Solvates and polymorphic modifications of steroid hormones. I

MICROCHEMICAL

JOURNAL

Solvates

16, 419-428

( 197 1)

and Polymorphic Modifications Steroid Hormones. I.

M. KUHNERT-BRANDST~TTER Institut

of

AND P. GASSER

fir Pharmakognosir der Univrrsitiit Innsbruck, Innsbruck, Austria Received March 1, 1971 INTRODUCTION

the attention of pharmaceutical technology has been directed toward the study of pseudopolymorphism (solvation) and polymorphism and their effect on the rate of dissolution and of resorption of medicinal agents. From this study, and from previous studies (I) on the differentiation of pseudopolymorphic and polymorphic modifications of steroid hormones, it is hoped that general relationships between the nature of the substituents and the tendency to form solvates will be found. In this investigation we used five solvents, which we shall call primary solvents. These solvents were methanol, ethanol, acetone, and chloroform, all in the anhydrous form, and aqueous ethanol for the preparation of hydrates. If the results of experiments with the five primary solvents were positive then a larger number of solvents were investigated. Investigations on polymorphism can never be considered to be completely exhaustive in that there is always the possibility that with some specific seeding condition a heretofore unknown crystal modification may appear. Examination of the crystals was carried out thermomicroscopically (2,3), and by infrared (ir) spectrophotometry. In some special casesdifferential scanning calorimetry (DSC) was employed. Recently

EXPERIMENTAL

Deoxycorticosteronc and Derivatives In all, 40 derivatives of deoxycorticosterone were investigated, In this paper shall be reported 13 derivatives which differ from the others by the absenceof the 17 a-hydroxy group. They are arranged in Table 1. The data in regard to polymorphism and solvates refer only to the stated experimental conditions and solvents. Again, it should be kept in mind that there is always the possibility of finding a new modification from some unique solvent and condition. 419

420

STEROID

HORMONES

TABLE

__Deoxycorticosterone acetate cyclopentylpropionate enanthate pivalate propionate Corticosterone acetate Fluocortolone pivalate Clocortolone capronate pivalate

1 Solvates

Polymorphism

+

-

+ + + + + +

+ + + -I+ + -

Deoxycorticosterone, and its esters exhibit little tendency to form crystalline solvates. No substance in this group yielded a crystalline complex with the primary solvents. Only in the case of deoxycorticosterone acetate with dimethylformamide was a system found that formed a solvate complex. Quantitative determination indicates that the complex has the mole proportion of 1:l deoxycorticosteronedimethylformamide. The complex is ucstable and starts to desolvate shortly after drying. Also, the commercial preparation, which we have used in our investigations, consists partly of crystals with secondary structure. From this it may be inferred that other solvents may also be bound in the crystal lattice of deoxycorticosterone acetate. Apart from the previously described deoxycorticosterone propionate (4), deoxycorticosterone pivalate is the only member of this group for which we have found a polymorphic modification. This modification was investigated both by thermomicroscopy and by ir spectrophotometry. On cooling the melt, modification II is found alongside of modification I, in the form of morphologically similar radiated or plane spherulites. The transformation of modification II to modification I takes place but slowly below 190°C only immediately before the melting of modification II at 195199°C does the rate of transformation increase. Although both modifications usually grow from the melt together, we were successful in preparing pure modification II for the recording of the ir spectrum. The ir spectra of both modifications were taken in Nujol 1 and by the KBr pellet technique. As deoxycorti1 “Nujol” is the registered trademark of Plough Laboratories, Inc., Memphis, Tennessee, for heavy white mineral oil.

