# Patent

## Title
Pseudopolymorphic forms of a HIV protease inhibitor

## Abstract

New pseudopolymorphic forms of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate and processes for producing them are disclosed.

## Background
This application is the national stage of Application No. PCT/EP03/50176, filed May 16, 2003, which application claims priority from European Patent Application No. 02076929.5, filed May 16, 2002.
TECHNICAL FIELD
This invention relates to novel pseudopolymorphic forms of (3R,3aS,6aR)-hexahydro-furo[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate, a method for their preparation as well as their use as a medicament.
BACKGROUND OF THE INVENTION
Virus-encoded proteases, which are essential for viral replication, are required for the processing of viral protein precursors. Interference with the processing of protein precursors inhibits the formation of infectious virions. Accordingly, inhibitors of viral proteases may be used to prevent or treat chronic and acute viral infections. (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate has HIV protease inhibitory activity and is particularly well suited for inhibiting HIV-1 and HIV-2 viruses.
The structure of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-phenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate, is shown below:




Compound of formula (X) and processes for its preparation are disclosed in EP 715618, WO 99/67417, U.S. Pat. No. 6,248,775, and in Bioorganic and Chemistry Letters, Vol. 8, pp. 687-690, 1998, “Potent HIV protease inhibitors incorporating high-affinity P2-ligands and (R)-(hydroxyethylamino)sulfonamide isostere”, all of which are incorporated herein by reference.
Drugs utilized in the preparation of pharmaceutical formulations for commercial use must meet certain standards, including GMP (Good Manufacturing Practices) and ICH (International Conference on Harmonization) guidelines. Such standards include technical requirements that encompass a heterogeneous and wide range of physical, chemical and pharmaceutical parameters. It is this variety of parameters to consider, which make pharmaceutical formulations a complex technical discipline.
For instance, and as example, a drug utilized for the preparation of pharmaceutical formulations should meet an acceptable purity. There are established guidelines that define the limits and qualification of impurities in new drug substances produced by chemical synthesis, i.e. actual and potential impurities most likely to arise during the synthesis, purification, and storage of the new drug substance. Guidelines are instituted for the amount of allowed degradation products of the drug substance, or reaction products of the drug substance with an excipient and/or immediate container/closure system.
Stability is also a parameter considered in creating pharmaceutical formulations. A good stability will ensure that the desired chemical integrity of drug substances is maintained during the shelf-life of the pharmaceutical formulation, which is the time frame over which a product can be relied upon to retain its quality characteristics when stored under expected or directed storage conditions. During this period the drug may be administered with little or no risk, as the presence of potentially dangerous degradation products does not pose prejudicial consequences to the health of the receiver, nor the lower content of the active ingredient could cause under-medication.
Different factors, such as light radiation, temperature, oxygen, humidity, pH sensitivity in solutions, may influence stability and may determine shelf-life and storage conditions.
Bioavailability is also a parameter to consider in drug delivery design of pharmaceutically acceptable formulations. Bioavailability is concerned with the quantity and rate at which the intact form of a particular drug appears in the systemic circulation following administration of the drug. The bioavailability exhibited by a drug is thus of relevance in determining whether a therapeutically effective concentration is achieved at the site(s) of action of the drug.
Physico-chemical factors and the pharmaco-technical formulation can have repercussions in the bioavailability of the drug. As such, several properties of the drug such as dissociation constant, dissolution rate, solubility, polymorphic form, particle size, are to be considered when improving the bioavailability.
It is also relevant to establish that the selected pharmaceutical formulation is capable of manufacture, more suitably, of large-scale manufacture.
In view of the various and many technical requirements, and its influencing parameters, it is not obvious to foresee which pharmaceutical formulations will be acceptable. As such, it was unexpectedly found that certain modifications of the solid state of compound of formula (X) positively influenced its applicability in pharmaceutical formulations.
SUMMARY OF THE INVENTION
Present invention concerns pseudopolymorphic forms of compound of formula (X) for the preparation of pharmaceutical formulations. Such pseudopolymorphic forms contribute to pharmaceutical formulations in improved stability and bioavailability. They can be manufactured in sufficient high purity to be acceptable for pharmaceutical use, more particularly in the manufacture of a medicament for inhibiting HIV protease activity in mammals.
In a first aspect, the present invention provides pseudopolymorphs of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate.
Pseudopolymorphs provided include alcohol solvates, more in particular, C1-C4 alcohol solvates; hydrate solvates; alkane solvates, more in particular, C1-C4 chloroalkane solvates; ketone solvates, more in particular, C1-C5 ketone solvates; ether solvates, more in particular, C1-C4 ether solvates; cycloether solvates; ester solvates, more in particular, C1-C5 ester solvates; and sulfonic solvates, more in particular, C1-4 sulfonic solvates, of the compound of formula (X). Preferred pseudopolymorphs are pharmaceutically acceptable solvates, such as hydrate and ethanolate.
Particular pseudopolymorphs are Form A (ethanolate), Form B (hydrate), Form C (methanolate), Form D (acetonate), Form E (dichloromethanate), Form F (ethylacetate solvate), Form G (1-methoxy-2-propanolate), Form H (anisolate), Form I (tetrahydrofuranate), Form J (isopropanolate) of compound of formula (X). Another particular pseudopolymorph is Form K (mesylate) of compound of formula (X).
In a second aspect, present invention relates to processes for preparing pseudopolymorphs. Pseudopolymorphs of compound of formula (X) are prepared by combining compound of formula (X) with an organic solvent, water, or mixtures of water and water miscible organic solvents, and applying any suitable technique to induce crystallization, to obtain the desired pseudopolymorphs.
In a third aspect, the invention relates to the use of the present pseudopolymorphs, in the manufacture of pharmaceutical formulations for inhibiting HIV protease activity in mammals. In relation to the therapeutic field, a preferred embodiment of this invention relates to the use of pharmaceutically acceptable pseudopolymorphic forms of compound of formula (X) for the treatment of an HIV viral disease in a mammal in need thereof, which method comprises administering to said mammal an effective amount of a pharmaceutically acceptable pseudopolymorphic form of compound of formula (X).
The following drawings provide additional information on the characteristics of the pseudopolymorphs according to present invention.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, FIG. 2 and FIG. 3 are the powder X-ray diffraction patterns of the Form A (1:1).
FIG. 4 depicts Form A (1:1) in three dimensions with the atoms identified.
FIG. 5 is a comparison of the Raman spectra of Forms A, B, D, E, F, H, (1:1) and the amorphous form at the carbonyl stretching region of 1800-100 cm−1 and the region 3300-2000 cm−1.
FIG. 6 is a comparison of the expanded Raman spectra of Forms A, B, D, E, F, H, (1:1) and the amorphous form at the carbonyl stretching region of 600-0 cm−1.
FIG. 7 is a comparison of the expanded Raman spectra of Forms A, B, D, E, F, H, (1:1) and the amorphous form at the carbonyl stretching region of 1400-800 cm−1.
In FIGS. 5, 6, and 7, P1 corresponds to Form A, P18 corresponds to Form B, P19 corresponds to amorphous form, P25 corresponds to Form E, P27 corresponds to Form F, P50 corresponds to Form D, P68 corresponds to Form H, P69 corresponds to Form C, P72 corresponds to Form I, and P81 corresponds to Form G.
FIG. 8 is the Differential Scanning Calorimetric (DSC) thermograph of Form A (1:1).
FIG. 9 is the Infrared (IR) spectrum that reflects the vibrational modes of the molecular structure of Form A as a crystalline product
FIG. 10 is the IR spectrum that reflects the vibrational modes of the molecular structure of Form B as a crystalline product
FIG. 11: IR spectrum of forms A, B, and amorphous form, at spectral range 4000 to 400 cm−1.
FIG. 12: IR spectrum of forms A, B, and amorphous form, at spectral range 3750 to 2650 cm−1.
FIG. 13: IR spectrum of forms A, B, and amorphous form, at spectral range 1760 to 1580 cm−.
FIG. 14: IR spectrum of forms A, B, and amorphous form, at spectral range 980 to 720 cm−1.
In FIGS. 11, 12, 13 and 14, curve A corresponds to Form A, curve B corresponds to Form B, and curve C corresponds to the amorphous form.
FIG. 15: DSC Thermograph curves of Form A (curve D), Form A after Adsorption/Desorption (ADS/DES) (curve E), and Form A after ADS/DES hydratation tests (curve F)
FIG. 16: Thermogravimetric (TG) curves of Form A (curve D), Form A after ADS/DES (curve E), and Form A after ADS/DES hydratation tests (curve F)
FIG. 17: TG curve of Form A at 25° C. under dry nitrogen atmosphere in function of time
FIG. 18: ADS/DES curves of Form A.
FIG. 19: ADS/DES curves of the hydratation test of Form A
FIG. 20: ADS/DES curves of Form B
FIG. 21: IR spectrum of Form K
FIG. 22: Raman spectrum of Form K
FIG. 23: DSC curve of Form K
FIG. 24: TG curve of Form K
FIG. 25: ADS/DES isotherm of Form K, batch 1
FIG. 26: ADS/DES isotherm of Form K, batch 2



