molecules Article
Synthesis and Characterization of Process-Related Impurities of Antidiabetic Drug Linagliptin Article Synthesis and Characterization of Process-Related Impurities of Antidiabetic Drug Linagliptin Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry,
Yiwen Huang, Xiaoqing He, Taizhi Wu * and Fuli Zhang *
285 Gebaini Road, Pudong, Shanghai 201203, China;
[email protected] (Y.H.);
[email protected] (X.H.) Yiwen Huang,
[email protected] Xiaoqing He, Taizhi Wu * and Fuli Zhang * * Correspondence: (T.W.);
[email protected] (F.Z.); Tel.:Shanghai +86-21-2057-2000 5036) (T.W.); +86-21-2057-2000 (ext. 5069) (F.Z.) Institute of (ext. Pharmaceutical Industry, China State Institute of Pharmaceutical Industry, 285 Gebaini Road, Pudong, Shanghai 201203, China;
[email protected] (Y.H.);
[email protected] (X.H.)
Academic Editor: Diego Muñoz-Torrero * Correspondence:
[email protected] (T.W.);
[email protected] (F.Z.); Received: 22+86-21-2057-2000 June 2016; Accepted: 5 August Published:(ext. 9 August 2016 Tel.: (ext. 5036) (T.W.); 2016; +86-21-2057-2000 5069) (F.Z.)
Linagliptin, a Muñoz-Torrero xanthine derivative, is a highly potent, selective, long-acting and orally Abstract: Academic Editor: Diego Received: 22 June 2016; Accepted: 5 August 2016; Published: bioavailable DPP-4 inhibitor for the treatment of type date 2 diabetes. During the process development of linagliptin, five new process-related impurities were detected by high performance liquid Abstract: Linagliptin, a xanthine derivative, is a highly potent, selective, long-acting and orally chromatography (HPLC). All these impurities were identified, synthesized, and subsequently bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes. During the process development 1 H-NMR, 13 C-NMR and IR) as described characterized by their spectral data (MS, HRMS, of linagliptin, fiverespective new process-related impurities were detected by high performance liquid in this article. The identification of these impurities be useful for quality control and the chromatography (HPLC). All these impurities wereshould identified, synthesized, and subsequently 1 13 validation of the analytical methodspectral in the manufacture of linagliptin. characterized by their respective data (MS, HRMS, H-NMR, C-NMR and IR) as described in this article. The identification of these impurities should be useful for quality control and the
Keywords: linagliptin; type 2 method diabetes; impurities; synthesis; characterization validation of the analytical in the manufacture of linagliptin. Keywords: linagliptin; type 2 diabetes; impurities; synthesis; characterization
1. Introduction 1. Introduction The presence of impurities in a drug substance can have a significant impact on the quality and safety of the product [1]. According the general guidelines on impurities substances Thedrug presence of impurities in a drugto substance can have a significant impact on in thedrug quality and recommended by the International Conference on Harmonization (ICH), any impurities present safety of the drug product [1]. According to the general guidelines on impurities in drug substancesin the drug recommended substance greater a level of 0.10% forondrugs with a maximum dose present equal toinor by thethan International Conference Harmonization (ICH), anydaily impurities thelesser substance greater than level of 0.10% for with a maximum daily dose equal to or lesser than 2drug g should be identified anda characterized [2].drugs On one hand, the identification and characterization than 2 g shouldimpurities be identified and characterized [2]. On one hand,impurities the identification of process-related can guide us in controlling these withinand thecharacterization acceptable level by of process-related impurities can guide us in controlling these impurities within the level to improving reaction conditions in turn. On the other hand, impurities in pure formacceptable are also needed by improving reaction conditions in turn. On the other hand, impurities in pure form are also needed validate the analytical method including checking the system suitability and relative correctiontofactor. validate the analytical method including checking the system suitability and relative correction factor. Linagliptin (1, Figure 1), a highly potent, selective, long-acting and orally bioavailable DPP-4 Linagliptin (1, Figure 1), a highly potent, selective, long-acting and orally bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes, is chemically known as (R)-8-(3-aminopiperidin-1-yl)-7inhibitor for the treatment of type 2 diabetes, is chemically known as (R)-8-(3-aminopiperidin-1-yl)(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione 7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione [3].[3]. CH 3
O N N
N N N CH 3
O
CH 3
CH 3
O N
N
N
N NH 2
O
CH 3
N
N N
N
N
O
N
N N
4
CH3
N
N N
O
CH3
O N
N H H N
N O
CH 3
O
N N
N NH HO
CH 3
O
O
3
O
N
O
N
N O
2
N
N
O
N
CH3
O N
N N
NH HN
CH 3
1
N
OH
N
N
CH 3
O
N
N
O
CH 3
NH N CH3
5
CH3
O N
HN
N
O OH
N N
N
NH 2
CH3
6
Figure 1. Chemical structures of linagliptin and process-related impurities. Figure 1. Chemical structures of linagliptin and process-related impurities. Molecules 2016, 21, 1041; doi:10.3390/molecules21081041
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In the industrial manufacturing process (Scheme 1) of linagliptin by the Boehringer2Ingelheim Molecules 2016, 21, 1041 of 9 company [4], cyclization of 1-(2-aminophenyl)ethanone (7) with 2-chloroacetonitrile (8) in the presence the industrial manufacturing process (Scheme 1) of linagliptin by the(9, Boehringer Ingelheim which of hydrogenIn chloride afforded 2-(chloromethyl)-4-methylqu inazoline yield 74%–85%) company [4], cyclization of 1-(2-aminophenyl)ethanone (7) with 2-chloroacetonitrile (8) in the condensed with 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione (10) in presence the presence of of hydrogen chloride afforded 2-(chloromethyl)-4-methylqu inazoline (9, yield 74%–85%) which sodium carbonate as a basic reagent, giving 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin condensed with 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione (10) in