KUHNERT-BRANDSTiTTER

AND GASSER

421

costerone pivalate contains no hydroxyl group, this usually valuable absorption region is of little value for the differentiation of the two modifications. Indeed, there are only slight differences of maximal 6 cm-l in all regions of the ir spectra. This hardly guarantees a rigorous differentiation. Thus it appears that the spectrum of modification I, reported by Mesley and Johnson (5), does not correspond completely to either of the two forms. Their deviations are well within those expected for instrumental and sample preparation differences. The following are some characteristic ir absorption frequencies: Modification I: 1420, 123% 1231, 1167, 781 cm-l; and modification II: 1415, 1234-1229, 1161 cm-l. Corticosterone, like deoxycorticosterone, does not itself exhibit any tendency to form solvates, at least with the primary solvents. Earlier, several polymorphic modifications have been reported (4). Corticosterone-21-acetate. The commercial preparation used in this investigation is a mixture of two polymorphic modifications, which differ from each other morphologically. Modification I forms rods and stems; modification II plates. Pure modification I may be prepared by tempering the commercial mixture, or modification II, at 145°C for 5 minutes, or by heating the hydrate. Pure modification II may be prepared by crystallization from solvents. It crystallizes from methanol, ethanol, and acetone in plates and coarse prisms which on heating, a& 140°C, slowly change to modification 1. Usually the melting temperature of modification II is obtainable. When crystallized from 70% ethanol on a watch glass, in open air, the hydrate is found in the form of Iong stems. It mehs between 105 and I 16°C inhomogeneously, forming modification I. All three forms are stable enough for the ir spectra to be recorded by the KBr pellet technique. In the hydroxyl absorption region, distinct differences are noticeable. For modification I the absorption maxima are found at 3540 and 3370 cm-l, for modification II at 3430 (sh) and 3370 cm-l. The hydrate absorbs at 3595 and 3460 cm-‘. The slight, though typical differences in the C-H region are of small value compared with the C=O and C=C frequency shifts: Modification Modification Hydrate

I II

Thermomicroscopic tion

1759, 1750 (sh), 1727, 1670, 1647, 1620 iw) cm-1 1748, 1727, 1647, 1616 (w) cm-1 1742, 1722-1713 id), 1660. 1611 (wj cm-1

and DSC Examination

of the Commercial Prepara-

If the material is heated on the microscope hot stage at a starting temperature of 140°C the melting temperature of the two modifications may be determined, Modification II melts at 14%148’C, the melt solid-

422

STEROID

HORMONES

ifies to modification I which melts together with the initially existing crystals of modification I at 153-155°C. On the contrary, if it is heated from ambient temperature, a transformation of the crystals of modification II to modification I results, and only the melting temperature of modification I is determined. The fact that the commercial preparation is a mixture is shown by the thermogram obtained by means of the Differential Scanning Calorimeter, Fig. 1. The first endotherm corresponds to the melting of modification II, the following, and smaller, exotherm indicates the resolidification of the melt, and the last endotherm corresponds to the melting of modification I. Fluocortolone, diene-3.20-dione) .

(6&uoro-1

lj3.21 -dihydroxy-I

6wnethyl-I

.4-pregna-

Of all the substances investigated, fluocortolone was the substance from which we were able to prepare the greatest number of solvates. For the 3 1 solvents tested, 19 positive reactions were observed. Solvates were successfully prepared from: methanol, anhydrous ethanol, 70% ethanol, l-propanol, acetone, methylene chloride, ethylene chloride, methyl acetate, tetrahydrofuran, dioxane, benzene, chlorobenzene, bromobenzene, m-chlorotoluene, toluene, xylene, benzyl alcohol, nitrobenzene, and N-methylaniline. Negative results were obtained from: 2-propanol, I-butanol, 1-hexanol, 1-octanol, methyl ethyl ketone, tetrachloroethane, ethyl acetate, diethyl ether, glacial acetic acid, acetonitrile, decalin, and carbon tetrachloride. In going from I-propanol to 2-propanol, from acetone to methyl ethyl ketone, from methyl acetate to ethyl acetate, and from chloroform to carbon tetrachloride, the limitation for solvate formation was clearly

