Supporting Information File 2 for Synthesis of ... - Beilstein Journals

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2. 4. 6. 8. 10. 12. 14. 3.7830 3.7646 3.6189 3.6010 3.4258 3.4116 3.3974 3.3029. 2.3407 2.3102 2.3061 2.2756 1.6748 1.6623 1.6546 1.6481 1.6413 1.6349 ...

Supporting Information File 2 for Synthesis of medronic acid monoesters and their purification by high-performance countercurrent chromatography or by hydroxyapatite Elina Puljula*, Jouko Vepsäläinen and Petri A. Turhanen

Address: School of Pharmacy, University of Eastern Finland, Biocenter Kuopio, P.O.Box 1627, FI-70211 Kuopio, Finland

Email: Elina Puljula - [email protected] * Corresponding author 1

H, 13C, and 31P NMR spectra and an example of

HPCCC chromatogram

S1

10 8 6 S2

4 2 8.8820

5.9463

5.9152

4

6

8

10

12

2.3407 2.3102 2.3061 2.2756 1.6748 1.6623 1.6546 1.6481 1.6413 1.6349 1.6213 1.4622 1.4497 1.4373 1.4250 1.0164 1.0042 0.9919

3.7830 3.7646 3.6189 3.6010 3.4258 3.4116 3.3974 3.3029

14

[rel] EP-071015-2 CDCl3

1

1.8734

2

10

5.8982 3.0000

5.6083 2.8229

0

EP-071015-2 "E:\NMR 2015"

600 MHz, CDCl3 General ------DATE = 2016/02/18 TIME = 17:34 INSTRUM = spect PULPROG = zg30

F1 (1H) ------------SI = 65536 SF = 600.18 SW_p = 12019.231

[ppm]

"E:\NMR 2015"

151 MHz, CDCl3

-0.0004

52.5116 52.4701 51.7962 51.7567 48.8811

61.0446

77.3133 77.1015 76.8895

General ------DATE = 2016/02/18 TIME = 18:05 INSTRUM = spect PULPROG = zgpg30

13.7425

EP-071015-2 CDCl3

1

25.4230 24.6480 24.5572 24.3605 23.7831 19.7287

12

10

[rel]

EP-071015-2

0

2

4

6

8

F1 (13C) ------------SI = 262144 SF = 150.915 SW_p = 36231.884

150

S3

100

50

[ppm]

EP-071015-2 CDCl3

"E:\NMR 2015"

202 MHz, CDCl3 General ------DATE = 2015/08/06 TIME = 10:12 INSTRUM = spect PULPROG = zgpg

8.6789 8.6442

1

18.7307

12

29.9247 29.8897

20

[rel]

150725-1

30

25

20

1.1429

0.0801

1.0000

0

5

10

15

F1 (31P) ------------SI = 32768 SF = 202.456 SW_p = 20325.203

S4

15

10

5

[ppm]

10 8 6 S5

4 2 2.8128

1.9020

6

8

-0.0001

2.4977 2.4555 2.4133 1.9065 1.7082 1.6948 1.6807 1.6649 1.6515 1.4560 1.4411 1.4260 1.4109 1.3961 1.3813 1.2554 0.9591 0.9443 0.9294

4.1426 4.1382 4.1292 4.1249 4.1137 4.1107 4.1005 4.0971 3.8342 3.8263 3.8116 3.8037

7.2808

10

[rel] EP-020914-1 lopputuote, CDCl3

1.9016

4

1

2.0000

2

41

8.1833

1.8859

0

140902-1 E:\nmr2014

500 MHz, CDCl3

[ppm]

0.5

1.0

1.5

2.0

2.5

150 S6 100

50

13.5945

25.2557 24.1628 23.0699 18.6766

EP-020914-1 lopputuote, CDCl3

32.4917 32.4418

1

53.2162 53.1650 53.1433 53.0926

45

66.5668 66.5143

77.3193 77.0651 76.8109

3.0 [rel] 140902-1 E:\nmr2014

126 MHz, CDCl3

[ppm]

42

1

E:\nmr2014

EP-020914-1 lopputuote, CDCl3

202 MHz, CDCl3

21.7741 21.7439

0

5

10

15

23.3862 23.3560

20

[rel]

140902-1

30

25

20

S7

15

10

5

[ppm]