the presence of -2-yl)methyl)-1H-puri yield 76%–83%). Subsequently, the condensation of 11 sodium carbonatene-2,6(3H,7H)-dione as a basic reagent, giving(11, 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolinwith (R)-2-(piperidin-3-yl)isoindoline-1,3-dione D-(´)-tartaric (12) using as 2-yl)methyl)-1H-puri ne-2,6(3H,7H)-dione (11, yield 76%–83%).acid Subsequently, thediisopropylethylamine condensation of 11 with (R)-2-(piperidin-3-yl)isoindoline-1,3-dione D-(−)-tartaric acid (12) using diisopropylethylamine as a basic reagent provided (R)-7-(but-2-yn-1-yl)-8-(3-(1,3-dioxoisoindolin-2-yl) pipe ridin-1-yl)-3-methyla basic reagent provided (R)-7-(but-2-yn-1-yl)-8-(3-(1,3-dioxoisoindolin-2-yl)pipe ridin-1-yl)-3-methyl1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione (13, yield 90%–94%). Compound 13 1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione (13, yield 90%–94%). Compound 13 finally converted to the desired linagliptin (1) in 81.9% yield via aminolysis using ethanolamine as the finally converted to the desired linagliptin (1) in 81.9% yield via aminolysis using ethanolamine as aminolysis agent. the aminolysis agent. a
NH2
CH3
N
Cl
C
9
CH3
O
O
Br N N CH3
O
CH3
11
N
HN
N
N N
CH3
8
7
N
b
Cl N
O
CH3
O
N
Br N CH3
N
10
HO
COOH
HO
COOH
HN
c
N
O
O 12
N O
CH3
N
d
N
N N
1
NH2
H2N
OH
N
N N
N N N CH3
CH3
O
CH3
O
CH3
O
N N N CH3
N
O
O
13
1. Synthesis of linagliptin Conditions:(a) (a) HCl, HCl, 1,4-dioxane, 6 °C (b) Na(b) 2CO3, SchemeScheme 1. Synthesis of linagliptin (1).(1). Conditions: 1,4-dioxane, 6 ˝, 2Ch,, 274%–85%; h, 74%–85%; Na2 CO3 , N-methyl-2-pyrrolidone (NMP), 140 °C, 2 h, 76%–83%; (c) diisopropanolamine, NMP, 140 °C, 2 h,˝ ˝ N-methyl-2-pyrrolidone (NMP), 140 C, 2 h, 76%–83%; (c) diisopropanolamine, NMP, 140 C, 2 h, 90%–94%; (d) ethanolamine, THF/H2O, 60 °C, 3 h, 81.9%. 90%–94%; (d) ethanolamine, THF/H2 O, 60 ˝ C, 3 h, 81.9%.
However, the literature search did not reveal much work on the impurity research of linagliptin.
However, the literature search did reveal much on the impurity research linagliptin. The impurity profile of linagliptin in not this synthetic route work is different from the earlier reportedof study The impurity profile of linagliptin in this synthetic is different from the earlier reported study [5,6], [5,6], which makes it more challenging to identifyroute the unknown impurities formed in small quantities in the drug substance. Since mosttoofidentify the time itthe is very difficult to identify andformed control impurities which makes it more challenging unknown impurities in small within quantities in levels in the process, some advanced purification forand an active drug substance within the drugacceptable substance. Since most of the time it is very difficulttechniques to identify control impurities may then be taken into account, such as the continuous-flow process [7], organic solvent nanofiltration acceptable levels in the process, some advanced purification techniques for an active drug substance (OSN) [8], molecularly imprinted membranes for OSN [9] and counter current chromatography may then be taken into account, aschromatography the continuous-flow [7], organic solvent nanofiltration (CCC), as a valuable additionsuch to the toolbox process [10]. (OSN) [8], molecularly imprinted membranes for OSN [9] and counter current the chromatography Hence, a comprehensive study was undertaken to identify and synthesize impurities in a (CCC), sampleaddition of linagliptin as described in this article. The study as a valuable to the chromatography toolbox [10].will help to understand the formation of the impurities in linagliptinstudy synthesis provide a clue how to obtain a pure drugthe substance. Hence, a comprehensive wasand undertaken to on identify and synthesize impurities in a sample of linagliptin as described in this article. The study will help to understand the formation of 2. Results and Discussion the impurities in linagliptin synthesis and provide a clue on how to obtain a pure drug substance. 2.1. Structures of Impurities
2. Results and Discussion
During the process development of linagliptin, five new process-related impurities were detected by high performance 2.1. Structures of Impuritiesliquid chromatography (HPLC) (Figure 2), varying from 0.15% to 0.5%, and the molecular weight of the respective impurities was identified through liquid chromatographic mass spectrometry (LC-MS). From the molecular weight information, an extensive study was undertaken During the process development of linagliptin, five new process-related impurities weretodetected predict and synthesize the five new impurities. Finally, all these impurities were synthesized by high performance liquid chromatography (HPLC) (Figure 2), varying from 0.15% toand 0.5%, and subsequently subjected to spectral analysis. The five predicted and synthesized impurities showed the
the molecular weight of the respective impurities was identified through liquid chromatographic mass spectrometry (LC-MS). From the molecular weight information, an extensive study was undertaken to predict and synthesize the five new impurities. Finally, all these impurities were synthesized and subsequently subjected to spectral analysis. The five predicted and synthesized impurities showed the
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samesame retention time with those detected in HPLC. Based on the spectral data, these impurities were retention time with those detected in HPLC. Based on the spectral data, these impurities were characterized as structures shown fivenew newimpurities impurities reported for the characterizedthe as the structures shownininFigure Figure1. 1. The The five areare all all reported herehere for the first time. first time. 2016, 21, 1041 Molecules 3 of 9 same retention time with those detected in HPLC. Based on the spectral data, these impurities were characterized as the structures shown in Figure 1. The five new impurities are all reported here for the first time.
typical sample blank control
typical sample
Figure 2. Typical HPLC chromatogram of linagliptin with impurities.
blank Figure 2. control Typical HPLC chromatogram of linagliptin with impurities.