DETAILED DESCRIPTION
The term “polymorphism” refers to the capacity of a chemical structure to occur in different forms and is known to occur in many organic compounds including drugs. As such, “polymorphic forms” or “polymorphs” include drug substances that appear in amorphous form, in crystalline form, in anhydrous form, at various degrees of hydration or solvation, with entrapped solvent molecules, as well as substances varying in crystal hardness, shape and size. The different polymorphs vary in physical properties such as solubility, dissolution, solid-state stability as well as processing behaviour in terms of powder flow and compaction during tabletting.
The term “amorphous form” is defined as a form in which a three-dimensional long-range order does not exist. In the amorphous form the position of the molecules relative to one another are essentially random, i.e. without regular arrangement of the molecules on a lattice structure.
The term “crystalline” is defined as a form in which the position of the molecules relative to one another is organised according to a three-dimensional lattice structure.
The term “anhydrous form” refers to a particular form essentially free of water. “Hydration” refers to the process of adding water molecules to a substance that occurs in a particular form and “hydrates” are substances that are formed by adding water molecules. “Solvating” refers to the process of incorporating molecules of a solvent into a substance occurring in a crystalline form. Therefore, the term “solvate” is defined as a crystal form that contains either stoichiometric or non-stoichiometric amounts of solvent. Since water is a solvent, solvates also include hydrates. The term. “pseudopolymorph” is applied to polymorphic crystalline forms that have solvent molecules incorporated in their lattice structures. The term pseudopolymorphism is used frequently to designate solvates (Byrn, Pfeiffer, Stowell, (1999) Solid-state Chemistry of Drugs, 2nd Ed., published by SSCI, Inc).
The present invention provides pseudopolymorphs of (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate.
In one embodiment pseudopolymorphs are alcohol solvates, more in particular, C1-C4 alcohol solvates; hydrate solvates; alkane solvates, more in particular, C1-C4 chloroalkane solvates; ketone solvates, more in particular, C1-C5 ketone solvates; ether solvates, more in particular C1-C4 ether solvates; cycloether solvates; ester solvates, more in particular C1-C5 ester solvates; or sulfonic solvates, more in particular, C1-C4 sulfonic solvates, of the compound of formula (X). The term “C1-C4 alcohol” defines straight and/or branched chained saturated and unsaturated hydrocarbons having from 1 to 4 carbon atoms substituted with at least a hydroxyl group, and optionally substituted with an alkyloxy group, such as, for example, methanol, ethanol, isopropanol, butanol, 1-methoxy-2-propanol and the like. The term “C1-C4 chloroalkane” defines straight and/or branched chained saturated and unsaturated hydrocarbons having from 1 to 4 carbon atoms substituted with at least one chloro atom, such as, for example, dichloromethane. The term “C1-C5 ketone” defines solvents of the general formula R′—C(═O)—R wherein R and R′ can be the same or different and are methyl or ethyl, such as, acetone and the like. The term “C1-C4 ether” defines solvents of the general formula R′—O—R wherein R and R′ can be the same or different and are a phenyl group, methyl or ethyl, such as, anisole and the like. The term “cycloether” defines a 4- to 6-membered monocyclic hydrocarbons containing one or two oxygen ring atoms, such as tetrahydrofuran and the like. The term “C1-C5 ester” defines solvents of the general formula R′—O—C(═O)—R wherein R and R′ can be the same or different and are methyl or ethyl, such as ethylacetate and the like. The term “C1-C4 sulfonic solvent” defines solvents of the general formula R—SO3H wherein R can be a straight or branched chained saturated hydrocarbon having from 1 to 4 carbon atoms, such as mesylate, ethanesulfonate, butanesulfonate, 2-methyl-1-propanesulfonate, and the like.
Pseudopolymorphs of the present invention, which are pharmaceutically acceptable, for instance hydrates, alcohol solvates, such as, ethanolate, are preferred forms.
Several pseudopolymorphs are exemplified in this application and include Form A (ethanolate), Form B (hydrate), Form C (methanolate), Form D (acetonate), Form E (dichloromethanate), Form F (ethylacetate solvate), Form G (1-methoxy-2-propanolate), Form H (anisolate), Form I (tetrahydrofuranate), Form J (isopropanolate), or Form K (mesylate) of compound of formula (X).
Solvates can occur in different ratios of solvation. Solvent content of the crystal may vary in different ratios depending on the conditions applied. Solvate crystal forms of compound of formula (X) may comprise up to 5 molecules of solvent per molecule of compound of formula (X), appearing in different solvated states including, amongst others, hemisolvate, monosolvate, disolvate, trisolvate crystals, intermediate solvates crystals, and mixtures thereof. Conveniently, the ratio of compound of formula (X) to the solvent may range between (5:1) and (1:5). In particular, the ratio may range from about 0.2 to about 3 molecules of solvent per 1 molecule of compound of formula (X), more in particular, the ratio may range from about 1 to about 2 molecules of solvent per 1 molecule of compound of formula (X), preferably the ratio is 1 molecule of solvent per 1 molecule of compound of formula (X).
Solvates may also occur at different levels of hydration. As such, solvate crystal forms of compound of formula (X) may in addition comprise under certain circumstances, water molecules partially or fully in the crystal structures. Consequently, the term “Form A” will be used herein to refer to the ethanolate forms of compound of formula (X) comprising up to 5 molecules of solvent per 1 molecule of compound of formula (X), intermediate solvates crystals, and the mixtures thereof; and optionally comprising additional water molecules, partially or fully in the crystal structures. The same applies for Form B through Form K. In case a particular “Form A” needs to be denoted, the ratio of solvation will follow the “Form A”, for instance, one molecule of ethanol per one molecule of compound (X) is denoted as Form A (1:1).
The X-ray powder diffraction is a technique to characterise polymorphic forms including pseudopolymorphs of compound of formula (X) and to differentiate solvate crystal forms from other crystal and non-crystal forms of compound of formula (X).
As such, X-ray powder diffraction spectra were collected on a Phillips PW 1050/80 powder diffractometer, model Bragg-Brentano. Powders of Form A (1:1), around 200 mg each sample, were packed in 0.5 mm glass capillary tubes and were analysed according to a standard method in the art. The X-ray generator was operated at 45 Kv and 32 mA, using the copper Ka line as the radiation source. There was no rotation of the sample along the chi axis and data was collected between 4 and 60° 2-theta step size. Form A (1:1) has the characteristic two-theta angle positions of peaks as shown in FIGS. 1, 2 and 3 at: 7.04°±0.50, 9.24°±0.5°, 9.96°±0.5°, 10.66°±0.5°, 11.30°±0.5°, 12.82°±0.5°, 13.80°±0.5°, 14.56°±0.5°, 16.66°±0.5°, 17.30°═0.5°, 18.28°±0.5°, 19.10°±0.5°, 20.00°+0.5°, 20.50°±0.5°, 21.22°±0.5°, 22.68°±0.5°, 23.08°±0.5°, 23.66°±0.5°, 25.08°±0.5°, 25.58°±0.5°, 26.28°±0.5°, 27.18°±0.5°, 28.22°±0.5°, 30.20°±0.5°, 31.34°±0.5°, 32.68°±0.5°, 33.82°±0.5°, 39.18°±0.5°, 41.20°±0.5°, 42.06°±0.5°, and 48.74°±0.5°.
In another set of analytical experiments, X-ray single diffraction was applied to Form A (1:1), which resulted in the following crystal configuration, listed in the table below.







TABLE 1







Crystal Data










Crystal shape
Prism



Crystal dimensions
0.56 × 0.38 × 0.24 mm



Crystal color
Colorless



Space Group
P 21 21 21 orthorhombic



Temperature
293 K



Cell constants
a = 9.9882(6) Å




b = 16.1697(8) Å




c = 19.0284(9) Å




alpha (α) = 90°




beta (β) = 90°




gamma (γ) = 90°



Volume
3158.7(3) Å3



Molecules/unit cell (Z)
4



Density, in Mg/m3
1.248



μ (linear absorption coefficient)
1.340 mm−1



F(000)
1272







Intensity Measurements










Diffractometer
Siemens P4



Radiation
Cu Kα (λ = 1.54184 {acute over (Å)})



Temperature
ambient



2θmax
138.14°



Correction
Empirical via Ψ-scans



Number of Reflections Measured
Total: 3912







Structure Solution and Refinement










Number of Observations
3467 [F2 > 2 σ(F2)]



Residual (R)
  0.0446










The resulting three-dimensional structure of Form A (1:1) is depicted in FIG. 4.
Table 2 shows the atomic coordinates (x 104) and equivalent isotropic displacement parameters ({acute over (Å)}2×103) for Form A (1:1). Atoms are numbered as exhibited in FIG. 4. The x, y and z fractional coordinates indicate the position of atoms relative to the origin of the unit cell. U(eq) is defined as one third of the trace of the orthogonalized Uij tensor.
















x
y
z
U(eq)






















O1
  7778(3)
  2944(2)
9946(1)
 70(1)



C2
  7171(4)
  3513(2)
9487(2)
 64(1)



C3
  6831(3)
  3046(2)
8823(2)
 52(1)



C3A
  7953(3)
  2411(2)
8793(2)
 55(1)



C4
  7527(4)
  1533(2)
8708(2)
 65(1)



C5
  7425(5)
  1241(2)
9457(2)
 70(1)



O6
  8501(3)
  1642(2)
9809(1)
 76(1)



C6A
  8582(4)
  2416(2)
9534(2)
 62(1)



O7
  5533(2)
  2702(1)
8945(1)
 51(1)



O8
  5168(2)
  2636(1)
7768(1)
 53(1)



C9
  4791(3)
  2534(1)
8368(1)
 42(1)



N10
  3590(2)
  2256(1)
8562(1)
 43(1)



C11
  2638(3)
  1916(2)
8068(2)
 44(1)



C12
  2223(3)
  1071(2)
8310(2)
 58(1)



C13
  3381(3)
   501(2)
8387(2)
 56(1)



C14
  3937(4)
   340(2)
9038(2)
 67(1)



C15
  4989(5)
 −200(2)
9111(3)
 80(1)



C16
  5494(5)
 −581(3)
8530(3)
 96(2)



C17
  4975(6)
 −413(3)
7881(3)
 98(2)



C18
  3926(5)
   126(2)
7810(2)
 78(1)



C19
  1423(3)
  2464(2)
7976(2)
 45(1)



O20
   494(2)
  2112(1)
7502(1)
 61(1)



C21
  1829(3)
  3307(2)
7740(2)
 48(1)



N22
   699(3)
  3880(1)
7721(1)
 49(1)



C23
   521(4)
  4312(2)
7048(2)
 58(1)



C24
 −61(4)
  3785(2)
6473(2)
 67(1)



