Supporting Information
Process development and optimization of linagliptin aided by Design of
Experiments (DoE)
Hailong Wanga,b,d, Kai Chena, Biyue Linb,d , Jingping Koud, Lijun Lid, Shuming Wub,d, Shouzhu Liaob,d,
Guodong Sunb,d, Junwen Pud, Hua Yanga, and Zhongqing Wangb,c,d
a College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, P. R.
China
b State Key Laboratory of Anti-Infective Drug Development, Sunshine Lake Pharma Co., Ltd., Dongguan
523871, P. R. China
c School of Pharmacy, Xiangnan University, Chenzhou 423000 Hunan, China, Xiangnan University,
Chenzhou 423000, China
d HEC Research and Development Center, HEC Pharm Group, Dongguan 523871, P. R. China
Table of Content
Table S1. The 1/4-fractional factorial DoE studies parameters and results of the residue of 2.......3
Table S2. Analysis of Variance (ANOVA) for the residual starting material 2..............................3
Table S3. Three process parameters were considered for the CCF.................................................4
Table S4. The RSM DoE studies parameters and results of the residue of 2 ..................................4
Table S5. ANOVA Analysis for the RSM DoE studies of the residue of 2 ....................................5
Table S6. Four verification experiments for synthesizing N7-substituted xanthine 3.....................5
Table S7. The half-fractional factorial DoE studies parameters and results of the residue of 3......6
Table S8. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of 3........7
Table S9. The RSM DoE studies parameters and results of the residue of 3 ..................................7
Table S10. The ANOVA for the RSM DoE studies of the residue of 3..........................................8
Table S11. Four verification experiments of 4................................................................................8
Table S12. The screen of the stoichiometry K2CO3 used in the preparation of 5............................8
Table S13. The half-fractional factorial DoE studies parameters and results of the residue of 4
and allene impurity..........................................................................................................................9
Table S14. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of 4....10
Table S15. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of allene
impurity.........................................................................................................................................10
Table S16. The RSM DoE studies parameters and results of the residue of 3, allene impurity and
the assay of 5.................................................................................................................................11
Table S17. The ANOVA Analysis for the RSM DoE studies of the residue of 4.........................12
Table S18. The ANOVA Analysis for the RSM DoE studies of the assay of 5............................12
Table S19. Four verification experiments for preparing N-Boc linagliptin 5................................13
 Corresponding author. e-mail: Hyangchem@csu.edu.cn
 Corresponding author. e-mail: Wangzhongqing@hec.cn
Table S20. Alternative deprotection methods to prepare linagliptin 1..........................................13
Table S21. The solubility screening of Linagliptin in different solvents ......................................13
1H NMR and 13C NMR spectra for compound 3...........................................................................14
1H NMR and 13C NMR spectra for compound 4...........................................................................15
1H NMR and 13C NMR spectra for compound 5...........................................................................16
1H NMR and 13C NMR spectra for compound 1...........................................................................17
HPLC chromatography for Compound 3......................................................................................18
HPLC chromatography for Compound 4......................................................................................18
HPLC chromatography for Compound 5......................................................................................19
HPLC chromatography for Compound 1......................................................................................20
Table S1. The 1/4-fractional factorial DoE studies parameters and results of the residue of 2
StdOrder RunOrder CenterPt Blocks
Volumes of
acetonitrile
BNB Temp. Time DIPEA
Residual
SM 2
(%)
1 1 1 1 10 1 50 20 1.5 12.9
6 2 1 1 16 1 80 8 1.5 10.7
4 3 1 1 16 1.3 50 20 1.1 1.7
9 4 0 1 13 1.15 65 14 1.3 1.7
7 5 1 1 10 1.3 80 8 1.1 1.2
3 6 1 1 10 1.3 50 8 1.5 2.1
10 7 0 1 13 1.15 65 14 1.3 2.0
11 8 0 1 13 1.15 65 14 1.3 2.2
8 9 1 1 16 1.3 80 20 1.5 1.0
5 10 1 1 10 1 80 20 1.1 3.3
2 11 1 1 16 1 50 8 1.1 7.5
Table S2. Analysis of Variance (ANOVA) for the residual starting material 2.
