Journal of Molecular Structure 1233 (2021) 130154

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Journal of Molecular Structure

journal homepage: www.elsevier.com/locate/molstr

Bromhexine and its fumarate salt: Crystal structures, Hirshfeld

surfaces and dissolution study
Ya-an Zhang a,∗, Cui-Min Yan b, Bai-Wang Sun b,∗, Lin-Xuan Wang a
a
School of Pharmaceutical and Chemical Engineering, Chengxian College, Southeast University, Nanjing 210088, PR China
b
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Bromhexine is an expectorant drug repurposing as a TMPRSS2 inhibitor, which has also been proposed for
Received 14 September 2020 potential treatment in COVID-19 infection. Multicomponent crystal strategy has been applied in bromhex-
Revised 3 February 2021
ine to improve its poor solubility, which limits its bioavailability and efficacy. A new bromhexine crystal
Accepted 15 February 2021
and its fumarate salt crystal have been successfully obtained by slow evaporation technique. Both com-
Available online 24 February 2021
pounds have been characterized by X-ray single-crystal diffraction, TGA and FT-IR spectroscopy. Hirsh-
Keywords: feld surface analysis has been carried out to further quantify the patterns of intermolecular interactions.
Bromhexine Compared with bromhexine, the multicomponent crystal with pharmaceutically acceptable conformer of
Crystal structure fumaric acid shows improved thermal stability and solubility in water.
Multicomponent crystal
Salt © 2021 Elsevier B.V. All rights reserved.
Hirshfeld surface
Solubility

1. Introduction However, the poor solubility of bromhexine gives rise to dif-

In the recent years, supramolecular chemistry together with
crystal engineering has become one of the research hotspots in
the field of pharmaceutical chemistry [1–4]. Preparation of mul-
ticomponent crystals including co-crystallization and salt forma-
tion promotes the design and synthesis of new solid type of Active
Pharmaceutical Ingredient (API) with the desired physicochemi-
cal properties by exploring the advantages of supramolecular in-
teractions [5–6]. Considerable researches have shown that both
cocrystals and salts obtained by co-crystallization of the API and
the GRAS (Generally Recognized as Safe) coformers have improved
solid state properties of API such as better stability, solubility,
bioavailability, and dissolution without altering the pharmacolog-
ical activity of the API [7–15].
Bromhexin is an effective and safe expectorant drug, which is
mainly used in patients with chronic bronchitis, asthma [16]. It
has shown that bromhexine is also a potent inhibitor of TMPRSS2,
a key protease in the transmission of SARS-CoV-2 [17–18]. There-
fore, several clinical trials on the prevention or treatment of Novel
Coronavirus Infectious Disease (COVID -19) with bromhexin are un-
derway in the world to evaluate its prophylactic potential against
SARS-CoV-2 [19–23].
∗
Corresponding authors.
E-mail addresses: yaanamm@126.com (Y.-a. Zhang), chmsunbw@seu.edu.cn (B.-
W. Sun).
ficulties in pharmaceutical formulation for oral use, which may
lead to variation in bioavailability [24]. To overcome these diffi-
culties, salt or co-crystal strategy has been applied in bromhexine
to develop novel salt or co-crystal with good solubility and sta-
bility bioavailability. Thus, screening experiments were performed
for bromhexin co-crystallization with a series of carboxylic acid
coformers by slow evaporation method. Finally, a new bromhex-
ine crystal and its fumarate salt crystal have been obtained, which
have not been found in the literature or Cambridge Crystallo-
graphic Data Centre (CCDC) so far [25–28]. The two crystals have
been characterized by single-crystal and powder X-ray diffraction,
IR spectroscopy, thermal analysis (TGA). Hirshfeld surfaces analysis
has been carried out in order to analyze the intermolecular con-
tacts. The equilibrium solubility and powder dissolution of the two
crystals have been also evaluated. The fumarate of bromhexine ex-
hibits better thermal stability and water solubility, compared with
the free bromhexine base.
2. Experiment section
2.1. Materials and physical measurements
Bromhexin (> 99%) was gifted from Chia Tai Qingjiang Phar-
maceutical Co., Ltd, Jiangsu, China. All the coformers and sol-
vents were analytically pure purchased from Sinopharm Chem-
ical Reagent and used as received without further purification.

