Synthesis and characterization of process-related impurities of antidiabetic drug linagliptin

Article abstract of DOI:10.3390/molecules21081041

Linagliptin, a xanthine derivative, is a highly potent, selective, long-acting and orally bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes. During the process development of linagliptin, five new process-related impurities were detected by high performance liquid chromatography (HPLC). All these impurities were identified, synthesized, and subsequently characterized by their respective spectral data (MS, HRMS, ¹H-NMR, ¹³C-NMR and IR) as described in this article. The identification of these impurities should be useful for quality control and the validation of the analytical method in the manufacture of linagliptin.

Full text of DOI:10.3390/molecules21081041

molecules
Article
Synthesis and Characterization of Process-Related
Impurities of Antidiabetic Drug Linagliptin
Yiwen Huang, Xiaoqing He, Taizhi Wu * and Fuli Zhang *
Shanghai Institute of Pharmaceutical Industry, China State Institute of Pharmaceutical Industry,
2
*
85 Gebaini Road, Pudong, Shanghai 201203, China; cpuhyw2010@163.com (Y.H.); xuesitu@163.com (X.H.)
Correspondence: wu.taizhi@sipi.com.cn (T.W.); zhangfuli1@sinopharm.com (F.Z.);
Tel.: +86-21-2057-2000 (ext. 5036) (T.W.); +86-21-2057-2000 (ext. 5069) (F.Z.)
Academic Editor: Diego Muñoz-Torrero
Received: 22 June 2016; Accepted: 5 August 2016; Published: 9 August 2016
Abstract: Linagliptin, a xanthine derivative, is a highly potent, selective, long-acting and orally
bioavailable DPP-4 inhibitor for the treatment of type 2 diabetes. During the process development
of linagliptin, five new process-related impurities were detected by high performance liquid
chromatography (HPLC). All these impurities were identified, synthesized, and subsequently
1
13
characterized by their respective spectral 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
validation of the analytical method in the manufacture of linagliptin.
Keywords: linagliptin; type 2 diabetes; impurities; synthesis; characterization
1
. Introduction
The presence of impurities in a drug substance can have a significant impact on the quality and
safety of the drug product [1]. According to the general guidelines on impurities in drug substances
recommended by the International Conference on Harmonization (ICH), any impurities present in the
drug substance greater than a level of 0.10% for drugs with a maximum daily dose equal to or lesser
than 2 g should be identified and characterized [2]. On one hand, the identification and characterization
of process-related impurities can guide us in controlling these impurities within the acceptable level by
improving reaction conditions in turn. On the other hand, impurities in pure form are also needed to
validate the analytical method including checking the system suitability and relative correction factor.
Linagliptin (
inhibitor for the treatment of type 2 diabetes, is chemically known as (R)-8-(3-aminopiperidin-1-yl)-7-
but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione [3].
1, Figure 1), a highly potent, selective, long-acting and orally bioavailable DPP-4
(
CH
3
O
CH
3
CH
3
O
O
N
N
N
N
N
N
OH
O
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
N
O
N
NH HN
NH
2
CH
3
CH
3
NH
HO
CH
3
CH
3
CH
3
CH
3
O
O
O
1
2
3
CH
3
O
N
N
N
O
O
CH
3
CH
3
N
O
O
N
N
O
N
N
N
N
H
N
N
N
N
N
HN
NH
N
H
N
N
O
N
N
O
N
O
N
N
OH
NH
2
CH
3
CH
3
CH
3
N
N
N
O
CH
3
4
5
6
Figure 1. Chemical structures of linagliptin and process-related impurities.
Molecules 2016, 21, 1041; doi:10.3390/molecules21081041
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1041
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In the industrial manufacturing process (Scheme 1) of linagliptin by the Boehringer Ingelheim
company [ ], cyclization of 1-(2-aminophenyl)ethanone ( ) with 2-chloroacetonitrile ( ) in the presence
of hydrogen chloride afforded 2-(chloromethyl)-4-methylqu inazoline ( , yield 74%–85%) which
4
7
8
9
condensed with 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1H-purine-2,6(3H,7H)-dione (10) in the presence of
sodium carbonate as a basic reagent, giving 8-bromo-7-(but-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin
-
2-yl)methyl)-1H-puri ne-2,6(3H,7H)-dione (11, yield 76%–83%). Subsequently, the 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-methyl-
-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6(3H,7H)-dione (13, yield 90%–94%). Compound 13
´
1
finally converted to the desired linagliptin (
aminolysis agent.
1) in 81.9% yield via aminolysis using ethanolamine as the
CH3
O
N
a
Cl
b
N
N
N
N
NH2
O
N
Br
N
O
N
Cl
CH3
C
CH3
CH3
CH3
CH3
O
N
7
8
9
N
11
HN
Br
HO COOH
HO COOH
O
N
N
CH3
1
0
HN
c
O
N
O
12
CH3
N
O
CH3
O
N
d
N
N
N
N
N
N
N
N
N
N
N
N
O
N
O
O
OH
H2N
CH3
CH3
NH2
CH3
CH3
O
1
13
˝
Scheme 1. Synthesis of linagliptin (
1
). Conditions: (
a
) HCl, 1,4-dioxane, 6 C , 2 h, 74%–85%; (
b) Na CO ,
2 3
˝
˝
N-methyl-2-pyrrolidone (NMP), 140 C, 2 h, 76%–83%; (
c
) diisopropanolamine, NMP, 140 C, 2 h,
˝
0%–94%; (d) ethanolamine, THF/H O, 60 C, 3 h, 81.9%.
2
9
However, the literature search did not reveal much work on the impurity research of linagliptin.
The impurity profile of linagliptin in this synthetic route is different from the earlier reported study [ ],
5,6
which makes it more challenging to identify the unknown impurities formed in small quantities in
the drug substance. Since most of the time it is very difficult to identify and control impurities within
acceptable levels in the process, some advanced purification techniques for an active drug substance
may then be taken into account, such as the continuous-flow process [7], organic solvent nanofiltration
(
OSN) [ ], molecularly imprinted membranes for OSN [ ] and counter current chromatography (CCC),
8
9
as a valuable addition to the chromatography toolbox [10].
Hence, a comprehensive study was undertaken to identify and synthesize the impurities in a
sample of linagliptin as described in this article. The study will help to understand the formation of
the impurities in linagliptin synthesis and provide a clue on how to obtain a pure drug substance.
2
. Results and Discussion
2
.1. Structures of Impurities
During the process development of linagliptin, five new process-related impurities were detected
by high performance liquid 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
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