2.2. Source of Impurities
2.2. Source of Impurities
2. Typical HPLC chromatogram of linagliptin with The synthesis ofFigure linagliptin involved the aminolysis of 13 using 10impurities. equivalents of ethanolamine
The synthesis of2 linagliptin involved thestep, aminolysis of 13incomplete using 10 equivalents ethanolamine [4]. Compound is the by-product in this formed from aminolysis. of Impurity 2 was [4]. 2.2. Source of Impurities further hydrolyzed to afford impurity due toformed water/tetrahydrofuran (THF) as a solvent.Impurity The formed Compound 2 is the by-product in this 3step, from incomplete aminolysis. 2 was linagliptin moiety (1)ofreacted with starting 13 orofwith 2, providing 4. During The synthesis linagliptin involved aminolysis 13 using 10 equivalents ethanolamine further hydrolyzed to afford impurity 3 due the tomaterial water/tetrahydrofuran (THF)impurity as aofsolvent. Thethe formed [4]. Compound 2 reacted is the12 by-product in this formed incomplete aminolysis. Impurity 2During was condensation 11 with to afford 13, the step, intermediate 11 cannot converted completely may the linagliptin moietyof(1) with starting material 13from or with 2,beproviding impurity 4.and further to afford 3 due to water/tetrahydrofuran (THF) as a in solvent. The formed13 remain inhydrolyzed the intermediate 13. impurity Unfortunately, the small amount of 11 remaining the intermediate condensation of 11 with 12 to afford 13, the intermediate 11 cannot be converted completely and may linagliptin moiety (1) reacted with material 13 orwhich with 2,explains providing the 5. reacted with ethanolamine in the nextstarting aminolysis process, the impurity formation4.ofDuring impurity remain condensation in the intermediate 13. Unfortunately, the small amount ofbe 11converted remaining in the intermediate 13 of 11 with 12 to afford 13, the intermediate 11 cannot completely and may Similarly, during the condensation of 10 with 9 to afford 11, the intermediate 10 cannot be converted reacted with ethanolamine in the next aminolysis process, which explains the formation of impurity 5. remain in and the intermediate 13. Unfortunately, the11. small amount of 11 remaining inin the 1311 completely may remain in the intermediate Subsequently, 10 remaining theintermediate intermediate Similarly, during the condensation of 10 with 9 to afford 11, the intermediate 10 cannot be converted reacted with ethanolamine in the next aminolysis process, which explains the formation of impurity 5. took part in the condensation reaction with 12 and in the aminolysis reaction in the next two steps, Similarly, during thethe condensation ofimpurity 10 with 9611. to(Scheme afford 11, 10 cannot be converted completely and may remain in the intermediate Subsequently, 10 remaining in the intermediate which finally leads to formation of 2).the As intermediate a result, the five impurities accounted 11 completely and may remain in the intermediate 11. Subsequently, 10 remaining in the intermediate 11 steps, took for part in the condensation with 12 and inin the reaction in the next two 0.4%, 0.32%, 0.27%, 0.33%reaction and 0.16%, respectively, theaminolysis drug substance. took part in the condensation reaction with 12 and in the aminolysis reaction in the next two steps, which finally leads to the formation of impurity 6 (Scheme 2). As a result, the five impurities accounted which finally leads to the formation of impurity 6 (Scheme 2). As a result, the five impurities accounted CH for 0.4%, 0.27%, 0.33% andand 0.16%, respectively, thedrug drugsubstance. substance. CH O in O for0.32%, 0.4%, 0.32%, 0.27%, 0.33% 0.16%, respectively, in the HN
3
HN O
N
3
O
N
N N CH 3
O
H 2N
N
O
OH
Br
impurity 5
N N CH 3
CH3
N
O
CH 3
O
O
impurity 6
N
N CH3
N N
OH
N
10
N
H 2N
N
HN
12
Br
THF/H2O
11
CH3
O N
N
N N
CH 3
O
H 2N
N N
N
N
CH3
O
O
OH
impurity 2
H2O
impurity 3
THF/H2O
13
linagliptin 1
impurity 4
linagliptin 1
impurity 4
Scheme 2. Source of impurities of linagliptin. Scheme 2. Source of impurities of linagliptin.
Scheme 2. Source of impurities of linagliptin.
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2.3. Preparation and Characterization of Impurities Compound Compound 2 was synthesized by incomplete aminolysis of 13. In order to prepare 2 in good yield, we replaced to obtain a homogeneous system we replaced the thesolvent solventof ofTHF/H THF/H2 O with dichloromethane (CH (CH22Cl22)) to and a smaller smaller inventory ratio of ethanolamine ethanolamine was used used for for more more moderate moderate aminolysis aminolysis conditions conditions (Scheme (Scheme 3). 3).
Scheme 3. Preparation of impurity 2 and 3. Scheme 3. Preparation of impurity 2 and 3.