https://doi.org/10.1016/j.molstruc.2021.130154
0022-2860/© 2021 Elsevier B.V. All rights reserved.
Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al. Journal of Molecular Structure 1233 (2021) 130154

Table 1 over an angular range from 5°to 50°(2θ ) in continuous scan mode
Crystal data and structure refinements for Compounds 1-2.
with a step size of 0.02°(2θ ) and a step time of 0.15 s. Mercury
Compound 1 2 CSD 3.5.1 program was used to calculate PXRD patterns with the
Formula C14 H20 Br2 N2 C18 H24 Br2 N2 O4 data of single crystal structures [33].
Formula weight 376.14 492.21
Crystal system monoclinic monoclinic 2.5. Hirshfeld surfaces analysis
Space group P 21 /n P 21 /n
Z 4 4
Molecular Hirshfeld Analysis for both compounds was per-
a/ Å 6.5271(1) 8.1069(12)
b/ Å 23.5874(2) 26.277(4) formed using the Crystal Explorer 3.1 software to investigate the
c/ Å 9.9592(1) 9.4104(13) nature of intermolecular interactions and their relative contribu-
α /° 90 90 tions in the crystals [34]. The distances from the Hirshfeld surface
β /° 94.190(1) 103.994(4) to the closest nucleus outside and inside the surface are defined
γ /° 90 90
as de and di, respectively. The normalized contact distance dnorm
V, Å3 1529.19(3) 1945.1(5)
T/K 293(2) 193 is based on di ,de and the van der Waals radii of the atoms. The
μ (mm−1 ) 6.596 3.790 dnorm values can be mapped onto the Hirshfeld Surface, which fa-
D calc (Mg m−3 ) 1.634 1.681 cilitates easy comparison of intermolecular contacts relative to van
Cryst dimensions(mm) 0.09 × 0.13 × 0.22 0.10 × 0.15 × 0.20
der Waals radii by a simple red-white-blue color scheme (contacts
No. of reflns collected 3110 3554
No. of unique reflns 2725 3502
shorter or longer than van der Waals contacts and equal to the
No. of params 173 251 sum of the van der Waals radii, are visualized as red, blue and
Goodness of fit on F2 1.095 1.207 white colours in the Hirshfeld surfaces respectively). [35]. In this
R1 ,wR2 ((I>2σ (I)) 0.0418, 0.1162 0.0621, 0.1472 study, a standard (high) surface resolution was used to generate
R1 ,wR2 (all data) 0.0478, 0.1261 0.0628, 0.1477
the Hirshfeld surfaces of bromhexine molecules in compounds 1–
CCDC No. 2025907 2025908
2. The 3-D dnorm surfaces were mapped with a fixed color scale of
0.76 Å (red) to 2.4 Å (blue). Using standard 0.6 Å–2.6 Å view, the
2-D fingerprint plots were displayed with the de and di distance
With the samples prepared as KBr pellets, infrared spectra were
scale.
performed using a Bruker-TENSOR27 FT-IR spectrometer in the
range 40 0 0-40 0 cm−1 . Thermo gravimetric analysis (TGA) were
2.6. Solubility and dissolution experiments
performed with a thermal analyzer TG209 F3 at a heating rate of
10˚C/ /min under an atmosphere of N2 .
To compare the equilibrium solubility values, an excess amount
2.2. Crystals growth of bromhexine and its fumarate was added in 10 ml distilled water
and the supersaturated solutions were shaken at 37°C. After 72 h,
Single crystal of bromhexine (Compound 1): 60 mg (0.160 the suspensions were filtered and the concentrations of the drugs
mmol) bromhexine and 23 mg (0.160mmol) L-glutamine were dis- were analyzed by high-performance liquid chromatography (HPLC).
solved in 10 ml ethanol and 2 ml distilled water by ultrasonic For each dissolution experiment, powdered sample containing 480
