Intensified Crystallization Processes for 1:1 Drug-Drug Cocrystals of Sulfathiazole-Theophylline, and Sulfathiazole-Sulfanilamide

Article abstract of DOI:10.1021/acs.cgd.7b01197

The chemical synthesis and crystallization steps were integrated successfully for directly producing a 1:1 cocrystal of sulfathiazole-theophylline and a 1:1 cocrystal of sulfathiazole-sulfanilamide. The benefits of this process intensification were the reduction of number of steps, and the amount of energy consumption and solvent used. In addition, the overall cocrystal yields by Intensified Method I were much higher than the ones by the conventional method. Intensified Method I also gave high-purity cocrystals of ≥99%. Sulfathiazole not forming cocrystals with sulfanilamide by Intensified Method I was dissolved in the mother liquor by taking advantage of the pH-dependent solubility of sulfathiazole. Cocrystals of both sulfathiazole-theophylline and sulfathiazole-sulfanilamide systems remained stable under conditions of 40 °C and 75% relative humidity for a month.

Full text of DOI:10.1021/acs.cgd.7b01197

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Article
Intensified Crystallization Processes for 1:1 Drug-Drug Co-crystals
of Sulfathiazole-Theophylline, and Sulfathiazole-Sulfanilamide
Kuan Lin Yeh, and Tu Lee
Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b01197 • Publication Date (Web): 16 Jan 2018
Downloaded from http://pubs.acs.org on January 16, 2018
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Intensified Crystallization Processes for 1:1 Drug-Drug
Co-crystals of Sulfathiazole-Theophylline,
and Sulfathiazole-Sulfanilamide
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Kuan Lin Yeh and Tu Lee*
Department of Chemical and Materials Engineering, National Central University,
300 Zhongda Road, Zhongli District, Taoyuan City 32001, Taiwan R.O.C.
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ABSTRACT
The chemical synthesis and crystallization step were integrated successfully
for directly producing 1:1 coꢀcrystal of sulfathiazoleꢀtheophylline and 1:1 coꢀcrystal
of sulfathiazoleꢀsulfanilamide. The benefits of this process intensification were the
reduction of number of steps, and the amount of energy consumption and solvent used.
In addition, the overall coꢀcrystal yields by Intensified Method I were much higher
than the ones by Conventional Method. Intensified Method I also gave highꢀpurity
coꢀcrystals of ≥ 99%. Sulfathiazole not forming coꢀcrystals with sulfanilamide by
*
Corresponding Author : Tel: +886ꢀ3ꢀ4227151 ext. 34204. Fax: +886ꢀ3ꢀ4252296, Email:
tulee@cc.ncu.edu.tw
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Intensified Method I, was dissolved in the mother liquor by taking advantage of the
pHꢀdependent solubility of sulfathiazole. Coꢀcrystals of both
sulfathiazoleꢀtheophylline and sulfathiazoleꢀsulfanilamide systems remained stable
under conditions of 40 oC and 75% relative humidity for a month.
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INTRODUCTION
Coꢀcrystal is defined as a crystalline solid composed of two or more distinct
compounds in a definite stoichiometric ratio. Those distinct components are
originally in solid state under ambient conditions. The crystalline framework of
coꢀcrystal is held together by nonꢀcovalent interactions such as van der Waals forces,
halogen bonding, πꢀπ stacking, and especially hydrogen bonding.1 Unlike salts,
pharmaceutically acceptable coꢀcrystals are not merely restricted to active
pharmaceutical ingredients (APIs) with an ionizable site. A wide range of coꢀcrystal
formers (i.e. coꢀformers according to the list of generally recognized as safe (GRAS))
offers more flexibility for tailoring the physiochemical properties of APIs such as
melting point,2 solubility,3 dissolution rate,4 bioavailability,5 compressibility6 and
moisture stability7 without changing the effective chemical entity of APIs. Since
about 70 % of new drug candidates and many commercial drugs suffer from
inadequate physicochemical properties such as solubility, dissolution rate and
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bioavailability,8 coꢀcrystallization has attracted more and more attention, and become
an important tool in pharmaceutical product design. 4,9,10
However, from a process design point of view, process intensification is a
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concept of transforming a conventional chemical process into a more economical,
productive and greener process.11 In the past decades, the same concept has evolved
into a paradigm for better process performance and higher product quality, and more
lucrative and safer technologies through the improvement of manufacturing process.
The utilization of new apparatus and technique as an alternative can reduce processing
times, solvent and energy consumption, number of processing steps and equipment
size.11,12 In general, APIs and fine chemicals are firstly made by chemical synthesis,
and then isolated and purified by crystallization.13,14 And yet, for most of the studies,
only coꢀcrystallization between API and coꢀformer was investigated without giving
consideration to chemical synthesis and coꢀcrystallization as a whole. Therefore, our
past success in integrating the synthesis of API and coꢀcrystallization by the approach
of direct coꢀcrystal assembly15 has prompted us to look into more complicated
coꢀcrystal systems, and finally implement process intensification method.
