# Patent

## Title
Short-acting benzodiazepine salts and their polymorphic forms

## Abstract

The invention relates to besylate salts of the compound of formula (I):




Methods of preparing the salts, and their use as medicaments, in particular for sedative or hypnotic, anxiolytic, muscle relaxant, or anticonvulsant purposes is also described.

## Background
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a continuation of currently pending U.S. application Ser. No. 12/373,472, filed Nov. 2, 2009, which is the U.S. National Stage of International Application No. PCT/GB2007/002565, filed Jul. 10, 2007, which designated the United States and has been published as International Publication No. WO 2008/007071 and which claims the priority of Great Britain Patent Applications, Serial Nos.: 0613694.9 and 0613692.3 of Jul. 10, 2006, pursuant to 35 U.S.C. 119(a)-(d). The contents of the aforementioned applications are hereby incorporated herein by reference.


BACKGROUND OF THE INVENTION
This invention relates to salts of a short acting benzodiazepine, and to use of the salts as medicaments, in particular for sedative or hypnotic, anxiolytic, muscle relaxant, or anticonvulsant purposes.
European Patent No. 1,183,243 describes short-acting benzodiazepines that include a carboxylic acid ester moiety and are inactivated by non-specific tissue esterases. An organ-independent elimination mechanism is predicted to be characteristic of these benzodiazepines, providing a more predictable and reproducible pharmacodynamic profile. The compounds are suitable for therapeutic purposes, including sedative-hypnotic, anxiolytic, muscle relaxant and anticonvulsant purposes. The compounds are short-acting CNS depressants that are useful to be administered intravenously in the following clinical settings: preoperative sedation, anxiolysis, and amnestic use for perioperative events; conscious sedation during short diagnostic, operative or endoscopic procedures; as a component for the induction and maintenance of general anesthesia, prior and/or concomitant to the administration of other anaesthetic or analgesic agents; ICU sedation.
One of the compounds disclosed in EP 1,183,243 (in Example Ic-8, page 36) is Methyl 3-[(4S)-8-bromo-1-methyl-6-(2-pyridinyl)-4H-imidazol [1,2-a][1,4]benzodiazepin-4-yl] propanoate, as shown in formula (I) below:




Whilst the free base of formula (I) is stable when stored at 5° C., samples stored at 40° C./75% relative humidity (open) are observed to deliquesce, become yellow to orange in colour, and show notable decreases in content relative to initial (see Example 1 below).
It has now surprisingly been found that the compound of formula (I) forms highly crystalline mono (benzenesulfonic acid) besylate salts that are easily isolated from a range of pharmaceutically acceptable solvents and show good thermal stability, low hygroscopicity and high aqueous solubility.
SUMMARY OF THE INVENTION
According to the invention there is provided a besylate salt of a compound of formula (I). Preferably the salt is a crystalline salt. Preferably the crystalline salt has a stoichiometry of 1:1 compound of formula (I):besylate. Preparation and characterisation of polymorphic forms of besylate salts is described in the Examples below.
According to the invention there is provided a crystalline polymorph of a besylate salt of a compound of formula (I) (herein designated besylate Form 1), that exhibits an X-ray powder diffraction (XRPD) pattern which comprises a characteristic peak at about 7.3, 7.8, 9.4, 12.1, 14.1, 14.4, 14.7, or 15.6 degrees two-theta.
Preferably the besylate Form 1 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at about 7.3, 7.8, 9.4, 12.1, 14.1, 14.4, 14.7, and 15.6 degrees two-theta.
More preferably the besylate Form 1 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at: 7.25 (10.60), 7.84 (72.60), 9.36 (12.10), 12.13 (32.50), 14.06 (48.50), 14.41 (74.30), 14.70 (50.70), 15.60 (26.90) [angle two-theta degrees (percentage relative intensity)].
Preferably the besylate Form 1 crystalline polymorph has a differential scanning calorimetry (DSC) onset melting temperature in the range 187-204° C., preferably about 191-192° C.
A crystal structure of Form 1 has been resolved at 190K (R factor of 6.3). Form I has a stoichiometry of 1:1 compound:besylate. Its crystallographic asymmetric unit contains two independent compound molecules and two besylate molecules. The two independent compound molecules are singly protonated on the imidazole ring. The crystal structure has unit cell dimensions of a=7.6868 Å, b=29.2607 Å, c=12.3756 Å, α=90°, β=97.7880°, γ=90°, and a space group of P21. The crystal structure is described in more detail in Example 9, and crystallographic coordinates are given in Table 17. Bond lengths and angles for Form 1 are given in Tables 19 and 20, respectively.
According to the invention there is provided a besylate salt of a compound of formula (I) which is a crystalline polymorph comprising a crystal with unit cell dimensions of a=7.6868 Å, b=29.2607 Å, c=12.3756 Å, α=90°, β=97.7880°, γ=90°.
There is also provided according to the invention a besylate salt of a compound of formula (I) which is a crystalline polymorph having a crystal structure defined by the structural coordinates as shown in Table 17.
There is further provided according to the invention a besylate salt of a compound of formula (I) with bond lengths and angles as shown in Tables 19 and 20, respectively.
There is further provided according to the invention a crystalline polymorph of a besylate salt of a compound of formula (I) (herein designated besylate Form 2), that exhibits an XRPD pattern which comprises a characteristic peak at about 8.6, 10.5, 12.0, 13.1, 14.4, or 15.9 degrees two-theta.
Preferably the besylate Form 2 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at about 8.6, 10.5, 12.0, 13.1, 14.4, and 15.9 degrees two-theta.
More preferably the besylate Form 2 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at: 8.64 (17.60), 10.46 (21.00), 12.03 (22.80), 13.14 (27.70), 14.42 (11.20), 15.91 (100.00) [angle two-theta degrees (percentage relative intensity)].
Preferably the besylate Form 2 crystalline polymorph has a differential scanning calorimetry (DSC) onset melting temperature in the range 170-200° C., preferably about 180° C.
A crystal structure of Form 2 has been resolved at 190K (R factor of 3.8). Form 2 has stoichiometry of 1:1 compound:besylate. Its crystallographic asymmetric unit contains one compound molecule and one besylate molecule. The compound molecule is singly protonated on the imidazole ring. The crystal structure has unit cell dimensions of a=8.92130 Å, b=11.1536 Å, c=25.8345 Å, α=90°, β=90°, γ=90°, and a space group of P212121. The crystal structure is described in more detail in Example 10, and crystallographic coordinates are given in Table 18. Bond lengths and angles for Form 2 are given in Tables 21 and 22, respectively.
According to the invention there is provided a besylate salt of a compound of formula (I) which is a crystalline polymorph comprising a crystal with unit cell dimensions of a=8.92130 Å, b=11.1536 Å, c=25.8345 Å, α=90°, β=90°, γ=90°.
There is also provided according to the invention a besylate salt of a compound of formula (I) which is a crystalline polymorph having a crystal structure defined by the structural coordinates as shown in Table 18.
There is further provided according to the invention a besylate salt of a compound of formula (I) with bond lengths and angles as shown in Tables 21 and 22, respectively.
There is further provided according to the invention a crystalline polymorph of a besylate salt of a compound of formula (I) (herein designated besylate Form 3), that exhibits an X-ray powder diffraction (XRPD) pattern which comprises a characteristic peak at about 7.6, 11.2, 12.4, 14.6, 15.2, 16.4, or 17.7 degrees two-theta.
Preferably the besylate Form 3 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at about: 7.6, 11.2, 12.4, 14.6, 15.2, 16.4, and 17.7 degrees two-theta.
More preferably the besylate Form 3 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at: 7.61 (65.70), 11.19 (33.20), 12.38 (48.70), 14.63 (30.60), 15.18 (33.20), 16.40 (29.60), 17.68 (51.30) [angle 2θ° (percentage relative intensity)].
Preferably the besylate Form 3 crystalline polymorph has a differential scanning calorimetry (DSC) onset melting temperature in the range 195-205° C., preferably about 200-201° C.
There is further provided according to the invention a crystalline polymorph of a besylate salt of a compound of formula (I) (herein designated besylate Form 4), that exhibits an XRPD pattern which comprises a characteristic peak at about 7.6, 10.8, 15.2, 15.9, or 22.0 degrees two-theta.
Preferably the besylate Form 4 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at about: 7.6, 10.8, 15.2, 15.9, and 22.0 degrees two-theta.
Preferably the besylate Form 4 crystalline polymorph exhibits an XRPD pattern which comprises characteristic peaks at: 7.62 (83.50), 10.75 (14.70), 15.17 (37.80), 15.85 (28.70), 22.03 (100) [angle 2θ° (percentage relative intensity)].
Preferably the besylate Form 4 crystalline polymorph has a differential scanning calorimetry (DSC) onset melting temperature in the range 180-185° C., preferably about 182° C.
A preferred salt is the besylate Form 1 based on the robustness of formation, yield, purity and chemical and solid form stability.
There is also provided according to the invention a method of making a besylate salt of a compound of formula (I), which comprises reacting a free base of a compound of formula (I) with benzene sulfonic acid.
Also according to the invention there is provided a method of making a salt of the invention, which comprises contacting a free base of a compound of formula (I) with benzene sulfonic acid in solution to cause formation of a precipitate of the besylate salt. Preferably the method further comprises isolating the precipitate.
Preferably the free base is dissolved in toluene, ethanol, ethyl acetate, MtBE, dichloromethane (DCM), isopropyl acetate, ethyl formate, methanol, or acetone. More preferably the free base is dissolved in toluene or ethyl acetate. Preferably the benzene sulfonic acid is dissolved in ethanol.
The besylate Form 1 may be prepared by contacting a solution of a free base of a compound of formula (I) in toluene, ethyl acetate, acetone, isopropyl acetate, or ethyl formate with a solution of benzene sulfonic acid in ethanol to cause formation of a precipitate of the salt.
There is also provided according to the invention a besylate salt of a compound of formula (I) which is obtainable by the above method.
The besylate Form 2 may be prepared by contacting a solution of a free base of a compound of formula (I) in methanol with a solution of benzene sulfonic acid in ethanol to cause formation of a precipitate of the salt. Preferably the mixture is cooled below ambient temperature (for example 4° C.).
There is also provided according to the invention a besylate salt of a compound of formula (I) which is obtainable by the above method.
The besylate Form 3 may be prepared by seeding liquor resulting from crystallisation of Form 1 from ethyl acetate/ethanol with Form 1. Preferably the liquor is cooled below ambient temperature (for example 4° C.).
In one embodiment the besylate Form 3 may be prepared by seeding, with a besylate Form 1 crystalline salt of a compound of formula (I), a filtrate solution separated from the precipitate formed by contacting a solution of a compound of formula (I) in ethyl acetate with a solution of benzene sulfonic acid in ethanol, to produce the besylate Form 3 crystalline polymorph.
There is also provided according to the invention a besylate salt of a compound of formula (I) which is obtainable by any of the above methods.
The besylate Form 4 may be prepared by re-crystallising besylate Form 1 from isopropyl acetate/ethanol, preferably 40% isopropyl acetate/ethanol.
There is also provided according to the invention a besylate salt of a compound of formula (I) which is obtainable by the above method.
Salts of the invention may also be prepared by crystallising compound of formula (I) besylate from a suitable solvent, or from a suitable solvent/anti-solvent or solvent/co-solvent mixture. The solution or mixture may be cooled and/or evaporated to achieve crystallisation if appropriate.
We have found that crystallisation of Form 2 is observed in conditions where there are extremes of either polarity (for example acetonitrile:water) or lipophilicity (n-nonane), or both (dimethyl sulfoxide:1,2-dichlorobenzene).
Examples of solvents for crystallisation of Form 2 are: nonane; methanol.
Examples of solvent/anti-solvent mixtures for crystallisation of Form 1 are: dimethylacetamide/methyl isobutyl ketone; dimethylacetamide/tetrachloroethylene; acetonitrile/3-methylbutan-1-ol; acetonitrile/1,2-dichlorobenzene; acetonitrile/pentylacetate; methanol/3-methylbutan-1-ol; methanol/methyl isobutyl ketone; 2,2,2-trifluoroethanol/1,4-dimethylbenzene; ethanol/methyl isobutyl ketone; ethanol/1,4-dimethylbenzene; propan-1-ol/1,2-dichlorobenzene; propan-1-ol/tetrachloroethylene; propan-2-ol/1,2-dichlorobenzene; propan-2-ol/n-nonane; 2-methoxy ethanol/water; 2-methoxy ethanol/pentyl acetate; 2-methoxy ethanol/1,4-dimethylbenzene; tetrahydrofuran/water; tetrahydrofuran/3-methylbutan-1-ol; tetrahydrofuran/1,2-dichlorobenzene; tetrahydrofuran/ethyl acetate; tetrahydrofuran/1,3-dimethylbenzene.
Examples of solvent/anti-solvent mixtures for crystallisation of Form 2 are: ethanol/ethyl acetate; ethanol/methyl isobutyl ketone; ethanol/p-cymene; dimethylsulfoxide/1,2-dichlorobenzene; acetonitrile/water; ethano/1,2-dichlorobenzene; ethanol/tetrachloroethylene; tetrahydrofuran/1,2-dichlorobenzene; tetrahydrofuran/ethyl acetate.
According to a preferred embodiment, Form 1 is crystallised from 2-methoxyethanol/pentyl acetate.
According to a preferred embodiment, Form 2 is crystallised from ethanol/ethyl acetate.
According to a preferred embodiment, Form 2 is crystallised from methanol/ethanol (preferably by cooling a solution of compound of formula (I) besylate in methanol/ethanol below ambient temperature, for example 4° C.).
According to a preferred embodiment, Form 3 is crystallised from ethanol/ethyl acetate (suitably by cooling the mixture below ambient temperature, for example 4° C.).
According to a preferred embodiment, Form 4 is crystallised from isopropyl acetate/ethanol (preferably by cooling a solution of compound of formula (I) besylate in isopropyl acetate/ethanol to ambient temperature).
There is also provided according to the invention a besylate salt of a compound of formula (I) obtainable by any of the above methods.
Methods of preparing salts of the invention are described in detail in the Examples below.
A salt of the invention may be used as a medicament, in particular for sedative or hypnotic, anxiolytic, muscle relaxant, or anticonvulsant purposes.
While it is possible for a salt of the invention to be administered as a bulk active chemical, it is preferably provided with a pharmaceutically acceptable carrier, excipient, or diluent in the form a pharmaceutical composition. The carrier, excipient, or diluent must, of course, be acceptable in the sense of being compatible with the other ingredients of the composition and must not be deleterious to the recipient.
Accordingly, the present invention provides a pharmaceutical composition comprising a salt of the invention and a pharmaceutically acceptable carrier, excipient, or diluent.
Pharmaceutical compositions of the invention include those suitable for oral, rectal, topical, buccal (e.g. sub-lingual) and parenteral (e.g. subcutaneous, intramuscular, intradermal or intravenous) administration.
Preferably a salt of the invention is provided in the form of a pharmaceutical composition for parenteral administration, for example, by intravenous or intramuscular injection of a solution. Where the pharmaceutical composition is for parenteral administration, the composition may be an aqueous or non-aqueous solution or a mixture of liquids, which may include bacteriostatic agents, antioxidants, buffers or other pharmaceutically acceptable additives.
A preferred formulation of a salt of the invention is in an aqueous acidic medium of pH 2-4 or in an aqueous solution of a cyclodextrin (CD). Cyclodextrins that can be used for these formulations are either the anionically charged sulfobutylether (SBE) derivatives of β-CD, specifically SBE7-β-CD, marketed under the tradename Captisol by CyDex, Inc. (Critical Reviews in Therapeutic Drug Carrier Systems, 14 (1), 1-104 (1997)), or the hydroxypropyl CD's.
A further preferred formulation of a salt of the invention is a lyophilised formulation comprising, in addition to the salt, at least one of the following agents: ascorbic acid, citric acid, maleic acid, phosphoric acid, glycine, glycine hydrochloride, succinic acid or tartaric acid. These agents are believed to be useful as buffering, caking or vizualisation agents. In some cases it may be beneficial to include sodium chloride, mannitol, polyvinylpyrrolidone, or other ingredients in the formulation.
The preferred method of formulation (i.e., acid buffer or CD-based) may depend on the physicochemical properties (e.g., aqueous solubility, pKa, etc.) of a particular salt. Alternatively the salt may be presented as a lyophilized solid for reconstitution with water (for injection) or a dextrose or saline solution. Such formulations are normally presented in unit dosage forms such as ampoules or disposable injection devices. They may also be presented in multi-dose forms such as a bottle from which the appropriate dose may be withdrawn. All such formulations should be sterile.
According to the invention there is provided a method for producing sedation or hypnosis in a subject, which comprises administering an effective sedative or hypnotic amount of a salt of the invention to the subject.
There is also provided according to the invention a method for inducing anxiolysis in a subject, which comprises administering an effective anxiolytic amount of a salt of the invention to the subject.
There is further provided according to the invention a method for inducing muscle relaxation in a subject, which comprises administering an effective muscle relaxant amount of a salt of the invention to the subject.
There is further provided according to the invention a method for treating convulsions in a subject, which comprises administering an effective anticonvulsant amount of a salt of the invention to the subject.
According to the invention there is also provided use of a sedative or hypnotic amount of a salt of the invention in the manufacture of a medicament for producing sedation or hypnosis in a subject.
According to the invention there is also provided a salt of the invention for producing sedation or hypnosis in a subject.
There is also provided according to the invention use of an anxiolytic amount of a salt of the invention in the manufacture of a medicament for producing anxiolysis in a subject.
There is also provided according to the invention a salt of the invention for producing anxiolysis in a subject.
There is further provided according to the invention use of a muscle relaxant amount of a salt of the invention in the manufacture of a medicament for producing muscle relaxation in a subject.
There is further provided according to the invention a salt of the invention for producing muscle relaxation in a subject.
There is further provided according to the invention use of an anticonvulsant amount of a salt of the invention in the manufacture of a medicament for treating convulsions in a subject.
There is further provided according to the invention a salt of the invention for treating convulsions in a subject.
The subject is suitably a mammal, preferably a human.
A suitable pharmaceutical parenteral preparation for administration to humans will preferably contain 0.1 to 20 mg/ml of a salt of the invention in solution or multiples thereof for multi-dose vials.
Intravenous administration can take the form of bolus injection or, more appropriately, continuous infusion. The dosage for each subject may vary, however, a suitable intravenous amount or dosage of a salt of the invention to obtain sedation or hypnosis in a mammal would be 0.01 to 5.0 mg/kg of body weight, and more particularly, 0.02 to 0.5 mg/kg of body weight, the above being based on the weight of the salt which is the active ingredient. A suitable intravenous amount or dosage of a salt of the invention to obtain anxiolysis in a mammal would be 0.01 to 5.0 mg/kg of body weight, and more particularly, 0.02 to 0.5 mg/kg of body weight, the above being based on the weight of the salt which is the active ingredient. A suitable intravenous amount or dosage of a salt of the invention to obtain muscle relaxation in a mammal would be 0.01 to 5.0 mg/kg of body weight, and more particularly, 0.02 to 0.5 mg/kg of body weight, the above being based on the weight of the salt which is the active ingredient. A suitable intravenous amount or dosage of a salt of the invention to treat convulsions in a mammal would be 0.01 to 5.0 mg/kg of body weight, and more particularly, 0.02 to 0.5 mg/kg of body weight, the above being based on the weight of the salt which is the active ingredient.
Salts of the invention are short-acting CNS depressants that are useful to be administered intravenously in the following clinical settings: preoperative sedation, anxiolysis, and amnestic use for perioperative events; conscious sedation during short diagnostic, operative or endoscopic procedures; as a component for the induction and maintenance of general anaesthesia, prior and/or concomitant to the administration of other anaesthetic or analgesic agents; ICU sedation.