treatment for 20 min, then left for slow evaporation at room tem- mg of bromhexine or its equivalent in fumarate was added in 100
perature after filtrating. The colorless plate-like crystals were ob- ml distilled water, and the resulting suspension stirred at 25°C and
tained after about 2 weeks with the total yield of 50.2%. 500 rpm. At a predetermined time interval, each aliquot was taken
Fumarate of bromhexine (Compound 2): 100 mg (0.266 mmol) out filtered through a 0.45 μm membrane filter, and then the con-
bromhexine and 31 mg (0.266 mmol) fumaric acid were dissolved centration of the drug was quantified by HPLC. The concentration
in 20 ml acetonitrile and 2 ml methanol. The mixture was stirred of bromhexine was determined by Agilent 1260 HPLC equipped
at room temperature for 30min followed by filtrating. The color- with a variable UV detector (set at 284 nm). A C18 column (100
less block-like crystals were obtained after about 2 weeks for slow mm × 4.6 mm, 5 μm) was used and the flow rate was set to 1.0
evaporation with the total yield of 45.0%. mL/min with the column temperature at 30°C. The mobile phase
consisted of 0.1% phosphoric acid buffer (adjusted to pH 3.5 with
2.3. X-Ray crystallography trimethylamine): acetonitrile (10:90 v/v). Solubility and dissolution
experiments for each sample were conducted three times to calcu-
Crystal structures of compounds 1–2 were determined by late the standard deviations.
single-crystal X-ray diffraction, the date of compound 1 were col-
lected at 293 K with Cu Kα radiation (λ = 1.54184 Å), while the 3. Results and discussion
date of compound 2 were collected at 193 K with Ga Kα radiation
(λ = 1.34138 Å), using ω-scan method. The structures were solved 3.1. Crystal structural analysis
by SHELXT 2015 program [29] in direct method and refined by
SHELXL-2015 program [30], respectively. A summary of the crystal- Compound 1 crystallizes in monoclinic space group P21 /n with
lographic data for compounds 1–2 was provided in Table 1. Molec- Z = 4 and the thermal ellipsoid plot at 50% probability is shown
ular graphics were prepared using DIAMOND [31] and Mercury in Fig. 1a. In the molecular structure, the C–N–C bond angle is
program [32]. CCDC reference numbers 2025907 and 2025908 con- 111.07(2) °and the cyclohexane ring adopts the most stable chair
tains the supplementary crystallographic data for this paper. These configuration. Br1, Br2, N1 are almost coplanar with benzene ring,
data can be obtained free of charge via http://www.ccdc.cam.ac.uk/ with slight deviations of 0.071(2) Å, 0.004(1) Å, 0.086(1) Å, re-
conts/retrieving.html. spectively. In addition, the molecule is stabilized by intramolec-
ular hydrogen bonds N1–H1B···N2 [N1···N2 distance of 2.855(4)
2.4. Powder X-ray diffraction (PXRD) Å, H1B···N2 distance of 2.115(3)Å, N1–H1B···N2 angle of 142.2(4)
°] (Table 2). A weak N1–H1A···Br1 interaction has been detected
Room temperature PXRD analyses were performed using a with the N1···Br1 distance of 3.098 Å significantly shorter than
Bruker D8 Discover diffractometer (Bruker, AXS) with Cu Kα radi- sum of the van der Waals radii (3.40 Å) [36], formed intramolecu-
ation (λ = 1.5406 Å) at 40 kV and 40 mA. The data were collected lar five-membered hydrogen bonds which have also been reported

