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Electronic Supplementary Information Fifty-year old samples of progesterone demonstrate the complex role of synthetic impurities in stabilizing a metastable polymorph Robert W. Lancaster, Lisa D. Harris, David Pearson

Contents

1. Reference IR spectra of Progesterone forms 1 and 2. 2. Details of polymorph nomenclature associated with the progesterone literature and associated

documented crystal structures. 3. Mass spectrum for form 1, conditions used for obtaining LC-MS data and information about

molecular ions found. 4. Conditions used for obtaining NMR spectra. 5. Solution state NMR spectrum of progesterone (for assignment and reference purposes), plus

spectrum of form 2 with impurities highlighted. 6. Impurity profile for Aldrich reference material. 7. Origin of reference samples used in spiking experiments in conjunction with NMR and LC-MS 8. LC-MS spiking experiments with reference standards. 9. Carbon-13 NMR spectra of reference materials.

1. Reference IR spectra of Progesterone forms 1 and 2.

Figure S1. Reference IR spectrum of progesterone form 1.

Supplementary Material (ESI) for CrystEngCommThis journal is © The Royal Society of Chemistry 2011

Figure S2. Reference IR spectrum of progesterone form 2.

2. Details of polymorph nomenclature associated with the progesterone literature and associated documented crystal structures.

Polymorph nomenclature / notation CSD refcodes Form 1 Refs: 1,2

Form I, high temperature phase M.pt. 129 °C Refs: 3-7

α form Refs: 8,9

Form A Ref: 10

PROGST103 PROGST034 PROGST027 PROGST1211

Form 2 Refs: 1,2

Form II, low temperature phase M.pt. 122 °C Refs: 5,6,12,13

β form Refs: 8,9

Form B Ref: 10

PROGST0114 EWACUA13 (ent progesterone) PROGST1311

References 1. R. W. Lancaster, P. G. Karamertzanis, A. T. Hulme, D. A. Tocher, D. F. Covey and S. L. Price, Chem.

Commun., 2006, 4921-4923. 2. J. A. Wachter. 2004. 3. H. Campsteyn, L. Dupont and O. Dideberg, Acta Crystallogr. , Sect. B., 1972, 28, 3032-3042. 4. B. A. Haner and D. A. Norton, Acta Crystallogr., 1964, 17, 1610. 5. M. Kuhnert-Brandstätter and A. Kofler, Mikrochim. Acta, 1959, 47, 847-853. 6. F. Wang, J. A. Wachter, F. J. Antosz and K. A. Berglund, Org. Process Res. Dev., 2000, 4, 391-395. 7. G. van den Bossche, Bull. Soc. R. Sci. Liege, 1971, 40, 142. 8. M. Muramatsu, M. Iwahashi and U. Takeuchi, J. Pharm. Sci., 1979, 68, 175-177. 9. R. S. Payne, R. J. Roberts, R. C. Rowe and R. Docherty, Int. J. Pharm., 1999, 177, 231-245. 10. R. J. Mesley and C. A. Johnson, J. Pharm. Pharmacol., 1965, 17, 329-340. 11. R. W. Lancaster, P. G. Karamertzanis, A. T. Hulme, D. A. Tocher, T. C. Lewis and S. L. Price, J.

Pharm. Sci., 2007, 96, 3419-3431.


12. R. Cameroni, G. Gamberin, M. T. Bernabei and M. Facchini, Farmaco-Edizione Pratica, 1973, 28, 621-635.

13. R. J. Auchus, A. S. Kumar, C. A. Boswell, M. K. Gupta, K. Bruce, N. P. Rath and D. F. Covey, Arch. Biochem. Biophys., 2003, 409, 134-144.

14. E. Foresti Serantoni, A. Krajewski, R. Mongiorgi, L. Riva de Sanseverino and R. Cameroni, Cryst. Struct. Commun., 1975, 4, 189-192.

