1-(4-aminophenyl)-3-morpholinopiperidin-2-one: A new process related impurity of Apixaban- An anticoagulant drug

1-(4-aminophenyl)-3-morpholinopiperidin-2-one: A new process related impurity of Apixaban- An anticoagulant drug | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article 1-(4-aminophenyl)-3-morpholinopiperidin-2-one: A new process related impurity of Apixaban- An anticoagulant drug Vipin Kumar, Sachin Kumar Sharma, Govind Singh, Shyambaran Dhuriya, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3992931/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this report, we describe the identification of new process related impurity observed during the preparation of Apixaban-An anticoagulant drug. As a part of the investigation, we isolated the impurity by column chromatography with > 96% purity and analyzed the sample of impurity by LC-MS, IR and NMR spectroscopy. The LC-MS analysis coupled with IR and NMR analysis suggested that the impurity is 1-(4-aminophenyl)-3-morpholinopiperidin-2-one, generated during preparation of Apixaban intermediate (APN-05). The impurity is a reduced form of APN-05 possibly formed due to reduction of ring double bond of dihydropyridin-2(1H)-one moiety of APN-05. Process related impurity Intermediate Isolation Synthesis Active Pharmaceutical Ingradients Figures Figure 1 Figure 2 Introduction Anticoagulants are commonly used for the prevention and treatment of thromboembolic disorders [ 1 ]. Warfarin, heparin and their derivative are traditional anticoagulants [ 2 , 3 ]. These anticoagulants are associated with several drawbacks including drug-drug and drug-food interactions, parenteral administration, and routine monitoring, suggesting the urgent need for the development of safe and efficacious oral anticoagulants [ 4 ]. The direct inhibition of factor Xa is a newly identified mechanism for controlling coagulation [ 5 ]. In this category, apixaban has evolved as a safe, efficacious, and convenient molecule for controlling coagulation [ 6 ]. Apixaban was approved for medical use in the European Union in May 2011 [ 7 ]. A new drug application (NDA) for the approval of apixaban was submitted to the US Food and Drug Administration (FDA) by Bristol Myers Squibb (BMS) and Pfizer jointly after the conclusion of the ARISTOTLE clinical trial in 2011 [ 8 ]. On 13 March 2014 it was approved for the additional indication of preventing deep vein thrombosis and pulmonary embolism in people who have recently undergone knee or hip replacement [ 9 , 10 ]. Apixaban is sold under the brand name Eliquis as an anticoagulant medication 7 . Other examples of direct factor (FXa) inhibitors include rivaroxaban [ 11 ], edoxaban,[ 12 ]. and betrixaban (Fig. 1), which have gained approval for several indications, most notably, for the prevention and treatment of venous thromboembolism (VTE) and for the prevention of stroke in patients with atrial fibrillation hepatic impairment. Various chemical syntheses of apixaban have been reported in the literature [ 13 – 17 ]. Among them, the most common synthesis is that reported by Bristol Mayor Squibb, an innovator of apixaban [ 6 ]. The innovator route involves the use of p-amino iodobenzene as a key starting material (Scheme 1 ,a). The key step in the synthesis of Apixaban involves C-N bond formation using Cu as a catalyst followed by ammonolysis to obtain apixaban. This route of synthesis of apixaban suffers from the disadvantages of (a) use of costly p-aminoiodobenzene, (b) a C-N coupling reaction involving costly metal catalyst, and (c) poor overall yield and thus has limitation of commercially viable. A modified syntheses of apixaban was reported that involved the use of p-amino nitrobenzene as a key starting material instead of p-iodo nitrobenzene [ 14 ] (Scheme 1 , b). The key advantages of this route are its low cost and catalyst free condition for C-N bond formation. Process related impurities are the impurities observed in drug substance and get generated in a manufacturing process [ 18 – 20 ]. The investigation of any process related impurity in drug substances is a crucial aspect of API process research and development [ 21 – 23 ]. Knowing the chemical structure of process related impurities is important for assessing their toxicological nature and understanding the mechanism of impurity formations [ 24 , 25 ]. This information help process scientists to categorize impurities according to the ICH classification, which further helps to define the limit of the impurity and its control strategy [ 26 , 27 ]. This work present, the isolation and characterization of new process related impurity, observed during the preparation of apixaban. This work would be helpful to the pharmaceutical scientist working on the development of Apixaban as an active pharmaceutical ingredient. Results and discussion 1. Apixaban synthesis We have developed a process for the preparation of apixaban by slight modification of literature known process. The optimized route of synthesis of apixaban consists of eleven chemical transformations (Scheme 1 ). The synthesis starts with the acylation of p-nitroaniline with 5-chlorovaleroyl chloride followed by intramolecular cyclization to obtain APN-02. Chlorination of APN-02 using PCl 5 followed by reaction with morpholine led to the formation of APN-04. A reduction of APN-04 using Raney Ni lead to the formation of APN-05. APN-05 again undergoes acylation and intramolecular cyclization to form APN-07, which is an intermediate required for the synthesis of apixaban. Finally, APN-07 and APN-08 react with each other to form APN-09 as an insitu intermediate, which leads to the formation of apixaban upon ammonolysis. 2. Identification of unknown impurity During the preparation of APN-05 from APN-04 by optimized process (Scheme 2 ), an unknown impurity was observed at 0.30 RRT in HPLC chromatograph of reaction monitoring samples. In all the R & D batches, this impurity was observed upto a level of ~ 1.0%. However, during scale up studies in pilot plant, the same impurity was found to be present in a reaction mass upto ~ 11.0%. Hence, we became interested to know the structure of impurity in order to understand the root cause of its formation and possible control strategy during scale up studies. The reaction monitoring data of APN-05 of R & D batches and pilot plant batches are provided in Table 1 . Table 1 Reaction monitoring profile of APN-04 to APN-05 reaction. Batch No. Source Batch size Reaction monitoring by HPLC (%) a % Conversion to APN-05 Unreacted APN-04 (%) Unknown impurity at 0.30 RRT (%) 001 R & D 10.0 g 96.70 0.70 0.88 002 R & D 10.0 g 93.46 0.65 0.98 003 R & D 10.0 g 94.30 0.65 1.0 004 R & D 10.0 g 96.22 0.72 0.88 005 R & D 10.0 g 96.72 0.74 0.88 006 R & D 10.0 g 92.89 0.67 1.0 007 R & D 10.0 g 93.78 0.65 1.0 011 Pilot plant 20 kg 93.03 0.02 4.37 012 Pilot plant 20 kg 86.69 0.15 11.43 a 3–4 kg/cm 2 H 2 pressure, 50–60°C temperature, 4–6 h. 3. Isolation of unknown impurity Next, we focused on the isolation of the impurity for its characterization. We isolated the impurity first dissolving crude APN V in water. The filtered mother liquor was analyzed which indicated the higher percent of impurity. The mother liquor was evaporated to get residue which was subjected to flash chromatography to get impurity with ~ 95% purity. A pure sample of impurity was used for analysis by LC-MS spectrometry, IR, 1 H NMR and 13 C NMR spectroscopic techniques. 