Triple Structural Transition below Room Temperature in the Antifilarial

DOI: 10.1021/cg100212q

Triple Structural Transition below Room Temperature in the Antifilarial Drug Diethylcarbamazine Citrate

2010, Vol. 10 3094–3101

Cecilia C. P. da Silva,† Felipe T. Martins,† Sara B. Honorato,‡ N ubia Boechat,§ Alejandro P. Ayala,‡ and Javier Ellena*,† †

Instituto de Fı´sica de S~ ao Carlos, Universidade de S~ ao Paulo, CP 369, S~ ao Carlos, S~ ao Paulo 13560-970, a, CP 6030, Fortaleza, Cear a 60455-970, Brazil, ‡Departamento de Fı´sica, Universidade Federal do Cear armacos-FarManguinhos, Brazil, and §Fundac-a~o Oswaldo Cruz - FioCruz, Instituto de Tecnologia em F Rua Sizenando Nabuco 100, Rio de Janeiro, Rio de Janeiro 21041-250, Brazil Received February 10, 2010; Revised Manuscript Received April 20, 2010

ABSTRACT: A very unusual triple structural transition pattern below room temperature was observed for the antifilarial drug diethylcarbamazine citrate. Besides the first thermal, crystallographic, and vibrational investigations of this first-line drug used in clinical treatment for lymphatic filariasis, a noteworthy behavior with three structural transformations as a function of temperature was demonstrated by differential scanning calorimetry, Raman spectroscopy, and single-crystal X-ray diffractometry. Our X-ray data on single crystals allow for a complete featuring and understanding of all transitions, since the four structures associated with the three solid-solid phase transformations were accurately determined. Two of three structural transitions show an order-disorder mechanism and temperature hysteresis with exothermic peaks at 224 K (T10 ) and 213 K (T20 ) upon cooling and endothermic ones at 248 K (T1) and 226 K (T2) upon heating. The other transition occurs at 108 K (T3) and it is temperature-rate sensitive. Molecular displacements onto the (010) plane and conformational changes of the diethylcarbamazine backbone as a consequence of the C-H 3 3 3 N hydrogen bonding formation/cleavage between drug molecules explain the mechanism of the transitions at T10 /T2. However, such changes are observed only on alternate columns of the drug intercalated by citrate chains, which leads to a doubling of the lattice period along the a axis of the 235 K structure with respect to the 150 and 293 K structures. At T20 /T1, these structural alterations occur in all columns of the drug. At T3, there is a rotation on the axis of the N-C bond between the carbamoyl moiety and an ethyl group of one crystallographically independent diethylcarbamazine molecule besides molecular shifts and other conformational alterations. The impact of this study is based on the fascinating finding in which the versatile capability of structural adaptation dependent on the thermal history was observed for a relatively simple organic salt, diethylcarbamazine citrate.

1. Introduction Filariasis is an endemic disease in 83 tropical countries of Asia, Africa, and Central and South Americas.1-5 About 120 million persons are infected worldwide.6 Among other disease manifestations, chronic stages of lymphatic filariasis (LF) often present with lymphoedema, known also as elephantiasis. This worst symptom causes serious psychological and social damage to infected persons.7,8 Diethylcarbamazine (1-(N,N-diethylcarbamoyl)-4-methylpiperazine, DEC) is the first-line antifilarial drug for LF treatment. It acts by killing the parasitic filarial worms (mainly Wuchereria bancrofti) lodged in the lymphatic system.9,10 DEC, in particular its citrate salt (DEC citrate) due to its most favorable solubility and stability compared to the free base and other salts, has been marketed for more than 50 years by several pharmaceutical companies under different trade names.11-14 Even though this active pharmaceutical ingredient (API) has been known and incorporated into drug formulations for a long time, its solid state physical behavior has not been investigated. Therefore, vibrational, crystallographic, and thermal studies of DEC citrate were our first goals in an attempt to pioneer the characterization and understanding of their solid state properties related to drug performance. Structure determination was the first step in featuring DEC citrate. In addition to the first solid state structure determination of this antifilarial drug reported here, we found three reversible structural transitions *To whom correspondence should be addressed. E-mail: javiere@ ifsc.usp.br. pubs.acs.org/crystal

Published on Web 05/28/2010

below room temperature that exhibit a very complex thermodynamics. Such triple structural transition behavior below room temperature is very rare.15-18 Even more surprising than discovering three low temperature structural transformations and determining the four related crystal phases of DEC citrate was the fact that this triple phenomenon is still more unusual in the case of molecular solids composed only by organic frameworks,19,20 mainly for molecular salts. Therefore, DEC citrate is an attractive case for solid state dynamics comprehension of such compounds. 2. Experimental Section 2.1. Crystal Growth. Transparent well-grown block-shaped DEC citrate crystal suitable for single-crystal X-ray diffraction analysis was selected after the crystallization procedure. To prepare such a crystal, raw material of DEC citrate (100 mg) from Fundac-~ ao Oswaldo Cruz (FioCruz, Instituto de Tecnologia em F armacos-FarManguinhos) was dissolved in ethanol (50 mL) by vigorous shaking of the mixture at 60 °C. Next, the mixture was filtered through a 0.25 μm filter (Millipore) at room temperature and the resulting solution was allowed to evaporate slowly for 15 days at 5 °C. 2.2. Single-Crystal X-ray Structure Determination. DEC citrate crystal structures of phase I, phase II, phase III, and phase IV were determined at 293, 235, 150, and 100 K, respectively. However, it is important to note that exothermic peaks in differential scanning calorimetry (DSC) traces (see below) associated with the three structural transitions were observed at 224 K (temperature T10 , from phase I to phase II) and 213 K (temperature T20 , from phase II to phase III) upon cooling DEC citrate crystals, while upon heating the endothermic ones were observed at 248 K (temperature T1, from phase II to phase I), 226 K (temperature T2, from phase III to phase II), and 108 K r 2010 American Chemical Society