10 8 6 S8

4 2 2.9656

10.7566

6

8

2.4971 2.4550 2.4128 1.9719 1.7176 1.7043 1.6904 1.6747 1.6611 1.3720 1.3567 1.2971 1.2871 1.2774 1.2689 0.8948 0.8813 0.8671

4.1276 4.1118 4.0984 4.0851 3.8342 3.8258 3.8116 3.8032

10

[rel] EP-020914-2 lopputuote, CDCl3

1.9902

4

1

1.9800

2

41

8.2554

2.0000

0

140902-2 E:\nmr2014

500 MHz, CDCl3

[ppm]

1

2

3

EP-020914-2 lopputuote, CDCl3

150 S9 100

50

31.7579 30.4891 30.4394 29.1629 29.1108 25.4344 25.2544 24.1616 23.0690 22.6180 14.0638

1

53.2095 53.1585 53.1312 53.0805

45

66.8780 66.8255

77.3180 77.0638 76.8095

[rel] 140902-2 E:\nmr2014

126 MHz, CDCl3

[ppm]

1

E:\nmr2014

202 MHz, CDCl3

23.3947 23.3645

EP-020914-2 lopputuote, CDCl3

0

5

10

21.7569 21.7267

42

15

[rel]

140902-2

30

25

20

S10

15

10

5

[ppm]

10 8 6 S11

8

10

4.8288 4.8165 4.8041 4.7888 4.7765 4.7640

4 2 5.9007

1.3692 1.3643 1.3569 1.3520 1.2560

1.9117

2.4827 2.4405 2.3983

6

12

[rel] EP-240914-2 lopputuote, CDCl3

2.0000

4

1

8.2248

2

51

3.8337 3.8253 3.8132 3.8112 3.8027 3.7905

0.8596

0

140924-2 E:\nmr2014

500 MHz, CDCl3

[ppm]

0.5

1.0

1.5

1

EP-240914-2 lopputuote, CDCl3

150 S12 100

50

25.8453 24.7503 24.0986 24.0694 23.8341 23.7906 23.6548

55

53.1806 53.1299 53.0977 53.0468 52.9716 52.9211

77.3112 77.0570 76.8028 71.9146 71.8624

[rel] 140924-2 E:\nmr2014

126 MHz, CDCl3

[ppm]

52

1

E:\nmr2014

EP-240914-2 lopputuote, CDCl3

202 MHz, CDCl3

20.6869 20.6558

0

5

10

15

23.4632 23.4322

20

[rel]

140924-2

30

25

20

S13

15

10

5

[ppm]

10 8 6 S14

4 4.0605

15

2.5080 2.4804 2.4659 2.4536 2.4402 2.4235

5.8482 5.8347 5.8275 5.8212 5.8141 5.8005 5.7933 5.7869 5.7798 5.7664 5.1718 5.1688 5.1375 5.1344 5.1246 5.1041 4.1958 4.1796 4.1662 4.1527 3.8350 3.8309 3.8249 3.8124 3.8083 3.8022

[rel] EP-271014-1 lopputuote, CDCl3

8.5124

10

1

2.0000

5

41

1.8075

0.8486

0

141027-1 E:\nmr2014

500 MHz, CDCl3

2 [ppm]

0.5

1.0

1.5

200 150 S15

100 50

25.3013 24.2076 23.1137

34.8496 34.8000

EP-271014-1 lopputuote, CDCl3

53.2303 53.1901 53.1809 53.1432 53.0943

1

65.8027 65.7508

[rel]

45

117.8162

133.3936

2.0

141027-1 E:\nmr2014

126 MHz, CDCl3

[ppm]

1

E:\nmr2014

EP-271014-1 lopputuote, CDCl3

202 MHz, CDCl3

0

5

10

15

21.9587 21.9297

42

23.3151 23.2862

20 [rel]

141027-1

30

25

20

S16

15

10

5

0

[ppm]

6 4 S17

1.0375

6

8

10

2.6260 2.6208 2.6126 2.6074 2.5992 2.5940 2.5366 2.4943 2.4520 2.0382 2.0331 2.0281

4.2499 4.2360 4.2200 4.2064 3.8432 3.8377 3.8339 3.8204 3.8151 3.8113

12

[rel] EP 051115-1 fr14-26 CDCl3

2.0057 2.2608

4

1

8.7000

2

51

2.0000

0

151105-1 "E:\NMR 2015"

500 MHz, CDCl3

2 0 [ppm]