Sulfathiazole (STZ) and sulfanilamide (SNM) were chosen as two model sulfa
drugs in our present study. Both drugs possessed a sulfonamide group, and were
used as antibacterial APIs through the history of human.16 Although sulfa drugs had
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already been replaced by antibiotics due to the drugꢀresistant strains, sulfa drugs are
still being investigated extensively because of their low cost, excellent activity and
popularity.17,18 Several coꢀcrystal systems of STZ and SNM had been discovered and
reported in the literature.19ꢀ21 They included 1:1 coꢀcrystal of STZꢀglutaric acid, 1:1
coꢀcrystal of STZꢀ4ꢀaminobenzamide, 1:1 coꢀcrystal of sulfathiazoleꢀtheophylline
(STZꢀTHE) and 1:1 coꢀcrystal of STZꢀSNM, which were prepared by grinding and
evaporation on a small scale. Among those coꢀcrystal systems, 1:1 coꢀcrystal of
STZꢀTHE and STZꢀSNM, were chosen as the two drugꢀdrug coꢀcrystals produced by
the intensified crystallization processes in our present study. Interestingly, STZ and
SNM can be synthesized from the same chemical reaction, and THE is a commonly
used API in pharmaceutical coꢀcrystals.22
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The drugꢀdrug coꢀcrystal system is a coꢀcrystal made up of two APIs.23 The
drug combo has been an approach to offer a synergistic therapy in recent years.24 It
shows potential advantages of reducing drug dosage, toxicity and resistance, and
enhancing the efficacy of therapy.25ꢀ27 Since the drugꢀdrug coꢀcrystals have a higher
degree of unpredictability than the one of drug combos and ordinary type of
coꢀcrystals, they are more patent eligible. As a consequence, there is a strong
incentive to design drugꢀdrug coꢀcrystals. For example,
valsartanꢀsacubitril·3Na·2.5H2O having a brand name of Entresto®,28 is a commercial
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product comprising of coꢀcrystals of valsartan salt and sacubitril salt. Entresto® has
shown a better effect on treating heart failure than the other traditional medicines.29
It is also a demonstration of using coꢀcrystal to prolong the lifeꢀcycle management of
an API.
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Since the development of intensified processes requires the integration of
chemical synthesis and coꢀcrystallization of STZ and SNM, the understanding of each
synthetic and coꢀcrystallizing step, and product analysis becomes essential.
Therefore, the aims in our study include the determination of: percent yield and purity
given by different processes, pHꢀdependent solubility, partition coefficient (log P) and
moisture stability of coꢀcrystals. The percent yield and purity for the coꢀcrystals of
STZꢀTHE, and STZꢀSNM, obtained by conventional methods and intensified
processes have been compared for evaluating the feasibility of process intensification.
It was reported that sulfonamideꢀinduced crystalluria (i.e. sulfa drugs) in urinary tract
is highly dependent on the solubility and concentration of the sulfa drugs in urine in
the pH range of 5.5 to 7.0.30,31 Therefore, the effect of pH on solubility for the
coꢀcrystals has been studied. In addition, the values of solubility, log P, moisture
stability for coꢀcrystals have also been determined.
MATERIALS AND METHODS
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Materials. 2ꢀAminothiazole (C3H4N2S; 97 % purity; MW, 100.14; Lot: 10163024)
was purchased from Alfa Aesar (Heysham, England). Nꢀacetylsulfanilyl chloride
(C8H8ClNO3S; 98% assay; MW, 233.67; Lot: STBD9067V) and theophylline
(C7H8N4O2; ≥99% purity; MW, 180.16; Lot: SLBF2355V) were purchased from
SigmaꢀAldrich (China). 1ꢀoctanol (C8H18O; 97% assay; M.W. 130.2; Lot: E39586),
ammonium hydroxide (NH4OH; 28ꢀ30% assay; MW, 35.05; Lot: E51053), sodium
bicarbonate (NaHCO3; 99.7ꢀ100.3% assay; MW, 84.01; Batch No: 0000085775) and
sodium chloride (NaCl, ≥ 99% assay; MW, 58.44; Batch No: 0000031282) were
purchased from J. T. Baker (USA). Sulfanilamide (C6H8N2O2S; MW, 172.20; Batch
No: 0000099327) was purchased from J. T. Baker (China). Sodium hydroxide
(NaOH; 96% assay; MW, 40.00; Lot: KXꢀ2842A) was purchased from Showa
Chemical Co., Ltd. (Tokyo, Japan). Sulfathiazole (C9H9N3O2S2; ≥ 98% assay; MW,
255.32; Lot: 410504) was purchased from Fluka (Steinheim, USA). Acetone
(CH3COCH3; 99.5% purity; MW, 58.0; Lot: EB8n311) and methanol (CH3OH; 99.9%
assay; MW, 32.04; Lot: 14050368) were purchased from Tedia Company Inc.
(Fairfield, OH, USA). Hydrochloric acid (HCl; 37% assay; MW, 36.46; Lot:
UN1789) was purchased from Scharlau Chemie S.A. (Barcelona, Spain). Reversible
osmosis (RO) water was clarified by a water purification system (model MilliꢀRO
Plus) bought from Millipore (Billerica, MA, USA). All of the chemicals were used
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without further purification.
Instruments. Optical Microscopy (OM). Crystal habit was observed by optical
microscopy (Olympus BXꢀ51, Tokyo, Japan) equipped with a digital camera
(Moticam 2000)) and a cross polarized filter. The micrograph was transformed
through Motic Images Plus (Version 2.0) into a digital photograph and analyzed by
Measure Tool (Version 4.10).
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Thermogravimetric Analysis (TGA). TGA analysis was carried out by TGA 7
(Perking Elmer, Norwalk, CT) to monitor sample weight loss as a function of
temperature. All samples were heated under nitrogen atmosphere to avoid oxidation.
About 3ꢀ6 mg of samples were weighed and placed on an open platinum pan
suspended in a heating furnace. The heating rate was 10 oC⋅minꢀ1 and the
temperature range of scan was from 40 oC to 280 oC.
Differential Scanning Calorimetry (DSC). Thermal analytical data were obtained
by Perkin Elmer DSCꢀ7 calorimeter (Shelton, CT, USA). Each sample was loaded
in a perforated aluminum pan, and characterized with a scanning rate of 10 oC⋅minꢀ1
from 40 oC to 240 oC under a constant 99.99% nitrogen purge.
