CN1328253C - Fumaric acid ibutilide synthesis method - Google Patents

Abstract

The present invention relates to a synthesis method for the fumaric acid ibutilide of an antiarrhythmic drug in a third class. Aniline serving as starting raw material reacts to methanesulfonyl chloride to obtain methylsulfonyl aniline, products react to succinic anhydride to obtain 4-oxo-4-(4-methanesulfonamidephenyl) butanoic acid under the existence of methylene chloride and anhydrous aluminium trichloride which is condensed into acid amide with N-ethyl heptyl amine under the existence of diisopropyl carbodiimide and tetrahydrofuran, the acid amide is reduced by lithium aluminum hydride to obtain products, namely ibutilide by using ultrasonic as reactive catalysts, and the ibutilide is salified with fumaric acid to obtain the fumaric acid ibutilide under the existence of the ultrasonic. The overall yield of the synthesis technology of the present invention achieves 60%, reaction yield and conversion rate are high, product purity is larger than 99.8%, raw material is easy to obtain, and the present invention is suitable for industrialized production.

Description

The synthetic method of ibutilide fumarate

(1) Technical field: the present invention relates to a novel synthesis method of a third type antiarrhythmic drug having type I activity, specifically a synthesis method of ibutilide fumarate.

(2) Background technology: Ibutilide (Ibutilide) is a new type III antiarrhythmic drug with type I activity. It is a new ion channel antagonist antiarrhythmic drug developed by Upjohn Company. Arrhythmia drugs. Its molecular formula is: C ₂₀ H ₃₆ N ₂ O ₃ S.1/2C ₄ H ₄ O ₄ , molecular weight: 442.62, chemical name: (±)-N-[4-[4-(ethylheptylamino) -1-Hydroxybutyl] phenyl] methanesulfonamide fumarate, the structural formula is:

It was launched in the United States for the first time in 1996 under the trade name Convert, and its clinical injection is mainly used for the conversion of atrial fibrillation and atrial flutter.

At present, the synthetic method that relates to product ibutilide fumarate of the present invention has:

Document 1 uses methanesulfonylanilide as the starting material, under the catalysis of anhydrous aluminum trichloride, reacts with succinic anhydride to prepare 4-oxo-4-(4-methanesulfonic acid by Friedel-Crafts) Aminophenyl)butyric acid, the product is condensed with N-ethylheptylamine under the catalysis of N,N-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole to form an amide, and the amide is then treated with lithium tetrahydrogen After being reduced to amine, it is then salted with fumaric acid to obtain ibutilide fumarate product, a total of four steps of reaction, the total yield is 17.6%, calculated using methanesulfonyl anilide as the starting material, but there is no methanesulfonate in China Acid aniline industrial production.

Document 2 uses aniline as a starting material, and pyridine, which is highly toxic and difficult to handle, is used as an acid-binding agent to react with methanesulfonyl chloride to obtain methanesulfonyl anilide. The second step is to react with succinic anhydride under the catalysis of anhydrous aluminum trichloride to prepare 4-oxo-4-(4-methanesulfonamidophenyl)butanoic acid. In the third step, N,N-dimethylformamide, which is highly toxic and difficult to handle, is used as a solvent, and N,N-dicyclohexylcarbodiimide is used as a shrinking agent, and the obtained product amide is not easy to handle. Recrystallized from ethyl acetate and n-hexane. In the fourth step, the recrystallized product is reduced with lithium tetrahydrogen to obtain the product ibutilide, and in the fifth step, a salt-forming reaction is carried out to generate the ibutilide fumarate product with a yield of 35%, calculated with aniline as the starting material.

(3) Summary of the invention: the task of the present invention is to provide a kind of method of synthesizing ibutilide fumarate.