Source DF Adj SS Adj MS F-Value P-Value
Model 3 142.276 47.425 9.99 0.006
Linear 2 122.051 61.026 12.86 0.005
BNB 1 100.848 100.848 21.25 0.002
DIPEA 1 21.203 21.203 4.47 0.072
2-Way Interactions 1 20.225 20.225 4.26 0.078
BNB*DIPEA 1 20.225 20.225 4.26 0.078
Error 7 33.219 4.746
Curvature 1 20.936 20.936 10.23 0.019
Lack-of-Fit 4 12.172 3.043 54.58 0.018
Pure Error 2 0.112 0.056
Total 10 175.496
Table S3. Three process parameters were considered for the CCF
Symbol Parameter Unite High Low
1 Temperature ºC 50 80
2 BNB eq. 1.05 1.2
3 DIPEA eq. 1.1 1.5
Table S4. The RSM DoE studies parameters and results of the residue of 2
StdOrder RunOrder CenterPt Blocks Temperature BNB DIPEA
The residue of
2
12 1 0 1 65 1.125 1.3 3.168
4 2 1 1 80 1.2 1.1 1.31
8 3 1 1 80 1.2 1.5 2.823
3 4 1 1 50 1.2 1.1 2.214
7 5 1 1 50 1.2 1.5 3.743
2 6 1 1 80 1.05 1.1 3.733
11 7 0 1 65 1.125 1.3 1.754
10 8 0 1 65 1.125 1.3 2.714
5 9 1 1 50 1.05 1.5 9.785
9 10 0 1 65 1.125 1.3 2.669
6 11 1 1 80 1.05 1.5 7.939
1 12 1 1 50 1.05 1.1 4.039
13 13 -1 2 50 1.125 1.3 2.361
15 14 -1 2 65 1.05 1.3 3.506
18 15 -1 2 65 1.125 1.5 3.383
17 16 -1 2 65 1.125 1.1 3.021
14 17 -1 2 80 1.125 1.3 1.578
16 18 -1 2 65 1.2 1.3 1.518
Table S5. ANOVA Analysis for the RSM DoE studies of the residue of 2
Source DF Adj SS Adj MS F-Value P-Value
Model 5 70.575 14.1150 18.01 0.000
Linear 3 50.358 16.7861 21.42 0.000
Temperature 1 2.265 2.2648 2.89 0.115
BNB 1 30.255 30.2551 38.61 0.000
DIPEA 1 17.838 17.8383 22.76 0.000
Square 1 14.248 14.2484 18.18 0.001
DIPEA*DIPEA 1 14.248 14.2484 18.18 0.001
2-Way Interaction 1 5.969 5.9685 7.62 0.017
BNB*DIPEA 1 5.969 5.9685 7.62 0.017
Error 12 9.404 0.7837
Lack-of-Fit 9 8.350 0.9278 2.64 0.229
Pure Error 3 1.054 0.3513
Total 17 79.979
Table S6. Four verification experiments for synthesizing N7-substituted xanthine 3
Entry BNB (eq.) DIPEA (eq.) Temperature (ºC) 2 (%)a 95% CI
1 1.20 1.27 80 0.748
2 1.20 1.27 80 0.810
3 1.20 1.27 80 0.915
4 1.20 1.27 80 0.909
(-0.937,1.261)
Average - - - 0.845 a
Determined by HPLC analysis
Table S7. The half-fractional factorial DoE studies parameters and results of the residue of 3
StdOrder RunOrder CenterPt Blocks CMQ K2CO3 Time Temperature Solvent
The
residue
of 3
4 1 1 1 1.2 2 4 50 14 0.419
15 2 1 1 1 2 14 90 8 0.631
13 3 1 1 1 0.5 14 90 14 3.195
9 4 1 1 1 0.5 4 90 8 5.329
19 5 0 1 1.1 1.25 9 70 11 0.68
11 6 1 1 1 2 4 90 14 0.506
17 7 0 1 1.1 1.25 9 70 11 0.532
6 8 1 1 1.2 0.5 14 50 14 2.999
20 9 0 1 1.1 1.25 9 70 11 0.408
5 10 1 1 1 0.5 14 50 8 3.875
3 11 1 1 1 2 4 50 8 2.058
7 12 1 1 1 2 14 50 14 1.032