Fourier-Transform Infrared (FT-IR) Spectroscopy. FTꢀIR spectroscopy was
conducted on Perkin Elmer Spectrum One (Norwalk, CT, USA.) to identify functional
groups and polymorphs. Each sample was ground gently with potassium bromide
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(KBr) powders in a ratio of 1 to 100 and then a hydraulic press was used to form a
tablet. The tablet was scanned 8 times with a resolution of 2 cmꢀ1 in the
wavenumber region of 4000 to 400 cmꢀ1.
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Powder X-ray Diffraction (PXRD). PXRD diffractograms were conducted by
Bruker D8 Advanced (Germany), whose source used was Cu Kα (λ = 1.542 Å), and
the diffractometer was operated at 40 kV and 40 mA. The Xꢀray passed through a
nickel filter with a divergence slit of 0.5o, a scattering slit of 0.5o, and a receiving slit
of 1 mm. The scanning rate was set at 0.05o 2θ⋅secꢀ1 ranging from 2θ = 5o to 35o.
Nuclear Magnetic Resonance (NMR). About 40 mg of dried sample powders were
dissolved in an adequate amount of deuterated dimethyl sulfoxide (DMSOꢀd6).
Around 0.8 mL of the sample solution was added into an NMR tube having a 5ꢀmm in
outer diameter and 178 mm in length for solution 1HꢀNMR analysis (Bruker
AscendTM 600 MHz). Chemical shifts were relative to the one of DMSO at 2.50
ppm. Resonance peak areas were integrated by MestReNova (Version 11.0.4)
software from Mestrelab Research S. L.
Ultraviolet and Visible (UV/vis) Spectrophotometer. UV/Vis spectrophotometer
(Lambda 25, Perkin Elmer, Norwalk, CT, USA) was used to measure the
concentrations of STZ, SNM, STZꢀTHE and STZꢀSNM at the characteristic
absorption peaks of 283, 258, 277 and 258 nm, respectively. The concentrations of
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STZ, SNM, STZꢀTHE, and STZꢀSNM were converted from the absorbance values to
the corresponding concentrations by the linear calibrations of: absorbance (au) =
49666 × concentration of STZ (M), absorbance (au) =15650 × concentration of
SNM (M), absorbance (au) =27728 × concentration of STZꢀTHE (M), absorbance
(au) =31807 × concentration of STZꢀSNM (M), respectively, as determined in water
at 25 oC.
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High-Performance Liquid Chromatography (HPLC). HPLC analysis was
performed using a Shimadzu Prominenceꢀi LCꢀ2030C 3D HPLC. Sample solutions
were prepared by dissolving 5 mg of sample powders in 25 mL of methanol. 2 ꢁL of
sample was withdrawn and injected into a column (YMCꢀPack ODSꢀAQ 150
×
3.0
mm 3 ꢁm 12nm). The mobile phase used was a gradient system composed of
×
×
methanol/0.05 % formic acid aqueous solution. The composition of the mobile
phase started at 5/95 (v/v), increased to 90/10 (v/v) in 15 min, and maintained for 5
min. The flow rate was 0.5 mL⋅minꢀ1 and the temperature was kept at 37 oC. The
detection wavelength for HPLC was set at λ = 254 nm.
Experiments.
Syntheses of STZ and SNM. For the synthetic processes of STZ and SNM, each
process was divided into two steps. In Step 1 (Scheme 1): 6 g (25.7 mmol) of
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Nꢀacetylsulfanilyl chloride were slowly added to the suspension containing 1.25 g
(12.5 mmol) of 2ꢀaminothiazole, 2.25 g (26.8 mmol) of sodium bicarbonate and 3 g
(51.3 mmol) of sodium chloride in 11 mL of water and 5.5 mL of acetone in a 100 mL
roundꢀbottom flask at 25 oC to 30 oC under agitation for 2.5 h. Nꢀacetylsulfanilyl
chloride was fed as six equal portions for avoiding the fast evolution of CO2 which
was a byꢀproduct of neutralization between HCl and NaHCO3. 3.1 mL of 28~30
wt% NH4OH solution were added to the reaction mixture. Afterwards, the resulting
mixture was heated to 65 oC with a total reflux for 2.5 h. After the reaction solution
was cooled to room temperature, N4ꢀacetyl SNM was precipitated, filtered and rinsed
by 15 mL of cold water twice. The mother liquor was treated with 0.25 g of Darco
for decolorization. It was then stirred for 1 h, and filtered for the removal of Darco
particles. The pH of filtrate was adjusted to 5 to 6 by adding 12~14 mL of 2M HCl
aqueous solution to precipitate out N4ꢀacetyl STZ. N4ꢀacetyl STZ solids were
filtered and rinsed with cold water twice. Solids of N4ꢀacetyl SNM and N4ꢀacetyl
STZ were oven dried at 40 oC overnight.
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In Step 2a (Scheme 1): 2.97 g (10 mmol) of N4ꢀacetyl STZ solids were added
into a 50 mL roundꢀbottom flask with a magnetic spin bar. 12.5 mL of 3.2 M NaOH
aqueous solution were then introduced into the flask for hydrolysis, and the reaction
solution was heated to 65 oC in a water bath for 2 h. The reaction solution was then
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acidified by adding 15 mL of 2 M HCl aqueous solution to produce STZ solids. On
the other hand, 1.07 g (5 mmol) of N4ꢀacetyl SNM solids were reacted with 10 mL of
1.5 M HCl aqueous solution for hydrolysis in a 25 mL roundꢀbottom flask, and heated
to 95 to 100 oC in an oil bath with a reflux for 4 h. Then, the reaction solution was
cooled to 20 oC, treated with 25 mg of Darco and stirred for 30 min, and then filtered
for the removal of Darco. The mother liquor was neutralized by adding 4M NaOH
aqueous solution to adjust the pH to 4~5 to precipitate SNM solids. All harvested
solids were filtered, rinsed with copious amounts of cold water twice and oven dried
at 40 oC overnight.
