Reducing the Hygroscopicity of the Anti-Tuberculosis Drug (S,S

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Reducing the hygroscopicity of the anti-tuberculosis drug (S,S)-ethambutol using multicomponent crystal forms Luan F. Diniz, Paulo S. Carvalho Jr, Cristiane C. de Melo, and Javier Ellena Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.7b00144 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on April 3, 2017

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Crystal Growth & Design

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Reducing the hygroscopicity of the anti-tuberculosis drug (S,S)-

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ethambutol using multicomponent crystal forms

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Luan F. Diniza, Paulo S. Carvalho Jra, Cristiane C. de Meloa, Javier Ellenaa*

4

a

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Carlos, SP, Brazil;

6

*e-mail address: [email protected]

Instituto de Física de São Carlos, Universidade de São Paulo, CP 369, 13560-970 - São

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ABSTRACT. Ethambutol (ETB) is a chiral dibasic compound formulated and marketed as

9

the (S,S)-ethambutol dihydrochloride, (S,S)-EDH, to treat tuberculosis. It is administered

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orally in a solid formulation composed by isoniazid, rifampicin and pyrazinamide as a fixed-

11

dose combination (FDC) tablet. Due its high hygroscopicity, (S,S)-EDH is known for

12

catalyzing the degradation of isoniazid by rifampicin to yield isonicotinyl hydrazone. In

13

order to avoid or even minimize these mutual drug-drug interactions, in this work we have

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focused on the development of less hygroscopic multicomponent solid forms of ETB. Four

15

salts of this drug, namely oxalate (ETBOXA), maleate (ETBMAL), terephthalate (ETBTRP)

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and trichloroacetate (ETBTCA) were prepared via supramolecular synthesis by slow

17

evaporation method and characterized by X-ray diffraction (SCXR, PXRD), spectroscopic

18

(FT-IR) and thermal (TGA, DSC, HSM) techniques. The hygroscopic nature of these salts,

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including the (S,S)-EDH were evaluated and all of them were found to be hygroscopic, with

20

exception of ETBOXA.

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Keywords: Tuberculosis, Ethambutol, Hygroscopicity, X-ray diffraction

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1. Introduction

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The development of multicomponent crystal forms, e.g. salts and cocrystals,

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represents an important branch of pharmaceutical sciences as alternative route to improve

4

drug’s physicochemical properties (aqueous solubility, dissolution rate, hygroscopicity and

5

thermal stability).1-5 Apart from pharmaceutical perspectives, salt or cocrystal formation is a

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process strictly governed by the acidity/basicity of the ionizable groups in the drug and in the

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salt formers (or coformers).1 In general, pharmaceutically acceptable strong acids are used to

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protonate a basic drug, such as ethambutol (ETB - Scheme 1), and thereby convert it into

9

salts.6 In cases of non-protonation or even non-ionizable drugs, a cocrystal can be formed.7

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However, for ionizable drugs, salt formation is still the most effective low-cost method and

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consequently the preferential one to increase the low solubility and bioavailability of the

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parent drug.1,4,5 As estimated, approximately 50% of all drugs available for medicinal use are

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marketed as salts.8

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Ethambutol is one of the first-line antimycobacterial drugs used to treat tuberculosis

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(TB).9 It is formulated using the active stereoisomer (S,S)-ethambutol in the form of a

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dihydrochloride salt, (S,S)-EDH.9,10 The mechanism of action of this drug is not well known,

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but there are some evidences that it acts by inhibition of the arabinosyl transferase. This

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enzyme catalyzes the polymerization of the arabinose into arabinan and then

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arabinogalactan, an essential component of the mycobacterial cell wall. 9-11

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The first phase of TB treatment is based on the use of ethambutol in combination

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with isoniazid, rifampicin and pyrazinamide as a fixed-dose combination (FDC) tablet.9-11

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The use of four drugs in a single tablet can simplify the TB treatment and limit the risk of

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drug-resistance.12,13 Besides its obvious benefits, one of the main concerns about the use of


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antitubercular FDCs lies in the high hygroscopicity of (S,S)-EDH, which increases the

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degradation of rifampicin and isoniazid by providing a favorable acidic environment.9,10,14 At

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low pH values, rifampicin can be converted to 3-formylrifamycin, which further reacts with

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isoniazid to form isonicotinyl hydrazone. To avoid drug-drug interactions and water uptake,

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two separated wet granulation process are employed, one for (S,S)-EDH and the other one

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for isoniazid and pyrazinamide. These two types of granules are coated with polymers before

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they are blended with rifampicin to make up the final product formulation15.

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In order to overcome the undesirable effects of (S,S)-EDH, an extensive amount of

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studies have been emerged in the last years, many of them dealing with the development of

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multicomponent crystal forms of this drug.16-22 Recently, Nangia et al. have been prepared

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and characterized five salts (sulfate, dimesylate, ditosylate, dibesylate and fumarate) and two

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ionic liquids (dibenzoate and adipate), but all of them were found to be hygroscopic.22 For

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this reason, herein we have been reported a screen of ethambutol with a series of

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carboxylic/dicarboxylic acids, trichloroacetic, maleic, oxalic and terephthalic acids. This

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reaction has given rise to four ETB salts, and this is supported not only by the X-ray

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structures, but also by the spectroscopic data (FT-IR). Finally, the hygroscopic nature of

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these salts and of (S,S)-EDH has been evaluated by thermogravimetric analysis (TGA), in

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which the samples, initially dried, have been exposed to an environment with high relative

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humidity and their mass losses measured for different exposure time.

