Mechanochemical Synthesis of a Multicomponent Solid Form: The

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Mechanochemical Synthesis of a Multicomponent Solid Form: The Case of 5-Fluorocytosine Isoniazid Codrug Matheus S. Souza, Luan F. Diniz, Lautaro Vogt, Paulo S. Carvalho Jr, Richard F. D'Vries, and Javier A. Ellena Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.8b00647 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 13, 2018

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Crystal Growth & Design

1

Mechanochemical Synthesis of a Multicomponent Solid Form: The

2

Case of 5-Fluorocytosine Isoniazid Codrug

3

Matheus S. Souza†, Luan F. Diniz†, Lautaro Vogt†, Paulo S. Carvalho-Jr†, Richard F. D’ Vries†,‡

4

and Javier Ellena†*

5

†

6

Carlos, SP, Brazil.

7

‡

Universidad Santiago de Cali, Facultad de Ciencias Básicas, Cali, Valle del Cauca, Colombia.

8

*

e-mail address: [email protected]

9

ABSTRACT. Mechanochemistry synthesis was applied to the supramolecular synthesis

10

and green scale-up production of a 1:1 drug-drug cocrystal involving the antimetabolite

11

prodrug 5-Fluorocytosine (5-FC) and the tuberculostatic drug Isoniazid (INH), namely

12

as 5FC-INH. Crystalline material, also obtained by traditional slow evaporation

13

methods, was analyzed by single-crystal X-ray diffraction. The crystal packing is

14

stabilized by a classical N–H•••N hydrogen bond interaction between the amine moiety

15

of 5-FC and the INH pyridine nitrogen. IR and Raman data provided spectroscopic

16

evidence about the hydrogen atom positions, thereby confirming the neutral nature of

17

the cocrystal. Furthermore, 5FC-INH codrug was also evaluated by a range of analytical

18

techniques such as powder X-ray diffraction (PXRD) and thermal (TGA, DSC, HSM)

19

analysis. A physical stability study was performed in high relative humidity (RH)

20

conditions to verify possible 5-FC solid-state hydration and/or INH degradation. The

21

equilibrium solubility of this codrug was compared to the anhydrous 5-FC and INH raw

22

materials, in pH 1.2 buffer media, and was found to be similar to 5-FC, a BCS class I

23

drug. The results show that the cocrystal has superior phase stability properties against

24

moisture when compared to the starting pharmaceutical ingredients, so it could be

Instituto de Física de São Carlos, Universidade de São Paulo, CP 369, 13.560-970 – São

ACS Paragon Plus Environment

Crystal Growth & Design 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

considered as a potential candidate for the treatment of concomitantly fungal infections,

2

tuberculosis and cancer.

3

Keywords: Drug-drug cocrystals or codrug, Antifungal and antineoplastic drugs,

4

Tuberculosis, 5-Fluorocytosine, Isoniazid, Solvent-drop grinding, X-ray diffraction.

5

1. Introduction

6

A particular type of crystal form is represented by multidrug cocrystals (MDC) or

7

drug-drug cocrystals (DDC, or codrug), where the structure must be composed by at

8

least two different API molecules fully dissociable and in a stoichiometric ratio within

9

the same crystal lattice.1–4 In the literature, discussion about DDC is quite scarce,

10

because the complexity in their rational design and synthetic procurement. Indeed, the

11

raw materials are thoroughly selected among the APIs that will be possible co-

12

administered in a specific therapy, rather than being chosen on the Principles of Crystal

13

Engineering.5 The exploration of solid forms should be a routine practice during the

14

drug development process, however, for some marketed substances, Research &

15

Development on their active ingredients has not been comprehensively performed, as is

16

the case of 5-Fluorocytosine (5-FC, Scheme 1a) and Isoniazid (INH, Scheme 1a).6–11

17

Anhydrous 5-FC (4-amino-5-fluoro-1,2-dihydropyrimidin-2-one) is a high-dose

18

antimetabolite nontoxic prodrug that is in the forefront on the antifungal treatment

19

against Candida spp. and Cryptococcus neoformans by the inhibition of the enzyme

20

thymidylate synthase.9,12 Furthermore, it has boosted studies on 5-FC on cancer

21

treatment.13 Apart from these features, one of the main concerns about the 5-FC is a

22

solid-state hydration phenomena induced by atmosphere conditions when exposed to

23

high relative humidity (RH), leading variable pharmacokinetic profile.14 On the other

24

hand, INH (pyridine-4-carbohydrazide) is a bacteriostatic drug that is stable over long

25

time periods at ambient as well as in accelerated stability conditions. However, some


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1

studies show that INH undergoes a degradation process due to drug-drug interactions in

2

the fixed-dose combination tablet used in the first phase of tuberculosis (TB) treatment.