1 13 1 1 1 0.5 4 50 14 6.615
8 14 1 1 1.2 2 14 50 8 0.138
16 15 1 1 1.2 2 14 90 14 0.138
18 16 0 1 1.1 1.25 9 70 11 0.443
12 17 1 1 1.2 2 4 90 8 0.138
10 18 1 1 1.2 0.5 4 90 14 4.428
2 19 1 1 1.2 0.5 4 50 8 5.276
14 20 1 1 1.2 0.5 14 90 8 3.397
Table S8. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of 3
Source DF Adj SS Adj MS F-Value P-Value
Model 4 67.4824 16.8706 15.84 0.000
Linear 3 64.4199 21.4733 20.17 0.000
CMQ 1 2.4869 2.4869 2.34 0.147
K2CO3 1 56.4527 56.4527 53.02 0.000
Time 1 5.4803 5.4803 5.15 0.038
2-Way Interactions 1 3.0625 3.0625 2.88 0.111
K2CO3*Time 1 3.0625 3.0625 2.88 0.111
Error 15 15.9719 1.0648
Curvature 1 12.7377 12.7377 55.14 0.000
Lack-of-Fit 11 3.1901 0.2900 19.71 0.016
Pure Error 3 0.0441 0.0147
Total 19 83.4543
Table S9. The RSM DoE studies parameters and results of the residue of 3
StdOrder RunOrder CenterPt Blocks CMQ K2CO3 Time The residue of 3
11 1 0 1 1.1 1.25 9 0.443
5 2 1 1 1 0.5 14 5.356
10 3 0 1 1.1 1.25 9 0.679
6 4 1 1 1.2 0.5 14 4.234
2 5 1 1 1.2 0.5 4 4.447
9 6 0 1 1.1 1.25 9 0.215
7 7 1 1 1 2 14 0.32
1 8 1 1 1 0.5 4 5.357
8 9 1 1 1.2 2 14 0.179
3 10 1 1 1 2 4 0.39
4 11 1 1 1.2 2 4 0.207
15 12 -1 2 1.1 2 9 0.242
16 13 -1 2 1.1 1.25 4 1.552
13 14 -1 2 1.2 1.25 9 0.71
17 15 -1 2 1.1 1.25 14 0.402
14 16 -1 2 1.1 0.5 9 5.066
12 17 -1 2 1 1.25 9 2.354
Table S10. The ANOVA for the RSM DoE studies of the residue of 3
Source DF Adj SS Adj MS F-Value P-Value
Model 6 67.5510 11.2585 53.75 0.000
Linear 3 55.2764 18.4255 87.96 0.000
CMQ 1 1.6000 1.6000 7.64 0.020
K2CO3 1 53.4627 53.4627 255.23 0.000
Time 1 0.2137 0.2137 1.02 0.336
Square 2 11.9099 5.9549 28.43 0.000
CMQ*CMQ 1 0.3994 0.3994 1.91 0.197
K2CO3*K2CO3 1 6.6802 6.6802 31.89 0.000
2-Way Interaction 1 0.3647 0.3647 1.74 0.216
CMQ*K2CO3 1 0.3647 0.3647 1.74 0.216
Error 10 2.0947 0.2095
Lack-of-Fit 8 1.9870 0.2484 4.61 0.190
Pure Error 2 0.1077 0.0538
Total 16 69.6456
Table S11. Four verification experiments of 4
Entry K2CO3 (eq.) CMQ (eq.) Time (h) 3(%) 95% CI
1 1.8 1.13 14 0.124
2 1.8 1.13 14 0.181
3 1.8 1.13 14 0.188
4 1.8 1.13 14 0.110
(-0.844,0.284)
average - - - 0.150 Table
S12. The screen of the stoichiometry K2CO3 used in the preparation of 5
Equev. of K2CO3 1.5 3 4
Allene impurity 0.03 0.13 0.21
Conditions:4 (2 g) , NMP (16 ml) ,75-80ºC,16 h. Determined by HPLC
Table S13. The half-fractional factorial DoE studies parameters and results of the residue of 4 and allene impurity
StdOrder RunOrder CenterPt Blocks Temperature Time RBP solvent Na2CO3
The
residue
of 4
allene
impurity
1 1 1 1 70 10 1.00 6 2.0 6.556 0.000