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2. Experimental Section

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2.1 Preparation of Ethambutol free base (ETB). (S,S)-EDH was purchased from Sigma-

23

Aldrich and used without further purification. All other chemicals were of analytical or



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chromatographic grade. ETB was obtained according to the method reported by Bhutani et

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al.9 To 2g of (S,S)-EDH were added 20 mL of a 5 mol L-1 aqueous NaOH solution. This

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mixture was stirred for 10-15 min and the product (ETB) extracted with CH2Cl2 (25 mL).

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The organic phase was further dried with Na2SO4 to remove traces of water. Block-shaped

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crystals of ETB were grown after one day by slow evaporation of CH2Cl2 at room

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temperature. The final product was characterized by PXRD (Figure S2, Supporting

7

Information).

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2.2 Supramolecular Synthesis

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a. Ethambutol oxalate (ETBOXA). 20 mg of ETB (0.098 mmols) were mechanically milled

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with 17.6 mg (0.195 mmols) of oxalic acid in a 1:2 (drug:acid) molar ratio. The resulting

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solid was dissolved in a 70% (v/v) methanol/ethanol solution and left to evaporate at room

12

temperature. Colorless prism crystals were obtained after a few days upon slow solvent

13

evaporation. The PXRD patterns from the milled and recrystallized samples are presented in

14

Figure S4.

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b. Ethambutol maleate (ETBMAL). 20 mg of ETB were dissolved in a 70% (v/v)

16

methanol/ethanol solution and stirred at 70 oC. To this solution were added 22.7 mg (0.196

17

mmols) of maleic acid. Colorless plate crystals were obtained after a few days upon slow

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solvent evaporation.

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c. Ethambutol terephthalate (ETBTRP). 32.5 mg (0.195 mmols) of terephthalic acid were

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dissolved in a 70% (v/v) methanol/ethanol solution and stirred at 70 oC. This solution was

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filtered and then 20 mg of ETB were added. After a few hours of solvent evaporation

22

colorless prism were grown.


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d. Ethambutol trichloroacetate (ETBTCA). 20 mg of ETB were dissolved in methanol and

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stirred at 50 oC. Next, 31.9 mg (0.196 mmols) of trichloroacetic acid were added to the drug

3

solution. Colorless prism crystals were formed after a few days upon slow solvent

4

evaporation.

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2.3 Single Crystal Structure Determination (SCSD). The X-ray diffraction data for the

6

ETB salts were collected at room temperature on a Agilent Super Nova diffractometer with

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CCD detector system equipped with a Mo source (λ = 0.71073Å). Data integration, Lorentz-

8

polarization effects and absorption corrections were performed with CrysAlisPro (version

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171.38.43b). Using Olex2,23 the structure was solved by direct methods and the model

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obtained was refined by full–matrix least squares on F2 (SHELXTL–97).24 All the hydrogen

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atoms were placed in calculated positions and refined with fixed individual displacement

12

parameters [Uiso(H) = 1.2Ueq or 1.5Ueq] according to the riding model (C–H bond lengths of

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0.97 Ǻ and 0.96 Ǻ, for methylene and methyl groups, respectively). Molecular

14

representations were generated by Olex223 and MERCURY 3.7.25 The CIF file of all

15

structures were deposit in the Cambridge Structural Data Base26 under the codes CCDC

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1529922, 1529925, 1529926 and 1529927. Copies of the data can be obtained, free of

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charge, via www.ccdc.cam.ac.uk.

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2.4 Powder X-ray Diffraction (PXRD). Powder X-ray diffraction was used to check the

19

purity of all solid forms reported here (Figure S2). Data were recorded at room temperature

20

on Rigaku Ultima IV diffractometer, in Bragg-Brentano reflective geometry, with CuKα

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radiation (λ = 1.5406 Å) at 40 kV-30 mA and Ni filter. The diffractograms were acquired in

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the 3-80° 2θ range with a step width of 0.02° and a constant counting time of 5 s per step.



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2.5 Vibrational Spectroscopy Analysis. Fourier Transform infrared (FTIR) spectra were

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recorded on an Alpha Bruker FT-IR spectrophotometer, using KBr pellets, in the range of

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4000-400 cm-1, with an average of 64 scans and 2 cm-1 of spectral resolution.

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2.6 Thermal Analysis. Differential Scanning Calorimetry (DSC) curves were obtained with a

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Schimadzu DSC-60 instrument. The samples (2.5 ± 0.5 mg) were placed in open aluminum

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pans and heated from 25 to 400 ºC under a N2. The heating rate was set to 10 ºC/min.

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Thermogravimetric analyses (TGA) were carried out on a Shimadzu TGA-60 thermobalance.

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Approximately 5.0 mg of the samples were placed in open alumina pans and heated under N2

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flow from 25 to 500 ºC at a heating rate of 10 ºC/min. The resulting data were analyzed using

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the Shimadzu TA-60 software (version 2.2).

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2.7 Hot-Stage Polarized Optical Microscopy (HSM). HSM experiments were performed

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on a Linkam T95-PE device coupled to a Leica DM2500P optical microscope. Images were

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recorded using a CCD camera attached to the microscope at time intervals of 10 s. Single

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crystals of ETB salts were heated at a constant rate of 10 ºC/min over a temperature range

15

from 30 ºC until the melting of the crystals. Both heating and acquisition of the images were

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controlled by the Lynksys 32 software package (version 1.96).

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3. Result and Discussion

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3.1 Structural Description. ETB is a dibasic drug that contains two ionizable amine groups

19

(pKa1 = 6.35 and pKa2 = 9.35) on its molecular structure.27 In order to reproduce the primary

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NH+…COO- synthon, only acids containing carboxylic/dicarboxylic groups were selected for

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the co-crystallization experiments with ETB. The salt formation was investigated using a

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range of acids with different pKa values: acid oxalic (pKa1 = 1.25; pKa2 = 4.27), maleic acid


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(pKa1 = 1.83; pKa2 = 6.07), trichloroacetic acid (pKa1 = 0.66) and terephthalic acid (pKa1 =

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3.55; pKa2 = 4.46).28 The calculated ∆pKa (pKa(ETB) − pKa(acid)) values for the reaction of

3

ETB with these acids are presented in Table 1. Even considering the pKa2, these reactions

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give ∆pKa values above the ones required by salt formation (∆pKa > 3). The only exception

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was the terephthalic acid, for which the ∆pKa values fell into the range from 0 to 3 (Table 1).