The structural formula of ibutilide fumarate of the present invention is as follows:

The following is the synthesis method of the compound of the present invention, wherein the starting materials aniline and methanesulfonyl chloride are commercially available. The synthetic method of the present invention comprises:

(1) react aniline with methanesulfonyl chloride to obtain compound methanesulfonyl anilide;

(2) Methanesulfonylanilide and succinic anhydride were carried out in the presence of dichloromethane and anhydrous aluminum trichloride to carry out the Fourier reaction, and heated to reflux for 24 hours to obtain 4-oxo-4-(4-methanesulfonamide phenyl) butanoic acid;

(3) Use tetrahydrofuran as a solvent, N-hydroxysuccinimide as an antioxidant, diisopropylcarbodiimide as a shrinking agent, and 4-oxo-4-(4-methylsulfonamidophenyl) butanoic acid Reaction with N-ethylheptylamine to obtain product N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl) butyramide;

(4) Using tetrahydrofuran as a solvent, under the ultrasonic wave as a catalyst for the reaction, reduce the product N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl) with lithium tetrahydrohydride Butanamide, obtain product ibutilide;

(5) Under the catalyzer of the reaction, the ibutilide and fumaric acid are salt-formed to obtain ibutilide fumarate.

The third step reaction product N-ethyl-N-heptyl-4-oxo-4-(4-methanesulfonamidophenyl) butyramide of the present invention, first use 480ml volume ratio to be 1: 1 Ethyl acetate and sherwood oil are recrystallized, then enter (4) step reaction.

Compared with the prior art, the present invention has produced substantive features and remarkable progress:

(1) In the first step reaction, there is no supply of methanesulfonamide industrial products in China at present, so the reaction of aniline and methanesulfonyl chloride is used to prepare methanesulfonyl anilide. In this reaction process, aniline is used as both a reactant and an acid Avoid using pyridine as the acid-binding agent, which is more toxic and difficult to handle, and the yield of this step reaches 95%, mp: 92 ° C ~ 93 ° C (document 2: yield 80.5%, mp: 91 ° C ~ 93°C; Document 3: Yield 81.1%, mp: 93°C～94°C);

(2) In the second step reaction, the literature uses carbon disulfide as a solvent, and anhydrous _ALCl3 catalyzes the Fournier reaction, and the yield is lower, and the present invention has adopted methylene chloride as a reaction solvent, in anhydrous aluminum chloride Under catalysis, heat and reflux for 24 hours to react with succinic anhydride to obtain 4-oxo-4-(4-methanesulfonylaminophenyl)butyric acid, and the yield of this step reaches 98%, mp: 200°C～ 202°C (document 1: yield 40%, mp: 198°C-200°C; document 2: yield 96.5%, mp: 199°C-200°C);

(3) In the third step reaction, document 1 uses silica gel column chromatography to separate the product, document 2 uses N,N-dimethylformamide which is more toxic and difficult to handle as solvent, and N,N-dimethylformamide with poor shrinkage Dicyclohexylcarbodiimide is a shrinking agent, and the obtained product amide is not easy to handle, and the cost is also relatively high. The present invention uses tetrahydrofuran as a solvent, diisopropylcarbodiimide as a shrinking agent, and the obtained product is recrystallized with ethyl acetate and petroleum ether to obtain N-ethyl-N-heptyl-4- Oxo-4-(4-methylsulfonamidophenyl)butanamide, the yield reaches 83.6%, mp: 100°C～102°C (document 1: yield 75%, mp: 100°C～102°C; document 2 : Yield 77.3%, mp: 102 ° C ~ 103 ° C);

(4) The principle of sonochemistry is adopted, and an ultrasonic generator is added in the fourth step of the reaction process, and the ultrasonic wave is used as a catalyst for the reaction, and the product ibutilide is obtained by reduction with lithium tetrahydrogen, and the yield of the product reaches 92%. (document 1: yield 87%; document 2: yield 83.3%);

(5) In the fifth step reaction, the principle of sonochemistry is adopted, and ibutilide and fumaric acid are carried out in the presence of ultrasonic waves to carry out a salt-forming reaction, which can reach the crude product of ibutilide fumarate with a higher yield (yield 91.6%), refining rate 85%, mp: 117°C-119°C (document 1: yield 85.5%, mp: 117°C-119°C; document 2: refining rate 81.8%, mp: 117°C-119°C);

(6) The total yield of the synthetic method of the present invention can reach about 60% (calculated with aniline as the starting material), the product purity is greater than 99.8%, and the reaction conditions in the whole reaction process are mild, the cost is reduced, and it is more conducive to industrial production.

(4) Specific embodiments: the following examples can make those skilled in the art understand the present invention more comprehensively, but do not limit the present invention in any way.