0.5

1.0

1.5

150 S18 100

50

25.3816 24.2863 23.1911 20.9056 20.8535

EP 051115-1 fr14-26 CDCl3

53.2581 53.2110 53.1637 53.1252 53.0734

1

64.3889 64.3396

55

70.3737

79.5957

[rel] "141027-2 esteri" "E:\NMR 2015"

126 MHz, CDCl3

[ppm]

52

1

"E:\NMR 2015"

EP 051115-1 fr14-26 CDCl3

202 MHz, CDCl3

0

5

10

15

20

25

21.7714 21.7449 20.9437 20.9171

30

[rel]

151105-1

30

25

20

S19

15

10

5

[ppm]

10

8

10

12

8 6 S20

4 2

0.9384 0.9251

2.4959 2.4537 2.4115 1.7729 1.7595 1.7460 1.7326 1.7192 1.6097 1.5962 1.5827 1.5691

4.1714 4.1673 4.1574 4.1537 4.1427 4.1399 4.1289 3.8348 3.8265 3.8122 3.8039

14

[rel]

fr. 12-22

6.0737

6

PT/MS-240414-1 cdcl3

0.9163 2.0118

4

1

2.0993

2

22

8.1635

2.0000

0

MS-240414-1 "E:\NMR 2015"

500 MHz, CDCl3

[ppm]

0.5

1.0

1.5

2.0

200

fr. 12-22

150 S21

100 50

25.2780 24.5744 24.1854 23.0930 22.3900 22.3516

PT/MS-240414-1 cdcl3

39.1686 39.1190

1

53.2187 53.1628 53.1345 53.1093 53.0841

33

65.3362 65.2840

77.1929 76.9369

[rel] MS-240414-1 "E:\NMR 2015"

126 MHz, CDCl3

[ppm]

1

PT/MS-240414-1 cdcl3

"E:\NMR 2015" fr. 12-22

202 MHz, CDCl3

0

5

10

15

21.7828 21.7525

11

23.3816 23.3513

20 [rel]

MS-240414-1

30

25

20

S22

15

10

5

[ppm]

10 8 6 S23

4 2.9404

4.0617

10

15

1.7689

2.4942 2.4590 2.4239 2.4158 2.4043

4.8376 4.7776 4.2528 4.2509 4.2394 4.2280 4.2164 3.8306 3.8255 3.8190 3.8118 3.8067 3.8000

[rel] EPNL-081215-1 CDCl3

1

8.7536

5

10

2.0000

1.9855

0

EPNL-081215-1 "E:\NMR 2015"

600 MHz, CDCl3

2 [ppm]

1

2

3

4

5

200 150 S24

100 50

-0.0056

25.1590 24.2479 23.3366 22.4372

38.4796 38.4380

53.2118 M 53.1687 53.1623 53.1167 53.0719

EPNL-081215-1 CDCl3

1

64.8534 64.8104

12

112.7457

141.0283

6 [rel] EPNL-081215-1 "E:\NMR 2015"

151 MHz, CDCl3

[ppm]

11

EPNL-081215-1 CDCl3

1

"E:\NMR 2015"

243 MHz, CDCl3

20.6615 20.6375

0

5

10

15

22.0716 22.0477

20

[rel]

EPNL-081215-1

30

25

20

S25

15

10

5

[ppm]

10 8 6 S26

4 2

2.8271

1.5

2.0

2.0945 2.0552 2.0159 1.6394 1.6257 1.6115 1.5961 1.5823 1.4158 1.4010 1.3858 1.3707 1.3560 1.3412 0.9339 0.9190 0.9042

3.9001 3.8864 3.8727 3.8591

2.5

[rel] EP-280515-1 HPCCC fr 28, D2O+NaOD

1.9346

1.0

1

1.9389

0.5

111

2.0354

2.0000

- 0.0

150528-1 "E:\NMR 2015"

500 MHz, D2O+NaOD

[ppm]

"E:\NMR 2015"

126 MHz, D2O+NaOD

5

10

15

20

25

15.9568

EP 280515-1 fr 28 D2O+NaOD+TSP

21.2416

1

35.1996 35.1524 31.8768 30.9173 30.8799 29.9208

116

67.4760 67.4311

[rel]

150528-1

150

S27 100

50

[ppm]

1

"E:\NMR 2015"