3

The limited stability of the aforementioned APIs has spurred the design of novel solid

4

forms, such as drug-coformer cocrystals (DCC)7–9,11,15–17 and codrugs, in order to

5

enhance its therapeutic behavior and solve its stability issue. In the literature there are cases of coexistence of invasive fungal infections (IFIs)

6 7

and

the

bacteria

that

cause TB

(Mycobacterium

tuberculosis), mainly in

8

immunocompromised patients.18,19 This type of coinfection represents a growing threat

9

with high morbidity and mortality, and has emerged due to the high use of steroids and

10

broad-spectrum antibiotics. Other studies have addressed a correlation between TB and

11

lung cancer (LC).20 Some researchers have suggested that inflammation and pulmonary

12

fibrosis caused by TB may be responsible for inducing genetic damage and,

13

consequently, causing a greater propensity to trigger a LC.21

14

In order to expand the range of new solid forms comprising two APIs, some

15

structures have been reported in the last few years.4 Recently, Lamivudine (β-L-2', 3'-

16

dideoxy-3'-thiacytidine; 3TC) and Emtricitabine, antiretroviral (anti-HIV) drugs, were

17

used by members of our group in collaboration for producing a solid solution.22 In

18

addition to this, a 5-FC cocrystal with the antineoplastic drug 5-Fluorouracil (5-FU) was

19

also reported aiming its potential future application in cancer therapy.8 Other notable

20

examples are 3TC and Zidovudine (anti-HIV drugs, commercialized as Combivir®),23

21

Theophylline (antiasthmatic) with 5-FU24 or Barbital (sleeping aid),25 Lamotrigine and

22

Phenobarbital (anticonvulsants),26 among few others research articles27–29 and

23

patents.30,31 Following this approach, herein we report a standardized protocol for

24

supramolecular synthesis as well as the main physical and chemical properties of a

25

codrug obtained from the reaction of the prodrug 5-FC with the anti-TB drug INH



1

(Scheme 1b). Initially, the sample was prepared by slow evaporation of solvent (SES)

2

(see Section 2.2). Afterwards, we develop a scale-up method based on the Principles of

3

Green Chemistry32 involving the solvent-drop grinding (SDG) method.5,33–36

4

2. Experimental section

5

2.1 Materials. 5-FC anhydrous and INH samples were purchased from Sigma-Aldrich

6

Brazil® and used without any further purification. The ultra-pure deionized water used

7

in this experiment was obtained from a Milli-Q® System (18.2 mΩ cm), while other

8

solvent used like isopropyl alcohol were HPLC grade and purchased from Acros

9

Organics.

10

2.2 Experimental Design and Supramolecular Cocrystal Synthesis. To obtain the

11

optimal reaction conditions several protocols involving different stoichiometric ratios of

12

5-FC:INH were design (1:0.5, 1:1 and 1:2). Also, different solvents were tested in the

13

cocrystal formation search. The proper selection of solvents or mixed solvents was

14

considered an important cocrystallization step in the protocol development. Slow

15

evaporation from solvent solution (SES) methods was preferred for screening

16

homogeneous single crystals suitable for structural characterization. The solvent-drop

17

grinding (SDG) mechanochemical method was employed aiming the possibility of a

18

large-scale codrug production under the Green Chemistry Principles.

19

5-Fluorocytosine Isoniazid Codrug – 5.00 mg (0.039 mmol) of 5-FC was dissolved in a

20

mixture of isopropyl alcohol/Milli-Q water (1:1, v/v) and stirred at 100°C. To this

21

solution were added 5.31 mg (0.039 mmol) of INH, in a 1:1 (drug:drug) molar ratio and

22

this system was stirred using a temperature controlled magnetic stirrer at 100°C until

23

complete dissolution of the API. Then, it was allowed to cool down slowly until room

24

temperature (~25°C) and covered with Parafilm® for slow evaporation of the solvent.


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Colorless prismatic crystals were obtained after 3-5 days. Additionally, the same 5FC-

2

INH phase was also obtained by SDG method using an oscillatory ball mill Mixer Mill

3

MM400 RETSCH. This experiment was performed by the addition of 50 mg of 5-FC

4

(0.39 mmol), 53 mg of INH (0.39 mmol) and 25µL of isopropyl alcohol/Milli-Q water

5

(1:1, v/v) mixture. A powder sample was placed in 1.5 mL volume stainless steel

6

milling jar containing two 7 mm diameter stainless steel balls. The final optimized

7

condition to obtain this new solid form was achieved by milling the system at 25 Hz for

8

60 minutes at room temperature. External temperature of the grinding jar did not exceed

9

25°C.

10

2.3 Single Crystal X-ray Diffraction (SCXRD). The crystallographic data for the

11

codrug 5FC-INH were collected at room temperature (293±2 K) on an Agilent Super

12

Nova diffractometer with CCD detector system equipped with a Mo source (λ =

13

0.71073 Å). Data integration, cell determination and final parameters were obtained

14

using the software CrysAlisPro.37 Using Olex2,38 the structure was solved by direct

15

methods and the model obtained was refined by full–matrix least squares on F2

16

(SHELXT).39 All the hydrogen atoms were placed in calculated positions and refined

17

with fixed individual displacement parameters [Uiso(H) = 1.2Ueq or 1.5Ueq] according to

18

the riding model. Molecular representations, tables and pictures were generated by

19

MERCURY 3.1040 and Olex2.38

20

2.4 Powder X-ray Diffraction (PXRD). The milled sample were analyzed by powder

21

X-ray diffraction at 25°C using a Rigaku RU200B Rotaflex diffractometer, in Bragg-

22

Brentano reflective geometry, with CuKα radiation (λ = 1.54 Å) from a voltage of 40

23

kV, current of 60 mA and Ni filter. The raw materials and the novel codrug were

24

scanned from 5 to 50° (2θ) with a step width of 0.02° θ min-1 and a constant counting



1

time of 3s per step, providing unique structural information about the crystallinity

2

degree of the samples.

