WO 2006/128150 PCT/US2006/020871
CRYSTALLINE SOLID FORMS OF TIGECYCLINE AND
METHODS OF PREPARING SAME
This application claims benefit of U.S. Provisional Application No.
60/684,955, filed May 27, 2005, the contents of which are incorporated herein by
reference.
[001] The present invention relates to crystalline solid forms of
tigecycline, compositions thereof, and processes for preparing them.
[002] Tigecycline is an antibiotic in the tetracycline family and a chemical
analog of minocycline. It has been used as a treatment against drug-resistant
bacteria, and has been shown to work where other antibiotics have failed. For
example, it is active against methicillin-resistant Staphylococcus aureus, penicillin-
resistant Streptococcus pneumoniae, vancomycin-resistant enterococci (DJ.
Beidenbach et al., Diagnostic Microbiology and Infectious Disease 40:173-177
(2001); H.W. Boucher et al., Antimicrobial Agents & Chemotherapy 44:2225-2229
(2000); PA Bradford Clin. Microbiol. Newslett. 26:163-168 (2004); D. Milatovic et
al., Antimicrob. Agents Chemother. 47:400-404 (2003); R. Patel et al., Diagnostic
Microbiology and Infectious Disease 38:177-179 (2000); P.J. Petersen et al.,
Antimicrob. Agents Chemother. 46:2595-2601 (2002); and P.J. Petersen et al.,
Antimicrob. Agents Chemother. 43:738-744(1999)), and organisms carrying either
of the two major forms of tetracycline resistance: efflux and ribosomal protection
(C. Betriu et al., Antimicrob. Agents Chemother. 48:323-325 (2004); T. Hirata et
al. Antimicrob. Agents Chemother. 48:2179-2184 (2004); and P.J. Petersen et al.,
Antimicrob. Agents Chemother. 43:738-744(1999)).
[003] Tigecycline has historically been administered intravenously
because it has exhibited generally poor bioavailability when given orally. The
intravenous solution may be prepared by reconstitutution of an amorphous
powder with sterile water, 0.9% Sodium Chloride Injection, USP, or 5% Dextrose
Injection, USP. Tigecycline is typically rendered into the amorphous powder via
lyophilization without excipients for sterilization purposes. Due to the propensity
for tigecycline to degrade, however, these powders are prepared and processed
under iow-oxygen and low-temperature conditions. Such processing is expensive
because it requires special equipment and handling. Additionally, amorphous
materials are generally less stable than crystalline forms of the same compound.
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WO 2006/128150 PCT/US2006/020871
(Polymorphism in Pharmaceutical Solids, H. G. Brittain (ed.), 1999, p.208). It
would be advantageous, therefore, if one were able to use and manufacture
crystalline solid forms of tigecycline without the need for special handling systems.
[004] Crystalline compounds are solids with ordered arrays of molecules,-
whereas amorphous compounds are composed of disordered molecules. These
arrays are also termed crystal lattices and are composed of repeating structural
segments called unit cells. When the same molecule, such as an organic
molecule, can order itself in a solid in more than one way, that molecule exhibits
what is called polymorphism. For example, the element carbon exhibits
polymorphism (in elements it is termed aliotropism). Solid carbon exists in three
known crystalline solid forms: graphite, diamond, and fullerenes. Although each
crystalline solid form is carbon, each has different properties because the solid-
state structure of each form differs. For example, whereas diamond is one of the
hardest substances known, graphite is extremely soft. Many organic compounds
are also known to be polymorphic in that their structures differ in how they pack
together to form crystalline solids. (See e.g., Stephenson, G. A; Stowell, J. G;
Toma, P.H; Dorman, D.E.; Greene, J.R.; Byrne, S. R.; "Solid state analysis of
polymorphic, isomorphic and solvated forms of Dirithromycin". J. Am. Chem. Soc,
1994,116,5766.)
[005] Based on a chemical structure, which is the chemical connectivity
of atoms to make a molecule, one cannot predict with any degree of certainty
whether a compound will crystallize, under what conditions it will crystallize, how
many crystalline solid forms of the compound might exist, or the solid-state
structure of any of those forms. The term "solid-state structure" as used herein
means the structure obtained when molecules pack together to form a solid.
[006] Sometimes solvent or water moiecuies become incorporated into
the crystal lattice of a crystalline solid. Such a crystalline solid may be referred to
as a solvate or hydrate, respectively. Solvates, hydrates, and polymorphs are
often called crystalline solid forms. Here, as in most of the solid-state chemical
arts, weakly bound solvates and hydrates are also included as crystalline solid
forms where the solvent or water molecules are in channels or not incorporated
into the crystal lattice. Amorphous forms are often referred to as solid forms but
they are not crystalline solid forms.
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[007] Different crystalline solid forms of the same compound often
possess different solid-state properties such as melting point, solubility, handling,
and stability. Thus, once different crystalline solid forms of the same compound
have been identified, the optimum crystalline solid form under any given set of
processing and manufacturing conditions may be determined as well as the
different solid-state properties of each crystalline solid form.
[008] There are a number of analytical methods one of ordinary skill in
the art in solid-state chemistry can use to analyze solid forms. The term "analyze"
as used herein means to obtain information about the solid-state structure of solid
forms. For example, X-ray powder diffraction is a suitable technique for
differentiating amorphous solid forms from crystalline solid forms and for
characterizing and identifying crystalline solid forms of a compound. X-ray
powder diffraction is also suitable for quantifying the amount of a crystalline solid
form (or forms) in a mixture. In X-ray powder diffraction, X-rays are directed onto
a crystal and the intensity of the diffracted X-rays is measured as a function of
twice the angle between the X-ray source and the beam diffracted by the sample.