Molecules 2016, 21, 1041
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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
Figure 2. Typical HPLC chromatogram of linagliptin with impurities.
2
.2. Source of Impurities
The synthesis of linagliptin involved the aminolysis of 13 using 10 equivalents of ethanolamine [
4
].
was
due to water/tetrahydrofuran (THF) as a solvent. The formed
) reacted with starting material 13 or with , providing impurity . During the
Compound
further hydrolyzed to afford impurity
linagliptin moiety (
2 is the by-product in this step, formed from incomplete aminolysis. Impurity 2
3
1
2
4
condensation of 11 with 12 to afford 13, the intermediate 11 cannot be converted completely and may
remain in the intermediate 13. Unfortunately, the small amount of 11 remaining in the intermediate 13
reacted with ethanolamine in the next aminolysis process, which explains the formation of impurity
5.
Similarly, during the condensation of 10 with to afford 11, the intermediate 10 cannot be converted
9
completely and may remain in the intermediate 11. Subsequently, 10 remaining in the intermediate 11
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
for 0.4%, 0.32%, 0.27%, 0.33% and 0.16%, respectively, in the drug substance.
Scheme 2. Source of impurities of linagliptin.

Molecules 2016, 21, 1041
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2
.3. Preparation and Characterization of Impurities
Compound was synthesized by incomplete aminolysis of 13. In order to prepare
2
2
in good yield,
we replaced the solvent of THF/H O with dichloromethane (CH Cl ) to obtain a homogeneous system
2
2
2
and a smaller inventory ratio of ethanolamine was used for more moderate aminolysis conditions
Scheme 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
1
mode, indicating the mass of this compound to be 663. The H-NMR of this compound revealed three
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
´
1
protons. In the IR spectrum, observed bands at 3473 and 3263 cm indicated the amide N-H and
O-H stretching. The 13C-NMR revealed 35 carbon atoms and further Distortionless Enhancement by
Polarization Transfer (DEPT) exhibited eight secondary carbon atoms. Based on all the spectral data
1
13
1
(
MS, HRMS, H-NMR, C-NMR and IR), the structure of impurity 2 was confirmed as (R)-N -(1-(7-(but-
2
-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)
2
piperidin-3-yl)-N -(2-hydroxyethyl)phthalamide.
Compound
3 was prepared by the alkali hydrolysis of 13 with phase transfer catalyst (Scheme 3).
The undesired product 1 from excessive hydrolysis can be removed by CH Cl .
2
2
+
The mass spectrum of
3
exhibited a molecular ion peak at m/z 643.25 (M + Na) in positive
´
ion mode and m/z 619.31 (M
´
H) in negative ion mode, indicating the mass of this compound
1
to be 620. The H-NMR of this impurity revealed two D O exchangeable signals at 12.86 and 8.35,
2
indicating one acid O-H proton and one amide N-H proton, and an additional eight proton signals
at 7.41–8.25 corresponding to aromatic protons. Interestingly, we found the methyl group (CH ) in
3
quinazoline at
δ 2.89 ppm is acidic at a certain degree, which explains the proton exchange of the
CH group with D O which takes place sometimes. The proton exchange’s resulting compound
3
2
(
3-d, Figure 3) also explains the proton splitting of the CH group, which showed a triplet peak
3
1
with one proton of 2.89 ppm in the H-NMR with D O added (Figure 3). The IR spectrum of
exhibited a band at 3527 cm corresponding to amide N-H stretching, and 3255 cm for acid O-H
stretching. The 13C-NMR revealed 33 carbon atoms and further DEPT showed six secondary carbon
3
2
´
1
´1
1
13
atoms. Impurity 3 was characterized by all respective spectral data (MS, HRMS, H-NMR, C-NMR

Molecules 2016, 21, 1041
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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-2,3,6,7-