3

2.5 Hot-stage Polarized Optical Microscopy (HSM). Polarized microscopy study was

4

performed on a Leica DM2500P microscope connected to the Linkam T95-PE hot-stage

5

equipment. Data were visualized with the Linksys 32 software for hot-stage control.

6

The single-crystal was placed on an individual 13mm glass coverslip, placed on a

7

22mm diameter pure silver heating block inside of the stage. The sample was heated at a

8

ramp rate of 10°C min-1 until the beginning of the degradation.

9

2.6 Thermal Analysis. Thermogravimetric analysis (TGA) was performed using a

10

Shimadzu TGA-50 thermobalance. An amount of approximately 5.0 mg ± 0.001 mg of

11

sample was placed in Al2O3 crucible and heated at 10°C min-1 under a N2 atmosphere

12

(50 mL min-1) between the 50 to 500°C range. The differential scanning calorimetry

13

(DSC) data acquisition was carried out on a Shimadzu DSC-60 calorimeter according to

14

the previously TGA data, that is, until the degradation temperature of the compound.

15

The sample (2.0 mg ± 0.01 mg) was heated from 50 to 350°C at a 10°C min-1 rate in a

16

crimped sealed aluminum pan. Nitrogen was used as purge gas under a 50 mL min-1

17

flow. The data were processed using the Shimadzu TA-60 thermal data analysis

18

software (version 2.2).

19

2.7 Vibrational Spectra. Fourier Transform Infrared (FT-IR) spectra were recorded on

20

an Alpha Bruker FT-IR spectrophotometer, using KBr pellets, in the range of 3600-600

21

cm-1, with an average of 64 scans and 2 cm-1 of spectral resolution. FT-Raman

22

spectroscopy was performed using a Bruker RFS 100 instrument with Nd3+/YAG laser

23

operating at 1064 nm in the near-infrared and a CCD detector cooled with liquid

24

nitrogen using a spectral resolution of 4 cm-1.


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1

2.8 Stability Study in Relative Humidity (RH). Powder samples of the anhydrous 5-

2

FC and 5FC-INH codrug (30 mg) were stored in a chamber containing water (100%

3

RH; 25°C). Further the milled materials were subjected to PXRD measurements to

4

monitor possible phase transitions.

5

2.9 Spectrophotometric measurements and calibration curves. A UV-1800

6

Shimadzu spectrophotometer was used to determine the absorbance of standard

7

solutions of the raw precursors of the cocrystal and construct pattern curves. These

8

curves were used to determine the unknown concentration of solutions of 5-FC, INH or

9

5FC-INH. The spectra were built in the range from 200 to 400 nm using 1 cm quartz

10

cuvettes in the medium scan speed at a 1.0 nm data interval and 1 nm bandwidth.

11

Standard stock solutions of 5-FC and INH were prepared separately at pH 1.2

12

hydrochloric buffer (Table S1, see ESI†). 5-FC standard solutions were prepared

13

dissolving 10.00 mg of the raw material in 1.5 mL of buffer into a 10 mL becker (24hs

14

agitation) and after that a conformation of dissolutions from 0.006 mg mL-1 to 0.024 mg

15

mL-1. On the other hand, INH solutions were prepared dissolving 20.00 mg of the solid

16

drug in 4 mL of buffer into a 50 mL volumetric flask. After complete dissolution (24hs

17

agitation process) different concentration points were made by appropriate dilutions in

18

concentrations ranging from 0.004 to 0.01 mg mL-1 (Table S2, see ESI†). UV-vis

19

spectra of this solutions were used to build individual calibration curves of the drugs

20

(Table S3, see ESI†).

21

2.10 Equilibrium solubility studies. Equilibrium solubility of 5-FC, INH and its

22

codrug were determined by the shake-flask method41 at 25°C in pH 1.2 buffer media.

23

Saturated solutions of the compounds were prepared stirring an excess amount of 5-FC,

24

INH and 5FC-INH, enough to reach saturation, into 2 mL of the dissolution media for a

25

48hs period. These solutions were prepared in triplicate according to the method



Page 8 of 43

1

outlined in the BCS guidance.42 After 48hs of sedimentation, the solutions were filtered

2

through a 0.45 mm PTFE hydrophilic filter (Millipore). The solid sediments identity

3

was checked by PXRD analysis (Figure S1, see ESI†). UV-Vis spectroscopy was

4

employed to analyze the supernatant concentration of the compounds. The samples were

5

diluted in the pH 1.2 buffer media before we start measuring. 5-FC, INH and its codrug

6

showed similar spectrum that could be resolved with the addition of absorbances

7

(Figure S2, see ESI†). Solubilities of 5-FC, INH as well as 5FC-INH codrug were

8

measured interpolating their maximum absorbance readings to the corresponding

9

calibration curves. After the equilibrium solubility measurements, the pH values in each

10

dissolution medium was determined (Table S4, see ESI†) using a pH meter QX 1500

11

Plus Qualxtron.