The intensity of these diffracted X-rays can be plotted on a graph as peaks with
the x-axis being twice the angle (this is known as the "29" angle) between the X-
ray source and the diffracted X-rays and with the y-axis being peak intensity of the
diffracted X-rays. This graph is called an X-ray powder diffraction pattern or
powder pattern. Different crystalline solid forms exhibit different powder patterns
because the location of the peaks on the x-axis is a property of the solid-state
structure of the crystal.
[009] Such powder patterns, or portions thereof, can be used as an
identifying fingerprint for a crystalline solid form. Thus, one could take a powder
pattern of an unknown sample and compare that powder pattern with a reference
powder pattern. A positive match would mean that the unknown sample is of the
same crystalline solid form as that of the reference. One could also analyze an
unknown sample containing a mixture of solid forms by adding and subtracting
powder patterns of known compounds.
[010] When selecting peaks in a powder pattern to characterize a
crystalline solid form or when using a reference powder pattern to identify a form,
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one identifies a peak or collection of peaks in one form that are not present in the
other solid forms.
[011] The term "characterize" as used herein means to select an
appropriate set of data capable of distinguishing one solid form from another.
That set of data in X-ray powder diffraction is the position of one or more peaks.
Selecting which tigecycline X-ray powder diffraction peaks define a particular form
is said to characterize that form.
[012] The term "identify" as used herein means taking a selection of
characteristic data for a solid form and using those data to determine whether that
form is present in a sample. In X-ray powder diffraction, those data are the x-axis
positions of the one or more peaks characterizing the form in question as
discussed above. For example, once one determines that a select number of X-
ray diffraction peaks characterize a particular solid form of tigecycline, one can
use those peaks to determine whether that form is present in a sample containing
tigecycline.
[013] When characterizing and/or identifying crystalline solid forms of the
same chemical compound with X-ray powder diffraction, it is often not necessary
to use the entire powder pattern, A smaller subset of the entire powder pattern
can often be used to perform the characterization and/or identification. By
selecting a collection of peaks that differentiate the crystalline solid form from
other crystalline solid forms of the compound, one can rely on those peaks to both
characterize the form and to identify the form in, for example, an unknown
mixture. Additional data can be added, such as from another analytical technique
or additional peaks from the powder pattern, to characterize and/or identify the
form should, for instance, additional polymorphs be identified later.
[014] Due to differences in instruments, samples, and sample
preparation, peak values are reported with the modifier "about" in front of the peak
values. This is common practice in the solid-state chemical arts because of the
variation inherent in peak values. A typical precision of the 20 x-axis value of a
peak in a powder pattern is on the order of plus or minus 0.2° 20. Thus, a powder
diffraction peak that appears at "about 9.2° 20," means that the peak could be
between 9.0° 20 and 9.4° 20 when measured on most X-ray diffractometers under
most conditions. Variability in peak intensity is a result of how individual crystals
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WO 2006/128150 PCT/US2006/020871
are oriented in the sample container with respect to the external X-ray source
(known as "preferred orientation"). This orientation effect does not provide
structural information about the crystal.
[015] X-ray powder diffraction is just one of several analytical techniques
one may use to characterize and/or identify crystalline solid forms. Spectroscopic
techniques such as Raman (including microscopic Raman), infrared, and solid-
state NMR spectroscopies may be used to characterize and/or identify crystalline
solid forms. These techniques may also be used to quantify the amount of one or
more crystalline solid forms in a mixture.
[016] Thermal techniques such as melting point do not necessarily, in
and of themselves, characterize and/or identify different crystalline solid forms of a
compound because it is possible that different crystalline solid forms of the same
compound would have indistinguishable melting points. In such circumstances,
however, melting points could be used together with another analytical method,
such as X-ray powder diffraction, to characterize and/or identify crystalline solid
forms.
[017] The present invention is directed to crystalline solid forms of
tigecycline identified as Form I, Form II. Form 111. Form IV. and Form V. The
invention is also directed to compositions, including pharmaceutical compositions,
containing one or more crystalline solid forms of tigecycline. The invention is
further directed to processes for preparing crystalline solid forms of tigecycline.
BRIEF DESCRIPTION OF THE DRAWINGS
[018] F1G.1 is the X-ray powder diffraction pattern and peak list for Form
I tigecycline.
[019] FIG. 2 is the X-ray powder diffraction pattern and peak list for Form
II tigecycline.
[020] FIG. 3 is the X-ray powder diffraction pattern and peak list for Form
III tigecycline.
[021 ] FIG. 4 is the X-ray powder diffraction and peak list for Form IV
tigecycline.
[022] FIG. 5 is the X-ray powder diffraction and peak list for Form V
tigecyciine.
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WO 2006/128150 PCT/US2006/020871
[023] FIG. 6 is an X-ray powder diffraction overlay of Forms I-V of
tigecycline.
[024] FIG. 7 is an expanded version of the X-ray powder diffraction
overlay of Forms I-V of tigecycline.
[025] FIG. 8 is a thermal gravimetric analysis ("TGA") of Form II
tigecycline.
[026] FIG. 9 is a TGA heat-cool cycle of Form II tigecycline.