BRIEF DESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described in the following Examples with reference to the accompanying drawings in which:
FIG. 1 shows a graph of compound of formula (I) content (% relative to initial) vs storage temperature;
FIG. 2 shows Differential Scanning calorimetry (DSC) of LJC-039-081-1;
FIG. 3 shows DSC of LJC-039-081-1 (solid) overlaid with LJC-039-081-2 (dotted);
FIG. 4 shows DSC of besylate forms (Form 1 solid, Form 2 dashed);
FIG. 5 shows DSC of besylate forms (Form 1 solid, Form 3 dotted and dashed);
FIG. 6A and FIG. 6B show chromatographs of LJC-039-037-1 at T0 and T4 (and relate to the results in Table 10);
FIG. 7 shows XRPD comparing LJC-039-037-1 (besylate salt) pre and post 4 week stability study;
FIG. 8A shows an XRPD comparison of besylate Form 1 and 2;
FIG. 8B shows Differential Scanning calorimetry (DSC) overlays of Form 1 and 2;
FIG. 9A shows an XRPD comparison of besylate Form 1 and 3, and FIG. 9B shows overlays of Form 1 and 3;
FIG. 10 shows DSC of LJC-039-086-1 (besylate Form 4);
FIGS. 11A-11I show results for besylate Form 1: FIG. 11A) XRPD for 100 mg batch LJC-039-037-1; FIG. 11B) DSC for 100 mg batch LJC-039-037-1; FIG. 11C) TGA for 100 mg batch LJC-039-037-1; FIG. 11D) 1H NMR for 100 mg batch LJC-039-037-1; FIG. 11E) GVS for 100 mg batch LJC-039-037-1; FIG. 11F) XRPD post GVS for 100 mg batch LJC-039-037-1; FIG. 11G) XRPD post stability at 40° C./75% RH for 100 mg batch LJC-039-037-1; FIG. 11H) VT XRPD for 100 mg batch LJC-039-037-1; FIG. 11I) light polarised microscopy for 100 mg batch LJC-039-037-1;
FIGS. 12A-12D show results for besylate Form 2: FIG. 12A) XRPD for 100 mg batch LJC-039-067-8; FIG. 12B) DSC for 100 mg batch LJC-039-067-8; FIG. 12C) DSC with ramp rate of 2° C./min; FIG. 12D) 1H NMR for LJC-039-067-8;
FIGS. 13A-13G show results for besylate Form 3: FIG. 13A) XRPD for LJC-039-081-2 (2nd crop from liquors of LJC-039-081-1); FIG. 13B) DSC for LJC-039-081-2; FIG. 13C) DSC for LJC-039-081-2 (2° C./min ramp rate); FIG. 13D) TGA for LJC-039-081-2; FIG. 13E)1H NMR for LJC-039-081-2; FIG. 13F) GVS for LJC-039-081-2; FIG. 13G) XRPD post GVS for LJC-039-081-2;
FIGS. 14A-14C show results for besylate Form 4: FIG. 14A) XRPD for LJC-039-086-1; FIG. 14B) DSC for LJC-039-086-1; FIG. 14C) 1H NMR for LJC-039-086-1;
FIGS. 15A and 15B show HPLC chromatographs for release batch of besylate salts, followed by FIGS. 15C and 15D showing Agilent ChemStation reports detailing results;
FIGS. 16A-16D show chiral chromatography for LJC-039-081-1, and LJC-039-083-1;
FIG. 17 shows exemplar images (ca. 4-8 mm diameter field of view) of the solid forms observed in crystallisations of compound of formula (I) besylate;
FIG. 18 shows content of the asymmetric unit in Form 1;
FIG. 19 shows molecular structure as determined by single-crystal X-ray diffraction of a crystal of compound of formula (I) besylate, Form 1, grown from a 2-methoxyethanol:pentyl acetate solution with atoms represented by thermal ellipsoids. Only hydrogens specifically located in the crystal structure are depicted;
FIG. 20 shows conformation adopted by the two independent molecules in Form 1;
FIG. 21 shows comparison of the conformation adopted by one independent molecule in Form 1 (top) and the conformation in Form 2 (bottom);
FIG. 22 shows comparison of the conformation adopted by the two independent besylates in Form 1, view along two different directions;
FIG. 23 shows comparison of the conformation adopted by one independent besylate in Form 1 (top) and the conformation in Form 2 (bottom);
FIG. 24A-24C show crystal structure, determined by single-crystal X-ray diffraction of a crystal of compound of formula (I) besylate grown from 2-methoxyethanol:pentyl acetate solution, viewed along the crystallographic a axis (FIG. 24A), b axis (FIG. 24B), and c axis (FIG. 24C);
FIG. 25 shows short contact C—O<3.6 Å, C—C<3.6 Å, and N—O<3.5 Å for Form 1;
FIG. 26 shows calculated powder pattern diffraction from single crystal X-ray diffraction data for Form 1;
FIG. 27 shows plate form crystals observed for compound of formula (I) besylate Form 2;
FIG. 28 shows content of the asymmetric unit in Form 2;
FIG. 29 shows molecular structure as determined by single-crystal X-ray diffraction of a crystal of compound of formula (I) besylate Form 2 with atoms represented by thermal ellipsoids. Only Hydrogens specifically located in the crystal structure are depicted;
FIG. 30 shows conformation adopted by the independent molecule in Form 2;
FIG. 31 shows conformation adopted by the independent besylate in Form 2, viewed along two different directions;
FIGS. 32A-32C show a crystal structure, determined by single-crystal X-ray diffraction of a crystal of compound of formula (I) besylate Form 2, viewed along the crystallographic a axis (FIG. 32A), b axis (FIG. 32B), and c axis (FIG. 32C);
FIG. 33 shows short contact C—O<3.6 Å, C—C<3.6 Å and N—O<3.5 Å for Form 2;
FIG. 34 shows calculated powder pattern diffraction from single crystal X-ray diffraction data for Form 2;
FIG. 35 shows labelling of atomic centres for Compound of formula (I) besylate Form 1; and
FIG. 36 shows labelling of atomic centres for Compound of formula (I) besylate Form 2.



DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Example 1
Solid-State Stability Study of Compound of Formula (I)
Method/Technique.
2 mg samples of compound of formula (I), accurately weighed, were placed in 4-mL clear glass screw-cap vials. Samples were tested at initial and after 34 days stored at 5° C./Ambient Relative Humidity (AMRH) Closed, 30° C./60% RH Closed, 40° C./75% RH Open and 60° C./AMRH Closed.







TABLE 1





HPLC Method Condition
















Column: Phase =
Phenomenex Luna C18(2)


Length × i.d =
100 × 4.6 mm


Particle size =
3 μm


Mobile phase:
A = 1000:1 Water/Trifluoroacetic Acid



B = 1000:0.5 Acetonitrile/Trifluoroacetic Acid


Flow rate:
1.0 mL/min


Column Temperature:
40° C.













Gradient
Time (min)
% A
% B






0.0
80
20



20.0
20
60



25.0
20
60



25.1
80
20



30.0
80
20











Detection Wavelength:
230 mm


Sample Mass Injected
μg, typically 1 μL injection of 1.0 mg compound



of formula (I)/mL in 60:40 Water/Acetonitrile


Retention Times
Compound of formula (I) elutes at



approximately 7.64 min









Results

Appearance.
Table 2 lists the appearance results.







TABLE 2







Summary of Compound of Formula (I) Appearance Data










Timepoint



Storage Condition
days
Appearance





RT
initial
Cream to light yellow powder


5 C./AMRH Closed
34
Cream to light yellow powder


30 C./60% RH Closed
34
Cream to light yellow powder


40 C./75% RH Open
34
Deliquesced yellow mass on bottom




of vial


60 C./AMRH Closed
34
Deliquesced dark yellow to orange




mass on bottom of vial









Compound of Formula (I) Content (% w/w).
The % w/w content values (see Table 3) show too much variability to detect differences between the initial value and those measured after 34 days at 5° C./AMRH Closed, 30° C./60% RH Closed or 40° C./75% RH Open. The average % w/w measured for the samples stored 34 days at 60° C./AMRH Closed show a 10% w/w decrease from the initial value.
Compound of Formula (I) Content (% Area).
The compound of formula (I) % area content (see Table 3 and FIG. 1) shows no significant change after 34 days stored at 5° C./AMRH Closed, but decreases steadily with increasing storage temperature for samples at 30° C./60% RH Closed, 40° C./75% RH Open or 60° C./AMRH Closed. Major degradation peaks are observed at RRT 0.68, 0.87 and RRT 0.90, but the chromatograms, which are relatively complex even at initial (23 peaks), also show many new small degradent peaks (e.g. 7 peaks at 30° C./60% RH Closed; 13-20 peaks at 60° C./AMRH Closed). These observations suggest multiple degradation pathways. The degradent at RRT 0.68 is tentatively identified as the ester hydrolysis product (the free acid of compound of formula (I)). It is most prevalent in the 40° C./75% RH Open samples, as would be expected for a hydrolysis product.







TABLE 3







Summary of Compound of Formula (I) HPLC Data












Compound of Formula
% Relative to



Timepoint
(I) Content
Avg. Initial %











Storage Condition
Days
% w/w
% area
area














RT
initial
100.5
95.14
Avg = 94.81


RT
initial
104.1
94.47


5 C./AMRH
34
102.6
95.30
100.52


Closed#11


30 C./60% RH
34
94.7
94.20
99.36


Closed #11


40 C./75% RH
34
105.4
93.45
98.57


Open #1


40 C./75% RH
34
100.3
93.39
98.50


Open #2


60 C./AMRH
34
93.4
87.77
92.57


Closed #1


60 C./AMRH
34
91.1
87.77
92.57


Closed #2





Notes


Only one sample was tested due to an autosampler sequencer error.






Conclusions

Compound of formula (I) is stable with respect to appearance and content for at least 34 days stored at 5° C./AMRH Closed. No change in appearance was noted at 30° C./60% RH Closed, but an approximately 0.6% drop in compound of formula (I) content relative to the initial % area was observed. Samples stored at 40° C./75% RH Open or 60° C./AMRH Closed deliquesced, became yellow to orange in colour and showed notable decreases (1.5 to 8%) in compound of formula (I) content relative to initial. Major degradation peaks at RRT 0.68, 0.87 and RRT 0.90 are observed along with numerous smaller peaks, suggesting multiple degradation pathways. The degradent at RRT 0.68 is tentatively identified as the ester hydrolysis product. These results indicate that compound of formula (I) should be stored refrigerated for long term storage.
Example 2
The solubility of the compound of formula (I) was determined in a wide range of organic solvents. The solubility data is shown in Table 4 below.