12

3. Results and Discussion

13

From the structural point of view, 5-FC and INH possess multiple donor

14

and acceptors hydrogen bond sites. 5-FC is a weak basic molecule (pka = 3.26)

15

that structurally presents two donors: NH(pirimidinic

16

acceptors: Nring and carbonyl (C═O). INH (pKa = 3.50) also is a weak base that

17

contains two main functional groups: hydrazide and pyridine ring. These groups are the

18

bases for the generation of the already reported cocrystals of these APIs with Generally

19

Recognized as Safe (GRAS) coformers containing COOH and NH2 functional

20

groups.8,11 As part of our ongoing studies in the development of stable multicomponent

21

cocrystal forms, we rationally designed (applying the ∆pKa approach, i.d ∆pKa = [pKa

22

(conjugate acid of the base) – pKa (acid)]) a cocrystal involving these two APIs. The

23

difference between the pKa of 5-FC and INH gives a ∆pKa value within the once

24

required for cocrystal formation (-0.24 = ∆pKa < 0). Suitable single crystals of 5FC-

25

INH were obtained by evaporation methods. Details of the data collection,

ring),


NH2(amine

group)

and two

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1

refinement and crystallographic parameters are summarized in Table 1. The main

2

intermolecular interactions geometric parameters are listed in Table 2. The

3

ORTEP43 type diagram of the asymmetric unit (ASU) is shown in Figure 1.

4

Crystalline purity of this codrug was assessed by PXRD (Figure S3, see ESI†). The

5

compound was also obtained as powder pure phase by solvent-drop grinding (SDG)

6

method (see Figure 2).

7

3.1 Structural Description

8

5FC-INH crystallize in the triclinic space group 1 with one molecule of 5-FC

9

and one of INH in the ASU (Figure 1). 5-FC molecules form a (8) homodimer

10

through N–H•••O (2.735(2) Å, 176.6(2) º) H-bonds. Likewise, INH molecules form a

11

(10) homosynthon via N–H•••O (2.965(2) Å, 120.52(2) º) H-bonds (Figure 3a). The

12

5-FC (colored in green in Figure 3a) and INH (colored in blue in Figure 3a)

13

homodimers are alternately arranged into infinite chains along the [121] direction via

14

N–H•••N (2.946(2) Å, 160.0(2) º) H-bonds (Figure 3a). Along [001] direction, adjacent

15

chains (color in orange and blue) are related by (7) heterosynthon (highlighted in red

16

in Figure 3b) formed between 5-FC of one chain and INH from another one. Is

17

interested to note that in a Cambridge Structural Database (CSD)44 survey we didn´t

18

found any structure of either 5-FC or INH presenting this (7) heterosynthon. Thus,

19

the molecules are arranged giving rise to planes as shown in Figure 3c. Such planes are

20

stacked through N–H•••O (3.151(2) Å, 160.78(2)°), C–H•••N (3.517(2) Å, 141.0(2)°)

21

and slip-stacked π•••π (centroid distance: 3.7385 Å for INH•••INH and 3.746 Å for 5-

22

FC•••5-FC) interactions that give to the 3D structure (Figure 3c).

23

3.2 Thermal Analysis

24

The phase purity of 5FC-INH codrug was also assessed by a combination of

25

DSC, TGA and HSM techniques. DSC and TGA curves of the sample are shown in



1

Figure 4a. The thermograms of pure 5-FC (anhydrous and monohydrate) as well as

2

those form the INH were included to characterize the starting materials (see Figure S4,

3

see ESI†). The DSC curve of 5-FC raw material (anhydrous) show an endothermic

4

peak at ~299.62°C coupled to an exothermic one, both attributed to the degradation

5

process. The DSC curve of the 5-FC exposed to highly humid environment (100%

6

relative humidity and 25°C), in turn, is very different from the anhydrous form, since it

7

is characterized by one extended and endothermic peak in the 70.19 to 131.91°C range,

8

which corresponds to the dehydration process, and one endothermic peak at 237.80°C,

9

related to the melting process. According to the TGA data, the 5FC-INH codrug is

10

thermally stable between 50°C and 157.45°C (Figure 4a). After this temperature it

11

shows a significant weight loss as observed in the TGA curve. The 5FC-INH DSC

12

curve (Figure 4a) presents an endothermic peak at 221.0°C followed by an exothermic

13

one at 224.06°C, both attributed to the codrug degradation. No traces of 5-FC/INH

14

peaks were observed in the thermogram, confirming the purity of the sample.