Ibutilide fumarate of the present invention is synthesized in the following way:

(1) Preparation of methanesulfonyl anilide:

Raw materials: aniline, methanesulfonyl chloride

synthetic route:

In the three-necked flask, add aniline (516g, 5.55mol), toluene 500ml, start mechanical stirring, add methanesulfonyl chloride (215ml, 2.78mol) dropwise, the internal temperature of the reaction does not exceed 60°C, after dropping, continue stirring for 3-4 After 1 hour, a large amount of solids precipitated out, and it was impossible to stir, so the stirring was stopped and left overnight. Stir slowly, filter, collect the solid, wash with water to obtain a white solid, and vacuum-dry under reduced pressure to obtain 456 g of the product methanesulfonylanilide, with a yield of 95% (calculated using aniline as the starting material).

Intermediate control:

Melting point: 92°C-93°C, literature 3 value: 93°C-94°C;

Developing solvent: ethyl acetate: petroleum ether = 1:1, R _f 0.6-0.7.

(2) Preparation of 4-oxo-4-(4-methanesulfonamidophenyl) butanoic acid:

synthetic route:

In a three-necked flask equipped with mechanical stirring, reflux condenser and thermometer, add 500ml of dichloromethane, add 360g of anhydrous aluminum trichloride under stirring, cool to about 0°C in an ice bath, add succinic anhydride (72g, 0.72 mol), after stirring for 30 minutes, add 120g methanesulfonanilide in two times, stir for 30 minutes, remove the ice bath, and heat to reflux in an oil bath for 24 hours. Stop heating, let it stand, pour off the upper layer of dichloromethane, under vigorous stirring, slowly pour the red sticky residue into ice water, let it stand, and filter with suction to obtain a white solid, wash it with water, add 10% The sodium carbonate solution was adjusted to a pH of about 10, left to stand, and suction filtered, and the filtrate was extracted with dichloromethane (200ml×2), and the aqueous layer was adjusted to a pH of about 1 with concentrated hydrochloric acid, left to stand, and suction filtered to obtain a white solid. Wash with water (500ml×2). Repeat the above operations for the obtained solid to obtain a white solid, which was dried in vacuo at 90° C. to obtain 182.2 g of the product 4-oxo-4-(4-methanesulfonamidophenyl)butanoic acid, with a yield of 98%.

Intermediate control:

Melting point: 200°C-202°C Literature 1 value: 198°C-200°C.

(3) Preparation of N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl)butanamide:

synthetic route:

In 4-oxo-4-(4-methanesulfonamidophenyl)butanoic acid (54g, 0.20mol), add anhydrous tetrahydrofuran (350ml), antioxidant N-hydroxysuccinimide 3g, ice-bath cooling , diisopropylcarbodiimide (DIC) (38 g, 0.30 mol) was added dropwise under stirring, the ice bath was removed, and the reaction was stirred for 1 hour. N-ethylheptylamine (35 g, 0.24 mol) was added dropwise, and the reaction was continued at room temperature for 24 hours. Suction filtration, wash the filter cake with a small amount of tetrahydrofuran, combine the tetrahydrofuran solution, evaporate to dryness under reduced pressure, dissolve the residue in ethyl acetate (600ml), wash with 5% hydrochloric acid (200ml), 5% sodium carbonate solution (200ml) and saturated salt successively Washed with water (150ml×2), dried over anhydrous sodium sulfate. Evaporate to dryness under reduced pressure to obtain a yellow solid, which was recrystallized from ethyl acetate:petroleum ether (480ml, 1:1, v/v) to obtain a yellowish white solid—N-ethyl-N-heptyl-4-oxo - 66 g of 4-(4-methylsulfonamidophenyl) butyramide, yield 83.6%.

