Crystal engineering of exemestane to obtain a co-crystal with enhanced urease inhibition activity

Co-crystals of the commercially available anticancer aromatase inhibitor exemestane (Ex) and guest co-former thiourea were synthesized and the difference in the biological properties of the co-crystal compared with Ex was investigated. To study the various intermolecular interactions and their contribution to the structure stability, Hirshfeld surface analysis was successfully employed, and thermal stability was evaluated by DSC and TGA.


Introduction
As defined by the FDA regulatory classification of pharmaceutical co-crystal guidance for industry, 'co-crystals are crystalline materials composed of two or more different molecules, typically an active pharmaceutical ingredient (API) and a co-crystal former in the same crystal structure'. The approval of co-crystals by the FDA as new drug applicants has opened up new vistas in both the academic and the industrial research sectors (Gadade & Pekamwar, 2016), resulting in a significant increase in research in the field of co-crystals. It has already been established that the physical and biological properties of an API can be altered by co-crystallization (Caira et al., 2012;Ghosh & Reddy, 2012;Issa et al., 2012). The literature has disclosed many examples of fine tuning the biological and physical properties of many bioactive compounds and key pharmaceutical ingredients with suitable co-formers by using a number of crystal engineering approaches (Chen et al., 2014;Charron et al., 2013;Castro et al., 2011). Examples include the co-crystallization of naturally occurring anti-leishmanial seselin (Hussain et al., 2018), anti-tumour drug temozolomide (Sanphui et al., 2013), antibiotic nalidixic acid (Gangavaram et al., 2012) and well known quercetin (Smith et al., 2011). Co-crystals are also known to have uses in thr agrochemical industry, as well as paint, electronic and optical materials (Blagden et al., 2008;Papaefstathiou et al., 2004;Sokolov et al., 2006). The stabilization of co-crystals is known to be influenced by classical hydrogen bonding andstacking. Non-covalent interactions (mainly hydrogen bonding) are involved in various biological systems due to their dynamic nature. The famous 'lock-and-key' model proposed by Emil Fischer (Fischer, 1894) for enzyme and substrate interactions has two main features: supramolecular chemistry and molecular recognition (Desiraju, 2001(Desiraju, , 2000Lehn, 1995). Therefore, it is worth studying both qualitatively and quantitatively the various interactions which contribute to crystal stability. Hirshfeld surface analysis (Spackman & Jayatilaka, 2009) is an effective approach for the qualitative and quantitative analysis of non-covalent interactions in crystal packing.
Enzyme inhibition is an important area of biomedical research contributing to the treatment of a wide range of disorders such as ulcers, cancer, inflammation, cardiovascular and central nervous system problems, and many infectious diseases. The binding of specific inhibitors to block the activity of an enzyme is the key to searching for potential drugs and to treating several physiological conditions associated with enzymes (Ramsay & Tipton, 2017). Urease is a pharmaceutically important and unique enzyme found in a wide variety of organisms and is known to catalyze the hydrolysis of urea into ammonia and carbonic acid (Amtul et al., 2002;Mobley et al., 1995). Urease also plays a key role in the formation of gastric ulcers (Mobley, 1996). The Helicobacter pylori infection (which can cause peptic ulcers and gastritis) is considered a worldwide problem with a high morbidity and mortality rate (Taha et al., 2015). It is estimated that about 50% of the world's population is infected with H. pylori (Rego et al., 2018). The urease-producing pathogen (H. pylori) is reported to survive in the highly acidic environment of the stomach because the urease-catalyzed hydrolysis of urea produces ammonia, which forms a protective shield around the pathogen to protect it from the acidic conditions, and is the actual damaging factor for the host tissues, resulting in gastritis and gastro-duodenal ulcers (Rego et al., 2018). Urea is a metabolic nitrogenous waste that is mainly produced in the liver, and the blood stream is responsible for carrying it towards the kidneys for excretion in urine. However, 20-25% of the urea remains in the body and is utilized as a substrate by the urolytic bacteria to produce high quantities of ammonia. During hepatic failure, the removal of toxic substances from the blood stream is affected and, as a result, the accumulation of toxic nitrogenous waste, including ammonia in the brain, is responsible for hepatic coma. The role of urease-producing pathogens is also reported in the progress of urinary catheter obstruction and in the production of kidney stones (urolithiasis) (Rego et al., 2018;Maroney & Ciurli, 2014). Therefore, the search for possible inhibitors of this key enzyme is an important therapeutic need, as the currently available inhibitor drugs are reported to have many side effects (Kosikowska & Berlicki, 2011).
Exemestane (EX), or 6-methylideneandrosta-1,4-diene-3,17-dione, is an anabolic steroid, used as an irreversible steroidal aromatase inhibitor. It is used clinically to treat breast cancer (Shiraki et al., 2008). To the best of our knowledge, only one co-crystal of EX with maleic acid has been synthesized and reported by Shiraki and co-workers in 2008 in order to study the change in dissolution rate of poorly soluble APIs, i.e. EX.
By considering all of the above facts, the present study was conducted to observe changes in the biological activities of synthesized co-crystals of EX. The objective was to assimilate the co-former (thiourea) in the molecular structure of the API (EX) to alter the crystal properties and therefore contribute to a change in the biological properties. Unfortunately, the synthesized exemestane:thiourea (EX:TH) (1:1) co-crystal was found to be inactive for anti-cancer activity evaluated against the breast cancer cell line. The discouraging results regarding anti-cancer activity prompted us to evaluate the synthesized co-crystal against urease inhibition activity as our co-former (thiourea) is already reported to be a tested standard in urease inhibition assays (Kanwal et al., 2018). Synthesized co-crystals were found to be several folds more active than tested standard thiourea. Therefore, as per the approved guidelines of the FDA, the synthesized co-crystal is a potential candidate as a urease inhibitor for further studies. The detailed structural features of the synthesized co-crystal were established on the basis of single-crystal X-ray diffraction (SCXRD) studies followed by qualitative and quantitative analyses of intermolecular interactions contributing to the co-crystal stability by Hirshfeld surface analysis. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) were also carried out to observe the thermal stability of the synthesized co-crystal.