15

The thermal behavior of the codrug was also observed in the HSM experiment

16

(Figure 4b) and was successfully confirmed by them. The crystal gradually gets dark as

17

the temperature rise, becoming opaque at ~160°C. From this temperature forward, the

18

sample continue reducing their mass in a degradation process.

19

3.3 Spectroscopy analysis

20

Fourier Transform Infrared (FT-IR) and Raman (FT-Raman) spectroscopy

21

provides crucial information about vibrational modes and molecular conformations of

22

the APIs.45,46 For this purpose, a comparative analysis of 5-FC and INH and their 5FC-

23

INH codrug FT-IR and FT-Raman spectra (Figure S5 and Figure 5) was carried out.

24

This study shows differences in some typical bands of 5-FC and INH when compared to

25

the bands present in the codrug spectra which indicates changes in the hydrogen


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1

bonding patterns.47,48 The spectra interpretation and band assignments (Table S5, see

2

ESI†) were performed taking into account the crystallographic study previously

3

described and using spectroscopic data available from related 5-FC and INH

4

compounds found in the literature.49–51

5

5-FC molecule exhibit IR and Raman stretching frequencies at 3375 cm-1 (amine

6

N−H stretch), 1682 cm-1 and 1677 cm-1 (amide C═O stretches), 1337 cm-1 and 1340 cm-

7

1

8

other hand, the functional groups present in the INH molecule (hydrazide, amide and

9

pyridine ring) exhibit IR and Raman stretching frequencies at 3303 cm-1 and 3212 cm-1

10

(primary amine N−H stretches), 3107 cm-1 (secondary amine N−H stretch), 1664 cm-1

11

and 1670 cm-1 (amide C═O stretch), 1331 cm-1 and 1334 cm-1 (pyridine ring C−N

12

stretch). As expected, the main 5-FC and INH stretching frequencies are observed in the

13

spectra of 5FC-INH. Moreover, these vibrational modes appear shifted (10−60 cm-1) in

14

the FT-IR and FT-Raman spectra (Table S5, see ESI†), in agreement with the cocrystal

15

formation.

16

3.4 Representativity, phase transition and stability test

(pyrimidine ring C−N stretches) and 1227 cm-1 and 1236 cm-1 (C−F stretches). On the

17

PXRD is the most suitable characterization tool to confirm the formation of

18

novel crystalline forms.52 The cocrystal obtained by both SES and SDG methods,

19

display experimental diffraction patterns in good agreement with the simulated ones,

20

obtained from SCXRD analysis (Figures S3 and 2, respectively). This confirm that, in

21

one hand the powder sample obtained from SDG is pure and present a high degree of

22

crystallinity and in the other that the single crystal used in the SCXRD experiment is

23

representative of the whole sample.

24

The PXRD was also used as a tool to verify the physical stability of the

25

reported cocrystal. Anhydrous 5-FC is converted to the monohydrate form after one



1

week of exposure to humid atmosphere, as can be seen in the diffractograms of Figure

2

6. It was possible to note that the major peaks of the 5-FC pattern were no longer

3

observed after one week giving rise to the characteristic peaks of the monohydrate form.

4

This phase transition does not occur in the novel codrug, since the main characteristic

5

peaks remain present after the same exposure time to a highly humid atmosphere.

6

3.5 Equilibrium Solubility

7

The solubility study of the codrug was performed in a buffer mimicking the

8

stomach pH. These tests show that the new solid form retains solubility values

9

statistically similar to the ones found for the 5-FC raw material (Figure 7 and Table S6,

10

see ESI†). PXRD also shows that the crystal structure of the solid obtained after the

11

solubility test remains the same as the original codrug (Figure S1, see ESI†). The same

12

results are obtained for INH. However, diffractograms before and after the solubility

13

study of 5-FC do not show the same profile. In this case the remnant solid shows

14

additional peaks that correspond to others 5-FC polymorphs as well as different

15

hydrates (Figure S6, see ESI†). When thermal analyses of these solids are made four

16

important facts are exposed: DSC contrast between the solids before and after the

17

solubility study shows an endothermic signal at 52.19ºC (Figure S7, see ESI†) that

18

correspond to a phase transition; Figure S7 also shows an endothermic signal at

19

104.42ºC that correlate to a water loss; TGA comparison between 5-FC monohydrate

20

and the 5-FC solid form obtained after the solubility studies reveals that the amount of

21

water in the crystal structure is reduced when compared to the monohydrate (Figure S8,

22

see ESI†); finally, DSC plot (Figure S7) shows the same signal at ~300ºC related to

23

the decomposition of the 5-FC anhydrous. The summary of these facts seems to indicate

24

that the anhydrous 5-FC solid form used in the solubility test lead to a different kind of


Page 12 of 43

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1

hydrates in a pH 1.2 buffer solution. The DSC profiles (Figure S7) show that all these

2

changes are reversible.

3

As expect, in the pH 1.2 hydrochloric buffer, the INH solubility is very high due

4

to the interconversion of INH into its hydrochloride salt. In fact, the hydrochloride salt

5

formation was confirmed by an increase of the final pH measured after the experiment,

6

from 1.20 to 4.40 (Table S4, see ESI†).