TABLE 4







Solvent
Min solvent required/mg/ml









Methanol
446



Ethanol
324



Propan-2-ol
454



Acetone
214



Toluene
460



Ethyl acetate
218



Tetrahydrofuran
311



Acetonitrile
362










The data clearly shows that the compound of formula (I) has high solubility in common organic solvents. The preferred solvents are ethanol and toluene.
Two basic centres of the free base of the compound were measured for pKa. However, the basic centre of the pyridine ring had a pKa of 1.99. The pKa of the basic centre of the imidazole ring was measured to be 4.53.
Benzene sulfonic acid was used to produce a besylate salt of the compound of formula (I). Experiments were conducted on a 20 mg scale using 6 volumes of solvent. All reactions were carried out at ambient temperature with acids charged as stock solutions in ethanol (1M) or as solids depending on solubility.
Solids isolated showed significant peak shifts in 1H NMR to confirm salt formation. X-Ray Powder Diffraction (XRPD) showed that the salt had crystalline indication. Table 5 summarises the isolated salt form.












TABLE 5







Entry
Salt
Solvent
ID









1
besylate
toluene
LJC-039-009-7










The salt was subsequently stored at 40° C./75% RH for two weeks then re-analysed by XRPD and HPLC for chemical purity to assess stability of the materials. The salt retained the same powder pattern after exposure to the humidity conditions, and also retained high chemical purity supporting improved stability.
It can be seen from the T1 purity results of the isolated salt (Table 6 below) that the besylate salt from toluene showed high purity values before and after the stability study.







TABLE 6







Summary of purity before and after 40° C./75% RH for 1 week











Entry
Salt
ID
Purity T0/%
Purity T1/%





1
besylate
LJC-039-009-7
95.9
95.9









The results above show that the besylate salt form showed high purity and favourable stability results.
Example 3
Scale up of the besylate salt to 100 mg was performed based on data in Example 2. Toluene was found to be the preferred solvent for isolating besylate salts.
Besylate Salt of Compound of Formula (I)
A scale up to 50 mg of input material was carried out in order to confirm whether or not the process would scale up, and to confirm that the material isolated was of the same crystalline form (Form 1) seen from the previous smaller scale experiment. Once the analysis confirmed the salt to be Form 1 and that the properties were in keeping with what was expected, another scale up was carried out with 100 mg of input material in order to carry out full characterisation and submit the sample for a 4 week stability study at 40° C./75% RH. Both the scaled up reactions were carried out in toluene with benzene sulfonic acid added as a solution in ethanol (1M).
Besylate Experimental Procedure
Compound of formula (I) free base (100 mg, batch 704-17) was charged to a vial and toluene (600 μl) was added at ambient temperature. To the solution benzene sulfonic acid (250 μl, 1M in ethanol) was added and the reaction mixture stirred for fifteen minutes, after which time a solid had precipitated from the solution which was filtered, washed with toluene and oven dried at 40° C. under vacuum. Analysis by XRPD showed the solid to be of identical powder pattern as other besylates generated, and the 1H NMR confirmed salt formation due to significant peak shifts.















TABLE 7










TGA


Chiral





GVS
Onset
weight
Solubility
Chemical
purity/%


Entry
ID
salt
uptake/%
melt/° C.
loss/%
mg/ml
purity/%
e.e







1
LJC-039-
besylate
2.0
201.3
4.9
8.3
97.1
94.4



037-1









The enantiomeric excess for LJC-039-037-1 was only 94.4 therefore the result was compared to another batch of besylate (LJC-039-081-1) that was isolated under identical conditions. The enantiomeric excess of this batch was 99.1%.
Process Optimisation
To improve further yields of besylate salt (Form 1) four solvents were screened (isopropyl acetate, ethyl formate, methanol and acetone). In total eight 100 mg scale reactions were conducted in these solvents with the relevant acid added as stock solution in ethanol for comparison to previous experiments.
Compound of formula (I) (batch 704-38, 100 mg) dissolved in solvent (600 μl) at ambient. Acid (250 μl, 1M stock solution in ethanol) added and all reaction mixtures stood for 48 hours at ambient. The results are summarised in Table 8.







TABLE 8







Results of process optimisation experiments





















Purity post


Table
Lab book




Purity/%
40° C./75% RH


entry
reference
Salt
Solvent
XRPD
Yield/%
area
for 4 weeks





1
LJC-039-067-2
besylate
acetone
Form 1
38
98.4
98.1


2
LJC-039-067-4
besylate
iPrOAc
Form 1
79
97.7
95.9


3
LJC-039-067-6
besylate
Ethyl
Form 1
40
98.6
98.3





formate






4
LJC-039-067-8
besylate
MeOH
Single
Not
98.1
Not






crystals,
recorded

recorded






Form 2









All reactions except that of besylate formation in methanol showed Form 1. The methanol reaction was stored at 4° C. The data obtained confirmed anhydrous besylate 1:1, and a powder pattern of the material confirmed the existence of a new form (Form 2).
It was concluded from the study that solvents such as isopropyl acetate increased the purity of the salts, however reduced the recovery. Because the previous choice of solvent (ethyl acetate) gave high yielding salts with high purity values, it was decided to use ethyl acetate for the final scale up experiments.
Besylate (Form 1) 1 g Scale-Up
A 1 g formation of the besylate salt was carried out. This successfully produced 950 mg (70% yield) of Form 1. The liquors were highly coloured (yellow) and therefore seeded with a small amount of Form 1, to assist recovery. The liquors were stored at 4° C. for 16 hours. The solid obtained displayed a new powder pattern (Form 3). The solid was analysed by thermal analysis and variable temperature XRPD to confirm whether or not it was a true polymorph or a solvate. Interpretation of the analysis concluded it not to be a solvate from the 1H NMR evidence, and the DSC showed two endothermic events confirmed by hotstage microscopy (FIG. 3). It was interpreted that the seeds of Form 1 melted at 187° C., with Form 3 melting at 200° C. The reason that Form 1 was not identified by XRPD is that this is a less sensitive technique than microscopy.
Form 3 precipitates at a lower temperature to Form 1.
Characterisation was carried out on the polymorphs to propose the relationship between them.







TABLE 9







Thermal data of besylate forms











Entry
ID
Form
Onset of Melt/° C.
ΔH/Jg−1





1
LJC-039-081-1
1
201
56


2
LJC-039-067-8
2
180
73


3
LJC-039-081-2
1, 3
187, 200
7.6, 37





The lower melting point of the small amount of Form 1 present in LJC-039-081-2 can be potentially attributed to lower purity (97.2% compared with 97.9% in LJC-039-081-1).






FIG. 4 shows the DSC of besylate forms 1 (solid) and 2 (dashed).
FIG. 5 shows the DSC of besylate forms 1 (solid) and 3 (dotted and dashed).
Example 4
Salt Stability Studies







TABLE 10







Summary Table of salt purities after 4 week stability study













Sample ID
salt
T0
T1
T2
T3
T4





LJC-039-037-1
besylate
97.1
97.3
97.4
96.7
96.7









Crystalline samples of besylate were stored at 40° C./75% RH for a total of four weeks and samples were taken for HPLC every seven days. The besylate hplc purity remained consistent up until T3 when it reached 96.7%. This value did however remain consistent to T4.
The hplc chromatographs for the besylate salt form are shown in FIG. 6 for time points week zero and week four.
It is suspected that the dominant peak prior to that of the parent is from contamination as the λmax does not match the λmax of the parent peak. It is also absent from the impurity profile of T1, T2, T3 and T4.
It can be seen from the powder patterns of the salts pre and post humidity studies that there are no changes in form.
FIG. 7 shows XRPD comparing LJC-039-037-1 (besylate salt) pre and post 4 week stability study.
Example 5
Polymorphism Investigation
In order to determine the propensity of besylate salts to exhibit polymorphism, a maturation experiment was set up using thirty solvents (fifteen neat plus their 2.5% aqueous counterparts). The solid was slurried in various solvents (see Table 11) for one week on a heat/cool cycle from ambient to 60° C. After one week the slurries were evaporated and the solids analysed by XRPD and HPLC.







TABLE 11







Results of polymorphism investigation for besylate (LJC-039-058-2)


starting hplc purity 97.7%










Entry
solvent
XRPD post 1 week
HPLC purity/% area













1
acetone
Form 1
97.5


2
THF
Form 1
97.6


3
IPA
amorphous
97.1


4
MtBE
Form 1
97.7


5
DCM
amorphous
97.4


6
EtOH
oil
not analysed


7
MEK
Form 1
97.2


8
1,4-Dioxane
Form 1
97.2


9
iPrOAc
Form 1
97.5


10
DMF
oil
not analysed


11
MeCN
Form 1
94.3


12
nBuOH
oil
not analysed


13
nPrOH
oil
not analysed


14
MlBK
Form 1
97.7


15
MeOH
oil
not analysed


16
2.5% aq acetone
Form 1
96.8


17
2.5% aq THF
amorphous
93.3


18
2.5% aq IPA
Form 1
76.1


19
2.5% aq MtBE
oil
not analysed


20
2.5% aq DCM
Form 1
97.4


21
2.5% aq EtOH
oil
not analysed


22
2.5% aq MEK
Form 1
93.9


23
2.5% aq 1,4-Dioxane
Form 1
86  


24
2.5% aq iPrOAc
oil
not analysed


25
2.5% aq DMF
oil
not analysed


26
2.5% aq MeCN
Form 1
93.3


27
2.5% aq nBuOH
oil
not analysed


28
2.5% aq nPrOH
oil
not analysed


29
2.5% aq MlBK
Form 1
97.3


30
2.5% aq MeOH
oil
not analysed









The maturation study using the besylate salt revealed no new forms. The purity results post maturation show that those slurried in acetonitrile, aqueous THF, aqueous IPA aqueous MEK, aqueous dioxane and aqueous acetonitrile degraded. This suggests that the besylate salt (Form 1) has good solution stability in neat organic solvents at high temperature.
Investigating New Forms of Besylate
Although no new forms of the besylate salt were seen from the maturation study, a new form was seen when crystals were grown in methanol. The single crystals obtained from methanol were ground in order to obtain a powder pattern. This pattern turned out to be different from Form 1. A repeat experiment was carried out in order to obtain a further supply of Form 2. It was only possible to isolate Form 2 from precipitation over 16 hours from the liquors, opposed to allowing the solvent to evaporate, this gave Form 1. Interestingly two habits were present; needles and blocks. Both showed the same powder pattern as the needle habit that was used for single crystal structure determination.
Full analysis was carried out on Form 2. It had been concluded that it was a true polymorph as the single crystal data confirmed anhydrous besylate 1:1.
FIG. 8A shows an XRPD comparison of besylate Form 1 and 2. There is an obvious difference between Form 1 (trace 1) and Form 2 (trace 2). As can be seen from the two powder patterns, both forms are very different. Thermal analysis was carried out to compare the melting points of the two forms and also thermodynamic solubility measurements recorded.
FIG. 8B shows overlays of Form 1 and 2. Form 1 and 2 show one endothermic event (melting).
Form 3 was identified when a second crop was isolated from the liquors of LJC-039-081-1 (the 1 g scale-up reaction). Analysis was carried out in order to determine whether or not it was a solvate and how the forms interconvert.
FIG. 9A shows an XRPD comparison of besylate Form 1 and 3. FIG. 9B shows overlays of Form 1, and 3.
Form 1 shows one endothermic event (melting), whereas Form 3 shows two events. Hotstage microscopy on Form 3 clearly shows two melts within 20° C. of each other. It is postulated that a small amount of the lower melting polymorph is present as it was not picked up in variable temperature XRPD, which is a less sensitive technique. It is quite possible that the first endothermic event represents Form 1 as it was used to seed the liquors that Form 3 was isolated from.
The solubility data shows that all three forms have very similar aqueous solubilities of 7.8 to 8.3 mg/ml at pH 3.
Besylate Salt Form 4
The release batch of besylate salt Form 1 (LJC-039-083-1) was of high purity (97.6%), but contained a small amount of impurity carried through from the free base (0.78%, 11.9 min RT). This impurity was observed in the DSC experiment showing an endothermic transition (onset at 130° C.). The peak was confirmed as having an unrelated λmax to that of the parent peak.
A 100 mg sample was taken for a re-crystallisation attempt from 40% isopropyl acetate/ethanol. The re-crystallisation was carried out traditionally by dissolving the salt in the minimum amount of hot solvent, then cooling slowly to ambient to yield a precipitate. The dried solid was analysed by XRPD which indicated a new form, and with thermal analysis and 1H NMR it was confirmed to be a polymorph and not a solvate. FIG. 10 shows DSC of LJC-039-086-1.
The salt screen investigations have shown that compound of formula (I) forms many salts within the appropriate pKa range, and that they are easily isolated from a range of solvents. From full characterisation of the salts, it has been determined that the besylate salts have good stability with respect to humidity. It has been concluded that there are two polymorphic forms of besylate. Form 3 came from the second crop of LJC-039-081-1 liquors after seeding with Form 1. Form 4 has been observed after a re-crystallisation of Form 1 was carried out from 40% isopropyl acetate/ethanol.
Full analytical data is shown in FIGS. 11-14 below.
Experimental Methods for Examples 2-5
Example 2
Compound of formula (I) (5 mg/well) was dissolved in solvent (ethanol, toluene, and acetonitrile) (30 μl) in HPLC vials. To the solutions, benzene sulfonic acid (11.4 μl, 1M in ethanol) was added and the reaction mixtures stood overnight at ambient. Those vials that contained solid were dried at 40° C. under vacuum, and those that remained as solutions were concentrated by evaporation and then treated with heptane. Those that precipitated were dried as mentioned, and those that oiled were stored at 4° C.
Besylate Form 1 Scale Up
Compound of formula (I) (100 mg) dissolved in ethyl acetate (600 μl) and benzene sulfonic acid (250 μl, 1M in ethanol) added. Precipitation occurred instantly and the reaction mixture was stirred for 24 hours at ambient. The solid was filtered, washed with ethyl acetate and oven dried at 40° C. under vacuum for 16 hours.
Analysis Methods
Differential Scanning Calorimetry (DSC)
DSC data was collected on a TA instrument Q1000 equipped with a 50 position autosampler. The energy and temperature calibration standard was indium. Samples were heated at a rate of 10° C./min between 25 and 350° C. A nitrogen purge at 30 ml/min was maintained over the sample.
Between 0.5 and 3 mg of sample was used, unless otherwise stated, and all samples ran in a pin holed aluminium pan.
Thermogravimetric Analysis (TGA)
TGA data was collected on a TA Instrument Q500 TGA, calibrated with Alumel and running at scan rates of 10° C./minute. A nitrogen purge at 60 ml/min was maintained over the sample.
Typically 5-10 mg of sample was loaded onto a pre-tared platinum crucible unless otherwise stated.
NMR
All spectra were collected on a Bruker 400 MHz equipped with autosampler. Samples were prepared in d6-DMSO, unless otherwise stated.
XRPD (X-Ray Powder Diffraction)
Bruker AXS C2 GADDS Diffractometer
X-ray powder diffraction patterns for the samples were acquired on a Bruker AXS C2 GADDS diffractometer using Cu Kα radiation (40 kV, 40 mA), automated XYZ stage, laser video microscope for auto-sample positioning and a HiStar 2-dimensional area detector. X-ray optics consists of a single Göbel multilayer mirror coupled with a pinhole collimator of 0.3 mm.
Beam divergence, i.e. the effective size of the X-ray beam on the sample, was approximately 4 mm. A θ-θ continuous scan mode was employed with a sample to detector distance of 20 cm which gives an effective 2θ range of 3.2-29.8°. A typical exposure time of a sample would be 120 s.
Samples run under ambient conditions were prepared as flat plate specimens using powder as received without grinding. Approximately 1-2 mg of the sample was lightly pressed on a glass slide to obtain a flat surface. Samples run under non-ambient conditions were mounted on a silicon wafer with heat conducting compound. The sample was then heated to the appropriate temperature at ca. 20° C./minute and subsequently held isothermally for ca 1 minute before data collection was initiated.
Purity Analysis:
Chemical Method
Purity analysis was performed on a HP1100 Agilent:
Method: Gradient, Reverse Phase
Method Duration/min: 34
Column: Phenomenex Gemini C18 5 μm (2.0×50 mm) (Guard cartridge Phenomenex
Gemini C18 guard cartridge 2×4 mm)
Column Temperature/° C.: 40
Injection/μl: 5
Flow Rate ml/min: 0.8
Detection: UV
Wavelength/nm: 255 (bandwidth of 90 nm), 240 (bandwidth of 80 nm), 254 (bandwidth of 8 nm)
Phase A: 2 mmol NH4HCO3 (adjusted to pH10 with NH3 solution)
Phase B: acetonitrile
Timetable:












Time/Min
% A
% B

















0
90
10


25
10
90


28.8
10
90


29
90
10


34
90
10









Chiral Method
Purity analysis was performed on a Gilson HPLC system:
Method: Isocratic, Normal Phase
Method Duration/min: 50
Column: Diacel Chiralcel OJ-H (5 μm) 4.6×250 mm (Guard cartridge Diacel Chiralcel
OJ-H analytical guard cartridge 5 μm 4.0×10 mm)
Column Temperature/° C.: 40
Injection/μl: 10
Flow Rate ml/min: 1.0
Detection: UV
Wavelength/nm: 225 (single wavelength detector)
Phase A: hexane
Phase B: ethanol
Timetable:












Time/Min
% A
% B







0
93
7









Gravimetric Vapour Sorption (GVS) Studies

All samples were run on a Hiden IGASorp moisture sorption analyser running CFRSorp software. Sample sizes were typically 10 mg. A moisture adsorption desorption isotherm was performed as outlined below (2 scans giving 1 complete cycle). All samples were loaded/unloaded at typical room humidity and temperature (40% RH, 25° C.). All samples were analysed by XRPD post GVS analysis. The standard isotherm was performed at 25° C. at 10% RH intervals over a 0-90% RH range unless otherwise stated.












Scan1
Scan2










Adsorption
Desorption
Adsorption












40
85
10


50
75
20


60
65
30


70
45
40


80
35



90
25




15




5




0









Solubility

This was measured by suspending sufficient compound in 0.25 ml of solvent (water) to give a maximum final concentration of 10 mg/ml of the parent free form of the compound. The suspension was equilibrated at 25° C. for 24 hrs followed by a pH check and filtration through a glass fibre C 96 well plate. The filtrate is then diluted down 101×. Quantitation was by HPLC with reference to a standard dissolved in DMSO at approx 0.1 mg/ml. Different volumes of the standard, diluted and undiluted tests were injected. The solubility was calculated by integration of the peak area found at the same retention time as the peak maximum in the standard injection. If there is sufficient solid in the filter plate the XRPD is normally checked for phase changes, hydrate formation, amorphization, crystallization etc.
Timetable:












Time/min
% Phase A
% Phase B

















0.0
95
5


1.0
80
20


2.3
5
95


3.3
5
95


3.5
95
5


4.4
95
5









pKa Determination

pKa determination was performed on a Sirius GlpKa instrument with D-PAS attachment. Measurements were made by potentiometric titration in MeOH:H2O mixtures at 25° C. The titration media was ionic strength adjusted with 0.15M KCl. The values found in the MeOH:H2O mixtures were extrapolated to 0% co-solvent via a Yasuda-Shedlovsky extrapolation.
Hot Stage Microscopy
Hot stage microscopy was studied using a Leica LM/DM polarised microscope combined with a Mettler-Toledo MTFP82HT hot-stage in the temperature range 25-350° C. with typical heating rates in the range 10-20° C./min. A small amount of sample was dispersed onto a glass slide with individual particles separated as well as possible. Samples were viewed under normal or cross-polarised light (coupled to a λ false-colour filter) with a ×20 objective lens.
Chiral Purity Method
System Setup
Pump: Gilson 322 binary pump
Detector: Gilson 152 UV/Vis
Autosampler: Gilson 233XL rack+Gilson 402 dual syringe pump
Column oven: Phenomenex Thermasphere TS-130
Software: Gilson Unipoint LC software
Column: Daicel Chiralcel OJ-H, 5 μm, 4.6×250 mm
Guard column: Daicel Chiralcel OJ-H analytical guard cartridge, 5 μm, 4.6×10 mm
HPLC Conditions
Channel A: Hexane (93%)
Channel B: Ethanol (7%)
Flow rate: 1.0 ml/min
Detector wavelength 225 nm
Column Temperature: 40° C.
Run time: 50.0 mins
Sample Conditions
Approximately 0.2 mg of sample was dissolved in the appropriate volume of Hexane:Ethanol 1:1 v/v to give a 0.2 mg/ml solution. This was capped and placed on a vortex mixer at high speed for a duration of  ̃15 seconds. If solid remained at this point, then the sample vial was sonicated for approximately 10 seconds followed by a further 10 to 15 seconds on the vortex mixer. 10 μl was injected onto the HPLC system. Samples were injected in duplicate following an initial duplicate injection of Hexane:Ethanol 1:1 v/v as a blank.
Example 5
Pharmacological Test Example
The anaesthetic and sedative effects of the besylate salt Form 1 of the present invention was evaluated. The besylate (benzenesulfonic acid) salt was dissolved in physiological saline for administration of the test composition to the animal. The test composition was administered to mice, placed in individual Plexiglas cages (20×10×10 cm). Mice were injected with either vehicle or test substance by the intravenous route. The latency to sleep and the duration of anaesthesia (maximum: 90 minutes after test-substance administration) were recorded. Anaesthesia is indicated by loss of the righting reflex (LRR). The righting reflex test was performed as soon as the animals appear sedated, approximately every 20-30 seconds. Once the righting reflex is absent, duration of loss of righting reflex was measured by testing for the return of the righting reflex approximately every 20-30 seconds thereafter. Eight mice were studied per group and the test was performed blind. Results from the study are given in the table below.















LATENCY




NUMBER
TO LRR
LRR DURATION (##)


TREATMENT
OF MICE
(min)
(min)











(mg/kg)
WITH
mean ± s.e.m.
mean ± s.e.m.



i.v.
LRR
(#)
(#)
p value





Vehicle
0
—
0.0 ± 0.0
—


CNS 7056X
2
—
1.7 ± 1.3 NS
0.1441


besylate (20.4)






CNS 7056X
5 +
3.0 ± 0.2
4.9 ± 1.6 *
0.0106


besylate (27.2)






CNS 7056X
6 ++
1.8 ± 0.2
6.0 ± 1.9 **
0.0038


besylate (34)






CNS 7056X
6 ++
1.6 ± 0.5
7.3 ± 2.5 **
0.0038


besylate (40.8)





Mann-Whitney U test: NS = Not Significant; *= p < 0.05; **= p < 0.01


Fisher's Exact test (number of mice with LRR): no indication = not significant; + = p < 0.05; ++ = p < 0.01


(#): not calculated if n < 3


(##): maximum = 90 minutes after injection






The results in the above table show that the besylate salt Form 1 has a short latency to loss of righting reflex and therefore a short induction time to anaesthesia in the animals. Additionally the mice recover rapidly from anaesthesia as indicated by the short duration of loss of righting reflex. Thus, this compound can provide rapid induction and recovery from anaesthesia.
Example 6
Additional Conditions for Crystallisation of Forms 2, 3, and 4
Additional conditions were tested in an attempt to reproduce the previously reported crystallisations of Forms 2, 3 and 4. However, the reported scales were substantially reduced and the methodology modified accordingly, as described below.
Form 2
5 mg of solid was dissolved in 25 ul of methanol and 10 ul of ethanol added; the solution was then chilled at 4° C. for 3 days.
Form 3
Three variants were attempted:
5 mg of solid was dissolved in 50 ul of ethanol and 120 ul of ethyl acetate added; the solution was then chilled at 4° C. for 3 days.
10.1 mg of solid was dissolved in 300 ul of ethanol and 120 ul of ethyl acetate added; the solution was then chilled at 4° C. for 3 days.
2.5 mg of solid was dissolved in 50 ul of ethanol in a silanized vial and 100 ul of ethyl acetate added; the solution was then chilled at 4° C. for 3 days.
Form 4
Three variants were attempted:
A warmed (70° C.) mixture isopropyl acetate:ethanol (40%:60% v/v) was added to 5 mg of warmed solid in 20 ul aliquots until the solid dissolved (60 ul of solvent mixture in total); the solution was then allowed to cool slowly to ambient in a thermostated waterbath initially at 70° C. over a period of hours.
5 mg of solid was dissolved in 180 ul of warmed (50° C.) isopropyl acetate:ethanol (40%:60% v/v) solvent and the solution allowed to cool slowly to ambient in a thermostated waterbath (initially at 50° C.) over a period of hours.
5 mg portion of solid was dissolved in 100 ul of warmed (50° C.) isopropyl acetate:ethanol (40%:60% v/v) solvent in a silanized vial and the solution allowed to cool slowly to ambient in a thermostated waterbath (initially at 50° C.) over a period of hours.
Each of the crystallisations yielded solid material with blade and plate-like habits, with the Form 4 crystallisations also yielding needle-like material.
Example 7
Characterisation of Compound of Formula (I) Besylate
Compound of formula (I) besylate is chiral and assumed to be of the single enantiomeric form below, i.e. the S enantiomer (consistent with the subsequently determined crystal structures):




The heterocyclic structure contains a basic Nitrogen in the imidazole ring (pKa of ca. 5), and a weaker basic Nitrogen in the pyridyl ring (pKa of ca. 2). The imidazole-Nitrogen will typically be protonated in the presence of the strongly acidic besylate (pKa ca. −0.6) in aqueous solution, with the pyridyl-Nitrogen also potentially being protonated under conditions of excess besylate.
The neutral free base form (i.e. unprotonated) of the compound is expected to be somewhat lipophilic (log Poctanol:water ca. 4.0) and thus would prefer some lipophilic environments over aqueous ones. Moreover, it is likely to retain a degree of lipophillicity even when monoprotonated (log Doctanol:water ca. 2 at pH3), although the effect of the besylate counter-ion is likely to ameliorate this tendency through its inherent hydrophilicity. The degree of lipophilicity further diminishes for the diprotonated form (log Doctanol:water ca. 0.6 at pH0).
The compound also has an excess of Hydrogen bond acceptors and therefore will be suitably partnered by Hydrogen bond donating solvents. It is thus expected that the compound will prefer solubilisation in a range of polar organic solvents such as the alcohols, particularly those which provide a partially lipophilic, Hydrogen bond donating environment. This has been borne out by experimental evidence (details of solvents used are given in Example 8):














Observed solubility



Solvent
(mg/ml)


















Formamide
350



Water
2



Dimethyl sulfoxide
500



Dimethylacetamide
200



1,2-ethanediol
60



Dimethylformamide
300



Acetonitrile
>20



Methanol
400



2-ethoxyethanol
20



2,2,2-trifluoroethanol
1000



Ethanol
100



Acetone
2



Propan-1-ol
15



Propan-2-ol
4.8



2-methoxyethanol
167



Hexafluoropropan-2-ol
>700



Dichloromethane
<<0.3



Tetrahydrofuran
2.5



Methylbenzoate
2



Ethyl acetate
0.2



Chloroform
<<0.4



1,4-dioxan
1





Soluble (>5 mg/ml), partially soluble (2.5-5 mg/ml), partially insoluble (0.5-2.5 mg/ml, insoluble (<0.5 mg/ml).


Values quoted are approximate, but experimentally confirmed.






These results highlight the good solubility of the compound in a wide variety of polar organic solvents. In particular, 2,2,2-trifluoroethanol and hexafluoropropan-2-ol are both identified as extremely good solvents for this compound. This is consistent with the considerations discussed above, both solvents being strong Hydrogen bond donors. Likewise, the more substantially lipophilic solvents are identified as poor solvents and thence potential anti-solvents for crystallisations.
Example 8
Compound of Formula (I) Besylate Crystallisations
Various conditions conducive to obtaining crystalline material of compound of formula (I) besylate Forms 1 and 2 are described. Crystallisation conditions which include alcohols or acetonitrile solvents as components, with their respectively compatible anti-solvents or co-solvents, are believed to provide the most promising conditions to yield useful crystalline material. Crystallisation using solvent/anti-solvent binary mixtures was primarily used. Crystallisations were performed by retarded evaporation from sub-saturated solutions of the compound in solvent/anti-solvent mixtures, at ambient and reduced (4° C.) temperature. Crystallisation was typically observed within 3-5 days of preparation.
Where sample quantity allowed, all crystallisation conditions were performed in duplicate in a glass 96-wellplate format; one half of each wellplate being used to duplicate the conditions in the other half of the wellplate. Cross-contamination between wells is minimised by design. All of the conditions tested behaved reproducibly in at least duplicate, most yielding solid material suitable for further analysis.
In all cases, equipment coming into contact with samples and crystallisation media were scrupulously cleaned with a variety of solvents and reagents before being bathed in ethanol and blown dry using copious evaporated nitrogen.
High quality solvents from commercial suppliers were employed, as described in Table 12.












TABLE 12





Solvent
Supplier
Cat. No.
Batch No.
Grade
Purity







1,2-dichlorobenzene
Romil
H177
E558470
SpS
>99.8%


1,4-dimethyl-
Fluka
95682
429739/1
puriss
  >99%


benzene



p.a.