Article

Crystal Growth & Design, Vol. 10, No. 7, 2010

3095

Table 1. Structure Determination of the DEC Citrate Crystal Phases at Different Temperatures and Refinement Statistics 100 K/phase IV

150 K/phase III

235 K/phase II

293 K/phase I

space group P1 a = 13.656(5) A˚ b = 10.259(5) A˚ c = 13.902(6) A˚ R = 90.140(3)° β = 95.389(3)° γ = 92.677(3)° V = 1937(1) A˚3 Z=4 Fcalc = 1.34 g/cm3 8042 unique reflns R(int) = 0.06 θmax = 25.5° 98.2% compltness R1[I>2σ(I)] = 0.06 wR2 = 0.17 S = 1.03

space group P21/c a = 13.915(2) A˚ b = 10.096(2) A˚ c = 14.089(3) A˚ R = 90° β = 103.56(1)° γ = 90° V = 1924.1(6) A˚3 Z=4 Fcalc = 1.35 g/cm3 3501 unique reflns R(int) = 0.05 θmax = 25.4° 98.8% compltness R1[I>2σ(I)] = 0.05 wR2 = 0.13 S = 1.03



(temperature T3, from phase IV to phase III). In this way, the occurrence of the crystal phases between the temperatures ranges below and above each structural transition upon either cooling or heating were also confirmed by determination of unit cell parameters by means of single-crystal X-ray diffractometry. As mentioned, DEC citrate crystal structures of phases I-IV were determined on X-ray diffraction data collected at both room and low temperatures using a Enraf-Nonius Kappa-CCD diffractometer (graphite-monochromated MoKR radiation with λ = 0.71073 A˚, data collecting strategy with set of j scans and ω scans and κ offsets, 95 mm CCD camera detector on a κ-goniostat). In the case of low temperature measurements, a cold N2 gas blower cryogenic device (Oxford Cryosystem) coupled to the diffractometer was used. The X-ray diffraction data were processed as described: the COLLECT21 and the HKL Denzo-Scalepack software22 for acquisition, indexing, integration, and scaling of Bragg reflections, no absorption correction due to small absorption coefficients (μ) ranging from 0.105 to 0.108 mm-1, solving by direct methods using SHELXS-9723 within the WinGX,24 refinement by full-matrix least-squares on F2 with SHELXL-9725 also within the WinGX,24 splitting of the carbon and hydrogen atoms of the ethyl group syn oriented relative to the carbonyl moiety over two positions in the structures of phases I and II, with major and extra sites of constrained 70% and 30% occupancies, respectively, C-H hydrogen atoms with constrained coordinates according to a riding model (C-H bond distances of 0.96 and 0.97 A˚ in methyl and methylene groups) and fixed isotropic thermal parameters (Uiso(H) = 1.2Ueq(Cmethylene) or 1.5Ueq(Cmethyl)), finding of Nþ-H and O-H hydrogens from the difference Fourier maps and free refinement of their positions with fixed isotropic thermal parameters (Uiso(H) = 1.2Ueq(N) or 1.5Ueq(O)). The crystallographic information files (CIF) of the four DEC citrate structures were deposited with the Cambridge Structural Data Base under the codes CCDC 757079-757082. Free of charge, copies of these files may be solicited from The Director, CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK, fax: þ44123-336-033; e-mail: [email protected]. uk or http:www.ccdc.cam.ac.uk. 2.3. Thermal Analysis. Thermal behavior of DEC citrate was investigated by differential scanning calorimetry (DSC) using a Netzsch DSC 204 F1 Phoenix CC 200 F1 system under nitrogen atmosphere. Several cooling/heating temperature cycles were performed at different constant rates. Sealed aluminum crucibles with pierced lids were used for both samples (about 5 mg each) and reference. 2.4. Raman Spectroscopy. The temperature dependence of the DEC citrate Raman spectra was recorded in a T64000 Jobin Yvon spectrometer equipped with an Olympus microscope and a liquid N2 cooled CCD to detect scattered beam from an argon ion laser (λ = 514.5 nm). A Nikon 20 objective with a focal distance of 20 mm and numeric aperture of 0.35 was used to focus the laser beam on the polished sample surface. Low-temperature measurements were performed using an Air Products closed-cycle He refrigeration system. A Lakeshore controller was used to control the temperature with a precision of 0.1 K.