7

4. Conclusions

8

Solvent-drop grinding was successfully applied as a synthetic pathway of the

9

codrug involving the antimetabolite prodrug 5-Fluorocytosine and the tuberculostatic

10

drug Isoniazid. This mechanochemical technique, as well as solvent evaporation one,

11

led to the 5FC-INH cocrystal formation, being the first one in agreement with the Green

12

Chemistry Principles.

13

Crystal structure analysis revealed that the crystal packing is stabilized by

14

N−H•••N and N−H•••O H-bonds. FT-IR and FT-Raman spectra confirm the cocrystal

15

formation by the concomitant appearing of the 5-FC and INH characteristic bands in the

16

codrug spectra. However, most of these typical bands appear shifted in both FT-IR and

17

FT-Raman spectra in agreement with the new intermolecular interactions formation.

18

The degradation point (~220°C) was found to be between similar to the ones of the

19

parent APIs (~170°C for INH and ~300°C for anhydrous 5-FC). The solubility profile

20

of INH in pH 1.2 buffer corroborate the high value previously reported for this drug in a

21

wide range of solvents.53,54 The 5-FC solubility, even when is statistically lower than the

22

INH one, is still high enough to classify this prodrug as a class I drug8 due to their

23

considerable solubilization in the stomach at low pH. Within this context, the new

24

codrug shows a solubility value very close to one of the 5-FC raw material, which



1

reinforces the idea of cocrystallization as an effective tool to resolve the hydration

2

problem of an API (as the 5-FC in this case) without severe impair its solubility.

3

Furthermore, this work introduces an important multi-API cocrystal as a

4

promising candidate for (1) concomitant treatment of fungal and bacteriological

5

infections; (2) concomitant treatment of fungal and/or cancer in patients undergoing

6

gene-directed enzyme prodrug therapy; (3) concomitant treatment of bacteriological

7

infections and/or cancer in patients undergoing gene therapy and last but not least (4)

8

increase the physical stability of the raw APIs, avoiding undesirable 5-FC phase

9

transitions (hydration) and/or INH degradability in pharmaceuticals containing them in

10

its formulations.

11

ASSOCIATED CONTENT

12

Supporting Information

13

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

14

at DOI:

15

Figure S1. Experimental powder X-ray diffraction patterns of 5-FC, INH and codrug

16

that remain non-solubilized after the solubility test. Figure S2. Absorption spectra of 5-

17

FC 0.0055 mg mL-1, INH 0.016 mg mL-1 and codrug (5FC-INH) 0.013 mg mL-1 in

18

buffer pH = 1.2. Figure S3. Simulated and experimental powder X-ray diffraction

19

patterns of 5FC-INH codrug. The diffractograms are in a good agreement indicating that

20

the sample present high crystallinity and purity. Figure S4. DSC curves of: (i) the

21

prodrug 5-Fluorocytosine (5-FC anhydrous), (ii) the 5-FC after one week in

22

environment with high relative humidity (5-FC monohydrate) and (iii) the drug

23

Isoniazid (INH). Figure S5. FT-IR spectra of 5-FC raw, INH raw and 5FC-INH codrug.

24

Figure S6. Powder X-ray diffraction patterns of 5-FC after solubility study (upper) and


Page 14 of 43

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1

different solid forms of 5-FC anhydrous and hydrated reported in literature. Figure S7.

2

DSC profiles of 5-FC before (red) and after (purple) solubility study. Figure S8. TGA

3

profiles of 5-FC monohydrate (blue) and 5-FC anhydrous after (purple) solubility study.

4

Table S1. Composition of the solution used for the preparation of the calibration curves

5

and solubility determinations. Table S2. Standard solutions of 5-FC and INH used to

6

construct the calibration curves. Table S3. Regression coefficients for the calibration

7

curves of 5-FC and INH at buffer media, pH = 1.2. Table S4. pH values measured after

8

solubility. Table S5. Main FT-IR and FT-Raman bands (cm-1) for 5-FC and INH, and

9

the codrug 5FC-INH and Table S6. Solubility values obtained for the three compounds.

10

Accession codes

11

One patent application was generated and deposited on November 16, 2017 at the

12

National

13

BR1020170245640 (Title of the invention: Pharmaceutical cocrystal and its use). The

14

CIF was deposited in the Cambridge Structural Data Base44 under the code CCDC

15

1817620. Copies of the data can be obtained, free of charge, via www.ccdc.cam.ac.uk.

16

AUTHOR INFORMATION

17

Corresponding Author

18

*Email: [email protected] ; Phone: +55 (016) 3373-8096 / 3373-9876.

19

Orcid

20

Javier Ellena: 0000-0002-0676-3098.

21

Matheus S. Souza: 0000-0003-1994-1145.

22

Paulo S. Carvalho-Jr: 0000-0002-5551-9155.

23

Richard F. D’Vries: 0000-0002-3655-1838.