C25
−1453(5)
  3497(3)
6654(2)
 86(2)



C26
 −47(7)
  4247(3)
5779(2)
102(2)



S27
   510(1)
  4414(1)
8440(1)
 50(1)



O28
   572(3)
  3860(1)
9015(1)
 61(1)



O29
 −693(2)
  4873(1)
8345(1)
 65(1)



C30
  1854(3)
  5080(2)
8509(2)
 50(1)



C31
  1803(3)
  5825(2)
8159(2)
 54(1)



C32
  2871(4)
  6341(2)
8195(2)
 56(1)



C33
  4033(4)
  6133(2)
8564(2)
 55(1)



C34
  4063(4)
  5385(2)
8909(2)
 59(1)



C35
  2998(4)
  4869(2)
8883(2)
 56(1)



N36
  5076(3)
  6667(2)
8596(2)
 72(1)



C37
  1920(10)
  2231(7)
5258(4)
232(6)



C38
  1310(10)
  1590(6)
5564(4)
191(5)



O39
  1768(4)
  1393(2)
6249(2)
 94(1)










Table 3 shows the anisotropic displacement parameters (Å2×103) for Form A (1:1). The anisotropic displacement factor exponent takes the formula:

−2π2[h2a*2U11+ . . . +2hka*b*U12]



















U11
U22
U33
U23
U13
U12






















O1
65(2)
89(2)
55(1)
−4(1)
−12(1)
−3(1)


C2
53(2)
68(2)
71(2)
−7(2)
−8(2)
−11(2)


C3
38(2)
63(2)
55(2)
4(1)
−2(1)
−12(1)


C3A
37(2)
78(2)
49(1)
9(1)
1(1)
−3(2)


C4
61(2)
74(2)
61(2)
−4(2)
−6(2)
10(2)


C5
72(3)
67(2)
71(2)
8(2)
−11(2)
−7(2)


O6
78(2)
80(2)
70(1)
16(1)
−21(1)
−8(2)


C6A
47(2)
80(2)
59(2)
5(2)
−6(2)
−7(2)


O7
34(1)
69(1)
50(1)
0(1)
−1(1)
−9(1)


O8
42(1)
68(1)
50(1)
3(1)
2(1)
−12(1)


C9
35(2)
41(1)
49(1)
1(1)
−3(1)
3(1)


N10
31(1)
50(1)
49(1)
−1(1)
1(1)
−2(1)


C11
32(2)
41(1)
57(1)
−4(1)
0(1)
−2(1)


C12
44(2)
42(1)
87(2)
2(1)
2(2)
−4(1)


C13
50(2)
39(1)
78(2)
0(1)
8(2)
0(1)


C14
64(2)
56(2)
80(2)
0(2)
5(2)
9(2)


C15
68(3)
72(2)
100(3)
18(2)
7(2)
12(2)


C16
77(3)
68(2)
143(4)
26(3)
34(3)
28(2)


C17
114(4) 
72(2)
109(3)
−6(2)
32(3)
38(3)


C18
89(3)
60(2)
85(2)
−4(2)
10(2)
10(2)


C19
30(2)
44(1)
61(1)
−3(1)
−5(1)
−5(1)


O20
44(1)
56(1)
83(1)
−6(1)
−18(1)
−6(1)


C21
36(2)
42(1)
64(2)
2(1)
−4(1)
−1(1)


N22
42(1)
47(1)
57(1)
1(1)
0(1)
3(1)


C23
59(2)
50(1)
64(2)
7(1)
−8(2)
1(2)


C24
79(3)
59(2)
62(2)
1(1)
−11(2)
6(2)


C25
75(3)
83(2)
101(3)
6(2)
−30(3)
−5(2)


C26
143(5) 
99(3)
65(2)
14(2)
−15(3)
−6(3)


S27
44(1)
47(1)
61(1)
2(1)
2(1)
1(1)


O28
64(2)
58(1)
61(1)
9(1)
3(1)
−7(1)


O29
46(1)
58(1)
92(2)
−4(1)
6(1)
10(1)


C30
50(2)
46(1)
54(1)
2(1)
1(1)
1(1)


C31
50(2)
48(1)
64(2)
6(1)
−4(2)
6(1)


C32
59(2)
45(1)
65(2)
4(1)
2(2)
1(1)


C33
57(2)
55(2)
52(1)
−4(1)
1(1)
−3(1)


C34
56(2)
63(2)
59(2)
6(1)
−13(2)
−3(2)


C35
63(2)
52(1)
53(1)
5(1)
−8(2)
−2(2)


N36
67(2)
70(2)
80(2)
4(2)
−5(2)
−19(2)


C37
290(10)
260(10)
145(7)
68(7)
67(8)
120(10)


C38
280(10)
187(7) 
104(4)
1(5)
−53(6)
−80(10)


O39
99(2)
91(2)
93(2)
1(2)
−13(2)
−28(2)









Raman spectroscopy has been widely used to elucidate molecular structures, crystallinity and polymorphism. The low-frequency Raman modes are particularly useful in distinguishing different molecular packings in crystal. As such, Raman spectra were recorded on a Bruker FT-Raman RFS 100 spectrometer equipped with a photomultiplier tube and optical multichannel detectors. Samples placed in quartz capillary tubes were excited by an argon ion laser. The laser power at the samples was adjusted to about 100 mW and the spectral resolution was about 2 cm−1. It was found that Forms A, B, D, E, F, and H, (1:1) and the amorphous form have the Raman spectra which appear in FIGS. 5, 6, and 7.
In addition, Forms A and B were characterized using a μTATR (Micro-Attenuated Total Reflectance) accessory (Harrick Split-Pea with Si crystal). The infrared spectra were obtained with a Nicolet Magna 560 FTIR spectrophotometer, a Ge on KBr beamsplitter, and a DTGS with KBr windows detector. Spectra were measured at 1 cm−1 resolution and 32 scans each, in a wavelength range of from 4000 to 400 cm−1, and application of baseline correction. The wavenumbers for Form A obtained are exhibited in the following Table 4.







TABLE 4





Wavenumbers (cm−1) and relative intensities of absorption bands (1)















3454w, 3429w, 3354w, 3301w, 3255w, 3089w, 3060w, 3041w, 3028w


2964w, 2905w, 2875w, 2856w, 2722vw, 2684vw, 2644vw, 2603vw,


2234vw


1704s, 1646w, 1595s, 1550m, 1503m, 1466w, 1453w, 1444w, 1413w


1373w, 1367w, 1340w, 1324m, 1314m, 1306m, 1290w, 1266m, 1244m,


1229m


1187w, 1146s, 1124m, 1104m, 1090m, 1076m, 1052m, 1042s, 1038m,


1024s


987s, 971m, 944m, 909w, 890w, 876w, 841m, 792w, 768s, 742s, 732w,


697m, 674s, 645w, 630m


598w, 593w, 574m, 564s, 553vs, 538m, 533m, 531m, 526m, 508m,


501m, 491m, 471m, 458w, 445w, 442w, 436w, 428w, 418w





vs = very strong, s = strong, m = medium, w = weak, vw = very weak, br = broad






The IR spectrum in FIG. 9 reflects the vibrational modes of the molecular structure as a crystalline product.
The wavenumbers obtained for Form B are exhibited in the following Table 5.







TABLE 5





Wavenumbers (cm−1) and relative intensities of absorption bands (1)















3614w, 3361m, 3291m, 3088w, 3061w, 3043w, 3028w


2967w, 2905w, 2872w, 2222vw


Wavenumbers (cm−1) and relative intensities of absorption bands (1)


1703s, 1631w, 1595s, 1553m, 1502w, 1467w, 1453w, 1444w, 1436w


1388vw, 1374vw, 1366w, 1355vw, 1340w, 1308m, 1291w, 1267m,


1245m


1187w, 1148s, 1125m, 1105m, 1091m, 1077m, 1052m, 1044m, 1025s


990m, 972w, 944m, 912w, 891w, 876vw, 862w, 843w, 836w, 792w,


769m, 757w, 743m, 717w, 699m, 672m


598w, 591w, 585w, 576m, 566m, 553vs, 536m, 509w, 502m, 484w,


471w, 432vw, 425w, 418w





(1) vs = very strong, s = strong, m = medium, w = weak, vw = very weak, br = broad






The IR spectrum in FIG. 10 reflects the vibrational modes of the molecular structure of Form B as a crystalline product.
Following the same analytical IR method, Form B and the amorphous form were also characterised and compared with Form A, as shown in FIGS. 11 to 14. IR spectra of the different physical forms showed distinct spectral differences, most relevant are those in Table 6:







TABLE 6







Wavenumbers (cm−1) and relative intensities of absorption bands (1)