The mass spectrum of 2 showed a molecular ion peak at m/z 686.23 (M + Na)+ in positive ion The mass spectrum showed a molecular ion The peak at m/z 686.23 + Na)+ in positivethree ion 1H-NMR of this(M compound revealed mode, indicating the massofof2this compound to be 663. 1 mode, indicating the mass of this compound to beand 663.3.70, Theindicating H-NMR of revealed additional D2O exchangeable signals at 8.35, 6.95 thethis twocompound amide N-H protonsthree and additional D O exchangeable signals at 8.35, 6.95 and 3.70, indicating the two amide N-H protons and 2 one O-H proton. Additional signals observed in the region of 8.04–7.47 indicated eight aromatic C-H one O-H proton. Additional observed region 8.04–7.47 indicatedthe eight aromatic protons. In the IR spectrum,signals observed bandsinatthe 3473 and of 3263 cm−1 indicated amide N-H C-H and ´ 1 protons. In the IR spectrum, observed bands at 3473 and 3263 cm indicated the amide N-H and 13 O-H stretching. The C-NMR revealed 35 carbon atoms and further Distortionless Enhancement by O-H stretching. The 13(DEPT) C-NMRexhibited revealed eight 35 carbon atomscarbon and further Distortionless by Polarization Transfer secondary atoms. Based on all Enhancement the spectral data Polarization Transfer (DEPT) exhibited eight secondary carbon atoms. Based on all the spectral data (MS, HRMS, 1H-NMR, 13C-NMR and IR), the structure of impurity 2 was confirmed as (R)-N1-(1-(7-(but(MS, HRMS, 1 H-NMR, 13 C-NMR and IR), the structure of impurity 2 was confirmed as (R)-N1 -(1-(7-(but2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl) 2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl) piperidin-3-yl)-N2-(2-hydroxyethyl)phthalamide. 2 piperidin-3-yl)-N Compound 3 -(2-hydroxyethyl)phthalamide. was prepared by the alkali hydrolysis of 13 with phase transfer catalyst (Scheme 3). Compoundproduct 3 was prepared by the alkali hydrolysis of 13 with phase transfer catalyst (Scheme 3). The undesired 1 from excessive hydrolysis can be removed by CH 2Cl2. The undesired product 1 from excessive hydrolysis can be removed by CH Cl . The mass spectrum of 3 exhibited a molecular ion peak at m/z 643.252 (M2 + Na)+ in positive ion The spectrum molecular ion peak the at m/z 643.25 (Mcompound + Na)+ intopositive mode andmass m/z 619.31 (M −ofH)3− exhibited in negativea ion mode, indicating mass of this be 620. ´ ion andofm/z 619.31 (M ´revealed H) in two negative mode, indicating the12.86 massand of this 1H-NMR The mode this impurity D2O ion exchangeable signals at 8.35,compound indicating 1 H-NMR of this impurity revealed two D O exchangeable signals at 12.86 and 8.35, to beacid 620.O-H Theproton 2 one and one amide N-H proton, and an additional eight proton signals at 7.41–8.25 indicating one acid O-H proton and one amide N-H proton, and an additional eight proton signals corresponding to aromatic protons. Interestingly, we found the methyl group (CH 3) in quinazoline at 7.41–8.25 corresponding to aromatic protons. Interestingly, we found the methyl group (CHwith 3 ) in at δ 2.89 ppm is acidic at a certain degree, which explains the proton exchange of the CH3 group quinazoline at δ 2.89 ppm is acidic The at a proton certain exchange’s degree, which explains the proton exchange ofalso the D2O which takes place sometimes. resulting compound (3-d, Figure 3) CH group with D O which takes place sometimes. The proton exchange’s resulting compound 3 2 explains the proton splitting of the CH3 group, which showed a triplet peak with one proton of 2.89 (3-d, 3) also explains proton splitting of the CH3 group, showed a triplet ppm Figure in the 1H-NMR with D2Othe added (Figure 3). The IR spectrum of 3which exhibited a band at 3527peak cm−1 1 with one proton of 2.89 ppm in the H-NMR with D O added (Figure 3). The IR spectrum of 3 −1 13 2 corresponding to amide N-H stretching, and 3255 cm for acid O-H stretching. The C-NMR revealed ´1 corresponding to amide N-H stretching, and 3255 cm´1 for acid O-H exhibited a band at 3527 cm 33 carbon atoms and further DEPT showed six secondary carbon atoms. Impurity 3 was characterized stretching. The 13spectral C-NMRdata revealed carbon atoms and furtherand DEPT six secondary carbon 1H-NMR, 13C-NMR by all respective (MS, 33 HRMS, IR) showed as (R)-2-((1-(7-(but-2-yn-1-yl)atoms. Impurity 3 was characterized by all respective spectral data (MS, HRMS, 1 H-NMR, 13 C-NMR 3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)piperidin3-yl)carbamoyl)benzoic acid.
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and IR) as (R)-2-((1-(7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid. Molecules 2016, 21, 1041 5 of 9 Molecules 2016, 21, 1041
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Figure3.3.Structures Structuresand andpartial partial11H-NMR H-NMR spectrum spectrum of of 3-d. 3-d. Figure Figure 3. Structures and partial 1H-NMR spectrum of 3-d.
We can only obtain compound 4 with the low yield of 1.5% directly via 13 reacting with the We We can can only only obtain obtain compound compound 4 with with the low yield of 1.5% directly via 13 reacting reacting with the linagliptin moiety (1). Instead, we have exploited a different concept for the synthesis of compound linagliptin moiety (1). Instead, we have exploited a different concept for the synthesis synthesis of compound compound 4 starting from phthaloyl dichloride (14). However, 12% of the single-benzoyl chloride compound 15 4 starting from from phthaloyl phthaloyldichloride dichloride(14). (14).However, However,12% 12%ofofthe the single-benzoyl chloride compound single-benzoyl chloride compound 15 and 11% of the cyclization product 13 would be generated meanwhile due to the steric hindrance from 15 and 11% of the cyclization product 13 would be generated meanwhile due to the steric hindrance and 11% of the cyclization product 13 would be generated meanwhile due to the steric hindrance from linagliptin, which influenced the purity of 4. We reduced them to below 0.3% and finally got compound from linagliptin, influenced theof purity 4. We reduced them 0.3% to below 0.3% and finally got linagliptin, whichwhich influenced the purity 4. Weof reduced them to below and finally got compound 4 with a high purity of 99.15% by washing the reaction mass with CH2Cl2 and aqueous sodium compound 4 with a high purity of by washing the reaction mass CH2 Cl and aqueous 4 with a high purity of 99.15% by99.15% washing the reaction mass with CHwith 2Cl2 and aqueous sodium 2 hydroxide solution instead of the column chromatography (Scheme 4). sodium hydroxide instead of the column chromatography 4). hydroxide solutionsolution instead of the column chromatography (Scheme(Scheme 4).