4. Characterization of impurity A systematic characterization study of impurity was carried out using spectrometric (LC-MS) and spectroscopic techniques ( 1 H and 13 C NMR). The LC-MS analysis of impurity showed m/z 276. The mass spectrum for parent compound i.e. APN-05 shows m/z 274 correspond to (M + H) + ion peak. Thus, the mass spectrum of impurity suggest the presence of two additional proton in the structure of APN-05. The structural feature of APN-05 suggest that, there is also a possibility of reduction of keto as well as double bond as a part of 2-piperidone moiety (Fig. 2 ).Then we analyzed the IR spectrum of an impurity. A stretching band at 1632.33 cm − 1 indicate the presence of ketone functional group. The IR spectrum rule out the possibility of reduction of ketone moiety and hint toward the possibility of reduction of double bond which is part of 2-piperidone moiety. Next, we analyzed the 1 H NMR spectrum of impurity and compared it with parent compound i.e. APN-05. The 1 H NMR spectrum of APN 05 showed a triplet at 5.61 ppm due to the presence of one vinylic proton (-C = C-H) which is coupled with two protons present at the adjacent carbon. In the 1H NMR spectrum of impurity, this signal was found to be absent and an additional triplet of one proton was observed at 3.2 ppm. This pattern of 1 H NMR spectrum clearly suggest the reduction of ring double bond of 5,6-dihydropyridin-2(1H)-one moiety of APN-05 leading to an impurity with piperidin-2-one moiety. Rest of the 1H NMR pattern of impurity is same as that of APN-06. The 13 C NMR of impurity also suggest that the ring double bond of 5,6-dihydropyridin-2(1H)-one moiety of APN-05 is reduced and thus only four signals in aromatic region was observed against six signals due to six aromatic protons in APN-05. Out of six signals, four signals are due to aromatic ring carbon and two signals are due to double bond carbon of 5,6-dihydropyridin-2(1H)-one moiety present in APN-05. And thus in 13 C NMR spectrum of impurity only four signals are observed due to the aromatic ring. The 13 C NMR spectrum of impurity showed a signal due to carbonyl carbon at 169.5 ppm thus rule out the possibility of reduction of carbonyl group. All the above data suggest that the impurity is hydrogenated product of APN-05 having proposed structure as shown in Fig. 2 . Conclusions This work demonstrates, the identification of new process related impurity generated during the synthesis of Apixaban. The unknown process related impurity was found to get generate during preparation of APN-05 by reduction of APN-04 with Raney Ni/H 2 under 3–6 kg/cm 2 pressure at 50–60°C. The unknown impurity is present to a level of ~ 1.0% in R & D batches of 10.0g scale, but during scale up study at 20 kg scale, the impurity was found to be present upto a level of ~ 11.0%. The unknown impurity was isolated by flash chromatography and characterized by spectroscopic techniques such as 1 H NMR, 13 C NMR, IR and ESI-MS spectrometry. Based on the spectral data, the unknown impurity was identified as 1-(4-aminophenyl)-3-morpholinopiperidin-2-one, which is a reduced form of APN-05. Experimental Materials and Methods All the reagents and solvents required for the synthesis were obtained from commercial vendors and had the desired purity. NMR spectra of the synthesized compounds were recorded on Bruker 400 MHz spectrometers with TMS as the internal standard. Chemical shift is represented with δ in parts per million . Mass spectra were recorded with a XEVO-TQSmicro#QEE0233 LC Mass Spectrometer. The IR spectra of synthesized compounds were recorded on Perkin Elmer Spectrum IR ES Version 10.6.0 by using the KBr pellet method. HPLC. The purity of apixaban and its intermediates was determined by HPLC using an area normalization method. Synthesis 5-chloro-N-(4-nitrophenyl)pentanamide ( APN-01 ) Two liters of clean and dry round bottom flask was placed in a water bath. About 800 g of Tetrahydrofuran (THF) was charged into the flask, followed by P-nitroaniline (150 g) at 25–30 o C under nitrogen atmosphere. The suspension was stirred for 10–15 min, and then the NaOH flakes (52.10 g) were charged at 25–30°C under a nitrogen atmosphere. The reaction mixture was cooled to -5 to 0°C. 5-Chlorovaleroylchloride (219.0 g) was slowly added over a period of 2–3 h, and the reaction mass was maintained for 1–2 h at -5 to 0°C. The progress of the reaction was monitored by HPLC. After the completion of reaction, the THF was distilled under vacuum below 65°C. The mixture was charged with water (750 g) at 25–30°C to obtain the suspension. The suspension was stirred for 1 h, after which the suspension was filtered. The material was dried in a hot air oven for 5–6 h to obtain dried APN-01 (235.0 g, 85%). 1 H NMR (400 MHz, DMSO- d 6 ), δ: 10.54 (s, 1H), 8.22 (d, J = 8.0 Hz, 2H,), 7.85 (d, J = 8.0 Hz, 2H,), 3.67 (t, J = 8 Hz, 2H,), 2.42 (t, J = 8.0, 2H), 1.72–1.78 (m, 4H); 13 C NMR (100 MHz, DMSO-d 6 ,) δ : 171.8, 145.4, 141.9, 124.9, 118.5, 45.0, 35.5, 31.5, 22.1; IR (KBr, cm − 1 ): 3300.58 (-NH), 1681.18 (-C = O), 1329.14 (-C-N); ESI-MS ( m/z ) Calculated for C 11 H 13 N 2 O 3 Cl, 256.0; found 257.0 (M + H) + . 1-(4-nitrophenyl)piperidin-2-one (APN-02) APN-01 (235.0 g) was dissolved in DMSO (800 gm), and K 2 CO 3 (191.20 kg, 1.4 mole equiv.) was charged. The temperature of the reaction mass was increased to 80–85°C and maintained for 6–8 h. The progress of the reaction was monitored by HPLC. The reaction mass was cooled to 25–30°C. Approximately 1200 mL of water was taken in another RBF and cooled to 0–5°C. The reaction mixture was poured into cold water, and the resulting suspension was stirred for 1–2 h. The reaction mixture was filtered, and the material was dried in a hot air oven at 50–60°C to obtain dried APN-02 (184.0 g, 70%). 1 H NMR (400 MHz, DMSO- d 6 ) δ : 8.24 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.8 Hz, 2H,), 3.71 (t, J = 5.8 Hz, 2H,), 2.46 (t, J = 8.0 Hz, 2H), 1.83–1.91 (m, 4H, m); 13 C NMR (100 MHz, DMSO- d 6 ) δ : 169.5, 149.3, 144.1, 125.9, 123.8, 50.0, 32.8, 22.7, 20.6; IR (KBr, cm − 1 ): 2951.41 (Ar-C-H), 1656.75 (-C = O), 1343.51 (-C-N); ESI-MS ( m/z ) calculated for C 11 H 12 N 2 O 3 , 220.08; found 221.00 (M + H) + . 3-morpholino-1-(4-nitrophenyl)-5,6-dihydropyridin-2(1H)-one (APN-04) A clean and dried four neck RBF was taken. Chloroform (1500 g) and APN-02 (184.0 g) were added to RBF, and the reaction mixture was stirred. The reaction mixture was allowed to cool to 10–15°C. PCl 5 was added to the reaction mass in lot wise manner. The reaction mixture was heated to 55–60°C and maintained 2–3 h. The reaction mixture was allowed to cool to 20–30°C. Another RBF was arranged with water (3000 g) and cooled to 0–5°C. The reaction mixture was poured into RBF containing pre chilled water. The reaction mixture was stirred at 25–30°C for 1 h. The organic layer was separated by a separatory funnel. The organic layer was washed with water (40 mL). The organic layer was distilled to obtain crude APN-03. Morpholine (1200 g) was charged at 25–30°C to the RBF containing crude APN-03. The reaction mixture was heated to 120–130°C and maintained for 2–4 h. The progress of the reaction was monitored by HPLC. Approximately 50% of the morpholine was distilled out under vacuum below 95°C. Water (1200 g) was slowly added to the reaction mixture over a period of 2–3 h at 90–95°C. The reaction mixture was stirred for 1–2 h, after which the reaction mixture was allowed to cool to 10–15°C. The reaction mixture was filtered through Buchner funnel. The wet material was purified using methanol (270 g) to obtain pure APN-04. The material was dried in a hot air oven at 50–55°C. to get dry APN-04 (141.0 g, 60%). 1 H NMR (400 MHz, DMSO- d 6 ) δ : 8.25 (d, J = 8.9 Hz, 2H), 7.60 (d, J = 8.9 Hz, 2H), 5.70 (t, J = 4.1 Hz, 1H), 3.84 (t, J = 8.0 Hz, 2H), 3.62 (t, J = 4.0 Hz, 4H), 2.76 (t, J = 4.0 Hz, 4H), 2.44–2.48 (m, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ : 160.6, 148.7, 143.7, 142.4, 125.2, 123.8, 116.4, 65.9, 50.0, 49.0, 22.7; IR (KBr, cm − 1 ): 2824.96 (Ar-C-H), 1671.12 (-C = O), 1505.88 (-C-O), 1214.19 (C-N), 1117.91 (C-N); ESI-MS ( m/z ) calculated for C 15 H 17 N 3 O 4 303.12; found 304.00 (M + H) + . 1-(4-aminophenyl)-3-morpholino-5,6-dihydropyridin-2(1H)-one (APN-05) APN-04 (141g) was dissolved in methanol (3000 mL) and charged in a clean and dry autoclave. Raney Nickel (1.41g, 1% w/w) was charged to the reaction mass, and then autoclaved was closed. A hydrogen pressure of 4–6 kg/cm 2 ) and a temperature of 55–60°C were applied. The reaction was maintained for 10–12 h, and the progress of the reaction was monitored by HPLC. After the reaction was complete, the reaction mass was filtered through a hyflow bed. Methanol was partially distilled below 60°C to obtain a suspension. The suspension was cooled to 0–5°C, filtered and dried to get APN-05 (102 g, 80%). 1 H NMR (400 MHz, DMSO- d 6 ) δ : 6.91 (d, J = 8.4 Hz, 2H), 6.52 (d, J = 8.4 Hz, 2H), 5.61 (t, J = 4.0 Hz, 1H), 5.06 (s, 2H), 3.63 (t, J = 4.0 Hz, 4H), 3.56 (t, J = 8.0 Hz, 2H), 2.77 (t, J = 4.0 Hz, 4H), 2.37 (q, J = 4.0 Hz, 2H); 13 C NMR (100 MHz, DMSO- d 6 ) δ :160.6, 146.7, 143.1, 131.9, 126.3, 113.7, 113.5, 65.9, 49.9, 48.9, 22.9; IR (KBr, cm − 1 ): 3425.59 and 3346.56 (NH 2 ), 2804.84 (Ar-CH), 1613.65 (C = O), 1515.95 (-C-O), 1221.37 (C-N), 1120.79 (C-N); ESI-MS: m/z calculated for C 15 H 19 N 3 O 2 273.15; found 274.29 (M + 1) + . 