2
Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al. Journal of Molecular Structure 1233 (2021) 130154

Fig. 1. Molecular structure of compound 1(a); One-dimensional chain structure of compound 1(b); Two-dimensional network of compound 1 (c).

3
Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al.
Table 2
Hydrogen bond parameters in compounds 1-2.
D-H···A D-H (Å)
compound 1
N1-H1A··· Br1 0.87(3)
N1-H1B···N2 0.87(3)
compound 2
N1-H1A···Br1 0.87(7)
N1-H1A···O2 0.87(7)
N1-H1B···O3 0.90(7)
N2+ -H2···O1− 0.90(6)
O4-H4···O2 0.840
C7-H7B···N1 0.990
C9-H9···O4 0.95(5)
in the crystal structures of bromhexine hydrochloride [25] and
other bromo-substituted aromatic amides [37]. As shown in Fig. 1b,
the bromhexine molecules are connected in a head-to-tail fash-
ion, resulting in the formation of a one dimensional zigzag chain
through C-H···Br [H···Br distance of 2.966(1) Å] contacts. The ad-
jacent chains are further linked by C-Br···Br halogen bonding con-
tacts [Br···Br distance of 3.6670(7) Å] shown in Fig. 1c, belonged to
type-I halogen-halogen interactions [38], with both C-Br···Br angles
of 148.04(9)°.
Compound 2 also crystallizes in monoclinic space group P21 /n
with Z = 4 and the basic structure unit consists of one proto-
nated bromhexine cation and one deprotonated fumaric acid anion,
where one proton transfers from carboxylate of fumaric acid to N-
methyl amino group of bromhexine(Fig. 2a). The C–N–C bond angle
is 111.6(4) ° and the cyclohexane ring also adopts the most sta-
ble chair configuration, similar to those in compound 2. Bromhex-
ine cations and fumaric acid anions are held together via N2+ –
H2···O1− [N2+ ···O1− distance of 2.692(5) Å, H2···O1 distance of
1.836(6) Å, N2–H2···O1 angle of 158.7(5)°] along with N1–H1···O2
[N1···O2 distance of 2.854(6) Å, H1···O2 distance of 2.144(7))Å, N1–
H1···O2 angle of 138.7(7) °] hydrogen bond interactions to give
infinite 1D chains (Fig. 2b). The 1D chains are further interact-
ing by R3 3 (9) and R3 2 (8) supramolecular heterosynthons through
C9–H9···O4 [C9···O4 distance of 3.267(6) Å, H9···O4 distance of
2.462(5) Å, C9–H9···O4 angle of 141.9(4)°], O4–H4···O2 [O2···O4 dis-
tance of 2.534(5) Å, H4···O2 distance of 1.710 Å, O4–H4···O2angle of
166.6°] and N1–H1B···O3 [N1···O3 distance of 3.011(6) Å, H1B···O3
distance of 2.167(7) Å, N1–H1B···O3 angle of 155.2(6)°] hydro-
gen bonds thereby form a 2D layer along the (010) plane(Fig. 2c,
Table 2). In addition, the weak π –π stacking interactions are de-
tected with distance of 3.580 (3) Å, which also involved in stabi-
lizing the crystal structure (Fig. 2d).
3.2. Fourier-transform infrared spectra (FT-IR) analysis
FT-IR spectroscopy is an effective tool used to differentiate dis-
tinct chemical structures and environments. In FT-IR spectra of
compounds 1-2, obvious distinctions have been exhibited in the N-
H stretching, N-H bending and carbonyl stretching vibrations fre-
quencies (Supplementary Fig.S1 and Table S1). Compound 1 shows
IR absorption frequencies at 3412 cm−1 (asymmetric N-H stretch-
ing) and 3134 cm−1 (symmetric N-H stretching) and at 1601 cm−1
(N-H bending) without carbonyl stretching. Compound 1 shows
N-H stretching at 3412 cm−1 , 3134 cm−1 and N-H bending at
1601 cm−1 attributed to aromatic amino groups, while no carbonyl
stretching has been observed. On the other hand, compound 2 dis-
plays blue-shift of N-H stretching frequencies at 3444 cm−1 and
3333 cm−1 , with N-H bending at 1633 cm−1 due to changes in
molecular conformations and hydrogen bonding. The N+ -H stretch-