Data collection and refinement
Crystals of suitable sizes were mounted on a Bruker SMART APEX II X-ray diffractometer for data collection and structure determination. X-ray diffraction data were collected using Mo K radiation ( = 0.71073 Å ). The program SAINT (Bruker, 2016) was used for data integration and reduction. Direct methods followed by Fourier transformation by employing full-matrix least-squares calculations were carried out to solve the structure by using SHELXTL and SHELXL97 (Sheldrick, 1997) in the case of EX; the APEX3 Suite combined with SHELXL2014/7 and SHELXL2016/6 (Sheldrick, 2015) was used in the case of the co-crystal. Intermolecular interactions were calculated using PLATON (Spek, 2009). The program ORTEP3 (Farrugia, 1997) was utilized to generate a structural representation. Mercury 2.0 was used to create molecular graphics of the interactions (Macrae et al., 2008). Crystallographic data, experimental details and structure-refinement parameters are summarized in Table 1.

Hirshfeld surface analysis
Qualitative and quantitative analyses of various hydrogen bonds contributing to the co-crystal stability were carried out research papers with the aid of Hirshfeld surface analysis, utilizing the software package Crystal Explorer. The two-dimensional Hirshfeld surface for the API is generated using d norm parameters in the range À0.580-1.325, with a shape index of À0.996-0.994 and a curvedness of À4.029-0.281. Whereas for the co-former, the d norm surface range is À0.612-1.059, the shape index is À0.998-0.996 and the curvedness is À4.000-0.40. The electrostatic potential surface has also been studied using the TONTO package incorporated in Crystal Explorer (Wolff et al., 2012).

Synthesis and crystallization
Commercially available solvents and chemicals were utilized without any further purification. Thiourea (TH) was purchased from Merck, Germany (index No. 612-082-0000). A small pestle and mortar was used for grinding. The commercially available drug Aromasin (1.411 g) was ground manually by using a pestle and mortar, and extracted in 100 ml dichloromethane (DCM) to obtain the API exemestane (0.350 g) after solvent evaporation under reduced pressure. The obtained API was further allowed to re-crystallize by dissolution in a 1:1 mixture of DCM and methanol after overnight evaporation. Manual grinding of crystallized EX (10 mg) and TH (20 mg) in a 1:1 stoichiometric ratio was carried out for 3 h, followed by methanol (1 ml Computer programs: SAINT, XPREP (Bruker, 2016), SADABS (Sheldrick, 2014), SHELXTL (Sheldrick, 2008), SHELXL2016/6 (Sheldrick, 2015).

Figure 1
Schematic representation of the co-crystal synthesis.
( Fig. 1). The shiny co-crystals obtained (8 mg) were found to be suitable for SCXRD analysis.