3. Results and Discussion 3.1. Room Temperature Structure Determination. At room temperature, DEC citrate crystallized in the centrosymmetric monoclinic space group P21/c with one (DEC)þ(citrate); ionic pair in the asymmetric unit (Table 1). The piperazine ring of (DEC)þ adopts a chair conformation. The plane passing through the four carbons of piperazine forms an angle of 75.0(2)° with the carbamoyl plane. This is due to a rotation on the axis of the N-C bond between the piperazine and carbamoyl moieties so that the carbonyl group of (DEC)þ can interact with the hydroxyl moiety of (citrate); through a classical O-H 3 3 3 O hydrogen bonding. The methylated nitrogen of piperazine is protonated. The protonation on this nitrogen can be rationalized on the basis of hyperconjugation effects from the methyl group. Furthermore, the other two nitrogens of (DEC)þ are hindered to be protonated because an electronic withdrawal by its neighboring carbonyl group. The protonated Nþ-H group of piperazine is a hydrogen bonding donor to the C-O; carboxylate and the C-OH hydroxyl groups of a same (citrate); fragment. Therefore, these two Nþ-H 3 3 3 O; and Nþ-H 3 3 3 O hydrogen bonds show a bifurcated pattern. To the best of our knowledge, this is the first structure determination of the antifilarial drug diethylcarbamazine in the form of its citrate salt. However, the two carbons of one of the two ethyl tails of (DEC)þ showed occupancy sites disordered over two positions. This ethyl group is syn oriented relative to the carbonyl moiety of carbamoyl. With the carbamoyl plane taken as a reference, the atomic fractions in the major occupancy sites (constrained to 70%) form an ethyl tail present with an anti conformation relative to the piperazine ring. On the other hand, a syn conformation relative to this six-membered cycle is described for the ethyl group formed by the carbon fractions in the extra occupancy sites (constrained to 30%). The other ethyl group that is anti oriented relative to the carbonyl group of carbamoyl is not disordered. Its CH3 end is held bonded to the carbonyl moiety of one of the two carboxyl groups of (citrate); through a nonclassical C-H 3 3 3 O hydrogen bonding. This ethyl tail adopts a syn conformation relative to piperazine. Therefore, it is anti oriented relative to another ethyl tail formed by atomic fractions in the major occupancy sites. Steric effects between these two ethyl groups are responsible for this anti conformation. In addition, there is a rotation on the axis of the carbamoyl N-C bond resulting in a deviation of the CH2 carbons of both ethyl groups from the carbamoyl plane. The torsions j1 and j2 (see the molecular structure representation of DEC in

3096


Figure 1. DEC conformers of the DEC citrate crystal structures determined at different temperatures. Thermal ellipsoids are at the 30% probability level. Hydrogens were hidden for clarity. Dashed lines represent bonds involving carbon fractions in extra occupancy sites. Values of torsions that largely vary according to temperature are shown (see Scheme 1 for definition of the torsions).

Scheme 1. DEC Torsions That Largely Vary According to Temperature

Scheme 1) on the carbamoyl N-C bond measure -24.0(5)° and -39.8(4)° at 293 K, respectively. One will see throughout the text that this study is focused on multiple structural transitions in DEC citrate as a function of temperature. For that reason, the torsion j3 (see Scheme 1) on the axis of the N-C bond between the carbamoyl and the ethyl group anti oriented relative to carbonyl helps describe the (DEC)þ conformers of the structures at different temperatures, mainly when comparing the 100 K structure to others (see below). At the 293 K structure, the torsion j3 measures 137.0(3)°. 3.2. First Evidence for Low Temperature Polymorphs. Although the positional disorder of (DEC)þ was solved and refined satisfactorily, we were interested to know if by decreasing the temperature on X-ray diffraction data collection each atom of the disordered ethyl group would occupy only one positional site. Upon performing single-crystal X-ray diffraction analysis on the same crystal after it was frozen at 150 K, we were able to observe for the first time on this compound a structural transition for which the low temperature monoclinic phase III (note that the crystal structure of phase II was determined at 235 K upon heating the crystal from 150 K, and the thermal evidence that allowed us to discover its existence will be presented in the sequence) shows three clear differences when compared to the room temperature structure (Figure 1): (1) an ordering phenomenon of the ethyl moiety that was disordered at 293 K, (2) conformational change of (DEC)þ, and (3)

da Silva et al.

rearrangement of the supramolecular architecture. At 150 K, there is a nonclassical intermolecular C-H 3 3 3 N hydrogen bonding between the CH2 methylene group of ethyl and the neutral nitrogen of piperazine, connecting (DEC)þ molecules along the [010] direction. This is responsible for ordering of the ethyl tail because this group is fixed through an intermolecular contact at 150 K. At 293 and 150 K, the interatomic C 3 3 3 N distances are 5.22(1) A˚ and 3.55(1) A˚, respectively, which show the occurrence of this weak C-H 3 3 3 N intermolecular interaction only at low temperature. Contrary to the room temperature structure, the weak nonclassical C-H 3 3 3 O hydrogen bonding through the CH3 group of the ethyl moiety anti oriented relative to carbamoyl and the carbonyl group of one of the two carboxyl groups of (citrate); does not exist at 150 K. The interatomic C 3 3 3 O separation is 5.590(3) A˚ at this low temperature, while at 293 K the corresponding measurement is 3.352(4) A˚. In addition, the rotation on the axis of the carbamoyl N-C bond differs between the structures determined at 293 and 150 K (j1, j2, and j3 measure -5.8(3)°, -20.6(3)°, and 121.3(3)° at 150 K). This is related to the C-H 3 3 3 N hydrogen bonding formation that holds the ethyl group on only one conformation (Figure 1). Similarly, the C-H 3 3 3 N hydrogen bonding formation forces a displacement of (DEC)þ onto the ac plane (Figure 2). As a consequence of a structural rearrangement, (citrate); anions are also displaced onto the (010) plane (Figure 2). From these structural shifts onto the ac plane, the unit cell angle β of the 150 K structure is more expanded than that of the 293 K structure. Therefore, crystal packing changes with temperature decreasing from 293 K to 150 K. Furthermore, the crystal packing at 150 K is more compact than that at 293 K (see the calculated density values in Table 1). At both temperatures, two-dimensional zigzag chains of (citrate); grow along the b and c axes through O-H 3 3 3 O hydrogen bonds between the anions. The packing of (citrate); anions gives rise to alternate holes that accommodate (DEC)þ molecules in a capsule-like fashion (Figure 3). The (DEC)þ molecules as guests are bonded to (citrate); chains through hydrogen bonds in which the protonated Nþ-H group of piperazine and the CdO group of carbamoyl are hydrogen bonding donor and acceptor groups, respectively. This assembles (DEC)þ columns parallel to the b axis. These columns are intercalated by (citrate); ribbons along the [100] direction. However, the aforementioned C-H 3 3 3 N hydrogen bonding between the guests within neighboring holes of the hosts contributes to the structural stabilization at 150 K. Indeed, the formation of this hydrogen bonding is related to the solid-solid phase transition (Figure 2). Fascinated by the structural transition of DEC citrate crystals in response to temperature decreasing, we decided to perform a unit cell determination at 100 K. At this temperature, the unit cell parameters were similar to those at 150 K when cooling the crystal from 150 to 100 K at a rate of 2 K/min. As an alternative procedure, the crystal was rapidly frozen at 100 K from room temperature and the unit cell was determined again. Unpredictably, another triclinic unit cell was found. This indicated that another structural transition exists between room temperature and 100 K. Thereby, X-ray diffraction data were collected to determine the crystal phase IV at 100 K. After data collection, the crystal was heated from 100 to 150 K at a rate of 2 K/ min in order to investigate the reversibility of the phase transitions. This procedure resulted in the crystal appearance going from transparent to opaque. During the X-ray diffraction data collection, it was possible to observe this alteration by direct inspection of the crystal mounted on the goniometer, since the cooled N2 flow does not cloud the crystal