Scheme 4. Preparation of impurity 4. Scheme 4. 4. Preparation Preparation of of impurity impurity 4. 4. Scheme
The mass spectrum of 4 exhibited a molecular ion peak at m/z 1075.24 (M + H)+, 1097.22 (M + Na)+ The mass spectrum of 4 exhibited a molecular ion peak at m/z 1075.24 (M + H)++, 1097.22 (M + Na)++ in positive ionspectrum mode, indicating the mass of this compound be 1075.24 1074. The formula this The mass of 4 exhibited a molecular ion peak attom/z (Mmolecular + H) , 1097.22 (M +ofNa) in positive ion mode, indicating the mass of this compound to be 1074. The molecular formula of this compound wasmode, C58H58N 16O6 as confirmed resolution spectrum (HRMS). In the IR in positive ion indicating the massby of the thishigh compound to mass be 1074. The molecular formula compound was C58H58N16O6 as confirmed by the high resolution mass spectrum (HRMS). In the IR −1 indicated the amide N-H. The 13C-NMR showing 29 carbon spectrum the observed band at 3243 cm of this compound was C H N O as confirmed by the high resolution mass spectrum (HRMS). 58 58 16 6 −1 spectrum the observed band at 3243 cm indicated the´amide N-H. The 13C-NMR showing 13 29 carbon 1 indicated atoms the the symmetry in the structure compound 4. Based on all the spectral (MS, In the indicated IR spectrum observed band at 3243ofcm the amide N-H. The data C-NMR atoms indicated the symmetry in the structure of compound 4. Based on all the spectral data (MS, 1 13 HRMS, H-NMR, and IR), the 4 was of confirmed as N,N-bis((R)-1-(7showing 29 carbon C-NMR atoms indicated the structure symmetryofinimpurity the structure compound 4. Based on all HRMS, 1H-NMR, 13C-NMR and IR), the structure of impurity 4 was confirmed as N,N-bis((R)-1-(7(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purinthe spectral data (MS, HRMS, 1 H-NMR, 13 C-NMR and IR), the structure of impurity 4 was confirmed (but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin8-yl)piperidin-3-yl)phthalamide. as N,N-bis((R)-1-(7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,78-yl)piperidin-3-yl)phthalamide. Compound 5 was independently provided via the condensation of compound 11 with ethanolamine. tetrahydro-1H-purin-8-yl)piperidin-3-yl)phthalamide. Compound 5 was independently provided via the condensation of compound 11 with ethanolamine. In order to prepare 5 in good yield, we replaced the solvent of tetrahydrofuran/water with toluene to In order to prepare 5 in good yield, we replaced the solvent of tetrahydrofuran/water with toluene to obtain a homogeneous system and a more violent reaction temperature. The increasing inventory obtain a homogeneous system and a more violent reaction temperature. The increasing inventory ratio of ethanolamine also contributed to the high yield of 94% (Scheme 5). ratio of ethanolamine also contributed to the high yield of 94% (Scheme 5).
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Compound 5 was independently provided via the condensation of compound 11 with ethanolamine. In order to prepare 5 in good yield, we replaced the solvent of tetrahydrofuran/water with toluene to obtain a homogeneous system and a more violent reaction temperature. The increasing inventory ratio of ethanolamine also contributed to the high yield of 94% (Scheme 5). Molecules 2016, 21, 1041 6 of 9
Scheme Preparation of of impurities impurities 55 and and 6. 6. Scheme V. 5. Preparation
The mass spectrum of 5 showed a molecular ion peak at m/z 456.51 (M + Na)+ in positive ion The mass spectrum of 5 showed a molecular ion peak1 at m/z 456.51 (M + Na)+ in positive ion mode, indicating the mass of this compound to be 433. The H-NMR of this compound revealed two mode, indicating the mass of this compound to be 433. The 1 H-NMR of this compound revealed two D2O exchangeable signals at 7.23 and 4.78, indicating the N-H and O-H protons, and an additional D2 O exchangeable signals at 7.23 and 4.78, indicating the N-H and O-H protons, and an additional four four proton signals at 7.62–8.25 corresponding to aromatic protons. In the IR spectrum the observed proton signals at 7.62–8.25 corresponding to aromatic protons. In the IR spectrum the observed bands bands at 3446 and 3364 cm−1 indicated the N-H and O-H stretching. The 13C-NMR revealed 22 carbon at 3446 and 3364 cm´1 indicated the N-H and O-H stretching. The 13 C-NMR revealed 22 carbon atoms atoms and DEPT showed four secondary carbon atoms. Based on all the spectral data (MS, HRMS, and DEPT 13showed four secondary carbon atoms. Based on all theconfirmed spectral data (MS, HRMS, 1 H-NMR, 1H-NMR, C-NMR and IR), the structure of impurity 5 was as 7-(but-2-yn-1-yl)-8-((213 C-NMR and IR), the structure of impurity 5 was confirmed as 7-(but-2-yn-1-yl)-8-((2-hydroxyethyl) hydroxyethyl)amino)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione. amino)-3-methyl-1-((4-methylquinazolin-2yl)methyl)-1H-purine-2,6(3H,7H)-dione. Compound 6 was independently synthesized, starting from compound 10, following a synthetic Compound 6 was independently synthesized, starting