ing vibration of the protonated aminomethyl group in compound 2
appears at 3232 cm−1 . The carbonyl stretching vibration for com-
Journal of Molecular Structure 1233 (2021) 130154
H···A (Å) D···A (Å) ࢬD-H···A (deg) Symmetry operation
2.65(3) 3.098(3) 114(3) x, y, z
2.12(3) 2.855(4) 142(4) x, y, z
2.71(8) 3.050(5) 105(6) x, y, z
2.14(7) 2.854(6) 139(7) x,y,-1+z
2.17(7) 3.011(6) 155(6) -1+x,y,-1+z
1.84(6) 2.692(5) 159(5) x, y, z
1.710 2.534(5) 167 1+x,y,z
2.560 2.926(7) 102 x, y, z
2.46(5) 3.267(6) 142(4) -1+x,y,z
pound 2 appears at 1688cm−1 , confirming the presence of carboxyl
groups associated with the fumaric acid coformer. From compari-
son the spectrum with respect to fumaric acid, an increase in car-
bonyl acid stretching frequency has been observed for compound
2, also indicated the formation of salt.
3.3. Thermal analysis
The thermal behaviors of compounds 1–2 have been investi-
gated and the TGA curves are shown in Fig.S2. Compound 1 under-
goes one step of mass loss from 125°C, with a continued weight
loss completed by 300°C. Compound 2 undergoes three steps of
mass loss, a first loss of 10% at approximately 152°C; the second
step is a loss of 23% at approximately 172°C, while the third step
is a loss of 44% at approximately 266°C. In addition, the melting
points of compounds 1–2 have been tested with X-5 precise mi-
cro melting point tester. Compound 1 melted at about 60°C, while
compound 2 melted at about 148°C. The results of thermal analy-
sis confirm that compound 2 as a novel solid-state form improved
the physical stabilities through higher decomposition and melting
point.
3.4. Hirshfeld surface analysis
In order to provide further insight into the intermolecular inter-
actions, Hirshfeld surfaces of bromhexine molecules in compounds
1–2 associated finger print plots have been calculated. The Hirsh-
feld surface mapped with dnorm of bromhexine molecule in com-
pound 1 is illustrated in Fig. 3a. Small red areas on the dnorm Hir-
shfeld surfaces of compound 1 represent the C–H···Br interactions,
while slight red area is in accordance with weaker Br···Br halogen
bonding interactions. It’s noticeable that there are more deep red
spots in the 3D-dnorm surfaces of bromhexine molecule in com-
pound 2 compared to that in compound 1, mainly due to close-
contact N−H···O interactions from fumaric acid(Fig. 3b).
On the shape index surface of bromhexine molecule in com-
pound 2, adjacent red and blue triangles have been observed, indi-
cating the presence of aromatic stacking (Fig.S3). However, the π –
π stacking interactions are absent in compound 1, because there
is no evidence of adjacent bow-tie patterns on the shape index
surfaces [39]. On the coincident curvedness surfaces of bromhex-
ine molecule in compound 2, the relatively large and green flat re-
gions, marked with ovals in Fig.S3, also provide proof for the exis-
tence of aromatic stacking interactions.
The 2D finger print plots for the main intermolecular contacts
of the two compounds are shown in Fig. 4. For compound 1, the
H···H interactions exhibit the most significant contribution (52.3%)
to the total Hirshfeld surfaces. The H···Br intermolecular interac-
tions are seen as sharp spikes in the 2D fingerprint plots, account-
ing for 28.6 % of the total Hirshfeld surfaces. Whereas in compound
2, the proportion of H···H interactions is decreased after salifica-
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Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al. Journal of Molecular Structure 1233 (2021) 130154