3-morpholino-1-(4-(2-oxopiperidin-1-yl)phenyl)-5,6-dihydropyridin-2(1H)-one (APN-07) Dried and cleaned four neck RBF was arranged with water bath. APN-05 (102 g) was charged onto the RBF and dissolved in the MDC (1300 g). The reaction mixture was stirred for 10–15 min at 25–30°C. Triethylamine (76 gm, 2.0 mole equiv.) was slowly added to the reaction mixture over a period of 10–15 min, followed by the slow addition of 5-chlorovaleroyl chloride (69.5gm, 1.20 mole equiv.) at 25–30°C. The reaction mixture was stirred and maintained for 2–3 h. The progress of the reaction was monitored by HPLC. Water (200 g) was charged to the reaction mixture, and the mixture was stirred for 20–30 min. The reaction mixture was then transferred to a separatory funnel. The layers were separated. The organic layer containing APN-06 proceeded insitu to the next stage. Next, the organic layer containing APN-06 was charged into another RBF, and KOH (41.66 gm, 2.0 mole equiv.) was charged. The temperature of the reaction mixture was raised to 40°C, and the reaction mass was maintained for 2–4 h. The progress of the reaction was monitored by HPLC. After the reaction is completed, MDC was distilled and degassed to get crude residue. The crude residue was charged with DM water (600 g), and the reaction mixture was stirred at 25–30°C for1-2 h. The suspension was filtered to obtain wet material. Wet material was dried in a hot air oven at 55–65°C to get APN-07 (109.20 g, 77%). 1 H NMR (400 M Hz, DMSO- d 6 ) δ : 7.35 (d, J = 8.5 Hz, 2H), 7.26 (d, J = 8.5 Hz, 2H), 5.70 (t, J = 4.3 Hz, 1H), 3.71 (t, J = 8.0 Hz, 2H), 3.64 (t, J = 4.0 Hz, 4H), 3.59 (t, J = 4.0 Hz, 2H), 2.78 (t, J = 4.0 Hz, 4H), 2.44 (q, J = 4.0 Hz, 2H), 2.38 (t, J = 8.0 Hz, 2H), 1.79–1.89 (m, 4H); 13 C NMR (100 MHz, DMSO- d 6 ) δ : 168.8, 160.5, 142.8, 140.9, 140.7, 126.2, 125.6, 114.8, 65.9, 50.8, 49.9, 48.3, 32.5, 23.0, 22.8, 20.8; IR (KBr, cm − 1 ): 2964.34 (Ar-C-H), 1646.70 (-C = O), 1525.10 (-C = O), 1514.50 (-C-O), 1311.90 (-C-N), 1214.14 (C-N), 1114, 1070, 1050 (C-O stretching). ESI-MS m/z calculate for C 20 H 25 N 3 O 3 355.19, found 356.31 (M + H) + . Ethyl 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate (APN-10) Dried and clean four neck RBF was arranged. APN-07 (100 g) and toluene (562 g) were charged onto the RBF. APN-08 (87.02 gm, 1.20 mole equiv.) was then charged onto the reaction mass, followed by addition of triethylamine (71.2 g, 2.50 mole equiv.). The reaction mixture was stirred for10-15 min at 25–30°C, after which the temperature of the reaction mass increased to 100–105°C. The reaction mass was maintained for 5–6 h, and the progress of the reaction was monitored by HPLC. Aq. HCl solution was added to the reaction mass at 25–30°C over a period of in 1–2 h. The reaction mixture was stirred and maintained for 4–6 h. The progress of reaction was monitored by HPLC. After the reaction is completed, toluene (350 g) was added to the reaction mixture. The temperature of reaction mixture was increased to 70–80°C. The reaction mixture was stirred and maintained for 1 h, after which layers were separated. The toluene layer was partially distilled under vacuum at 55–60°C. The reaction mixture was then cooled to 10–15°C. Then, the mixture was stirred for 60 min. The reaction mixture was filtered and the wet cake was washed with prechilled toluene (50 g). The wet material was dried in hot air oven at 55–60°C to obtain APN-10 (113.0 g, 82.70%). 1 H NMR (400M Hz, DMSO-d 6 ) δ: 7.49 (d, J = 8.8Hz, 2H), 7.47 (d, J = 8.8Hz, 2H), 7.35 (d, J = 8.8Hz, 2H), 7.01 (d, J = 8.8Hz, 2H), 4.35 (q, J = 8.0 Hz, 2H), 4.07 (t, J = 8.0 Hz, 2H), 3.80 (s, 3H), 3.59 (t, J = 8.0 Hz, 2H), 3.20 (t, J = 8.0 Hz, 2H), 2.38 (t, J = 8.0 Hz, 2H), 1.79–1.88 (m, 4H), 1.33 (t, J = 8.0 Hz, 3H), 13 C NMR (100 MHz, DMSO-d 6 ) δ:168.8, 161.3, 159.3, 156.3, 141.4, 139.6, 138.4, 133.0, 132.4, 126.7, 126.7, 126.3, 126.0, 113.4, 60.6, 55.4, 50.8, 50.7, 32.5, 22.9, 21.1, 20.8, 14.1; IR (KBr, cm − 1 ): 2941.35 (Ar-C-H), 1737.22 (C = N), 1671.12 (C = O), 1651.01 (-C = O), 1515.94 (-C = N), 1467.08 (-C-O), 1250.11 (-C-N), 1142.34 (-C-N). (ESI-MS) m/z calculate for C 27 H 28 N 4 O 5 488.21, found 489.00 (M + H) + . 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (Apixaban) 2 litre clean and dry autoclave reactor was arranged. PAN-10 (110 g) and methanol (2178 g) were charged into the autoclave, after which the autoclave was closed. Ammonia gas was purged into the reactor (4–6 kg/cm 2 ) and then the temperature was increased to 70–75°C. The reaction mixture was maintained for 6–8 h. The progress of reaction was monitored by HPLC. After the reaction complies on HPLC, reaction mass was filtered through hyflow bed. Methanol was partially distilled to get suspension which upon filtration and drying yielded pure apixaban (83.0 g, 80%). 1 H NMR (400M Hz, DMSO-d 6 ) δ: 7.73 (br s, 1H), 7.51 (d, J = 8.0 Hz, 2H), 7.45 (br s, 1H), 7.35(d, J = 6.4 Hz, 2H), 7.28 (d,. J = 8.0 Hz, 2H), 7.00 (d, J = 8.0 Hz, 2H), 4.04 (t, J = 4.0 Hz, 2H), 3.79 (s, 3H), 3.58 (t, J = 8.0 Hz, 2H), 3.20 (t, J = 4.0 Hz 2H), 2.38 (t, J = 4.0 Hz, 2H), 1.79–1.88 (m, 4H), 13 C NMR (100 MHz, DMSO-d 6 ) δ:168.8, 163.2, 159.1, 156.6, 141.4, 141.3, 139.7, 132.9, 132.5, 126.8, 126.3, 125.9, 125.2, 113.3, 55.4, 50.9, 50.8, 32.5, 23.0, 21.0, 20.9; IR (KBr, cm − 1 ): 3300.04 (N-H ), 2866.47 (Ar-C-H), 1682.59 (C = O), 1630.14 (C = O), 1595.08 (-C = O), 1519.24 (-C = N), 1343.85 (C-N), 1310.09 (-C-N). M/S m/z calculate for m/z C 25 H 25 N 5 O 4 459.19, found 460.16 (M + H) + . 1-(4-aminophenyl)-3-morpholinopiperidin-2-one (APN-05 impurity) 1 H NMR (400M Hz, DMF-d 7 ) δ: 6.91 (d, J = 8.0 Hz, 2H), 6.66 (d, J = 8.4 Hz, 2H), 5.15 (s, 2H), 3.57–3.63 (m, 4H), 3.49–3.55 (m, 2H), 3.47–3.52 (m, 2H), 3.21 (t, 1H), 2.62–2.66 (m, 2H), 2.91–2.93 (m, 2H), 1.85–2.01 (m, 4H); 13 C NMR (100 MHz, DMF-d 7 ) δ: 169.51, 147.76, 133.21, 127.36, 114.24, 67.65, 65.09, 51.53, 50.47, 25.68, 21.94; IR (KBr, cm − 1 ): 3398.29 (-NH 2 ), 1632.33 (C = O stretching), 1514.50 (C-O stretching), 1398.11 (C-N stretching). ESI-MS: m/z calculated for C 15 H 21 N 3 O 2 275.16; found 276.21 (M + 1) + . Declarations Supporting information The supporting information file contains details of HPLC methods used for the analysis of reaction mass and purity of compounds. Scan copy of HPLC chromatogram indicating peak of APN V along with impurity peak. Scan copies of 1 H NMR, 13 C NMR, IR and ESI-MS spectra of synthesized compound along with isolated impurity are provided. Details of Flash chromatographic condition used for the isolation of impurity are also provided. Conflict of Interest There is no conflict of interest declared by authors of the manuscript. Author Contribution Vipin Kumar, Sachin Kumar Sharma,Govind Singh and Shyambaran Dhuriya: Synthetic lab work and collection of dataLingaiah Balthu: Reviewed the manuscriptSushilkumar,Rashmi Devi: Isolation of impurity by flash chromatography Krishna Kumar Yadav: Reviewed analytical dataSumit S. Chourasiya: Conception and drafting of manuscript Acknowledgements The authors acknowledge the management of IOL Chemicals and Pharmaceuticals Ltd for giving permission to publish the research work. References Y.H. Mekaj, A.Y. Mekaj, S. B. Duci, E. I. Miftari, Ther. Clin. Risk Manage. 24, 967–77 (2015) B.E. Pollock, J. Am. Med. Assoc. 12, 1094–7 (1955) G.D. Murray, Br. J. Surg. 27, 567 – 98 (1940) D. Hawkins, Pharmacotherapy. 24, 62S-5S (2004) J. Leadley, Curr. Trends Med. Chem. 1, 151–9 (2001) D.J. Pinto, M.J. Orwat, S. Koch, K.A. Rossi, R.S. Alexander, A. Smallwood, P.C.Wong, A.R. Rendina, J. M. Luettgen, R.M. Knabb, K. He J. Med. Chem. 50, 5339–56. (2007) J. Watson, G. Whiteside, C. Perry, Drugs. 71, 2079–89 (2011) Z. Hijazi, S.H. Hohnloser, U. Andersson, J. H. Alexander, M. Hanna, M. Keltai, A. Parkhomenko, J. L. Lopez-Sendon, R.D. Lopes, A. Siegbahn, C.B. Granger, JAMA cardiology . 1, 451 – 60 (2016) M.W. Mandernach, R.J. Beyth, A. Rajasekhar, Ther. Clin. Risk Manage. 26, 1273–82 (2015) G. Agnelli, H.R. Buller, A. Cohen, M. Curto, A. S. Gallus, M. Johnson, A. Porcari, G.E. Raskob, J. I. Weitz, N. Engl. J. Med. 21, 699–708 (2013) E. Perzborn, S. Roehrig, A. Straub, D. Kubitza, W. Mueck, V. Laux, Arterioscler., Thromb., Vasc. Biol., 30, 376–81 (2010) K. Ogata, J. Mendell-Harary J, M. Tachibana, H. Masumoto, T. Oguma, M. Kojima, S. Kunitada, J. Clin. Pharmacol. 50, 743–53 (2010) P. Zhang, W. Huang, L.Wang, L. Bao, Z.J. Jia, S. M. Bauer, E. A. Goldman, G.D. Probst, Y. Song, T. Su, J. Fan. Bioorg. Med. Chem. Lett. 19, 2179–85 (2009) J. A. Jiang, Y. Ji, Synth. Commun.43, 72 – 9. (2013) B.D. Maxwell, S. B. Tran, S.Y. Chen, D. Zhang, B.C. Chen, H. Zhang, S. J. Bonacorsi Jr. J. Labelled Compd. Radiopharm. 54, 418–25 (2011) S.V. Rao, R.K. Dubey, G. Nagarajuna G, G.S. Mallikarjuna, G.S. Krishna, N. Ganesh, N. A. Babu, Journal of Chemistry and Chemical Sciences. 8, 204–211 (2018) N.R. Nevuluri, R.K. Rapolu, J. Iqbal, B. Kandagatla, S. Sen, V. H. Dahanukar, S. Oruganti. Monatshefte für Chemie-Chemical Monthly. 148, 1477–82 (2017) N. Rahman, S. N. Azmi, H. F. Wu, Accredit. Qual. Assur. 69–74 (2006) S. B. Bari, B. R. Kadam, Y. S. Jaiswal, A. A Shirkhedkar, Eurasian J Anal Chem., 2, 32–53 (2007) F. Qiu, D.L. Norwood, J. Liq. Chromatogr. Relat. Technol. 