Intermediate control:

Melting point: 100°C-102°C, document 1 value: 100°C-102°C

Developing agent: pure ethyl acetate R _f 0.6～0.7

(4) Preparation of ibutilide:

synthetic route:

Put the three-necked flask into an ultrasonic generator device, add anhydrous tetrahydrofuran (200ml) in the three-necked flask cooled by an ice bath, add LiAlH ₄ (14.5g, 0.38mol), add dropwise amide (50.0g , 0.126mol) of anhydrous tetrahydrofuran (500ml) solution, control the drop rate, so that the reaction temperature is not higher than 5 ° C, after the drop is completed, continue for 1 hour, remove the ice bath, and stir at room temperature for 12 hours. At 20°C, 600 ml of 0.5 mol sodium potassium tartrate was slowly added dropwise, and stirring was continued for 4-5 hours after dropping, and left to stand overnight. Suction filtration, wash the filter cake (100ml×3) with ethyl acetate, extract the filtrate with ethyl acetate (400ml×4), combine the organic phases and wash with saturated brine (200ml×2), dry with anhydrous sodium sulfate, reduce Evaporate under pressure to obtain a light yellow oil, which was eluted with chloroform, chloroform:methanol:ammonia=100:5:0.5, chloroform:methanol:ammonia=100:10:1 through column chromatography, and the product was collected and concentrated to shallow Yellow oil, the oily product was dissolved in 300ml of ether, washed with 100ml of saturated sodium bicarbonate and saturated sodium chloride, dried, and concentrated to give 44.7g of the product light yellow oil (ibutilide), with a yield of 92%.

Intermediate control:

Developing solvent: chloroform:methanol=5:1, R _f 0.3-0.4, single spot

(5) Preparation of ibutilide fumarate:

synthetic route:

Dissolve ibutilide (34g, 0.089mol) in absolute ethanol (150ml) in a device equipped with an ultrasonic generator, add fumaric acid (4.60g, 0.040mol) in ethanol (200ml) and stir at room temperature 0.5h. Ethanol was distilled off under reduced pressure, acetone (150ml) was added to the residue, heated to reflux until dissolved, and activated carbon was decolorized. The filtrate was cooled to room temperature and refrigerated overnight for crystallization. Filter and wash the solid with 50ml of acetone to obtain a crude white solid. Heat and dissolve with 200ml of acetone, decolorize and recrystallize with activated carbon to obtain ibutilide fumarate as a white powder, and vacuum-dry at 50°C to obtain 33.8g of pure product with a refining rate of 85%.

QC:

Melting point: 116°C-117°C, literature 1 value: 117°C-119°C

Through elemental analysis, infrared absorption spectrum (IR), ultraviolet absorption spectrum (UV), nuclear magnetic resonance hydrogen spectrum ( ¹ HNMR) and carbon spectrum ( ¹³ CNMR), mass spectrum, determination of powder X-ray diffraction patterns, the results are analyzed and compared with The chemical structure is consistent, the sample is also consistent with the reference substance, and is also consistent with the results reported in the literature.

Elemental analysis: C ₂₀ H ₃₆ N ₂ O ₃ S.1/2C ₄ H ₄ O ₄ , molecular weight: 442.62 Theoretical value (%): C (59.70%), H (8.65%), N (6.33%), S (7.24%); Found (%): C (59.71%), H (8.66%), N (6.50%), S (7.17%). The elemental analysis results show that the elemental analysis results of the sample and the reference substance are basically consistent, and the error is less than 0.3% compared with the theoretical calculation value.

Melting point determination, elemental analysis, ultraviolet absorption spectrum (UV), infrared absorption spectrum (IR), hydrogen nuclear magnetic resonance spectrum ( ¹ H-NMR) (H-HCOSY) and carbon nuclear magnetic resonance spectrum were carried out on ibutilide fumarate ( ¹³ C-NMR), decoupling carbon nuclear magnetic resonance spectrum (DEPT), C-HCOSY spectrum, mass spectrum and powder X-diffraction determination, the comprehensive analysis is as follows:

1. Confirmation of the molecular formula of the sample (C ₂₀ H ₃₆ N ₂ O ₃ S·1/2C ₄ H ₄ O ₄ )

a. The elemental analysis results show that the C, H, N, and S percentage compositions of the sample are 59.71%, 8.66%, 6.50%, and 7.17%, respectively, and the calculation of the C, H, N, and S compositions of ibutilide fumarate The value errors are all less than 0.3%, and the elemental composition of the sample is consistent with that of ibutilide fumarate molecule;

b. Proton NMR spectrum (CD ₃ OD is the solvent, TMS is the internal standard) gives 35 proton absorption peaks in total, consistent with the protons contained in the molecular structure of ibutilide fumarate (active protons (-COOH, OH , NH except).