DETAILED DESCRIPTION OF THE INVENTION
[027] Two X-ray diffractometers were used in the work leading to the
present invention. The data in FIG. 1 and FIG. 2 were collected using a Rigaku
Miniflex Diffraction System (Rigaku MSC Inc., Tokyo, Japan). The powder
samples were deposited on a zero-background polished silicon sample holder. A
normal focus copper X-ray tube is operated at 30kV and 15 mA, and the
instrument is equipped with a Ni KD filter. Sample scanning is at 0.027step from
3.00 to 40.00°29. The data processing is done using Jade 6.0 software
(Molecular Data Systems Inc., Livermore, CA). The data in FIG. 3 and FIG. 4,
and FIG. 5 were collected using a Scintag Advanced Diffraction System Model X2
(Scintag, Inc. Cupertino, CA) with a quartz sample holder. The copper X-ray
generator is operated at 45 kV and 40 mA, and scanning is done.from 3 to 40° 20
at 0.02° per step. Data processing is done using Jade 6.0 software (Molecular
Data Systems). FIGS. 1-5 each contains two parts: a peak-picked X-ray powder
diffractogram and a peak list. The peak lists were generated using standard
parameters and commercial software.
[028] When those of ordinary skill in the solid-state chemistry art analyze
powder patterns to discern whether the seemingly different patterns actually
represent different crystalline solid forms, they often overlay the powder patterns
on, for example, a light box or on a computer screen. An example of just such an
overlay can be found at FIG. 6 which is the overlay representing the five
crystalline solid forms of tigecycline that were characterized and identified. An
expanded version of the overlay is at FIG. 7.
[029] The entire X-ray powder diffraction may be used to characterize
each crystalline solid form of tigecycline, however, one may select a smaller
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WO 2006/128150 PCT/US2006/020871
subset of peaks in each pattern to characterize each crystalline solid form of
tigecycline. Those selected peaks may then be used to identify the presence of
particular crystalline solid forms of tigecycline in an unknown sample of or
containing tigecycline.
[030] To illustrate, Table 1 lists the first 6 peaks in the diffractograms of
Form I and Form II.
Table 1

Tigecycline
Form °2θ °2θ °2θ °2θ °2θ °2θ
Form I 5.2 8.3 10.4 11.1. 13.2 13.7
Form II 9.2 9.7 11.6 13.3 15.4 17.7
[031] Based on a comparison of these data, and only considering these
two forms of tigecycline, it is apparent that one could rely on the 6 listed Form I
peaks to characterize Form I because the collection of 6 peaks is not present to +
0.2° 2θ in Form II. However, it is not necessary to rely on all 6 peaks to conclude
that Form I differs from Form II. In fact, the single peak at about 5.2° 2θ in Form I
uniquely characterizes Form I because the nearest Form II peak to about 5.2° 28
is found at about 9.2° 2θ,4 degrees 20 away. This 4° 20 difference is significantly
greater than the 0.4° 2θ obtained by combining the variability (0.2° 20) in any two
peaks. In other words, so long as a peak in one sample is more than 0.4° 2θ
away from any peak in another sample, then those represent different crystalline
solid forms because the chance that any given peak in a crystalline solid form
would vary by more than 0.4° 20 from sample to sample and/or instrument to
instrument is extremely small. Therefore, in a system that contains only Form I
and Form II, a tigecycline powder pattern containing a peak at about 5.2° 20
characterizes Form I tigecycline and the presence of that peak may be used to
identify Form I. Similarly, when characterizing Form II, one could use just the
peak at about 9.2° 20 because there is no Form I peak within 0.4° 20 of that peak.
[032] Not all peaks in Table 1 could be used to characterize Form I. For
example, the peak at about 13.2° 29 in Form I could not in and of itself be used to
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WO 2006/128150 PCT/US2006/020871
characterize Form I because Form II possesses a peak at about 13.3° 2θ which is
only 0.1° 2θ away.
[033] In accordance with the invention, X-ray powder diffraction data
were collected on the five crystalline tigecycline forms, Forms I, II, III, IV, and V,
and the peaks below around 26° 2θ appear in Table 2. It is determined that peaks
of higher intensity were more susceptible to preferred orientation effects.
Furthermore, the peaks listed in Table 2 were selected by considering the peak
lists in Figures 1-5. Thus, the data in Table 2 may be used to find the subsets of
peaks to characterize and/or identify the crystalline solid forms of tigecycline that
are the subject of the present invention.
Table 2

Forms (peaks listed in °2θ)
I II III IV V
5.2 9.2 5.2 4.6 4.3
8.3 9.7 6.0 8.8 8.6
10.4 11.6 8.3. 9.2 11.4
11.1 13.3 9.3 11.9 12.9
13.2 15.4 10.6 12.6 13.2
13.7 17.7
11.8 13.1 14.9
14.7 . 18.4 13.1 15.0 15.5
15.6 19.8 13.7 15.7 16.2
16.6 20.4 14.4 16.1 16.6
19.0 21.4 15.0 16.8 17.3
19.3 22.3 15.5 18.0 18.9
19.9 17.8 19.5 19.9
21.2 21.4 19.9 20.5
22.4 24.8 .20.4 21.1
23.1 2i.2 21.6
24.8 22.0 22.0
22.9 22.9
23.4 24.1
24.4 25.9
2-5.3
[034] In accordance with the invention, because none of the peaks listed
for Form I is greater than 0.4° 29 from every other peak of each of Forms II, III, IV,
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WO 2006/128150 PCT/US2006/020871
and V, no single Form I peak characterizes Form I from each of Forms II, III, IV,
and V. For example, whereas the peak at about 5.2° 2θ in Form I could be used
to distinguish between Form I and Form II, it alone could not be used to
distinguish between Form I and Form ill because Form III also has a peak at 5.2°
2θ. However, the subset of Form I peaks at about 5.2° 2θ and about 11.1 ° 2θ
could be used to distinguish Form I from both Form II and Form III because the
peak at about 11.1 ° 2θ is more than 0.4° 2θ from any peak in Form III. By the
same reasoning, the peak at 5.2° 2θ could also be used to distinguish Form.I from
Form IV and Form V. Thus, X-ray diffraction peaks at about 5.2° 2θ and about
11.1° 2θ are characteristic of Form I and may be used to identify Form I in a
sample. In characterizing and identifying Form I, one may also rely on some or ail
of the other peaks from the Form I powder pattern peak list in FIG. 1.
