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High-performance liquid chromatographic enantioseparation of methanobenzazocines
Journal of Chromatography A, 1216 (2009) 7708–7714
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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High-performance liquid chromatographic enantioseparation of methanobenzazocines William M. Barker ∗ , Karin Worm, Roland E. Dolle Department of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
a r t i c l e
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Article history: Received 3 April 2009 Received in revised form 6 August 2009 Accepted 12 August 2009 Available online 18 August 2009 Keywords: HPLC Chiral separation Enantioselective Benzazocine Benzomorphans Racemic Cyclodextrin Polysaccharide and Pirkle-type chiral stationary phases
a b s t r a c t Chiral recognition and resolution of methanobenzazocines was investigated by HPLC using polysaccharide, Pirkle-type, native and derivatized -cyclodextrin chiral stationary phases. Enantioseparation of phenyl substituted 2,6-methanobenzazocines was achieved with multiple chiral stationary phases throughout the classes described. Chiral resolution of the enantiomers of 1,5-methano-3-methyl-6-oxo1,2,3,4,5,6-hexahydro-3-benzazocine was produced on both polysaccharide and Pirkle-type phases. In the case of 1,5-methano-3-methyl-6-phenyl-1,2,3,4,5,6-hexahydro-3-benzazocine only a dinitrophenyl substituted -cyclodextrin produced a separation of enantiomers. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Benzo-fused heterocyclic ring systems have received a lot of attention over the years because of their ubiquitous appearance in natural products and modern pharmaceuticals [1,2]. Benzazocines are a class of benzo-fused nitrogen-containing eight-membered heterocyclic rings and include the 1-benzazocine, 2-benzazocine and 3-benzazocine skeletons. 1,5-Methano-3benzazocine 1 [3] and 2,6-methanobenzazocines 3–5 [4] (also called benzomorphans) shown in Fig. 1, were reported to have analgesic activity but to be devoid of physical dependence liability, the most serious side effect of morphine like drugs. Several synthetic procedures for compound 1, its precursor 2 and compounds 3–5, both racemic and enantioselective, have been reported [5–8]. For compounds 4 and 5 a method for optical resolution via d- and l-mandelic acid salts has been described [8]. Enantiomers of pharmaceutically active agents are often shown to display key differences in activity and efficacy [9,10]. Investigation of the pharmacological differences between the enantiomers of these compounds required that we develop chromatographic methods capable of discriminating each form. To the best of our
∗ Corresponding author. Tel.: +1 484 712 5767. E-mail address: [email protected] (W.M. Barker). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.033
knowledge the work presented here is the first study systematically investigating the chiral separation of methanobenzazocines 1–5 by high-performance liquid chromatography (HPLC) on a number of chiral stationary phases (CSPs).
2. Experimental 2.1. Materials Racemic methanobenzazocines were prepared according to the methods described in the literature [3,4,11]. The relative stereochemistry accessible via the synthetic route employed only yielded one enantiomeric pair for each compound described. Samples were prepared in 50/50 water/acetonitrile with glacial acetic acid added at 1% (v/v) for use in reverse-phase chromatography. Ethanol was used as the sample diluent for chromatographic screening in the polar-organic and normal-phase modes. All solvents used as diluents and mobile phases were HPLC grade. Hexanes (Hex), 2-propanol (IPA), ethanol (EtOH), methanol (MeOH), acetonitrile (ACN), water, and d6 -DMSO were purchased from Sigma–Aldrich (St. Louis, MO, USA), Fisher Scientific (Pittsburgh, PA, USA), and Pharmco (Brookfield, CT, USA). The reagents used to buffer, acidify, or basify the solutions were ammonium acetate (NH4 OAc), glacial acetic acid (HOAc), and triethylamine (TEA), respectively. These were of reagent grade, ACS or spectroscopic grade and were pur-
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Fig. 1. Benzazocine nomenclature and compounds investigated.