endotherm

I

exotherm

L

410 FIG.

420

L30

T’K

1. Thermogram of corticosterone acetate.

KUHNERT-BRANDSTiTTER

AND GASSER

423

demonstrated. In most cases the transformation to the solvate took place in seconds, in some cases, as with ethanol and acetone, within minutes. Figure 2 shows crystals of fluocortolone which are starting to change to the solvate. The solvent is methanol. On heating, the solvate crystals give off the solvent above 40°C for methanol, and 115°C for benzene. In all cases the desolvation may be observed on crystals that are mounted on the hot stage in Nujol or silicone gel, by the characteristic formation of gas bubbles. Pseudomorphosis occurs in the form of modification II. For the solvates from

FIG. 2. Fluocortolone:

growing of the solvate from a methanol suspension.

FIG. 3. Clocortolone capronate: growing of coarse grains of the methanol solvate and thin needles of the hydrate from a suspension.

424

STEROID

HORMONES

chloroform, benzene, anhydrous ethanol, and 70% ethanol, Ihe mole proportion of steroid to solvent was found to be 2:l. The other solvates were not measured quantitatively. Noteworthy is the fact that two different solvates can be crystallized from ethanol, from absolute ethanol stems, form CY,from 70% ethanol needles and rods, form ,L?,which differ in their ir spectra and in their thermomicroscopic behavior. Since in both cases the same mole proportion of steroid to solvent was found, it appears to be a phenomenon of dimorphy of the solvate. The investigation of polymorphism in fluocortolone has been less successful than that of solvation. Other than the two forms already present in the commercial preparation, no other forms were found. The commercial preparation consists of metastable crystals of modification II which exhibit secondary structure and certainly must have resulted from desolvation. Also, a small quantity of crystals of modification 1 are present. Both forms melt one after the other. Modification II melts inhomogeneously with the separation of crystals of modification I and, therefore, results in a very long melting interval for the commercial preparation. Pure modification II melts at 184-19O”C, modification I melts at 200-213°C. The ir spectra of the polymorphic modifications were recorded by the KBr pellet technique. As was to be expected, they differ from each other in the hydroxyl absorption region. Modification I possesses a broad strong absorption at 3490 cm-*, modification II a shoulder at 3545 cm-l and a strong band at 342,Ocm-‘. In Nujol, the shoulder appears as a weak absorption. In the region of the C-H stretching, modification I exhibits a weak absorption at 2910 cm-l, modification II at 2923 cm-l. Further significant absorption frequencies are noted in Table 2. The ir spectra of six of the 19 solvates prepared were recorded. The solvates of chloroform, methylene chloride, methyl acetate, benzene, and dioxane clearly exhibit solvent absorption which, with the exception of benzene, are compared with the free solvent (in Nujol) absorption shifted to lower frequencies. In the solvate of benzene, the C-H absorption due to “wagging” (out of plane deformation) is shifted from 674 to 682 cm-l. In the solvate of chloroform, the two C-Cl absorptions at 765 and 755 cm-l stand opposite to that of the free chloroform absorption at 761 cm-l. Methyl acetate exhibits shifting from 1757 to 1748 cm-l, and methylene chloride from 745 to 737 cm-l. The dioxane solvate exhibits characteristic solvent absorption at 1251, 1122, and 872 cm-l which, when compared with the absorption of free dioxane in Nujol, 1255, 1122, and 874 cm-l, are shifted only slightly. All ir spectra of solvates were recorded in Nujol. For the purpose of comparison,

KUHNERT-BRANDSTLTTER

AND

TABLE

425

GASSER

2

FLUOCORTOLONE

Frequencies (cm-‘) OH

Form Modification I Modification II Ethanol solvate-ol Ethanol solvate-B

Chloroform solvate

Benzene solvate

Methyl acetate solvate

Methylene chloride solvate

Dioxane solvate

3490 3545 3420 3505 3400 3510 3430 3330 3490 3515 3460 3390 3535 3440 3400 3540 3500 3470 3390 3490 3520 3400 3535 3440

