Structural studies of five novel bile acid-4-aminopyridine conjugates Kari V. Ahonen a,⇑ , Manu K. Lahtinen b , Miika S. Löfman a , Anniina M. Kiesilä a , Arto M. Valkonen a , Elina I. Sievänen a , Nonappa a , Erkki T. Kolehmainen a a Department of Chemistry, Laboratory of Organic Chemistry, P.O. Box 35, FIN-40014 University of Jyväskylä , Finland b Department of Chemistry, Laboratory of Inorganic and Analytical Chemistry, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland article info Article history: Received 3 May 2012 Accepted 27 June 2012 Available online 16 July 2012 Keywords: 4-Aminopyridine Bile acids Optical spectroscopy X-ray diffraction Solid-state NMR Thermoanalysis abstract Synthesis and solid-state structural characterization of five bile acid amides of 4-aminopyridine (4-AP) are reported. Systematic crystallization experiments revealed a number of structural modifications and/or solvate/hydrate systems for these conjugates. Particularly, cholic acid conjugate exhibited five dis- tinct structure modifications, including one anhydrous form, mono- and dihydrates, as well as ethanol and 2-butanol solvates. The obtained crystal forms were examined extensively with various analytical methods, including solid-state NMR, Raman, and IR spectroscopies, powder and single crystal X-ray dif- fraction methods, thermogravimetry, and differential scanning calorimetry. After releasing their crystal solvent molecules, the resulted non-solvated structure forms showed 50–75 °C higher melting points than corresponding bile acids, and thermal degradation occurred for all conjugates at about 300– 330 °C. Moreover, the single crystal X-ray structure of the ursodeoxycholic acid-4-aminopyridine conju- gate is reported. Ó 2012 Elsevier Inc. All rights reserved. 1. Introduction Different crystal modifications of compounds can have distinct physicochemical properties, such as solubility and chemical stabil- ity [1], and therefore solid form screening is a regulatory require- ment for new pharmaceuticals [2]. Polymorphism is defined as the ability of any element or compound to crystallize as more than one distinct crystalline form [3]. Unfortunately, nomenclature for describing different crystal forms, where solvent molecule(s) are present in the crystal lattice, is still indecisive. Terms pseudopoly- morphism and solvatomorphism are frequently used to describe systems having mutually inconsistent elemental composition due to solvent intake [4]. In our opinion, however, simple solvate/hy- drate nomenclature is a better suited terminology for those sys- tems, and the ‘‘solvate/hydrate polymorph’’ should be applied only when an equal stoichiometry of solvent molecules incorpo- rated in different crystal modifications holds up [5]. For dalfampridine (4-aminopyridine, 4-AP), which has been ap- proved by the U.S. Food and Drug Administration (FDA) for the treatment of multiple sclerosis (MS) on January 22nd 2010 [6], one anhydrous crystal structure is known [7]. In addition to its anti-MS activity, 4-AP has been examined for the treatment of pa- tients with Lambert–Eaton syndrome [8], myasthenia gravis [9], human botulism [10], spinocerebellar ataxia type 1 [11], and Alz- heimer’s disease [12]. It affects by blocking the potassium channels leading to promotion of axonal conduction in the central nervous system (CNS) [13]. Although the efficacy and safety of this drug is well documented in many clinical trials [14], overdosing caused by formulation errors can lead to severe, even lethal, complications [15]. Benefits of 4-AP in the treatment of spinal cord injury have been modest due to the low tolerability; a concentration of 100 times higher compared to the maximal tolerated dose would be needed for the most efficient influence [16]. Moreover, convul- sive side effects in doses higher than 1 mg/kg limit its use in attenuating the symptoms of Parkinson’s disease [17]. Recently, 4-aminopyridine-3-methanol has in an animal model shown higher potency in the treatment of MS compared to 4-AP [18,19]. Bile acids are endogenous steroids with a wide variety of bio- logical functions [20,21] also present in the CNS [22–24]. As ther- apeutics, promising results have been obtained, i.a. with ursodeoxycholic acid, which has been used in the treatment of neurological dysfunctions caused by bilirubin [25], cisplatin [26], Huntington’s disease [27], and Alzheimer’s disease [28]. In addi- tion, it is used in the treatment of autosomal recessive ataxia to re- duce plasma concentration of neurotoxic bile acid intermediates [29]. Since 1999, when bile acids were discovered to act as physi- ological ligands to the farnesoid X receptor (FXR) [30–32], other nuclear receptors sensitive to bile acids have been reported, open- ing a new and active field of bile acid research [21]. In order to decrease the undesirable side effects and/or to improve the activity of dalfampridine, we have investigated its conjugation with bile acids. In a previous report by us we describe the preparation and structural characterization of lithocholyl 0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.steroids.2012.06.003 ⇑ Corresponding author. Tel.: +358 503483168; fax: +358 142602501. E-mail addresses: kari.v.ahonen@jyu.fi, [email protected](K. Ahonen). Steroids 77 (2012) 1141–1151 Contents lists available at SciVerse ScienceDirect Steroids journal homepage: www.elsevier.com/locate/steroids