Article


3097

Figure 2. Nonclassical hydrogen bonds (green dashed lines) associated with the three structural transitions of DEC citrate crystals. Views onto the ac plane are depicted. Thermal ellipsoids are at the 30% probability level. Hydrogens of DEC molecules as hydrogen bonding acceptors and the atomic fractions in extra occupancy sites of phase I and phase II were hidden for clarity. Red arrows illustrate the displacement of DEC and citrate molecules of phase II and phase III onto the (010) plane with respect to phase I and phase IV.

Figure 3. DEC columns assembled due to accommodation of the drug molecules into holes created on citrate chains change according to temperature. Views along the c axis are depicted (a axis in the horizontal and b axis in the vertical). Hydrogens and the atomic fractions in extra occupancy sites of phase I and phase II were hidden for clarity. Boxed regions detach the relative orientation of ethyl tails under different temperatures.

visualization. Accompanying this visual alteration, the single-crystal X-ray diffraction pattern at 100 K goes to a powder X-ray diffraction pattern at 150 K. The 100 K structure (phase IV) is present with two (DEC)þ and two (citrate); molecules in the asymmetric unit (Figure 1). At this temperature, the 21-screw axis symmetry relating (DEC)þ molecules in all other structures is quenched. Consequently, the structure goes from a monoclinic unit cell (space group P21/c) to a triclinic one (space group P1). This symmetry loss occurs because of conformational changes. One crystallographically independent (DEC)þ molecule shows a rotation of about 180° on the axis of the N-C bond between the carbamoyl group and the ethyl moiety anti oriented relative to carbonyl when compared to another asymmetric (DEC)þ molecule (Figure 1). This configures both ethyl tails with an

anti conformation relative to piperazine, while each alkyl tail shows a syn conformation relative to one another. The torsions j1, j2, and j3 respectively measure -16.0(3)°, -42.6(3)°, and -47.6(3)° in this conformer. This rotation results from the nonclassical C-H 3 3 3 O hydrogen bonding formation in which the CH2 moiety of ethyl is a hydrogen bonding donor to the carbonyl moiety of one of two carboxyl groups of (citrate);. When compared to the hydrogen bonding pattern of the other (DEC)þ conformer occurring at 100 K and the 293 K structure, this Cmethylene-H 3 3 3 O hydrogen bonding substitutes the one between the CH3 end of ethyl and the same carboxyl of (citrate);. The interatomic Cmethylene 3 3 3 Ocitrate distance measures 3.672(2) A˚ at 100 K and is somewhat larger than the Cmethyl 3 3 3 Ocitrate separation (3.352(4) A˚) in the room temperature structure. The other asymmetric (DEC)þ molecule

3098


da Silva et al. Table 2. Unit Cell Metrics and Crystal Phases of DEC Citrate above and below the Structural Transitions Observed at 226 and 248 K upon Heating from 150 to 293 K at 2 K/min T(K)

a (A˚)

b (A˚)

c (A˚)

β (deg)

phase

150 215 235 293

13.905(13) 13.928(13) 27.4386(7) 13.84 (4)

10.042(16) 10.075(16) 10.1644(2) 10.27(2)

14.025(16) 14.054(16) 14.0536(3) 14.01 (2)

103.41(2) 103.41(3) 95.033(1) 93.72 (6)

III III II I

Table 3. Unit Cell Metrics and Crystal Phases of DEC Citrate above and below the Structural Transitions Observed at 213 and 224 K upon Cooling from 293 to 150 K at 2 K/min

Figure 4. Low temperature DSC traces of DEC citrate recorded during cycles (a) cooling (b) heating at 10 K/min and fast cooling (∼80 K/min) and (c) heating at 10 K/min.