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Cooling Co-crystallization of STZ-THE. Synthesized STZ crystals were replaced
by the use of purchased STZ crystals for the control experiment. 1 g of purchased
THE was totally dissolved in 160 mL of methanol in a 0.5 L flatꢀbottom flask with an
inner diameter of 7.4 cm equipped with four equally spaced vertical baffles, a four
bladed turbine impeller made of stainless steel, and a total reflux condenser. The
temperature was maintained at 60 oC. An equimolar amount of purchased STZ
crystals was dissolved in 60 mL of methanol, and added into the flask, and mix with
the solution of THE. The coꢀcrystallization was carried out by cooling the solution
from 60 oC to 20 oC at a stirring rate of 600 rpm.
Cooling Co-crystallization of STZ-SNM. Synthesized SNM crystals were replaced
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by the use of purchased SNM crystals for the control experiment. 1.29 g of
purchased SNM and 1.53 g of purchased STZ were added together in a 100 mL
scintillation vial. 60 mL of preheated methanol were introduced into the vial to
simultaneously dissolve SNM and STZ at 60 oC using a magnetic spin bar. Then,
the solution was cooled to 20 oC to form 1:1 coꢀcrystal of STZꢀSNM.
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Intensified Method I. For direct preparation of 1:1 coꢀcrystal of STZꢀTHE, 5 mL
of 4 M HCl aqueous solution were added right after hydrolysis. An equimolar
amount of purchased THE with respect to N4ꢀacetyl STZ, and 3.5 mL of 4 M HCl
aqueous solution were added to the reaction solution to produce 1:1 coꢀcrystal of
STZꢀTHE. For direct preparation of 1:1 coꢀcrystal of STZꢀSNM, an equimolar
amount of purchased STZ with respect to N4ꢀacetyl SNM was introduced immediately
after hydrolysis. The solution was treated with 25 mg of Darco, stirred for 30 min,
and then filtered for the removal of Darco particles. The pH value of the resulting
solution was adjusted to 9 for producing the 1:1 coꢀcrystal of STZꢀSNM. All solids
harvested were filtered, rinsed with cold water twice, and then dried at 40 oC
overnight.
Intensified Method II. 2.14 g (10 mmol) of N4ꢀacetyl SNM and 20 mL of 1.5 M
HCl aqueous solution were added into a 25 mL roundꢀbottom flask. The reaction
solution was heated to 95 to 100 oC in an oil bath equipped with a reflux condenser
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for 4h to give a SNMꢀcontaining solution. Afterwards, the SNMꢀcontaining solution
was cooled to room temperature for the ease of operation, treated with 25 mg of
Darco, stirred for 30 min, and then filtered for the removal of Darco. Additionally,
2.97 g (10 mmol) of N4ꢀacetyl STZ and 20 mL of 3.2 M NaOH aqueous solution were
added together into a 50 mL roundꢀbottom flask. The solution was heated to 65 oC
for 2 h to give a STZꢀcontaining solution. 1:1 coꢀcrystal of STZꢀSNM was
precipitated out by introducing the SNMꢀcontaining solution into the STZꢀcontaining
solution at 65 oC. All solids were filtered, rinsed with cold water twice, and oven
dried at 40 oC overnight.
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o
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Solubility Test. Solubility test was performed at 15 C, 25 C, 40 C and 60 C in
water. Each unsieved solid sample was weighed into a 20 mL scintillation vial.
Water was titrated dropwise in a vial at a fixed temperature in a water bath. The
solution was shaken intermittently by hand. Water was gradually added within 6
hours until all solids were just dissolved as determined by eyes. This titration
method was simple and robust, provided solubility values, and minimized the
possibility of solvate and hydrate formation during the measurements. The total
volume of water added was recorded and the solubility was also calculated in terms of
molarity (M).32
pH Dependence in Solubility. Each solid sample was weighed and suspended in
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water in a 100 mL scintillation vial. The pH value of each suspension was adjusted
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by adding HCl and NaOH aqueous solutions. The suspension was kept at 37 C for
a day to achieve equilibrium, and then filtered through a 0.22 ꢁm PVDF membrane
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(PALL Corporation, PN).
The mother liquors were assayed by a UV/vis
spectrophotometer, and the solids were characterized by DSC.
Partition Coefficient Determination. Around 2ꢀ3 mg of sample powders were
weighed into a 100 mL scintillation vial comprising of 15 mL of water with an equal
volume of 1ꢀoctanol at 25 oC. The solutions were shaken for 40 h. Afterwards,
only the aqueous phase was assayed by HPLC.
Moisture Stability Test. Each solid sample was weighed into a 7 mL open vial,
which was placed in a capped 100 mL glass bottle filled with 10 mL of saturated NaCl
aqueous solution to have a closed system at 40 oC and 75% relative humidity (RH) for
a month.7 The sample was then characterized by DSC.