[035] A similar analysis is done for Forms II, III, IV, and V to construct
sets of characteristic X-ray powder diffraction peaks that can be used to
characterize and identify the different crystalline solid forms of tigecycline of the
present invention.
[036] In accordance with the invention, with respect to Form II, the peak
at about 9.2° 2θ distinguishes Form II from Form I and Form V. It does not, in and
of itself, however, distinguish Form II from Form III or from Form IV. The peak at
about 9.7° 2θ is distinguishable over Form IV, but is 0.4° 2θ from a peak in Form
III. The peak at about 20.4° 2θ in Form II distinguishes it from Form HI.
Therefore, Form II peaks at about 9.2° 2θ, about 9.7° 2θ, and about 20.4° 2θ
distinguish Form II over Forms I, III, IV, and V and thus characterize Form II in a
sample. Further, these peaks make be used to identify Form II. In characterizing
and identifying Form II, one may also rely on some or all of the other peaks from
the Form II powder pattern peak list in FIG. 2.
[037] In accordance with the invention, in Form III, the peak at about 6.0°
29 distinguishes Form III over Forms I, II, IV, and V and further characterizes
Form III. Further, this peak may be used to identify Form III in a sample. In
characterizing and identifying Form III, one may also rely on some or all of the
other peaks from the Form III powder pattern peak list in FIG. 3.

WO 2006/128150 PCT/US2006/020871
[038] In accordance with the invention, in Form IV, the peak at about 4.6°
2θ distinguishes Form IV over Forms I, II, and III, and the peak at about 9.2° 2θ is
more than 0.4° 2θ from peaks in Form V. Thus, peaks at about 4.6° 2θ and about
9.2° 2θ distinguish Form IV from Forms I, II, III, and V and thus characterize Form'
IV. Further, these peaks may be used to identify Form IV in a sample. In
characterizing and identifying Form IV, one may also rely on some or all of the
other peaks from the Form IV powder pattern peak list in FIG. 4.
[039] In accordance with the invention, for Form V, the peak at about
4.3° 2θ distinguishes Form V from Forms I, II, and III. The Form V peak at 11.4°
2θ further distinguishes Form V from Form IV. Thus, peaks at about 4.3° 2θ and
about 11.4° 2θ distinguish Form V over Forms I, II, III, and IV and therefore
characterize Form V. Further, these peaks may be used to identify Form V in a
sample. In characterizing and identifying Form V, one may also rely on some or
all of the other peaks from Form V powder pattern peak list in FIG. 5.
[040] Other analytical techniques may also be useful in analyzing
crystalline solid forms of tigecycline. Spectroscopic techniques such as Raman
(including microscopic Raman), infrared, and solid-state NMR spectroscopies may
be used to characterize and/or identify crystalline solid forms. These techniques
may also be used to quantify the amount of one or more crystalline solid forms in
a mixture. .
[041] Table 3 sets forth data showing melting point onset by hot stage
microscopy of several samples of Forms I, II, III, IV and V of the invention.
Thermal techniques, such as melting point onset by hot stage microscopy, do not
necessarily, in and of themselves, characterize or identify the different crystalline
solid forms of tigecycline based on the data shown in Table 3. For example, Form
I and Form IV each include a measured melting point onset by hot stage
microscopy of about 170°C and thus cannot be distinguished from one another by
this technique. In contrast, Form III is distinguishable from Form V by using this
technique because Form V has a melting point onset by hot stage at about 174°C
and Form III at about 167°C. Melting point onsets by hot stage microscopy may
be used together with another analytical method, such as X-ray powder diffraction,
to characterize and/or identify crystalline solid forms of tigecycline.
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Table 3

Tigecycline
Form Melting Point Onset by Hot Stage
Microscopy
Form 1 (3
samples) 170°C, 172°C, 172°C
Form II 169°C
Form III 167°C
Form IV 170°C
Form V 174°C
[042] In the hot stage microscopy measurements, the stage temperature
for the hot stage microscope is controlled by a Creative Devices, Inc (Neshanic
Station, NJ), Model 50-600 Controller. The sample heating rate is 10°C/min. The
microscope used is a Nikon (Tokyo, Japan) DIAPHOT 300 system. Images were
processed with the Image-Pro Plus software. Melting point onset values are
reported with the modifier "about," which is standard terminology in the solid-state
chemical arts and is meant to account for changes in melting point due to the
presence of water, solvent, or chemical impurities, as well as variability introduced
into melting point measurements by the analytical instrument and methodology
employed.
[043] Thermal measurements by a TA Instruments (New Castle, DE)
thermal gravimetric analysis ("TGA") indicate that Form II is a hydrate. In FIG. 7,
Form II exhibited a weight loss of about 5.3% when a Form II sample is heated at
10pC/minute beginning from between approximately 25°C to approximately
100°C. That the majority of this weight loss is likely due to water is further
indicated by a heat-cool cycle TGA experiment in FIG. 8. In such a cycle, the
sample is heated first to about 55°C and then cooled to about 37°C and heated
again, cooled, and heated again. Weight loss of about 4.4% is observed in the
first heat-cool cycle, with little weight loss observed in the subsequent cycles.