chased from Sigma–Aldrich, Fisher Scientific, and EMD (Gibbstown, USA). Aqueous ammonium acetate buffers were prepared at 50 mM concentration and the adjustment of pH was performed by titration with acetic acid while monitoring pH with a calibrated pH meter. All aqueous buffers were filtered before use with a Phenomenex (Torrance, CA, USA) filter apparatus and Phenomenex nylon and PTFE filters of 0.45 pore size. Mobile phase mixtures for the normal-phase, polar-organic, and reverse-phase HPLC chromatography were mixed at use on the HPLC system. Columns packed with the CSPs evaluated were of 250 mm × 4.6 mm in dimension. The polysaccharide based CSPs were the cellulose and amylose derived phases from Chiral Technologies (Daicel, Tokyo, Japan) and Phenomenex. These included the Chiralcel OD, Chiralcel OJ, Chiralpak AD, Chiralpak AS, and the LUX Cellulose-2 trademarked names. The Pirkle-type phase CSPs were Regis WHELK-0 1 (S,S), ULMO (S,S), PIRKLE 1-J (3S,4R) and LUECINE, from Regis Technologies (Morton Grove, IL, USA). The -cyclodextrin (-CD) based phases employed were Cyclobond I 2000, Cyclobond 2000 DMP, and Cyclobond I 2000 DNP from Sigma–Aldrich. 2.2. Equipment NMR spectroscopy was performed using a Bruker Avance 400 controlled with the Bruker TopSpin software package (Bruker BioSpin Corp., Billerica, MA, USA). Normal-phase HPLC chromatography was carried out on a Waters system (Waters Corp., Milford, MA, USA) composed of an Waters Alliance 2695 pump and injection module, a Waters 996 photo-diode array detector and Waters Millennium chromatography software. Reverse-phase and polarorganic phase chromatographic separations were performed on a Waters system composed of an Waters Alliance 2795 pump and injection module, a Waters 996 photo-diode array detector, a Micromass ZQ2000 mass spectrometer and Waters MassLynx chromatography software (Waters Corp., Milford, MA, USA). UV detection with photo-diode array detectors was carried out scanning from 200 to 300 nm. For reverse-phase and polar-organic chromatographic separations the chromatograms were plotted at 225 nm and for normal-phase chromatography 280 nm was selected. 2.3. Column evaluation All CSPs, except for the native -CD, were evaluated in the normal-phase mode (Hex-EtOH or Hex-IPA). Mobile phase mixtures containing TEA at 0.2% (v/v) were also evaluated when screening normal-phase conditions on the polysaccharide and Pirkle-type CSPs. The performance of -CDs was evaluated in reverse-phase mode using acetonitrile-buffer and methanol-buffer mobile phases. Zhong et al. [12] report that aqueous buffers prepared with triethylamine-acetic acid (TEAA) systems are favored in reverse-phase separations when using cyclodextrin based CSPs. In our experience, aqueous buffers prepared with TEAA produce
significant baseline noise on the UV detector. Issues with sample purity resulted in the desire to confirm peak identity using mass spectrometry as a secondary detection system, thus leading to the choice of ammonium acetate to create a buffer system which is compatible with the mass detector. This buffer was prepared at pH 4.1, 5.0, and neutral pH. The 50 mM concentration of ammonium acetate was selected to ensure the buffer was of sufficient ionic strength while still allowing for mass detection with an electrospray ion source. Polar-organic mode separations providing an alternative to reverse-phase separations with cyclodextrin based CSPs have been described by Chen et al. [13]. The polar-organic mode separations on the -CD columns utilized a pre-mixed mobile phase containing 95% acetonitrile and 5% methanol. This mixture was further modified with TEA and HOAc at 0.2% (v/v) and 0.3% (v/v) respectively. Further optimization of the mobile phase was made upon evidence of chiral recognition. A flow rate of 1.0 ml min−1 was used for all chromatographic systems. The retention factor (k) for bands representing the racemates was determined as k = (tR − t0 )/t0 , where tR and t0 were the retention times of retained and unretained compounds, respectively. In these studies, t0 was estimated based on void marker. The selectivity was calculated as ˛ = k2 /k1 . 3. Results and discussion The results of the trial separations are summarized in Table 1. The analytes were chromatographed and detected without precolumn or post-column derivatization. The degree of resolution describing the best results obtained for each chromatographic mode in which a column was evaluated is presented. 3.1. Polysaccharide based CSPs Literature reports state that upwards of 80% of racemic compounds may be resolved analytically on polysaccharide based CSPs [14]. Therefore the polysaccharide based CSPs, particularly the tris-(3,5-dimethylphenylcarbamate) of cellulose (Chiralcel OD) and amylose (Chiralpak AD), are generally our first choice when beginning development of a new chiral HPLC method. In this case, they were very effective at resolving the enantiomers of 4 out of the 5 analytes described here. Figs. 2 and 3 demonstrate the effectiveness of OD and AD at producing baseline separation of the enantiomers of compounds 2–5. As shown in Table 1, the Chiralcel OJ and Chiralpak AS CSPs were far less successful in providing chiral recognition of the analytes tested. The ester linked cellulose tris-(4-methyl benzoate) CSP (Chiralcel OJ) performed better than the tris-(S)-1-methylphenylcarbamate derivative of amylose (Chiralpak AS). The least successful polysaccharide based CSP screened was the Phenomenex Lux Cellulose-2, a tris-(3)chloro-4-methylphenyl carbamate of cellulose. First described by Chankvetadze et al. [15] in 1994, this CSP was capable of producing an enantiomeric separation of only one compound 2.