Form A
Form B
Amorphous form





3454m, 3429m, 3353m,
3615m, 3356m, 3291m,
3462m, 3362m, 3249m,


3255m, 3089w, 3060m,
3089m, 3061m, 3043w,
3062m, 3026m


3041w, 3028w
3027w


2963m, 2905m, 2869m,
2966m, 2905m, 2873m
2959m, 2871m


2856m


1704s, 1646m, 1596s,
1703s, 1630m, 1595s,
1704s, 1628s, 1596s, 1525s,


1549s, 1503s
1552s, 1502m
1502s


1306s, 1266s, 1244s
1308s, 1267s, 1245s
1312s, 1259s


1146s, 1104s, 1090s, 1076s,
1148s, 1105s, 1090s, 1077s,
1143s, 1090s, 1014s


1052s, 1042s, 1038s, 1023s
1052s, 1044s, 1024s


987s, 971s, 954s, 945s,
989s, 972s, 944s, 925m,
960s, 953s, 950s, 944s, 937s,


912m, 909m, 891s, 876s,
915m, 912s, 891s, 862s,
922s, 832s


841s, 827s
843s


792m, 768s, 742s, 697s,
792s, 769s, 744s, 699s, 672s
750br, 702s, 672s


674s





(1) s = strong, m = medium, w = weak, vw = very weak, br = broad






The physical Forms A, B, and amorphous form are identified through spectral interpretation, focused on absorption bands specific for each form. Unique and specific spectral differences between forms are noticed in 3 spectral ranges: from 3750 to 2650 cm−1 (range 1), from 1760 to 1580 cm−1 (range 2) and from 980 to 720 cm−1 (range 3).
Range 1 (from 3750 to 2650 cm−1)
FIG. 11: Form A shows a double band with absorption maxima at 3454 cm−1 and 3429 cm−1. Form B shows a single absorption band at 3615 cm−1 and amorphous form shows a single absorption band at 3362 cm−1.
Range 2 (from 1760 to 1580 cm−1)
FIG. 12: Form A shows a single absorption band at 1646 cm−1, Form B shows a single absorption band at 1630 cm−1 and amorphous form shows a single absorption band at 1628 cm−1 with a clearly higher intensity compared to the Form B band. Additionally, amorphous form shows a less intense, broad band at 1704 cm−1 compared to both forms A and B bands at about 1704 cm−1.
Range 3 (from 980 to 720 cm−1)
FIG. 13: Form A shows a distinct set of 5 absorption bands at 911, 890, 876, 862 and 841 cm−1. Form B shows a similar set but the 876 cm−1 band is missing. Amorphous form shows a single broad band at about 750 cm−1, both forms A and B show two maxima at about 768 cm−1 and 743 cm−1.
Thermomicroscopy is another useful technique in the study of solid-state kinetics. The kinetics of nucleation processes from solutions or melts, including the analysis of the nucleation speed, can be quantified. The simplest and most widely used method is the melting point determination. As such, a Mettler model FP 82 controller with heating stage was used on a Leitz microscope. A few particles of Form A were placed on a glass slide and observed while heating at 10° C. per minute. The melting range for Form A (1:1) was found to be between 90° and 110° C.
On another means of characterization, the solubility of Form A (1:1) was also a matter subject to study. Its solubility in different solvents at approximate 23° C. was determined to be as follows:







TABLE 7







Approximate solubility for Form A (1:1), in mg/ml











Approximate solubility



Solvent
Form A (mg/ml)







Acetone
106-211



Dichloromethane
105-209



1-Methoxy-2-propanol
160-213



Ethylmethylketone
102-204



Ethylacetate
 71-107



Ethanol absolute
<3.4



Heptane
<3.4



Water
<3.5



Isopropylether
<3.4



Methacyanate
>200



Methanol
<3.4



2-Propanol
<3.4



Tetrahydrofurane
102-203



Toluene
<3.5










Further solubility investigations were performed in function of pH. As such, the aqueous solubilities of Form A (1:1) were measured in solvents with different pH. An excess of the solute was equilibrated with the solvent at 20° C. for at least 24 hours. After removing the undissolved compound, the concentration in solution was determined using UV spectrometry.







TABLE 8







Solubility for Form A (1:1) in function of pH











Solubility



Solvent
(mg/100 ml solution)







Water
 16 (pH 5.9)



Buffer pH 2 (citrate/HCl)
 18 (pH 2.0)



Buffer pH 3 (citrate/HCl)
 10 (pH 3.0)



Buffer pH 4 (citrate/HCl)
 9 (pH 4.0)



0.01 N HCl
 18 (pH 2.1)



0.1 N HCl
 83 (pH 1.1)



1.0 N HCl
620 (pH 0.2)










Solubility of Form A (1:1) in function of HPβCD (hydroxypropyl-β-cyclodextrin) was measured. An excess of product was equilibrated with the solvent during 2 days at 20° C. After removing the undissolved compound, the concentration in solution was determined using UV spectrometry.







TABLE 9







Solubility for Form A (1:1) in function of HPβCD











Solubility in



solvent
mg/ml solution







Water
0.16 (pH = 5.9)



 5% HPβCD in water
 2.4 (pH = 5.8)



10% HPβCD in water
 6.5 (pH = 6.0)



20% HPβCD in water
  17 (pH = 6.0)



40% HPβCD in water
  40 (pH = 5.9)