30, 877–935 (2007) M.D. Argentine, P.K. Owens, B. A. Olsen, Adv. Drug Delivery Rev. 59, 12–28 (2007) B.A. Olsen, A. Sreedhara, S. W. Baertschi, TrAC., 101, 17–23 (2018) B.A Olsen, S. W. Baertschi, Sep. Sci. Technol. (Philadelphia, PA, U. S.). 5 , 89–117 (2004) D. J. Snodin, Org. Process Res. Dev. 14, 960–76 (2010) K.L. Dobo, N. Greene, M. O. Cyr, S. Caron, W. W. Ku, Regul. Toxicol. Pharmacol. 44, 282–93 (2006) D.J. Snodin, S. D. McCrossen. Regul. Toxicol. Pharmacol. 63, 298–312 (2012) Guideline IH. Impurities in new drug substances Q3A (R2). InProceedings of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland 2006 Oct 25 (Vol. 25). Schemes Schemes 1 and 2 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files ApixabanManuscriptSupportinginformationfile.docx scheme1.png Scheme 1: Commercial route of syntheses of Apixaban; (A) Innovator route; (B) Alternate route scheme2.png Scheme 2: Synthesis of Apixaban (this work) by slight modification of literature process. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3992931","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275109634,"identity":"810648d8-91d1-4674-8419-bbdca05b6028","order_by":0,"name":"Vipin Kumar","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Vipin","middleName":"","lastName":"Kumar","suffix":""},{"id":275109635,"identity":"d742d7be-556d-4e3e-acd8-f57661ea324e","order_by":1,"name":"Sachin Kumar Sharma","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Sachin","middleName":"Kumar","lastName":"Sharma","suffix":""},{"id":275109636,"identity":"54b89ad1-56df-404d-801d-49886f63347c","order_by":2,"name":"Govind Singh","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Govind","middleName":"","lastName":"Singh","suffix":""},{"id":275109637,"identity":"7d3a8566-3797-4745-9872-625114d15e17","order_by":3,"name":"Shyambaran Dhuriya","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Shyambaran","middleName":"","lastName":"Dhuriya","suffix":""},{"id":275109638,"identity":"9e56504b-dad5-40a6-92d3-f3589b2c60d9","order_by":4,"name":"Lingaiah Balthu","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Lingaiah","middleName":"","lastName":"Balthu","suffix":""},{"id":275109640,"identity":"f7c35a4c-35b1-49f7-a8db-6ea8a918de1b","order_by":5,"name":"Sushil Kumar","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Sushil","middleName":"","lastName":"Kumar","suffix":""},{"id":275109642,"identity":"52b2ec5a-7525-4779-a68a-685b9b3ac678","order_by":6,"name":"Rashmi Devi","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Rashmi","middleName":"","lastName":"Devi","suffix":""},{"id":275109643,"identity":"2d8e139f-8535-434e-b2ef-c310940d3459","order_by":7,"name":"Krishna Kumar Yadav","email":"","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":false,"prefix":"","firstName":"Krishna","middleName":"Kumar","lastName":"Yadav","suffix":""},{"id":275109644,"identity":"9dc75544-5cae-4c8c-955f-0d5a86c7d133","order_by":8,"name":"Sumit S. Chourasiya","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA90lEQVRIiWNgGAWjYJCCA2AEAgkMDHJsDMwNhLUcQNJizMbASFgLA1wLECQ2ENIiPyM78fCHmjtyBrcPP/7wsM0uvY+9sfFzAYOdnC4OnQY3cjccOHDsmbHBuTQzicS25Nw2noPN0jMYko3NDuDQIgHUcrDhcOKGMwxmDIltzLltQI3MPAwHErfh0CI/A66F/fOHxLb6dDZCWhhuwLXwGAAVH04gqMXgzNsNB84cO2wseYanTCLh3HFDiF8McPtFvj1384eKmsNyfGfYN3/8UVYtL9/efPBzQYWdHC4tqICRDUIzMxgQoxwM/sC0jIJRMApGwShAAACzdmeJDgLltAAAAABJRU5ErkJggg==","orcid":"","institution":"IOL Chemicals and Pharmaceuticals Ltd","correspondingAuthor":true,"prefix":"","firstName":"Sumit","middleName":"S.","lastName":"Chourasiya","suffix":""}],"badges":[],"createdAt":"2024-02-27 05:29:43","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3992931/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3992931/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51798414,"identity":"242c2fee-80ac-4ad8-ab23-dae69f9aa63a","added_by":"auto","created_at":"2024-02-29 08:18:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":26112,"visible":true,"origin":"","legend":"\u003cp\u003e2D Structures of rivaroxaban, apixaban, edoxaban and betrixaban, the oral factor Xa inhibitors.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/731c20096081041f312a3950.png"},{"id":51799090,"identity":"a2253361-5544-49b0-9f90-dfe601e7868a","added_by":"auto","created_at":"2024-02-29 08:26:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":11733,"visible":true,"origin":"","legend":"\u003cp\u003eApixaban intermediate (APN-05) and its corresponding impurity.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/a846d4c77868ff10428d15c3.png"},{"id":52562041,"identity":"519d67d9-0429-4112-a8a1-32181bf2de10","added_by":"auto","created_at":"2024-03-13 02:55:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":449354,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/fdd2f55f-4ebe-483e-8749-86de7eb9e404.pdf"},{"id":51798419,"identity":"47dd3428-e683-46c7-bb25-a476ae57e306","added_by":"auto","created_at":"2024-02-29 08:18:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":7216505,"visible":true,"origin":"","legend":"","description":"","filename":"ApixabanManuscriptSupportinginformationfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/294b01e7fd00b97dd026b77a.docx"},{"id":51798415,"identity":"2ef08679-34e5-46dc-aa0a-8364408da11a","added_by":"auto","created_at":"2024-02-29 08:18:40","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e: Commercial route of syntheses of Apixaban; (A) Innovator route; (B) Alternate route\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/cc78efd6c2e3a78abbf058f7.png"},{"id":51798417,"identity":"9607b161-0f31-4b64-a390-6699c3c35488","added_by":"auto","created_at":"2024-02-29 08:18:40","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2: \u003c/strong\u003eSynthesis of Apixaban (this work) by slight modification of literature process.\u003c/p\u003e","description":"","filename":"scheme2.png","url":"https://assets-eu.researchsquare.com/files/rs-3992931/v1/db780d1c44a8d3fb5b2d66d0.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"1-(4-aminophenyl)-3-morpholinopiperidin-2-one: A new process related impurity of Apixaban- An anticoagulant drug","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnticoagulants are commonly used for the prevention and treatment of thromboembolic disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Warfarin, heparin and their derivative are traditional anticoagulants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These anticoagulants are associated with several drawbacks including drug-drug and drug-food interactions, parenteral administration, and routine monitoring, suggesting the urgent need for the development of safe and efficacious oral anticoagulants [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe direct inhibition of factor Xa is a newly identified mechanism for controlling coagulation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In this category, apixaban has evolved as a safe, efficacious, and convenient molecule for controlling coagulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Apixaban was approved for medical use in the European Union in May 2011 [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. A new drug application (NDA) for the approval of apixaban was submitted to the US Food and Drug Administration (FDA) by Bristol Myers Squibb (BMS) and Pfizer jointly after the conclusion of the ARISTOTLE clinical trial in 2011 [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. On 13 March 2014 it was approved for the additional indication of preventing deep vein thrombosis and pulmonary embolism in people who have recently undergone knee or hip replacement [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Apixaban is sold under the brand name Eliquis as an anticoagulant medication\u003csup\u003e7\u003c/sup\u003e. Other examples of direct factor (FXa) inhibitors include rivaroxaban [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], edoxaban,[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. and betrixaban (Fig.\u0026nbsp;1), which have gained approval for several indications, most notably, for the prevention and treatment of venous thromboembolism (VTE) and for the prevention of stroke in patients with atrial fibrillation hepatic impairment.