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Table 1 Summary of enantioresolution of compounds 1–5 with various CSPs and mobile phase conditions. Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Polysaccharide Chiralcel OD Chiralpak AD Chiralcel OJ Chiralpak AS LUX Cellulose-2
k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 = 2.6c , ˛ = 1.2 k1 = 1.8g , ˛ = 1.1 k1 = 1.2g , ˛ = 1.1 k1 > 0.5c , ˛ = 1.0 k1 = 1.3i , ˛ = 2.2
k1 = 1.3d , ˛ = 1.6 k1 = 2.9h , ˛ = 1.5 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0
k1 = 2.8e , ˛ = 1.5 k1 = 0.6h , ˛ = 1.4 k1 = 1.3f , ˛ = 1.9 k1 > 0.5a , ˛ = 1.0 k1 = 1.1i , ˛ = 1.1
k1 = 4.0f , ˛ = 1.4 k1 = 1.7f , ˛ = 1.3 k1 = 3.9f , ˛ = 1.4 k1 = 1.5f , ˛ = 1.2 k1 > 0.5a , ˛ = 1.0
Pirkle-type Regis WHELK-O 1 Regis ULMO Regis PIRKLE 1-J Regis LEUCINE
k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 = 2.6e , ˛ = 1.3 k1 > 0.5i , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0
k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0
k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0
k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0
Cyclobond I 2000 Reverse-phase Polar-organic Normal-phase
k1 > 0.5j , b , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 N/A
k1 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 0.85l , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 2.3k , ˛ = 1.2 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 1.3k , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 N/A
Cyclobond I 2000 DMP Reverse-phase Polar-organic Normal-phase
k1 > 0.5j , b , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 > 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 4.0j , ˛ = 1 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
k1 = 2.5n , ˛ = 1.2 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 3.8o , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
Cyclobond I 2000 DNP Reverse-phase Polar-organic Normal-phase
k1 = 3.1j , ˛ = 1.3 k1 < 0.5m , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 > 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 0.98n , ˛ = 1.9 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , b , ˛ = 1.0
k1 = 1.0p , ˛ = 1.4 k1 < 0.5q , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0
k1 = 0.95p , ˛ = 1.5 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
Note: Mobile phases a and c–i all contain TEA at 0.2% (v/v). Font legend for separation of racemate at indicated ˛ value: bold = full baseline resolution; italic = partial resolution; normal = no chiral recognition. Best results obtained during screening campaign have been shown. N/A = not applicable. a Mobile phase: 80/20 Hex/IPA. b Minimum polar-organic solvent needed for compound solubility. c Mobile phase: 99.5/0.5 HX/IPA. d Mobile phase: 80/20 Hex/EtOH. e Mobile phase; 95/5 Hex/IPA. f Mobile phase: 90/10 Hex/EtOH. g Mobile phase: 98/2 Hex/EtOH. h Mobile phase: 85/15 Hex/EtOH. i Mobile phase: 90/10 Hex/IPA. j Mobile phase: 65/35 pH 7 NH4 OAc buffer/ACN. k Mobile phase: 65/35 pH 7 NH4 OAc buffer/MeOH. l Mobile phase: 50/50 pH 4.1 NH4 OAc buffer/MeOH. m Mobile phase: 95/5 ACN/MeOH with (v/v) 0.2% TEA and 0.3% HOAc. n Mobile phase: 55/45 pH 5 NH4 OAc buffer/MeOH. o Mobile phase: 65/35 pH 5 NH4 OAc buffer/MeOH. p Mobile phase: 65/35 pH 5 NH4 OAc buffer/ACN.
3.2. Pirkle-type CSPs Pirkle-type CSPs, primarily Regis WHELK-O 1, are often our next choice when screening for HPLC methods to resolve enantiomeric pairs. Pirkle-type CSPs generally perform best when the solute is aromatic in nature, thus providing the possibility of donor/acceptor interactions leading to formation of diastereomeric complexes having different binding energies [16,17]. The compounds in our series meet this condition and these CSPs were screened extensively. The four Pirkle-type chiral stationary phases examined were 1-(3,5-dinitrobenzamido)1,2,3,4,-tetrahydrophenanthrene based Regis WHELK-O 1, a 3,5-dinitrobenzyol derivative of diphenylethylenediamine (Regis ULMO), a 3-(3,5-dinitrobenzamido)-4-phenyl--lactam (Regis PIRKLE 1-J), and a 3,5-dinitrobenzoyl derivative of leucine (Regis LEUCINE). As can be seen in Table 1, the Pirkle-type CSPs were almost completely ineffective in chiral recognition of the study compounds. Only compound 2, 1,5-methano-3-methyl-6-oxo-
1,2,3,4,5,6-hexahydro-3-benzazocine, was shown to be optically resolved on the Pirkle-type phases investigated. 3.3. Cyclodextrin CSPs The primary mode of chiral interaction with cyclodextrin based CSPs is thought to involve formation of an inclusion complex wherein a portion of the solute is accommodated inside the cavity of the cyclodextrin. Also, it is believed that these phases perform best when the enantiomeric pairs contain chiral centers juxtaposed between systems [18]. While the use of cyclodextrin based CSP’s is often cited in enantiomeric separations employing capillary electrophoresis, we still make it a practice to screen for HPLC separations using cyclodextrin based columns. Derivatized cyclodextrins can provide modes of interaction that do not require the formation of an inclusion complex between the solute and stationary phase and have been shown to exhibit optical resolving power in both polar-organic and normal-phase modes of
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Fig. 2. Enantioseparation of compounds 2–5 on Chiralcel OD. Mobile phase conditions: compound 2, 99.5/0.5 Hex/IPA with 0.2% TEA (v/v); 3 80/20 Hex/EtOH with 0.2% TEA (v/v); 4 95/5 Hex/IPA with 0.1% TEA (v/v); 5 90/10 Hex/EtOH with 0.1% TEA (v/v).
Fig. 3. Enantioseparation of compounds 2–5 on Chiralpak AD. Mobile phase conditions: compound 2, 98/2 Hex/EtOH with 0.2% TEA (v/v); 3 85/15 Hex/EtOH with 0.2% TEA (v/v); 4 85/15 Hex/EtOH with 0.2% TEA (v/v); 5 90/10 Hex/IPA with 0.2% TEA (v/v).