In a second aspect, the present invention relates to processes for preparing pseudopolymorphs. Pseudopolymorphs of compound of formula (X) are prepared by combining compound of formula (X) with an organic solvent, or water, or mixtures of water and water miscible organic solvents, applying any suitable technique to induce crystallization, and isolating the desired pseudopolymorphs.
By techniques for inducing crystallization are to be understood those processes for the production of crystals, which include amongst others, dissolving or dispersing compound of formula (X) in a solvent medium, bringing the solution or dispersion of compound of formula (X) and the solvent(s) to a desired concentration, bringing the said solution or dispersion to a desired temperature, effecting any suitable pressure, removing and/or separating any undesired material or impurities, drying the formed crystals to obtain the pseudopolymorphs in a solid state, if such state is desired.
Bringing the solution or dispersion of compound of formula (X) and solvents to a desired concentration does not necessarily imply an increase in the concentration of compound of formula (X). In certain cases, a decrease or no change in concentration could be preferable. By bringing the said solution or dispersion to a desired temperature, one will understand the acts of heating, cooling or leaving at ambient temperature.
The techniques used for obtaining a desired concentration are those common in the art, for instance, evaporation by atmospheric distillation, vacuum distillation, fractioned distillation, azeotropic distillation, film evaporation, other techniques well known in the art and combinations thereof. An optional process for obtaining a desired concentration could as well involve the saturation of the solution of compound of formula (X) and solvent, for example, by adding a sufficient volume of a non-solvent to the solution to reach the saturation point. Other suitable techniques for saturating the solution include, by way of example, the introduction of additional compound of formula (X) to the solution and/or evaporation of a portion of the solvent from the solution. As referred to herein, saturated solution encompasses solutions at their saturation points or exceeding their saturation points, i.e. supersaturated.
Removing and/or separating any undesired material or impurities may be performed by purification, filtering, washing, precipitation or similar techniques. Separation, for example, can be conducted by known solid-liquid separation techniques. Filtering procedures known to those skilled in the art can as well be used in the present process. The filtrations can be performed, amongst other methods, by centrifugation, or using Buchner style filter, Rosenmund filter or plates, or frame press. Preferably, in-line filtration or safety filtration may be advantageously intercalated in the processes disclosed above, in order to increase the purity of the resulting pseudopolymorphic form. Additionally, filtering agents such as silica gel, Arbocel®, dicalite diatomite, or the like, may also be employed to separate impurities from the crystals of interest.
Crystals obtained may be also dried, and such drying process may optionally be used in the different crystallization passages, if more than one crystallization passage is applied. Drying procedures include all techniques known to those skilled in the art, such as heating, applying vacuum, circulating air or gas, adding a desiccant, freeze-drying, spray-drying, evaporating, or the like, or any combination thereof.
Processes for crystallization of pseudopolymorphs of compound of formula (X) embrace multiple combinations of techniques and variations thereof. As such, and by way of example, crystallization of pseudopolymorphs of compound of formula (X) may be executed by dissolving or dispersing compound of formula (X) at a suitable temperature in the solvent whereby portion of the said solvent evaporates increasing the concentration of the compound of formula (X) in the said solution or dispersion, cooling the said mixture, and optionally washing and/or filtering and drying resulting solvate crystals of compound of formula (X). Optionally, pseudopolymorphs of compound of formula (X) may be prepared by dissolving or dispersing compound of formula (X) in a solvent medium, cooling said solution or dispersion and subsequently filtering and drying the obtained pseudopolymorph. Another example of preparation of solvates of compound of formula (X) could be by saturating compound of formula (X) in the solvent medium, and optionally filtering, washing and drying obtained crystals.
Crystal formation may as well involve more than one crystallization process. In certain cases, one, two or more extra crystallization steps may be advantageously performed for different reasons, such as, to increase the quality of the resulting solvate. For instance, pseudopolymorphs of the present invention could also be prepared by adding a solvent to an initial starting base material of compound of formula (X), stirring the solution at a fixed temperature until the substances would be fully solved, concentrating the solution by vacuum distillation, and cooling. A first crystallization would take place and the formed crystals would be newly washed with a solvent, and followed by dissolution of compound of formula (X) with the solvent to form the desired pseudopolymorph. Recrystallization of the reaction mixture would occur, followed by a cooling step from reflux. The formed pseudopolymorph would optionally be filtered and allowed to dry.
By dissolving or dispersing compound of formula (X) in the organic solvent, water or a mixture of water and water miscible organic solvents, one may obtain different degrees of dispersion, such as suspensions, emulsions, slurries or mixtures; or preferably obtain homogeneous one-phase solutions.
Optionally, the solvent medium may contain additives, for example one or more dispersing agents, surfactants or other additives, or mixtures thereof of the type normally used in the preparation of crystalline suspensions and which are well documented in the literature. The additives may be advantageously used in modifying the shape of crystal by increasing the leniency and decreasing the surface area.
The solvent medium containing the solution may optionally be stirred for a certain period of time, or vigorously agitated using, for example, a high shear mixer or homogeniser or a combination of these, to generate the desired droplet size for the organic compound.
Examples of organic solvents useful for the present invention include C1-C4 alcohols such as methanol, ethanol, isopropanol, butanol, 1-methoxy-2-propanol, and the like; C1-C4 chloroalkanes such as dichloromethane; C1-C4 ketones such as acetone; C1-C4 ethers such as anisole, and the like; cycloethers such as tetrahydrofuran; C1-C4 esters such as ethylacetate; C1-C4 sulfonates such as mesylate, ethanesulfonate, butanesulfonate, 2-methyl-1-propanesulfonate; and the like.
Examples of mixtures of water and water miscible organic solvents include, mixtures of water with all organic solvents listed above provided they are miscible in water, e.g. ethanol/water, for instance in a 50/50 ratio.
Preferred solvents are those pharmaceutically acceptable solvents. However, pharmaceutically non-acceptable solvents may also find their use in the preparation of pharmaceutically acceptable pseudopolymorphs.
In a preferred method, the solvent is a pharmaceutically acceptable solvent since it results in a pharmaceutically acceptable pseudopolymorph. In a more preferred method, the solvent is ethanol.
In a particular embodiment, pharmaceutically acceptable pseudopolymorphs of compound of formula (X) can be prepared starting from pseudopolymorphic forms of compound of formula (X), which may not be necessarily pharmaceutically acceptable. For instance, Form A may be prepared starting from Form J. Pseudopolymorphs may also be prepared starting from the amorphous form.
In the mixtures of water and water miscible organic solvents, the amount of water can vary from about 5% by volume to about 95% by volume, preferably from about 25% to about 75% by volume, more preferably from about 40% to about 60% by volume.
It should also be noted that the quality of selected organic solvent (absolute, denaturated, or other) also influences the resulting quality of the pseudopolymorph.
Control of precipitation temperature and seeding may be additionally used to improve the reproducibility of the crystallization process, the particle size distribution and form of the product. As such, the crystallization can be effected without seeding with crystals of the compound of the formula (X) or preferably in the presence of crystals of the compound of the formula (X), which are introduced into the solution by seeding. Seeding can also be effected several times at various temperatures. The amount of the seed material depends on the amount of the solution and can readily be determined by a person skilled in the art.
The time for crystallization in each crystallization step will depend on the conditions applied, the techniques employed and/or solvents used.
Breaking up the large particles or aggregates of particles after crystal conversion may additionally be performed in order to obtain a desired and homogeneous particle size. Accordingly, the solvate crystal forms of compound of formula (X) are optionally milled after undergoing conversion. Milling or grinding refers to physically breaking up the large particles or aggregates of particles using methods and apparatus well known in the art for particle size reduction of powders. Resulting particle sizes may range from millimeters to nanometers, yielding i.e. nanocrystals, microcrystals.
The yield of the preparation process of the pseudopolymorphs of compound of formula (X) may be 10% or more, a more preferred yield would vary from 40% to 100%. Interestingly, the yield varies between 70% and 100%.
Suitably, pseudopolymorphs of the present invention have a purity greater than 90 percent. More suitably, the present pseudopolymorphs have a purity greater than 95 percent. Even more suitably, the present pseudopolymorphs have a purity greater than 99 percent.
In a third aspect, the present invention relates to a pharmaceutical formulation comprising a therapeutically effective amount of a pseudopolymorph of compound of formula (X), and a pharmaceutically acceptable carrier or diluent thereof.
In one embodiment, present invention relates to the use of pharmaceutically acceptable pseudopolymorphic forms of compound of formula (X), preferably Form A, in the manufacture of a medicament for treating diseases caused by retroviruses, such as HIV infections, for example, Acquired Immune Deficiency Syndrome (AIDS) and AIDS-related complex (ARC).
In another embodiment, present invention provides a method for the treatment of a retroviral infection, for example an HIV infection, in a mammal such as a human, which comprises administering to the mammal in need thereof an effective antiretroviral amount of a pharmaceutically acceptable pseudopolymorphic form of compound of formula (X), preferably Form A.
Present invention also relates to a method in which the treatment of a HIV viral infection comprises the reduction of HIV load. Present invention also relates to a method in which the treatment of said HIV viral infection comprises the increase of CD4+ cell count. Present invention relates as well to a method in which the treatment of said HIV viral infection comprises inhibiting HIV protease activity in a mammal.
Pharmaceutically acceptable pseudopolymorphic forms of compound of formula (X), preferably Form A, also referred to herein as the active pharmaceutical ingredients, may be administered by any route appropriate to the condition to be treated, preferably orally. It will be appreciated however, that the preferred route may vary with, for example, the condition of the recipient.
For each of the above-indicated utilities and indications the amount required of the active ingredient will depend upon a number of factors including the severity of the condition to be treated and the identity of the recipient and will ultimately be at the discretion of the attendant physician or veterinarian. The desired dose preferably may be presented as one, two, three or four or more subdoses administered at appropriate intervals throughout the day.
For an oral administration form, pseudopolymorphs of the present invention are mixed with suitable additives, such as excipients, stabilizers or inert diluents, and brought by means of the customary methods into the suitable administration forms, such as tablets, coated tablets, hard capsules, aqueous, alcoholic, or oily solutions. Examples of suitable inert carriers are gum arabic, magnesia, magnesium carbonate, potassium phosphate, lactose, glucose, or starch, in particular, corn starch. In this case the preparation can be carried out both as dry and as moist granules. Suitable oily excipients or solvents are vegetable or animal oils, such as sunflower oil or cod liver oil. Suitable solvents for aqueous or alcoholic solutions are water, ethanol, sugar solutions, or mixtures thereof. Polyethylene glycols and polypropylene glycols are also useful as further auxiliaries for other administration forms.
For subcutaneous or intravenous administration, the pseudopolymorphs of compound of formula (X), if desired with the substances customary therefor such as solubilizers, emulsifiers or further auxiliaries, are brought into solution, suspension, or emulsion. The pseudopolymorphs of compound of formula (X) can also be lyophilized and the lyophilizates obtained used, for example, for the production of injection or infusion preparations. Suitable solvents are, for example, water, physiological saline solution or alcohols, e.g. ethanol, propanol, glycerol, in addition also sugar solutions such as glucose or mannitol solutions, or alternatively mixtures of the various solvents mentioned.
Suitable pharmaceutical formulations for administration in the form of aerosols or sprays are, for example, solutions, suspensions or emulsions of the pseudopolymorphs of compound of formula (X) in a pharmaceutically acceptable solvent, such as ethanol or water, or a mixture of such solvents. If required, the formulation can also additionally contain other pharmaceutical auxiliaries such as surfactants, emulsifiers and stabilizers as well as a propellant. Such a preparation customarily contains the active compound in a concentration from approximately 0.1 to 50%, in particular from approximately 0.3 to 3% by weight.
Pseudopolymorphs of the present invention may also be presented in a formulation comprising micrometer-, nanometer- or picometer-size particles of the pseudopolymorph of compound of formula (X), which formulation may contain other pharmaceutical agents and may optionally be converted to solid form.
It may be convenient to formulate the present pseudopolymorphs in the form of nanoparticles which have a surface modifier adsorbed on the surface thereof in an amount sufficient to maintain an effective average particle size of less than 1000 nm. Useful surface modifiers are believed to include those that physically adhere to the surface of the antiretroviral agent but do not chemically bind to the antiretroviral agent.
It may be further convenient to store the pseudopolymorphs of compound of formula (X) in packaging materials which are protective to mechanical, environmental, biological or chemical hazards, or degradation. Conditioning drug substances can be achieved by employing packaging materials impermeable to moisture, such as sealed vapour lock bags. Conditioning drug products, such as tablets, capsules, can be achieved by employing for instance, aluminium blisters.
It should be understood that in addition to the ingredients particularly mentioned above, formulations of this invention includes other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents or taste masking agents.
The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.
EXAMPLE 1
The industrial scale synthesis of Form A (1:1) was performed using the following steps. First a solution was prepared with isopropanol and (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate. The solution was concentrated by vacuum distillation at 70° C. and 200-500 mbar pressure and cooled from a T>35° to a T between 15°and 20° C. for about 10 hours. The crystals formed were newly washed with 13 liters isopropanol and filtered. A subsequent recrystallization from ethanol/water (90 liters/90 liters) was performed. This was followed by a new dissolution step, but with 60 liters ethanol instead. Recrystallization of the reaction mixture from ethanol occurred, followed by a cooling step from reflux to −15° C. approximately and during 10 hours. The ethanolate formed was filtered and let to dry at about 50° C. and about 7 mbar. The yield of this process was at least 75%.
EXAMPLE 2
In another example a mixture of Form D and Form B were prepared. Acetone was used as a solvent during the crystallisation process to form Form D. The crystallisation process then comprised the step of stirring the initial starting compound (10 g) in 70 ml acetone. The solution was subsequently refluxed until the compound was completely solved. 40 ml of water were added and the solution was subsequently cooled slowly until room temperature and stirred overnight. Formed crystals were filtered and dried in the vacuum oven at 50° C. 7.6 g of product resulted from the crystallization, being the yield of this process of about 75%.
EXAMPLE 3
In another example Form J crystals were prepared. Isopropanol was used as a solvent during the crystallisation process to form Form J. The crystallisation process then comprised the step of solving the initial starting material in the hot solvent. The solution was subsequently cooled until room temperature. Formed crystals were filtered and dried in the vacuum oven at 50° C. The crystals contained about 50 mol % isopropanol.
EXAMPLE 4
In this example, the mass losses for different pseudopolymorphs in thermogravimetric (TG) experiments were calculated. Thermogravimetry is a technique that measures the change in mass of a sample as it is heated, cooled or held at constant temperature. Approximately 2 to 5 mg of sample were placed on a pan and inserted into the TG furnace, model Netzsch Thermo-Microbalance TG 209 coupled to a Bruker FTIR Spectrometer vector 22. The samples were heated in a nitrogen atmosphere at a rate of 10° C./min, up to a final temperature of 250° C. The detection limit of residual solvents was in the order of 0.1% for distinct stepwise solvent loss over a narrow temperature range (few degrees Celsius).
The following TG data were obtained:
Form A: a weight loss of 4.2% was observed in the temperature range of 25-138° C. (ethanol+little water) and of 6.9% (ethanol+CO2) in the temperature range of 25-200° C. Ethanol loss rate was maximal at 120° C. CO2 loss was due to chemical degradation and was visible at around 190° C.
Form B: a weight loss of 3.4% was observed in the temperature range 25-78° C. (water) and of 5.1% in the temperature range 25-110° C. (ethanol+water for T>78° C.). From 110-200° C. further 1.1% weight was lost (ethanol).
Form C: a weight loss of 2.1% was observed in the temperature range 25-83° C. (water+methanol) and of 4.2% in the temperature range 25-105° C. (methanol for T>83° C., distinct step). From 105-200° C. further 2.1% weight was lost (methanol). No ethanol was observed in the gas phase.
Form D: a weight loss of 0.1% was observed in the temperature range 25-50° C., of 4.2% in the temperature range 25-108° C. (acetone+ethanol for T>50° C.), of 8.2% in the temperature range 25-157° C. (acetone+ethanol for T>108° C.) and of 10.5% in the temperature range 25-240° C. (acetone+ethanol for T>157° C.).
Form E: a weight loss of 0.2% was observed in the temperature range 25-75° C. (water), of 1.8% in the temperature range 25-108° C. (dichloromethane+ethanol for T>75° C.), of 6.8% in the temperature range 25-157° C. (dichloromethane+ethanol for T>108° C.) and of 8.8% in the temperature range 25-240° C. (dichloromethane+ethanol for T>157° C.).
Form F: a weight loss of 0.1% was observed in the temperature range 25-50° C. (probably water), of 1.7% in the temperature range 25-108° C. (ethylacetate+ethanol for T>50° C.), of 6.6% in the temperature range 25-157° C. (ethylacetate+ethanol for T>108° C.) and of 9% in the temperature range 25-240° C. (ethylacetate+ethanol for T>157° C.).
Form G: a weight loss of 0.0% was observed in the temperature range 25-50° C., of 3.7% in the temperature range 25-108° C. (1-methoxy-2-propanol+ethanol for T>50° C., distinct step), of 8% in the temperature range 25-157° C. (1-methoxy-2-propanol+ethanol for T>108° C.) and of 12.5% in the temperature range 25-240° C. (1-methoxy-2-propanol+ethanol for T>157° C.).
Form H: a weight loss of 0.8% was observed in the temperature range 25-100° C. (anisole+little ethanol) and of 8.8% in the temperature range 25-200° C. (anisole+ethanol for T>100° C.).
Form I: a weight loss of 0.3% was observed in the temperature range 25-89° C. (water) and of 11.0% in the temperature range 25-200° C. (tetrahydrofurane for T>89° C.). No ethanol was observed in the gas phase.
Table 10 shows approximate expected mass losses for different Forms in thermogravimetric (TG) experiments.