at 100 K, and the supramolecular architecture, are similar to those of phase I, although there are other differences in the conformation and packing (Figures 2 and 3). The torsions j1, j2, and j3 measure -13.6(3)°, -39.4(3)°, and 137.8(3)° in the 293 K structure-like (DEC)þ conformer of phase III. Interestingly, the C-H 3 3 3 N hydrogen bonding present in phase III is not formed at 100 K (Figure 2). The interatomic C 3 3 3 N distance is 5.288(3) A˚ at 100 K, while in the structure of phase III this value is 3.55(1) A˚. Furthermore, no displacement of (DEC)þ onto the (010) plane is observed when compared to the room temperature structure. Additionally, the phase IV density is lower than that of phase III. This reveals that the packing of phase IV is less compact than the phase III, in agreement with the assembly differences of the structures determined at these temperatures. The similarities between phase I and phase IV are in good agreement with the fact that the last one is just obtained by quenching. On the other hand, the structural transition into phase III implies not only the displacement of (DEC)þ but also the ordering of the alkyl tail around the j3 dihedral angle. 3.3. Thermal Analysis. To understand better the complex energy exchanges involved in this structure transformation, we also studied the thermal behavior of DEC citrate by differential scanning calorimetry (DSC). Surprisingly, two structural transitions were observed when cooling a DEC citrate sample from room temperature characterized by exothermic peaks at (onset temperature) 224 and 213 K (Figure 4). These events are reversible, independent of the heating/cooling rate and exhibit a considerably large hysteresis (23 and 13 K, respectively). The presence of ca. 20 K hysteresis of the phase transition temperatures and heat flow anomaly sharpness suggest that these two phase transitions have a first-order character. Contrary to the welldefined behaviors of these two transitions, a third endothermic event was only observed on heating. By cooling the sample at 10 K/min, a weak and wide endothermic peak at 108 K was observed on heating. This transition is not observed upon slower cooling rates, but it is very well-defined if the sample is cooled down at ∼80 K/min. Our results suggest that the low temperature phase IV is partially stabilized using slow cooling rates. As a consequence, the solid-solid phase transition at 108 K depends on the thermal history of the sample. Similarly, this transition was observed by single-crystal X-ray diffractometry only upon quenching the crystal at 100 K. The crystallographic and

T(K)

a (A˚)

b (A˚)

c (A˚)

β (deg)

phase

293 235 215 150

13.87(2) 13.81(3) 27.10(8) 13.95(2)

10.295(14) 10.262(11) 10.094(16) 10.102(9)

14.040(19) 13.993(16) 13.95(2) 14.098(13)

93.81(5) 93.68(5) 94.85(8) 103.51(4)

I I II III

calorimetric data infer that one of three transitions is temperature-rate sensitive, suggesting that the low temperature phase IV is metastable and transforms into a stable one at 108 K. 3.4. Structure Determination of Phase II. On the basis of previous observations, another DEC citrate crystal structure remained to be determined, that of phase II. First, another DEC citrate crystal than that previously analyzed by X-ray diffractometry was frozen at 150 K and the temperature was increased to 235 at 2 K/min. So, the X-ray diffraction experiment was performed again. As in the other three structure determinations, the fourth structure of DEC citrate (phase II) was successfully determined using X-ray diffraction intensities collected at 235 K. This procedure also included unit cell measurements at 150 and 215 K. At both temperatures, unit cell dimensions were very similar, in agreement with the occurrence of the same crystal phase (phase III) under such thermal conditions (Table 2). However, the 235 K unit cell was different from those at 150 and 215 K, in agreement with the structural transition occurring at 226 K upon heating. After X-ray diffraction data collection at 235 K, the temperature was increased from 235 to 293 K using the same heating rate and the unit cell was determined once again. From this procedure, it was possible to observe the structural transition at 248 K by crystallography, since the unit cell parameters measured at 293 K (phase I) after heating of the crystal were different from those at 235 K (phase II) (Table 2). In addition, the monoclinic unit cell of the room temperature structure directly determined without temperature increasing procedure was the same one measured at 293 K after heating from 235 K (Tables 2 and 3). Therefore, three structural transitions and the four related structures were completely characterized, allowing us to understand the structural and thermal changes occurring in DEC citrate crystals as a function of temperature. Similar to the heating procedure in which the unit cell was measured sometimes, X-ray diffraction analyses on the same crystal were carried out to determine the unit cell at 293, 235, 215, and 150 K upon cooling at 2 K/min, in an attempt to observe the reversible and hysteretic behaviors of two of the three phase transitions by the crystallographic technique (Table 3). At temperatures of 293 and 235 K, unit cells were practically equal, confirming the occurrence of phase I at both temperatures upon cooling. The unit cell dimensions measured at 215 K after cooling from 293 K were similar to those of the 235 K structure determined on data collected after heating practice from 150 K, demonstrating that phase II exists at different temperatures depending on the cooling or heating procedure. In addition, the unit cell measured at 150 K (phase III) by direct freezing of the crystal without any