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Fig. 4. Enantioseparation of 2,6-methanobenzazocines 3–5 on Cyclobond I 2000. Mobile phase conditions: compound 3, 50/50 pH 4.1 NH4 OAcbuffer/MeOH; 4 and 5, 65/35 pH 7 NH4 OAcbuffer/MeOH.
chromatography [12,19,20]. The utility of preparative chromatography to produce enantiomerically pure material is frequently a foremost consideration when we set out to develop chromatographic methods for chiral molecules and since the majority of chiral separations described using cyclodextrin based CSPs occur in reverse-phase, our laboratories generally examine cyclodextrin phases after the polysaccharide and Pirkle-type phases have been evaluated. The native -CD column first screened, Cyclobond I 2000, did not produce chiral separation of 1,5-methano-3-benzazocines (1, 2) in any chromatographic mode. However, the 2,6 methano-3benzazocines (3–5) all demonstrated chiral interaction with this CSP under reverse-phase conditions (Fig. 4). Similarly when the 3,5dimethylphenyl carbamate derivative of -CD (Cyclobond I 2000 DMP) was utilized, chiral recognition of analytes occurred only in reverse-phase conditions (compounds 3–5). The polar-organic and normal-phase modes screened on the Cyclobond I 2000 DMP column did not produce enantiomeric separation of any compounds. Use of Cyclobond I 2000 DNP, a 3-trifluromethyl-1,5-dinitrophenyl derived -CD, under reverse-phase conditions yielded the best results, exhibiting chiral resolution with 4 out of the 5 compounds (1, 3–5) (Fig. 5). As observed with the other -CD CSPs investigated, use of the polar-organic or normal-phase modes did not resolve any of the mixtures. Interestingly, none of the -CD CSPs were able to resolve compound 2. This finding supports the assertion that resolution of enantiomeric mixtures on these CSPs is most probable when the chiral center is juxtaposed between two aromatic moieties.
Fig. 5. Separation of enantiomers of compounds 1, 3, 4, and 5 on Cyclobond I 2000 DNP. Peak identities are confirmed by mass spectrometry. Mobile phase conditions: 1, 65/35 pH 7 NH4 OAcbuffer/ACN; 2, 65/35 pH 7 NH4 OAcbuffer/MeOH; 3, 55/45 pH 5 NH4 OAcbuffer/MeOH; 4 and 5, 65/35 pH 5 NH4 OAcbuffer/ACN.
3.3.1. Evaluation of ˇ-CD interactions: influence of pH upon reverse-phase separations Concentration and the ionic strength of the buffer are known to have significant influence upon retention and separation of chiral analytes tested on cyclodextrin CSPs [14]. The charge state of analyte may also effect its affinity for the hydrophobic cavity of a neutral cyclodextrin and alter the equilibrium constant for inclusion into the cyclodextrin cavity [21]. The pH of the mobile phase buffer was considered when evaluating reverse-phase separations with these CSPs because of its effect on the polarity and charge of solutes. Variation of the buffer concentration to levels above 50 mM was not considered because of issues this might cause with the inlet of the mass detector. While preparing sample solutions it was observed that, as neutral species, these solutes exhibited poor aqueous solubility. For compounds incorporating amine functionalities, solubility in 50/50 water/acetonitrile was markedly improved upon the addition of a small quantity of glacial acetic acid. For a few compounds issues with analyte solubility appeared to produce deleterious results, particularly in the case of methanol as the organic modifier of the mobile phase. In these cases poor peak efficiency and reduced UV detection were observed. As the pH of the buffer component used to prepare the mobile phase is lowered from ∼7 (neutral buffer) to 4.1, the compounds with an amine function change from predominately neutral to ionized species and become less hydrophobic. This change produced significant chromatographic changes in the retention, peak shape and enantioseparation
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Fig. 6. (Left) Retention and chiral resolution of compound 5 decreases as pH is lowered. Mobile phase conditions A, 65/35 pH 7 NH4 OAcbuffer/MeOH; B, 65/35 pH 5 NH4 OAcbuffer/MeOH; C, 65/35 pH 4.1NH4 OAcbuffer/MeOH. (Right) compound 3, a neutral analyte is not significantly influenced by mobile phase pH. Mobile phase conditions A, 50/50 pH 7 NH4 OAcbuffer/MeOH; B, 50/50 pH 5 NH4 OAcbuffer/MeOH; C, 50/50 pH 4.1 NH4 OAcbuffer/MeOH.
for ionizable compounds in both the 1,5-methano-3-benzazocine and 2,6-methano-3-benzazocine series on each type of -CD. An illustration of the influence that buffer pH and the resulting charge state of the solute has upon the interactions between the -CD CSP and an ionizable compound 5 in comparison to the chromatographs of a non-ionizable compound 3 is shown in Fig. 6. As expected, compound 3, a neutral molecule whose charge, polarity, or solubility should not be altered by changes in mobile phase pH, is observed to retain relatively constant peak shape, retention factor, and enantiomeric resolution factor. Identification of optimized mobile phase conditions was challenged by the need to balance the solubility of ionizable compounds in our series and their interaction with the cyclodextrin cavity for a given charge state brought about by pH of the mobile phase. 