RESULTS AND DISCUSSION
Syntheses of STZ and SNM
There were five polymorphs for STZ (Forms I to V), and four modifications
for SNM (Forms α to δ).33ꢀ35 According to the literature, Form III is the most stable
form for STZ, and Form β is the most stable form for SNM under the ambient
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condition.36,37 The transition temperature of STZ from Form III is reported at 175 oC
followed by a melting point of Form I at 202 oC.36,38 Form β had a phase transition
to Formγat 131 oC to 141 oC before melting at 166 oC.39 For the integration of
chemical synthesis and coꢀcrystallization, a good understanding of process and crystal
forms was necessary. Therefore, STZ and SNM were synthesized and analyzed as
controls. The preparation processes of STZ, SNM, 1:1 coꢀcrystal of STZꢀTHE and
1:1 coꢀcrystal of STZꢀSNM were shown in Scheme 1. Step 2 in Scheme 1 was
categorized into three methods: Conventional Method, Intensified Method I and
Intensified Method II.40,41 As indicated by Step 2a, STZ and SNM were obtained via
hydrolyzing N4ꢀacetyl STZ by the base addition, and N4ꢀacetyl SNM by the acid
addition, respectively. They were both crystallized out by neutralization. In
addition to the reactionꢀrelated factors such as percent yield and purity, polymorphism
of API solids is equally important, and needs to be controlled due to its profound
effect on the quality of a final product.
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The IR assignments for Form III STZ crystals were shown in Figure 1. The
bands at 3279 cmꢀ1 and 3320 cmꢀ1 were assigned for the –NH2 symmetric and
asymmetric stretching vibrations, respectively. The –SO2– symmetric and
asymmetric stretching vibration frequencies are found at 1136 cmꢀ1 and 1323 cmꢀ1,
respectively. The bands at 1531 cmꢀ1 can be contributed to C=N stretching vibration,
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and 631 cmꢀ1 to C–S stretching frequency.19,42 The FTꢀIR spectrum of synthesized
STZ in Figure 1(b) matched well with the one of purchased STZ crystals in Figure
1(a), indicating that the synthesized STZ crystals were chemically identical to the
purchased STZ crystals, which were Form III STZ crystals.43 The band assignments
for Form β SNM crystals were as follows: 3477 cmꢀ1 and 3370 cmꢀ1 for –NH2
symmetric and asymmetric stretching vibrations, respectively, 1313 cmꢀ1 and 1146
cmꢀ1 for –SO2– symmetric and asymmetric stretching vibrations, respectively, and 900
cmꢀ1 for S–N stretching vibration.42,44 Synthesized SNM in Figure 1(d) gave a
different IR spectrum from the one of purchased SNM crystals in Figure 1(c). The
characteristic bands at 3375 cmꢀ1 and 3266 cmꢀ1, and the ones at 3383 cmꢀ1, 3318 cmꢀ1
and 3243 cmꢀ1 were corresponding to Form β and Form γ SNM crystals,
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respectively.39
The absorption region of 3235 cmꢀ1 to 3380 cmꢀ1 was used to
distinguish Form γ crystals from the other polymorphs of SNM crystals. The
synthesized SNM crystals are not always the most stable Form β. Although the
purchased SNM crystals and synthesized SNM crystals possess an identical molecule,
the packing arrangements of molecules are different.
There was a good agreement among the PXRD patterns of purchased and
synthesized STZ crystals in Figures 2(a) and 2(b), respectively, and the theoretical
pattern of Form III STZ crystals based on singleꢀcrystal Xꢀray experiment in Figure
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2(c). This was also evidenced by the IR spectra in Figures 1(a) and (b). The
slightly shifted diffraction peaks for synthesized STZ crystals in Figure 2(b) were
most likely attributed to the buildꢀin strain of crystal originated from the lengthening
or shortening of the dꢀspacing45 when the synthesized STZ crystals were precipitated
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out rapidly by the addition of HCl aqueous solution into the reactor.
The
diffraction pattern of purchased SNM in Figure 3(a) was consistent with the
theoretical one of Form β SNM crystals in Figure 3(c). The PXRD diffractogram of
synthesized SNM crystals in Figure 3(b) matched with the theoretical one of Form γ
SNM crystals in Figure 3(d). However, several additional peaks at 2θ = 18.97o,
20.61o, 21.45o and 22.85o designated for Form β SNM crystals were also detected for
the synthesized SNM crystals in Figure 3(b), meaning that the synthesized SNM
crystals were adulterated with a trace amount of Form β SNM crystals.
According to the Ostwald’s Rule of Stages,46 the metastable synthesized Form
γ SNM crystals would eventually transform to the most stable Form β SNM crystals if
given with a long enough residence time in the reactor or a stimulus from downstream
processes and storage. Drug properties will be altered when API is transformed into
a different polymorph. Since the presence of different polymorphs in a final product
is deemed as contamination, this kind of uncontrolled situation should be avoided.47
Controlling the polymorphism of synthesized SNM crystals may be challenging in a
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scaleꢀup process because of the local variations in concentration and temperature.
This gave us the motivation to employ Intensified Method to turn the polymorphic
SNM solids into isomorphic coꢀcrystals directly during synthesis to avoid
polymorphic impurity.
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Cooling Co-crystallization of STZ-THE and STZ-SNM.
The discovery of 1:1 coꢀcrystal of STZꢀTHE and 1:1 coꢀcrystal of STZꢀSNM
were first reported in 1971.20 However, the physicochemical data about those two
coꢀcrystal systems such as melting point, crystal habit, solubility and stability were
unavailable. Due to the lack of physicochemical data in the literature, STZꢀTHE
coꢀcrystals and STZꢀSNM coꢀcrystals were prepared in methanol by cooling
coꢀcrystallization as control. Synthesized STZ and SNM crystals were replaced by
the use of purchased STZ and SNM crystals in Conventional Method for the
controlled experiments. The two coꢀcrystals produced by cooling were abbreviated
as CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM. The weight losses of CocryꢀSTZꢀTHE
and CocryꢀSTZꢀSNM coꢀcrystals started at about 230 oC due to chemical degradation
(Figure S1). Therefore, the temperature scanning range for DSC was set from 40 oC
to 240 oC. Other data for CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM coꢀcrystals would
be discussed in detail along with the ones for the coꢀcrystals produced by Intensified
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Method I in the next section.