\u003c/p\u003e \u003cp\u003eVarious chemical syntheses of apixaban have been reported in the literature [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Among them, the most common synthesis is that reported by Bristol Mayor Squibb, an innovator of apixaban [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The innovator route involves the use of p-amino iodobenzene as a key starting material (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e,a). The key step in the synthesis of Apixaban involves C-N bond formation using Cu as a catalyst followed by ammonolysis to obtain apixaban. This route of synthesis of apixaban suffers from the disadvantages of (a) use of costly p-aminoiodobenzene, (b) a C-N coupling reaction involving costly metal catalyst, and (c) poor overall yield and thus has limitation of commercially viable. A modified syntheses of apixaban was reported that involved the use of p-amino nitrobenzene as a key starting material instead of p-iodo nitrobenzene [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, b). The key advantages of this route are its low cost and catalyst free condition for C-N bond formation.\u003c/p\u003e\u003cp\u003eProcess related impurities are the impurities observed in drug substance and get generated in a manufacturing process [\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The investigation of any process related impurity in drug substances is a crucial aspect of API process research and development [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Knowing the chemical structure of process related impurities is important for assessing their toxicological nature and understanding the mechanism of impurity formations [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This information help process scientists to categorize impurities according to the ICH classification, which further helps to define the limit of the impurity and its control strategy [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This work present, the isolation and characterization of new process related impurity, observed during the preparation of apixaban. This work would be helpful to the pharmaceutical scientist working on the development of Apixaban as an active pharmaceutical ingredient.\u003c/p\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Apixaban synthesis\u003c/h2\u003e \u003cp\u003eWe have developed a process for the preparation of apixaban by slight modification of literature known process. The optimized route of synthesis of apixaban consists of eleven chemical transformations (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The synthesis starts with the acylation of p-nitroaniline with 5-chlorovaleroyl chloride followed by intramolecular cyclization to obtain APN-02. Chlorination of APN-02 using PCl\u003csub\u003e5\u003c/sub\u003e followed by reaction with morpholine led to the formation of APN-04. A reduction of APN-04 using Raney Ni lead to the formation of APN-05. APN-05 again undergoes acylation and intramolecular cyclization to form APN-07, which is an intermediate required for the synthesis of apixaban. Finally, APN-07 and APN-08 react with each other to form APN-09 as an \u003cem\u003einsitu\u003c/em\u003e intermediate, which leads to the formation of apixaban upon ammonolysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2. Identification of unknown impurity\u003c/h2\u003e \u003cp\u003eDuring the preparation of APN-05 from APN-04 by optimized process (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), an unknown impurity was observed at 0.30 RRT in HPLC chromatograph of reaction monitoring samples. In all the R \u0026amp; D batches, this impurity was observed upto a level of ~\u0026thinsp;1.0%. However, during scale up studies in pilot plant, the same impurity was found to be present in a reaction mass upto\u0026thinsp;~\u0026thinsp;11.0%. Hence, we became interested to know the structure of impurity in order to understand the root cause of its formation and possible control strategy during scale up studies. The reaction monitoring data of APN-05 of R \u0026amp; D batches and pilot plant batches are provided in Table \u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReaction monitoring profile of APN-04 to APN-05 reaction.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e\u003cp\u003e\u003cimg 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\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eBatch No.\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eSource\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eBatch size\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003eReaction monitoring by HPLC (%)\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e% Conversion\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eto APN-05\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eUnreacted\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eAPN-04 (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eUnknown impurity\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003eat 0.30 RRT (%)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e001\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e002\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e003\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e004\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e005\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e96.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e006\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e92.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e007\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eR \u0026amp; D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10.0 g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e011\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePilot plant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e93.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e012\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePilot plant\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e20 kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e86.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003ea\u003c/b\u003e \u003c/sup\u003e 3\u0026ndash;4 kg/cm\u003csup\u003e2\u003c/sup\u003eH\u003csub\u003e2\u003c/sub\u003e pressure, 50\u0026ndash;60\u0026deg;C temperature, 4\u0026ndash;6 h.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e3. Isolation of unknown impurity\u003c/h3\u003e\n\u003cp\u003eNext, we focused on the isolation of the impurity for its characterization. We isolated the impurity first dissolving crude APN V in water. The filtered mother liquor was analyzed which indicated the higher percent of impurity. The mother liquor was evaporated to get residue which was subjected to flash chromatography to get impurity with ~\u0026thinsp;95% purity. A pure sample of impurity was used for analysis by LC-MS spectrometry, IR, \u003csup\u003e1\u003c/sup\u003eH NMR and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopic techniques.\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4. Characterization of impurity\u003c/h2\u003e \u003cp\u003eA systematic characterization study of impurity was carried out using spectrometric (LC-MS) and spectroscopic techniques (\u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR). The LC-MS analysis of impurity showed m/z 276. The mass spectrum for parent compound i.e. APN-05 shows m/z 274 correspond to (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e ion peak. Thus, the mass spectrum of impurity suggest the presence of two additional proton in the structure of APN-05. The structural feature of APN-05 suggest that, there is also a possibility of reduction of keto as well as double bond as a part of 2-piperidone moiety (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).Then we analyzed the IR spectrum of an impurity. A stretching band at 1632.33 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e indicate the presence of ketone functional group. The IR spectrum rule out the possibility of reduction of ketone moiety and hint toward the possibility of reduction of double bond which is part of 2-piperidone moiety.\u003c/p\u003e \u003cp\u003eNext, we analyzed the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of impurity and compared it with parent compound i.e. APN-05. The \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of APN 05 showed a triplet at 5.61 ppm due to the presence of one vinylic proton (-C\u0026thinsp;=\u0026thinsp;C-H) which is coupled with two protons present at the adjacent carbon. In the 1H NMR spectrum of impurity, this signal was found to be absent and an additional triplet of one proton was observed at 3.2 ppm. This pattern of \u003csup\u003e1\u003c/sup\u003eH NMR spectrum clearly suggest the reduction of ring double bond of 5,6-dihydropyridin-2(1H)-one moiety of APN-05 leading to an impurity with piperidin-2-one moiety. Rest of the 1H NMR pattern of impurity is same as that of APN-06. The \u003csup\u003e13\u003c/sup\u003eC NMR of impurity also suggest that the ring double bond of 5,6-dihydropyridin-2(1H)-one moiety of APN-05 is reduced and thus only four signals in aromatic region was observed against six signals due to six aromatic protons in APN-05. Out of six signals, four signals are due to aromatic ring carbon and two signals are due to double bond carbon of 5,6-dihydropyridin-2(1H)-one moiety present in APN-05. And thus in \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of impurity only four signals are observed due to the aromatic ring. The \u003csup\u003e13\u003c/sup\u003eC NMR spectrum of impurity showed a signal due to carbonyl carbon at 169.5 ppm thus rule out the possibility of reduction of carbonyl group.