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At this range, it is not possible to predict the proton transfer between the two species

7

(ETB/acid) and consequently the salt formation, being both forms salts and cocrystals equally

8

likely to occur.

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The reaction between ETB and the acids resulted in the following crystal

10

stoichiometries: [ETBH2]2+/OXA- 0.5:1, [ETBH2]2+/MAL- 1:2, [ETBH2]2+/TRP2- 1:1 and

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[ETBH2]2+/TCA- 1:2 salts. A single deprotonation for the maleic and oxalic acids is expected

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due to the large differences (> 3 units) on their pKa values. On the other hand, for the

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therephtalic acid, the first and second ionization constants (expressed by pKa1/pKa2 values)

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are close, which suggest that a double deprotonation could occurs concomitantly (Table 1).

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For all ETB X-ray structures, the identification of the crystalline form was performed

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measuring the C−O bond length differences (∆DC-O).7,28 The expected equality of the C−O

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bond distances (∆DC-O < 0.03 Å) was used as an indicative of the electronic conjugation and

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partial double-bond character normally expected after the proton loss. Even so, spectroscopy

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techniques were also applied to confirm the salt formation.7,28 The asymmetric unit (ASU) of

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ETB salts is depicted in Figure S1 (see Supporting Information). To evaluate if the crystal

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chosen for the single crystal data collection was representative of the whole sample, PXRD

22

measurements were carried out (Figure S2). A detailed description of each crystal structure is



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1

provided below. In Table 2 and 3 are summarized, respectively, the crystallographic details

2

and the geometric parameters of the H-bonds.

3

Ethambutol oxalate (ETBOXA, 0.5:1). This salt crystallizes in the orthorhombic space

4

group P21212 with Z’= ½. The ASU (Figure S1a) consists of one oxalate anion partially

5

deprotonated (OXA-) and a half of ethambutol cation ([ETBH2]2+). The [ETBH2]2+ cation lies

6

on a 2-fold axis, which gives rise to a 0.5:1 ([ETBH2]2+/OXA-) stoichiometry. The partial

7

deprotonation of the acid could not be proven by analysis of the ∆DC-O values (0.049 and

8

0.086 Å). ETBOXA adopts a 2D layered structure defined not only by the expected

9

NH+…COO- synthon, but also by the OH…COO- and COOH…COO- ones. In the crystal

10

structure (Figure 1a), the cations are associated to the OXA- anions via N+−H···O- H-bonds

11

(N1+−H1A···O4A-, 2.956 Å, 140.1º and N1+−H1B···O2A-, 2.859 Å, 169.1º) to form 1D

12

chains along the [001] direction (Figure 1b). Beyond these interactions, the oxalate is also

13

connected to the hydroxyl group of [ETBH2]2+ through the O1−H1···O1A- (2.787 Å; 161.7º)

14

H-bond. Furthermore, the anions are also linked to each other via O−H···O-

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(O3A−H3AA···O1A-, 2.465 Å, 175.5º) H-bonds, to form two independent 1D chains running

16

in opposite directions along the [100] (Figure 1c). In these chains, the OH hydrogens might

17

be shared for both oxygen or even located in the center of the O···O atoms. These

18

supramolecular features might explain the differences found for the C−O and C=O bond

19

lengths.34,35 These chains are perpendicularly stacked along the [010] direction, as shown in

20

Figure 1a.

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Ethambutol maleate (ETBMAL, 1:2). This salt crystalizes in the orthorhombic space group

22

P212121 with one [ETBH2]2+ cation and two MAL- anions in a 1:2 stoichiometry. The ASU


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1

containing the ion-pair is shown in Figure S1b. Proton transfer from two acid molecules to a

2

single ETB molecule confirms the salt formation with protonation of both amine groups.

3

However, this finding agrees only with the ∆DC-O values calculated for one anion (0.024 Å).

4

The other one shows C−O bonds distances very unequal (1.273/1.215 and 1.281/1.225 Å),

5

which gives ∆DC-O (0.058 and 0.056 Å) values above the defined cut-off (∆DC-O < 0.03 Å). As

6

expected, the anions display a planar configuration stabilized by the intramolecular O−H···O-

7

(O2A−H4AA···O4A-, 2.418 Å, 172.7º and O2B−H4BA···O4B-, 2.416 Å, 173.56º) H-bonds.

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A similar phenomenon to the one found in ETBOXA could explain the single deprotonation

9

of the acid.34,35 Despite the unit cell differences (Table 1), this salt has some similarities at

10

supramolecular level with the oxalate salt (see Figure 1a and 2a). The OH−COO- interactions

11

are preserved in both salts, as well as the NH+…COO- synthons. The MAL- anions act as a

12

bridge, connecting the cations via N+−H···O- H-bonds (see Table 2) to form 1D chains along

13

the [010] direction (Figure 2b). The 1D chains are further connected to each other into 2D

14

layers parallel to the (001) plane by H-bonds involving the OH groups (O2−H2C···O-, 2.765

15

Å, 174.3º) of the drug (Figure 2a). In addition, the anions also make an infinite and

16

independent 1D chain along the [100] direction through C−H···O- interactions, thus giving

17

rise to a (7) motif (Figure 2c). As observed in the ETOXA structure, these chains run in

18

opposite directions along the [100] and are perpendicularly stacked along the [001] direction

19

(Figure 2a).