Preparation of STZ-THE and STZ-SNM and by Intensified Method I
The 1:1 coꢀcrystal of STZꢀTHE and 1:1 coꢀcrystal of STZꢀSNM produced by
the Intensified Method I were abbreviated as IntenꢀIꢀSTZꢀTHE and IntenꢀIꢀSTZꢀSNM
coꢀcrystals, respectively, in Scheme 1, Step 2b. The purchased THE and STZ
crystals were fed to the reaction solution consisting of STZ and SNM species after
hydrolysis, respectively, and formed STZꢀTHE and STZꢀSNM coꢀcrystals after
neutralization. The products were changed from the original APIs to the coꢀcrystals.
Figures 4 and 5 showed the PXRD patterns of STZꢀTHE and STZꢀSNM coꢀcrystals
obtained from cooling coꢀcrystallization (i.e. Conventional Method) and Intensified
Method I, and their theoretical patterns based on singleꢀcrystal Xꢀray
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crystallography.20 They matched well with each other. The relative intensity of the
diffraction peaks in Figures 4 and 5 varied from method to method. We speculated
that this was caused by the solvent effect on growth rate of different crystallographic
direction.48
CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM coꢀcrystals exhibited the endotherms
of melting at 227.7 oC and 182.3 oC in Figures 6 (b) and 6(d), respectively. The
coꢀcrystals produced by Intensified Method I showed the same endothermic peak in
Figures 6(b) and 6(d). Both DSC and PXRD results indicated that STZꢀTHE and
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STZꢀSNM coꢀcrystals were successfully prepared by Intensified Method I using a
oneꢀpot process. Moreover, no detectable trace of SNM polymorph was present in
the DSC scans for IntenꢀIꢀSTZꢀSNM coꢀcrystals. The possibility of forming
different SNM polymorphs was minimized by forming coꢀcrystals using Intensified
Method I. Crystallinity for all solids was determined by DSC based on Equation
(1):49
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Crystallinity (%) = (ꢀHsfp / ꢀH pfp )×100%
(1)
f
where ꢀHsfp = enthalpy of fusion for samples, andꢀH pp = enthalpy of fusion for a
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standard. CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM coꢀcrystals were defined as the
calculation standards. STZ and SNM crystals can be dissolved in either a strong
basic or acidic aqueous solution, and then precipitated out by neutralization.40 STZ
crystals were precipitated out from the basic mother liquor instantly upon the addition
of HCl aqueous solution, indicating of a relatively high degree of supersaturation due
to the drastic decrease of solubility. In general, a high degree of supersaturation
gives solids with a low degree of crystallinity.50 The enthalpies of fusion for both
IntenꢀIꢀSTZꢀTHE and IntenꢀIꢀSTZꢀSNM coꢀcrystals were close to the ones for
CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM coꢀcrystals, meaning that the coꢀcrystals
produced by Intensified Method I have a high degree of crystallinity. FTꢀIR spectra
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of STZꢀTHE and STZꢀSNM coꢀcrystals were shown in Figures S2 and S3 of
Supporting Information, respectively. The spectra of coꢀcrystals generated by
Intensified Method matched well with the ones by cooling coꢀcrystallization in
Conventional Method.
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Various loading ratios (LRs) of N4ꢀacetyl STZ to THE, and N4ꢀacetyl SNM to
STZ were attempted to further investigate the effect of addition amount of coꢀformer.
The melting point of IntenꢀIꢀSTZꢀTHE coꢀcrystals in Figure S4a could vary while the
LR of N4ꢀacetyl STZ to THE, was changed from 1:1.4 to 1:0.6. Noticeably, a new
endothermic peak appeared at 167.3 oC (Figure S4a(i)) as the LR was lowered to 1:0.6.
We speculated that the original endothermic peak of 175 °C for Form III STZ crystals
o
might be depressed to a lower temperature of 167.3 C due to the presence of
o
STZꢀTHE coꢀcrystals. In addition, a new endothermic peak of 157.7 C was
observed for the sample with the LR of 1:0.5 (Figure S4b(i)), whereas another new
endothermic peak of 165.8 oC was seen in the DSC scan for the sample having the LR
of N4ꢀacetyl SNM to STZ of 1:1.5 (Figure S4b(iii)). The appearance of the two
endothermic peaks in Figures S4b(i) and S4b(iii) was ascribed to the excess amount
of SNM and STZ crystals, respectively. However, the original melting points of
o
o
Form γ SNM crystals and Form III STZ crystals are 166 C and 175 C,
respectively.36,39 A possible explanation for the shift of the endothermic peaks was
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due to eutectic behavior of mixing of SNM crystals with STZꢀSNM coꢀcrystals, and
STZ crystals with STZꢀSNM coꢀcrystals. This kind of eutectic behavior leads to the
depression of melting point, which had been reported in other coꢀcrystal systems.51ꢀ53
This was further evidenced by performing the DSC scans on the physical blend of
purchased SNM crystals and CocryꢀSTZꢀSNM coꢀcrystals with a molar ratio of 1:1,
and the physical blend of purchased STZ crystals and CocryꢀSTZꢀSNM coꢀcrystals
with a molar ratio of 1:1, as displayed in Figure S5. It seems that the LR of 1:1 is
the best ratio to produce the pure coꢀcrystals of STZꢀTHE, and STZꢀSNM.
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Agreeing with the DSC scan in Figure S4, when the LR was varied, one of the
coꢀcrystal components was present as an isolated component in the coꢀcrystal product.