24

Author Contributions

Institute

of

Industrial

Property

(INPI,


Brazil)

under

the

code


1

The manuscript was written through contributions of all authors. All authors have given

2

approval to the final version of the manuscript.

3

Notes

4

The authors declare no competing financial interest.

5

ACKNOWLEDGEMENTS

6

The authors acknowledge the Brazilian funding agencies CAPES (M.S.S.),

7

FAPESP (L.F.D. grant 15/25694-0) and CNPq (J.E. grant #305190/2017-2) for financial

8

support. L. Vogt thanks IFSC/USP for being granted with a Salazar Scholarship which

9

made possible the active collaboration in this work. The authors would also like to

10

thank Dra. Charlane C. Correa, Dr. Luiz F. C. de Oliveira (Federal University of Juiz de

11

Fora) for allowing access to the Single-crystal X-ray diffraction and Raman

12

spectroscopy facilities.

13

14

ABBREVIATIONS

15

5-FC, 5-Fluorocytosine; INH, Isoniazid; IR, Infrared; PXRD, powder X-ray diffraction;

16

TGA, thermogravimetric analysis; DSC, differential scanning calorimetry; HSM, hot-

17

stage microscopy; RH, relative humidity; BCS, Biopharmaceutics Classification

18

System; MDC, multidrug cocrystal; DDC, drug-drug cocrystal; API, active

19

pharmaceutical ingredient; TB, tuberculosis; DCC, drug-coformer cocrystal; IFIs,

20

invasive fungal infections; LC, lung cancer; 3TC, β-L-2', 3'-dideoxy-3'-thiacytidine;

21

HIV, human immunodeficiency virus; 5-FU, 5-Fluorouracil; SES, slow evaporation of

22

solvent; SDG, solvent-drop grinding; SCXRD, single-crystal X-ray diffraction; HPLC,

23

high performance liquid chromatography; CCD, charge-coupled device; UV-Vis,

24

ultraviolet–visible; PTFE, polytetrafluoroethylene; GRAS, Generally Recognized as


Page 16 of 43

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1

Safe; ORTEP, Oak Ridge Thermal-Ellipsoid Plot; ASU, asymmetric unit; CSD,

2

Cambridge Structural Database.

3

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FIGURE CAPTIONS

1 2

3

Scheme 1. (a) Chemical structure of the drugs 5-Fluorocytosine and Isoniazid; (b)

4

Heterosynthon presented in 5FC-INH novel codrug.

5

Figure 1. ORTEP type diagram with atomic numbering scheme showing 50% of

6

probability ellipsoids for 5FC-INH.

7

Figure 2. Experimental powder X-ray diffraction pattern of 5FC-INH obtained from

8

solvent-drop grinding (SDG) compared to the simulated one from SCXRD of the crystal

9

obtained by slow evaporation (SES) method.

10

Figure 3. (a) Partial view down [100] direction of 5FC-INH crystal packing

11

highlighting the formation of alternating 5-FC and INH dimer chains stabilized by N–

12

H•••N and N–H•••O interactions and forming (8) and (10) graph sets motifs. Black

13

dotted lines indicate hydrogen bonds. (b) View along [001] direction: adjacent chains

14

are related by N– H ••• N (7) heterosynthon formed between 5-FC of one chain and

15

INH from another one. (c) Three-dimensional arrangement of crystalline 5FC-INH. The

16

5-FC and INH molecules are arranged giving rise to plane parallel to the (110 ) plane.

17

Such planes are stacked through N–H•••O, C–H•••N and slip-stacked π•••π interactions

18

leading to 3D structure formation.

19

Figure 4. (a) TGA and DSC curves of the 5FC-INH codrug. (b) Crystal behavior as a

20

function of temperature increase visually checked by HSM of the novel crystalline

21

form.

22

Figure 5. FT-Raman spectra of the raw materials and the 5FC-INH codrug.



1

Figure 6. PXRD pattern of 5FC-INH codrug showing the same solid phase after one

2

week in humid environment, which not occurs with the 5-FC raw material – currently

3

marketed.

4

Figure 7. Solubility concentration of 5-FC, INH and it codrug in pH 1.2 dissolution

5

media.

6

7

8

9

10

11

12

13

14

15

16

17 18 19 20 21 22 23


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1

TABLE CAPTIONS

2 3

Table 1. Crystal data and structure refinement of the 5FC-INH codrug.

4 5

Table 2. Geometric parameters of the Hydrogen Bonds for 5FC-INH.

6

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29



1

Scheme 1.

2

3 4 5 6 7 8 9 10


Page 24 of 43

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1

Figure 1.

2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24



1

Figure 2.

2

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


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1

Figure 3.

2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23



1

Figure 4.

2

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17


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1

Figure 5.

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



1

Figure 6.

2

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


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1

Figure 7.

2

3



1

Table 1.