3. Mass spectrum of form 1 conditions used for obtaining LC-MS data and information

about molecular ions found. ALPHA_081010154255 #7224-7410 RT: 21.20-21.68 AV: 187 NL: 1.05E7T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

315.36

316.26313.45

317.29311.39297.39271.37 318.31253.42245.38 326.38279.46 291.44263.41239.46 349.44227.44 338.34

Figure S3. Mass Spectrum (APCI) of progesterone form 1 (the mass spectrum of form 2 bears a close resemblance to form 1). LC-MS data was collected on a Thermo Finnigan LTQ Ion-Trap operating in Atmospheric Pressure Chemical Ionization (APCI) positive mode equipped with an Agilent 1100 series LC. The LC conditions were as follows: Column: Agilent Eclipse XDB-C18 150 mm x 4.6 mm, Flow: 0.5 mL/min, Mobile Phase: (Isocratic) 50:50 MeCN:H2O (0.25Mm NH4OAc), Injection Volume: 10µL. The Mass Spectrometer conditions were as follows: Mode: APCI+, Vaporizer Temperature (°C): 475, Capillary Temperature (°C): 275, Tube Lens (V): 80, Sheath Gas Flow Rate (arb): 40, Aux Gas Flow Rate (arb): 5, Sweep Gas Flow Rate (arb): 5, Discharge Current (μA): 5. The molecular ions associated with some of these components are [M+H]+ = 288.9, 331.2, 329.2, 317.2, 313.2, and 315.2. The last corresponds to progesterone and 317.2 appears to correspond to pregnenolone. However, if a sample of progesterone is spiked with pregnenolone it co-elutes with the former, suggesting that [M+H]+ = 317.2 is an isomer of pregnenolone. The possibility of an isomer of pregnenolone present as an impurity in pregnenolone when used in the early syntheses has been considered. Plausible identification of some of the remaining impurities are as follows: [M+H]+ = 331.2 - hydroxyprogesterone (11-OH or 17-OH?); [M+H]+ = 329.2 – bisnoraldehyde (from ergosterol?); [M+H]+ = 313.2 - 1- dehydroprogesterone. The latter is considered to be a potential impurity present at a level at greater than 0.1% whilst hydroxyprogesterone, if present, would probably exist at levels below 0.1%.1 It should be stressed that there is absolutely no inference that any modern synthesis corresponds to that used to produce the 50-year old Innsbruck sample, indeed we feel that to be a very unlikely scenario. Reference. 1. Progesterone USP Regulatory filing. PfizerCentreSource.com, 2004.


4. Conditions used for obtaining solution state NMR spectra. NMR spectra were obtained using a Bruker DRX400 system with a 5 mm DUL 1H/13C probe under ambient conditions. The probe was tuned and matched for all samples prior to data acquisition and referenced to the residual solvent signal of CDCl3. 5. Solution state NMR spectrum of progesterone (for assignment and reference purposes), plus spectrum of form 2 with impurities highlighted.

O

H

H H

O

2030405060708090100110120130140150160170180190200210 ppm

13.47

17.51

21.16

22.98

24.50

31.62

32.03

32.91

34.08

35.69

35.87

38.71

38.81

44.06

53.79

56.17

63.64

76.84

77.04

77.16

77.36

77.48

124.08

171.04

199.56

209.41

Figure S4. Progesterone form 2 solution-state 13C NMR. Progesterone shows peaks at (Assignments in brackets) δ 209.4 (C20), 199.6 (C3), 171.0 (C5), 124.1 (C4), 63.6 (C17), 56.2 (C14), 53.8 (C9), 44.1 (C13), 38.8, 38.7 (C12, C10), 35.9, 35.7, 34.1 (C1, C8, C7), 32.9, 32.0 (C2, C6), 31.6 (C21), 24.5, 23.0, 21.2, (C15,11,16), 17.5 (C19), 13.5 (C18).