3.3.2. Evaluation of ˇ-CD interactions: influence of organic solvent selection in reverse-phase conditions The polarity of solvents used to prepare the mobile phase may change the enantioselectivity of cyclodextrins by altering interactions (e.g., non-polar, electrostatic, and dipolar) between the cyclodextrin and its guest [21]. Additionally, organic modifiers are known to compete with solutes for residence in the cyclodextrin cavity and therefore interfere in the formation of an inclusion complex [22]. Change of the organic modifier from methanol to acetonitrile sometimes produced dramatic effects upon the enantioseparation of solutes by the -CD phases, while in other cases enantioseparation was maintained. The interaction between compound 4 and the native -CD changes dramatically when the organic content of the mobile phase is altered from 35% MeOH to 35% ACN. Under the methanol condition compound 4 is well retained and resolved. Replacement of methanol with acetonitrile leads to a decrease in retention of the solutes and resolution of the enantiomers collapses. These effects are also observed on the derivatized -CD CSPs 4. Conclusions We had hoped to identify generic chromatographic conditions to support any further medicinal chemistry efforts in this series, however none of the columns surveyed was able to achieve enantio-separation for all five analytes. Columns with a carbohydrate backbone performed better than silica based CSPs containing
chiral appendages. The polysaccharide Chiralcel OD and Chiralpak AD columns baseline separated 4 analytes (compounds 2–5) and 3 analytes (compounds 3–5), respectively. The modified -CD based Cyclobond I 2000 DNP column also provided separation for 4 of the analytes and was the only column tested demonstrating chiral recognition of compound 1. This emphasizes the necessity for the formation of an inclusion complex to achieve baseline separation for this molecule. Interestingly, compound 2 was not resolved by any -CD CSP, but separation was observed on 4 out of 5 polysaccharide columns examined. In grouping the analogous phases and reviewing the performance of each under equivalent chromatographic conditions we find support for the notion that no single chiral stationary phase will resolve every solute and that even minor changes within a chemical series can dramatically alter the chromatographic conditions necessary for optical resolution of enantiomers. References [1] [2] [3] [4] [5] [6] [7]
[8] [9] [10] [11]
A.A. Patchett, R.P. Nargund, Ann. Rep. Med. Chem. 35 (2000) 289. D.A. Horton, G.T. Bourne, M.L. Smythe, Chem. Rev. 103 (2003) 893. L.A. Walter, W.K. Chang, R.I. Taber, J. Med. Chem. 14 (10) (1971) 1011. F.B. Block, F.H. Clarke Jr., J. Med. Chem. 12 (5) (1969) 845. T. Kometani, S. Shiotani, K. Mitsuhashi, Chem. Pharm. Bull. 24 (3) (1976) 541. J. Boivin, J. Pothier, S.Z. Zard, Tetrahedron Lett. 40 (19) (1999) 3701. C.G. Bashore, M.G. Vetelino, M.C. Wirtz, P.R. Brooks, H.N. Frost, R.E. McDermott, D.C. Whritenour, J.A. Ragan, J.L. Rutherford, T.W. Makowski, S.J. Brenek, J.W. Coe, Org. Lett. 8 (26) (2006) 5947. N. Yokoyama, P.I. Almaula, F.B. Block, F.R. Granat, N. Gottfried, R.T. Hill, E.H. McMahon, W.F. Munch, H. Rachlin, et al., J. Med. Chem. 22 (5) (1979) 537. A.M. Krstulovic, J. Chromatogr.: Biomed. Appl. 488 (1) (1989) 53. M.C. Nunez, M.E. Garcia-Rubino, A. Conejo-Garcia, O. Curz-Lopez, M. Kimatrai, M.A. Gallo, A. Espinosa, J.M. Campos, Curr. Med. Chem. 16 (16) (2009) 2064. NMR data (400 MHz): compound 1: 1 H NMR (DMSO), ı 1.81 (dd, J = 12 Hz and 1 Hz, 1H), 1.88 (m, 1H), 1.91 (s, 3H), 2.01 (m, 2H), 2.23 (dd, J = 12 Hz and 1 Hz, 1H), 2.29 (d, J = 12, 1H), 2.74 (d, J = 12 Hz, 1H), 2.95 (br s, 1H), 4.35 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 7.03 (m, 3H), 7.22 (m, 2H), 7.29 (br s, 4H). Compound 2: 1 H NMR (DMSO), ı 1.87 (dt, J = 13 Hz and 3 Hz, 1H), 1.99 (s, 3H), 2.21 (d, J = 13 Hz, 1H), 2.27 (dd, J = 12 Hz and 3 Hz, 1H), 2.35 (dd, J = 12 Hz and 3 Hz, 1H), 2.57 (m, 1H), 2.67 (d, J = 12 Hz, 1H), 2.94 (d, J = 12 Hz, 1H), 3.15 (m, 1H), 7.36 (m, 2H), 7.53 (dt, J = 8 Hz and 2 Hz, 1H), 7.80 (m, J = 8 Hz and 2 Hz, 1H). Compound 3: 1 H NMR (DMSO), ı 1.65 (dd, J = 13 Hz and 3 Hz, 1H), 1.83 (dd, J = 12 Hz and 1 Hz, 1H), 2.12 (dt, J = 13 Hz and 5 Hz, 1H), 2.29 (d, J = 13 Hz, 1H), 2.45 (m, 1H), 2.64 (d, J = 17 Hz, 1H), 3.10 (dd, J = 17 Hz and 5 Hz, 1H), 3.78 (br s, 1H), 4.50 (br s, 1H), 5.86 (d, J = 2 Hz, 1H), 5.96 (s, 2H), 6.50 (dd, J = 8 Hz and 3 Hz, 1H), 6.91 (d, J = 8 Hz, 1H), 7.25 (m, 1H), 7.36 (m, 4H), 8.91 (s, 1H). Compound 4: 1 H NMR (DMSO), ı 1.74 (m, 2H), 2.04 (m, 2H), 2.23 (m, 2H), 2.31 (s, 3H), 2.56 (m, 1H), 2.70 (dd, J = 17 Hz and 6 Hz, 1H), 3.03 (d, J = 18 Hz, 1H), 5.79 (d, J = 3 Hz, 1H), 6.47 (dd, J = 8 Hz and 3 Hz, 1H), 6.92 (d, J = 8 Hz, 1H), 7.26 (m, 1H), 7.34 (m, 4H), 8.83 (br s, 1H). Compound 5: 1 H NMR (DMSO), ␦ 2.03 (d, J = 13 Hz, 1H), 2.13 (d,
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[12] [13] [14] [15] [16]
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J = 13 Hz, 1H), 2.50 (m, 2H), 2.87 (dt, J = 13 Hz and 2 Hz, 1H), 3.07 (d, J = 18 Hz, 1H), 3.35 (m, 1H), 3.49 (dd, J = 18 Hz and 8 Hz, 1H), 4.04 (m, 1H), 5.98 (d, J = 2 Hz, 1H), 6.64 (dd, J = 8 Hz and 2 Hz, 1H), 7.05 (d, J = 8 Hz, 1H), 7.29 (m, 1H), 7.38 (m, 4H). Q. Zhong, L. He, T.E. Beesley, W.S. Trahanovsky, P. Sun, C. Wang, D.W. Armstrong, J. Chromatogr. A 1115 (2006) 19. S.C. Chang, G.L. Reid, S. Chen, C.D. Chang, D.W. Armstrong, Trends Anal. Chem. 12 (1993) 144. H.Y. Aboul-Enein, I. Ali, Chiral Separations by Liquid Chromatography and Related Technologies, CRC Press, Inc., 2003. B. Chankvetadze, E. Yashima, Y. Okamoto, J. Chromatogr. A 670 (1994) 39. C.J. Welch, J. Chromatogr. A 666 (1994) 3.