20

Ethambutol terephthalate (ETBTRP, 1:1). This salt belongs to the triclinic space group P1

21

and its ASU consists of one [ETBH2]2+ cation and one TRP2- anion (Figure S1c). Protonation

22

of the two secondary amines of the drug by the diprotic terephthlalic acid is confirmed by

23

∆DC-O values (0.024 and 0.031 Å). The corresponding NH+…COO- synthons lead to the



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formation of a (12) motif defined by four N+−H···O- (see Table 2) H-bonds (Figure 3b).

2

Such motifs further assemble into a 3D arrangement via O1−H1···O3- (2.826 Å, 145.9º) and

3

O2−H2C···O5- (2.751Å, 162.4º) H-bonds (Figure 3a). Beyond these interactions, ETB+

4

cations and TRP- anions are also connected along the [010] direction by non-classical

5

C−H···π (C4−H4C···Cg, 3.647 Å, 147.3º) interactions (Figure 3c).

6

Ethambutol trichloroacetate (ETBTCA, 1:2). This salt was solved and refined in the

7

monoclinic space group P21 with one [ETBH2]2+ cation and two TCA- anions (A and B) in

8

the ASU (Figure S1d). Proton transfer from two monoprotic acids (∆DC-O = 0.035 Å, ∆DC-O =

9

0.005 Å) to a single ETB molecule resulted in protonation of both N1 and N2 amine atoms

10

and in the formation of NH+…COO- synthons. However, as expected from structures with Z' >

11

1, each TCA- anion has its own supramolecular pattern.29 While the anion A is associated to

12

both amine groups by the bifurcated N1+−H1B···O1A- (2.780 Å, 147.7º) and

13

N2+−H2A···O1A- (2.861 Å, 160.6º) interactions, the anion B binds only to one via the

14

N2+−H2B···O1B- interaction (Figure 4b). These ion-pairs propagate along the [001] direction

15

through O1−H1···O2B- (2.801 Å, 160.6º) H-bonds (Figure 4c). The overall 3D packing is

16

characterized by the presence of a hydrophilic cavity established by TCA- anions running

17

along the ac plane (Figure 4a).

18

ETB is a flexible molecule and therefore can adopt different conformations by

19

rotation of C-C and C-N bonds. In Figure S3 is presented the overlay of the five [ETBH2]2+

20

cations (one arising from the (S,S)-EDH salt and the other ones from the salts studied here),

21

obtained by the superimposing of N1, C5, C6 and N2 atoms. The cations differ in the spatial

22

orientation of the ethyl and hydroxyl groups, which is implicit by the different values


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founded for the N1−C2−C1−O1 and N1−C2−C3−C4 torsion angles (Table 4). For ETBTCA,

2

it is also observed a significant variation on the value of the respective N1−C5−C6−N2

3

torsion angle. Such deviation could be a consequence of the bifurcated N+−H···O- H-bonds

4

presented only in its structure.

5

3.2 Spectroscopy Analysis. FT-IR spectra of ETB free base and its salts are presented in

6

Figure 5. Analysis and band assignments were performed using the spectroscopic data

7

available for the carboxylic/dicarboxylic acids and ETB solid forms.22,30,31 Salt formation

8

was identified by evaluation of changes in the vibrational modes of specific functional

9

groups of the drug and the salt formers.32,33 After the salt formation, the strong C=O

10

absorption band (~1700 cm-1) of COOH group should disappear giving rise to two bands in

11

the FT-IR spectrum. These two bands are associated to the antisymmetric (~1550 cm-1) and

12

symmetric (~1400 cm-1) stretching vibrations of COO- group and can be used as a fingerprint

13

to check the acid deprotonation. Consequently, this should also affect the NH vibration

14

modes of the secondary amine groups of ETB. According to the literature, secondary amine

15

shows only one NH stretch (3300-3150 cm-1), while its salt, or protonated form( ⋰⋱NH ),

16

shows NH stretches in the region of 3100-2800 cm-1. In the FT-IR spectrum of ETB free

17

base, the NH stretching vibration appears as a single band at 3270 cm-1. However, due to its

18

protonation, bands related to the NH2+ stretching modes are observed shifted to lower

19

wavenumbers in the salts spectra: 3000 cm-1 (ETBOXA), 3004 cm-1 (ETBMAL), 2977 cm-1

20

(ETBTRP) and 2983 cm-1 (ETBTCA). With respect to the acids, it is worth mentioning that

21

the partial deprotonation of the oxalic and maleic acids could be identified by the presence of

22

bands at 3447 and 3350 (O−H stretching) and 1702 and 1698 cm-1 (C=O stretching),

23

respectively. The complete deprotonation of the acid molecules in the ETBTRP and



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ETBTCA structures is confirmed by the presence of bands arising only from the

2

antisymmetric and symmetric stretching of COO- groups (bands assigned from 1620 to 1330

3

cm-1).

4

3.3 Thermal Characterization. DSC curves show distinct melting points for all salts,

5

however for ETBOXA and ETBTRP, the melting temperatures are found to be quite close

6

(Figure 6). DSC curves of ETBOXA and ETBTRP are characterized by an endothermic

7

melting peak at 204.1 oC (Tonset = 200.04 oC) and 207.43 oC (Tonset = 206.29 oC), respectively.

8

These values are in agreement with the TGA curves, since that any mass loss was observed

9

for these salts in the range of 50-208 oC. The first mass losses in the TGA curves of

10

ETBOXA (210-240 oC) and ETBTRP (209-263 oC) were attributed to the beginning of

11

sample decomposition.