65 60 55 50 45 40 35 30 25 20 15 10 5 ppm

11.56

13.47

17.51

21.16

22.98

24.50

29.16

29.79

31.62

32.03

32.91

34.08

35.69

35.87

38.71

38.81

44.06

44.79

46.80

53.79

56.17

56.65

63.64

Figure S5. Expanded spectrum of form 2 with carbon signals associated with impurities arrowed. The spectrum of form 1 is identical, though on a similar scale of expansion there is no obvious evidence for impurities. The proton spectrum of form 2 did not offer any evidence for impurities but the 13C spectrum of the metastable form 2 (Figure S2) showed evidence for trace impurity(s) with extra signals, possibly associated with skeletal carbon atoms proximal to the junction of the B and C rings. Alternatively, the impurity peaks could be attributed to a fairly simple aliphatic compound, though there is no evidence to suggest that such a compound would be associated with the synthesis, or if it were, carried through it. The S/N associated with the spectrum was poor owing to the number of low level impurities present. There were no obvious impurities present in the spectrum of form 1. 6. Impurity profile for Aldrich reference material. RT: 3.00 - 45.00 SM: 7B

5 10 15 20 25 30 35 40 45Tim e (m in)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

Relative Abu

ndan

ce

20.14

23.7013.47 25.1017.917.24 27.549.82 32.003.71 33.79 36.7214.1412.73 41.15 44.17

NL:1.42E7TIC MS PROGTEST_081008172357

Figure S6. LC trace for Aldrich reference material (1.2% impurities).


7. Origin of reference samples used in characterization and spiking experiments in conjunction with NMR and LC-MS analysis. Samples of 16-dehydroprogesterone, 4-Pregnen-20β-ol-03-one, 4,6-Pregnadiene-3,20-di-one, 4-Pregnen-3-one-20-β−carboxyaldehyde, and 4-Pregnen-20α-ol-3-one were obtained from Steraloids and used as reference standards. 8. LC-MS spiking experiments with reference standards. From the Mass Spec data several potential steroids were considered. LC traces and Mass Spectra for each are shown below. PROGESTERONE (PURE) RT: 3.00 - 40.00 SM: 15B

5 10 15 20 25 30 35 40Tim e (m in)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

Relative Abu

ndan

ce

20.15

23.35

23.7013.50

24.2925.0917.91

26.30 27.557.23 29.589.81 31.9910.423.72 5.00 33.69 35.0613.89 39.28

NL:1.39E7TIC MS PROGTEST_081008172357

Figure S7. Expanded LC trace for progesterone (pure). Peaks are seen at 7.23, 9.81, 10.42, 13.5, 17.91 and 20.15 min (peak at 21.4 min being progesterone). Peaks at 7.23, 9.81, 10.42 and 17.91 are also seen in the ‘beta-progesterone’ LC and have the same mass spectra. PROGTEST_081008172357 #4353-4515 RT: 13.13-13.62 AV: 163 SB: 2167 3.62-6.64 , 36.49-40.00 NL: 1.24E4T: ITMS + c APCI corona Full m s [220.00-390.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

317.24

318.29

299.27

319.31300.30 315.29297.27 339.56230.28 281.26255.17 271.17 328.40243.18 261.22 346.32

Figure S8. Mass Spectrum of peak at 13.5 min.


CHOLESTEROL (PURE) RT: 0.40 - 31.00 SM: 15B

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Tim e (m in)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

2.722.62

22.12

28.8625.290.45 17.09 18.6915.6714.534.39 11.9610.854.74 5.78

NL:2.46E5TIC MS CHOL_081010093818

Figure S9. LC Trace for Cholesterol (pure). CHOL_081010093818 #691-964 RT: 2.10-2.92 AV: 274 SB: 1145 27.97-30.33 , 0.72-1.82 NL: 3.71E4T: ITMS + c APCI corona Full m s [220.00-395.00]

220 240 260 280 300 320 340 360 380m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

352.24

387.30370.40

244.18 388.35226.17 385.38313.31 358.37343.26328.41283.26255.19 271.22 297.27

Figure S10. Mass Spectrum for peak at 2.7 min (cholesterol).