Evaluation of biological activity
Urease enzyme inhibition activity in vitro against the urease enzyme from Jack bean (Canavalia ensiformis, EC 3.5.1.5) was evaluated by adopting the same procedure as that described by Kanwal and co-workers (Kanwal et al., 2018).
In vitro cytotoxicity and anti-cancer activity of the API and synthesized co-crystals were evaluated by using a standard MTT colorimetric assay as per the protocol adopted and explained by Park et al. (2008).

Result and discussions
SCXRD analysis revealed that EX crystallizes in the orthorhombic space group P2 1 2 1 2 1 (Gö rlitzer et al., 2006), whereas the co-crystal crystallizes in the monoclinic space group P2 1 ; hence EX and TH are independent asymmetric moieties in the unit cell with Z = 2, as depicted in Table 1. Structural analysis revealed that EX and its co-crystal are both composed of four transfused rings (A-D). As a result of the C1 C2 and C4 C5 olefinic bonds in conjugation with the C3 carbonyl, ring A is planar in nature, with a maximum deviation of 0.05 (1) and 0.03 (3) Å from the root-mean-square plane (r.m.s.) for the C3 atom in EX and the co-crystal, respectively.  ORTEP drawing of the co-crystal of exemestane (6-methylideneandrost-4-ene-3,17-dione) and thiourea [(C 20 H 24 O 2 )(CH 4 N 2 S)] showing 30% probability ellipsoids.

Figure 3
Overlapped ORTEP drawing of EX and the co-crystal with 30% probability ellipsoids.  and (Q = 0.510 Å , = 12.40 , ' = 253 ) and (Q = 0.546 Å , = 5.19 , ' = 276.96 ) for EX and the co-crystal (Boeyens, 1978), respectively, whereas the five-membered ring D is folded like an envelope, with a maximum deviation of 0.262 and 0.265 Å for C14 with puckering parameters of Q = 0.4116 Å , ' = 206.59 and Q = 0.4124 Å , ' = 210.11 for EX and the coformer (Cremer & Pople, 1975). The ,-unsaturated carbonyl carbon (C3 O1) was found to be involved in the strong conjugation with the C1 C2 and C4 C5 olefinic bonds. Two methyl groups were found to be axially oriented (Fig. 2). The conformational features of the EX molecule in the co-crystal were found to be similar to that of the individual molecule. In comparison, the difference between the bond length of the C3 O1 olefinic bond was found to deviate between 1.194 and 1.189 Å , with the orientation of the angle C2-C3-O1 being 121.85 and 122.54 , and C3-C4-O1 being 120.97 and 121.09 for EX and the co-crystal, respectively (Fig. 3).  Table 2. A two-dimensional Hirshfeld surface (Spackman & Jayatilaka, 2009) has been generated using d norm [ Fig. 5(a)]. In the Hirshfeld surface, the question of 'compact packing' arises (Spackman & Jayatilaka, 2009), as shown in the encircled area and empty s spaces (A) in the unit cell. This is answered by the expansion of crystal packing, revealing that the neighbouring molecule is encapsulating this gap [ Fig. 5(b)]. The above situation is the main reason for analysing the API and coformer separately in qualitative and qualitative analyses, as they are involved in intermolecular interactions with neighbouring molecules. Fig. 6 shows the intermolecular interactions of the API and co-former with the neighbouring moieties. SÁ Á ÁH and the OÁ Á ÁH are important interactions responsible for forming strong hydrogen bonds as indicated by the red spots on the Hirshfeld surface.

Supramolecular features
Two-dimensional fingerprint plots (McKinnon et al., 2007) (FPs) for the API [ Fig. 7(a)] show the percentage contribution of contacts towards the crystal packing in which conventionally HÁ Á ÁH contributes the most, amounting to 65.5%. OÁ Á ÁH was found to be the strongest interaction among them all as shown by the sharp spike pointing to the origin and a 17.9% contribution to the unit-cell packing, followed by a 10.2% contribution from CÁ Á ÁH. SÁ Á ÁH and NÁ Á ÁH contribute 4.2 and 2.1%, respectively; usually FPs generate the percentage contribution as a reciprocal for both distances d e and d i . Here in the case of SÁ Á ÁH and NÁ Á ÁH for the API, cyan dots are only research papers IUCrJ (2020). 7, 105-112 Syeda et al. Crystal engineering to form a co-crystal with urease inhibition activity 109 Table 2 Hydrogen bonds for the co-crystal (Å and ). Symmetry transformations used to generate equivalent atoms: (i) Àx + 1, y À 1/2, Àz; (ii) x + 1, y, z À 1; (iii) Àx, y + 1/2, Àz + 1.