Article

cooling procedure was the same one determined at 150 K after temperature decreasing from 293 K (Tables 2 and 3). Therefore, when the DEC citrate crystal is cooled from room temperature to 150 K, the unit cell dimensions at 293, 215, and 150 K, respectively, correspond to those determined at 293, 235, and 150 K if the crystal is heated from 150 K to room temperature. It is important to state that the unit cell at 293 K is the same one at 235 K after cooling of the crystal, and the unit cell at 150 K matches that determined at 215 K after heating of DEC citrate. These demonstrate that two structural transitions depend on either cooling or heating DEC citrate crystals. In this way, the hysteretic feature of two phase transitions dependent on temperature was doubtless confirmed by single-crystal X-ray diffraction analysis. Likewise, these structural transitions are reversible. Two (DEC)þ and two (citrate); fragments were found in the asymmetric unit of the structure determined using data collected at 235 K (phase II) (Figure 1). These two (DEC)þ molecules are those found in the asymmetric units determined at 293 K (phase I) and 150 K (phase III). At 235 K, the torsions j1, j2, and j3 measure -4.9(3)°, -20.8(4)°, and 121.5(3)° in the crystallographically independent cation of the drug corresponding to that of the 150 K structure and -24.0(5)°, -40.6(4)°, and 138.0(3)° in the another one related to the (DEC)þ molecule of the 293 K structure. This reflects a partial transition from the 150 K structure to the 293 K structure in which a stable intermediate exists between either 226 and 248 K when heating the DEC citrate sample or 213 and 224 K when cooling it. In addition, the packing compactness and the unit cell angle β of phase II are intermediate between those of phase I and phase III. Concerning the compactness of packing, this is stated on the basis of the density value calculated for phase II that lies between the densities calculated for phase I and phase III. Partial displacements onto the ac plane are responsible for the expansion of the unit cell angle β of phase II when compared to the β value of phase I and for its contraction with respect to that of phase III. Indeed, the supramolecular architecture of phase II shows alternate holes on each (citrate); chain that accommodate different conformers of (DEC)þ (Figure 3). Similarly to phase III, in phase II there is the CH 3 3 3 N hydrogen bonding between neighboring (DEC)þ guests within columns self-assembled along the b axis that partially orders the drug molecules. Into these columns, the interatomic C 3 3 3 N distance is 3.61(1) at 235 K, similar to the structure of phase III in which this measurement is 3.55(1) A˚. Likewise, the weak nonclassical C-H 3 3 3 O hydrogen bonding between the CH3 end of the ethyl group anti oriented relative to carbamoyl and the carbonyl group of one of the two carboxyl groups of (citrate); does not occur into these columns at 235 K because the interatomic C 3 3 3 O separation is 5.602(4) A˚ in this structure section, similar to phase III whose corresponding C 3 3 3 O distance measures 5.590(3) A˚. However, another adjacent (DEC)þ column separated by a (citrate); ribbon along the [100] direction from a phase III-like hydrogen-bonded column resembles closely phase I, in which any intermolecular interaction does not occur between the guests and one ethyl group of (DEC)þ is disordered (into these columns, the corresponding interatomic C 3 3 3 N distance is 5.17(1) in phase II, a value similar to 5.22(1) A˚ in the room temperature structure). Accompanying the hydrogen bonding pattern of phase I, in this 235 K structure part there is the weak nonclassical Cmethyl-H 3 3 3 Ocitrate hydrogen bonding which does not take place in the other phase III-like structure section. The related interatomic C 3 3 3 O distances are 3.324(3) A˚ in the phase I-like


3099

region of the 235 K structure and 3.352(4) A˚ in the room temperature structure. Therefore, in phase II there is a doubling of the lattice period along the a axis with respect to phase I and phase III (Table 1 and Figure 2). This is a result of alternating ordered hydrogen bonded and disordered non-hydrogen bonded columns of (DEC)þ along this axis (Figure 3). While the transformation from phase III to phase II features partial molecular disordering and change of conformation and hydrogen bonding pattern, the phase transition from phase II to phase I shows a complete alteration of the (DEC)þ conformation in which all columns of the drug are not hydrogen bonded through the C-H 3 3 3 N atoms and the (DEC)þ molecules are disordered on one of their two ethyl tails. 3.5. Thermodynamic Relationships between DEC Citrate Phases. Similar transition enthalpies (ΔH) values for the two structural transformations at T2 and T1 are in agreement with the gradual change from an ordered hydrogen-bonded structure (phase III) first to a partially disordered nonhydrogen-bonded one (phase II) and after to an entirely disordered non-hydrogen-bonded system (phase I) when heating DEC citrate crystals and vice versa when cooling them (Figure 4). However, it is interesting to observe that the measured ΔH value associated with the transition from phase III to phase II upon heating from 150 to 235 K is lower than that related to the transformation from phase II to phase III. Similarly, the |ΔH| value for the transition from phase I to phase II by cooling the crystal is lower than that for the complete transformation to a hydrogen-bonded system (from phase II to phase III) upon temperature decreasing from 215 to 150 K (Figure 4). This effect is directly related to the corresponding transition entropies (ΔS = ΔH/T) which, on heating, are ΔS1 = 7.23 J/(mol K) and ΔS2 = 5.56 J/(mol K), whereas ΔS10=-5.98 J/(mol K) and ΔS20=-6.42 J/(mol K) were measured on cooling. The molecular mechanism of the reversible phase transformations involves two processes: (i) the appearance of an intermolecular nonclassical hydrogen bond associated with a conformational change and a molecular displacement onto the (010) plane and (ii) the ordering of one of the alkyl tails also related to the hydrogen bonding formation. Calorimetric measurements are able determine if displacive or orderdisorder mechanisms are involved in the transition. If in a displacive case the transition takes place between two ordered structures, then the transition entropy and enthalpy are small. Such transformations should be followed by a rather small entropy change (ΔS ≈ 0.1R, R: gas constant). On the other hand, in the order-disorder case, since the transition takes place from an ordered low temperature phase to a disordered high temperature phase, the conformation contribution to the entropy is the dominant term. If in the ordered and disordered phases the total number of configurations is N1 and N2, respectively, then ΔS=R ln(N2/N1).26 In our case, in the first phase transformation, one column of (DEC)þ is ordered giving rise to an 1:1 organization of ordered and disordered columns along the a axis (phase II). The remaining disordered molecules are ordered after the second transition (phase III). Remembering that alkyl tails are disordered between two orientations (N2=2 and N1=1), the statistical molar entropy for the complete disordering of a system with two energetically equivalent states should be R ln 2. According to our DSC results, the transition entropies accompanying the first and second transformations can be rewritten as ΔS10 =R ln 2.05 and ΔS20 =R ln 2.16 on cooling and ΔS1=R ln 2.39 and ΔS2 = R ln 1.96 on heating. These values are is excellent

3100


da Silva et al.