A solid mixture was produced once the LR was deviated from 1:1 significantly. For
example, the characteristic diffraction peaks of THE crystals were detected when the
LR was increased to 1:1.2, and 1:1.4 in the IntenꢀIꢀSTZꢀTHE system (Figure S6(d)
and (e)). As for the IntenꢀIꢀSTZꢀSNM in Figure S7, the PXRD pattern of STZꢀSNM
system with the LR of 1:1.5 in Figure S7(c) consisted of the diffraction peaks of
STZꢀSNM coꢀcrystals and a trace amount of Form III STZ crystals, and yet, the
diffraction peaks of pure SNM were absent for the system using LR of 1:0.5 in Figure
S7(a). The trace level of SNM crystals might be below the detection limit of PXRD,
which had been used to quantify the amount of coꢀcrystals in a mixture with the limit
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of 1.23 wt%.54 Besides, we had tested the PXRD detection limit by powder blends
having different weight percentages of purchased SNM crystals to CocryꢀSTZꢀSNM
coꢀcrystals of 4 wt%, 5 wt% and 10 wt%. The diffraction peak at 2θ = 10.6o
representing SNM crystals was found for the powder blends containing 5 wt% and 10
wt% SNM crystals, and disappeared for powder blends having 4 wt% SNM crystals
as shown in Figure S8. Therefore, only 4 wt% or less of SNM crystals were present
if there was any in the product of IntenꢀIꢀSTZꢀSNM coꢀcrystals using LR of 1:0.5.
The 1H NMR spectra in Figure S9 showed that all resonance peaks could be
assigned to the specific protons of STZ or THE molecules, and STZ or SNM
molecules,55 for IntenꢀIꢀSTZꢀTHE, and IntenꢀIꢀSTZꢀSNM coꢀcrystals, respectively.
No byꢀproduct was generated upon the formation of coꢀcrystals in Intensified Method
I.
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Figure 7 displayed the crystal morphology for STZ and SNM crystals, and
STZꢀTHE and STZꢀSNM coꢀcrystals. The synthesized STZ, and SNM crystals had
rhombic, and thin plateꢀlike habits, respectively. The STZꢀTHE coꢀcrystals also
exhibited a rhombic morphology regardless of the method used. The rhombic
morphology of the STZꢀTHE coꢀcrystals (Figure 7(c) and S7(d)) was more regular
than the one of synthesized STZ crystals. In general, a more regular shape can lead
to better manufacturability such as flowability, which can benefit downstream
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processes including blending, granulation and tableting.
However,
CocryꢀSTZꢀSNM and IntenꢀIꢀSTZꢀSNM coꢀcrystals had a rodꢀlike habit, which was
unfavorable for the downstream processes. The rodꢀlike or needleꢀlike crystals
generally leads to a low power flow, poor compressibility and high filtration
resistance.56,57
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According to Scheme 1, Conventional Method was comprised of the steps of
reaction, crystallization of API, two sets of filtration and drying, addition of coꢀformer
and coꢀcrystallization. For Intensified Method I, crystallization of API, and a set of
filtration and drying can be omitted. Generally speaking, the number of processing
steps is directly proportional to time, energy, waste and labor intensity. In this regard,
Intensified Method I is more advantageous over Conventional Method. Table 1
revealed the percent yields of synthesized STZ crystals, synthesized SNM crystals,
CocryꢀSTZꢀTHE, CocryꢀSTZꢀSNM, and IntenꢀIꢀSTZꢀTHE and IntenꢀIꢀSTZꢀSNM
coꢀcrystals produced with LR of 1:1. We found that the coꢀcrystal yield of 81.0% for
STZꢀTHE solids by Intensified Method I was higher than the one of 68.1% by cooling
coꢀcrystallization.
On the other hand, the coꢀcrystal yields for STZꢀSNM by
Intensified Method I and the cooling coꢀcrystallization were almost the same.
Remarkably, the overall coꢀcrystal yields by Intensified Method I were much higher
than the ones by Conventional Method when including the synthetic steps. In
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addition, when the feeding rate of the HCl aqueous solution was too fast, fine STZ
crystals were produced and tended to agglomerate into large lumps. However, if
THE crystals were introduced prior to neutralization, no agglomerates were formed
even with the same fast addition rate of HCl aqueous solution.
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HPLC data were provided in Figure S10 and the results were tabularized in
Table 2. The retention times of STZ, SNM and THE molecules were 6.4, 3.4 and 7.2
min, respectively. According to the HPLC results, Intensified Method I gave all
harvested samples a purity of ≥ 99%. The stoichiometric ratios of STZ to THE, and
STZ to SNM crystals were calculated based on the ratios of the integration peak areas
of the dissolved STZ to THE species, and the dissolved STZ to SNM species,
respectively. All ratios were very close to one. The results indicated that the 1:1
coꢀcrystals produced had a very high purity.
Preparation of STZ-SNM by Intensified Method II
According to Scheme 1, Step 2b, the reaction solutions of STZ, and SNM
were basic and acidic, respectively, and their crystallizations were carried out through
neutralization, by adding HCl, and NaOH aqueous solution, respectively. Since the
solids were coꢀcrystallized through neutralization in Intensified Method I which gave
us the idea that those two processes could be further combined to prepare STZꢀSNM
coꢀcrystals. Therefore, the two reaction solutions of N4ꢀacetyl STZ and N4ꢀacetyl
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SNM after hydrolysis were mixed together to yield STZꢀSNM coꢀcrystals (Scheme 1,
Step 2c). This approach was called Intensified Method II.
STZ and SNM species
were present separately in the aqueous solutions before mixing for coꢀcrystallization.
The solution phase was more convenient for feeding and mixing than the powder
form.