1,4-dioxan
Romil
H297
H540480
SpS
>99.9%


2,2,2-trifluoro-
Romil
H860
M538412
SpS
>99.9%


ethanol







acetonitrile
Romil
H049
D531490
SpS
>99.9%


dimethylacetamide
Romil
H249
B540480
SpS
>99.9%


dimethylsulfoxide
Romil
H280
W530480
SpS
>99.9%


ethanol
Romil
H314
O533480
SpS
>99.8%


ethyl acetate
Romil
H346
T533480
SpS
>99.9%


methyl iso-butyl
Romil
H446
M539430
SpS
>99.9%


ketone







n-nonane
Romil
H568
O558450
SpS
>99.9%


pentylacetate
Fluka
46022
13248/1
puriss
>98.5%






p.a.



propan-1-ol
Romil
H624
G531460
SpS
>99.9%


propan-2-ol
Romil
H625
O530480
SpS
>99.9%


tetrachloroethylene
Romil
H702
W536450
SpS
>99.9%


tetrahydrofuran
Romil
H718
B532470
SpS
>99.9%


Acetone
Romil
H031
E559470
SpS
>99.9%


Chloroform
Romil
H135
B554470
SpS
>99.9%


Dichloromethane
Romil
H202
O554460
SpS
>99.9%


Dimethylformamide
Romil
H253
T546460
SpS
>99.9%


Formamide
Romil
H351
Q537480
BioPure
>99.9%


Hexafluoropropan-
Romil
H359
H559470
SpS
>99.9%


2-ol







Methylbenzoate
Fluke
12460
417868/1
purum
  >98%


water
Romil
H950
D537480
SpS
>99.9%









Visual analysis of the resulting crystalline morphologies was achieved using a binocular microscope (ca. 10×-40× magnification) with digital camera attached, employing both transmitted and reflected lighting as appropriate.
Visual characterisation of the solid material is summarised in Table 14 below. A predominance of blade or tabular/plate morphologies, either as unique crystals or as spherulites, was observed. Over all, there was little morphological difference between the crystallisations performed at ambient temperatures and those at 4° C., with the exception of those with ethanol as solvent where the tendency for spherulite and interface type growth diminished with lowered temperature. It is notable that the use of anti-solvent can improve the quality of the crystalline material substantially.
Example images of the crystalline material observed are presented in FIG. 17. As illustrated in this Figure, acetonitrile has a tendency to produce spherulite growth, typically seen as a consequence of poor nucleation and thence growth from poor quality crystal surfaces. In contrast, 2-methoxyethanol has a tendency to produce unique crystals of blade/needle-like morphology.
There appears to be a general preference for Form 1 to crystallise from many of the conditions. However, it is notable that Form 2 has also been observed from several crystallisation conditions, including the scaled-down analogues for obtaining Forms 3 and 4 (described in Example 6). Form 2 is observed in conditions where there are extremes of either polarity (acetonitrile:water) or lipophilicity (n-nonane) or both (dimethyl sulfoxide:1,2-dichlorobenzene). In general, the crystals of Form 2 were notable in their superior quality and distinctive well-formed plate/tabular habit.
Single Crystal X-Ray Diffraction Cell Determinations
To provide corroborative evidence of the crystalline forms generated, the cell parameters of a number of crystals of suitable quality were determined using single crystal X-ray diffraction. Crystal unit cell parameters were determined using a Kappa CCD diffractometer with Mo radiation, the crystals mounted on a glass fibre with oil and held at 260K. The parameters for Form 1 and Form 2 have been determined as summarised in Table 13.







TABLE 13







Cell parameters determined for crystals of


compound of formula (I) besylate.










Form 1
Form 2














Crystal State





Solvent
2-methoxyethanol
ethanol



Anti-solvent/Co-
pentyl acetate
ethyl acetate



solvent





Crystal Morphology
needle
plate



Crystal Size (mm)
0.8 × 0.04 × 0.02
0.7 × 0.3 × 0.25



Colour
colourless
colourless



Crystal Structure





System
monoclinic
orthorhombic



Unit Cell a (Å)
7.6868(1)
8.92130(10)



b (Å)
29.2607(5)
11.1536(2)



c (Å)
12.3756(3)
25.8345(4)



α (°)
90
90



β (°)
97.7880(8)
90



γ (°)
90
90



Volume (Å3)
2757.86(9)
2570.65(7)









The crystallisation results from solvent/co-solvent and solvent/anti-solvent conditions for compound of formula (I) besylate with single crystal X-ray diffraction unit cell results are tabulated in Table 14.







TABLE 14







Experimental crystallisation results from solvent/co-solvent and solvent/anti-solvent


conditions for compound of formula (I) besylate, with single crystal X-ray diffraction


unit cell results (X-ray results for ambient crystallisations unless otherwise stated).













X-ray Form



Co/Anti-solvent
Observed
(No & habit


Solvent
(& conditions)
Crystallisations Habit
of crystals)





methanol
ethanol (at 4° C., 3 days)
blades & plates
2 (hex, blade)


ethanol
ethyl acetate (at 4° C.,
blades & plates
2 (4 plates)



3 days)




ethanol
ethyl acetate
blades & plates
2 (6 plates)


isopropyl acetate
ethanol (70° C. → 20° C.)
blades, plates & needles
2 (2 plates)


isopropyl acetate
ethanol (50° C. → 20° C.)
blades & plates
2 (2 hex plates,





2 plates, 2





blades)


ethanol
methyl isobutyl ketone
tabular plates
2 (3 plates)



(at 4° C., 3 days,





silanized vial)




ethanol
p-cymene (at 4° C., 3
plate & tabular
2 (2 tabular)



days, silanized vial)




nonane
none (silanized vial)
blades & plates
2 (plate)


dimethylsulfoxide
1,2-dichlorobenzene
intergrown blades
2 (tabular)




dendrite, one huge tabular



dimethylacetamide
methyl isobutyl ketone
plate-like fragments
1 (blade)


dimethylacetamide
tetrachloroethylene
intergrown blades
1 (2 blades)


acetonitrile
water
interface
2 (2 tabular)


acetonitrile
3-methylbutan-1-ol
triangular plates,
1 (blade)




fragments & dendrite



acetonitrile
1,2-dichlorobenzene
spherulite blades
1 (2 blades)


acetonitrile
pentyl acetate
spherulite blades
1 (blade)


methanol
none
interface plates
2 (plate)


methanol
3-methylbutan-1-ol
triangular plates &
1 (2 blades)




fragments



methanol
methyl isobutyl ketone
fragments & blade
1 (blade)


2,2,2-
1,2-dichlorobenzene
interface & blade
1 (trans, blade)


trifluoroethanol

opaque & translucent





blades



2,2,2-
1,4-dimethylbenzene
plate-like fragments
1 (sph, plate)


trifluoroethanol





ethanol
methyl isobutyl ketone
interface plates
1 (interface),




(5° C.: tabular & plate)
2 (tabular)


ethanol
1,2-dichlorobenzene
interface plates,
2 (plate)




(5° C.: needles)



ethanol
tetrachloroethylene
interface (5° C.:
2 (blade 4° C.)




hexagonal tabular)



ethanol
1,4-dimethylbenzene
interface blades
1 (blade)


propan-1-ol
none
plate-like fragments
1 (plate)


propan-1-ol
1,2-dichlorobenzene
interface
1 (blade)


propan-1-ol
tetrachloroethylene
plate-like fragments &
1 (blade)




interface



propan-2-ol
1,2-dichlorobenzene
fan needles & dendrite
1 (blade)


propan-2-ol
n-nonane
blades, needles &
1 (needle)




spherulite needles



2-methoxy ethanol
water
blade
1 (2 blades)


2-methoxy ethanol
pentyl acetate
needles
1 (blade)


2-methoxy ethanol
1,4-dimethylbenzene
blades & needles
1 (blade)


2-methoxy ethanol
n-nonane
blades & dendrite
1 (blade)


tetrahydrofuran
water
plate
1 (plate)


tetrahydrofuran
3-methylbutan-1-ol
intergrown blades
1 (plate)


tetrahydrofuran
1,2-dichlorobenzene
prismatic tabular,
2 (3 tabular)




fragments, powder



tetrahydrofuran
ethyl acetate
dendrite, interface
2 (plate 4° C.)


tetrahydrofuran
isopropyl acetate
intergrown plates
1 (plate)




& intergrown blades



tetrahydrofuran
1,3-dimethylbenzene
intergrown blades
1 (blade)


1,4-dioxane
pentyl acetate
triangular plates,
1 (2 tri plate)




some part of spherulite



1,4-dioxane
1,4-dimethylbenzene
blade
1 (blade)









A variety of crystals of suitable quality for full single crystal X-ray diffraction crystal structure determination were achieved and the full structure obtained for Forms 1 and 2. These crystal structures are reported in Examples 9 and 10.
Example 9
Crystal Structure of Form 1
Crystals of compound of formula (I) besylate grown from a 2-methoxyethanol:pentyl acetate solution which have a needle habit, are imaged in FIG. 17.
A single needle habit crystal (ca. 0.8×0.04×0.02 mm in size) was selected and its cell parameters determined at 260K and then at 190K. No transition was observed on lowering the temperature between 260-190K. The structure analysed here is for the data at 190K; parameters of the crystal and the X-ray diffraction refinement are given in Table 15.







TABLE 15





Data of the 2-methoxyethanol:pentyl acetate grown crystal of


compound of formula (I) besylate, Form 1.
















Crystal State



Code
CNS7056 besylate


Solvent
2-methoxyethanol


Anti-solvent/Co-solvent
pentyl acetate


Crystal Morphology
needle


Crystal Size (mm)
0.8 × 0.04 × 0.02


Colour
colourless


Crystal Structure



Formula
C54H50Br2N8O10S2


Formula Weight
1194.98


System
monoclinic


Space Group
P 21


Unit Cell a (Å)
7.6868(1)


b (Å)
29.2607(5)


c (Å)
12.3756(3)


α (°)
90


β (°)
97.7880(8)


γ (°)
90


Volume (Å3)
2757.86(9)


Z (No. molecules in unit)
2


Z′ (No. molecules in asymmetric unit)
2


Density (g cm3)
1.439


Absorption μ [MoKα] (mm−1)
1.610


F(000)
1224


Data Collection



Temperature, (K)
190


Instrument
Kappa CCD diffractometer


Scan Type
ω


Absorption Correction Type
multi-scan


No. of Measured Reflections
9868


No. of Independent Reflections
9848


θ min/max (°)
 1.80/27.49


h min/max
−9/9


k min/max
−37/36


l min/max
−15/15


Refinement



Refinement On
F


I/σ(I) Cut-off
3


No. of Used Reflections
6821


No. of Parameters
686


R factor (%)
6.34


Rw factor (%)
6.39


S
1.00


Δρ(min) Å−3
−0.8


Δρ(max) Å−3
0.8


Max Shift/Error
0.0005


Flack Parameter
0.027(11)









The content of the asymmetric unit is displayed in FIG. 18. It consists of two independent molecules of the compound and two independent besylate counter ions. Each compound has the imidazole-Nitrogen protonated.
The Flack “Enantiopole” parameter was determined as 0.03(1) and thus the stereochemistry of the structures depicted here are well established and are consistent with the purported stereochemistry for the compound:




Crystallographic co-ordinates and other relevant data are tabulated in the form of a SHELX file in Table 17.
The conformational disorder can be represented (in first approximation) by the “thermal ellipsoids” of the atomic positions, as presented on FIG. 19. It can be seen that the major regions of disorder lie in the methyl groups and in the besylate.
The difference between the two independent molecules comes mainly from the ester chains as seen in FIG. 20. One molecule has the ester chain being coplanar with the imidazole ring, whereas the other molecule has the ester chain being orthogonal.
The conformation of the ester chains are different to that adopted in Form 2 (FIG. 21). The orthogonal conformation observed in Form 1 bears the greatest similarity to that found in Form 2.
The two independent besylates have staggered conformations (FIG. 22). No substantial differences in bond lengths are apparent.
One besylate adopts the conformation observed for the besylate in Form 2 (FIG. 23).
The resolved crystal structure, viewed along the crystallographic a, b and c axes, is illustrated in FIGS. 24a, b and c respectively. FIG. 25 summarises the shortest contacts observed in the crystal packing.
Each compound interacts with the two independent besylates. In particular, a short distance (hydrogen-bond type) is established between one oxygen atom of one besylate and the protonated nitrogen of the imidazole ring of the compound. The second independent compound interacts similarly, but with the second independent besylate.
Other close contacts (C—O, H—O) are observed between the compounds and the besylates mainly in the vicinity of the imidazole and pyridyl ring. Some close contacts are also observed between the two compounds themselves (Br—N, C—C, O—H) and the two besylate themselves (O—H contacts) but to a lesser extent for the latter.
Using the crystal structure determined experimentally, a powder diffraction pattern for Form 1 has been calculated using CrystalDiffract® (CrystalDiffract is a registered TradeMark of CrystalMaker Ltd) and is depicted in FIG. 26. This powder pattern matches the experimental powder pattern reported for Form 1.
Example 10
Crystal Structure of Form 2
A crystal of compound of formula (I) besylate Form 2, which has a plate habit, is imaged in FIG. 27.
A single plate habit crystal (ca. 0.7×0.30×0.25 mm in size) was selected and its cell parameters determined at 260K then at 190K. No transition was observed on lowering the temperature between 260-190K. The structure analysed here is for the data at 190K; parameters of the crystal and the X-ray diffraction refinement are given in Table 16.







TABLE 16





Data of the ethanol:ethyl acetate grown crystal of compound


of formula (I) besylate, Form 2.
















Crystal State



Code
CNS7056 besylate


Solvent
ethanol


Anti-solvent/Co-solvent
ethyl acetate


Crystal Morphology
plate


Crystal Size (mm)
0.7 × 0.30 × 0.25


Colour
colourless


Crystal Structure



Formula
C27H25Br1N4O5S1


Formula Weight
597.49


System
Orthorhombic


Space Group
P 212121


Unit Cell a (Å)
8.92130(10)


b (Å)
11.1526(2)


c (Å)
25.8345(4)


α (°)
90


β (°)
90


γ (°)
90


Volume (Å3)
2570.65(7)


Z (No. molecules in unit)
4


Z′ (No. molecules in asymmetric unit)
1


Density (g cm3)
1.544


Absorption μ [MoKα] (mm−1)
1.727


F(000)
1224


Data Collection



Temperature, (K)
190


Instrument
Kappa CCD diffractometer


Scan Type
ω


Absorption Correction Type
multi-scan


No. of Measured Reflections
5750


No. of Independent Reflections
5727


θ min/max (°)
 5.15/27.48


h min/max
−11/11


k min/max
−14/14


l min/max
−33/33


Refinement



Refinement On
F


I/σ(I) Cut-off
3


No. of Used Reflections
4067


No. of Parameters
344


R factor (%)
3.85


Rw factor (%)
3.66


S
1.12


Δρ(min) Å−3
−0.6


Δρ(max) Å−3
0.5


Max Shift/Error
0.0003


Flack Parameter
0.011(9)









The content of the asymmetric unit is displayed in FIG. 28. It consists of one independent molecule of the compound and one independent besylate. The compound has the imidazole-Nitrogen protonated.
The Flack “Enantiopole” parameter was determined as 0.011(9) and thus the stereochemistry of the structures depicted here are well established and are consistent with the purported stereochemistry for the compound. Crystallographic co-ordinates and other relevant data are tabulated in the form of a SHELX file in Table 18.
The conformational disorder can be represented (in first approximation) by the “thermal ellipsoids” of the atomic positions, as presented on FIG. 29. It can be seen that the major regions of disorder lie in the besylate.
As discussed above, the conformation of the ester chain in Form 2, depicted in FIG. 30, is different to that adopted in Form 1.
However, the conformation of the besylate is similar to the one observed for one of the besylate in Form 1 (FIG. 31).
The resolved crystal structure, viewed along the crystallographic a, b and c axes, is illustrated in FIGS. 32a, b and c respectively with FIG. 33 summarising the shortest contacts observed in the crystal packing. The compound establishes a short contact (hydrogen-bond type) with one oxygen atom of the besylate through its protonated nitrogen of the imidazole ring. Other short contacts (C—C, C—O, H—O) are observed between the compound and the besylate through the imidazole ring.
Some close contacts are also observed between the two compounds themselves (Br—C, C—C, O—C, O—H), most of which are via the ester chain. There are no close contacts between the besylate themselves.
Using the crystal structure determined experimentally, a powder diffraction pattern for Form 2 has been calculated using CrystalDiffract® (FIG. 34). This powder pattern matches the experimental powder pattern reported for Form 2.







TABLE 17





Crystallographic co-ordinates and other relevant data tabulated in the form


of a SHELX File for Compound of formula (I) besylate Form 1.

