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Steroids 77 (2012) 1141–1151
Contents lists available at SciVerse ScienceDirect
Structural studies of five novel bile acid-4-aminopyridine conjugates
Kari V. Ahonen a,⇑, Manu K. Lahtinen b, Miika S. Löfman a, Anniina M. Kiesilä a, Arto M. Valkonen a,Elina I. Sievänen a, Nonappa a, Erkki T. Kolehmainen a
a Department of Chemistry, Laboratory of Organic Chemistry, P.O. Box 35, FIN-40014 University of Jyväskylä , Finlandb Department of Chemistry, Laboratory of Inorganic and Analytical Chemistry, P.O. Box 35, FIN-40014 University of Jyväskylä, Finland
a r t i c l e i n f o
Article history:Received 3 May 2012Accepted 27 June 2012Available online 16 July 2012
Synthesis and solid-state structural characterization of five bile acid amides of 4-aminopyridine (4-AP)are reported. Systematic crystallization experiments revealed a number of structural modificationsand/or solvate/hydrate systems for these conjugates. Particularly, cholic acid conjugate exhibited five dis-tinct structure modifications, including one anhydrous form, mono- and dihydrates, as well as ethanoland 2-butanol solvates. The obtained crystal forms were examined extensively with various analyticalmethods, including solid-state NMR, Raman, and IR spectroscopies, powder and single crystal X-ray dif-fraction methods, thermogravimetry, and differential scanning calorimetry. After releasing their crystalsolvent molecules, the resulted non-solvated structure forms showed 50–75 �C higher melting pointsthan corresponding bile acids, and thermal degradation occurred for all conjugates at about 300–330 �C. Moreover, the single crystal X-ray structure of the ursodeoxycholic acid-4-aminopyridine conju-gate is reported.
� 2012 Elsevier Inc. All rights reserved.
1. Introduction
Different crystal modifications of compounds can have distinctphysicochemical properties, such as solubility and chemical stabil-ity [1], and therefore solid form screening is a regulatory require-ment for new pharmaceuticals [2]. Polymorphism is defined asthe ability of any element or compound to crystallize as more thanone distinct crystalline form [3]. Unfortunately, nomenclature fordescribing different crystal forms, where solvent molecule(s) arepresent in the crystal lattice, is still indecisive. Terms pseudopoly-morphism and solvatomorphism are frequently used to describesystems having mutually inconsistent elemental composition dueto solvent intake [4]. In our opinion, however, simple solvate/hy-drate nomenclature is a better suited terminology for those sys-tems, and the ‘‘solvate/hydrate polymorph’’ should be appliedonly when an equal stoichiometry of solvent molecules incorpo-rated in different crystal modifications holds up [5].
For dalfampridine (4-aminopyridine, 4-AP), which has been ap-proved by the U.S. Food and Drug Administration (FDA) for thetreatment of multiple sclerosis (MS) on January 22nd 2010 [6],one anhydrous crystal structure is known [7]. In addition to itsanti-MS activity, 4-AP has been examined for the treatment of pa-tients with Lambert–Eaton syndrome [8], myasthenia gravis [9],human botulism [10], spinocerebellar ataxia type 1 [11], and Alz-heimer’s disease [12]. It affects by blocking the potassium channels
leading to promotion of axonal conduction in the central nervoussystem (CNS) [13]. Although the efficacy and safety of this drugis well documented in many clinical trials [14], overdosing causedby formulation errors can lead to severe, even lethal, complications[15]. Benefits of 4-AP in the treatment of spinal cord injury havebeen modest due to the low tolerability; a concentration of100 times higher compared to the maximal tolerated dose wouldbe needed for the most efficient influence [16]. Moreover, convul-sive side effects in doses higher than 1 mg/kg limit its use inattenuating the symptoms of Parkinson’s disease [17]. Recently,4-aminopyridine-3-methanol has in an animal model shownhigher potency in the treatment of MS compared to 4-AP [18,19].
Bile acids are endogenous steroids with a wide variety of bio-logical functions [20,21] also present in the CNS [22–24]. As ther-apeutics, promising results have been obtained, i.a. withursodeoxycholic acid, which has been used in the treatment ofneurological dysfunctions caused by bilirubin [25], cisplatin [26],Huntington’s disease [27], and Alzheimer’s disease [28]. In addi-tion, it is used in the treatment of autosomal recessive ataxia to re-duce plasma concentration of neurotoxic bile acid intermediates[29]. Since 1999, when bile acids were discovered to act as physi-ological ligands to the farnesoid X receptor (FXR) [30–32], othernuclear receptors sensitive to bile acids have been reported, open-ing a new and active field of bile acid research [21].