This weight loss behavior in a heat-coo! cycle, where substantially no further
weight loss is observed after the first cycle, is consistent with the sample being a
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hydrate. Thus, Form II is believed to be a hydrate with a hydration level of about
4.4%.
[044] That tigecycline crystallizes into five different crystalline solid forms
could not have been predicted from the tigecycline chemical formula. Nor would it
have been possible to predict the structure or properties of any of the crystalline
solid forms. The five crystalline solid forms of tigecycline of the invention were
prepared by determining the appropriate set of conditions that would enable those
forms to crystallize.
[045] The present invention also provides processes for making
crystalline solid forms of tigecycline. By slurrying Form I in organic solvents under
varying conditions, Forms Il-V can be prepared. In the slurries, Form I is treated
with one or more organic solvents in such amounts wherein the solid Form I did
not completely dissolve and mixed in the solvent or mixtures of solvents for some
period of time as a slurry. Sometimes, the slurries were heated and on other
occasions the slurries were treated with another solvent. Prior to analysis, the
slurries were filtered to isolate the solid, followed by drying under vacuum. The
resulting solids could then be analyzed by a number of analytical techniques.
[046] Where it has been determined that a solvent can aid in converting
the starting form into another form, that solvent is referred to herein as a "suitable
solvent" for that conversion. For example, it is determined that by slurrying Form I
in dichloromethane, Form I is converted into Form ill. Therefore, dichloromethane
is a suitable solvent for that conversion. Also, Form I can be converted to Form
III by crystallizing from tetrahydrofuran (THF) and stirring with dichloromethane.
Form II can be generated by slurrying Form I in methanol. Accordingly, methanol
is a suitable solvent for that conversion. Optionally, Form II can be generated by
slurrying Form I in methanol/dichloromethane wherein the methanol content is
greater than 10%. Likewise, Form I is converted into Form IV by slurrying Form I
in acetonitrile. Thus, acetonitrile is a suitable solvent for that conversion. Further,
slurrying Form I in acetonitrile/n-heptane generates Form IV. Additionally,
slurrying Form ! in tetrahydrofuran results in the conversion of Form I into Form V.
As a result, tetrahydrofuran is a suitable solvent for that conversion. Slurrying of
form I in the acetone/methanol (1:1 v/v) results in form I as does crystallization
from acetone/methanol.
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[047] In addition to slurrying, other treatments with suitable solvents can
afford the conversion of one form to another. For example, tigecycline Form II can
be prepared by crystallizing tigecycline out of methanol. Additionally, tigecycline
Form II can be prepared by the slow addition of methanol to a solution of
tigecycline in water which gives Form II particles > 20 urn. Further, by the addition
of a solution of tigecycline in water to methanol gives Form II particles which are <
20 urn. Accordingly, methanol, methanol/water or water/methanol are suitable for
the formation of Form II by crystallizing tigecycline.
[048] Particle length data is collected from optical images of tigecycline
particles. Optical microscopy analysis is performed using a Nikon Eciipse E600
microscope capable of 5x to 100x magnification, fitted with a digital camera (Nikon
DXM 1200) and a calibrated image acquisition system (Nikon ACT-1 v 2.12).
Images are processed using ImagePro plus image processing software (Media
Cybernetics, Silver Spring, MD). The software used a contrast- differentiation
algorithm to isolate tigecyciine particles from a uniform background. The results
of data analysis is shown in the following Table 4.

Table 4
Sample Particle length

Mean Median 90th percentile

microns microns microns
Addition of
methanol to an
aqueous solution of 22.67 18.16 47.87
tigecycline
Addition of an
aqueous solution of
tigecycline to 7.28 3.97 18.1
methanol
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[049] Form 111 can be obtained by crystallizing tigecycline out of
dichloromethane. Form IV can be obtained by crystallizing tigecycline out of
acetonitrile, and Form V can be obtained by crystallizing tigecycline out of
tetrahydrofuran.
[050] In accordance with the invention, formulations of tigecycline for use
in animals or humans can be made from Forms I-V of the invention. The
solubilities of the five crystalline solid forms of tigecycline are all greater than 25
mg/ml in water, thus they are all expected to be bioequivalent with one another.
[051] Pharmaceutical compositions for parenteral use can also be
prepared with any of Forms I-V with or without a lyophiiization step.
Pharmaceutical compositions of crystalline tigecycline using one or more of Forms
I-V can also be prepared in accordance with the invention. Such compositions
can be used to deliver pharmaceutically effective amounts of one or more of
Forms I-V of tigecycline. The composition may comprise the crystalline form of
tigecycline in combination or association with a pharmaceuticaily suitable carrier.
[052] The following non-limiting examples illustrate several methods of
making Forms I-V of tigecycline.