Figure 5. Low temperature Raman spectra of DEC citrate recorded on cooling.

agreement with the previsions based on the crystalline structure, which show that each phase transition is driven by the freezing of two disordered states into an ordered one. However, a detailed analysis of the transition entropies shows that the transformations leading to phase II (T10 and T2) exhibit similar transition temperatures of ∼225 K and transition entropies |ΔS| ∼ R ln 2. Conversely, transitions leaving phase II (T1 and T20 ) have in both cases |ΔS| higher than R ln 2. The excess entropy over the theoretical value is |ΔS| ∼ 0.15 R ln 2 suggesting that a displacive effect also contributes to the transition mechanism. This phenomenon could be addressed to the displacement of the (DEC)þ and (citrate); ions onto the (010) plane (Figure 2), but further investigations need to be performed in order to clarify this process. From phase IV to phase III, the DSC traces show broad endothermic peaks associated with this phase transition, which is characterized by large entropy variation (≈ 44.0 J/(mol K) from curve (c) in Figure 4). These excess entropy values support an order-disorder process, but the transformation extended over a wide temperature range and depending on the thermal prehistory of the sample can be interpreted as nonsimultaneous changes in the dynamics and the orientation of the alkyl tail in the structure.26 The metastable nature of the low temperature phase IV combined with a complex structural relationship do not allow us to clearly define the main mechanism of this phase transition. 3.6. Temperature-Dependent Raman Spectroscopy. Once the crystal structures of DEC citrate at low temperatures were investigated by DSC and single-crystal X-ray diffraction techniques, resulting in the observation of several phase transitions, this compound was also investigated by Raman spectroscopy as a function of temperature on cooling. In Figure 5, we show the temperature dependence of the Raman spectra. From the analysis of these spectra, the first two phase transitions were identified. Thus, two relatively abrupt modifications around 220 and 200 K can be easily identified characterizing the transformations at 224 and 213 K. Unfortunately, the experimental setup was not able to reach cooling rates fast enough to stabilize the low temperature phase IV, but Raman spectra of phases I, II, and III were recorded. Comparing these spectra, the major changes

correspond to bands at approximately 1400 cm-1 and 2900 cm-1. These bands are associated with vibrations of the alkyl tails, δ(CH) at ∼1400 cm-1 and ν(CH) around 2950 cm-1. These observations are in excellent agreement with the X-ray diffraction results. As discussed earlier, the crystal structures of room and low temperatures differ with respect to the orientation of these groups. In addition, the results confirm that the changes observed by the X-ray diffraction were not associated with a simple effect of freezing a thermally activated phenomenon, but correspond to well-defined structural phase transitions. Two vibrational modes related to the stretching of the carbonyl of groups can be identified in the Raman spectra of DEC citrate at approximately 1724 and 1630 cm-1. The large difference between the carbonyl stretching modes of these molecules can be addressed to differences in the intramolecular environment and intermolecular interactions. The first and most intense one can be associated to the non-hydrogen bonded carbonyl group of the citrate anion. The second stretching mode is a very weak band due to the carbonyl group of the DEC molecule. This assignment is supported by the infrared and Raman spectrum of the DEC base compound.27 This carbonyl stretching is particularly low because the neighbor nitrogens induce a relevant mesomeric effect, which is combined with a moderate hydrogen bond with the citrate anion. At low temperatures, the carbonyl stretching of DEC splits in phase II (220 K) and is observed alone but shifted (1615 cm-1) in phase III. This temperature dependence is directly correlated to doubling of the number of DEC molecules per unit cell in phase II. The bands below 1200 cm-1 exhibit almost no influence of the structural changes. This effect can be understood in terms of the crystalline structures since this spectral region is dominated by the stretchings and deformations of the piperazine ring and backbone of the tails which are less sensitive to the phase transformations. The structural phase transitions are also evidenced by the analysis of the low energy region of the Raman spectrum which also suffer sudden changes around 220 and 200 K. Below 200 cm-1, a clear insight into the intermolecular interaction and molecular conformations is provided by the vibrational modes associated with the

Article


crystalline network and molecular torsions, like the one around j3, which plays a determining role in the phase transition mechanism of DEC citrate. 4. Conclusion In summary, a very unusual triple structural transition pattern was observed in an organic structure of the antifilarial drug diethylcarbamazine citrate by only decreasing/increasing the temperature. Besides the first thermal, vibrational, and crystallographic characterizations of this first-line drug used in clinical treatment for elephantiasis and other manifestations of lymphatic filariasis, a remarkable behavior with three structural transformations as a function of temperature was demonstrated by single-crystal X-ray diffractometry, differential scanning calorimetry, and Raman scattering. Our X-ray data on single crystals allow for a complete featuring and understanding of all transitions, since the four structures associated with the three solid-solid phase transformations were accurately determined. Furthermore, the DSC traces were correlated with the crystallographic data, allowing us to understand the phase transition mechanisms. At room temperature, no hydrogen bond between (DEC)þ molecules occurs in a structure of phase I in which the atoms of one ethyl tail are disordered. By cooling DEC citrate crystals, the partial ordering of the 215 K structure of phase II as a consequence of the C-H 3 3 3 N hydrogen bonding formation between (DEC)þ molecules explains the mechanism of the transition at 224 K. Partial molecular displacements onto the (010) plane and conformational changes of (DEC)þ also feature this transition. From phase II to phase III, these structural alterations are completed. On the other hand, the transition from the triclinic 100 K structure of phase IV to the monoclinic 150 K structure of phase III involves a rotation on the axis of the N-C bond between carbamoyl and ethyl of one crystallographically independent (DEC)þ molecule besides molecular shifts and other conformational alterations resulting in different dihedral angles. Two of three structural transformations, at 248 and 226 K, are reversible and show a significantly large temperature hysteresis with endothermic peaks, which allow us to classify them as first-order transitions. In addition, the corresponding enthalpy excesses clearly suggest order-disorder transitions, in excellent agreement with the structural data. The other transition at 108 K is sensitive to temperature rate. The main impact of this study is based on the fascinating finding in which the enormously versatile capability of structural adaptation dependent on the thermal history was observed for a relatively simple organic salt, diethylcarbamazine citrate. It is surprising how a drug with a changeable structure may guide the further development of materials that sensitively respond to temperature decreasing/increasing. Acknowledgment. We thank CAPES (C.C.P.S.), FAPESP (F.T.M.), and CNPq (J.E., A.P.A.) for research fellowships.