In order to decrease the undesirable side effects and/or toimprove the activity of dalfampridine, we have investigated itsconjugation with bile acids. In a previous report by us we describethe preparation and structural characterization of lithocholyl
1142 K. Ahonen et al. / Steroids 77 (2012) 1141–1151
conjugates of isomeric aminopyridines [33]. In the current study,we report the conjugation of five less toxic bile acids, namelydeoxycholic acid (DCA), cholic acid (CA), chenodeoxycholic acid(CDCA), ursodeoxycholic acid (UDCA), and hyodeoxycholic acid(HDCA), with 4-aminopyridine. The structural and thermalproperties of the prepared conjugates are examined. In addition,polymorph/solvate screening for the conjugates is performed byextensive crystallization experiments from a wide variety ofsolvents or binary solvent mixtures resulting in total in twelvedifferent crystalline modifications. The preliminary biologicaltests performed for these compounds reveal promising neuronalactivity [34], and will be presented in the near future. Since bileacids are amphiphilic molecules, they could transport pharmaco-logically active agents through biological barriers [35] allowingCNS-targeted drug administration through olfactory epithelium[36].
2. Experimental
2.1. Synthesis
All reagents were purchased from Sigma–Aldrich Chemical Co,TCI Europe, or Acros Organics and used without further purifica-tion. Compounds 1–5 (Scheme 1) were synthesized by followingthe previously reported procedure [37] with slight modifications.Seven millimole of triethylamine (TEA) was added to a solutionof 6.4 mmol of the bile acid (BA) in 45 mL of dry tetrahydrofuran(THF) at room temperature (RT). Seven millimole of ethyl chloro-formate was added and the resulting solution stirred for 45 min.4-Aminopyridine (7.0 mmol) was introduced into the flask. After24 h, the TEA salt was filtered off and the solvent evaporated underreduced pressure. The unreacted 4-AP and bile acid were removedby aqueous bicarbonate solution followed by refluxing in acetoni-trile, and the resulting white powder dried in vacuum. The yieldsfor compounds 1–5 were 67% (DCA-4-AP), 80% (CA-4-AP), 66%(CDCA-4-AP), 73% (UCDA-4-AP), and 64% (HDCA-4-AP),respectively.
2.2. Crystallization
Twenty milligrams (1, 2, and 5) or 10 mg (3 and 4) of the exam-ined compound was placed to a glass vial, and 0.5–2 mL of solventadded. If the compound dissolved into the solvent at RT, 0.25–3 mLof anti-solvent (water or acetone) was added in order to createenvironment not capable of dissolving the compound at RT, butsoluble after ultrasonic radiation and/or heating. If the compoundremained insoluble into the tested solvent despite of ultrasonicradiation and heating treatment, 0.2–2 mL of ethanol or dimethyl-sulfoxide (DMSO) was added to amend the solubility. In addition tothe solvents presented in Table 2, experiments were carried out inTHF, hexane, 1,4-dioxane, 1,1,2,2-tetrachloroethane, and xylene.
Scheme 1. Partial numbering and structures of compounds 1–5.
2.3. NMR spectroscopy
Liquid state 1H and 13C NMR spectra were recorded for all con-jugates in DMSO-d6 at 30 �C using a Bruker Avance DRX 500 spec-trometer equipped with a 5 mm diameter broad band inversedetection probehead operating at 500.13 MHz in 1H and125.77 MHz in 13C experiments. The solid-state NMR measure-ments were recorded with Bruker Avance 400 spectrometer using4.0 mm od rotor or Bruker AvanceII 500 spectrometer with 3.2 mmod rotor. The contact time for the basic 1D experiment was 2 ms,the relaxation delay 4 s, and the spinning rate 10 or 12 kHz. Forthe short CP experiments, the contact time was 50 ls. The 13Cchemical shift was calibrated using glycine’s carbonyl peak at176.03 ppm as an external standard.
2.4. Mass spectrometry
Applied Biosystems/MDS Sciex QSTAR-ELITE mass spectrometerwas used for IS–TOF MS runs in a positive ionization mode usingmethanol as a solvent.
2.5. Single crystal X-ray diffraction
The data for 4 were collected at 123(2) K on a Bruker–NoniusKappaCCD diffractometer with an APEX-II detector and Mo Ka radi-ation (k = 0.71073 Å). COLLECT [38] data collection software wasutilized and data were processed with DENZO-SMN [39]. The struc-ture was solved by direct methods using SIR-2004 [40] and refinedon F2 using SHELXL-97 [41] in a WinGX [42] program package. Thereflections were corrected for Lorenz polarization effects withoutabsorption correction. The hydrogen atoms, except N–H and O–H, were calculated to their idealized positions with isotropic tem-perature factors (1.2 or 1.5 times the C temperature factor) and re-fined as riding atoms. Hydrogens attached to N or O were locatedfrom electron density maps and fixed to their ideal distance fromtheir parent atoms (0.88 Å for N–H and 0.84 Å for O–H at 123 K),with isotropic temperature factors of 1.2 (N–H) or 1.5 (O–H) timesthe parent atom factor. The Friedel pairs were merged due to pooranomalous scattering effects (light atom structure). The figureswere drawn with ORTEP-3 [43] and Mercury [44]. Other experi-mental X-ray data are shown in Table 1. Crystallographic data(excluding structure factors) for the structure in this paper havebeen deposited with the Cambridge Crystallographic Data Centreas supplementary publication number CCDC-872741. Copies ofthe data can be obtained, free of charge, on application to CCDC,12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44 (0)1223336033 or e-mail: [email protected]).