Example 1 - Preparation of Form I
[053] 300 grams of crude 9-chIoroacetamidominocycIine is added at
room temperature (25-28°C) slowly with efficient stirring to 2000 mL of t-
butylamine in a 5 liter three-necked round-bottom flask fitted with a stirrer and
thermometer. Forty-eight grams of sodium iodide is added and the reaction
mixture is stirred at 35-40°C for 6 hours. The reaction is monitored by HPLC and .
when <2% starting material remained, it is treated with 500 mL of methanol and
the solvent is stripped off on a rotary evaporator at 40°C. To the residue is added
1100 mL of methanol and 1700 mL of water. The solution is cooled to 0-2°C and
adjusted to pH 7.2 with concentrated HCI (-250 mL). The total volume of the
reaction mixture at this point is 3500 mL It is diluted to 8.5 liters with water and
the pH adjusted to 4.0-4.2 with concentrated HCI (12 mL). 1.6 kg of washed
Amber-chrom® (CG 161 cd) (NVM 27%) resin is added to the solution and stirred
for 30 minutes adjusting the pH to 4.0-4.2. The resin is filtered off and the spent
aqueous solution is assayed for product and stored at 4-8°C. The resin is slurried
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in 2.0 liters of 20% methanol in water (vol./vol.) The suspension is stirred for 15
minutes adjusting the pH to 4.0-4.2.. The resin is again filtered off and the filtrate
is assayed for product. The extraction of the resin is repeated 2 more times with
2.0 liters of 20% methanol in water. All the resin extracts and the spent aqueous
solution from above were pooled and the pH adjusted to 7-7.2 with 30%
ammonium hydroxide. The aqueous solution is extracted with 6X3.6 liters of.
methylene chloride adjusting the pH to 7.0-7.2 between extractions. The pooled
methylene chloride extract is filtered through 250 grams of anhydrous sodium
sulfate, concentrated to 500 mL and cooled to 0-3°C. The product crystallized
out. The slurry is stirred for 1 hour at 0-3°C and the solids were filtered and
washed with 2X 50 mL of cold methylene chloride and dried at 40°G in vacuum
(2θ inches of Hg). The weight of solid obtained is 110 grams. The solid obtained
can be identified by X-ray powder diffraction as Form I tigecycline. (HPLC area %
97.9, 0.3 % epimer).
Example 1A - Preparation of Resin from Example 1
[054] The resin of Example 1 is washed prior to use by taking 1 kg of
Amberchrom® (CG 161 CD from Toso Haas) and siurrying it in 6-7 liters of 12% 2-
propanol in water (voL/vol.) and stirring.for 12-16 hours. It is filtered and stirred in
6-7 liters of 50% acetonitrile-water (voL/vol.) for 30 minutes. The slurry is filtered
and then reslurried in 7 liters of acetonitrile, stirred for 30 minutes, and filtered.
The acetonitrile slurry is repeated twice and filtered. The resin is then slurried in 6
liters of methanol, stirred for 1 hour, and filtered. The resin is then slurried in 7
liters of deionized water, stirred for 16 hours, and filtered. The resin is then
slurried again in 7 liters of water, stirred for 1 hour, and filtered. The water slurry
is repeated 3 more times. The resin is filtered and as much water as possible is
removed on the filter funnel for 10-12 hours. It is bottled and stored cold (5-89C).
Example 2-Preparation of Form 1
[055] 10.00 grams of 9-aminorninocyc!ine is added portionwise to 60 ml
of water at 0-5 °C. 10.98 g of t-butylglycine acid chloride hydrochloride is added
portionwise keeping the temperature at 0-5 °C. After stirring for 40-60 minutes,
30% ammonium hydroxide is added dropwise to the reaction mixture keeping
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temperature at 0-5 °C to adjust the pH to 7.2. To the solution is added 83 ml of
methanol followed by 60 ml of methyiene chloride. After stirring for 15 minutes,
the phases were separated. The aqueous phase is extracted with 4 X 40 ml of
methyiene chloride adjusting the pH to 7.2 before each extraction. To the
combined organics is added 10 ml of methanol and the solution is dried over
sodium sulfate and then filtered. The solution is concentrated. The resulting
suspension is stirred at 5-10 °C for 1 hour and then filtered. The solid is washed
with 2 X 10 mL of cold methyiene chloride and then dried to give 8.80 g of
product. (Yield 76.8%); Purity by HPLC area % 98.4 and C-4 epimer 0.1 %;
MS(FAB): m/z 586 (M+H); 585 (M+).
Example 3—Preparation of Form II
[056] 0.5 g of Form I is slurried in 8 mL of methanol at 22°C. The slurry
is warmed to about 35°C to obtain a clear solution which is cooled to 22°C and
held for 30 minutes to yield a thick red slurry. The slurry is filtered and dried under
vacuum at 25°C overnight. The resulting solid is analyzed by thermal gravimetric
analysis ("TGA") and powder X-ray diffraction. The TGA plot (FIG. 8) shows a
weight loss of 5.3% at 87°C and a standard ramp rate of 10°C per minute is used
in the experiment.
[057] A heat-cool cycle is run on a fresh sample in a nitrogen
atmosphere inside the TGA furnace (FIG. 9). In the first heat-cycle, a 4.4% weight
loss is observed which is not observed in the second and third cycles indicating
the weight loss is likely water. After the third cycle, the resulting powder is red in
appearance.
Example 4 - Preparation of Form II
[058] 30 mg of crude tigecycline is dissolved in 500 uL of deionized
water at 40°C and the solution is stirred at 23°C. To this solution, 500 uL of
methanol is added in 100 μL increments over 15 min. After complete addition, a
hazy solution is stirred for about 10 min. and then a red crystalline precipitate is
obtained. The material is isolated by filtration in air and then dried under vacuum
at 40°C for 8 hours to yield 21.4 mg of Form II as determined by X-ray powder
diffraction.