202 MHz, D2O+NaOD

20.4708 20.4310

EP-280515-1 HPCCC fr 28, D2O+NaOD

0

5

10

15

20

13.4690 13.4269

112

25

[rel]

150528-1

30

25

20

S28

15

10

5

0

[ppm]

10 8 6 S29

4 2

2.6841

0.6

0.8

2.0243 M 1.9853 1.9463 1.6382 1.6242 1.6098 1.3287 1.3048 1.2888 0.8841 0.8705 0.8567

3.8681 3.8543 3.8406 3.8268

1.0

[rel] EP-280515-2 HP-CCC fr 11-13, D2O+NaOD+TSP

9.7694

0.4

1

1.9126

0.2

91

2.0000

1.9476

- 0.0

150528-2 "E:\NMR 2015"

500 MHz, D2O+NaOD

[ppm]

5

10

15

1

EP-280515-2 HP-CCC fr 11-13, D2O+NaOD+TSP

150 S30 100

50 16.2486

94

33.9521 33.1309 33.0834 32.3428 31.3853 M 31.3474 31.2653 30.4010 27.8887 24.8516

67.5928 67.5484

20 [rel] 150528-2 "E:\NMR 2015"

126 MHz, D2O+NaOD

[ppm]

1

"E:\NMR 2015"

202 MHz, D2O+NaOD

22.3918 22.3499

EP-280515-2 HP-CCC fr 11-13, D2O+NaOD+TSP

0

5

10

15

20

12.1681 12.1264

92

25

[rel]

150528-2

30

25

20

S31

15

10

5

[ppm]

10 8 6 S32

4

0.08

4.4931 4.4827 4.4724 4.4689 4.4620 4.4586 4.4482 4.4379

0.10

1.2642 1.2539

0.06

[rel] EP-051114-1 fr 6, D2O+NaOD

6.1351

0.04

1

2.0262 1.9935 1.9604

0.02

10

2.0229

1.0000

0.00

EP-051114-1 "E:\NMR 2015"

600 MHz, D2O+NaOD

2 [ppm]

1

"E:\NMR 2015"

EP-051114-1 fr 6, D2O+NaOD

151 MHz, D2O+NaOD

33.3565 32.5688 32.5108 31.7232 26.5206 26.4949

71.0997 71.0615

General ------DATE = 2016/02/18 TIME = 09:42 INSTRUM = spect PULPROG = zgpg30

-0.0006

20

20

[rel]

EP-051114-1

0

5

10

15

F1 (13C) ------------SI = 262144 SF = 150.915 SW_p = 36231.884

150

S33 100

50

[ppm]

11

1

"E:\NMR 2015"

EP-051114-1 fr 6, D2O+NaOD

243 MHz, D2O+NaOD

12.0191 11.9860

0

5

10

15

21.0017 20.9685

20

[rel]

EP-051114-1

30

25

20

S34

15

10

5

[ppm]

10 8 6 S35

4 2.0000

0.3

2.4266 2.4132 2.3997 2.3863 2.0538 2.0147 1.9755

5.9607 5.9473 5.9401 5.9339 5.9265 5.9128 5.9054 5.8993 5.8920 5.8786 5.2123 5.2084 5.2054 5.1776 5.1737 5.1283 5.1262 5.1243 5.1076 5.1055 5.1036 4.8096 4.8026 4.7895 4.7784 3.9539 3.9402 3.9265 3.9128

[rel] EP-031214-1 HP-CCC ajettu 160 mg, fr 12-15, D2O+ NaOD

1.8991

0.2

1

2.0458

0.1

31

1.6203

0.7413

0.0

141027-1 "E:\NMR 2015"

500 MHz, D2O+NaOD

2 [ppm]

36

1

"E:\NMR 2015"

EP-031214-1 HP-CCC ajettu 160 mg, fr 12-15, D2O+ NaOD

126 MHz, D2O+NaOD

37.5700 37.5224 32.2935 31.3464 31.2955 30.3446

66.5369 66.4936

119.5965

138.3927

2

4

6

8

10

12

163.6565

14

[rel]

141027-1

200

150

S36

100

50

[ppm]

122

1

"E:\NMR 2015"

0

5

10

15

11.8255 11.7844

202 MHz, D2O+NaOD

22.3216 22.2796

EP-031214-1 HP-CCC ajettu 160 mg, fr 12-15, D2O+ NaOD

20

[rel]

141203-1

30

25

20

S37

15

10

5

[ppm]