Mass loss in % (M+x.LM=100%)


















Hemisol-
Monosol-
Disol-
Trisol-


Pseudopolymorph
BP[° C.]
vate
vate
vate
vate




















Form D
56
5.0
9.6
17.5
24.1


Form H
152
9.0
16.5
28.3
37.2


Form E
40
7.2
13.4
23.7
31.8


Form G
119
7.6
14.1
24.8
33.1


Form F
76
7.4
13.9
24.3
32.6


Form A
78
4.0
7.8
14.4
20.2


Form B
100
1.6
3.2
6.2
9.0


Form C
65
2.8
5.5
10.5
14.9


Form I
66
6.2
11.6
20.8
28.3









In another set of thermogravimetric methods, Form A, Form A after Adsorption/Desorption, and Form A after Adsorption/Desorption hydratation tests, were all transferred into an aluminum sample pan. The TG curve was recorded on a TA Instrument Hi-Res TGA 2950 thermogravimeter at the following conditions:
















initial temperature:
room temperature



heating rate:
20° C./min



resolution factor:
4



final condition:
300° C. or <80[(w/w)%]










The TG curves of the samples are collected in FIG. 16.
Table 11 shows mass losses for the forms tested:










TABLE 11









TG (% weight change)












Form A
Up to 80° C.
>80° C.







Form A
0.3
7.1



Form A after ADS/DES
2.9
4.0



Form A after A/D hydratation test
5.4
0.5










The loss of weight at temperatures up to 80° C. is mainly due to the evaporation of solvent (water) present in the sample. The loss of weight at temperatures above 80° C. is mainly due to the evaporation of solvent (ethanolate) present in the sample.
A TG curve of form A at 25° C. under dry nitrogen atmosphere in function of time is collected in FIG. 17. The loss of weight at 25° C. after 10 hours was around 0.6%. This was due to the evaporation of solvent.
EXAMPLE 5
In another example, measurements of differential scanning calorimetry (DSC) were also performed. For such purpose, a Perkin Elmer DSC 204 thermal analysis system was used. From 2 to 5 mg sample of Form A were accurately weighed into a DSC pan. The experiments were performed in an open pan. The sample was equilibrated to approximately 30° C. and then heated at a rate of 10° C. per minute, up to a final temperature of 200° C. The DSC data was obtained following a standard method in the art. The Form A was characterized by differential scanning calorimetry (DSC) in which it showed a sharp endotherm in the range 80-119° C., showing a peak at about 105.6° C., with a delta H=−98.33 J/g onset. Accordingly, the ethanol solvate crystal Form A of compound of formula (X) (1:1) showed the thermograph pattern, which appears in FIG. 8.
In another set of DSC measurements, Form A, Form A after Adsorption/Desorption, and Form A after Adsorption/Desorption hydratation tests were examined. About 3 mg of the samples were transferred into a 30 μl perforated aluminum Perkin Elmer sample pan. The sample pan was closed with the appropriate cover and the DSC curve recorded on a Perkin Elmer Pyris DSC, at the following conditions:
















initial temperature:
25° C.



heating rate:
10° C./min



final temperature:
150° C.



nitrogen flow:
30 ml/min










Form A showed an endothermic signal at about 104.6° C. and a heat of fusion of 95.8 J/g caused by the evaporation of the ethanolate and the melting of the product. Form A after ADS/DES showed a broad endothermic signal due to a mixture of ethanolate Form A and hydrated Form B. Form A after ADS/DES hydratation test showed an endothermic signal at about 73.5° C. and a heat of fusion of 126 J/g caused by the evaporation of water and the melting of the product. Thermograph curves are depicted in FIG. 15.
EXAMPLE 6
In another example stability studies of the Form A in three different conditions were tested out. They included conditions of 25° C. and 60% RH, 40° C. and 75% RH, and 50° C. These studies revealed that at 25° C. and 60% RH long-term stability, the amount of ethanol and water is stable.
Table 12 shows the Stability study for Form A. Long term stability at 25° C./60% RH (Relative Humidity), with brown glass bottles as sample container.














Test
Release data
0 month
1 month
3 month



















Residual solvent:
7.5
7.6
7.6
7.1


% (w/w) ethanol


% (w/w) Water
0.10
0.27
0.26
0.55









EXAMPLE 7
Adsorption-Desorption Tests
About 23 mg of Form A were transferred into a VTI vapor sorption analyzer model SGA100 and the weight change with respect to the atmospheric humidity was recorded at the following conditions:













drying temperature:
40° C.


equilibrium:
≦0.05% in 5 min. or 60 min.


data interval:
0.05% or 2 min.


temperature:
25° C.


first cycle
RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70,



80, 90, 95



RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30,



20, 10, 5


second cycle
RH (%) adsorption: 5, 10, 20, 30, 40, 50, 60, 70,



80, 90, 95



RH (%) desorption: 95, 90, 80, 70, 60, 50, 40, 30,



20, 10, 5









At the drying step about 0.6% weight loss was registered. The obtained dried product was not hygroscopic, it adsorbed up to 0.7% water at high relative humidity. During the desorption cycle a loss of weight of 1.4% was registered, this indicated that the product was losing ethanolate. The obtained product after ADS/DES was a mixture of ethanolate form and hydrated form.
The ADS/DES curve is collected in FIG. 18.
Adsorption-Desorption Hydratation Tests
About 23 mg of Form A were transferred into a VTI vapor sorption analyzer model SGA100 and the weight change with respect to the atmospheric humidity was recorded at the following conditions:
















equilibrium:
≦0.0005% in 5 min. or 90 min.



data interval:
0.05% or 2 min



temperature:
25° C.



cycle
RH (%) adsorption/desorption: 5.95




repeat the cycle 11 times










At the end of this test a loss of weight of 5.2% was registered. This was comparable with the TG result (TG 5.4% up to 80° C.). The ethanolate form was transferred into a hydrated form. The ADS/DES hydratation test curves are collected in FIG. 19.
EXAMPLE 8
The stability of Form A was studied after storage of the compound in a sample container with an inner cover made of single LD-PE (string sealed), and outer cover made of PETP/Alu/PE (Moplast) heat sealed. A long term stability study at 25° C./60% RH, and an accelerated stability study at 40° C./75% RH, were performed for a period of 6 months, and the samples analysed at different time points as shown in following tables.







TABLE 13







Long term stability at 25° C./60% RH

















Release






tests
Remark
Specification
data
0 month
1 month
3 month
6 month

















Polymorphism
° C. (onset)
For information only
97.3
97.3
95.5
97.9
97.5


DSC
° C. max
For information only
104
104.2
103.5
104.2
104


Residual
% (w/w) ethanol
<=10.0%
6.71
6.31
6.33
6.40
6.33


solvents
% (w/w) 2-propanol
<=0.5%
0.04
0.04
0.05
0.05
0.05



% (w/w) THF
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) acetone
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) CH2Cl2
<=0.06%
<0.01
<0.01
<0.01
<0.01
<0.01


Water (KF)
% (w/w)
<=7.0%
0.63
0.23
0.34
0.32
0.46


X-Ray

For information only
C
C
—
—
—


powder


diffraction





C: chrystal













TABLE 14







Accelerated stability at 40° C./75% RH

















Release
0
1
3
6


Tests
Remark
Specification
data
month
month
month
month

















Polymorphism
° C. (onset)
For information only
97.3
97.3
97.5
98.0
97.8


DSC
° C. max
For information only
104
104.2
103.4
1039
104.3


Residual
% (w/w) ethanol
<=10.0%
6.71
6.31
6.73
6.32
6.50


solvents
% (w/w) 2-propanol
<=0.5%
0.04
0.04
0.05
0.05
0.05



% (w/w) THF
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) acetone
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) CH2Cl2
<=0.06%
<0.01
<0.01
<0.01
<0.01
<0.01


Water (KF)
% (w/w)
<=7.0%
0.63
0.23
0.37
0.34
0.42


X-Ray

For information only
C
C
—
—
—


powder


diffraction









Form A exhibited chemical and crystallographic stability at the conditions mentioned in tables 13 and 14.
EXAMPLE 9
The stability of Form A was studied after storage of the compound in a sample container with an inner cover made of single LD-PE (string sealed), and outer cover made of vapor loc bag (LPS) heat sealed. A long term stability study at 25° C./60% RH, and an accelerated stability study at 40° C./75% RH, were performed for a period of 6 months, and the samples analysed at different time points as shown in following tables.