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Example 5 - Preparation of Form II
[059] 30 mg of crude tigecycline is dissolved in 500 uL of deionized .
water at 40 °C and the solution stirred at 23°C. This solution is added to 800 uL
methanol and stirred at 10 °C over about 15 minutes. A crystalline precipitate is
obtained during addition. The material is isolated by filtration in air and then dried
under vacuum at 40 °C for 8 hours to yield 20.2 mg of form II as determined by X-
ray powder diffraction.
Example 6 - Preparation of Form III
[060] 0.25 g of Form ! is slurried in 2 mL of dichloromethane at 22°C to
form a suspension. 0.01 g of Form V is added as a seed. The suspension is
stirred for 96 hours at 22°C. The slurry is subsequently filtered and dried under
vacuum at 22°C. The resulting solid Form III is analyzed by hot stage microscopy,
X-ray powder diffraction, HPLC (99.98% pure), and optical microscopy.
Example 7 - Preparation of Form IV
[061] 0.15 g of Form I is added to 2 mL of acetonitrile at 22°C. A clear
solution is obtained which is stirred for 30 minutes to obtain a suspension. The
slurry is filtered and dried under vacuum at 22°C. The resulting off-white solid
Form IV is analyzed by hot stage microscopy, X-ray powder diffraction, HPLC
(93.39% pure), and optical microscopy.
Example 8—Preparation of Form IV
[062] 0.167 g of Form I is slurried in acetonitriie at 22°C. The slurry is
warmed to 30°C to obtain a clear solution. 2 mL of n-heptane is added to the
solution over the course of 5 minutes. A suspension is formed which is cooled to
22°C and held at that temperature for 30 minutes. The slurry is filtered and the
resulting solid is washed with 5 mL of n-heptane and dried under vacuum at 22°C.
The resulting solid Form IV is analyzed by X-ray powder diffraction, HPLC
(96.39% pure), and optical microscopy.
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Example 9 - Preparation of Form V
[063] 0.22 g of Form I is added to 2mL of tetrahydrofuran at 22°C and
stirred for 5 minutes. A clear solution is obtained to which 2 mL n-heptane is
added. The resulting slurry is stirred at 22°C for 30 minutes. The slurry is filtered
and dried under vacuum at 22°C. The resulting solid Form V is analyzed by hot
stage microscopy, X-ray powder diffraction, HPLC (93.57% pure), and optical
microscopy.
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We claim:
1. . Form I tigecycline having X-ray powder diffraction peaks at about
5.2° 2θ and about 11.1° 2θ.
2. Form I tigecycline of claim 1 further having an X-ray powder
diffraction peak at about 8.3° 20.
3. Form I tigecycline of claim 1 further having an X-ray powder
diffraction peak at about 24.8° 20.
4. Form I tigecycline having an X-ray powder diffraction pattern .
substantially as in Figure 1.
5. Form 1 tigecycline of claim 1 having a hot stage melting point onset
temperature of about 170°C to about 172°C.
6. Form I tigecycline of claim 3 having a hot stage melting point onset
temperature of about 170°C to about 172°C.
7. Form I prepared by crystallizing tigecycline out of a solution.
8. Form I of claim 7 wherein the solution contains methylene chloride.
9. Form II tigecycline having X-ray powder diffraction peaks at about
9.2° 20, about 9.7° 2θ, and about 20.4° 20.
10. Form II tigecycline of claim 9 further having an X-ray powder
diffraction peak at about 18.4° 2θ.
11. " Form II tigecycline of claim 9 further having an X-ray powder
diffraction peak at about 17.7° 20.
12. Form II tigecycline having an X-ray powder diffraction pattern
substantially as in Figure 2.
13. Form II tigecycline of claim 9 having a hot stage melting point onset
temperature of about 169°C.
14. Form 11 tigecycline of claim 12 having a hot stage melting point onset
temperature of about 169°C.
15. Form II tigecycline prepared by slurrying Form I tigecycline in one or
more suitable solvents.
16. Form II tigecycline of claim 15 wherein one suitable solvent is
methanol.
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17. Form II of claim 15 wherein the slurry contains more than one
suitable solvent.
18. Form II of claim 15 wherein the suitable solvents are
methanol/methylene chloride
19. Form II prepared by crystallizing tigecycline out of a solution.
20. Form II of claim 19 wherein the solution contains methanol.
21. Form II of claim 19 wherein the solution contains
methanol/methylene chloride.
22. Form III tigecycline having an X-ray powder diffraction peak at about
6.0° 2θ.
23. Form III tigecycline of claim 22 further having an X-ray powder
diffraction peak at about 5.2° 26.
24. Form III tigecycline of claim 22 further having an X-ray powder
diffraction peak at.about 9.3° 2θ.
25. Form III tigecycline having an X-ray powder diffraction pattern
substantially as in Figure 3.
26. Form III tigecycline of claim 22 having a hot stage melting point
onset temperature of about 167°C.
27; Form III tigecycline of claim 25 having a hot stage melting point
onset temperature of about 167°C.
28. Form III tigecycline prepared by slurrying Form I tigecycline in one or
more suitable solvents.
2θ. Form III tigecycline of claim 25 wherein one suitable solvent is
dichloromethane.
30. Form 111 of claim 28 wherein the suitable solvents are methylene
chloride/tetrahydrofuran.
31. Form IV tigecycline having X-ray powder diffraction peaks at about
4.6° 2θ and about 9.2° 2θ.
32. Form IV tigecycline of claim 31 further having an X-ray powder
diffraction peak at about 8.8° 20.
33. Form IV tigecycline of claim 31 further having an X-ray powder
diffraction peak at about 16.8° 2θ.
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34. Form IV tigecycline of claim 33 further having an X-ray powder
diffraction peak at about 18.0° 2θ.
