoule with volume of 0.3 ml/tube, vacuum tubes, the whole process was carried out at factory X61. The product in ampoule was recorded NMR every 1, 2, 3 and 4 weeks. Results are presented in Table 4. Table 4. % IAN storaged under vacuum ampoule and under vacuum ampoule/K2CO3. Time (weeks) % IAN (vacuum ampoule) % IAN (vacuum ampoule, K2CO3 2 %) Content (benzyl benzoate, %) 0 97.4 97.4 99.7 1 95.7 97.1 99.7 2 93.4 96.9 99.7 3 91.1 96.5 99.7 4 89.5 96.3 99.7 Some common stabilizer were used: sodium phosphate, potassium carbonate, magnesium oxide, pyridine, epoxol 9-5, diphenylamine, etc. Potassium carbonate was used in examination (a cheap, safe and non-toxic stabilizer). Results are shown in Table 4, Figure 2. Approximately (some cases, the relationship is non-linear), it was possible to construct a linear equation of the form y=ax+b. The results showed that the product stored in vacuum ampoule could be used as a drug less than three months, whereas using potassium carbonate as stabilizer (2 %), the shelf life could be up to more than 12 months. Chemistry & Environment V.N. Toan, N.M. Tri, L.H. Minh, “Synthesis and evaluate the degradation of isoamyl nitrite.” 92 Figure 1. Linear regression between content and time (without stabilizer). Figure 2. Linear regression between content and time (stabilizer,K2CO3, 2 %). 4. CONCLUSION By simple way, we synthesized IAN from isoamyl alcohol, 85-88 % yields. The structure of this product was confirmed by modern physical methods such as IR, 1H NMR, 13C NMR and MS spectra. We also evaluated the degradation of product in four conditions: photochemical environment, sealed glass jars, vacumm ampoule with and without stabilizer. The results showed that the best storage condition of IAN was in vacuum ampoule, with stabilizer K2CO3, 2 %. In this condition, IAN shelf life was more than 12 months. REFERENCES [1]. Nguyễn Đình Thành, “Cơ sở các phương pháp phổ ứng dụng trong hóa học”, NXB Khoa học và Kỹ thuật, Hà Nội (2011), 671 tr. [2]. A. N. P. Van Heijst et al, “Antidotes for cyanide poisoning”, In Basic science in toxicology edited by G.N. Volanis, J. Sims, F. Sullivan & P. Turner, Brighton, Taylor & Francis, 1990, pp. 558-566. [3]. A. N. P. Van Heijst et al, “Therapeutic problems in cyanide poisoning”, Clin Toxicol, Vol. 25 (1987), pp. 383-398. [4]. B. J. Jandorf et al, “Therapeutic and prophylactic effect of methemoglobinemia in inhalation poisoning by hydrogen cyanide and cyanogen chloride”, J Indust Toxicol, Vol. 28 (1946), pp. 124-129. [5]. F.R. Sidell, “Medical aspects of chemical and biological warfare”, Department of the Army, United States of America (1997), 722 pages. [6]. J. A. Vick et al, “Studies on cyanide poisoning”, Arch. Int. Pharmacodyn., Vol. 273 (1985), pp. 314-322. [7]. J. L. Way et al, “Cyanide intoxication and its mechanism of antagonism”, Ann Rev Pharmacol Toxicol, Vol. 24 (1984), pp.451-481. [8]. J. L. Way et al, “Recent perspectives on the toxicodynamic basis of cyanide antagonism”, Fundam Appl Toxicol, Vol. 4(1984), pp. 231-239. Research Journal of Military Science and Technology, Special Issue, No.54A, 05 - 2018 93 [9]. J. W. Turczan et al, “NMR analysis of pharmaceuticals XIV: determination of amyl nitrite in its inhalant dosage form”, Journal of pharmaceutical sciences, (1976), pp. 235-238. [10]. S. J. Moore et al, “Antidote use of methemoglobin forming cyanide antagonist in concurrent carbon monoxide/cyanide intoxication”, J. Pharmacol. Exp. Ther., Vol. 242(1987), pp. 70-73. TÓM TẮT NGHIÊN CỨU TỔNG HỢP VÀ ĐÁNH GIÁ MỨC ĐỘ PHÂN HỦY CỦA HỢP CHẤT ISOAMYL NITRIT Isoamyl nitrit (IAN) là một trong các thuốc giải độc đặc hiệu đối với nhiễm độc xyanua. IAN có thể được tổng hợp qua nhiều con đường khác nhau. Đây là một sản phẩm dễ phân hủy, đòi hỏi phải nghiên cứu đánh giá mức độ phân hủy sau khi tổng hợp. Kết quả tổng hợp và đặc trưng hóa lý cũng như kết quả đánh giá mức độ phân hủy của IAN đã được trình bày cụ thể trong bài báo này. Kết quả cho thấy, khi sản phẩm được bảo quản trong ampoul chân không với K2CO3 2 % (w/w) làm chất ổn định có thể làm tăng thời gian bảo quản lên hơn 12 tháng, hiệu suất tổng hợp đạt 85-88 %. Từ khóa: Isoamyl nitrit, Antidot, Xyanua. Received 1st November 2017 Revised 25th February 2018 Accepted 15 thApril 2018 Author affiliations: Institute of Chemistry and Materials, Academy of Military Science and Technology. * Email: vntoanchem@gmail.com.