\u003c/p\u003e \u003cp\u003eAll the above data suggest that the impurity is hydrogenated product of APN-05 having proposed structure as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis work demonstrates, the identification of new process related impurity generated during the synthesis of Apixaban. The unknown process related impurity was found to get generate during preparation of APN-05 by reduction of APN-04 with Raney Ni/H\u003csub\u003e2\u003c/sub\u003e under 3\u0026ndash;6 kg/cm\u003csup\u003e2\u003c/sup\u003e pressure at 50\u0026ndash;60\u0026deg;C. The unknown impurity is present to a level of ~\u0026thinsp;1.0% in R \u0026amp; D batches of 10.0g scale, but during scale up study at 20 kg scale, the impurity was found to be present upto a level of ~\u0026thinsp;11.0%. The unknown impurity was isolated by flash chromatography and characterized by spectroscopic techniques such as \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, IR and ESI-MS spectrometry. Based on the spectral data, the unknown impurity was identified as 1-(4-aminophenyl)-3-morpholinopiperidin-2-one, which is a reduced form of APN-05.\u003c/p\u003e "},{"header":"Experimental","content":"\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eAll the reagents and solvents required for the synthesis were obtained from commercial vendors and had the desired purity. NMR spectra of the synthesized compounds were recorded on Bruker 400 MHz spectrometers with TMS as the internal standard. Chemical shift is represented with \u003cem\u003eδ in parts per million\u003c/em\u003e. Mass spectra were recorded with a XEVO-TQSmicro#QEE0233 LC Mass Spectrometer. The IR spectra of synthesized compounds were recorded on Perkin Elmer Spectrum IR ES Version 10.6.0 by using the KBr pellet method. HPLC. The purity of apixaban and its intermediates was determined by HPLC using an area normalization method.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis\u003c/h2\u003e \u003cp\u003e \u003cb\u003e5-chloro-N-(4-nitrophenyl)pentanamide\u003c/b\u003e (\u003cb\u003eAPN-01\u003c/b\u003e)\u003c/p\u003e \u003cp\u003eTwo liters of clean and dry round bottom flask was placed in a water bath. About 800 g of Tetrahydrofuran (THF) was charged into the flask, followed by P-nitroaniline (150 g) at 25\u0026ndash;30 \u003csup\u003eo\u003c/sup\u003eC under nitrogen atmosphere. The suspension was stirred for 10\u0026ndash;15 min, and then the NaOH flakes (52.10 g) were charged at 25\u0026ndash;30\u0026deg;C under a nitrogen atmosphere. The reaction mixture was cooled to -5 to 0\u0026deg;C. 5-Chlorovaleroylchloride (219.0 g) was slowly added over a period of 2\u0026ndash;3 h, and the reaction mass was maintained for 1\u0026ndash;2 h at -5 to 0\u0026deg;C. The progress of the reaction was monitored by HPLC. After the completion of reaction, the THF was distilled under vacuum below 65\u0026deg;C. The mixture was charged with water (750 g) at 25\u0026ndash;30\u0026deg;C to obtain the suspension. The suspension was stirred for 1 h, after which the suspension was filtered. The material was dried in a hot air oven for 5\u0026ndash;6 h to obtain dried APN-01 (235.0 g, 85%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e), δ: 10.54 (s, 1H), 8.22 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H,), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H,), 3.67 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H,), 2.42 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 2H), 1.72\u0026ndash;1.78 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e,) \u003cem\u003eδ\u003c/em\u003e: 171.8, 145.4, 141.9, 124.9, 118.5, 45.0, 35.5, 31.5, 22.1; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3300.58 (-NH), 1681.18 (-C\u0026thinsp;=\u0026thinsp;O), 1329.14 (-C-N); ESI-MS (\u003cem\u003em/z\u003c/em\u003e) Calculated for C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003eCl, 256.0; found 257.0 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1-(4-nitrophenyl)piperidin-2-one\u003c/b\u003e \u003cb\u003e(APN-02)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAPN-01 (235.0 g) was dissolved in DMSO (800 gm), and K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (191.20 kg, 1.4 mole equiv.) was charged. The temperature of the reaction mass was increased to 80\u0026ndash;85\u0026deg;C and maintained for 6\u0026ndash;8 h. The progress of the reaction was monitored by HPLC. The reaction mass was cooled to 25\u0026ndash;30\u0026deg;C. Approximately 1200 mL of water was taken in another RBF and cooled to 0\u0026ndash;5\u0026deg;C. The reaction mixture was poured into cold water, and the resulting suspension was stirred for 1\u0026ndash;2 h. The reaction mixture was filtered, and the material was dried in a hot air oven at 50\u0026ndash;60\u0026deg;C to obtain dried APN-02 (184.0 g, 70%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 8.24 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 7.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H,), 3.71 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H,), 2.46 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 1.83\u0026ndash;1.91 (m, 4H, m); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 169.5, 149.3, 144.1, 125.9, 123.8, 50.0, 32.8, 22.7, 20.6; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 2951.41 (Ar-C-H), 1656.75 (-C\u0026thinsp;=\u0026thinsp;O), 1343.51 (-C-N); ESI-MS (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e11\u003c/sub\u003eH\u003csub\u003e12\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e, 220.08; found 221.00 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-morpholino-1-(4-nitrophenyl)-5,6-dihydropyridin-2(1H)-one\u003c/b\u003e \u003cb\u003e(APN-04)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA clean and dried four neck RBF was taken. Chloroform (1500 g) and APN-02 (184.0 g) were added to RBF, and the reaction mixture was stirred. The reaction mixture was allowed to cool to 10\u0026ndash;15\u0026deg;C. PCl\u003csub\u003e5\u003c/sub\u003e was added to the reaction mass in lot wise manner. The reaction mixture was heated to 55\u0026ndash;60\u0026deg;C and maintained 2\u0026ndash;3 h. The reaction mixture was allowed to cool to 20\u0026ndash;30\u0026deg;C. Another RBF was arranged with water (3000 g) and cooled to 0\u0026ndash;5\u0026deg;C. The reaction mixture was poured into RBF containing pre chilled water. The reaction mixture was stirred at 25\u0026ndash;30\u0026deg;C for 1 h. The organic layer was separated by a separatory funnel. The organic layer was washed with water (40 mL). The organic layer was distilled to obtain crude APN-03. Morpholine (1200 g) was charged at 25\u0026ndash;30\u0026deg;C to the RBF containing crude APN-03. The reaction mixture was heated to 120\u0026ndash;130\u0026deg;C and maintained for 2\u0026ndash;4 h. The progress of the reaction was monitored by HPLC. Approximately 50% of the morpholine was distilled out under vacuum below 95\u0026deg;C. Water (1200 g) was slowly added to the reaction mixture over a period of 2\u0026ndash;3 h at 90\u0026ndash;95\u0026deg;C. The reaction mixture was stirred for 1\u0026ndash;2 h, after which the reaction mixture was allowed to cool to 10\u0026ndash;15\u0026deg;C. The reaction mixture was filtered through Buchner funnel. The wet material was purified using methanol (270 g) to obtain pure APN-04. The material was dried in a hot air oven at 50\u0026ndash;55\u0026deg;C. to get dry APN-04 (141.0 g, 60%).