4-PREGNEN-20α-OL-3-ONE (PURE) Steraloids Lot: TL1792 RT: 0.14 - 30.76 SM: 15B

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Tim e (m in)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

4.0

4.2

4.4

Relative Abu

ndan

ce

20.23

7.0015.54

15.91

16.4017.64

21.81

4.48 22.5211.31 23.68 25.69 30.646.132.69 7.731.24 8.44

NL:1.87E7TIC MS 09_10_08_1

Figure S11. LC Trace for 4-PREGNEN-20α-OL-3-ONE. 09_10_08_1 #4898 RT: 14.23 AV: 1 SB: 2355 0.92-4.83 , 25.92-28.92 NL: 7.49E5T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

317.23

318.26

299.25

315.37

300.33 319.29 339.74297.30281.35273.31 302.32263.32 328.37257.38239.07229.29 348.68

Figure S12. Mass Spectrum for peak at 13.53 min.


4-PREGNEN-20β-OL-3-ONE Steraloids Lot: TB0536 RT: 0.00 - 39.97 SM: 15B

0 5 10 15 20 25 30 35Tim e (m in)

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

36

38

40

Relative Abu

ndan

ce

13.54

21.47

22.9624.34 26.835.74 30.248.82 32.61 34.75 37.132.66 14.50 17.2810.09

NL:1.82E7TIC MS 09_10_08_2

Figure S13. LC Trace for 4-PREGNEN-20β-OL-3-ONE. Major peak at 20.1 min is beta, and small amount of alpha present at 13.54 min 09_10_08_2 #6957 RT: 20.31 AV: 1 SB: 2445 0.79-2.96 , 35.49-40.49 NL: 1.56E7T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

317.41

318.35315.69

299.53319.34300.59297.54281.57 340.09273.58 328.51255.49229.53 241.55 346.65267.62

Figure S14. Mass Spectrum of major peak at 20.1 min.


16-DEHYDROPROGESTERONE Steraloids Lot: 03114EM RT: 2.60 - 30.00 SM: 15B

4 6 8 10 12 14 16 18 20 22 24 26 28 30Tim e (m in)

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

Relative Abu

ndan

ce

5.92

16.97

5.6122.38

6.90 22.867.23 9.40 10.35 19.114.04 24.228.67 12.61 14.25 25.28 27.01

NL:1.85E7TIC MS 09_10_08_3

Figure S15. LC Trace for 16-DEHYDROPROGESTERONE. 09_10_08_3 #7132 RT: 20.85 AV: 1 SB: 1105 3.08-4.75 , 28.42-30.00 NL: 1.24E7T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

313.48

312.13

315.29310.15295.97 324.37 349.43263.95 271.89253.98 283.80231.70 336.58247.88

Figure S16. Mass Spectrum for major peak at 20.7 min.


4,6-PREGNADIENE-3,20-DIONE RT: 2.50 - 31.12 SM: 15B

4 6 8 10 12 14 16 18 20 22 24 26 28 30Tim e (m in)

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

Relative Abu

ndan

ce

21.72

16.268.70

9.6911.358.245.784.82 24.88 25.9814.8913.50 27.72 29.61

NL:2.16E7TIC MS 09_10_08_4

Figure S17. LC Trace for 4,6-PREGNADIENE-3,20-DIONE. 09_10_08_4 #6008-6446 RT: 17.60-18.75 AV: 439 SB: 1790 3.23-5.61 , 28.05-30.91 NL: 9.05E6T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

313.34

311.45315.25295.41 309.42269.37253.34 324.30277.42 349.37243.39227.36 337.45287.32



4-PREGNEN-3-ONE-20β-CARBOXALDEHYDE Steraloids Lot: TG482 RT: 2.50 - 33.00 SM: 15B