TABLE 15







Long term stability at 25° C./60% RH

















Release






Tests
Remark
Specification
data
0 month
1 month
3 month
6 month

















Polymorphism
° C. (onset)
For information only
97.3
97.3
96.3
96.2
98.5


DSC
° C. max
For information only
104
104.2
103.1
103.8
103.9


Residual
% (w/w) ethanol
<=10.0%
6.71
6.31
6.42
6.35
6.52


solvents
% (w/w) 2-propanol
<=0.5%
0.04
0.04
0.06
0.05
0.05



% (w/w) THF
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) acetone
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) CH2Cl2
<=0.06%
<0.01
<0.01
<0.01
<0.01
<0.01


Water (KF)
% (w/w)
<=7.0%
0.63
0.23
0.32
0.38
0.49


X-Ray

For information only
C
C
—
—
—


powder


diffraction
















TABLE 16







Accelerated stability at 40° C./75% RH

















Release
0
1
3
6


Tests
Remark
Specification
data
month
month
month
month

















Polymorphism
° C. (onset)
For information only
97.3
97.3
97.8
97.5
97.9


DSC
° C. max
For information only
104
104.2
103.4
103.7
104.0


Residual
% (w/w) ethanol
<=10.0%
6.71
6.31
6.35
6.31
6.30


solvents
% (w/w) 2-propanol
<=0.5%
0.04
0.04
0.06
0.05
0.05



% (w/w) THF
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) acetone
<=0.5%
<0.01
<0.01
<0.01
<0.01
<0.01



% (w/w) CH2Cl2
<=0.06%
<0.01
<0.01
<0.01
<0.01
<0.01


Water (KF)
% (w/w)
<=7.0%
0.63
0.23
0.31
0.36
0.51


X-Ray

For information only
C
C
—
—
—


powder


diffraction









Form A exhibited chemical and crystallographic stability at the conditions mentioned in tables 15 and 16.
EXAMPLE 10
For the purpose of chemical stability testing, Form A was stored for a period of 1, 4 and 8 weeks under different conditions. These conditions were 40° C./75% RH, 50° C., RT/<5% RH, RT/56% RH, RT/75% RH and 0.3 da ICH light. The compound was analysed after storage by HPLC and by visual inspection. The HPLC method used in this study was HPLC method 909. The results of the tests are reported in the following table.










TABLE 17









HPLC




Sum of impurities
Appearance













Conditions
1 week
4 week
8 week
1 week
4 weeks
8 weeks





Reference
1.07
—
—
slightly-yellow
—
—


0.3 da ICH light
1.01
—
—
slightly-yellow
—
—


40° C./75% RH
1.03
0.98
0.99
slightly-yellow
slightly-yellow
slightly-yellow


50° C.
1.05
1.08
1.06
slightly-yellow
slightly-yellow
slightly-yellow


RT/<5% RH
—
1.02
1.04
—
slightly-yellow
slightly-yellow


RT/56% RH
—
1.02
0.99
—
slightly-yellow
slightly-yellow


RT/75% RH
—
1.00
1.01
—
slightly-yellow
slightly-yellow









It was concluded that Form A is chemically stable after storage in all investigated conditions.
EXAMPLE 11
Different fractions of Form B were characterized with thermogravimetry (TG), differential scanning calorimetry (DSC) and infrared spectroscopy (IR). The results of the tests are reported in the following table.











TABLE 18









TG %

DSC












weight change

Max
Extra


Fractions
<100° C.
IR
(° C.)
(° C.)





Form B fraction 1
5.65
Hydrate, Ref
69.1
—


after ADS/DES
4.30
±Hydrate, Ref,
—
—




+amorphous


Form B fraction 2
5.91
~Hydrate, Ref
75.6
—


after 5 d 40° C./75% RH
3.56
~Hydrate, Ref
74.1
—


Form B fraction 3
3.13
±Hydrate, Ref,
77.0
67.8




+amorphous


after 5 d 40° C./75% RH
2.33
±Hydrate, Ref,
77.4
62.8




+amorphous





~hydrate, Ref: identical with reference






EXAMPLE 12
The adsorption and desorption of water at 25° C. at different conditions of relative humidity was investigated on 38 mg of Form B. The weight change as a function of relative humidity was registered. The results are displayed in FIG. 20. At the drying step about 5.6% weight loss was registered for Form B. The obtained dried product was hygroscopic, it adsorbed up to 6.8% water at high relative humidity. After the desorption cycle about 1.2% water remained on the sample. The obtained product after ADS/DES was a mixture of hydrate and amorphous product.
EXAMPLE 13
Aqueous solubilities of Form B were measured in solvents with different pH. An excess of the solute was equilibrated with the solvent at 20° C. for at least 24 hours. After removing the undissolved compound, the concentration in solution was determined using UV spectrometry.










TABLE 19








Solubility



Solvent
(mg/100 ml solution)









Water
 10 (pH 5.1)



Buffer pH 2 (citrate/HCl)
 23 (pH 2.0)



Buffer pH 3 (citrate/HCl)
 13 (pH 3.0)



Buffer pH 4 (citrate/HCl)
 12 (pH 4.0)



0.01N HCl
 18 (pH 2.1)



0.1N HCl
150 (pH 1.1)



1.0N HCl
510 (pH 0.14)










EXAMPLE 14
The stability of the crystal structure of Form B was studied after storage of the compound for a period of two weeks at room temperature (RT) under <5%, 56% and 75% relative humidity (RH), 50° C. and 40° C./75% RH. The samples were analyzed with thermogravimetry (TG), differential scanning calorimetry (DSC), infrared spectroscopy (IR) and X-ray diffraction (XRD). The results of the tests are reported in the following table.













TABLE 20









TG


DSC














condition
<100° C.
<225° C.
IR
XRD
Max (° C.)
Appearance





0 days
5.65
0.16
Ref
Ref
69.1
slightly yellow-orange


after ADS/DES
4.30
0.18
≠Ref
—
—
slightly yellow-orange


RT/<5% RH
0.32
0.07
≠Ref
≠Ref
71.2
slightly yellow-orange


RT/56% RH
5.71
0.25
~Ref
~Ref
71.0
slightly yellow-orange


RT/75% RH
6.20
0.10
~Ref
~Ref
71.5
slightly yellow-orange


50° C.
0.23
0.06
≠Ref
≠Ref
76.4
slightly yellow-orange


40° C. 75% RH
5.77
0.07
~Ref
±Ref
70.4
slightly yellow-orange





~Ref: identical with reference


±Ref: similar with reference


≠Ref: different with reference






EXAMPLE 15
In the chemical stability test program Form B was stored for a period of 1, 4 and 9 weeks under different conditions. These conditions were 40° C./75% RH, 50° C., RT/<5% RH, RT/56% RH, RT/75% RH and 0.3da ICH light. The compound was analysed after storage by HPLC and by visual inspection. The HPLC method used in this study was HPLC method 909. The results of the tests are reported in the following table, from which it was concluded that Form B is chemically stable.










TABLE 21









HPLC




Sum of impurities
Appearance













Condition
1 week
4 week
9 week
1 week
4 weeks
9 weeks





Reference
1.35
—
—
lightly yellow-
—
—






orange


0.3 da ICH
1.30
—
—
light-orange
—
—


light


40° C./75% RH
1.43
1.38
1.41
lightly yellow-
Orange
light-orange






orange


50° C.
1.46
1.50
1.46
lightly yellow-
light-orange
light-orange






orange


RT/<50% RH
—
1.48
1.37
—
light-orange
light-orange


RT/56% RH
—
1.11
1.35
—
lightly yellow-
light-orange







orange


RT/75% RH
—
1.34
1.29
—
light-orange
light-orange









EXAMPLE 16
Form K was prepared by adding neat methanesulfonic acid to a solution of Form A in THF at r.t. Form K was subsequently mixed with alkali halide and pressed to a pellet (Ph. Eur.) and analyzed by Infrared spectrometry (IR) at the following conditions:













apparatus:
Nicolet Magna 560 FTIR spectrophotometer


number of scans:
32


resolution:
1 cm−1


wavelength range:
4000 to 400 cm−1


baseline correction:
yes


detector:
DTGS with KBr windows


beamsplitter:
Ge on KBr


alkali halide:
KBr (potassium bromide)









The IR spectrum of Form K, as shown in FIG. 21, reflects the vibrational modes of the molecular structure of the mesylate solvate as a crystalline product.







TABLE 22





Wavenumbers (cm−1) and relative intensities of absorption bands (1)







3362m, 3064w


2985m, 2964m, 2906m, 2873m, 2632w, 2585w


1687s, 1627w, 1601w


1554m, 1495m, 1480w, 1470w, 1452w, 1443w, 1421w


1383w, 1373w, 1369w, 1345m, 1324m, 1314m, 1299w, 1268m, 1245m,


1221m, 1202s


1190s, 1166vs, 1122m, 1091m, 1077m, 1051s, 1043s, 1023m, 1002m


992m, 969w, 943w, 912w, 888w, 867vw, 836w, 813vw


773m, 754w, 743m, 711w, 700m, 658m, 634w


581w, 556m, 505w, 472vw, 452vw, 435vw, 417vw





(1) vs = very strong, s = strong, m = medium, w = weak, vw = very weak, br = broad






EXAMPLE 17
Form K was transferred to a glass capillary cell and analyzed by Raman spectrometry at the following conditions:













Raman mode:
Nondispersive Raman


apparatus:
Nicolet FT-Raman module


number of scans:
64


resolution:
4 cm−1


wavelength range:
3700 to 100 cm−1


laser:
Nd:YVO4


laser frequency:
1064 cm−1


detector:
InGaAs


beamsplitter:
CaF2


sample geometry:
180° reflective


polarization:
no









The Raman spectrum of Form K, as shown in FIG. 22, reflects the vibrational modes of the molecular structure of the mesylate as a crystalline product.