Figure 6
Intermolecular interactions with the two-dimensional Hirshfeld surface generated for the co-crystal.  in the region of de, which means S and N atoms are present only outside of the generated surface. In the case of the coformer [ Fig. 7(b)], the size of the molecule is small, which is why the percentage contribution of HÁ Á ÁH is equal to SÁ Á ÁH, i.e. a 36.3% contribution to the unit cell packing. The OÁ Á ÁH contribution is 15.2%, followed by contributions of 6.6 and 5.5% for NÁ Á ÁH and CÁ Á ÁH. In Fig. 7(c), FPs for the asym-metric unit have been drawn, which reveals that HÁ Á ÁH contacts are at a maximum of 60.3%, whereas OÁ Á ÁH and SÁ Á ÁH are 14.2 and 12.5%, respectively; in addition to these contacts, CÁ Á ÁH and NÁ Á ÁH show contribution of 9.3 and 3.4% to the unit cell packing. Two valuable tools of the Hirshfeld surface, namely the shape index and curvedness, reveal information about thestacking and the probability of forming interactions with neighbouring molecules, respectively. The shape index [ Fig. 8(a)] surface showed blurred patches which reveal the presence of weakstacking of the co-former moiety with neighbouring molecules. Curvedness [ Fig. 8(b)] shows the packing probabilities at various positions of the surface generated, these bumps and hollows on the surface reflect the packing behaviour. The ab initio electrostatic potential (Fig. 9) (Spackman et al., 2008) surface generated over the Hirshfeld surface shows the positive and negative potential sites of the molecule. In Fig. 9, blue dots over the surface relate the positions of the strong contacts to hydrogen-bond acceptors, whereas the red regions relate positions on the surface to hydrogen-bond donors (Fig. 9). The electrostatic potential map shows that the region of the    Shape index and curvedness mapped over the Hirshfeld surface.  surface where C S of the TH moiety is more electronegative than the C O attached to ring D.

Thermogravimetric analysis
From TGA studies (Steed, 2013) it is evident that the co-crystal (EX:TH) demonstrated a thermal stability up to 176.18 C, with a percentage weight loss of 2.384%. When the temperature was increased from 176.18 to 334.21 C, frequent percentage weight loss up to 51.396% was observed for the cocrystal (EX:TH) (Fig. 10). DSC spectra of the co-crystal (Fig. 11) clearly show three endothermic peaks observed at 170.27, 198.2 and 231.50 C due to the fusion of the TH crystal, thiourea:exemestane co-crystal (EX:TH) and the degradation of the thiourea polymer, respectively.

Biological activity
EX has been reported as an anticancer agent against breast cancer, therefore, the synthesized co-crystal was first evaluated in vitro to observe any changes in its anti-cancer activity against the MCF-7 breast cancer cell line; however, it was found to be ineffective. On the other hand, the coformer (thiourea) is reported as a tested standard to check urease inhibition activity in vitro (Kanwal et al., 2018). Therefore, both the API (EX) and the synthesized co-crystal (EX:TH) were evaluated for their urease enzyme inhibition activity (in vitro) and interesting results were obtained. EX was found to be inactive against the urease enzyme. However, its co-crystal showed potent urease inhibition activity (IC 50 = 3.86 AE 0.31mM) and was found to be severalfold more active than standard TH (IC 50 = 21.0 AE 1.45mM). These research papers IUCrJ (2020). 7, 105-112 Syeda et al. Crystal engineering to form a co-crystal with urease inhibition activity 111 Table 3 Bioactivity of exemestane and the co-crystal.
promising results clearly indicate that the significant change in activity can be attributed to the synergistic effects of both the API and co-former; however, further studies are required to be able to comment on that. Results of the biological activity evaluation are summarized in Table 3.

Conclusions
A co-crystal of the commercially available anti-cancer drug exemestane with thiourea has been successfully synthesized with enhanced urease inhibition activity (in vitro) compared with that of the API (EX) and co-former (thiourea) individually. The promising results of urease inhibition activity clearly demonstrate the possibility of the EX:TH (1:1) cocrystal as a new potential lead against peptic ulcers and urolithiasis. The contribution of non-covalent interactions to the structural stability of the co-crystal was also evaluated quantitatively with the aid of Hirshfeld surface analysis, and clearly demonstrated the role of the amine and carbonyl functionalities of the co-former and API in forming various contacts in the crystal structure. Electrostatic potential analysis clearly identified the sites of the molecule that can be targeted for structural modification. The promising results of the present study clearly demonstrate the role of co-crystallization as an effective tool in the search for potential leads against various health disorders.