12

The melting point of ETBMAL can be associated to a sharp endothermic peak

13

observed at 152.24 oC (Tonset = 151.08 oC) in the DSC curve. This salt is thermally stable up

14

to ~176 oC; after this temperature, a gradual mass loss can be observed in the TGA curve. In

15

comparison with the other salts reported here, ETBTCA is the one with the lowest melting

16

temperature. DSC curve of ETBTCA showed an endothermic peak at 126.23 oC (Tonset =

17

125.26 oC), which was assigned to the melting process.

18

The interpretation of DSC/TGA results were successfully confirmed by HSM

19

experiments (Figure 7). According to the HSM images, a single-crystal of ETBOXA,

20

ETBMAL, ETBTRP and ETBTCA starts to melt at 204 oC, 154 oC, 200 oC and 123 oC,

21

respectively. As a result, it is worth to mention two points: (1) these salts are thermally more

22

stable than the corresponding ETB free base (melting point of ∼88 oC) and (2) in all cases

23

there is no evidence of a phase transition.


Page 13 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

3.4 Hygroscopicity Measurements. Hygroscopicity of ETB salts were evaluated by

2

thermogravimetric analysis. Firstly, the samples were dried in an oven and then placed in a

3

desiccator. Distilled water was employed to maintain the relative humidity (100% RH)

4

inside the desiccator. The time that each salt was kept inside the desiccator can be related to

5

how fast it absorbs water. Therefore, the more hygroscopic the salt is, the more quickly it

6

absorbs water, and the less time it will be on the desiccator. The TGA curves were used to

7

check the amount of water adsorbed, since the mass losses before 150 oC were due to the

8

content of water molecules previously adsorbed by the dried samples.

9

As expected, (S,S)-EDH proved to be very hygroscopic. After one hour in a humid

10

environment, the hydrochloride form had a mass loss of about 25% (Figure 8a). Moreover,

11

this salt became liquid when remained more than eight hours into the desiccator (Figure 8d).

12

However, ETBOXA shown to be non-hygroscopic, even after 48 hours in the desiccator

13

(Figure 8e). This result suggests that the packing arrangement of ETBOXA molecules is so

14

cohesive that it would avoid penetration by water molecules. For the other salts (ETBMAL,

15

ETBTRP and ETBTCA), the TGA curves (Figures 8a-c) show significant mass losses owing

16

to their hygroscopic nature.

17

4. Conclusions Within our continuing efforts to develop new solid forms of anti-tuberculosis drugs,

18 19

in

this

work

we

presented

four

salts

of

ETB

20

carboxylic/dicarboxylic (oxalic, maleic, terephtalic and trichloroacetic) acids. These salts

21

were prepared aiming to get a non-hygroscopic solid form of ETB to reduce the influence of

22

the high hygroscopicity of (S,S)-EDH which is responsible for the degradation of isoniazid

23

and rifampicin in the FDC tablet used in the TB treatment. Analysis of the crystal structures


obtained

by

reaction

with


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 42

1

(and packing) revealed that the ion-pairs are stabilized by the expected NH+… COO-

2

synthons, and that in all cases the two amine groups of the drug are protonated. Similarly,

3

their salt natures were confirmed by the appearing of two bands in the FT-IR spectra (3400-

4

3200 cm-1) related to the NH2+ stretching modes.

5

The salts were found to melt between 123 and 204 oC, i.e. above the melting

6

temperature of ETB (~88 oC). For all salts, DSC curves showed only a single melting

7

endothermic peak, which rule out any phase transition in this temperature range. In addition,

8

with exception of the oxalate salt, all the other ones show to be hygroscopic. The reason for

9

this is not totally clear, but it seems to be related to the differences in the supramolecular

10

arrangements, mainly to the formation of OXA-… OXA- infinite chains through O−H···O-

11

H-bonds that give rise to the formation of a more compact 3D crystal packing. This

12

supramolecular feature might explain the non-hygroscopic nature of the ETBOXA salt.

13

Interestingly, the oxalate salt was also the one with higher melting temperature (204 oC).

14

Supporting Information

15

The Supporting Information is available free of charge on the ACS Publications

16

website at DOI:

17

PXRD patterns, ORTEP view of the asymmetric units, crystallographic data and Figures and

18

Tables related to single crystal studies for ethambutol (ETB) salts.

19

Acknowledgements

20

The authors acknowledge the Brazilian funding agencies FAPESP (L.F.D. grant

21

15/25694-0 and P.S.C.-Jr. grant 12/05616-7), CAPES (C.C.M) and CNPq for financial

22

support. The authors would also like to thank Dra. Renata Diniz and Dra. Charlane C. Correa

23

(Federal University of Juiz de Fora) for allowing access to the X-ray diffraction facilities.


Page 15 of 42

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46


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Johan, Wouters, Q. L. Pharmaceutical Salts and Co-crystals; 2011.

(2)

Berge, S. M.; Bighley, L. D.; Monkhouse, D. C. J. Pharm. Sci. 1977, 66, 1–19.

(3)

Babu, N. J.; Nangia, A. 2011, 2662–2

(4)

Serajuddin, A. T. M. Advanced Drug Delivery Reviews. 2007, p 603–616.

(5)

De Melo, C. C.; Da Silva, C. C. P.; Pereira, C. C. S. S.; Rosa, P. C. P.; Ellena, J. Eur. J. Pharm. Sci. 2016, 81, 149–156.

(6)

Carvalho, P. S.; De Melo, C. C.; Ayala, A. P.; Da Silva, C. C. P.; Ellena, J. Cryst. Growth Des. 2016, 16, 1543–1549.

(7)

Da Silva, C. C. P.; Pepino, R. de O.; De Melo, C. C.; Tenorio, J. C.; Ellena, J. Cryst. Growth Des. 2014, 14, 4383–4393.