[17] M.H. Hyun, Y.J. Cho, in: G. Gübitz, M.G. Schmid (Eds.), Chiral Separations: Methods and Protocols, Humana Press, Totowa, 2004, p. 197. [18] A.M. Stalcup, in: G. Subramanian (Ed.), A Practical Approach to Chiral Separations by Liquid Chromatography, Weinheim, NewYork, 1994, p. 95. [19] T. Hargitai, Y. Okamoto, J. Liquid Chromatogr. 16 (4) (1993) 843. [20] D.W. Armstrong, A.M. Stalcup, M.L. Hilton, J. Duncan, J.R. Faulkner Jr., S. Chang, Anal. Chem. 62 (1990) 1610. [21] Anon, Chromatographia 54 (Suppl.) (2001) S59. [22] R.A. Menges, D.W. Armstrong, in: S. Ahuga (Ed.), Chiral Separation by Liquid Chromatography, ACS Symposium Series 471, American Chemical Society, Washington, 1991, p. 67.
Contents lists available at ScienceDirect
Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma
High-performance liquid chromatographic enantioseparation of methanobenzazocines William M. Barker ∗ , Karin Worm, Roland E. Dolle Department of Chemistry, Adolor Corporation, 700 Pennsylvania Drive, Exton, PA 19341, USA
a r t i c l e
i n f o
Article history: Received 3 April 2009 Received in revised form 6 August 2009 Accepted 12 August 2009 Available online 18 August 2009 Keywords: HPLC Chiral separation Enantioselective Benzazocine Benzomorphans Racemic Cyclodextrin Polysaccharide and Pirkle-type chiral stationary phases
a b s t r a c t Chiral recognition and resolution of methanobenzazocines was investigated by HPLC using polysaccharide, Pirkle-type, native and derivatized -cyclodextrin chiral stationary phases. Enantioseparation of phenyl substituted 2,6-methanobenzazocines was achieved with multiple chiral stationary phases throughout the classes described. Chiral resolution of the enantiomers of 1,5-methano-3-methyl-6-oxo1,2,3,4,5,6-hexahydro-3-benzazocine was produced on both polysaccharide and Pirkle-type phases. In the case of 1,5-methano-3-methyl-6-phenyl-1,2,3,4,5,6-hexahydro-3-benzazocine only a dinitrophenyl substituted -cyclodextrin produced a separation of enantiomers. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Benzo-fused heterocyclic ring systems have received a lot of attention over the years because of their ubiquitous appearance in natural products and modern pharmaceuticals [1,2]. Benzazocines are a class of benzo-fused nitrogen-containing eight-membered heterocyclic rings and include the 1-benzazocine, 2-benzazocine and 3-benzazocine skeletons. 1,5-Methano-3benzazocine 1 [3] and 2,6-methanobenzazocines 3–5 [4] (also called benzomorphans) shown in Fig. 1, were reported to have analgesic activity but to be devoid of physical dependence liability, the most serious side effect of morphine like drugs. Several synthetic procedures for compound 1, its precursor 2 and compounds 3–5, both racemic and enantioselective, have been reported [5–8]. For compounds 4 and 5 a method for optical resolution via d- and l-mandelic acid salts has been described [8]. Enantiomers of pharmaceutically active agents are often shown to display key differences in activity and efficacy [9,10]. Investigation of the pharmacological differences between the enantiomers of these compounds required that we develop chromatographic methods capable of discriminating each form. To the best of our
∗ Corresponding author. Tel.: +1 484 712 5767. E-mail address: [email protected] (W.M. Barker). 0021-9673/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2009.08.033
knowledge the work presented here is the first study systematically investigating the chiral separation of methanobenzazocines 1–5 by high-performance liquid chromatography (HPLC) on a number of chiral stationary phases (CSPs).