3 2 1 1 70 16 1.00 6 0.5 4.346 0.000
13 3 1 1 70 10 1.12 12 2.0 2.757 0.000
8 4 1 1 100 16 1.12 6 0.5 0.01 0.063
19 5 0 1 85 13 1.06 9 1.25 1.744 0.000
7 6 1 1 70 16 1.12 6 2.0 0.23 0.000
11 7 1 1 70 16 1.00 12 2.0 4.267 0.000
17 8 0 1 85 13 1.06 9 1.25 1.005 0.000
2 9 1 1 100 10 1.00 6 0.5 2.946 0.048
14 10 1 1 100 10 1.12 12 0.5 0.028 0.000
4 11 1 1 100 16 1.00 6 2.0 2.285 0.037
15 12 1 1 70 16 1.12 12 0.5 1.915 0.000
12 13 1 1 100 16 1.00 12 0.5 3.966 0.057
9 14 1 1 70 10 1.00 12 0.5 10.601 0.000
10 15 1 1 100 10 1.00 12 2.0 4.109 0.050
6 16 1 1 100 10 1.12 6 2.0 0.007 0.00
5 17 1 1 70 10 1.12 6 0.5 2.964 0.000
18 18 0 1 85 13 1.06 9 1.25 0.794 0.000
16 19 1 1 100 16 1.12 12 2.0 0.04 0.000
Table S14. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of 4
Source DF Adj SS Adj MS F-Value P-Value
Model 4 104.604 26.1509 14.91 0.000
Linear 3 96.579 32.1931 18.35 0.000
Temperature 1 25.616 25.6163 14.60 0.002
Time 1 10.415 10.4151 5.94 0.029
RBP 1 60.548 60.5479 34.51 0.000
2-Way Interactions 1 8.024 8.0245 4.57 0.051
Temperature*Time 1 8.024 8.0245 4.57 0.051
Error 14 24.562 1.7544
Curvature 1 7.809 7.8094 6.06 0.029
Lack-of-Fit 11 16.255 1.4777 5.94 0.153
Pure Error 2 0.498 0.2489
Total 18 129.165
Table S15. ANOVA Analysis for the half-fractional factorial DoE studies of the residue of allene impurity
Source DF Adj SS Adj MS F-Value P-Value
Model 3 0.006144 0.002048 8.03 0.002
Linear 2 0.005104 0.002552 10.01 0.002
Temperature 1 0.004064 0.004064 15.94 0.001
RBP 1 0.001040 0.001040 4.08 0.062
2-Way Interactions 1 0.001040 0.001040 4.08 0.062
Temperature*RBP 1 0.001040 0.001040 4.08 0.062
Error 15 0.003824 0.000255
Curvature 1 0.000642 0.000642 2.82 0.115
Lack-of-Fit 12 0.003183 0.000265 * *
Pure Error 2 0.000000 0.000000
Total 18 0.009969
Table S16. The RSM DoE studies parameters and results of the residue of 4, allene impurity and the assay of 5
StdOrder RunOrder CenterPt Blocks Temperature Time RBP
The
residue
of 4 (%)
allene
impurit
y
The
assay of
5 (%)
4 1 1 1 90 16 1.00 3.218 0 95.452
3 2 1 1 70 16 1.00 5.1 0 94.831
1 3 1 1 70 10 1.00 6.128 0 92.565
10 4 0 1 80 13 1.08 1.175 0 97.674
6 5 1 1 90 10 1.16 0.000 0 98.674
7 6 1 1 70 16 1.16 0.233 0 98.779
11 7 0 1 80 13 1.08 0.612 0 98.27
8 8 1 1 90 16 1.16 0.000 0 98.775
9 9 0 1 80 13 1.08 0.517 0 98.338
5 10 1 1 70 10 1.16 0.936 0 98.13
2 11 1 1 90 10 1.00 3.534 0 95.067
16 12 -1 2 80 13 1.00 2.918 0 95.952
15 13 -1 2 80 16 1.08 0.203 0 98.662
13 14 -1 2 90 13 1.08 0.413 0 98.237
17 15 -1 2 80 13 1.16 0.252 0 98.883
14 16 -1 2 80 10 1.08 0.742 0 98.244
12 17 -1 2 70 13 1.08 2.698 0 96.144