TITL 12161316 Compound CNS7056 Form 1



CELL 0.71073 7.687 29.261 12.376 90.000 97.788 90.000



ZERR 2 0.0001 0.0005 0.0003 0.0000 0.0008 0.0000



LATT −1



SYMM −X, Y + 0.500, −Z



SFAC C 2.3100 20.8439 1.0200 10.2075 1.5886 0.5687 0.8650 =



51.6512 0.2156 0.0033 0.0016 1.15 0.7700 12.0110



SFAC H 0.4930 10.5109 0.3229 26.1257 0.1402 3.1424 0.0408 =



57.7998 0.0030 0.0000 0.0000 0.06 0.3200 1.0079



SFAC O 3.0485 13.2771 2.2868 5.7011 1.5463 0.3239 0.8670 =



32.9089 0.2508 0.0106 0.0060 3.25 0.7700 15.9994



SFAC BR 17.1789 2.1723 5.2358 16.5796 5.6377 0.2609 3.9851 =



41.4328 2.9557 −0.2901 2.4595 1000.00 1.1000 79.9040



SFAC N 12.2126 0.0057 3.1322 9.8933 2.0125 28.9975 1.1663 =



0.5826 −11.5290 0.0061 0.0033 1.96 0.7700 14.0067



SFAC S 6.9053 1.4679 5.2034 22.2151 1.4379 0.2536 1.5863 =



56.1720 0.8669 0.1246 0.1234 53.20 1.1100 32.0660



UNIT 108. 100. 20. 4. 16. 4.



S80 6 0.23964 0.43139 0.09908 11.00000 0.04634 0.03299 =



0.04052 0.00002 0.01880 −0.00340



O81 3 0.16028 0.39374 0.15143 11.00000 0.06864 0.04111 =



0.05255 −0.00210 0.02801 0.00002



O82 3 0.14598 0.47435 0.11207 11.00000 0.08099 0.03603 =



0.04614 0.00545 0.03373 −0.00236



O83 3 0.42589 0.43401 0.12925 11.00000 0.05754 0.08564 =



0.05198 −0.01536 0.01792 −0.00644



C84 1 0.20581 0.41866 −0.04324 11.00000 0.05949 0.04444 =



0.02903 0.00359 0.01728 0.00704



C85 1 0.03624 0.41100 −0.09142 11.00000 0.06649 0.10092 =



0.05586 0.01088 0.01751 0.00507



C86 1 0.00323 0.39810 −0.20187 11.00000 0.08670 0.14765 =



−0.02096 −0.03160 −0.00004



C87 1 0.14311 0.39209 −0.25693 11.00000 0.07916 0.11651 =



0.06238 −0.01696 0.00195 0.02481



C88 1 0.30473 0.39806 −0.20987 11.00000 0.09246 0.09710 =



0.04155 0.00157 0.01795 0.02685



C89 1 0.33456 0.41126 −0.10133 11.00000 0.05999 0.09817 =



0.07178 −0.01451 0.00886 0.02173



S90 6 0.68868 0.81145 0.51625 11.00000 0.04072 0.02869 =



0.05437 0.00158 0.00214 0.00223



O91 3 0.79129 0.77464 0.57315 11.00000 0.08025 0.03751 =



0.04867 −0.00213 −0.00954 0.01626



O92 3 0.52601 0.81933 0.56122 11.00000 0.04778 0.05360 =



0.06934 −0.00642 0.01702 0.00039



O93 3 0.78935 0.85213 0.50763 11.00000 0.07515 0.04369 =



0.05025 −0.01354 0.01764 −0.01547



C94 1 0.62446 0.78970 0.38130 11.00000 0.04232 0.04028 =



0.05049 0.00898 0.00929 0.00525



C95 1 0.74659 0.76959 0.32396 11.00000 0.06194 0.06998 =



0.03238 0.00341 −0.00103 0.00990



C96 1 0.69911 0.75023 0.22476 11.00000 0.12417 0.10337 =



0.03441 0.01537 0.02421 0.03314



C97 1 0.51941 0.75295 0.17732 11.00000 0.11897 0.11939 =



−0.01324 −0.00963 −0.00586



C98 1 0.40301 0.77268 0.23169 11.00000 0.06106 0.10242 =



0.00570 −0.01263 −0.00283



C99 1 0.45446 0.79193 0.33547 11.00000 0.05307 0.07089 =



0.00728 −0.00426 −0.01944



BR1 4 0.06011 0.52462 0.55140 11.00000 0.04153 0.05204 =



0.07369 −0.00524 0.02434 0.00670



C2 1 0.25757 0.50395 0.49005 11.00000 0.02832 0.04536 =



0.03350 −0.00752 0.01511 0.00763



C3 1 0.28921 0.45781 0.47911 11.00000 0.03135 0.03107 =



0.04579 0.00145 0.00221 −0.00479



C4 1 0.42954 0.44393 0.43174 11.00000 0.03767 0.03461 =



−0.00320 −0.00151 −0.00125



C5 1 0.54674 0.47556 0.39943 11.00000 0.03535 0.02939 =



0.03479 −0.00390 0.00647 0.00183



C6 1 0.51907 0.52242 0.41134 11.00000 0.04226 0.03479 =



0.04333 −0.00172 0.00236 0.00188



C7 1 0.37213 0.53602 0.45794 11.00000 0.03598 0.02793 =



0.04586 −0.00044 0.01652 0.00336



C8 1 0.64321 0.55824 0.38118 11.00000 0.03964 0.02453 =



0.02719 0.00516 0.00457 0.00373



C9 1 0.68998 0.59645 0.46059 11.00000 0.03743 0.03694 =



0.04454 −0.00375 0.01588 0.00649



N10 5 0.69097 0.58514 0.56581 11.00000 0.06070 0.03116 =



0.04918 −0.00640 0.02020 −0.00054



C11 1 0.74090 0.61847 0.63822 11.00000 0.06804 0.05787 =



0.04752 −0.00600 0.01695 −0.00669



C12 1 0.78515 0.66221 0.61053 11.00000 0.05480 0.04458 =



0.05526 −0.02125 0.01554 −0.00787



C13 1 0.77550 0.67229 0.50132 11.00000 0.04463 0.03102 =



0.05452 0.00407 0.01432 −0.00038



C14 1 0.73186 0.63955 0.42553 11.00000 0.04272 0.03021 =



0.04282 −0.00243 0.01499 0.00270



N15 5 0.71451 0.55972 0.29408 11.00000 0.04979 0.02502 =



0.03692 0.00975 0.01748 0.00775



C16 1 0.67500 0.52204 0.21324 11.00000 0.04463 0.02346 =



0.04948 −0.00464 0.01738 0.00561



C17 1 0.75857 0.47996 0.26673 11.00000 0.04549 0.02673 =



0.01954 −0.00693 0.00506 −0.00121



N18 5 0.70009 0.45973 0.35317 11.00000 0.03293 0.02806 =



0.02597 −0.00088 0.00321 0.00207



C19 1 0.81334 0.42409 0.39181 11.00000 0.03678 0.02848 =



0.03351 −0.00426 0.00585 0.00488



C20 1 0.93968 0.42402 0.32661 11.00000 0.03371 0.02802 =



0.03711 0.00202 0.00106 0.00680



N21 5 0.90585 0.45925 0.25315 11.00000 0.04775 0.03416 =



0.02231 −0.01051 0.01052 −0.00308



C22 1 0.79597 0.39511 0.48941 11.00000 0.03997 0.03711 =



0.04548 0.01039 0.00508 0.00197



C23 1 0.74788 0.53407 0.10940 11.00000 0.05650 0.04712 =



0.03514 0.00836 0.00449 0.00605



C24 1 0.68780 0.50047 0.01647 11.00000 0.08242 0.04077 =



0.03001 −0.00046 0.01385 0.00523



C25 1 0.71419 0.51690 −0.09234 11.00000 0.06429 0.06543 =



0.03392 0.00018 0.00559 −0.00499



O26 3 0.76261 0.55440 −0.11450 11.00000 0.12347 0.08282 =



0.04188 0.01501 0.01658 −0.04001



O27 3 0.65910 0.48459 −0.16756 11.00000 0.10340 0.06919 =



0.03191 0.00253 0.01824 −0.00449



C28 1 0.66642 0.49760 −0.27953 11.00000 0.19131 0.12699 =



0.01390 −0.01417 0.02134 −0.05279



BR51 4 1.06737 0.71057 0.98743 11.00000 0.03812 0.08781 =



0.06774 0.00566 −0.00531 0.00447



C52 1 0.84276 0.73306 0.93243 11.00000 0.03132 0.05952 =



0.03819 0.00358 0.00226 −0.00263



C53 1 0.81293 0.77906 0.93249 11.00000 0.04627 0.06820 =



0.03723 −0.00581 0.00481 −0.00474



C54 1 0.65043 0.79579 0.88269 11.00000 0.04551 0.03939 =



0.04858 −0.00084 0.00376 −0.01071



C55 1 0.51946 0.76552 0.84226 11.00000 0.04294 0.03573 =



0.03413 0.00062 0.00952 −0.00208



C56 1 0.54512 0.71765 0.84581 11.00000 0.02688 0.03659 =



0.04586 −0.00025 0.00561 0.00047



C57 1 0.71139 0.70186 0.88914 11.00000 0.03105 0.04840 =



0.04447 −0.00668 −0.00429 0.00504



C58 1 0.40956 0.68443 0.79765 11.00000 0.03348 0.02893 =



0.04334 0.00070 0.00351 0.00421



C59 1 0.38048 0.64253 0.86694 11.00000 0.03165 0.03488 =



0.04951 0.00002 0.00425 0.00528



N60 5 0.42879 0.64650 0.97247 11.00000 0.03542 0.05694 =



0.03178 0.00872 0.00154 0.00467



C61 1 0.38962 0.61026 1.03529 11.00000 0.04457 0.06338 =



0.05765 0.01416 0.00707 0.00171



C62 1 0.30187 0.57202 0.98967 11.00000 0.06548 0.04957 =



0.11303 0.03456 0.03582 0.00696



C63 1 0.25733 0.56863 0.88018 11.00000 0.07395 0.04664 =



0.09803 0.00115 0.01240 −0.01007



C64 1 0.29561 0.60475 0.81590 11.00000 0.08355 0.04152 =



0.05459 −0.00010 0.00128 −0.02308



N65 5 0.31344 0.68797 0.70771 11.00000 0.03846 0.03072 =



0.04952 −0.00160 0.00032 0.00597



C66 1 0.33129 0.72953 0.64125 11.00000 0.03574 0.02676 =



0.05519 0.00406 0.00580 0.00330



C67 1 0.26347 0.76733 0.70231 11.00000 0.03803 0.03316 =



0.04166 0.01528 0.00868 0.00029



N68 5 0.35122 0.78274 0.79764 11.00000 0.03387 0.03259 =



0.05055 0.00549 0.00427 0.00218



C69 1 0.24763 0.81583 0.84108 11.00000 0.05345 0.03305 =



0.04570 0.00005 0.02067 −0.00546



C70 1 0.09873 0.81841 0.77077 11.00000 0.04465 0.03799 =



0.06107 0.00794 0.01464 0.00936



N71 5 0.10819 0.78841 0.68720 11.00000 0.03892 0.03266 =



0.05306 0.00974 0.01063 0.00803



C72 1 0.30218 0.84064 0.94469 11.00000 0.08091 0.04934 =



0.08052 −0.01505 0.02392 −0.00661



C73 1 0.22541 0.72388 0.52948 11.00000 0.04039 0.05583 =



0.03295 0.00047 0.00724 −0.00165



C74 1 0.30154 0.68566 0.46508 11.00000 0.05896 0.05343 =



0.05504 −0.00576 0.00667 0.02016



C75 1 0.18003 0.67204 0.36587 11.00000 0.05296 0.05447 =



0.04241 0.00546 0.01355 0.00171



O76 3 0.06782 0.69497 0.31818 11.00000 0.05552 0.07543 =



0.05719 −0.00702 −0.00194 0.02108



O77 3 0.22119 0.62976 0.33149 11.00000 0.08466 0.04267 =



0.04376 −0.00714 0.00726 0.00488



C78 1 0.10717 0.61220 0.23887 11.00000 0.06302 0.09312 =



0.07465 −0.02449 0.02418 −0.00980



H611 2 10.42342 10.61111 11.10933 11.00000 0.06582



H621 2 10.27371 10.54835 11.03412 11.00000 0.09086



H631 2 10.20282 10.54235 10.84949 11.00000 0.08585



H641 2 10.26600 10.60396 10.74163 11.00000 0.07058



H661 2 10.45616 10.73494 10.63683 11.00000 0.04658



H701 2 10.00528 10.83765 10.77749 11.00000 0.05724



H721 2 10.20390 10.85662 10.96784 11.00000 0.10482



H722 2 10.39143 10.86250 10.93477 11.00000 0.10500



H723 2 10.34863 10.81975 11.00178 11.00000 0.10479



H731 2 10.22647 10.75279 10.49048 11.00000 0.05050



H732 2 10.10462 10.71635 10.53573 11.00000 0.05107



H741 2 10.41143 10.69632 10.44327 11.00000 0.06599



H742 2 10.32279 10.65905 10.51273 11.00000 0.06616



H571 2 10.73613 10.67093 10.88928 11.00000 0.04893



H531 2 10.89874 10.79871 10.96543 11.00000 0.05990



H541 2 10.63029 10.82681 10.87790 11.00000 0.05285



H161 2 10.54702 10.51731 10.19609 11.00000 0.04687



H201 2 11.03302 10.40374 10.33036 11.00000 0.03977



H221 2 10.90306 10.37871 10.51025 11.00000 0.06107



H222 2 10.77354 10.41394 10.54853 11.00000 0.06102



H223 2 10.70245 10.37370 10.47387 11.00000 0.06087



H231 2 10.71028 10.56434 10.08666 11.00000 0.05487



H232 2 10.87494 10.53365 10.12431 11.00000 0.05471



H241 2 10.56546 10.49241 10.01723 11.00000 0.06095



H242 2 10.75795 10.47323 10.02815 11.00000 0.06099



H111 2 10.74728 10.61186 10.71244 11.00000 0.06882



H121 2 10.81997 10.68398 10.66349 11.00000 0.06182



H131 2 10.79812 10.70154 10.48020 11.00000 0.05215



H141 2 10.72939 10.64544 10.35226 11.00000 0.04595



H71 2 10.35042 10.56684 10.46668 11.00000 0.04408



H31 2 10.21444 10.43638 10.50355 11.00000 0.04223



H41 2 10.44931 10.41280 10.42055 11.00000 0.04056



H891 2 10.44977 10.41481 9.93226 11.00000 0.09285



H881 2 10.39917 10.39332 9.75106 11.00000 0.09266



H871 2 10.12372 10.38356 9.66972 11.00000 0.10194



H861 2 9.88808 10.39388 9.76390 11.00000 0.11607



H851 2 9.94416 10.41466 9.94909 11.00000 0.08904



H951 2 10.86472 10.76918 10.35546 11.00000 0.06580



H961 2 10.78321 10.73544 10.18942 11.00000 0.10497



H971 2 10.48493 10.74055 10.10914 11.00000 0.10604



H981 2 10.28646 10.77378 10.20054 11.00000 0.08719



H991 2 10.37377 10.80653 10.37249 11.00000 0.07037



H781 2 10.14480 10.58182 10.22240 11.00000 0.11588



H782 2 10.11102 10.63197 10.17669 11.00000 0.11581



H783 2 9.98883 10.61082 10.25546 11.00000 0.11600



H711 2 10.01359 10.78308 10.62464 11.00000 0.05205



H211 2 10.98261 10.46785 10.19729 11.00000 0.04161



H281 2 10.62358 10.47180 9.67092 11.00000 0.11566



H282 2 10.59036 10.52501 9.70225 11.00000 0.11566



H283 2 10.79029 10.50514 9.71088 11.00000 0.11566
















TABLE 18





Crystallographic co-ordinates and other relevant data tabulated in the


form of a SHELX File for Compound of formula (I) besylate Form 2.

