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Heinrich, S.; Wermuth, C. Wiley InterScience. 2008, p 388. Bhutani, H.; Singh, S.; Jindal, K. C.; Chakraborti, A. K. J. Pharm. Biomed. Anal. 2005, 39, 892–899.

(10)

Singh, S.; Mohan, B. Int. J. Tuberc. Lung Dis. 2003, 7, 298–303.

(11)

Bastos, M. L.; Hussain, H.; Weyer, K.; Garcia-Garcia, L.; Leimane, V.; Leung, C. C.; Narita, M.; Penã, J. M.; Ponce-de-Leon, A.; Seung, K. J.; Shean, K.; SifuentesOsornio, J.; Van der Walt, M.; Van der Werf, T. S.; Yew, W. W.; Menzies, D. Clin. Infect. Dis. 2014, 59, 1364–1374.

(12)

WHO; The World Health Organization. 4Th Ed. 2010, 160.

(13)

Bastos, M. L.; Hussain, H.; Weyer, K.; Garcia-Garcia, L.; Leimane, V.; Leung, C. C.; Narita, M.; Penã, J. M.; Ponce-de-Leon, A.; Seung, K. J.; Shean, K.; SifuentesOsornio, J.; Van der Walt, M.; Van der Werf, T. S.; Yew, W. W.; Menzies, D.; Hong-min, W.; Xiao-Hong, Z. Clin. Infect. Dis. 2015, 60, 1285–1286.

(14)

Nh, C. H. C. H. O. C. H. O.; Tarasiewicz, J.; Ga, A.; Bhutani, H.; Singh, S.; Jindal, K. C.; Chakraborti, A. K.; Inst, R.; Lutz, A.; Swamy, N.; Prashanth, K. N.; Basavaiah, K. 2015, 1002, 28–36.

(15)

Part 6 Rifampicin/Isoniazid/ Pyrazinamide/Ethambutol 150mg/75mg/400mg/275mg film-coated Tablets Part 6 Rifampicin/Isoniazid/ Pyrazinamide/Ethambutol 150mg/75mg/400mg/275mg film-coated Tablets. Bai, G.-Y.; Zhang, C.-F.; Zhang, Y.-C.; Zeng, T.; Li, J.-S. Acta Crystallogr. Sect. E Struct. Reports Online 2006, 62, 2173–2174.

(16)



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Bai, G. Y.; Zhang, C. F.; Simpson, J.; Ning, H. S.; Peng, H. W. Acta Crystallogr. Sect. E-Structure Reports Online 2006, 62, 5580–5581.

(18)

Godfrey, R.; Hargreaves, R.; Hitchcock, P. B. Acta Crystallogr. Sect. C Cryst. Struct. Commun. 1992, 48, 79–81.

(19)

Bai, G.; Ning, H.; Qin, X. 2006, 4567–4568.

(20)

Faria, A. F.; Marcellos, L. F.; Vasconcelos, J. P.; De Souza, M. V. N.; Júnior, A. L. S.; Do Carmo, W. R.; Diniz, R.; De Oliveira, M. A. L. J. Braz. Chem. Soc. 2011, 22, 867–874.

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Cherukuvada, S.; Nangia, A. CrystEngComm 2012, 14, 7840. Cherukuvada, S.; Nangia, A. Cryst. Growth Des. 2013, 13, 1752–1760.

(23)

Dolomanov, O. V.; Bourhis, L. J.; Gildea, R. J.; Howard, J. A. K.; Puschmann, H. J. Appl. Crystallogr. 2009, 42, 339–341.

(24)

Sheldrick, G. M. Acta Crystallogr. Sect. A Found. Crystallogr. 2008, 64, 112–122.

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Macrae, C. F.; Edgington, P. R.; McCabe, P.; Pidcock, E.; Shields, G. P.; Taylor, R.; Towler, M.; Van De Streek, J. Journal of Applied Crystallography. 2006, p 453–457.

(26)

Allen, F. H.; Motherwell, W. D. S. Acta Crystallogr. B. 2002, 58, 407–422.

(27)

Tuberculosis 2008, 88, 102–105.

(28)

Childs, S. L.; Stahly, G. P.; Park, A. Mol. Pharm. 2007, 4, 323–338.

(29)

Steed, J. W. W. CrystEngComm 2003, 5, 169–179.

30)

Giffin, G. A.; Boesch, S.; Bopege, D. N.; Powell, D. R.; Wheeler, R. A.; Frech, R. 2009, 15914–15920.

(31)

Heacock, R. A.; Marion, L. Can. J. Chem. 1956, 34, 1782–1795.

(32)

Heinz, A.; Strachan, C. J.; Gordon, K. C.; Rades, T. J. Pharm. Pharmacol. 2009, 61, 971–988.

(33)

Lin, H. L.; Huang, Y. T.; Lin, S. Y. J. Therm. Anal. Calorim. 2016, 123, 2345–2356.

(34)

Steiner, T. Chemical Communications, 1999, 22, 2299–2300.

(35)

Bartoszak, E.; Dega-Szafran, Z.; Jaskólski, M.; Szafran, M. Journal of the Chemical Society, Faraday Transactions, 1995, 91, 87–92.


Page 17 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1


FIGURE CAPTIONS

2 3

Scheme 1. Molecular structure of ETB and salt formers used in this study.

4 5

Figure 1. Crystal packing of ETBOXA along the [100] direction (a). H-bond 1D chain

6

pattern highlighting the NH+… COO- synthon (b) and partial view of packing of the anion

7

chains anions along the [100] direction (c).

8 9

Figure 2. Crystal packing of ETBMAL along the [100] direction (a). H-bond 1D chain

10

pattern highlighting the NH+… COO- synthon (b) and partial view of packing of the anion

11

chains along the [001] direction (c).