2.6. Pxrd
Powder X-ray diffraction analyses were carried out systemati-cally for all synthetic products as well as for the recrystallized sam-ples wherein Raman spectroscopic evaluation indicated existenceof different crystal forms. The diffraction data was recorded withPANalytical XPert Pro diffractometer in Bragg–Brentano geometryusing Johansson monochromatized Cu Ka1 radiation (45 kV,30 mA, k = 1.5406 Å) and XCelerator detector. Step size of 0.017�2h in data range of 3–71� 2h with counting time of 200 s per stepwere used for acquiring the diffraction intensities from hand-ground powder samples. The simulated powder diffraction patternwas generated by the program MERCURY 3.0.1 using the structureparameters of herein reported structure modification of 4.
Formula weight 468.66Crystal system, space group Orthorhombic, P212121
Unit cell dimensionsa/Å 7.03840(10)b/Å 17.9476(3)c/Å 20.5958(4)Volume/Å3 2601.71(8)Z 4Density (calculated)/Mg/m3 1.196Absorption coefficient/mm�1 0.077F(000) 1024Crystal size/mm3 0.20 � 0.18 � 0.06h range for data collection/� 2.48–28.29Index ranges �9 6 h 6 9,�23 6 k 6 23,�27 6 l 6 27Reflections collected 6453Data/restraints/parameters 3660/3/316Reflections > 2r(I) 3101Rint 0.0294Completeness to theta /% 99.8Goodness-of-fit on F2 1.056Final R indices [I > 2r(I)] R1 = 0.0472, wR2 = 0.0965R indices (all data) R1 = 0.0617, wR2 = 0.1033Largest diff. peak and hole/e.Å�3 0.258 and �0.201
K. Ahonen et al. / Steroids 77 (2012) 1141–1151 1143
2.7. Thermoanalysis
Thermal decomposition paths were obtained by Perkin ElmerSTA 6000 simultaneous thermal analyzer (measuring both thermo-gravimetric and differential temperature signals; TG/DTA). Mea-surements were carried out in an open platinum pan under air
Table 2A selection of crystallization solvents used in the current study. Solvent mixtures are show
Compound
Main solvent 1 2c 3
Synthetic product 1a 2a 3aEthanol 1aa (2:1:2
ethanol:acetone:water)
2d (1:1 ethanol: acetone) 3a
Methanol 1a (4:1 methanol:water)
2c (2:1 methanol: water) 3a
1-Propanol 1a (4:1 1-propanol: water)
2c (1:1 1-propanol: water) 3a
2-Propanol 1a (4:1 2-propanol: water)
– 3a
2-Butanol 1b 2c, 2ed (2:1 2-butanol: water) –
Anisole 1cb – Am
Chloroform 1a (6:1chloroform:ethanol)
2b, 2dd (4:1 chloroform:ethanol)
Amstru
Toluene 1a (4:1 toluene:ethanol)
Amorphous, toluene in thestructure (2:1 toluene: ethanol)
Amstru
Acetonitrile 1a (2:1acetonitrile:ethanol)
2b (2:1 acetonitrile: ethanol) 3a3be
– = No clear results were obtained.a In addition to solvents presented in the table, form 1a was also obtained from follo
tonitrile, 2:1 xylene:ethanol and 10:1 toluene:DMSO.b Obtained from 2% w/v concentration.c Gel was formed in 1,1,2,2-tetrachloroethane (2% w/v).d Different forms were obtained in second crystallization experiment. Form 2d was also
2-butanol:ethanol solution.e Form 3b was obtained from saturated acetonitrile solution and confirmed by soli
reproduce the form 3b resulting in incomplete characterization of this form.f Form 4a was also obtained from 1:1 ethanol:acetone solution.
atmosphere (flow rate of 45 mL/min) with a heat rate of 10 �C/min in the range of 25–600 �C. The temperature calibration ofthe analyzer was performed using melting points of the indium(156.6 �C) and zinc (419.5 �C) standards. The weight balance wascalibrated by measuring the standard weight of 50.0 mg at roomtemperature. The sample weights used in the measurements var-ied between 2 and 4 mg.
Thermal transitions of the compounds were examined bypower-compensation type Perkin Elmer Pyris Diamond differentialscanning calorimeter (DSC). Each measurement was carried outunder nitrogen atmosphere (flow rate 50 mL/min) using 50 lL alu-minum pan that was sealed by 30 lL aluminum pan having an ex-tra pinhole in the middle to ease the solvent evaporation andthereby preventing pressurized conditions inside the pan. The tem-perature calibration was made using two standard materials(n-decane and In) and the energy calibration by an indiumstandard (28.45 Jg�1). The samples were heated and cooled witha rate of 10 �C/min, from 0 �C to close proximity of the pre-definedthermal degradation temperature (by TG/DTA) of each substance.All thermal transitions were obtained as extrapolated onsets,except the glass transition temperature, which was taken athalf-step temperature of DCp change. The sample weights usedin the analyses varied between 1 and 4 mg.