TABLE 23





Wavenumbers (cm1) and relative intensities of absorption bands (1)







3080m, 3068m, 3059m, 3043w, 3022w, 3006m


2989s, 2978s, 2962s, 2933vs, 2906m, 2871m


1685vw, 1628w, 1603s, 1585w, 1495w, 1479w, 1466w, 1450m, 1423w


1381w, 1346w, 1336w, 1313w, 1290w, 1271w, 1244w, 1230w, 1209m


1190w, 1182m, 1163vs, 1122w, 1105w, 1090m, 1049vs, 1032w, 1003s


968w, 955w, 941w, 914w, 897w, 877w, 866w, 845w, 823m, 814m


783m, 771m, 742w, 658w, 634m, 621w


577w, 561m, 534w, 524w, 497w, 451w, 436w


337w, 308w, 287m, 247w, 206w, 162m, 129m





(1) vs = very strong, s = strong, m = medium, w = weak, vw = very weak






EXAMPLE 18
About 3 mg of Form K were transferred into a standard aluminium TA-Instrument sample pan. The sample pan was closed with the appropriate cover and the DSC curve recorded on a TA-Instruments Q1000 MTDSC equipped with a RCS cooling unit, at the following conditions:
















initial temperature:
25° C.



beating rate:
10° C./min



final temperature:
200° C.



nitrogen flow:
50 ml/min










The DSC curve as depicted in FIG. 23, shows the melting with decomposition of a crystalline product. The melting of Form K occurs at 158.4° C. Due to the decomposition, the heat of fusion calculation can only be used to indicate the crystalline property of the product.
EXAMPLE 19
Form K was transferred into an aluminum sample pan. The TG curve was recorded on a TA Instruments Hi-Res TGA 2950 thermogravimeter at the following conditions:
















initial temperature:
room temperature



heating rate:
20° C./min



resolution factor:
4



final condition:
300° C. or <80[(w/w)%]










The TG curve is exhibited in FIG. 24. The loss of weight of around 0.2% up to .60° C. was due to the evaporation of solvent. The loss of weight at temperatures above 140° C. was due to the evaporation and decomposition of the product.
EXAMPLE 20
Adsorption-Desorption
About 21 mg of Form K were transferred into a VTI vapor sorption analyzer model SGA100 and the weight change with respect to the atmospheric humidity was recorded at the following conditions:













drying temperature:
40° C.


equilibrium:
≦0.05% in 5 min. or 60 min.


data interval:
0.05% or 2.0 min.


temperature:
25° C.


first cycle
RH (%) adsorption: 5,10,20,30,40,50,60,70,80,



90,95



RH (%) desorption: 95,90,80,70,60,50,40,30,20,



10,5


second cycle
RH (%)adsorption: 5,10,20,30,40,50,60,70,80,



90,95



RH (%) desorption: 95,90,80,70,60,50,40,30,20,



10,5









The Adsorption-Desorption isotherm is shown in FIG. 25. Form K is hygroscopic. At the initial drying step a loss of weight of 0.3% was registered, comparable to the TG result. Form K adsorbed up to 1.5% water at high relative humidity. The product dried completely during the desorption cycle.
A different study of the adsorption and desorption of water by Form K at 25° C. at different conditions of relative humidity was investigated on an amount of about 18 mg of the mesylate solvate. The weight change as a function of relative humidity was registered. The result is displayed in FIG. 26.
At the drying step about 0.6% weight loss is registered for Form K. The obtained dried product is slightly hygroscopic, it adsorbed up to 1.7% water at high relative humidity. The product dried completely during the desorption cycle.
EXAMPLE 21
Aqueous solubilities of Form K were measured in solvents with different pH. An excess of the solute was equilibrated with the solvent at 20° C. for at least 48 hours. After removing the undissolved compound, the concentration in solution was determined using UV spectrometry.










TABLE 24








Solubility



Solvent
(mg/100 ml solution)









Water
 19 (pH 3.3)



Buffer pH 2 (citrate/HCl)
 21 (pH 2.0)



Buffer pH 3 (citrate/HCl)
 12 (pH 3.0)



Buffer pH 4 (citrate/HCl)
 11 (pH 4.0)



0.01N HCl
 24 (pH 2.0)



20% HPβCD in water
2100 (pH 1.6)










EXAMPLE 22
The stability of the crystal structure of Form K batch 1 was studied after storage of the compound for a period of four weeks at room temperature (RT) under 75% relative humidity (RH), 50° C. and 40° C./75% RH. The stability of the crystal structure of Form K batch 2 was studied after storage of the compound for a period of four weeks at room temperature (RT) under <5%, 56% and 75% relative humidity (RH), 50° C. and 40° C./75% RH. The samples were analyzed with thermogravimetry (TG), differential scanning calorimetry (DSC) and infrared spectroscopy (IR). The results of the tests are reported in the following table.












TABLE 25









TG

DSC















compound
conditions
<80° C.
<125° C.
IR
Max (° C.)
Extra (° C.)
Appearance





Form K
0 days
0.47
0.15
Ref
143.7
—
slightly orange


Batch 1
RT/75% RH
2.87
0.19
≠Ref
146.6
64.3
slightly orange



50° C.
0.32
0.14
~Ref
140.6
45.6
orange



40° C./75% RH
1.48
3.71
—
—
—
brown oil


Form K
0 days
0.16
0.11
Ref
155.8
—
slightly orange


Batch 2
RT/<5% RH
0.00
0.03
~Ref
156.9
—
slightly orange



RT/56% RH
0.27
0.03
±Ref
154.6
—
slightly orange



RT/75% RH
1.82
0.07
≠Ref
149.2
67.0
slightly orange



50° C.
0.12
0.12
~Ref
156.8
—
slightly orange



40° C./75% RH
3.26
3.08
—
—
—
brown oil





~Ref: identical with reference


±Ref: similar with reference


≠Ref: different with reference






EXAMPLE 23
In the chemical stability test program Form K batch 1 was stored for a period of 1 and 4 weeks under different conditions. These conditions were 40° C./75% RH, 50° C., RT/75% RH and 0.3da ICH light. Form K batch 2 was also stored for a period of 1 and 4 weeks under different conditions. These conditions were 40° C./75% RH, 50° C., RT/<5% RH, RT/56% RH, RT/75% RH and 0.3 da ICH light. The compound was analysed after storage by HPLC and by visual inspection. The HPLC method used in this study was HPLC method 909. The results of the tests are reported in the following table.










TABLE 26









HPLC




Sum of



impurities
appearance












compound
conditions
1 week
4 weeks
1 week
4 weeks





Form K batch 1
Reference
3.57
—
slightly-orange
—



0.3 da ICH light
2.93
—
slightly-orange
—



40° C./75% RH
5.36
>90*
slightly-orange
brown oil



50° C.
3.99
 27.53
slightly-orange
orange



RT/75% RH
—
 3.61
—
slightly-orange


Form K Batch 2
Reference
1.50
—
slightly-orange
—



0.3 da ICH light
1.17
—
slightly-orange
—



40° C./75% RH
1.75
>85*
slightly-orange
brown oil



50° C.
1.46
 1.25
slightly-orange
slightly-orange



RT/<5% RH
—
 1.58
—
slightly-orange



RT/56% RH
—
 1.45
—
slightly-orange



RT/75% RH
—
 1.46
—
slightly-orange









EXAMPLE 24
A randomized, placebo-controlled, double-blind, multiple dose escalation trial was performed to examine the safety, tolerability and pharmacokinetics of Form A after oral administration twice or three times daily, in healthy subjects. Four dosages of Form A (400 mg b.i.d., 800 mg b.i.d., 800 mg t.i.d., and 1200 mg t.i.d.) were tested in 4 panels of 9 healthy subjects. Within each panel, 6 subjects were treated with Form A and 3 subjects with placebo for 13 days with a single intake in the morning of day 14. (b.i.d.=twice daily, t.i.d.=three times daily).
Form A was readily absorbed and concentration-time profiles of Form A after repeated dosing were dependent on the dose administered. Steady-state plasma concentrations were reached generally within 3 days, although C0h (conc. at administration time) and AUC24h (area under de curve or bioavailability) slightly decreased over time at all dose levels. AUC24h and Css,av (conc. at average steady-state) were dose-proportional (daily dose) at 400 mg b.i.d., 800 mg t.i.d. and 1200 mg t.i.d., but was more than dose-proportional at 800 mg b.i.d. Cmax (maximum conc.) was dose-proportional with respect to dose per intake. Less than 2% of unchanged Form A was excreted in the urine at all dose levels.

## Claims

1. An ethanolate solvate of the compound (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate, in which the ratio of compound to ethanol is about 1:1.

2. A solvate having the formula:

3. A composition comprising an ethanolate solvate of the compound (3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl(1S,2R)-3-[[(4-aminophenyl)sulfonyl](isobutyl)amino]-1-benzyl-2-hydroxypropylcarbamate, in which the ratio of compound to ethanol is about 1:1, and an inert carrier.

4. The composition of claim 3 wherein the inert carrier is a pharmaceutically acceptable carrier.

5. The composition of claim 4 wherein the pharmaceutically acceptable carrier is a solid inert carrier.

6. A composition comprising a solvate having the formula:



and an inert carrier.

7. The composition of claim 6 wherein the inert carrier is a pharmaceutically acceptable earner.

8. The composition of claim 7 wherein the pharmaceutically acceptable carrier is a solid inert carrier.