\u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 8.25 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 7.60 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.9 Hz, 2H), 5.70 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.1 Hz, 1H), 3.84 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 3.62 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 2.76 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 2.44\u0026ndash;2.48 (m, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 160.6, 148.7, 143.7, 142.4, 125.2, 123.8, 116.4, 65.9, 50.0, 49.0, 22.7; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 2824.96 (Ar-C-H), 1671.12 (-C\u0026thinsp;=\u0026thinsp;O), 1505.88 (-C-O), 1214.19 (C-N), 1117.91 (C-N); ESI-MS (\u003cem\u003em/z\u003c/em\u003e) calculated for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e17\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e 303.12; found 304.00 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1-(4-aminophenyl)-3-morpholino-5,6-dihydropyridin-2(1H)-one\u003c/b\u003e \u003cb\u003e(APN-05)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAPN-04 (141g) was dissolved in methanol (3000 mL) and charged in a clean and dry autoclave. Raney Nickel (1.41g, 1% w/w) was charged to the reaction mass, and then autoclaved was closed. A hydrogen pressure of 4\u0026ndash;6 kg/cm\u003csup\u003e2\u003c/sup\u003e) and a temperature of 55\u0026ndash;60\u0026deg;C were applied. The reaction was maintained for 10\u0026ndash;12 h, and the progress of the reaction was monitored by HPLC. After the reaction was complete, the reaction mass was filtered through a hyflow bed. Methanol was partially distilled below 60\u0026deg;C to obtain a suspension. The suspension was cooled to 0\u0026ndash;5\u0026deg;C, filtered and dried to get APN-05 (102 g, 80%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 6.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 6.52 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 5.61 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 1H), 5.06 (s, 2H), 3.63 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 3.56 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 2.77 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 2.37 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 2H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e:160.6, 146.7, 143.1, 131.9, 126.3, 113.7, 113.5, 65.9, 49.9, 48.9, 22.9; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3425.59 and 3346.56 (NH\u003csub\u003e2\u003c/sub\u003e), 2804.84 (Ar-CH), 1613.65 (C\u0026thinsp;=\u0026thinsp;O), 1515.95 (-C-O), 1221.37 (C-N), 1120.79 (C-N); ESI-MS: \u003cem\u003em/z\u003c/em\u003e calculated for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e19\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 273.15; found 274.29 (M\u0026thinsp;+\u0026thinsp;1)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3-morpholino-1-(4-(2-oxopiperidin-1-yl)phenyl)-5,6-dihydropyridin-2(1H)-one\u003c/b\u003e \u003cb\u003e(APN-07)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDried and cleaned four neck RBF was arranged with water bath. APN-05 (102 g) was charged onto the RBF and dissolved in the MDC (1300 g). The reaction mixture was stirred for 10\u0026ndash;15 min at 25\u0026ndash;30\u0026deg;C. Triethylamine (76 gm, 2.0 mole equiv.) was slowly added to the reaction mixture over a period of 10\u0026ndash;15 min, followed by the slow addition of 5-chlorovaleroyl chloride (69.5gm, 1.20 mole equiv.) at 25\u0026ndash;30\u0026deg;C. The reaction mixture was stirred and maintained for 2\u0026ndash;3 h. The progress of the reaction was monitored by HPLC. Water (200 g) was charged to the reaction mixture, and the mixture was stirred for 20\u0026ndash;30 min. The reaction mixture was then transferred to a separatory funnel. The layers were separated. The organic layer containing APN-06 proceeded \u003cem\u003einsitu\u003c/em\u003e to the next stage. Next, the organic layer containing APN-06 was charged into another RBF, and KOH (41.66 gm, 2.0 mole equiv.) was charged. The temperature of the reaction mixture was raised to 40\u0026deg;C, and the reaction mass was maintained for 2\u0026ndash;4 h. The progress of the reaction was monitored by HPLC. After the reaction is completed, MDC was distilled and degassed to get crude residue. The crude residue was charged with DM water (600 g), and the reaction mixture was stirred at 25\u0026ndash;30\u0026deg;C for1-2 h. The suspension was filtered to obtain wet material. Wet material was dried in a hot air oven at 55\u0026ndash;65\u0026deg;C to get APN-07 (109.20 g, 77%). \u003csup\u003e1\u003c/sup\u003eH NMR (400 M Hz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 7.35 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 7.26 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5 Hz, 2H), 5.70 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.3 Hz, 1H), 3.71 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 3.64 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 3.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 2H), 2.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 4H), 2.44 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 2H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 1.79\u0026ndash;1.89 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003c/em\u003e\u003csub\u003e6\u003c/sub\u003e) \u003cem\u003eδ\u003c/em\u003e: 168.8, 160.5, 142.8, 140.9, 140.7, 126.2, 125.6, 114.8, 65.9, 50.8, 49.9, 48.3, 32.5, 23.0, 22.8, 20.8; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 2964.34 (Ar-C-H), 1646.70 (-C\u0026thinsp;=\u0026thinsp;O), 1525.10 (-C\u0026thinsp;=\u0026thinsp;O), 1514.50 (-C-O), 1311.90 (-C-N), 1214.14 (C-N), 1114, 1070, 1050 (C-O stretching). ESI-MS \u003cem\u003em/z\u003c/em\u003e calculate for C\u003csub\u003e20\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e 355.19, found 356.31 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEthyl 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxylate\u003c/b\u003e \u003cb\u003e(APN-10)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eDried and clean four neck RBF was arranged. APN-07 (100 g) and toluene (562 g) were charged onto the RBF. APN-08 (87.02 gm, 1.20 mole equiv.) was then charged onto the reaction mass, followed by addition of triethylamine (71.2 g, 2.50 mole equiv.). The reaction mixture was stirred for10-15 min at 25\u0026ndash;30\u0026deg;C, after which the temperature of the reaction mass increased to 100\u0026ndash;105\u0026deg;C. The reaction mass was maintained for 5\u0026ndash;6 h, and the progress of the reaction was monitored by HPLC. Aq. HCl solution was added to the reaction mass at 25\u0026ndash;30\u0026deg;C over a period of in 1\u0026ndash;2 h. The reaction mixture was stirred and maintained for 4\u0026ndash;6 h. The progress of reaction was monitored by HPLC. After the reaction is completed, toluene (350 g) was added to the reaction mixture. The temperature of reaction mixture was increased to 70\u0026ndash;80\u0026deg;C. The reaction mixture was stirred and maintained for 1 h, after which layers were separated. The toluene layer was partially distilled under vacuum at 55\u0026ndash;60\u0026deg;C. The reaction mixture was then cooled to 10\u0026ndash;15\u0026deg;C. Then, the mixture was stirred for 60 min. The reaction mixture was filtered and the wet cake was washed with prechilled toluene (50 g). The wet material was dried in hot air oven at 55\u0026ndash;60\u0026deg;C to obtain APN-10 (113.0 g, 82.70%). \u003csup\u003e1\u003c/sup\u003eH NMR (400M Hz, DMSO-d\u003csub\u003e6\u003c/sub\u003e) δ: 7.49 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H), 7.47 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H), 7.35 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H), 7.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8Hz, 2H), 4.35 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 4.07 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 3.80 (s, 3H), 3.59 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 3.20 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 1.79\u0026ndash;1.88 (m, 4H), 1.33 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 3H), \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e) δ:168.8, 161.3, 159.3, 156.3, 141.4, 139.6, 138.4, 133.0, 132.4, 126.7, 126.7, 126.3, 126.0, 113.4, 60.6, 55.4, 50.8, 50.7, 32.5, 22.9, 21.1, 20.8, 14.1; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 2941.35 (Ar-C-H), 1737.22 (C\u0026thinsp;=\u0026thinsp;N), 1671.12 (C\u0026thinsp;=\u0026thinsp;O), 1651.01 (-C\u0026thinsp;=\u0026thinsp;O), 1515.94 (-C\u0026thinsp;=\u0026thinsp;N), 1467.08 (-C-O), 1250.11 (-C-N), 1142.34 (-C-N). (ESI-MS) m/z calculate for C\u003csub\u003e27\u003c/sub\u003eH\u003csub\u003e28\u003c/sub\u003eN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e5\u003c/sub\u003e 488.21, found 489.00 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide\u003c/b\u003e \u003cb\u003e(Apixaban)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003e2\u003c/b\u003e litre clean and dry autoclave reactor was arranged. PAN-10 (110 g) and methanol (2178 g) were charged into the autoclave, after which the autoclave was closed. Ammonia gas was purged into the reactor (4\u0026ndash;6 kg/cm\u003csup\u003e2\u003c/sup\u003e) and then the temperature was increased to 70\u0026ndash;75\u0026deg;C. The reaction mixture was maintained for 6\u0026ndash;8 h. The progress of reaction was monitored by HPLC. After the reaction complies on HPLC, reaction mass was filtered through hyflow bed. Methanol was partially distilled to get suspension which upon filtration and drying yielded pure apixaban (83.0 g, 80%). \u003csup\u003e1\u003c/sup\u003eH NMR (400M Hz, DMSO-d\u003csub\u003e6\u003c/sub\u003e) δ: 7.73 (br s, 1H), 7.