c. The carbon NMR spectrum gives a total of 20 carbon absorption peaks, of which, due to the symmetry between the benzene ring and the fumaric acid structure in the structure, only 6 carbon atom absorption peaks are given at 10 carbon atoms, which is different from that of ibutilide fumarate The molecular structure matches.

d. The decoupled carbon NMR spectrum shows that the sample has 3 quaternary carbon atoms, 10 methylene carbon atoms, 4 methine carbon atoms (representing 7 carbon atoms), and 3 methyl carbon atoms. The decoupled carbon NMR spectrum of the ibutilide maleate molecule is consistent.

e. The molecular ion peak of ibutilide determined by EI mass spectrometry is m/z384.2352, which is consistent with the molecular weight of ibutilide in the sample.

In summary, the molecular composition of the sample was determined to be C ₂₀ H ₃₆ N ₂ O ₃ S·1/2C ₄ H ₄ O ₄ .

2. Confirmation of molecular structure

2.1 Confirmation of the structural part of phenylcyclomethanesulfonamide

a. In the IR spectrum, 3383.4cm ^-1 shows the existence of NH stretching vibration, 3099.8 and 3025.7cm ^-1 show the existence of aromatic hydrogen, 1513.0 and 1452.4cm ^-1 show the existence of benzene ring skeleton, and 838.6cm ^-1 shows the existence of benzene ring as 1, 4 substitution type, 1358.1, 1150.6cm ^-1 are characteristic absorptions of asymmetric and symmetric stretching vibrations of the sulfonyl group in the sulfonamide group, and the infrared spectrum preliminarily shows the existence of the benzene ring methanesulfonamide structure.

b. Proton NMR spectrum δ7.345 (2H, d), δ7.229 (2H, d), δ2.931 (3H, s) is consistent with the structure of benzene ring methanesulfonamide.

c. C NMR spectrum δ121.882, 128.203, 138.750, 142.632, 39.343 are the carbon atoms of the benzene ring and the methyl carbon atoms in methanesulfonamide, among which δ121.882 and 128.203 actually represent 4 carbons due to the influence of structural symmetry factors Atom, decoupled carbon NMR spectrum shows that δ121.882, 128.203 four carbon atoms are CH carbon atoms, δ39.343 is methyl carbon atom, carbon NMR spectrum, decoupled carbon NMR spectrum, H-HCOSY, C- The HCOSY spectrum is partly consistent with the structure of phenylcyclomethanesulfonamide.

d. In the UV spectrum, λ _max =228nm is consistent with the K absorption band of the substituted benzene ring.

Based on comprehensive analysis of IR, ¹ H-NMR, ¹³ C-NMR, DEPT and UV spectra, it was preliminarily confirmed that there is a phenylcyclomethanesulfonamide structure in the structure of ibutilide fumarate.

2.2 Confirmation of 4-(ethyl-heptyl-amino)-1-hydroxy-butyl moiety

a. 3383.4cm ^-1 in the IR spectrum shows the presence of hydroxyl groups, 2953.5～2857.0cm ^-1 shows the presence of methyl groups and methylene groups in this structural part, 1466.4cm ^-1 shows the presence of methylene groups, and the infrared spectrum initially shows that there are Hydroxyl, methyl, methylene moieties are present.

b. Proton NMR spectrum δ0.913(3H, t), 1.245(3H, t), 1.323～1.359(8H, m), 1.60～1.90(6H, m), 2.967～3.096(6H, m), 4.670 (1H,t), showing that the moiety has 27 protons (except the active proton OH),

c. NMR carbon spectrum δ9.276, 14.578, 21.742, 23.736, 24.951, 27.869, 30.072, 32.922, 37.076, 48.671, 53.398, 53.451, 73.749, showing that this part of the structure has 13 carbon atoms, DEPT spectrum shows that there are 2 CH ₃ carbon atoms, 10 CH ₂ carbon atoms, 1 CH carbon atom, δ73.749 shows the carbon atom connected to the hydroxyl group.

d. H-HCOSY and C-HCOSY spectra show that the above-mentioned hydrogen-hydrogen correlation and carbon-hydrogen correlation are all consistent with this structure.

e. m/z 156.1609 in the EIMS mass spectrum is a fragment ion formed by β-cleavage of N atom in this part of the structure containing aliphatic amine.