35. Form IV tigecycline having an X-ray powder diffraction pattern .
substantially as in figure 4.
36. Form IV tigecycline of claim 31 having a hot stage melting point
onset temperature of about 170°C.
37. Form IV tigecycline of claim 35 having a hot stage melting point
onset temperature of about 170°C.
38. Form IV prepared by slurrying Form I in one or more suitable
solvents.
39. Form IV of claim 38 wherein the solvent is acetonitrile.
40. Form IV of claim 38 wherein the suitable solvents are acetonitrile/n-
heptane.
41. Form IV prepared by crystallizing tigecycline out of a solution.
42. Form IV of claim 36 wherein the solution contains acetonitrile.
43. Form V tigecycline having X-ray powder diffraction peaks at about
4.3° 2θ and about 11.4° 2θ.
44. Form V tigecycline of claim 43 further having an X-ray powder
diffraction peak at about 8.6° 2θ.
45. . Form V tigecycline of claim 43 further having an X-ray powder
diffraction peak at about 17.3° 2θ.
46. Form V tigecycline of claim 45 further having an X-ray powder
diffraction peak at about 25.9° 2θ.
47. Form V tigecycline having an X-ray powder diffraction pattern
substantially as in Figure 5.
48. Form V tigecycline of claim 43 having a hot stage melting point
onset temperature of about 174°C.
49. Form V tigecycline of claim 47 having a hot stage melting point
onset temperature of about 174°C.
50. Form V tigecycline prepared by siurrying Form i in one or more
suitable solvents.
51. Form V of claim 50 wherein the suitable solvent is tetrahydrofuran.
52. Form V prepared by crystallizing tigecycline out of a solution.
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53. Form V of claim 52 wherein the solution contains tetrahydrofuran.
54. Form V of claim 50 wherein trie suitable solvents are
tetrahydrofuran/ n-heptane.
55. A composition comprising at least one crystalline solid form of
tigecycline.
56. The composition of claim 55 comprising Form I tigecycline and Form
II tigecycline.
57. The composition of claim 55 comprising Form I tigecycline and Form
III tigecycline.
58. The composition of claim 55 comprising Form I tigecycline, Form II
tigecycline, and Form III tigecycline.
59. A pharmaceutical composition comprising at least one crystalline
solid form of tigecycline.
60. The pharmaceutical composition of claim 59 wherein at least one
solid form is Form I.
61. The pharmaceutical composition of claim 59 wherein at least one
solid form is Form II.
62. The pharmaceutical composition of claim 59 wherein at least one
solid form is Form III.
63. The pharmaceutical composition of claim 59 wherein at least one
solid form is Form IV.
64. The pharmaceutical composition of claim 59 wherein at least one .
solid form is Form V.
65. A composition comprising a pharmaceutically effective amount of
Form I tigecycline.
66. A composition comprising a pharmaceutically effective amount of
Form II tigecycline.
67. A composition comprising a pharmaceutically effective amount of
Form III tigecycline
68. A composition comprising a pharmaceutically effective amount of
Form IV tigecycline
69. A composition comprising a pharmaceutically effective amount of
Form V tigecycline.
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70. A process for preparing Form II tigecycline comprising slurrying
Form I tigecycline in one or more suitable solvents.
71. The process of claim 70 wherein one suitable solvent is methanol.
72. The process of claim 70 wherein the suitable solvents are
methanol/methylene chloride.
73. A process for preparing Form III tigecycline comprising slurrying
Form I tigecycline in one or more suitable solvents.
74. The process of claim 73 wherein one suitable solvent is
dichloromethane.
75. The process of claim 73 wherein the suitable solvents are methylene
chioride/ietrahydrofuran.
76. A process for preparing Form IV tigecycline comprising slurrying
Form I in one or more suitable solvents.
77. The process of claim 76 wherein one suitable solvent is acetonitrile.
78. The process of claim 76 wherein the suitable solvents are
acetonitrile/n-heptane.
79. A process for preparing Form V tigecycline comprising slurrying
Form I in one or more suitabie solvents.
80. The process of claim 79 wherein one suitable solvent is
tetrahydrofuran.
81. A process for preparing Form II comprising crystallizing tigecycline
out of a solution to make Form II.
82. The process of claim 81 wherein the solution contains methanol. •
83. A process for preparing Form IV comprising crystallizing tigecycline
out of a solution to make Form IV.
84. The process of claim 83 wherein the solution contains acetonitrile.
85. A process for preparing Form V comprising crystallizing tigecycline
out of a solution to make Form V.
86. The process of claim 85 wherein the solution contains
tetrahydrofuran.
87. A process for preparing Form I comprising crystallizing tigecycline
out of a solution to make Form I.
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88. The process of claim 87 wherein the solution contains methylene
chloride.
89. A process for preparing Form II tigecycline comprising adding
methanol to a solution of tigecycline in water to make Form II.
90. The process of claim 89 wherein the tigecycline of Form II has a
mean particle size of >20 urn.
91. A process for preparing Form II tigecycline comprising adding a
solution of tigecycline in water to methanol to make Form II.
92. The process of claim 91 wherein the tigecycline of Form II has a
mean particle size of <20 μm.
93. Use of a form of tigecycline as claimed in any one of claims 1 to 54
to prepare a pharmaceutical composition for parenteral use.
24

Crystalline solid forms of tigecycline, Form I, Form II, Form III, Form IV, and Form V, compositions comprising
these crystalline solid forms, and processes for preparing these crystalline solid forms are described herein.