TITL 1142055 Compound CNS7056 form 2



CELL 0.71073 8.921 11.154 25.834 90.000 90.000 90.000



ZERR 4 0.0001 0.0002 0.0004 0.0000 0.0000 0.0000



LATT −1



SYMM X + 0.500, −Y + 0.500, −Z



SYMM −X, Y + 0.500, −Z + 0.500



SYMM −X + 0.500, −Y, Z + 0.500



SFAC C 2.3100 20.8439 1.0200 10.2075 1.5886 0.5687 0.8650 =



51.6512 0.2156 0.0033 0.0016 1.15 0.7700 12.0110



SFAC H 0.4930 10.5109 0.3229 26.1257 0.1402 3.1424 0.0408 =



57.7998 0.0030 0.0000 0.0000 0.06 0.3200 1.0079



SFAC BR 17.1789 2.1723 5.2358 16.5796 5.6377 0.2609 3.9851 =



41.4328 2.9557 −0.2901 2.4595 1000.00 1.1000 79.9040



SFAC N 12.2126 0.0057 3.1322 9.8933 2.0125 28.9975 1.1663 =



0.5826 −11.5290 0.0061 0.0033 1.96 0.7700 14.0067



SFAC O 3.0485 13.2771 2.2868 5.7011 1.5463 0.3239 0.8670 =



32.9089 0.2508 0.0106 0.0060 3.25 0.7700 15.9994



SFAC S 6.9053 1.4679 5.2034 22.2151 1.4379 0.2536 1.5863 =



56.1720 0.8669 0.1246 0.1234 53.20 1.1100 32.0660



UNIT 108. 100. 4. 16. 20. 4.



BR1 3 −0.04819 −0.10880 −0.27710 11.00000 0.07032 0.03277 =



0.00144 −0.01238 −0.02224



C2 1 −0.15018 −0.21830 −0.32054 11.00000 0.02777 0.02177 =



−0.00009 −0.00209 −0.00471



C3 1 −0.17401 −0.18875 −0.37205 11.00000 0.02963 0.01861 =



0.02702 0.00623 0.00188 −0.00107



C4 1 −0.24491 −0.26965 −0.40362 11.00000 0.02825 0.02442 =



0.01718 0.00327 0.00106 −0.00145



C5 1 −0.29275 −0.37943 −0.38401 11.00000 0.02223 0.01822 =



0.01875 −0.00067 0.00141 0.00066



C6 1 −0.27139 −0.40894 −0.33163 11.00000 0.02028 0.01967 =



0.01926 0.00182 0.00105 −0.00153



C7 1 −0.20042 −0.32532 −0.29979 11.00000 0.02809 0.02763 =



0.01685 0.00206 0.00190 −0.00055



C8 1 −0.32197 −0.52600 −0.30927 11.00000 0.01670 0.02233 =



0.00135 −0.00476 −0.00144



C9 1 −0.39853 −0.52353 −0.25770 11.00000 0.01623 0.02317 =



0.00259 −0.00384 −0.00281



N10 4 −0.46099 −0.41943 −0.24363 11.00000 0.02251 0.02613 =



0.02353 −0.00189 0.00408 0.00155



C11 1 −0.52777 −0.41652 −0.19697 11.00000 0.02617 0.03441 =



0.02357 −0.00451 0.00365 0.00346



C12 1 −0.53610 −0.51390 −0.16425 11.00000 0.02740 0.04329 =



0.02040 −0.00335 0.00652 −0.00779



C13 1 −0.47518 −0.62062 −0.17997 11.00000 0.03584 0.03200 =



0.02405 0.00767 0.00645 −0.00687



C14 1 −0.40334 −0.62685 −0.22730 11.00000 0.02879 0.02223 =



0.02565 0.00090 0.00272 −0.00057



N15 4 −0.30040 −0.62781 −0.33049 11.00000 0.02151 0.02416 =



0.01713 0.00287 −0.00002 0.00182



C16 1 −0.21928 −0.62991 −0.38036 11.00000 0.02330 0.02286 =



0.01602 0.00057 0.00417 0.00450



C17 1 −0.32510 −0.57975 −0.41920 11.00000 0.02824 0.02308 =



0.01704 −0.00121 0.00336 −0.00285



N18 4 −0.36294 −0.46298 −0.41818 11.00000 0.02482 0.02037 =



0.01483 0.00150 −0.00070 0.00079



C19 1 −0.46920 −0.44117 −0.45641 11.00000 0.03022 0.02725 =



0.01634 0.00325 0.00039 −0.00224



C20 1 −0.49445 −0.54753 −0.47911 11.00000 0.03071 0.03401 =



0.00110 −0.00174 −0.00215



N21 4 −0.40440 −0.63226 −0.45591 11.00000 0.03619 0.02354 =



0.02146 −0.00463 0.00147 −0.00154



C22 1 −0.54310 −0.32298 −0.46595 11.00000 0.03636 0.03429 =



0.00778 −0.00982 −0.00011



C23 1 −0.15995 −0.75547 −0.39193 11.00000 0.03430 0.02640 =



0.01793 −0.00359 0.00177 0.00554



C24 1 −0.06166 −0.79435 −0.34621 11.00000 0.04707 0.03881 =



0.02350 0.00041 0.00034 0.01530



C25 1 0.06625 −0.87542 −0.35603 11.00000 0.03182 0.02650 =



0.00340 −0.00125 −0.00016



O26 5 0.17233 −0.88334 −0.32760 11.00000 0.03778 0.06570 =



0.03313 −0.01160 −0.01173 0.00417



O27 5 0.05245 −0.94265 −0.39885 11.00000 0.03130 0.03874 =



0.02467 −0.00799 −0.00330 0.01418



C28 1 0.17574 −1.02443 −0.40865 11.00000 0.05622 0.08123 =



0.03697 −0.01153 −0.00496 0.04396



S80 6 −0.94275 −0.52899 −0.49624 11.00000 0.03340 0.02679 =



0.02442 0.00000 0.00210 −0.00075



O81 5 −0.83867 −0.47114 −0.53020 11.00000 0.05118 0.08336 =



0.02297 −0.00622 −0.02476



O82 5 −1.08156 −0.46260 −0.49186 11.00000 0.04015 0.07788 =



0.05503 −0.01022 −0.00539 0.01721



O83 5 −0.97025 −0.65272 −0.50726 11.00000 0.13945 0.03230 =



0.06071 −0.01467 0.01447 −0.00725



C84 1 −0.86288 −0.52210 −0.43343 11.00000 0.02735 0.05893 =



0.02832 0.01509 0.00686 −0.00534



C85 1 −0.87781 −0.41462 −0.40588 11.00000 0.03763 0.08695 =



0.03855 −0.01799 0.00427 −0.00754



C86 1 −0.81420 −0.39965 −0.35764 11.00000 0.05438 0.16315 =



0.04455 −0.02905 0.00147 −0.02905



C87 1 −0.73766 −0.49241 −0.33773 11.00000 0.06202 0.20226 =



0.03510 −0.02105 −0.05062



C88 1 −0.71835 −0.60444 −0.36221 11.00000 0.04217 0.17120 =



0.11388 0.10762 −0.01320 −0.03729



C89 1 −0.78500 −0.61610 −0.41251 11.00000 0.03725 0.08786 =



0.05538 −0.00772 −0.01074



H891 2 9.22557 9.31210 9.56883 11.00000 0.08027



H881 2 9.33331 9.33306 9.65289 11.00000 0.13097



H851 2 9.06867 9.64846 9.57936 11.00000 0.06577



H861 2 9.17563 9.67239 9.66111 11.00000 0.10509



H161 2 9.86530 9.42517 9.62245 11.00000 0.02469



H111 2 9.42959 9.65626 9.81326 11.00000 0.03383



H121 2 9.41618 9.49292 9.86839 11.00000 0.03606



H131 2 9.51614 9.31066 9.84059 11.00000 0.03697



H141 2 9.64103 9.30191 9.76144 11.00000 0.03108



H231 2 9.89972 9.24922 9.57680 11.00000 0.03066



H232 2 9.75764 9.18723 9.60372 11.00000 0.03099



H241 2 9.87585 9.16237 9.67759 11.00000 0.04434



H242 2 9.97980 9.27746 9.67100 11.00000 0.04489



H281 2 10.15353 8.92912 9.56085 11.00000 0.08666



H282 2 10.18989 8.92278 9.62053 11.00000 0.08723



H283 2 10.26566 9.02166 9.58620 11.00000 0.08710



H201 2 9.44027 9.43682 9.49457 11.00000 0.03327



H221 2 9.36727 9.66624 9.51370 11.00000 0.05146



H222 2 9.52479 9.72860 9.51527 11.00000 0.05104



H223 2 9.43193 9.71611 9.56601 11.00000 0.05131



H41 2 9.73983 9.74902 9.56204 11.00000 0.02807



H31 2 9.85823 9.88568 9.61518 11.00000 0.03001



H71 2 9.81367 9.65791 9.73490 11.00000 0.02870



H871 2 9.30621 9.51762 9.69480 11.00000 0.13226



H211 2 9.59801 9.29339 9.53630 11.00000 0.03270
















TABLE 19





Bond lengths for Compound of formula (I) besylate Form 1.






