tetrahydro-1H-purin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid.
1
Figure 3. Structures and partial H-NMR spectrum of 3-d.
We can only obtain compound
linagliptin moiety ( ). Instead, we have exploited a different concept for the synthesis of compound
starting from phthaloyl dichloride (14). However, 12% of the single-benzoyl chloride compound
and 11% of the cyclization product 13 would be generated meanwhile due to the steric hindrance
from linagliptin, which influenced the purity of . We reduced them to below 0.3% and finally got
compound with a high purity of 99.15% by washing the reaction mass with CH Cl and aqueous
4 with the low yield of 1.5% directly via 13 reacting with the
1
4
1
5
4
4
2
2
sodium hydroxide solution instead of the column chromatography (Scheme 4).
Scheme 4. Preparation of impurity 4.
The mass spectrum of
in positive ion mode, indicating the mass of this compound to be 1074. The molecular formula
of this compound was C H N O as confirmed by the high resolution mass spectrum (HRMS).
4 ,
exhibited a molecular ion peak at m/z 1075.24 (M + H)+ 1097.22 (M + Na)+
5
8
58 16
6
´
1
13
In the IR spectrum the observed band at 3243 cm
indicated the amide N-H. The C-NMR
showing 29 carbon atoms indicated the symmetry in the structure of compound
the spectral data (MS, HRMS, H-NMR, C-NMR and IR), the structure of impurity
4
. Based on all
1
13
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-purin-8-yl)piperidin-3-yl)phthalamide.

Molecules 2016, 21, 1041
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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).
Scheme 5. Preparation of impurities 5 and 6.
+
The mass spectrum of
5
showed a molecular ion peak at m/z 456.51 (M + Na) in positive ion
1
mode, indicating the mass of this compound to be 433. The H-NMR of this compound revealed two
D O exchangeable signals at 7.23 and 4.78, indicating the N-H and O-H protons, and an additional four
2
proton signals at 7.62–8.25 corresponding to aromatic protons. In the IR spectrum the observed bands
´
1
13
at 3446 and 3364 cm indicated the N-H and O-H stretching. The C-NMR revealed 22 carbon atoms
1
and DEPT showed four secondary carbon atoms. Based on all the spectral data (MS, HRMS, H-NMR,
13
C-NMR and IR), the structure of impurity 5 was confirmed as 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.
Compound was independently synthesized, starting from compound 10, following a synthetic
6
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).
+
The mass spectrum of
6
showed a molecular ion peak at m/z 317.14 (M + H) in positive
ion mode, indicating the mass of this compound to be 316, which is 156 amu less than that of
1
linagliptin (
1
). The H-NMR of this compound revealed two D O exchangeable signals at 10.94 and
2
8
.27, indicating the imide N-H proton and N-H proton, and no additional aromatic protons were
2
´
1
observed. In the IR spectrum the observed band at 3021 cm indicated the imide N-H stretching
´
1
and double peaks of 3115 and 3074 cm , corresponding to primary amine N-H stretching. The
13
C-NMR revealed 15 carbon atoms and DEPT showed five secondary carbon atoms. Impurity
1
13
6
was characterized by all respective spectral data (MS, HRMS, H-NMR, 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
General Information
The compound 11 (chemical purity 98.36%), 13 (chemical purity 97.92%; chiral purity 99.97%)
(chemical purity 98.35%; chiral purity 99.97%) were prepared according to the literature
procedure [ ]. Other materials, solvents and reagents were of commercial origin and used without
additional operations.
and
1
4
1
13
The H- and C-NMR spectra were recorded on a Bruker Arance III 400 MHz spectrometer. The
1
solvents used were DMSO-d or CDCl . The H-NMR chemical shift values were reported as
δ
ppm
6
3