fromexploited compounda 10, following a synthetic process analogous to that of linagliptin. Differently, we have one-pot process for the process analogous to that of linagliptin. Differently, we have exploited a one-pot process for the synthesis of compound 6 instead of the two-step operation in greater yield (Scheme 5). synthesis of compound of the two-step ion operation greater yield 5). The mass spectrum6ofinstead 6 showed a molecular peak atinm/z 317.14 (M +(Scheme H)+ in positive ion mode, The mass spectrum of 6 showed a molecular ion peak at m/z 317.14 (M + H)+ in positive indicating the mass of this compound to be 316, which is 156 amu less than that of linagliptin (1). The ion mode,ofindicating the mass of this compound to be 316, which is 156 amu less than thatthe of 1H-NMR this compound revealed two D2O exchangeable signals at 10.94 and 8.27, indicating 1 linagliptin The and H-NMR this compound revealed two D2 O exchangeable at 10.94 imide N-H(1). proton N-H2of proton, and no additional aromatic protons were signals observed. In theand IR 8.27, indicating the imide N-H proton and N-H proton, and no additional aromatic protons were 2 spectrum the observed band at 3021 cm−1 indicated the imide N-H stretching and double peaks of ´1 indicated the imide N-H stretching observed. In the IR spectrum the observed band at 3021 cm −1 13 3115 and 3074 cm , corresponding to primary amine N-H stretching. The C-NMR revealed 15 carbon ´1 , corresponding to primary amine N-H stretching. The and double peaks of 3115 and 3074 cmcarbon atoms and DEPT showed five secondary atoms. Impurity 6 was characterized by all respective 13 C-NMR revealed 15 carbon atoms and DEPT showed five secondary carbon atoms. Impurity spectral data (MS, HRMS, 1H-NMR, 13C-NMR and IR) as (R)-8-(3-aminopiperidin-1-yl)-7-(but-2-yn61-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione. was characterized by all respective spectral data (MS, HRMS, 1 H-NMR, 13 C-NMR and IR) as (R)-8-(3-aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione. 3. Experimental Section 3. Experimental Section General Information Information General The compound 1313 (chemical purity 97.92%; chiral purity 99.97%) and The compound11 11(chemical (chemicalpurity purity98.36%), 98.36%), (chemical purity 97.92%; chiral purity 99.97%) 1 (chemical puritypurity 98.35%; chiral purity were prepared accordingaccording to the literature and 1 (chemical 98.35%; chiral 99.97%) purity 99.97%) were prepared to the procedure literature [4]. Other materials, solvents and reagents were of commercial origin and used without additional procedure [4]. Other materials, solvents and reagents were of commercial origin and used without operations. additional operations. 13 The 11HThe The H- and and 13C-NMR C-NMRspectra spectrawere were recorded recorded on on aa Bruker Bruker Arance Arance III III 400 400 MHz MHz spectrometer. spectrometer. The solvents used were DMSO-d 6 or CDCl3. The 11H-NMR chemical shift values were reported as δ ppm solvents used were DMSO-d6 or CDCl3 . The H-NMR chemical shift values were reported as δ ppm relative to tetramethylsilane (TMS) and the 13C-NMR chemical shift values were reported on δ ppm relative to DMSO-d6 or CDCl3. DEPT spectra revealed the presence of methyl and methine groups as positive peaks and methylene as negative peaks. The IR spectra were recorded in the solid state as KBr dispersion using a NICOLET 670 FT-IR spectrophotometer. Mass spectra and high resolution mass spectrum were recorded on Agilent 6120B series single quadrupole LC-MS and Q-Tof micro YA019 instrument. Melting points were measured on a WRS-1B apparatus. The specific rotation was
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relative to tetramethylsilane (TMS) and the 13 C-NMR chemical shift values were reported on δ ppm relative to DMSO-d6 or CDCl3 . DEPT spectra revealed the presence of methyl and methine groups as positive peaks and methylene as negative peaks. The IR spectra were recorded in the solid state as KBr dispersion using a NICOLET 670 FT-IR spectrophotometer. Mass spectra and high resolution mass spectrum were recorded on Agilent 6120B series single quadrupole LC-MS and Q-Tof micro YA019 instrument. Melting points were measured on a WRS-1B apparatus. The specific rotation was calculated from an optical rotation measurement performed on the Autopol IV, serial number 80799 (Rudolph, Hackettstown, NJ, USA) at the wavelength of 589 nm (D line of a sodium lamp), at 20 ˝ C. The HPLC analyses were recorded on a Dionex UItiMate 3000 HPLC instrument using Agilent Eclipse XDB C18 column (150 mm ˆ 4.6 mm, 5 µm) in a thermostated column heater at 55 ˝ C. The mobile phases consisting of A (0.1% methanoic acid, pH 2.5) and B (acetonitrile) were used with the gradient mode at the flow rate of 1.5 mL/min. The UV detection at 225 nm was used. Initial gradient starts with 5% of B and at 18 min it was set to 40%. The ratio had been set to 70% at 30 min and at 30.1 min it was 5%, which continued up to 35 min. The