12 13

Figure 3. View of ETBTRP crystal packing along the [100] (a). The NH+…COO- synthons

14

giving rise to a (12) motif (b). These motifs are further assembled into a 3D packing by

15

O−H···O- and C−H···π interactions (c).

16 17

Figure 4. View of ETBTCA packing along the ac plane showing the hydrophilic channels in

18

which the chlorine atoms are inserted (a). The ion-pairs formed by the N+−H···O- H-bonds

19

(b) are extended into a 1D chain via O−H···O- H-bonds along the [001] direction (c).

20 21

Figure 5. FT-IR spectra of ETB free base (black) and ETB salts: ETBOXA (blue),

22

ETBMAL (red), ETBTRP (violet) and ETBTCA (green).

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 6. DSC and TGA curves of ETB salts: (a) ETBOXA, (b) ETBMAL, (c) ETBTRP

2

and (d) ETBTCA.

3 4

Figure 7. Hot-stage microscopy images of ETBOXA (a), ETBMAL (b), ETBTRP (c) and

5

ETBTCA (d) single crystals.

6 7

Figure 8. TGA curves of ETB salts after one hour (a), six hours (b) and twelve hours (c) in

8

humid atmosphere. Images of (S,S)-EDH after eight hours (d) and ETBOXA after forty-eight

9

hours (e) in humid atmosphere.

10 11 12 13 14 15 16 17 18 19 20 21 22 23


Page 18 of 42

Page 19 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TABLE CAPTIONS

1 2 3

Table 1. Calculated ∆pKa values for the reaction of ETB with the dicarboxylic/carboxylic

4

acids studied here.

5 6

Table 2. Crystal data, data collection and structure refinement parameters of ETB salts.

7 8

Table 3. Geometric parameters of the H-bonds in the ETB salts.

9 10

Table 4. Selected torsion angles for (S,S)-EDH and ETB salts.

11 12 13 14 15 16 17 18 19 20 21 22 23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Scheme 1.

2 3

4


Page 20 of 42

Page 21 of 42

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1


Figure 1.

2 3

4 5 6 7 8 9 10 11 12 13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 2.

2 3

4 5 6 7 8 9 10 11 12 13


Page 22 of 42

Page 23 of 42

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1


Figure 3.

2 3

4 5 6 7 8 9 10 11 12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 4.

2 3

4 5 6 7 8 9 10 11 12


Page 24 of 42

Page 25 of 42

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1


Figure 5.

2 3

4 5 6 7 8 9 10 11 12 13 14



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 6.

2 3

4 5 6 7 8 9 10 11 12 13


Page 26 of 42

Page 27 of 42

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1


Figure 7.

2 3

4 5 6 7 8 9 10 11 12



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Figure 8.

2 3

4 5 6 7 8 9 10 11


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Table 1.

1

Compound

pKa1/pKa2

Ethambutol Oxalic acid Maleic acid Terephtalic acid Trichloroacetic acid

6.35/9.35 1.25/4.27 1.83/6.07 3.55/4.46 0.66

∆pKa = (pKa(ETB) − pKa(acid)) 5.10/5.08 4.52/3.28 2.80/4.89 5.69

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20


Stoichiometry 0.5:1 salt 1:2 salt 1:1 salt 1:2 salt


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 42

Table 2.

1

Identification code Empirical formula Formula weight Temperature (K) Crystal system Space group a (Å) b (Å) c (Å) α (°) β (°) γ (°) Volume (Å)3 Z / Z’ ρcalc (g cm3) µ (mm-1) F(000) Reflections collected Independent reflections Unique reflections R1 [I≥2σ(I)] wR2 [all data] Goodness-of-fit on F2 Flack parameter

ETBOXA C7H14NO5 192.19 293(2) Orthorhombic P21212 5.5391(4) 21.3637(19) 7.9772(5) 90 90 90 943.99(12) 2/½ 1.352 0.115 412.0 5775

ETBMAL C18H32N2O10 174.18 293(2) Orthorhombic P212121 5.7004(6) 19.2444(19) 20.694(4) 90 90 90 2270.1(5) 4/1 1.274 0.104 932.0 10447

ETBTRP C18H30N2O6 185.22 293(2) Triclinic P1 5.7153(2) 8.3805(4) 10.7486(5) 70.141(4) 85.652(3) 88.231(4) 482.81(4) 1/1 1.274 0.095 200.0 9861

ETBTCA C14H26Cl6N2O6 212.43 293(2) Monoclinic P21 11.0388(8) 8.7807(5) 12.8540(9) 90 104.999(7) 90 1203.47(15) 2/1 1.466 0.744 548.0 11591

1875

3904

3897

5315

1538 0.0468 0.1125 1.086 0.0(10)

2802 0.0589 0.1507 1.088 0.1(9)

3713 0.0460 0.1231 1.054 -0.3(5)

4509 0.0501 0.1280 1.045 0.02(3)

2 3 4 5 6 7 8


Page 31 of 42

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Table 3.