(b) (sh)

C=O and C=mC 1707, 1675, 1666 (sh), 1630. 1609 171lL1704 (d). 1665, 1623, 1604

(b)

1705. 1664, 1622, 1603 (sh)

1720, 1705, 1667, 1626, 1607 (sh) (sh) 1708, 1696 (w), 1668, 1626, 1603 (sh) (sh) (sh) 1701, 1660, 1622, 1632 (w)

(sh) (w)

1748, 1706,1626, 1696 (sh), 1666

1603 (wl

(sh) (sh) 1707,1669,1630-1622

(sh) (sh)

(d)

1603 (w) 1701, 1627. 1604 1663-1669 (d)

Notes: (s) strong, (m) medium, (w) week, (b) broad, (sh) shoulder, (di double band. spectra corded

of solvates

of dioxane,

chloroform,

and benzene

were

also re-

by the KBr pellet technique. No significant differences were ob-

served. Fluocortolone-21-pivalate, possesses, like fluocortolone, a pronounced tendency to form solvates. As the status of this substance is particularly complicated, having at least four polymorphic modifications and 12 solvates, a detailed study is reported in a separatepaper (6). Clocortolone, (&fluoro-9a-chloro-11 p,21 -dihydroxy-16a-methyl-l,4pregnadiene-3.20-dione). Clocortolone differs from fluocortolone

in

that it has less tendency to form solvates. The solvent-free form, which melts at 240-265°C with decomposition, is always found when recrystallized from completely anhydrous solvents. This is typical of the commercial preparation. The hydrate, which differs from the anhydrous form morphologically, is prepared by recrystallization from aqueous sol-

426

STEROID

HORMONES

vents. The hydrate forms needles, stems, and beams. The anhydrous form crystallizes in four- and six-sided leaflets or prisms. Clocortolone hydrate is unusually stable. If heated rapidly in Nujol or silicone gel, pseudomorphosis does not occur until a temperature of 160-190°C has been reached. At first it was thought that with some solvents, solvates were being formed; however, it was learned subsequently that in all cases, hydrates were being formed from the even slight amounts of water in “pure” solvents. Since the substance melts with much decomposition, it was impossible to use the melt for the investigation of polymorphism. This may be the reason that no polymorphic modifications have been found. The anhydrous form, A, and the hydrate, B, differ in their ir spectra also. As the hydrate is very stable, the KBr pellet technique was used to record the ir spectra. The hydroxyl and carbon-to-carbon frequencies for both forms show considerable differences. A B

3600, 3450, 3390 (sh), 1635, 1612 (w), 1606 (w) cm-’ 1607 cm- 1 3585, 3515, 3360 (b), 1625,

Form A also has characteristic absorption at 1298 and 815 cm-l, and form B, the hydrate, at 909 and 837 cm-*. Clocortolone-%I-capronate. This substance forms solvates, however, no polymorphic forms have been found. From anhydrous methanol, the solvate crystallizes as coarse prisms. From aqueous methanol, thin needle crystals of the hydrate are found along with the crystals of the methanol solvate. Figure 3 was taken at the time that the hydrate crystals were forming along side of the already present methanol solvate crystals. From the other primary solvents, the same modification always resulted, melting temperature 156-160°C. On heating, the methanol solvate undergoes pseudomorphosis between 60 and 120°C. Also, in Nujol inhomogeneous melting may occur. The hydrate melts between 90 and 120°C inhomogeneously with separation of the anhydrous form. As the methanol solvate cannot be subjected to pressure, the spectra were recorded in Nujol. The hydroxyl stretchings show great differences, so that they alone would be sufficient for differentiation. In addition, there are frequency shifts in other regions which, except for the C---O and C=C stretchings will not be discussed. Clocortolone-21-pivalate. Initially it appeared that this substance always crystallized from anhydrous solvents as the solvent complex. However, upon more detailed investigation, it was found that in all cases the hydrate was being formed. It was found that the small amount of water