S80
O81
1.454(5)
Å
S80
O82
1.468(5)
Å


S80
O83
1.432(6)
Å
S80
C84
1.784(7)
Å


C84
C85
1.376(12)
Å
C84
C89
1.318(12)
Å


C85
C86
1.408(14)
Å
C85
H851
0.927
Å


C86
C87
1.360(16)
Å
C86
H861
0.936
Å


C87
C88
1.310(15)
Å
C87
H871
0.934
Å


C88
C89
1.386(14)
Å
C88
H881
0.935
Å


C89
H891
0.932
Å
S90
O91
1.459(5)
Å


S90
O92
1.454(6)
Å
S90
O93
1.431(5)
Å


S90
C94
1.793(8)
Å
C94
C95
1.383(11)
Å


C94
C99
1.354(11)
Å
C95
C96
1.356(13)
Å


C95
H951
0.938
Å
C96
C97
1.428(17)
Å


C96
H961
0.934
Å
C97
C98
1.323(15)
Å


C97
H971
0.924
Å
C98
C99
1.409(13)
Å


C98
H981
0.927
Å
C99
H991
0.924
Å


Br1
C2
1.886(6)
Å
C2
C3
1.382(9)
Å


C2
C7
1.381(9)
Å
C3
C4
1.358(10)
Å


C3
H31
0.928
Å
C4
C5
1.388(9)
Å


C4
H41
0.937
Å
C5
C6
1.398(9)
Å


C5
N18
1.454(8)
Å
C6
C7
1.394(9)
Å


C6
C8
1.498(9)
Å
C7
H71
0.926
Å


C8
C9
1.500(9)
Å
C8
N15
1.274(8)
Å


C9
N10
1.343(9)
Å
C9
C14
1.386(9)
Å


N10
C11
1.345(10)
Å
C11
C12
1.379(11)
Å


C11
H111
0.933
Å
C12
C13
1.375(11)
Å


C12
H121
0.927
Å
C13
C14
1.351(10)
Å


C13
H131
0.918
Å
C14
H141
0.921
Å


N15
C16
1.492(9)
Å
C16
C17
1.500(9)
Å


C16
C23
1.511(9)
Å
C16
H161
0.988
Å


C17
N18
1.352(8)
Å
C17
N21
1.315(8)
Å


N18
C19
1.400(8)
Å
C19
C20
1.344(9)
Å


C19
C22
1.496(9)
Å
C20
N21
1.376(8)
Å


C20
H201
0.927
Å
N21
H211
1.000
Å


C22
H221
0.958
Å
C22
H222
0.950
Å


C22
H223
0.953
Å
C23
C24
1.536(11)
Å


C23
H231
0.962
Å
C23
H232
0.969
Å


C24
C25
1.470(11)
Å
C24
H241
0.971
Å


C24
H242
0.962
Å
C25
O26
1.202(10)
Å


C25
O27
1.354(10)
Å
O27
C28
1.445(10)
Å


C28
H281
1.000
Å
C28
H282
1.000
Å


C28
H283
1.000
Å
Br51
C52
1.886(7)
Å


C52
C53
1.366(11)
Å
C52
C57
1.412(10)
Å


C53
C54
1.404(11)
Å
C53
H531
0.927
Å


C54
C55
1.383(10)
Å
C54
H541
0.921
Å


C55
C56
1.414(9)
Å
C55
N68
1.427(9)
Å


C56
C57
1.396(9)
Å
C56
C58
1.489(9)
Å


C57
H571
0.925
Å
C58
C59
1.530(10)
Å


C58
N65
1.254(8)
Å
C59
N60
1.314(9)
Å


C59
C64
1.391(10)
Å
N60
C61
1.372(10)
Å


C61
C62
1.386(14)
Å
C61
H611
0.918
Å


C62
C63
1.355(15)
Å
C62
H621
0.928
Å


C63
C64
1.378(13)
Å
C63
H631
0.932
Å


C64
H641
0.917
Å
N65
C66
1.485(8)
Å


C66
C67
1.474(9)
Å
C66
C73
1.516(10)
Å


C66
H661
0.982
Å
C67
N68
1.354(9)
Å


C67
N71
1.334(8)
Å
N68
C69
1.406(9)
Å


C69
C70
1.343(11)
Å
C69
C72
1.484(12)
Å


C70
N71
1.366(10)
Å
C70
H701
0.925
Å


N71
H711
1.000
Å
C72
H721
0.964
Å


C72
H722
0.958
Å
C72
H723
0.965
Å


C73
C74
1.535(10)
Å
C73
H731
0.975
Å


C73
H732
0.967
Å
C74
C75
1.493(12)
Å


C74
H741
0.972
Å
C74
H742
0.977
Å


C75
O76
1.185(9)
Å
C75
O77
1.360(9)
Å


O77
C78
1.440(11)
Å
C78
H781
0.965
Å


C78
H782
0.966
Å
C78
H783
0.960
Å
















TABLE 20





Angles for Compound of formula (I) besylate Form 1






















O81
S80
O82
111.0(3)°
O81
S80 
O83 
112.9(4)°


O82
S80
O83
114.4(4)°
O81
S80
C84
105.5(3)°


O82
S80
C84
106.2(3)°
O83
S80
C84
106.0(4)°


S80
C84
C85
117.7(6)°
S80
C84
C89
123.6(7)°


C85
C84
C89
118.3(8)°
C84
C85
C86
120.0(9)°


C84
C85
H851
119.626°
C86
C85
H851
120.377°


C85
C86
C87
118.1(10)°
C85
C86
H861
120.636°


C87
C86
H861
121.303°
C86
C87
C88
121.8(10)°


C86
C87
H871
119.251°
C88
C87
H871
118.984°


C87
C88
C89
119.3(10)°
C87
C88
H881
120.392°


C89
C88
H881
120.264°
C84
C89
C88
122.5(10)°


C84
C89
H891
118.485°
C88
C89
H891
119.061°


O91
S90
O92
111.7(3)°
O91
S90
O93
112.8(4)°


O92
S90
O93
113.5(3)°
O91
S90
C94
104.5(3)°


O92
S90
C94
105.7(3)°
O93
S90
C94
108.0(3)°


S90
C94
C95
120.6(6)°
S90
C94
C99
120.1(6)°


C95
C94
C99
119.3(8)°
C94
C95
C96
121.6(9)°


C94
C95
H951
118.566°
C96
C95
H951
119.820°


C95
C96
C97
118.4(10)°
C95
C96
H961
119.911°


C97
C96
H961
121.695°
C96
C97
C98
119.9(8)°


C96
C97
H971
119.699°
C98
C97
H971
120.397°


C97
C98
C99
120.8(9)°
C97
C98
H981
119.080°


C99
C98
H981
120.094°
C94
C99
C98
119.9(9)°


C94
C99
H991
119.276°
C98
C99
H991
120.819°


Br1
C2
C3
121.0(5)°
Br1
C2
C7
118.5(5)°


C3
C2
C7
120.5(5)°
C2
C3
C4
119.7(6)°


C2
C3
H31
120.203°
C4
C3
H31
120.109°


C3
C4
C5
120.6(6)°
C3
C4
H41
120.600°


C5
C4
H41
118.766°
C4
C5
C6
120.6(6)°


C4
C5
N18
119.6(5)°
C6
C5
N18
119.8(6)°


C5
C6
C7
117.8(6)°
C5
C6
C8
123.3(6)°


C7
C6
C8
118.8(6)°
C2
C7
C6
120.6(6)°


C2
C7
H71
119.721°
C6
C7
H71
119.679°


C6
C8
C9
117.5(5)°
C6
C8
N15
126.6(6)°


C9
C8
N15
115.9(6)°
C8
C9
N10
114.9(6)°


C8
C9
C14
121.2(6)°
N10
C9
C14
123.9(6)°


C9
N10
C11
115.5(6)°
N10
C11
C12
124.4(7)°


N10
C11
H111
118.526°
C12
C11
H111
117.061°


C11
C12
C13
117.4(7)°
C11
C12
H121
121.279°


C13
C12
H121
121.289°
C12
C13
C14
120.4(6)°


C12
C13
H131
119.499°
C14
C13
H131
120.125°


C9
C14
C13
118.3(6)°
C9
C14
H141
120.274°


C13
C14
H141
121.419°
C8
N15
C16
118.0(5)°


N15
C16
C17
105.9(5)°
N15
C16
C23
109.4(5)°


C17
C16
C23
112.4(5)°
N15
C16
H161
110.723°


C17
C16
H161
109.539°
C23
C16
H161
108.851°


C16
C17
N18
122.7(6)°
C16
C17
N21
130.3(6)°


N18
C17
N21
106.5(5)°
C5
N18
C17
123.1(5)°


C5
N18
C19
127.0(5)°
C17
N18
C19
109.8(5)°


N18
C19
C20
105.2(5)°
N18
C19
C22
125.3(6)°


C20
C19
C22
129.4(6)°
C19
C20
N21
108.0(5)°


C19
C20
H201
126.017°
N21
C20
H201
126.026°


C17
N21
C20
110.5(5)°
C17
N21
H211
124.840°


C20
N21
H211
124.681°
C19
C22
H221
109.508°


C19
C22
H222
109.778°
H221
C22
H222
108.808°


C19
C22
H223
110.905°
H221
C22
H223
108.786°


H222
C22
H223
109.018°
C16
C23
C24
112.3(6)°


C16
C23
H231
109.392°
C24
C23
H231
108.812°


C16
C23
H232
108.378°
C24
C23
H232
109.105°


H231
C23
H232
108.825°
C23
C24
C25
114.3(7)°


C23
C24
H241
109.968°
C25
C24
H241
110.030°


C23
C24
H242
108.195°
C25
C24
H242
105.346°


H241
C24
H242
108.752°
C24
C25
O26
126.4(7)°


C24
C25
O27
109.4(7)°
O26
C25
O27
123.9(7)°


C25
O27
C28
115.2(7)°
O27
C28
H281
109.674°


O27
C28
H282
109.261°
H281
C28
H282
109.475°


O27
C28
H283
109.465°
H281
C28
H283
109.476°


H282
C28
H283
109.476°
Br51
C52
C53
119.3(6)°


Br51
C52
C57
119.0(5)°
C53
C52
C57
121.7(7)°


C52
C53
C54
118.9(7)°
C52
C53
H531
120.141°


C54
C53
H531
120.985°
C53
C54
C55
119.8(7)°


C53
C54
H541
120.227°
C55
C54
H541
120.000°


C54
C55
C56
122.1(6)°
C54
C55
N68
119.4(6)°


C56
C55
N68
118.5(6)°
C55
C56
C57
117.2(6)°


C55
C56
C58
123.2(6)°
C57
C56
C58
119.5(6)°


C52
C57
C56
120.2(7)°
C52
C57
H571
119.709°


C56
C57
H571
120.138°
C56
C58
C59
116.5(6)°


C56
C58
N65
126.7(6)°
C59
C58
N65
116.8(6)°


C58
C59
N60
116.3(6)°
C58
C59
C64
118.5(7)°


N60
C59
C64
125.0(7)°
C59
N60
C61
116.1(7)°


N60
C61
C62
121.7(8)°
N60
C61
H611
119.342°


C62
C61
H611
118.993°
C61
C62
C63
120.6(8)°


C61
C62
H621
120.029°
C63
C62
H621
119.353°


C62
C63
C64
118.4(9)°
C62
C63
H631
120.452°


C64
C63
H631
121.124°
C59
C64
C63
118.1(8)°


C59
C64
H641
120.844°
C63
C64
H641
121.057°


C58
N65
C66
118.2(6)°
N65
C66
C67
105.4(5)°


N65
C66
C73
109.7(5)°
C67
C66
C73
111.5(6)°


N65
C66
H661
109.122°
C67
C66
H661
108.890°


C73
C66
H661
112.017°
C66
C67
N68
121.8(6)°


C66
C67
N71
130.3(7)°
N68
C67
N71
107.4(6)°


C55
N68
C67
122.5(6)°
C55
N68
C69
128.7(6)°


C67
N68
C69
108.7(6)°
N68
C69
C70
105.5(6)°


N68
C69
C72
124.0(7)°
C70
C69
C72
130.5(7)°


C69
C70
N71
109.1(6)°
C69
C70
H701
125.444°


N71
C70
H701
125.502°
C67
N71
C70
109.2(6)°


C67
N71
H711
125.400°
C70
N71
H711
125.366°


C69
C72
H721
110.667°
C69
C72
H722
109.838°


H721
C72
H722
108.539°
C69
C72
H723
110.831°


H721
C72
H723
108.455°
H722
C72
H723
108.445°


C66
C73
C74
111.0(6)°
C66
C73
H731
108.535°


C74
C73
H731
110.248°
C66
C73
H732
110.751°


C74
C73
H732
108.249°
H731
C73
H732
108.042°


C73
C74
C75
112.4(6)°
C73
C74
H741
108.496°


C75
C74
H741
109.125°
C73
C74
H742
108.155°


C75
C74
H742
108.578°
H741
C74
H742
110.035°


C74
C75
O76
126.2(7)°
C74
C75
O77
110.7(7)°


O76
C75
O77
123.0(7)°
C75
O77
C78
115.6(7)°


O77
C78
H781
109.214°
O77
C78
H782
109.848°


H781
C78
H782
109.923°
O77
C78
H783
109.687°


H781
C78
H783
109.026°
H782
C78
H783
109.127°
















TABLE 21





Bond Lengths for Compound of formula (I) besylate Form 2.






















Br1
C2
1.892(3)
Å
C2
C3
1.387(5)
Å


C2
C7
1.383(5)
Å
C3
C4
1.371(5)
Å


C3
H31
0.938
Å
C4
C5
1.392(5)
Å


C4
H41
0.921
Å
C5
C6
1.406(4)
Å


C5
N18
1.428(4)
Å
C6
C7
1.395(5)
Å


C6
C8
1.497(4)
Å
C7
H71
0.924
Å


C8
C9
1.497(4)
Å
C8
N15
1.276(4)
Å


C9
N10
1.338(4)
Å
C9
C14
1.395(5)
Å


N10
C11
1.345(4)
Å
C11
C12
1.378(5)
Å


C11
H111
0.935
Å
C12
C13
1.370(5)
Å


C12
H121
0.948
Å
C13
C14
1.382(5)
Å


C13
H131
0.936
Å
C14
H141
0.934
Å


N15
C16
1.478(4)
Å
C16
C17
1.487(5)
Å


C16
C23
1.527(5)
Å
C16
H161
0.976
Å


C17
N18
1.346(4)
Å
C17
N21
1.320(4)
Å


N18
C19
1.391(4)
Å
C19
C20
1.342(5)
Å


C19
C22
1.494(5)
Å
C20
N21
1.378(5)
Å


C20
H201
0.912
Å
N21
H211
0.854
Å


C22
H221
0.965
Å
C22
H222
0.966
Å


C22
H223
0.960
Å
C23
C24
1.534(5)
Å


C23
H231
0.969
Å
C23
H232
0.981
Å


C24
C25
1.478(5)
Å
C24
H241
0.960
Å


C24
H242
0.988
Å
C25
O26
1.201(4)
Å


C25
O27
1.342(4)
Å
O27
C28
1.451(5)
Å


C28
H281
0.964
Å
C28
H282
0.965
Å


C28
H283
0.962
Å
S80
O81
1.431(3)
Å


S80
O82
1.447(3)
Å
S80
O83
1.430(3)
Å


S80
C84
1.774(4)
Å
C84
C85
1.400(7)
Å


C84
C89
1.369(7)
Å
C85
C86
1.380(7)
Å


C85
H851
0.932
Å
C86
C87
1.342(13)
Å


C86
H861
0.943
Å
C87
C88
1.410(13)
Å


C87
H871
0.934
Å
C88
C89
1.433(10)
Å


C88
H881
0.925
Å
C89
H891
0.940
Å
















TABLE 22





Angles for Compound of formula (I) besylate Form 2.






















Br1
C2
C3
119.3(3)°
Br1
C2
C7
118.9(3)°


C3
C2
C7
121.8(3)°
C2
C3
C4
119.0(3)°


C2
C3
H31
120.033°
C4
C3
H31
120.959°


C3
C4
C5
120.3(3)°
C3
C4
H41
119.485°


C5
C4
H41
120.261°
C4
C5
C6
121.0(3)°


C4
C5
N18
118.9(3)°
C6
C5
N18
120.1(3)°


C5
C6
C7
118.2(3)°
C5
C6
C8
122.3(3)°


C7
C6
C8
119.5(3)°
C2
C7
C6
119.7(3)°


C2
C7
H71
120.432°
C6
C7
H71
119.874°


C6
C8
C9
117.7(3)°
C6
C8
N15
124.4(3)°


C9
C8
N15
117.9(3)°
C8
C9
N10
116.6(3)°


C8
C9
C14
120.0(3)°
N10
C9
C14
123.4(3)°


C9
N10
C11
116.7(3)°
N10
C11
C12
123.7(3)°


N10
C11
H111
117.041°
C12
C11
H111
119.278°


C11
C12
C13
118.8(3)°
C11
C12
H121
120.443°


C13
C12
H121
120.783°
C12
C13
C14
119.3(3)°


C12
C13
H131
120.694°
C14
C13
H131
119.952°


C9
C14
C13
118.1(3)°
C9
C14
H141
120.942°


C13
C14
H141
120.983°
C8
N15
C16
117.6(3)°


N15
C16
C17
105.7(3)°
N15
C16
C23
110.8(3)°


C17
C16
C23
115.7(3)°
N15
C16
H161
107.681°


C17
C16
H161
107.726°
C23
C16
H161
108.910°


C16
C17
N18
120.7(3)°
C16
C17
N21
131.2(3)°


N18
C17
N21
108.0(3)°
C5
N18
C17
122.3(3)°


C5
N18
C19
128.6(3)°
C17
N18
C19
109.0(3)°


N18
C19
C20
105.7(3)°
N18
C19
C22
124.9(3)°


C20
C19
C22
129.3(3)°
C19
C20
N21
108.6(3)°


C19
C20
H201
127.007°
N21
C20
H201
124.433°


C17
N21
C20
108.7(3)°
C17
N21
H211
125.926°


C20
N21
H211
125.351°
C19
C22
H221
110.223°


C19
C22
H222
109.368°
H221
C22
H222
108.664°


C19
C22
H223
111.184°
H221
C22
H223
109.452°


H222
C22
H223
107.885°
C16
C23
C24
107.9(3)°


C16
C23
H231
107.712°
C24
C23
H231
110.073°


C16
C23
H232
111.123°
C24
C23
H232
109.430°


H231
C23
H232
110.583°
C23
C24
C25
118.8(3)°


C23
C24
H241
107.661°
C25
C24
H241
104.516°


C23
C24
H242
109.365°
C25
C24
H242
106.503°


H241
C24
H242
109.671°
C24
C25
O26
123.3(3)°


C24
C25
O27
114.4(3)°
O26
C25
O27
122.4(3)°


C25
O27
C28
115.2(3)°
O27
C28
H281
108.952°


O27
C28
H282
110.269°
H281
C28
H282
109.738°


O27
C28
H283
108.681°
H281
C28
H283
110.225°


H282
C28
H283
108.963°
O81
S80
O82
111.9(2)°


O81
S80
O83
115.1(2)°
O82
S80
O83
111.2(3)°


O81
S80
C84
106.30(18)°
O82
S80
C84
104.5(2)°


O83
S80
C84
107.0(2)°
S80
C84
C85
117.6(4)°


S80
C84
C89
122.1(4)°
C85
C84
C89
120.2(5)°


C84
C85
C86
121.6(6)°
C84
C85
H851
119.148°


C86
C85
H851
119.275°
C85
C86
C87
117.5(8)°


C85
C86
H861
121.859°
C87
C86
H861
120.606°


C86
C87
C88
124.9(7)°
C86
C87
H871
117.763°


C88
C87
H871
117.376°
C87
C88
C89
116.0(7)°


C87
C88
H881
122.592°
C89
C88
H881
121.435°


C84
C89
C88
119.8(8)°
C84
C89
H891
120.080°


C88
C89
H891
120.078°

## Claims

1. A pharmaceutical composition, comprising a besylate salt of the compound of formula (I)



and at least one pharmaceutically acceptable carrier, excipient or diluent, wherein said composition maintains at least 96.7% of the original amount of the compound of formula (I) in said composition following storage of said composition at 40° C. and 75% relative humidity for 4 weeks, and wherein said amount of the compound of formula (I) in said composition is measured by high-performance liquid chromatography.

2. The pharmaceutical composition according to claim 1, wherein said composition maintains at least 97.3% of the original amount of the compound of formula (I) in said composition following storage of said composition at 40° C. and 75% relative humidity for 2 weeks, and wherein said amount of the compound of formula (I) in said composition is measured by high-performance liquid chromatography.

3. The pharmaceutical composition according to claim 1, wherein said composition maintains at least 97.3% of the original amount of the compound of formula (I) in said composition following storage of said composition at 40° C. and 75% relative humidity for 1 week, and wherein said amount of the compound of formula (I) in said composition is measured by high-performance liquid chromatography.