2. Experimental 2.1. Materials Racemic methanobenzazocines were prepared according to the methods described in the literature [3,4,11]. The relative stereochemistry accessible via the synthetic route employed only yielded one enantiomeric pair for each compound described. Samples were prepared in 50/50 water/acetonitrile with glacial acetic acid added at 1% (v/v) for use in reverse-phase chromatography. Ethanol was used as the sample diluent for chromatographic screening in the polar-organic and normal-phase modes. All solvents used as diluents and mobile phases were HPLC grade. Hexanes (Hex), 2-propanol (IPA), ethanol (EtOH), methanol (MeOH), acetonitrile (ACN), water, and d6 -DMSO were purchased from Sigma–Aldrich (St. Louis, MO, USA), Fisher Scientific (Pittsburgh, PA, USA), and Pharmco (Brookfield, CT, USA). The reagents used to buffer, acidify, or basify the solutions were ammonium acetate (NH4 OAc), glacial acetic acid (HOAc), and triethylamine (TEA), respectively. These were of reagent grade, ACS or spectroscopic grade and were pur-
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Fig. 1. Benzazocine nomenclature and compounds investigated.
chased from Sigma–Aldrich, Fisher Scientific, and EMD (Gibbstown, USA). Aqueous ammonium acetate buffers were prepared at 50 mM concentration and the adjustment of pH was performed by titration with acetic acid while monitoring pH with a calibrated pH meter. All aqueous buffers were filtered before use with a Phenomenex (Torrance, CA, USA) filter apparatus and Phenomenex nylon and PTFE filters of 0.45 pore size. Mobile phase mixtures for the normal-phase, polar-organic, and reverse-phase HPLC chromatography were mixed at use on the HPLC system. Columns packed with the CSPs evaluated were of 250 mm × 4.6 mm in dimension. The polysaccharide based CSPs were the cellulose and amylose derived phases from Chiral Technologies (Daicel, Tokyo, Japan) and Phenomenex. These included the Chiralcel OD, Chiralcel OJ, Chiralpak AD, Chiralpak AS, and the LUX Cellulose-2 trademarked names. The Pirkle-type phase CSPs were Regis WHELK-0 1 (S,S), ULMO (S,S), PIRKLE 1-J (3S,4R) and LUECINE, from Regis Technologies (Morton Grove, IL, USA). The -cyclodextrin (-CD) based phases employed were Cyclobond I 2000, Cyclobond 2000 DMP, and Cyclobond I 2000 DNP from Sigma–Aldrich. 2.2. Equipment NMR spectroscopy was performed using a Bruker Avance 400 controlled with the Bruker TopSpin software package (Bruker BioSpin Corp., Billerica, MA, USA). Normal-phase HPLC chromatography was carried out on a Waters system (Waters Corp., Milford, MA, USA) composed of an Waters Alliance 2695 pump and injection module, a Waters 996 photo-diode array detector and Waters Millennium chromatography software. Reverse-phase and polarorganic phase chromatographic separations were performed on a Waters system composed of an Waters Alliance 2795 pump and injection module, a Waters 996 photo-diode array detector, a Micromass ZQ2000 mass spectrometer and Waters MassLynx chromatography software (Waters Corp., Milford, MA, USA). UV detection with photo-diode array detectors was carried out scanning from 200 to 300 nm. For reverse-phase and polar-organic chromatographic separations the chromatograms were plotted at 225 nm and for normal-phase chromatography 280 nm was selected. 2.3. Column evaluation All CSPs, except for the native -CD, were evaluated in the normal-phase mode (Hex-EtOH or Hex-IPA). Mobile phase mixtures containing TEA at 0.2% (v/v) were also evaluated when screening normal-phase conditions on the polysaccharide and Pirkle-type CSPs. The performance of -CDs was evaluated in reverse-phase mode using acetonitrile-buffer and methanol-buffer mobile phases. Zhong et al. [12] report that aqueous buffers prepared with triethylamine-acetic acid (TEAA) systems are favored in reverse-phase separations when using cyclodextrin based CSPs. In our experience, aqueous buffers prepared with TEAA produce
significant baseline noise on the UV detector. Issues with sample purity resulted in the desire to confirm peak identity using mass spectrometry as a secondary detection system, thus leading to the choice of ammonium acetate to create a buffer system which is compatible with the mass detector. This buffer was prepared at pH 4.1, 5.0, and neutral pH. The 50 mM concentration of ammonium acetate was selected to ensure the buffer was of sufficient ionic strength while still allowing for mass detection with an electrospray ion source. Polar-organic mode separations providing an alternative to reverse-phase separations with cyclodextrin based CSPs have been described by Chen et al. [13]. The polar-organic mode separations on the -CD columns utilized a pre-mixed mobile phase containing 95% acetonitrile and 5% methanol. This mixture was further modified with TEA and HOAc at 0.2% (v/v) and 0.3% (v/v) respectively. Further optimization of the mobile phase was made upon evidence of chiral recognition. A flow rate of 1.0 ml min−1 was used for all chromatographic systems. The retention factor (k) for bands representing the racemates was determined as k = (tR − t0 )/t0 , where tR and t0 were the retention times of retained and unretained compounds, respectively. In these studies, t0 was estimated based on void marker. The selectivity was calculated as ˛ = k2 /k1 . 3. Results and discussion The results of the trial separations are summarized in Table 1. The analytes were chromatographed and detected without precolumn or post-column derivatization. The degree of resolution describing the best results obtained for each chromatographic mode in which a column was evaluated is presented. 3.1. Polysaccharide based CSPs Literature reports state that upwards of 80% of racemic compounds may be resolved analytically on polysaccharide based CSPs [14]. Therefore the polysaccharide based CSPs, particularly the tris-(3,5-dimethylphenylcarbamate) of cellulose (Chiralcel OD) and amylose (Chiralpak AD), are generally our first choice when beginning development of a new chiral HPLC method. In this case, they were very effective at resolving the enantiomers of 4 out of the 5 analytes described here. Figs. 2 and 3 demonstrate the effectiveness of OD and AD at producing baseline separation of the enantiomers of compounds 2–5. As shown in Table 1, the Chiralcel OJ and Chiralpak AS CSPs were far less successful in providing chiral recognition of the analytes tested. The ester linked cellulose tris-(4-methyl benzoate) CSP (Chiralcel OJ) performed better than the tris-(S)-1-methylphenylcarbamate derivative of amylose (Chiralpak AS). The least successful polysaccharide based CSP screened was the Phenomenex Lux Cellulose-2, a tris-(3)chloro-4-methylphenyl carbamate of cellulose. First described by Chankvetadze et al. [15] in 1994, this CSP was capable of producing an enantiomeric separation of only one compound 2.