Molecules 2016, 21, 1041
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relative to tetramethylsilane (TMS) and the 13C-NMR chemical shift values were reported on
δ
ppm
relative to DMSO-d or CDCl . DEPT spectra revealed the presence of methyl and methine groups
6
3
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
µ
m) 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
3
0.1 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 L. Limit of Detection (LOD) was 0.10 g/mL
or 0.02% for impurities and 0.15 g/mL or 0.03% for impurity . Limit of Quantity (LOQ) was
.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
µ
µ
2,
3
,
4,
5
µ
6
0
˝
capillary voltage was maintained at 3.0 kV. The drying gas temperature was at 350 C.
1
13
The detailed spectral data (IR, H-NMR, 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.
1
(
R)-N -(1-(7-(But-2-yn-1-yl)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-2,6-ioxo-2,3,6,7-tetrahydro-1H-
2
purin-8-yl)piperidin-3-yl)-N -(2-hydroxyethyl)phthalamide (
2
). To a stirred solution of compound 13 (5.0 g,
0
.008 mol) in CH Cl (50.0 mL) was added ethanolamine (1.5 g, 0.024 mol) and maintained the reaction
2 2
mass at room temperature for 24 h. Then added 15 mL H O stirring for 30 min to dilute ethanolamine.
2
The isolated solid was collected by filtration and washed with 10 mL H O and 20 mL CH Cl . Then
2
2
2
˝
20
D
dried to yield
32.567 (c = 1 g/100 mL, DMSO); IR (KBr) νmax 3473, 3263 (amide N-H
C-H ); 2937, 2850 (C-H ); 2230 (C ); 1698, 1662 (C=O ); 1522 (aromatic C=C
440, 1400 (C-H ); 1228 (C-O ); 763 (aromatic C-H
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),
.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,
H), 1.75 (not resolved, 4H); 13C-NMR (100 MHz, DMSO-d6)
169.29, 168.75, 168.46, 161.44, 156.37,
2
as a light yellow solid (1.5 g, 27.3%), HPLC purity 97.52%; m.p. 189–192 C; rαs
´
ν
, O-H
ν
); 3062 (aromatic
, aromatic C=N );
) cm 1; H-NMR (400 MHz, CDCl )
8.35 (d,
ν
ν
”C
ν
ν
´
ν
ν
1
1
ν
γ
δ
3
δ
7
(
2
δ
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,
1
22.94, 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 C H N O , 664.2996 [M + H] ).
35
38
9
5
(
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-1H-
purin-8-yl)piperidin-3-yl)carbamoyl)benzoic acid ( ). To a stirred solution of compound 13 (5.0 g,
.008 mol) in CH Cl (50.0 mL) was added aqueous sodium hydroxide solution (1.0 g of NaOH in
3
0
5
2
2
0.0 mL of H O) and tetra-n-butylammonium bromide (0.27 g, 0.0008 mol). Then maintained the
2
reaction mass at rt for 72 h. Then water phase was collected and washed with CH Cl (25.0 mL) for
2
2
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
CH Cl (20.0 mL). Then dried to yield
3
as a light yellow solid (3.2 g, 62.2%), HPLC purity 97.34%;
49.700 (c = 1 g/100 mL, DMSO); IR (KBr) νmax: 3527 (amide N-H ); 3255
); 2944, 2856 (C-H ); 2224 (C ); 1700, 1654 (C=O ); 1573, 1519
2
2
˝
20
D
m.p. 144–148 C; rαs
acid O-H
´
ν
(
ν
); 3068 (aromatic C-H
ν
ν
”C
ν
ν

