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