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 7.45 (br s, 1H), 7.35(d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 2H), 7.28 (d,.\u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 7.00 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 4.04 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 2H), 3.79 (s, 3H), 3.58 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 3.20 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz 2H), 2.38 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 2H), 1.79\u0026ndash;1.88 (m, 4H), \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-d\u003csub\u003e6\u003c/sub\u003e) δ:168.8, 163.2, 159.1, 156.6, 141.4, 141.3, 139.7, 132.9, 132.5, 126.8, 126.3, 125.9, 125.2, 113.3, 55.4, 50.9, 50.8, 32.5, 23.0, 21.0, 20.9; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3300.04 (N-H ), 2866.47 (Ar-C-H), 1682.59 (C\u0026thinsp;=\u0026thinsp;O), 1630.14 (C\u0026thinsp;=\u0026thinsp;O), 1595.08 (-C\u0026thinsp;=\u0026thinsp;O), 1519.24 (-C\u0026thinsp;=\u0026thinsp;N), 1343.85 (C-N), 1310.09 (-C-N). M/S m/z calculate for m/z C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e25\u003c/sub\u003eN\u003csub\u003e5\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e 459.19, found 460.16 (M\u0026thinsp;+\u0026thinsp;H)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1-(4-aminophenyl)-3-morpholinopiperidin-2-one\u003c/b\u003e \u003cb\u003e(APN-05 impurity)\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003csup\u003e1\u003c/sup\u003eH NMR (400M Hz, DMF-d\u003csub\u003e7\u003c/sub\u003e) δ: 6.91 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 2H), 6.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 5.15 (s, 2H), 3.57\u0026ndash;3.63 (m, 4H), 3.49\u0026ndash;3.55 (m, 2H), 3.47\u0026ndash;3.52 (m, 2H), 3.21 (t, 1H), 2.62\u0026ndash;2.66 (m, 2H), 2.91\u0026ndash;2.93 (m, 2H), 1.85\u0026ndash;2.01 (m, 4H); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMF-d\u003csub\u003e7\u003c/sub\u003e) δ: 169.51, 147.76, 133.21, 127.36, 114.24, 67.65, 65.09, 51.53, 50.47, 25.68, 21.94; IR (KBr, cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3398.29 (-NH\u003csub\u003e2\u003c/sub\u003e), 1632.33 (C\u0026thinsp;=\u0026thinsp;O stretching), 1514.50 (C-O stretching), 1398.11 (C-N stretching). ESI-MS: \u003cem\u003em/z\u003c/em\u003e calculated for C\u003csub\u003e15\u003c/sub\u003eH\u003csub\u003e21\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 275.16; found 276.21 (M\u0026thinsp;+\u0026thinsp;1)\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eSupporting information\u003c/h2\u003e \u003cp\u003eThe supporting information file contains details of HPLC methods used for the analysis of reaction mass and purity of compounds. Scan copy of HPLC chromatogram indicating peak of APN V along with impurity peak. Scan copies of \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR, IR and ESI-MS spectra of synthesized compound along with isolated impurity are provided. Details of Flash chromatographic condition used for the isolation of impurity are also provided.\u003c/p\u003e\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eThere is no conflict of interest declared by authors of the manuscript.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eVipin Kumar, Sachin Kumar Sharma,Govind Singh and Shyambaran Dhuriya: Synthetic lab work and collection of dataLingaiah Balthu: Reviewed the manuscriptSushilkumar,Rashmi Devi: Isolation of impurity by flash chromatography Krishna Kumar Yadav: Reviewed analytical dataSumit S. Chourasiya: Conception and drafting of manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors acknowledge the management of IOL Chemicals and Pharmaceuticals Ltd for giving permission to publish the research work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eY.H. Mekaj, A.Y. Mekaj, S. B. Duci, E. I. Miftari, Ther. Clin. Risk Manage. 24, 967\u0026ndash;77 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eB.E. Pollock, J. Am. Med. Assoc. 12, 1094\u0026ndash;7 (1955)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG.D. Murray, Br. J. Surg. 27, 567\u0026thinsp;\u0026ndash;\u0026thinsp;98 (1940)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. Hawkins, Pharmacotherapy. 24, 62S-5S (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Leadley, Curr. Trends Med. Chem. 1, 151\u0026ndash;9 (2001)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD.J. Pinto, M.J. Orwat, S. Koch, K.A. Rossi, R.S. Alexander, A. Smallwood, P.C.Wong, A.R. Rendina, J. M. Luettgen, R.M. Knabb, K. He J. Med. Chem. 50, 5339\u0026ndash;56. (2007)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJ. Watson, G. Whiteside, C. Perry, Drugs. 71, 2079\u0026ndash;89 (2011)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZ. Hijazi, S.H. Hohnloser, U. Andersson, J. H. Alexander, M. Hanna, M. Keltai, A. Parkhomenko, J. L. Lopez-Sendon, R.D. Lopes, A. Siegbahn, C.B. Granger, \u003cem\u003eJAMA cardiology\u003c/em\u003e. 1, 451\u0026thinsp;\u0026ndash;\u0026thinsp;60 (2016)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM.W. Mandernach, R.J. Beyth, A. Rajasekhar, Ther. Clin. Risk Manage. 26, 1273\u0026ndash;82 (2015)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG. Agnelli, H.R. Buller, A. Cohen, M. Curto, A. S. Gallus, M. Johnson, A. Porcari, G.E. Raskob, J. I. Weitz, N. Engl. J. Med. 21, 699\u0026ndash;708 (2013)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eE. Perzborn, S. Roehrig, A. Straub, D. Kubitza, W. Mueck, V. Laux, Arterioscler., Thromb., Vasc. Biol., 30, 376\u0026ndash;81 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK. Ogata, J. Mendell-Harary J, M. Tachibana, H. Masumoto, T. Oguma, M. Kojima, S. Kunitada, J. Clin. 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S.). \u003cem\u003e5\u003c/em\u003e, 89\u0026ndash;117 (2004)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD. J. Snodin, Org. Process Res. Dev. 14, 960\u0026ndash;76 (2010)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eK.L. Dobo, N. Greene, M. O. Cyr, S. Caron, W. W. Ku, Regul. Toxicol. Pharmacol. 44, 282\u0026ndash;93 (2006)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eD.J. Snodin, S. D. McCrossen. Regul. Toxicol. Pharmacol. 63, 298\u0026ndash;312 (2012)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuideline IH. Impurities in new drug substances Q3A (R2). InProceedings of the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Geneva, Switzerland 2006 Oct 25 (Vol. 25).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 and 2 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Process related impurity, Intermediate, Isolation, Synthesis, Active Pharmaceutical Ingradients","lastPublishedDoi":"10.21203/rs.3.rs-3992931/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3992931/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this report, we describe the identification of new process related impurity observed during the preparation of Apixaban-An anticoagulant drug. As a part of the investigation, we isolated the impurity by column chromatography with \u0026gt;\u0026thinsp;96% purity and analyzed the sample of impurity by LC-MS, IR and NMR spectroscopy. The LC-MS analysis coupled with IR and NMR analysis suggested that the impurity is 1-(4-aminophenyl)-3-morpholinopiperidin-2-one, generated during preparation of Apixaban intermediate (APN-05). The impurity is a reduced form of APN-05 possibly formed due to reduction of ring double bond of dihydropyridin-2(1H)-one moiety of APN-05.\u003c/p\u003e","manuscriptTitle":"1-(4-aminophenyl)-3-morpholinopiperidin-2-one: A new process related impurity of Apixaban- An anticoagulant drug","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 08:18:35","doi":"10.21203/rs.3.rs-3992931/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d548b19-6631-4d1a-b894-ffc6cd10e5e3","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T02:47:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 08:18:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3992931","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3992931","identity":"rs-3992931","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}