samples were diluted in acetonitrile with a concentration of 0.5 mg/mL. The injection volume was 5 µL. Limit of Detection (LOD) was 0.10 µg/mL or 0.02% for impurities 2, 3, 4, 5 and 0.15 µg/mL or 0.03% for impurity 6. Limit of Quantity (LOQ) was 0.25 µg/mL or 0.05% for impurities 2, 3, 4, 5 and 0.33 µg/mL or 0.07% for impurity 6. This HPLC method further subjected for LC-MS. Samples were run in Electro-Spray Ionization positive mode (ESI+) and 12 L/min nebulizer gas flow rate. The fragmentor voltage was 70 V and capillary voltage was maintained at 3.0 kV. The drying gas temperature was at 350 ˝ C. The detailed spectral data (IR, 1 H-NMR, 13 C-NMR, MS, HRMS) of the compounds 2–6 as well as the HPLC chromatogram of the compounds 1–6 are provided in the Supplementary Materials. (R)-N1 -(1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-ioxo-2,3,6,7-tetrahydro-1Hpurin-8-yl)piperidin-3-yl)-N2 -(2-hydroxyethyl)phthalamide (2). To a stirred solution of compound 13 (5.0 g, 0.008 mol) in CH2 Cl2 (50.0 mL) was added ethanolamine (1.5 g, 0.024 mol) and maintained the reaction mass at room temperature for 24 h. Then added 15 mL H2 O stirring for 30 min to dilute ethanolamine. The isolated solid was collected by filtration and washed with 10 mL H2 O and 20 mL CH2 Cl2 . Then dried to yield 2 as a light yellow solid (1.5 g, 27.3%), HPLC purity 97.52%; m.p. 189–192 ˝ C; rαs20 D ´32.567 (c = 1 g/100 mL, DMSO); IR (KBr) νmax 3473, 3263 (amide N-Hν , O-Hν ); 3062 (aromatic C-Hν ); 2937, 2850 (C-Hν ); 2230 (C”Cν ); 1698, 1662 (C=Oν ); 1522 (aromatic C=Cν , aromatic C=Nν ); 1440, 1400 (C-Hδ ); 1228 (C-Oν ); 763 (aromatic C-Hγ ) cm´1 ; 1 H-NMR (400 MHz, CDCl3 ) δ 8.35 (d, J = 6.6 Hz, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.82–7.76 (m, 1H), 7.64–7.52 (m, 3H), 7.47 (m, 2H), 6.95 (t, J = 5.6 Hz, 1H), 5.56 (s, 2H), 4.89 (q, J = 2.0 Hz, 2H), 4.31 (s, 1H), 3.78 (m, 2H), 3.70 (m, 2H), 3.60 (m, 2H), 3.52 (m, 2H), 3.40 (m, 1H), 3.23 (s, 3H), 2.91 (s, 3H), 2.17–2.10 (m, 1H), 1.92 (m, 2H), 1.75 (not resolved, 4H); 13 C-NMR (100 MHz, DMSO-d6 ) δ 169.29, 168.75, 168.46, 161.44, 156.37, 153.80, 151.41, 149.51, 148.03, 136.58, 136.51, 134.51, 129.80, 129.67, 128.33, 128.26, 128.02, 127.57, 126.13, 122.94, 103.90, 81.74, 74.21, 60.22, 53.84, 50.47, 46.13, 46.08, 42.55, 35.90, 29.90, 29.59, 23.53, 22.01, 3.55; HRMS (ESI) m/z 664.3019 (calcd for C35 H38 N9 O5 , 664.2996 [M + H]+ ). (R)-2-((1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1Hpurin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid (3). To a stirred solution of compound 13 (5.0 g, 0.008 mol) in CH2 Cl2 (50.0 mL) was added aqueous sodium hydroxide solution (1.0 g of NaOH in 50.0 mL of H2 O) and tetra-n-butylammonium bromide (0.27 g, 0.0008 mol). Then maintained the reaction mass at rt for 72 h. Then water phase was collected and washed with CH2 Cl2 (25.0 mL) for three times. To the stirred water phase was added 1 mol/L aq. HCl (25.0 mL) and maintained at rt for 30 min. The isolated solid was collected by filtration and washed with water (20.0 mL) and CH2 Cl2 (20.0 mL). Then dried to yield 3 as a light yellow solid (3.2 g, 62.2%), HPLC purity 97.34%; m.p. 144–148 ˝ C; rαs20 D ´49.700 (c = 1 g/100 mL, DMSO); IR (KBr) νmax : 3527 (amide N-Hν ); 3255 (acid O-Hν ); 3068 (aromatic C-Hν ); 2944, 2856 (C-Hν ); 2224 (C”Cν ); 1700, 1654 (C=Oν ); 1573, 1519
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(aromatic C=Cν , aromatic C=Nν ); 1440, 1400 (C-Hδ ); 763 (aromatic C-Hγ ) cm´1 ; 1 H-NMR (400 MHz, DMSO-d6 ) δ 12.86(s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 7.96–7.89 (m, 1H), 7.85–7.78 (m, 2H), 7.68 (t, J = 7.6 Hz, 1H), 7.59 (td, J = 7.5, 1.1 Hz, 1H), 7.51 (td, J = 7.6, 1.1 Hz, 1H), 7.41 (d, J = 7.4 Hz, 1H), 5.34 (s, 2H), 4.92 (q, J = 2.1 Hz, 2H), 4.10–4.01 (m, 1H), 3.82 (m, 1H), 3.63 (m, 1H), 3.40 (s, not resolved, CH3 and water), 3.14–2.98 (m, 2H), 2.89 (s, 3H), 2.02–1.86 (m, 2H), 1.76 (not resolved, 4H), 1.59 (m, 1H); 13 C-NMR (100 MHz, DMSO-d6 ) δ 169.44, 169.28, 168.79, 161.43, 156.46, 153.79, 151.40, 149.50, 148.06, 137.82, 134.51, 133.49, 130.68, 129.64, 129.51, 128.35, 128.32, 127.57, 126.12, 122.93, 103.88, 81.70, 74.26, 53.98, 50.43, 46.06, 46.01, 35.92, 29.91, 29.56, 23.56, 22.01, 3.56; HRMS (ESI) m/z 621.2599 (calcd for C33 H33 N8 O5 , 621.2574 [M + H]+ ). N,N-bis((R)-1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro1H-purin-8-yl)piperidin-3-yl)phthalamide (4). Phthaloyl dichloride (1.0 g, 0.005 mol) was added slowly to a stirred solution of 1 (4.8 g, 0.010 mol) and triethylamine (5.0 g, 0.05 mol) in CH2 Cl2 (50.0 mL) at rt and refluxed for 24 h. Then the mixture was cooled to rt and 1 mol/L aq. NaOH (30 mL) was added, and maintained for 2 h. The isolated solid was collected by filtration and washed with water (20.0 mL), CH2 Cl2 (20.0 mL) and methanol (20.0 mL). Then dried to yield 4 as a light yellow solid (3.5 g, 66.1%), HPLC purity 99.15%; rαs20 D = ´18.133 (c = 1 g/100 mL, CHCl3 ); IR (KBr) νmax 3243 (amide N-Hν ); 3068 (aromatic C-Hν ); 2945, 2858 (C-Hν ); 2224 (C”Cν ); 