1. Prior to performing each reaction, a thorough hazard analysis and risk assessment should be carried out with regard to each chemical substance and experimental operation on the scale planned and in the context of the laboratory where the procedures will be carried out. Guidelines for carrying out risk assessments and for analyzing the hazards associated with chemicals can be found in references such as Chapter 4 of "Prudent Practices in the Laboratory" (The National Academies Press, Washington, D.C., 2011; the full text can be accessed free of charge at https://www.nap.edu/catalog/12654/prudent-practices-in-the-laboratory-handling-and-management-of-chemical . See also "Identifying and Evaluating Hazards in Research Laboratories" (American Chemical Society, 2015) which is available via the associated website "Hazard Assessment in Research Laboratories" at https://www.acs.org/content/acs/en/about/governance/committees/chemicalsafety/hazard-assessment.html. In the case of this procedure, the risk assessment should include (but not necessarily be limited to) an evaluation of the potential hazards associated with ((2S, 3R)-Noscapine, concentrated nitric acid, cotarnine, acetophenone, methanol, potassium hydroxide , acetone, ethyl acetate, 1,4-diiodobenzene and formic acid as well as the proper procedures for flame drying glassware and vacuum filtration.
2. (S,R)-Noscapine (97%), concentrated nitric acid (69% (wt/wt); KOH pellets (EMPLURA brand by EMD Millipore), acetophenone (99%, ACS reagent grade); 1,4-diiodobenzene (99%), deuterated chloroform (99.8 atom% deuterated); and glass-backed, extra-hard layer TLC plates (60 Å, 250 μm thickness containing F-254 indicator by EMD Millipore) were all purchased from Sigma-Aldrich and were used as received.
3. The 18% (v/v) aqueous nitric acid stock solution is obtained from concentrated nitric acid (69% (wt/wt) in H2O) by dilution (N1V1=N2V2). To a 250 mL volumetric flask, 170 mL of distilled water is added, followed by 65.2 mL of 69% concentrated nitric acid and an additional 14.8 mL of distilled water to bring the meniscus of the solution to the appropriate line on the volumetric flask.
4. Noscapine is not fully soluble in the reaction medium at room temperature, but upon heating and reaching an internal temperature of 38 °C (ca. 5-10 min after immersion in the oil bath) full dissolution occurs.
5. To avoid evaporation of nitric acid solution (which may change the HNO3 concentration), a condenser with a nitrogen inlet and cooled water circulation is employed until the reaction is complete.
6. The temperature is maintained below 50 °C, and the submitters state that care should be taken to maintain the reaction temperature between 50-55 °C. The submitters reported that the reaction temperature increases to 80 °C upon the formation of the precipitate; however, the checkers did not observe this temperature increase, as the maximum internal temperature reached was 48 °C.
7. These color changes occur over a 10-30 minute time period and a white precipitate (by-product) is observed after 30-35 minutes of heating (Figure 8).
Figure 8. Color change throughout heating the Noscapine in HNO3, A. Before heating, B. After 10 minutes, C. After 15 minutes, D. Color change and initial precipitate formation after 30 minutes
8. The precipitate begins to form ca. 20 min into the reaction.
9. The yellow color of the solution is observed at the extreme end of the reaction, at which point, no more precipitate is observed to form. The TLC analysis was performed on an acetone solution of the starting material and an aliquot (~2 drops) of the reaction mixture using a solution of EtOAc/MeOH (1:3) containing 1% formic acid as eluent and visualized with ceric ammonium molybdate (CAM) stain. Under these conditions, the product was observed to have an Rf=0.28 (Figure 9).
Figure 9. TLC of the noscapine with nitric acid and stained with CAM stain (Left: Front of TLC plate, Right: Back of TLC plate); Lane 1: starting material; Lane 3: reaction mixture; Lane 2: co-spot
10. Preparation of 40% (wt/wt) KOH solution: In a 200 mL volumetric flask that contains 100 mL of distilled water, 80 g of solid KOH pellets was added in two 40 g portions. After dissolution, additional distilled water is added to bring the meniscus of the solution to the appropriate line on the volumetric flask.
11. A precipitate is formed after addition of potassium hydroxide.
Figure 10. Precipitate crashing out of solution at pH 11 upon completion of reaction
12. Yellow crystalline solid. mp 126-128 °C; 1H NMR pdf(600 MHz, CDCl3) δ: 2.33-2.36 (m, 1H), 2.59 (s, 3H), 2.60-2.64 (m, 1H), 2.84-2.91 (m, 1H), 3.06 (td, J =11.5, 4.2 Hz, 1H), 4.04 (s, 3H), 5.41 (s, 1H), 5.88 (s, 2H), 6.31 (s, 1H); 13C NMR pdf(150 MHz, CDCl3) δ: 29.0, 41.1, 43.6, 59.8, 79.2, 100.9, 102.7, 122.5, 129.0, 134.2, 140.6, 149.1; HRMS (ESI) C12H14NO3 [M-wOH]+: calc. 220.0968, found 220.0967; FTIR (neat, ATR) 3082, 2953, 2870, 1617, 1477, 1445, 1261, 1091, 1036, 1091, 1036, 982, 970, 932, 791 cm-1; Rf=0.23, eluent: EtOAc: MeOH (3:7) with 1% formic acid. The purity of the compound was calculated by qNMR pdf with a delay of relaxation of 30 seconds using 11.4 mg of 1,4-diiodobenzene (purity 99%) and 8.2 mg of cotarnine (1) and was found to be of 99.4% purity.
13. A reaction performed on half scale provided 5.02 g (88%) of the identical product.
14. In most of the cases, the product precipitated during 10 min of stirring. If a precipitate does not appear in 5-10 min, precipitation can be induced via addition of a "seed crystal". This material is prepared in a separate 5-mL vial by reacting 0.5 mmol of cotarnine and 0.5 mmol of acetophenone in 0.5 mL methanol. A crystal is removed from the solution by use of a spatula and placed into the reaction mixture to facilitate crystallization.
15. White crystalline solid. mp 124-125 °C; 1H NMR pdf(600 MHz, CDCl3) δ: 2.37 (s, 3H), 2.41-2.47 (m, 1H), 2.75-2.80 (m, 1H), 2.85-2.92 (m, 1H), 3.10-3.27 (m, 3H), 3.94 (s, 3H), 4.47 (dd, J = 8.6, 3.9 Hz, 1H), 5.86 (s, 2H), 6.32 (s, 1H), 7.46 (t, J = 7.5 Hz, 2H), 7.54 (t, J = 6.9 Hz, 1H), 8.01 (d, J = 8 Hz, 2H); 13C NMR pdf(150 MHz, CDCl3) δ: 23.9, 42.0, 43.7, 44.6, 55.2, 59.3, 100.6, 102.7, 122.4, 128.2, 128.4, 128.6, 132.7, 134.0, 137.4, 140.3, 147.9, 199.0; HRMS (ESI) C20H22NO4, [M+H]+: calc. 340.1549, found 340.1546. FTIR (neat, ATR) 2915, 2838, 2794, 1678, 1619, 1597, 1498, 1318, 1283, 1257, 1062, 1041, 766, 696 cm-1; Rf=0.84, eluent: EtOAc/MeOH (3:1). The purity of the compound was calculated by qNMR pdf with a delay of relaxation of 30 seconds using 12.5 mg of 1,4-diiodobenzene (purity 99%) and 10 mg of product 3a and was found to be of 98.1% purity.
16. A reaction performed on half scale provided 2.78 g (82%) of the identical product.