Optical spectroscopy: The Raman measurements were per-formed using Bruker SENTERRA dispersive Raman microscopeoperating under OPUS version 6 software package (Bruker OpticsInc.). The microscope was equipped with a diode laser(k = 785 nm) and a charge-coupled device (CCD) detector operatingat �53 �C. The spectral data were collected in the range of 90–3300 cm�1 with 9–15 cm�1 resolution using a 50 � 1000 lm aper-
wing primary solvents or their mixtures: acetone, THF, 1,4-dioxane, 1:1 DMF:ace-
obtained from 50:1 CHCl3:ethanol solution. Form 2e was also crystallized from 2:1
d-state NMR. Unfortunately, despite of numerous attempts, we were not able to
1144 K. Ahonen et al. / Steroids 77 (2012) 1141–1151
ture. The data integration time was 10 s with two co-additions.‘‘Cosmic spike removal’’-setting and Sure_Cal� automated wave-length calibration were used. The baselines of the Raman spectrawere corrected with the concave rubberband method. The vacuumdried samples were placed on a glass slide and the spectra acquiredtwo to three times per sample from different locations using Olym-pus 100� objective for focusing. The measurement locations werevisually inspected both before and after the measurement undermicroscope in order to confirm that no directly observable damagewas caused by the laser exposure.
The ATR–FTIR spectra were measured with Bruker Tensor 27spectrometer using Pike Technologies GladiATR™ accessory. Thedata acquisition for the vacuum dried samples was performed with32 scans in the range of 400–4000 cm�1, and with 4 cm�1
resolution.Analytical data for 1 (3a,12a-dihydroxy-5b-cholan-24-oic N-
(C20,C60,C40), 129.0 (anisole m-CH), 116.1–112.7 (C30,C50, anisoleo-CH,p-CH), 74.2–70.8 (C12,C3), 55.4/54.9 (anisole CH3), 14.4/13.6,10.9⁄⁄ (C18). MS calc. for C29H44N2O3 (468.34):[M + H+] = 469.34; found = 469.35.⁄Overlapping with water resonance,⁄⁄Main peaks.Analytical data for 2 (3a,7a,12a-trihydroxy-5b-cholan-24-oic N-
Plausible structural modifications and/or solvate/hydrate sys-tems were screened by re-crystallization of the bulk powders of1–5 from different solvents or solvent mixtures. In order to assureoptimal dissolution conditions for the crystallization, followingtechniques were applied to fine-tune the solubility conditions:when the conjugate did not dissolve into the solvent, a step-by-step addition of a ‘‘pro-solvent’’ (ethanol or DMSO) was performedfor creating a miscible solution to which the substance dissolvedby heating; when the dissolution took place instantly an anti-sol-vent (water or acetone) was added in order to reduce solubility(Table 2).
Preliminary screening of the samples was performed byRaman spectroscopy and the differences observed therein werethen examined by powder X-ray diffraction. The identifieddifferent structural forms were further investigated by ATR–FTIR,TG, DSC, and SS-NMR techniques in order to unravel the chem-ical composition and the quantity of the solvents wherever sol-vated structure forms were detected. Moreover, single crystalsof 4 were obtained by slow evaporation of solvent mixtures(Table 2) and subjected for single crystal X-ray diffraction anal-ysis. As the conventional crystallization techniques were unsuc-cessful for the rest of the conjugates, by following theguidelines reported recently [45], vapor diffusion crystallizationwas applied for conjugates 1–3 and 5, unfortunately withoutsuccess.
Quite small sample amounts (10–20 mg) were used in the pre-liminary crystallization screening experiments, and because of thatalso crystallization tests in larger amounts were made in order tohave sufficient amount of the substance for SS-NMR measure-ments. ATR–FTIR spectroscopy was used to ensure the chemicaland structural integrity between the smaller and the larger crystal-lization patches.
By Raman spectroscopy four different forms of compound 1were observed, of which three distinct crystalline modificationswere confirmed (Fig. 1A). The existence of these forms was alsoverified by distinctively different X-ray powder diffraction patternsthat are shown in Fig. 1C. As shown by XRD and confirmed by DSCand TG analyses (Fig. 1E and F), the less crystalline synthetic prod-uct 1 (bulk) has a tendency to crystallize as a hydrate 1a, obtain-able from a variety of solvent combinations (Table 2). The secondform 1b crystallizes as a 2-butanol solvate along with traces ofthe hydrate 1a. The semi-crystalline anisole solvate 1c, on theother hand, could be obtained from a 2% w/v anisole solution. Withlower concentrations of 1 in anisole and slower crystallizationkinetics either the neat form 1a (0.25% w/v) or mixture of forms1a and 1c (1% w/v) were obtained (See ESI Fig. S3). SS-NMR analy-
Fig. 1. Raman (A) and IR (B) spectra, PXRD patterns (C), SS-NMR spectra (D), and TG (E) and DSC scans (F) of different crystal modifications of compound 1.