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Although the PXRD pattern of IntenꢀIIꢀSTZꢀSNM coꢀcrystals matched with
the theoretical pattern of STZꢀSNM coꢀcrystals (Figure 8(b), the characteristic peaks
at 2θ = 23.2o and 28.0o for Form γ were detected. Also, the DSC scan in Figure 9
exhibited a eutectic behavior for the mixture of SNM crystals and STZꢀSNM
coꢀcrystals showing a melting point of STZꢀSNM coꢀcrystal at 179.8 oC and another
smaller endothermic peak at 156.2 oC. Also, according to the HPLC data (Figure
S11), IntenꢀIIꢀSTZꢀSNM coꢀcrystals had the ratio of STZ to SNM of 0.87:1 verifying
that the amount of SNM species was more than the one for STZ species, and the
excess amount of SNM species was precipitated out as SNM solids along with the 1:1
coꢀcrystal STZꢀSNM product. Intensified Method II will need to be optimized in the
future.
Solubility, Partition Coefficient and Moisture Stability
The titration method was used to measure the solubility of purchased STZ
crystals, purchased SNM crystals, CocryꢀSTZꢀTHE coꢀcrystals, CocryꢀSTZꢀSNM
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coꢀcrystals, and the blends of purchased STZ and purchased THE crystals, and the
blends of purchased STZ and purchased SNM crystals with a molar ratio of 1:1 in
water at a variety of temperatures (Figure 10). The solubility values of STZ crystals
and STZꢀTHE coꢀcrystals were not too different from one another (Figure 10(b)).
On the other hand, STZꢀSNM coꢀcrystals had an obvious increase in the solubility
value (Figure 10 (b)). Noticeably, the blends of individual components with STZ
crystals exhibited a similar solubility value as the one of STZ crystals, meaning that
making a simple drug combo cannot affect the solubility of STZ too much.
Table 3 listed the solubility values of purchased STZ, purchased SNM,
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CocryꢀSTZꢀTHE and CocryꢀSTZꢀSNM coꢀcrystals at a variety of pH values. UV/vis
spectrophotometry was employed to measure the solubility value. To confirm the
coꢀcrystals were dissolved in water with a 1 to 1 stoichiometric ratio, the suspended
solids were filtered and oven dried, and characterized by DSC.
If only the endothermic peak of coꢀcrystal was detected in DSC scan, the ratio
of dissolved species of STZ to THE for STZꢀTHE coꢀcrystals, and the ratio of
dissolved species of STZ to SNM for STZꢀSNM coꢀcrystals should be equal to 1 to 1.
As expected, the DSC scans (not shown) of STZ crystals, SNM crystals and
STZꢀTHE coꢀcrystals exhibited only one melting point meaning that the integrity of
the crystal structure was maintained at the pH range of 1 to 8. However, the DSC
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scan indicated that STZ crystallized out separately from the solution of STZꢀSNM
system in an acidic environment. Therefore, the pH solubility values of STZꢀSNM
coꢀcrystals at low pH could not be measured.
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STZꢀTHE coꢀcrystals exhibited a slightly higher solubility than the one of STZ
crystals at the pH range from 3 to 6. The sulfonamide–induced crystalluria
frequently occurred during the therapy of sulfonamide, which would cause the kidney
lesions.30 The low solubility of sulfonamide in water is one of the reasons why it
deposits in urine, so that the enhancement in its solubility should help to prevent
crystalluria formation of sulfonamide.31 At the pH range from 5.5 to 7, the solubility
of STZꢀTHE coꢀcrystals was higher than the one of STZ crystals. The solubility
value of STZ enhanced by coꢀcrystal formation may reduce the likelihood of forming
crystalluria of STZ.
Interestingly, the pHꢀdependent solubility of STZ crystals was altered after
coꢀcrystal formation. The solubility value of STZ crystals increased drastically
when the pH value was < 2 or >7, and was higher than the solubility value of
STZꢀTHE coꢀcrystals. However, the degree of increase in the solubility value of
STZꢀTHE coꢀcrystals was not as much as the one of STZ crystals. As for STZꢀSNM
coꢀcrystals, its solubility value was higher than the one of STZꢀTHE coꢀcrystals but
lower than the one of STZ crystals in a basic environment. By taking the advantage
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of this property, the degree of separation of the product can be enhanced. Since STZ,
SNM, STZꢀTHE and STZꢀSNM species were crystallized out by neutralization, the
final pH value was adjusted to 4 or 5 for getting the maximum percent yield of STZ or
SNM crystals. However, the final product of IntenꢀIꢀSTZꢀSNM coꢀcrystals was
adulterated with a trace of STZ crystals when the final pH was lower to 4 or 5. The
endothermic peak of nonꢀcoꢀcrystallized STZ solids at around 165.8 oC was detected
by DSC. Since the solubility value of STZ crystals was much higher than the one of
STZꢀSNM coꢀcrystals when the pH value was > 8, the final pH was raised to 9 for
increasing the purity of IntenꢀIꢀSTZꢀSNM coꢀcrystals by allowing the STZ species to
remain dissolved in the mother liquor, and only to produce the STZꢀSNM coꢀcrystals.
The oilꢀwater partition coefficient, log P, of lornoxicamꢀsaccharin sodium
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coꢀcrystals had been investigated in the literature.58 Log P had a significant effect to
the absorption, distribution, metabolism and excretion (ADME), and it was
determined by the distribution of API molecules in the 1ꢀoctanol phase and the
aqueous phase. HPLC was used to measure the coꢀcrystal concentration in the
aqueous phase. The log P values of STZꢀTHE and STZꢀSNM coꢀcrystals in
1ꢀoctanol/water based on the concentration of dissolved STZ species were ꢀ2.1
×
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×
species.59 The log P value of STZꢀSNM coꢀcrystals based on the concentration of
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