3101

The authors thank CNPq, IPDI, FUNCAP, and FAPESP for financial support. Supporting Information Available: Crystallographic information files (CIF) for the four DEC citrate crystal structures. This material is available free of charge via the Internet at http://pubs.acs.org.

References (1) Bockarie, M. J.; Pedersen, E. M.; White, G. B.; Michael, E. Annu. Rev. Entomol. 2009, 54, 469–487. (2) Shenoy, R. K.; Suma, T. K.; Kumaraswami, V.; Rahmah, N.; Dhananjayan, G.; Padma, S. Ann. Trop. Med. Parasit. 2009, 103, 235–247. (3) Beyrer, C.; Villar, J. C.; Suwanvanichkij, V.; Singh, S.; Baral, S. D.; Mills, E. J. Lancet 2007, 370, 619–627. (4) Weil, G. J.; Ramzy, R. M. R. Trends Parasitol. 2007, 23, 78–82. (5) McPherson, T.; Persaud, S.; Singh, S.; Fay, M. P.; Addiss, D.; Nutman, T. B.; Hay, R. Brit. J. Dermatol. 2006, 154, 933–941. (6) Ottesen, E. A.; Hooper, P. J.; Bradley, M.; Biswas, G. PLoS Neglect. Trop. Dis. 2008, 2, e317. (7) Tripathi, R. P.; Katiyar, D.; Dwivedi, N.; Singh, B. K.; Pandey, J. Curr. Med. Chem. 2006, 13, 3319–3334. (8) Duke, B. O. L. Brit. Med. J. 1981, 283, 1036–1037. (9) Ismail, M. M.; Jayakody, R. L.; Weil, G. J.; Fernando, D.; Silva, M. S. G.; Silva, G. A. C.; Balasooriya, W. K. Trans. R. Soc. Trop. Med. Hyg. 2001, 95, 332–335. (10) Hawking, F.; Sewell, P.; Thurston, J. P. Lancet 1948, 255, 730–731. (11) Kasim, N. A.; Whitehouse, M.; Ramachandran, C.; Bermejo, M.; Lennernaes, H.; Hussain, A. S.; Junginger, H. E.; Stavchansky, S. A.; Midha, K. K.; Shah, V. P.; Amidon, G. L. Mol. Pharmaceut. 2004, 1, 85–96. (12) Sklaver, L.; Murray, C. Am. J. Hosp. Pharm. 1986, 43, 2987. (13) Partono, F.; Purnomo, O. S.; Soewarta, A. Acta Trop. 1981, 38, 217–225. (14) Hewitt, R. Nature 1949, 164, 1135. (15) Shakhvorostov, D.; Muser, M. H.; Mosey, N. J.; Song, Y.; Norton, P. R. Phys. Rev. B 2009, 79, 094107/1–094107/9. (16) Liu, Y.; Her, J.-H.; Dailly, A.; Ramirez-Cuesta, A. J.; Neumann, D. A.; Brown, C. M. J. Am. Chem. Soc. 2008, 130, 11813–11818. (17) Gagor, A.; Wojtas, M.; Pietraszko, A.; Jakubas, R. Acta Crystallogr. Sect. B 2008, 64, 558–566. (18) Mir, M.; Guimaraes, R. B.; Fernandes, J. C.; Continentino, M. A.; Doriguetto, A. C.; Mascarenhas, Y. P.; Ellena, J.; Castellano, E. E.; Freitas, R. S.; Ghivelder, L. Phys. Rev. Lett. 2001, 87, 147201/ 1–147201/4. (19) Minkov, V. S.; Tumanov, N. A.; Kolesov, B. A.; Boldyreva, E. V.; Bizyaev, S. N. J. Phys. Chem. B 2009, 113, 5262–5272. (20) Bordallo, H. N.; Kolesov, B. A.; Boldyreva, E. V.; Juranyi, F. J. Am. Chem. Soc. 2007, 129, 10984–10985. (21) COLLECT Data Collection Software; Nonius: Delft, The Netherlands, 1998. (22) Otwinowski, Z.; Minor, W. In Methods in Enzymology: Macromolecular Crystallography, Part A; Carter, C. W., Jr., Sweet, R. M., Eds.; Academic Press: New York, 1997; Vol. 276, pp 307-326. (23) Sheldrick, G. M. SHELXS-97: Program for Crystal Structure Resolution; University of G€ottingen: G€ottingen, Germany, 1997. (24) Farrugia, L. J. J. Appl. Crystallogr. 1999, 32, 837–838. (25) Sheldrick, G. M. SHELXL-97: Program for Crystal Structure Analysis; University of G€ottingen: G€ottingen, Germany, 1997. (26) Strukov, B. A.; Levanyuk, A. P., Eds.; In Ferroelectric Phenomena in Crystals: Physical Foundations; Springer: New York, 1997. (27) Honorato, S. B. Polymorphism in pharmaceuticals, their hydrates and salts: study of solid state properties, Ph.D. Thesis, Federal University of Ceara, Fortaleleza, CE, Brazil, 2009.

Triple Structural Transition below Room Temperature in the Antifilarial

Recommend Documents