1

Interaction

2

D···A(Å)

O1−H1···O1AO3A−H3AA···O1AN1+−H1A···O4AN1+−H1B···O2A-

2.787 2.465 2.956 2.859

O2−H2C···O1 O1−H1···O2BN1+−H1A···O1BN1+−H1B···O3AN2+−H2B···O3BN2+−H2A···O1AC2A−H2AA···O2AC3A−H3AA···O4AC2B−H2BA···O2BC3B−H3BA···O4B-

2.765 2.763 2.735 2.748 2.783 2.785 3.384 3.479 3.541 3.412

O1−H1···O3O2−H2C···O5N1+−H1A···O5N2+−H2A···O6N2+−H2B···O3N1+−H1B···O4C4−H4C···Cg *

2.826 2.751 2.744 2.777 2.735 2.761 3.647

N1+−H1B···O1AN2+−H2A···O1AN2+−H2B··· O1BN1+−H1A··· O2AO1−H1···O2BO2−H2···O2B-

2.780 2.862 2.747 2.796 2.801 2.798

H···A(Å) ETBOXA 1.996 1.647 2.217 1.980 ETBMAL 1.948 1.954 1.891 1.858 1.913 1.944 2.532 2.576 2.676 2.591 ETBTRP 2.109 1.957 1.868 1.926 1.854 1.916 2.802 ETBTCA 1.987 2.007 1.869 1.935 2.025 2.052

D−H···A(º)

Symmetry Code

161.72 175.56 140.13 169.12

x,y,z x,y,z x+1,+y,+z x+1,+y,+z+1

174.37 168.91 157.65 178.19 165.08 156.96 152.47 163.62 155.15 147.54

-x+1/2+2,-y+1, +z-1/2 -x+1/2+2,-y+1,+z+1/2 -x+1/2+1,-y+1,+z+1/2 -x+1,+y+1/2,-z+1/2+1 x+1/2,-y+1/2+1,-z+1 x+1,+y,+z x+1, +y, +z x+1, +y, +z x-1,+y,+z x-1,+y,+z

145.95 162.44 167.58 159.46 170.19 157.92 147.35

x,y,z x,+y+1,+z+1 x,+y,+z+1 x-1,+y,+z+1 x-1,+y+1,+z x,+y,+z+1 x,y,z

147.72 160.66 168.66 162.28 157.70 151.11

x,y,z x,y,z x,y,z -x+1,+y+1/2,-z+1 x-1,+y,+z -x+2,+y-1/2,-z+1

* Cg: C12→C17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Table 4.

1

N1−C2−C1−O1 N1−C2−C3−C4 N2−C8−C7−O2 N2−C8−C9−C10 N1−C5−C6−N2 2

Page 32 of 42

1

(S,S)-EDH1,2 58.6(7) 168.6(5) 58.6(7) 168.6(5) 171.0(4)

ETBOXA1,2 76.0(3) -163.9(3) 76.0(3) -163.9(3) 172.0(2)

ETBMAL -61.7(4) -170.4(5) -56.9(6) -168.4(5) -175.5(3)

ETBTRP -70.2(4) -178.0(4) -80.1(4) -170.9(3) 177.1(3)

N1−C2−C1−O1 = N2−C8−C7−O2 and 2 N1−C2−C3−C4 = N2−C8−C9−C10

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


ETBTCA 54.2(6) -161.7(7) -57.9(6) -164.4(5) 78.1(5)

Page 33 of 42

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1

FOR TABLE OF CONTENTS USE ONLY

2

Reducing the hygroscopicity of the anti-tuberculosis drug (S,S)-ethambutol using

3

multicomponent crystal forms

4

Luan F. Diniza, Paulo S. Carvalho Jra, Cristiane C. de Meloa, Javier Ellenaa*

5 6

7 8 9

We investigate novel multicomponent crystal forms of the anti-tuberculosis drug (S,S)-

10

ethambutol (ETB) with a series of carboxylic acids: trichloroacetic, maleic, oxalic and

11

terephthalic acids. This reaction has given rise to four ETB salts. The hygroscopic nature of

12

these salts, including the (S,S)-EDH, were evaluated and all of them were found to be

13

hygroscopic, with exception of the Ethambutol oxalate.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Scheme 1. Molecular structure of ETB and salt formers used in this study Scheme 1. 254x190mm (96 x 96 DPI)


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Figure 1. Crystal packing of ETBOXA along the [100] direction (a). H-bond 1D chain pattern highlighting the NH+… COO- synthon (b) and partial view of packing of the anion chains anions along the [100] direction (c). Figure 1. 254x190mm (96 x 96 DPI)



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Figure 2. Crystal packing of ETBMAL along the [100] direction (a). H-bond 1D chain pattern highlighting the NH+… COO- synthon (b) and partial view of packing of the anion chains along the [001] direction (c). Figure 2. 254x190mm (96 x 96 DPI)


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Figure 3. View of ETBTRP crystal packing along the [100] (a). The NH+…COO- synthons giving rise to a R(_4^4)(12) motif (b). These motifs are further assembled into a 3D packing by O−H···O- and C−H···π interactions (c). Figure 3. 279x190mm (96 x 96 DPI)



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Figure 4. View of ETBTCA packing along the ac plane showing the hydrophilic channels in which the chlorine atoms are inserted (a). The ion-pairs formed by the N+−H···O- H-bonds (b) are extended into a 1D chain via O−H···O- H-bonds along the [001] direction (c). Figure 4. 279x190mm (96 x 96 DPI)


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Figure 5. FT-IR spectra of ETB free base (black) and ETB salts: ETBOXA (blue), ETBMAL (red), ETBTRP (violet) and ETBTCA (green). Figure 5. 355x215mm (300 x 300 DPI)



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Figure 6. DSC and TGA curves of ETB salts: (a) ETBOXA, (b) ETBMAL, (c) ETBTRP and (d) ETBTCA. Figure 6. 254x190mm (96 x 96 DPI)


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Figure 7. Hot-stage microscopy images of ETBOXA (a), ETBMAL (b), ETBTRP (c) and ETBTCA (d) single crystals. Figure 7. 254x190mm (96 x 96 DPI)



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Figure 8. TGA curves of ETB salts after one hour (a), six hours (b) and twelve hours (c) in humid atmosphere. Images of (S,S)-EDH after eight hours (d) and ETBOXA after forty-eight hours (e) in humid atmosphere. Figure 8. 339x239mm (96 x 96 DPI)


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