Comprehensive analysis of IR, ¹ H-NMR, ¹³ C-NMR, DEPT spectrum, H-HCOSY, C-HCOSY spectrum and mass spectrum confirmed the existence of 4-(ethyl-heptyl-amino group in the structure of ibutilide fumarate )-1-hydroxy-butyl moiety.

2.3 Confirmation of the structure of fumaric acid

a. 1613.4 and 1573.9cm ^-1 in the IR spectrum show the presence of double bonds and carboxylate groups

b. The H NMR spectrum δ6.658 (1H, s) is an ethylenic bond proton, the C NMR spectrum δ137.393 is a double bond carbon atom, the DEPT spectrum shows a CH carbon atom, and δ174.698 shows a carbonyl carbon atom.

c. m/z 116.0572 in the EIMS spectrum is consistent with the molecular weight of fumaric acid.

Comprehensive analysis of IR, ¹ H-NMR, ¹³ C-NMR, and EIMS spectra indicated that the fumaric acid structure existed in the sample.

2.4 The powder X-diffraction results show that the crystal form of the sample is completely consistent with that of the reference substance. Since there is no literature report on the existence of polymorphs of ibutilide fumarate, the powder X-diffraction data of the sample is recorded as the sample nature characteristics.

2.5 Identification of product purity of the present invention

The detection and non-aqueous titration method are carried out according to the chromatographic conditions of the application materials, and the purity is >99.8%. Put 25g of refined ibutilide fumarate sample in a 250ml round-bottomed flask, add 75ml of acetone, heat to dissolve, add 5g of activated carbon, heat and reflux for 30min, filter while it is hot, place it under cooling, crystallize and filter, use a small amount of solid Washed twice with acetone, drained, and dried under reduced pressure at 50°C for 24 hours to constant weight to obtain 22.4 g of the reference substance for structure confirmation, the purity of which was determined by HPLC to be 99.8%.

The present invention provides the synthetic method of the example, the total yield of ibutilide fumarate is: 95%×98%×83.6%×92%×85%=60.86%, calculated with aniline as the starting material.

references

1: Hester JB, Gibson JK, Cimini MG.N-[(ω-Amino-1-hydroxyalkyl)phenyl]methanesylfonamide derivatives with class III antiarrhythmic activity[J].J.Med.Chem.1991,34(1):308- 315

2: Chinese Journal of Pharmaceutical Industry 2003, 34(5): 209-210

3: Hester JB, Gibson JK, Antiarrhythmic methanesulfonamides [P]. US 5155268, 1992-08-13 (CA 1986, 104: 109269m)

Claims (2)

1. a kind of synthetic method of the third class antiarrhythmic drug ibutilide fumarate with I type activity concurrently, it is characterized in that: (1) react aniline with methanesulfonyl chloride to obtain compound methanesulfonyl anilide; (2) Methanesulfonylanilide and succinic anhydride were carried out in the presence of dichloromethane and anhydrous aluminum trichloride to carry out the Fourier reaction, and heated to reflux for 24 hours to obtain 4-oxo-4-(4-methanesulfonamide phenyl) butanoic acid; (3) Use tetrahydrofuran as a solvent, N-hydroxysuccinimide as an antioxidant, diisopropylcarbodiimide as a shrinking agent, and 4-oxo-4-(4-methylsulfonamidophenyl) butanoic acid Reaction with N-ethylheptylamine to obtain product N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl) butyramide; (4) Using tetrahydrofuran as a solvent, under the ultrasonic wave as a reaction catalyst, reduce N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl) butyl with lithium tetrahydrogen Amide, obtain product ibutilide; (5) Under the catalyzer of the reaction, the ibutilide and fumaric acid are salt-formed to obtain ibutilide fumarate. 2. method according to claim 1, it is characterized in that in the 3rd step reaction, N-ethyl-N-heptyl-4-oxo-4-(4-methylsulfonamidophenyl) butanamide first Recrystallize with 480ml of ethyl acetate and sherwood oil with a volume ratio of 1:1, then enter step (4) for reaction.