KUHNERT-BRANDSTKTTER AND GASSER TABLE

427

3

CLOCORTOLONECAPRONATE Frequencies

Form ModificationI Hydrate Methanolsolvate

OH 3350

3610 3535 3340 3550 3420

(cm-l)

C=O

and C-C

1747, 1734, 1668, 1626, 1615 1750, 1731, 1629 (w), 1613 1722 (w), 1670

(b) 1721, 1663, 1621, 1605

contained in the substance as an impurity was sufficient for hydrate formation. Similar behavior was observed previously in the case of p-estradiol and related compounds (I). At that time it was supposed that this behavior was due to different solvates as the ir spectra indicated certain differences and the solvents were demonstrated by gas chromatography. After this experience of forming hydrates of clocortolone and clocortolone-21-pivalate from anhydrous solvents, the formation of solvates from anhydrous organic solvents may be accepted only if the ir spectra demonstrates shifts of the solvent absorption when compared with that of the free solvent. Otherwise, it is a question of solvent that is not held in the crystal lattice. In keeping with this new knowledge, a program for the reinvestigation of the data previously reported (I) is planned. As in the case of clocortolone, the hydrate is relatively stable. When heated in Nujol or silicone gel, pseudomorphosis and partly inhomogeneous melting may be observed between 130 and 150°C. The anhydrous form crystallized at the same time exhibits leafy habitus differing from the hydrate which crystallizes in long needles and thin stems. If the hydrate is heated without mounting in Nujol or silicone gel, the crystals soften up to about 120°C part undergoes pseudomorphosis and the remainder melts at about 140°C inhomogeneously. The melting temperature of the solvent-free crystal form is 230-245°C with so much decomposition that the use of the melt for the investigation of polymorphism is impossible. Spectra, in Nujol and by the KBr pellet technique, were recorded for the two crystalline forms, A = anhydrous form, B = hydrate. Differences in the hydroxyl absorption region of A were observed, depending on the technique. In Nujol, the absorption lies at 3350 cm-1 compared to that in KBr at 3420 cm-l. In the other regions the spectra by both

428

STEROID

HORMONES

techniques correspond. The differences in the hydroxyl region between form A and form B are so great that identification can be guaranteed: A: 3350 (3420 KBr), 1744, 1731. 1668, 1630. 1614 cm-‘; and B: 3630, 3375. 3220 (w), 1735 (w), 1722, 1665, 1622. 1609 cm-‘. SUMMARY The ability of deoxycorticosterone and 12 derivatives of deoxycorticosterone to form solvates and polymorphic modifications was investigated. It appears that fluocortolone and its ester form solvates with numerous solvents. Also, some new polymorphic modifications are reported. REFERENCES 2. Kuhnert-Brandstatter, M., and Grimm, H., Zur Unterscheidung von liisungsmittelhaltigen pseudopolymorphen Kristallformen und polymorphen ModifiActa, 115 (1968). kationen bei Steroidhormonen. Mikrochim. 2. Kofler, L., and Kofler, A., “Thermomikromethoden,” Wagner, Innsbruck, 1954. 3. Kuhnert, Brandstltter, M., “Thermomicroscopy in the Analysis of Pharmaceuticals,” Pergamon, London, 197 1. 4. Kuhnert-Brandstltter, M., Junger, E., and Kofler, A., Thermomicroscopic and spectrophotometric determination of steroid hormones. Microchem. J. 9, 105 (1965). 5. Mesley, R. J., and Johnson, C. A., “Infrared identification of pharmaceutically important steroids with particular reference to the occurance of polymorphism. J. Pharmacol. 17, 329 (I 965). 6. Kuhnert-Brandstatter, M., and Gasser, P., “Polymorphe Modifikationen und Solvate von Fluocortolonpivalat. Arch. Pharm., in press.