1700, 1662 (C=Oν ); 1634, 1563, 1519 (aromatic C=Cν , aromatic C=Nν ); 1440, 1400 (C-Hδ ); 763 (aromatic C-Hγ ) cm´1 ; 1 H-NMR (400 MHz, DMSO-d6 ) δ 8.37 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 8.1 Hz, 1H), 7.92–7.85 (m, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.67–7.62 (m, 1H), 7.50 (m, 2H), 5.30 (s, 2H), 4.90 (s, 2H), 4.10–4.00 (m, 1H), 3.81 (d, J = 9.0 Hz, 1H), 3.65 (m, 1H), 3.36 (s, 3H), 3.15–2.99 (m, 2H), 2.88 (s, 3H), 1.98–1.84 (m, 2H), 1.74 (not resolved, 4H), 1.65–1.55 (m, 1H); 13 C-NMR (100 MHz, CDCl3 ) δ 168.68, 168.52, 161.01, 155.73, 154.36, 151.71, 149.89, 147.29, 135.04, 133.23, 130.18, 128.80, 128.38, 126.71, 124.83, 123.10, 104.60, 81.53, 73.01, 53.57, 51.11, 46.32, 45.83, 35.63, 29.54, 28.76, 21.74, 21.67, 3.60; HRMS (ESI) m/z 1075.4800 (calcd for C58 H59 N16 O6 , 1075.4803 [M + H]+ ). 7-(But-2-yn-1-yl)-8-((2-hydroxyethyl)amino)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6 (3H,7H)-dione (5). Ethanolamine (8.0 g, 0.13 mol) was added to a stirred solution of 11 (2.0 g, 0.0044 mol) in toluene (32.0 mL) at reflux temperature and maintained for 2 h. Then the mixture was cooled to rt and 20 mL H2 O was added, and stirred for 30 min. The isolated solid was collected by filtration and washed with toluene (10.0 mL). Then dried to yield 5 as a white solid (1.8 g, 94.2%), HPLC purity 99.61%; m.p. 238–239 ˝ C; IR (KBr) νmax 3446, 3364 (N-Hν , O-Hν ); 2936, 2867 (C-Hν ); 1702, 1652 (C=Oν ); 1619, 1581, 1540 (aromatic C=Cν , aromatic C=Nν ); 1448, 1397 (C-Hδ ); 1225 (C-Oν ); 764 (aromatic C-Hγ ) cm´1 ; 1 H-NMR (400 MHz, DMSO-d6 ) δ 8.25 (d, J = 8.0 Hz, 1H), 7.95–7.88 (m, 1H), 7.82 (d, J = 8.2 Hz, 1H), 7.72–7.62 (m, 1H), 7.23 (t, J = 5.6 Hz, 1H), 5.31 (s, 2H), 4.89 (q, J = 2.2 Hz, 2H), 4.78 (t, J = 5.5 Hz, 1H), 3.61 (t, J = 6.0 Hz, 2H), 3.46 (t, J = 6.0 Hz, 2H), 3.39 (s, 3H), 2.89 (s, 3H), 1.77 (t, J = 2.1 Hz, 3H); 13 C-NMR (100 MHz, DMSO-d6 ) δ 169.19, 161.68, 154.38, 152.99, 151.50, 149.54, 149.52, 134.45, 128.31, 127.51, 126.11, 122.92, 101.31, 81.06, 74.27, 60.20, 45.89, 45.56, 33.13, 29.80, 21.99, 3.57; HRMS (ESI) m/z 434.1937 (calcd for C22 H24 N7 O3 , 434.1941 [M+H]+ ). (R)-8-(3-Aminopiperidin-1-yl)-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione(6). Diisopr opylethyl amine (9.8 g, 0.076 mol) was added to a stirred solution of 10 (5.0 g, 0.017 mol) and 12 (9.6 g, 0.025 mol) in NMP (50 mL) and maintained the reaction mass at 100 ˝ C for 13 h. Then ethanolamine (10.2 g, 0.17 mol) was added at 65 ˝ C and stirred for 4 h at 65 ˝ C. The isolated solid was collected by filtration and washed with NMP (30 mL). Then dried to yield 6 as a off-white solid (4.4g, 82.7%), HPLC purity 99.36%; m.p. 299–302 ˝ C; rαs20 D ´3.467 (c = 1 g/100 mL, DMSO); IR (KBr) νmax 3115, 3074 (primary amine N-Hν ); 3020 (imide N-Hν ); 2947, 2793 (C-Hν ); 2242 (C”Cν ); 1706 (C=Oν ); 1660 (C=Oν , primary amine N-Hδ ); 1610, 1519 (C=Cν , C=Nν ); 1445, 1384 (C-Hδ ) cm´1 ; 1 H-NMR (400 MHz, DMSO-d6 ) δ 10.94 (s, 1H), 8.27 (s, 2H), 4.92 (q, J = 2.4 Hz, 2H), 3.65 (m, 1H), 3.48–3.40 (m, 1H), 3.33 (incompletely resolved, 4H), 3.15 (m, 2H), 2.00 (m, 1H), 1.96–1.85 (m, 1H), 1.81 (t, J = 2.2 Hz, 3H), 1.67 (m, 2H);
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13 C-NMR
(100 MHz, DMSO-d6 ) δ 154.97, 153.99, 150.76, 148.37, 103.93, 81.20, 73.75, 51.43, 50.33, 46.09, 35.16, 28.49, 27.27, 21.85, 3.14; HRMS (ESI) m/z 317.1714 (calcd for C15 H20 N6 O2 , 317.1721 [M + H]+ ).
4. Conclusions Five new process-related impurities detected by HPLC varying from 0.15% to 0.5% were identified, synthesized, and subsequently characterized by HPLC, MS, HRMS, 1 H-NMR, 13 C-NMR (DEPT) and IR techniques. We have developed appropriate in-process checks and strategies in order to control these impurities within the acceptable level. For example, impurities 3, 4 and 5 could be reduced to below the identification threshold of 0.1% by salifying linagliptin with hydrochloric acid while impurities 2 and 6 could be controlled within 0.1% by recrystallization from toluene. The detection limit of impurities 2, 3, 4 and 5 was 0.10 µg/mL or 0.02% while the detection limit of impurity 6 was 0.15 µg/mL or 0.03%. Our efforts to synthesize and characterize them effectively prove to be valuable when it comes to complying with the regulatory norms as well as assessing the quality of linagliptin. Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 8/1041/s1. Acknowledgments: The authors are thankful to the teachers of the China State Institute of Pharmaceutical Industry for supporting this study and the cooperation from other colleagues is also highly appreciated. Author Contributions: Yiwen Huang designed and carried out the synthetic experiments, analyzed the data and wrote the paper. Xiaoqing He performed HPLC analysis and other analysis work. Taizhi Wu and Fuli Zhang reviewed and edited the manuscript. All authors read and approved the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–6 are available from the authors. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