Molecules 2016, 21, 1041
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(
aromatic C=C
DMSO-d6) 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, CH and water), 3.14–2.98 (m, 2H), 2.89 (s, 3H), 2.02–1.86 (m, 2H), 1.76 (not resolved, 4H),
ν
, aromatic C=N
ν
); 1440, 1400 (C-H ); 763 (aromatic C-H
γ
) cm 1; 1H-NMR (400 MHz,
´
δ
δ
(
3
1
1
.59 (m, 1H); 13C-NMR (100 MHz, DMSO-d6)
49.50, 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,
δ 169.44, 169.28, 168.79, 161.43, 156.46, 153.79, 151.40,
8
1.70, 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 C H N O , 621.2574 [M + H] ).
33
33
8
5
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-
H-purin-8-yl)piperidin-3-yl)phthalamide ( ). Phthaloyl dichloride (1.0 g, 0.005 mol) was added slowly to
a stirred solution of (4.8 g, 0.010 mol) and triethylamine (5.0 g, 0.05 mol) in CH Cl (50.0 mL) at rt
1
4
1
2
2
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),
CH Cl (20.0 mL) and methanol (20.0 mL). Then dried to yield
4
as a light yellow solid (3.5 g, 66.1%),
18.133 (c = 1 g/100 mL, CHCl ); IR (KBr) νmax 3243 (amide N-H );
); 1634, 1563, 1519 (aromatic
) cm ; H-NMR (400 MHz, DMSO-d )
2
2
20
HPLC purity 99.15%; rαs
=
´
ν
D
3
3
068 (aromatic C-H
C=C , aromatic C=N
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),
.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,
ν
); 2945, 2858 (C-H
ν
); 2224 (C
”C
ν
); 1700, 1662 (C=O
ν
´
1 1
ν
ν
); 1440, 1400 (C-H ); 763 (aromatic C-H
δ
γ
6
δ
(
3
13
1
1
H); C-NMR (100 MHz, CDCl3) δ 168.68, 168.52, 161.01, 155.73, 154.36, 151.71, 149.89, 147.29, 135.04,
33.23, 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 C H N O , 1075.4803 [M + H] ).
58
59 16
6
7
-(But-2-yn-1-yl)-8-((2-hydroxyethyl)amino)-3-methyl-1-((4-methylquinazolin-2-yl)methyl)-1H-purine-2,6
). 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 H O was added, and stirred for 30 min. The isolated solid was collected by filtration
(
3H,7H)-dione (5
2
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-H , O-H ); 2936, 2867 (C-H ); 1702,
652 (C=O ); 1619, 1581, 1540 (aromatic C=C , aromatic C=N ); 1448, 1397 (C-H ); 1225 (C-O ); 764
aromatic C-H ) cm ; H-NMR (400 MHz, DMSO-d ) 8.25 (d, J = 8.0 Hz, 1H), 7.95–7.88 (m, 1H),
.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),
.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,
˝
ν
ν
ν
1
(
ν
ν
ν
ν
δ
´
1 1
γ
δ
6
7
4
13
J = 2.1 Hz, 3H); C-NMR (100 MHz, DMSO-d ) δ 169.19, 161.68, 154.38, 152.99, 151.50, 149.54, 149.52,
6
1
34.45, 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 C H N O , 434.1941 [M+H] ).
22
24
7
3
(
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
3.467 (c = 1 g/100 mL, DMSO); IR (KBr) νmax 3115, 3074 (primary
); 2947, 2793 (C-H ); 2242 (C ); 1706 (C=O ); 1660 (C=O , primary
, C=N ); 1445, 1384 (C-H ) cm ; H-NMR (400 MHz, DMSO-d )
0.94 (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);
˝
20
D
9
9.36%; m.p. 299–302 C; rαs
´
amine N-H
ν
); 3020 (imide N-H
ν
ν
”C
ν
´
ν
ν
1 1
amine N-H ); 1610, 1519 (C=C
ν
ν
δ
6
δ
δ
1

Molecules 2016, 21, 1041
9 of 10
13C-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,
+
3
5.16, 28.49, 27.27, 21.85, 3.14; HRMS (ESI) m/z 317.1714 (calcd for C H N O , 317.1721 [M + H] ).
15
20
6
2
4
. Conclusions
Five new process-related impurities detected by HPLC varying from 0.15% to 0.5% were identified,
1
13
synthesized, and subsequently characterized by HPLC, MS, HRMS, H-NMR, 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
identification threshold of 0.1% by salifying linagliptin with hydrochloric acid while impurities
3
,
4
and
5
could be reduced to below the
and
could be controlled within 0.1% by recrystallization from toluene. The detection limit of impurities
and was 0.10 g/mL or 0.02% while the detection limit of impurity was 0.15 g/mL or
.03%. Our efforts to synthesize and characterize them effectively prove to be valuable when it comes
2
6
2
0
,
3
,
4
5
µ
6
µ
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/).