K. Ahonen et al. / Steroids 77 (2012) 1141–1151 1145
ses of conjugate 1 indicated that two non-equivalent moleculespossibly exist in the asymmetric unit of 1a, since doublet reso-nance patterns were observed in the spectrum (Fig. 1D). This pack-ing motif is rather common for bile acids [46] and their derivatives[33,47,48]. For the 2-butanol solvate 1b, only single resonanceswere visible in the spectrum including those assigned for the sol-vent. In the spectrum of the anisole solvate 1c, the carbonyl signalwas a triplet possibly indicating the existence of three conforma-tionally different conjugates in the asymmetric unit along withanisole, whose signals were also observable. The weight loss seen
in the TG analysis equals to 1/3 moles of anisole, supporting theassumption according to which three distinct moieties exist inthe asymmetric unit. This kind of packing pattern has also been ob-served for some of the bile acids, such as cholic acid hemihydrate[49] and 7-ketolithocholic acid [50].
In the Raman spectra of 1a–1c (Fig. 1A), appreciable differenceswere observed throughout the spectra (including the low wave-number area, where the lattice vibrations are located), suggestingdifferent crystal packing between these forms [51]. Profound dif-ferences were also visible in the IR spectra of 1a–1c, in which the
Fig. 2. Raman (A) and IR (B) spectra, PXRD patterns (C), SS-NMR spectra (D), and TG (E) and DSC scans (F) of different crystal modifications of compound 2.
1146 K. Ahonen et al. / Steroids 77 (2012) 1141–1151
amide I bands were observed at clearly different wavenumbers(1683, 1697, and 1704 cm�1, respectively) (Fig. 1B) indicating dif-ferences in the hydrogen bonding environments of these forms.
As indicated by the distinct Raman spectra and XRD patterns(Fig. 2A and C), conjugate 2 can crystallize at least in five differentcrystal forms, including two hydrates (2b and 2c), one ethanol sol-vate 2d, and one 2-butanol solvate 2e. The main synthetic productseems to have tendency to crystallize as an anhydrous form (2a),whereas 2b and 2c represent two different hydrate forms (see alsoTG and DSC analyses and Fig. 2E and F) that can be crystallizedfrom two different binary solvent mixtures (Table 2). Since theSS-NMR spectrum of the aged hydrate 2c resembled that of the
anhydrous form 2a, the XRD patterns were re-measured for agedsamples in order to rule out the possibility of a solid state phasetransition from a kinetically stable to a thermodynamically morestable form. XRD of the hydrate was reproduced yielding the origi-nal pattern, suggesting that care has to be taken in interpreting theSS-NMR spectra and the findings preferably confirmed by othermethods. Also the ethanol solvate 2d (frequently containing tracesof 2b, see Fig. 2C) and the 2-butanol solvate 2e were obtainedreproducibly from binary solvent mixtures. Some of the crystalliza-tion attempts from the ethanol/acetone mixture resulted in aphase mixture of the hydrate 2b and the ethanol solvate 2d (⁄ESIFig. S1).
Fig. 3. Raman (A) and IR (B) spectra, PXRD patterns (C), SS-NMR spectra (D), and TG (E) and DSC scans (F) of different crystal modifications of compounds 3, 4, and 5. Becauseof unsuccessful repetition of the crystallization of 3b, Raman and IR spectra were not measured.
K. Ahonen et al. / Steroids 77 (2012) 1141–1151 1147
As evidenced by the SS-NMR, XRD, and TG analyses (Fig. 3),the bulk synthetic product 3 corresponds to the anhydrous form3a obtained from five different solvent mixtures (Table 2). Onthe other hand, the hydrate 3b with a small amount of 3a incor-porated was obtained from a saturated acetonitrile solution onlyonce by using freshly water-washed sample for the crystallizationexperiment. Interestingly, the recrystallization of 3b from aceto-nitrile resulted in 3a instead of 3b. Moreover, 3b was never ob-tained from solvent mixtures commonly containing residualwater (ethanol, acetone, etc.). These observations suggest thatthe hydrate 3b is highly metastable, and thereby can only beobtained randomly when proper water content is present. Its
exceptionally low melting point (Table 4) and low crystallinity(Fig. 3C) indicate metastability as well. In the SS-NMR spectrum,resonances originating from the pyridine carbons 2 and 6 werewell separated, indicating quite similar electronic environmentcompared to that of compound 4 (Fig. 3D). The conjugate 4 crys-tallized as an anhydrous form regardless of the solvent systemused. Similarly the conjugate 5 crystallized as an anhydrous formfrom all of the solvents and solvent combinations tested (Fig. 3).Notably, the solubility of conjugate 5 to alcohols used in thisstudy was clearly lower than in the case of other conjugates.Therefore no anti-solvents were needed in the crystallizationexperiments (Table 2).