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32Tim e (m in)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Abu

ndan

ce

3.63

4.7921.76

20.24

13.479.956.10

32.967.70 31.2126.0913.04 29.7618.4615.15 25.17

NL:2.34E6TIC MS 09_10_08_5

Figure S19. LC Trace for 4-PREGNEN-3-ONE-20β-CARBOXALDEHYDE. 09_10_08_5 #6728-6959 RT: 19.74-20.41 AV: 232 SB: 390 3.05-4.17 NL: 6.58E4T: ITMS + c APCI corona Full m s [220.00-350.00]

220 230 240 250 260 270 280 290 300 310 320 330 340 350m /z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Relative Ab

unda

nce

329.24

343.24

317.26

330.28344.22

318.28

303.28341.93



9. Carbon-13 NMR spectra of reference materials

O

O

H

H H

2030405060708090100110120130140150160170180190200210 ppm

15.92

17.32

20.86

27.23

31.92

32.22

32.82

33.97

34.05

34.58

35.66

38.84

46.19

54.20

55.79

76.84

77.16

77.48

124.10

144.23

155.26

171.06

196.81

199.63

Figure S21. 16-dehydroprogesterone shows peaks at δ 199.6, 196.8, 171.1, 155.3, 144.2, 124.1, 55.8, 54.2, 46.2, 38.8, 35.7, 34.6, 34.1, 34.0, 32.8, 32.2, 31.9, 27.2, 20.9, 17.3, 15.9.


OH

H

H H

O

2030405060708090100110120130140150160170180190200210 ppm

15.92

17.32

20.86

27.23

31.92

32.22

32.82

33.97

34.05

34.58

35.66

38.84

46.19

54.20

55.79

76.84

77.16

77.48

124.10

144.23

155.26

171.06

196.81

199.63

Figure S22. 4-Pregnen-20β-ol-03-one shows peaks at δ 199.6, 171.5, 123.8, 70.4, 58.3, 55.4, 53.8, 42.36, 39.6, 38.6, 35.7, 35.5, 34.0, 32.9, 32.1, 25.6, 24.4, 23.8, 20.9, 17.4, 12.4.


O

H

H H

O

2030405060708090100110120130140150160170180190200210 ppm

13.37

16.41

20.76

23.02

24.01

31.62

34.03

36.16

37.72

38.69

44.83

50.68

53.79

63.37

76.84

77.15

77.47

123.91

128.25

140.63

163.53

199.57

209.13

Figure S23. 4,6 Pregnadiene-3,20-di-one shows peaks at δ 209.1, 199.6, 163.5, 140.6, 128.3, 123.9, 63.4, 53.8, 50.7, 44.8, 38.7, 37.7, 36.2, 34.0*, 31.6, 24.0, 23.0, 20.8, 16.4, 13.4 * Intense peak, probably 2 peaks overlapping.


C

H

H H

O

H

O

2030405060708090100110120130140150160170180190200 ppm

12.45

13.54

17.49

21.08

24.64

27.11

32.07

32.94

34.06

35.70

35.81

38.68

39.41

43.11

49.53

51.07

53.86

55.27

76.84

77.15

77.36

77.47

124.00

171.27

199.61

Figure S24. 4-Pregnen-3-one-20-β−carboxyaldehyde shows peaks at δ 205.0, 199.6, 171.3, 124.0, 55.3, 53.9, 51.1, 49.5, 43.1, 39.4, 38.7, 35.8, 35.7, 34.1, 32.9, 32.1, 27.1, 24.6, 21.1, 17.5, 13.5, 12.4.


OH

H

H H

O

2030405060708090100110120130140150160170180190200 ppm

12.68

17.51

20.87

23.68

24.21

25.69

32.11

33.01

34.09

35.38

35.83

38.72

38.77

41.79

53.88

55.82

58.44

70.25

76.84

77.15

77.36

77.47

123.97

171.48

199.69

Figure S25. 4-Pregnen-20α-ol-3-one shows peaks at δ 199.8, 171.4, 124.0, 70.3, 58.5, 55.8, 53.9, 41.8, 38.8, 35.8, 35.4, 34.1, 33.0, 32.1, 25.7, 24.2, 23.7, 20.9, 17.5, 12.7.


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