Fig. 2. Molecular structure of compound 2(a); One-dimensional chain structure of compound 2(b); Two-dimensional network of compound 2(c); crystal packing stabilized
by π –π stacking interactions are detected with distance of 3.580 Å viewed from c-axis.

tion, account for 41.2% of the total Hirshfeld surfaces. However,
close-contact O···H intermolecular interactions appear as a distinct
spike in the 2D fingerprint plot, obviously increasing up to 16.3%
of the total Hirshfeld surfaces. The other observed interactions are
summarized in Table 3.
In summary, the results of Hirshfeld surface analysis indicate
that there are stronger intermolecular forces in compound 2. It is
found that forming the salt multicomponent crystal changed the
bonding mode and intermolecular force of the two compounds.
3.5. Solubility and dissolution study
It has been reported that the solubility of bromhexine hy-
drochloride is increased by 5 fold at 15 mmol/L of methylated β -
cyclodextrin [40], while the stability and safety of inclusion com-
plexes need to be further studied [41]. It is also found that amino
acid prodrugs can increase the solubility of bromhexine hydrochlo-
ride by about 3-5 times [42], but with complicated chemical syn-
thesis and difficult purification. Therefore, development of new sta-
ble cocrystals/salts with simple processing and lower cost is still

5
Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al. Journal of Molecular Structure 1233 (2021) 130154

Fig. 3. Hirshfeld surfaces mapped with dnorm of bromhexin molecules in compound 1 (a) and 2 (b), respectively.

Fig. 4. 2D fingerprint plots of bromhexin molecules in compounds 1-2.

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Y.-a. Zhang, C.-M. Yan, B.-W. Sun et al. Journal of Molecular Structure 1233 (2021) 130154

Fig. 5. Powder dissolution profiles of compounds 1-2 in water.

Table 3 there are stronger intermolecular forces in compound 2. Moreover,

Relative percentage contributions (in %)
compound 2 exhibits higher thermostability and also shows an
of various intermolecular contacts to
the Hirshfeld surface area for bromhexin enhanced solubility in water and a better dissolution profile. By
molecules in compounds 1-2. forming the salt multicomponent crystal, thermostability and sol-
ubility of compound 2 has been improved from the parent drug.
Compound l Compound 2
Therefore, with a pharmaceutically acceptable coformer of fumaric
H···H 53.2 41.2 acid, compound 2 is a promising candidate worthy of further re-
H···Br 28.6 20.3
search.
C···H 11.5 8.6
O···H 0 16.3
C···C 0 3.4 Declaration of Competing Interest
Br···Br 2.6 4.3
C···Br 1.8 2.5 The authors declared that they have no conflicts of interest to
others 3.2 3.4
this work. We declare that we do not have any commercial or as-
sociative interest that represents aconflict of interest in connection
with the work submitted.
necessary. In this study, the equilibrium solubility value of com-
pound 2 at 37°C in water (4.9 ± 0.20 mg/ml) is more than 10-fold CRediT authorship contribution statement
higher than that of compound 1, and slightly higher than that of
bromhexine hydrochloride used in clinical (4.4± 0.25 mg/ml re- Ya-an Zhang: Data curtion, Formal analysis, Writing – orig-
ported in the literature) [42].The powder dissolution profiles of inal draft, Visualization. Cui-Min Yan: Methodology, Validation.
compound 1-2 in water are depicted in Fig. 5. Compound 1 is a Bai-Wang Sun: Conceptualization, Supervision, Funding acquisi-
practically insoluble in water, and the salt formation results in a tion. Lin-Xuan Wang: Investigation.
significantly improvement in the case of compound 2. A close ex-
amination of the remaining powder after dissolution experiments Acknowledgments
showed that no phase transition occurs for both compounds (Sup-
plementary Fig.S4). In addition, fumaric acid is a GRAS molecule This work was supported by the Research and development
according to US Food and Drug Administration and is easy to ob- Fund for young teachers of Southeast University Chengxian College
tain by industrial preparation with lower cost. Thus, the present (No. Z0022). We also give our sincere thanks to Dr. Yang-hui Luo
study offers a new perspective and possibility to improve the sol- and Dr. Yu-Heng Ma, who offered generous assistance throughout
ubility of bromhexine by forming the salt multicomponent crystal. our whole work.

4. Conclusions
Bromhexine is a powerful expectorant and has been repurposed
as a TMPRSS2 inhibitor also proposing for COVID-19 therapy. In
this study, a new bromhexine crystal and its fumarate salt crys-
tal have been obtained and characterized by single crystal X-ray
diffraction, TGA and FT-IR spectroscopy. It is found that both com-
pounds feature different crystal structures and proton transfer oc-
curred from fumaric acid to bromhexine molecule in compound
2. The results of Hirshfeld surface analysis further confirm that
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.130154.
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