Table 3Selected geometric parameters of compound 4.
1148 K. Ahonen et al. / Steroids 77 (2012) 1141–1151
3.1. Single crystal X-ray diffraction
High quality crystals of conjugate 4 for single crystal X-ray dif-fraction were obtained from ethanol/acetone and ethanol/watersolutions. The absolute configuration of the starting material(UDCA) is known and in the performed reactions the chiral centersremain unchanged. With radiation used in this study determina-tion of the absolute configuration reliably would have been unfea-sible due to low scattering effects of the light atom structure 4 andthe consequent low fraction of Friedel-related reflections, which iswhy the absolute structure parameter was not determined. Thequality of the obtained data was high, as can be seen from theparameters in Table 1.
Some of the most characteristic geometric parameters for com-pound 4 are collected in Table 3. As can be seen, the covalent bonddistances and bond angles show normal values. The C13–C17–C20–C22 dihedral angle (Table 1) of the bile acid side chain showstrans-conformation. The dihedral angles C13–C17–C20–C22, C17–C20–C22–C23, C20–C22–C23–C24, and C22–C23–C24–N24, forone, show an overall ttgi conformation (t = trans, g = gauche,i = intermediate) deviating from the conformations of the previ-ously analyzed lithocholic acid 2-pyridyl- and 4-pyridylaminoderivatives, which possessed overall conformations of tttg and ttti,respectively [33]. Additionally, in the case of compound 4 the sidechain conformation is more twisted compared to the previouslyanalyzed ones. The dihedral angle (C24–N24–C4–C5) of theamide–pyridine junction (165.0(4)�) is close to that found for litho-cholic acid 4-pyridylamino derivative [33], indicating a rather pla-nar orientation with respect of the pyridine ring and the adjacentamide group. Three hydrogen bond donors interact intermolecularly
Table 4Thermal properties of compounds 1–5.
1–5 Dwt-% (TG) Solvate a Therm
exp. calc. Tds (D
1a 4.32 3.70 w (1) 99 (11b 8.65 7.33 b (½) 124 (1c 4.37 10.35 as (x) 134 (2a an2b 7.03 6.92 w (2) 60 (92c 4.59 3.59 w (1) 101 (2d 8.29 8.69 e (1) 136 (2e 12.40 13.27 b (1) 125 (3a an3b 3.67 3.70 w (1) 84 (14 an5 an
a = solvents are abbreviated as follows: w = water, b = 2-butanol, as = anisole, an = anhydTm = melting T (�C), Td = decomposition T (�C) and their enthalpies H (J g�1); Tg = glass tranimmediately after desolvation.
with the adjacent molecules (see Table 3) strongly influencing onthe packing of the crystal. A common intermolecular hydrogen-bonded chain motif in bile acid derivatives is an interactionbetween the side chain donor/acceptor of one molecule and thesubstituent donor/acceptor in position 3 of the steroidal ring ofanother molecule. This motif is observed also for compound 4 run-ning along the ab plane (head-to-tail, O3–H� � �N1) in contrast to thelithocholic acid derivative [33], in which the 3b-H was hydrogen-bonded to N1. The additional two strong hydrogen bonds in com-pound 4 organize the molecules into right-handed helical chainsalong a (N24–H� � �O3) and b (O7–H� � �O24) axes (Fig. 4).
3.2. Thermal analysis
In order to examine the solvent composition and thermal stabil-ity of the conjugates 1–5, thermogravimetric analyses (TG) werecarried out both for non- and solvated structure modifications. Dif-ferential scanning calorimetry (DSC) was applied to examine thethermal transitions (solvent evaporation, recystallization etc.) ofthe conjugates prior to their thermal degradation temperature (Ta-ble 4, graphs E and F in Figs. 1–3). As stated earlier, conjugate 1exhibits three different structure forms, of which the first form1a turned out to be a monohydrate. The first major weight loss isvisible at about 99 �C (DSC onset) with D wt.% of 4.32 (calc. Dwt.% 3.70), which corresponds relatively well to a loss of one moleof water. Similarly, desolvation of the 2-butanol solvate 1b, occursat �124 �C with 8.65 Dwt-% also indicating the release of one moleof the solvent. In the case of the anisole solvate 1c, the solvent con-tent is assumed to be low, as the overall weight loss observed at134 �C is less than 5 wt.%. Theoretical loss for instance for 1/3 molesof anisole would be 7.14 wt.%. The observed weight gap may orig-inate from the low crystallinity of the sample having amorphouscontent with not necessarily stoichiometric amount of anisolebound. Melting points of all of these three forms resides within220–230 �C, and the desolvated conjugates degrade generallyabove 300 �C, having slight variations form-dependently (Table 4).In the case of forms 1a and 1c, the recrystallization is visible read-ily after the desolvation process, whereas the 2-butanol solvate 1bremains solid during desolvation, eventually melting at 230 �C.