2

5FC-INH Identification code C10H11FN6O2 Empirical formula 266.25 Molecular weight 293(2) Temperature / K Triclinic Crystal system Space group 1 3.7464(3) a/Å 9.6648(6) b/Å 16.3882(13) c/Å 76.142(6) α/° 88.948(6) β/° 82.040(5) γ/° 570.49(7) Volume / Å3 2/1 Z / Z' 3 1.550 ρcalc g/cm 0.125 µ / mm-1 276.0 F (000) 9602 Reflections collected 2495 Independent reflections 2092 Unique reflections 2495/0/173 Data / restraints / parameters 0.0457 R1 [I≥2σ(I)] 0.1233 wR2 [all data] 1.069 Goodness-of-fitness on F2 Agilent Super Nova X-ray diffractometer 3 4 5 6 7 8 9 10 11 12 13 14 15


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Table 2.

1 2 Interaction N5-H5···N3 N41-H41A···N5 N41-H41A···N4 C9-H9···O21 N1-H1···O21 N1-H1···N1 N41-H41B···N6 N4-H4A···O2 N4-H4B···O2 N4-H4B···N4 N4-H4B···O2 C6-H6···F51 C9-H9···N3 C10-H10···021 C10-H10···F51

d(D•••A) (Å) 3.027(2) 3.520(2) 2.963(2) 3.225(2) 2.735(2) 3.589(2) 2.946(2) 3.151(2) 3.659(2) 3.668(2) 2.965(2) 3.331(2) 3.517(2) 3.401(2) 3.532(2)

d(H•••A) (Å) 2.271(2) 2.824(2) 2.118(2) 2.606(2) 1.876(2) 2.992(2) 2.123(2) 2.211(2) 2.735(2) 2.863(2) 2.303(2) 2.536(2) 2.746(2) 2.814(2) 2.986(2)

∠D-H•••A (º) 147(1) 139(1) 167(1) 124(1) 176(1) 128(1) 160(1) 160(1) 149(1) 135(2) 120(1) 143(1) 141(1) 122(1) 119(1)

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


Symmetry codes x,y,z x,y,z x,y,z x,y,z -x+1,-y+1,-z+2 -x+1,-y+1,-z+2 x-1,+y-1,+z -x+2,-y+1,-z+1 x-1,+y,+z -x+1,-y+1,-z+1 -x+1,-y+1,-z+1 -x,-y,-z+2 x+1,+y,+z x+1,+y,+z x+1,+y+1,+z


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FOR TABLE OF CONTENTS USE ONLY

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Mechanochemical Synthesis of a Multicomponent Solid Form: The

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Case of 5-Fluorocytosine Isoniazid Codrug

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Matheus S. Souza†, Luan F. Diniz†, Lautaro Vogt†, Paulo S. Carvalho-Jr†, Richard F. D’ Vries†,‡

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and Javier Ellena†*

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In this manuscript we introduce mechanochemistry for supramolecularly

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synthesizing a stable codrug involving the antimetabolite prodrug 5-Fluorocytosine (5-

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FC) and the tuberculostatic drug Isoniazid (INH).

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Graphical Abstract 400x270mm (96 x 96 DPI)



Scheme 1. (a) Chemical structure of the drugs 5-Fluorocytosine and Isoniazid; (b) Heterosynthon presented in 5FC-INH novel codrug. 254x300mm (96 x 96 DPI)


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Figure 1. ORTEP type diagram with atomic numbering scheme showing 50% of probability ellipsoids for 5FCINH. 254x190mm (96 x 96 DPI)



Figure 2. Experimental powder X-ray diffraction pattern of 5FC-INH obtained from solvent-drop grinding (SDG) compared to the simulated one from SCXRD of the crystal obtained by slow evaporation (SES) method. 289x202mm (300 x 300 DPI)


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Figure 3. (a) Partial view down [100] direction of 5FC-INH crystal packing highlighting the formation of alternating 5-FC and INH dimer chains stabilized by N–H•••N and N–H•••O interactions and forming R(_2^2)(8) and R(_2^2)(10) graph sets motifs. Black dotted lines indicate hydrogen bonds. (b) View along [001] direction: adjacent chains are related by N–H•••N〖 R〗_2^2 (7) heterosynthon formed between 5-FC of one chain and INH from another one. (c) Three-dimensional arrangement of crystalline 5FC-INH. The 5FC and INH molecules are arranged giving rise to plane parallel to the plane. Such planes are stacked through N–H•••O, C–H•••N and slip-stacked π•••π interactions leading to 3D structure formation. 400x190mm (96 x 96 DPI)



Figure 4. (a) TGA and DSC curves of the 5FC-INH codrug. (b) Crystal behavior as a function of temperature increase visually checked by HSM of the novel crystalline form. 499x399mm (96 x 96 DPI)


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Figure 5. FT-Raman spectra of the raw materials and the 5FC-INH codrug. 289x202mm (300 x 300 DPI)



Figure 6. PXRD pattern of 5FC-INH codrug showing the same solid phase after one week in humid environment, which not occurs with the 5-FC raw material – currently marketed. 289x202mm (300 x 300 DPI)


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Figure 7. Solubility concentration of 5-FC, INH and it codrug in pH 1.2 dissolution media. 254x190mm (96 x 96 DPI)


Mechanochemical Synthesis of a Multicomponent Solid Form: The

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