20

EP-271014-2 D2O

1

"E:\NMR 2015"

600 MHz, D2O

2.5755 2.5711 2.5646 2.5602 2.5536 2.5492 2.3909 2.3865 2.3821 2.3544 2.3207 2.2870

4.0272 4.0162 4.0037 3.9927

0.8

[rel]

EP-271014-2

General ------DATE = 2016/02/16 TIME = 14:22 INSTRUM = spect PULPROG = zg30

8

6

S38

4

1.9880 0.7426 2.1575

2.0000

- 0.0

0.2

0.4

0.6

F1 (1H) ------------SI = 65536 SF = 600.18 SW_p = 12019.231

2

[ppm]

"E:\NMR 2015"

151 MHz, D2O

-0.0007

30.6374 29.8019 28.9759 23.3143 23.2733

General ------DATE = 2016/02/23 TIME = 23:41 INSTRUM = spect PULPROG = zgpg30

65.8501 65.8175

EP-271014-2 D2O

1

73.5217

80

84.9877

20 [rel]

EP-271014-2

0

5

10

15

F1 (13C) ------------SI = 262144 SF = 150.915 SW_p = 36231.884

150

S39 100

50

[ppm]

1

EP-271014-2 D2O+NaOD

"E:\NMR 2015"

243 MHz, D2O+NaOD General ------DATE = 2016/02/16 TIME = 13:24 INSTRUM = spect PULPROG = zgpg30

11.6804 11.6435

11

22.5863 22.5490

[rel]

EP-271014-2

30

25

20

S40

0.9546

0.0509

1.0000

0

5

10

15

F1 (31P) ------------SI = 262144 SF = 242.957 SW_p = 59523.81

15

10

5

[ppm]

10

0.3

0.4

8 6 S41

4

2.0272 1.9877 1.9488 1.7281 1.7145 1.7009 1.6875 1.6747 1.5463 1.5325 1.5185 1.5045 0.9201 0.9068

3.9238 3.9098 3.8959 3.8823

0.5

[rel]

fr 21, D2O+NaOD+TSP

2

5.8893

0.2

EP-031214-3

1

0.8690 1.9488

0.1

71

2.0000

1.9592

0.0

141203-3 "E:\NMR 2015"

500 MHz, D2O+NaOD

[ppm]

"E:\NMR 2015"

126 MHz, D2O+NaOD

2

4

6

8

10

12

32.4577 31.5147 31.4561 30.5118 27.0695 24.7441

fr 21, D2O+NaOD+TSP

66.0308 65.9864

EP-031214-3

1

42.0156 41.9692

76

14

[rel]

141203-3

200

150

S42

100

50

[ppm]

EP-031214-3

1

"E:\NMR 2015"

fr 21, D2O+NaOD+TSP

202 MHz, D2O+NaOD

0

5

10

15

20

11.9310 11.8890

72

22.3325 22.2905

[rel]

141203-3

30

25

20

S43

15

10

5

[ppm]

10 8 6 S44

4

5

4 2

2.6317

2.0000

1.8926

2.3992 2.3862 2.3733 2.0319 1.9930 1.9538 1.7835

4.0150 4.0013 3.9877 3.9742

3

6

[rel] EP-211015-1 Fr 16-26 D2O+NaOD+TSP

1.9741

2

1

4.8713 4.8429

1

71

2.0823

-0

151021-1 "E:\NMR 2015"

500 MHz, D2O+NaOD

[ppm]

"E:\NMR 2015"

32.4294 31.4882 31.4286 30.4864 24.6077

41.1768 41.1277

126 MHz, D2O+NaOD

65.3849 65.3418

EP-211015-1 Fr 16-26 D2O+NaOD+TSP

114.2701

1

146.9392

73

5

10

15

20

[rel]

151021-1

150

S45 100

50

[ppm]

1

"E:\NMR 2015"

EP 211015-1 HPCCC fr 16-26 D2O+NaOD

202 MHz, D2O+NaOD

0

5

10

15

20

11.9529 11.9102

72

22.4241 22.3814

25 [rel]

151021-1

30

25

20

S46

15

10

5

0 [ppm]

HPCCC chromatogram from the purification of 3g Detection by UV at 200 (red), 220 (blue) and 254 (green) nm. Fractions 1-8: 3g and medronic acid Fractions 9-23: 3g Fractions 24-26: 3g and 3g with one methyl ester remaining

S47

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