Cholic acid conjugate 2 shows the broadest variety of structuremodifications, of which conjugate 2a proved to be anhydrousshowing only a melting transition at 276 �C and degrading at323 �C. Two of the structure modifications were determined asdi- and monohydrates, 2b and 2c respectively, having experimen-tal weight losses (Dwt-% 7.03 and 4.59) sufficiently closely equiv-alent to the corresponding calculated values (D wt.% 6.92 and 3.59,
rous, e = ethanol, and their mole fraction is in parentheses; Tds = desolvation T (�C),sition T taken from the 2nd heating scan and its Cp (J g�1 �C�1); b = recrystallization
Fig. 4. Single crystal X-ray structure and crystal packing of compound 4.
K. Ahonen et al. / Steroids 77 (2012) 1141–1151 1149
respectively). Similarly, the weight losses observed at 136 and125 �C for the ethanol and 2-butanol solvates, 2d and 2e, agreewell with the calculated values, as can be seen in Table 4. The sol-vates 2b and 2d recrystallize readily after desolvation, then melt at278 �C, and degrade similarly as 2a at about 325 �C. The solvates 2cand 2e resemble 1b, as the solvent release occurs in the solid state.The melting and degrading temperatures are comparable with theother solvates in this group. The observed melting transitions ofthe conjugates seem to be generally 50–75 �C higher than theirnon-conjugated bile acid analogues. It can also be noted that theCA conjugates in general bear significantly higher melting temper-atures in contrast to the other conjugates, whereas the CDCA con-jugates have the lowest melting points (Table 4). This can be, atleast partially, explained by the structural properties of the bileacid scaffold; with three hydroxyl groups the CA derivatives areable to form the most extensive hydrogen bonding networks intheir crystal lattices.
The almost uniformly obtained form of conjugate 3 was theanhydrous structure modification 3a, which melts at 216 �C andthermally degrades at 315 �C. The monohydrate 3b isolated onlyonce (D wt.% exp. 3.67 and calc. 3.70) shows an exceptionallylow melting point in contrast to the other conjugates, with themelting point onset at 143 �C, but still with equal thermal stabilitywith 3a. The anhydrous forms of conjugates 4 and 5 show also rel-
atively high melting points (254 and 260 �C, respectively) and sim-ilar degradation temperatures than conjugates 1–3, being around300 �C. The cooling scan and consecutive heating scan of all conju-gates reveal only glass transitions, generally between a tempera-ture range of 127–141 �C except for 1a (105 �C), indicating theirpoor tendency to crystallize from a melt.
4. Discussion
In order to decrease the undesirable side effects and/or to im-prove the activity of dalfampridine, a compound approved fortreating multiple sclerosis as well as investigated for a wide varietyof additional pharmacological purposes, we have conjugated itwith bile acid molecules. As a continuation of our previous reportdescribing the preparation and structural characterization of litho-cholyl conjugates of isomeric aminopyridines, the bile acids em-ployed in the current study, namely deoxycholic acid, cholic acid,chenodeoxycholic acid, ursodeoxycholic acid, and hyodeoxycholicacid, are less toxic. In addition to their preparation, extensive solidstate structural characterization for the compounds was per-formed. By SS-NMR and optical spectroscopies, XRD methods,and thermal analysis three, five, and two distinct solid-state formsfor the deoxycholic acid, cholic acid, and chenodeoxycholic acidconjugates, respectively, were identified. Ursodeoxycholic and
1150 K. Ahonen et al. / Steroids 77 (2012) 1141–1151
hyodeoxycholic acid conjugates yielded a single crystalline form.Crystals of ursodeoxycholic acid conjugate obtained from ethanolmixtures were of high quality, enabling the determination of itssingle crystal X-ray structure.
Because different crystalline or amorphous forms of the samecompound can possess divergent physical, chemical, and biologicalproperties, solid state studies of active pharmaceutical ingredientsare essential. Untangling the solid state behavior of dalfampridine-bile acid conjugates is significant regarding both the current useand the development of formulations with less side effects and/or increased activity of 4-AP.
Acknowledgements
E.S. (Proj. nos. 119616, 218178, and 255648), M. Löfman (Proj.nos. 119616 and 255648), E.K. (Proj. no. 121805), K.A. (Proj. no.121805), and N. (Proj. no. 121805) gratefully acknowledge Acad-emy of Finland for financial support. Academy Professor Kari Rissa-nen and the Academy of Finland (Proj. nos. 130629, 122350, and140718) are thanked for financial support to A.V. Laboratory of Ap-plied Chemistry of University of Jyväskylä is acknowledged for pro-viding the facilities for Raman spectroscopy. Spec. Lab. Tech. MirjaLahtiperä is thanked for measuring the mass spectra. Dr. DavidŠaman, Dr. Radek Pohl, and Institute of Organic Chemistry and Bio-chemistry, ASCR, Prague, Czech Republic, are gratefully acknowl-edged for providing solid-state NMR facilities to K.A.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.steroids.2012.06.003.
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