Table S17. The ANOVA Analysis for the RSM DoE studies of the residue of 4
Source DF Adj SS Adj MS F-Value P-Value
Model 6 55.6814 9.2802 48.41 0.000
Linear 3 44.8926 14.9642 78.07 0.000
Temperature 1 6.2885 6.2885 32.81 0.000
Time 1 0.6687 0.6687 3.49 0.091
RBP 1 37.9354 37.9354 197.91 0.000
Square 2 9.4218 4.7109 24.58 0.000
Temperature*Temperature 1 2.2109 2.2109 11.53 0.007
RBP*RBP 1 2.3662 2.3662 12.34 0.006
2-Way Interaction 1 1.3670 1.3670 7.13 0.023
Temperature*RBP 1 1.3670 1.3670 7.13 0.023
Error 10 1.9168 0.1917
Lack-of-Fit 8 1.6639 0.2080 1.64 0.432
Pure Error 2 0.2530 0.1265
Total 16 57.5982
Table S18. The ANOVA Analysis for the RSM DoE studies of the assay of 5
Source DF Adj SS Adj MS F-Value P-Value
Model 6 52.1410 8.6902 28.60 0.000
Linear 3 42.3068 14.1023 46.40 0.000
Temperature 1 3.3132 3.3132 10.90 0.008
Time 1 1.4585 1.4585 4.80 0.053
RBP 1 37.5352 37.5352 123.51 0.000
Square 2 9.0002 4.5001 14.81 0.001
Temperature*Temperature 1 2.7946 2.7946 9.20 0.013
RBP*RBP 1 1.6299 1.6299 5.36 0.043
2-Way Interaction 1 0.8340 0.8340 2.74 0.129
Temperature*RBP 1 0.8340 0.8340 2.74 0.129
Error 10 3.0390 0.3039
Lack-of-Fit 8 2.7721 0.3465 2.60 0.308
Pure Error 2 0.2669 0.1335
Total 16 55.1800
Table S19. Four verification experiments for preparing N-Boc linagliptin 5
HPLC assay (%)
Entry
Temperature
(ºC)
RBP
(equiv)
Time (h)
4 5
Allene
impurity
95% CI
1 82 1.09 16 0.150 98.777 ND
2 82 1.09 16 0.065 98.748 ND
3 82 1.09 16 0.257 98.599 ND
4 82 1.09 16 0.088 98.741 ND
(-0.440, 0.564)
(98.229, 99.574)
average - - - 0.140 98.716 Table
S20. Alternative deprotection methods to prepare linagliptin 1
Reaction temperature Reaction time The residue of 5 1
HCla -10ºC 5 h ND 62.5%
Zinc chlorideb 30ºC 64 h 29.7% 69.1%
MeOH/H2Oc Reflux 18 h 100%
a 2 (1.5 g), DCM (7.5 ml), Con HCl (2.2 ml); b 2 (1.0 g), DCM (10 ml), Zinc chloride (1.3 g, 5 eq); c 2 (5.0 g),
MeOH/H2O (V/V=3:2, 15 ml)
Table S21. The solubility screening of Linagliptin in different solvents
Solvent Solubility (25 ºC) Solubility (reflux)
Tertiary butanol >200 mL/g 30 mL/g
EA >200 mL/g 30 mL/g
THF 60 mL/g 15 mL/g
Toluene >200 mL/g 10 mL/g
Isopropyl acetate >200 mL/g 60 mL/g
2-Me-THF >200 mL/g 35 mL/g
MTBE >200 mL/g >200 mL/g
1H NMR and 13C NMR spectra
1H NMR and 13C NMR spectra for compound 3
1H NMR and 13C NMR spectra for compound 4
1H NMR and 13C NMR spectra for compound 5
1H NMR and 13C NMR spectra for compound 1
HPLC chromatography for Compound 3
HPLC chromatography for Compound 4
HPLC chromatography for Compound 5
HPLC chromatography for Compound 1