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Table 1 Summary of enantioresolution of compounds 1–5 with various CSPs and mobile phase conditions. Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Polysaccharide Chiralcel OD Chiralpak AD Chiralcel OJ Chiralpak AS LUX Cellulose-2
k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 = 2.6c , ˛ = 1.2 k1 = 1.8g , ˛ = 1.1 k1 = 1.2g , ˛ = 1.1 k1 > 0.5c , ˛ = 1.0 k1 = 1.3i , ˛ = 2.2
k1 = 1.3d , ˛ = 1.6 k1 = 2.9h , ˛ = 1.5 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0
k1 = 2.8e , ˛ = 1.5 k1 = 0.6h , ˛ = 1.4 k1 = 1.3f , ˛ = 1.9 k1 > 0.5a , ˛ = 1.0 k1 = 1.1i , ˛ = 1.1
k1 = 4.0f , ˛ = 1.4 k1 = 1.7f , ˛ = 1.3 k1 = 3.9f , ˛ = 1.4 k1 = 1.5f , ˛ = 1.2 k1 > 0.5a , ˛ = 1.0
Pirkle-type Regis WHELK-O 1 Regis ULMO Regis PIRKLE 1-J Regis LEUCINE
k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 = 2.6e , ˛ = 1.3 k1 > 0.5i , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0
k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0 k1 > 0.5d , ˛ = 1.0
k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0
k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0 k1 > 0.5a , ˛ = 1.0
Cyclobond I 2000 Reverse-phase Polar-organic Normal-phase
k1 > 0.5j , b , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 N/A
k1 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 0.85l , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 2.3k , ˛ = 1.2 k1 < 0.5m , ˛ = 1.0 N/A
k1 = 1.3k , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 N/A
Cyclobond I 2000 DMP Reverse-phase Polar-organic Normal-phase
k1 > 0.5j , b , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 > 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 4.0j , ˛ = 1 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
k1 = 2.5n , ˛ = 1.2 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 3.8o , ˛ = 1.4 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
Cyclobond I 2000 DNP Reverse-phase Polar-organic Normal-phase
k1 = 3.1j , ˛ = 1.3 k1 < 0.5m , ˛ = 1.0 k1 < 0.5a , b , ˛ = 1.0
k1 > 0.5k , ˛ = 1.0 k1 < 0.5m , ˛ = 1.0 k1 < 0.5e , ˛ = 1.0
k1 = 0.98n , ˛ = 1.9 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , b , ˛ = 1.0
k1 = 1.0p , ˛ = 1.4 k1 < 0.5q , ˛ = 1.0 k1 > 0.5i , ˛ = 1.0
k1 = 0.95p , ˛ = 1.5 k1 < 0.5m , ˛ = 1.0 k1 < 0.5i , ˛ = 1.0
Note: Mobile phases a and c–i all contain TEA at 0.2% (v/v). Font legend for separation of racemate at indicated ˛ value: bold = full baseline resolution; italic = partial resolution; normal = no chiral recognition. Best results obtained during screening campaign have been shown. N/A = not applicable. a Mobile phase: 80/20 Hex/IPA. b Minimum polar-organic solvent needed for compound solubility. c Mobile phase: 99.5/0.5 HX/IPA. d Mobile phase: 80/20 Hex/EtOH. e Mobile phase; 95/5 Hex/IPA. f Mobile phase: 90/10 Hex/EtOH. g Mobile phase: 98/2 Hex/EtOH. h Mobile phase: 85/15 Hex/EtOH. i Mobile phase: 90/10 Hex/IPA. j Mobile phase: 65/35 pH 7 NH4 OAc buffer/ACN. k Mobile phase: 65/35 pH 7 NH4 OAc buffer/MeOH. l Mobile phase: 50/50 pH 4.1 NH4 OAc buffer/MeOH. m Mobile phase: 95/5 ACN/MeOH with (v/v) 0.2% TEA and 0.3% HOAc. n Mobile phase: 55/45 pH 5 NH4 OAc buffer/MeOH. o Mobile phase: 65/35 pH 5 NH4 OAc buffer/MeOH. p Mobile phase: 65/35 pH 5 NH4 OAc buffer/ACN.
3.2. Pirkle-type CSPs Pirkle-type CSPs, primarily Regis WHELK-O 1, are often our next choice when screening for HPLC methods to resolve enantiomeric pairs. Pirkle-type CSPs generally perform best when the solute is aromatic in nature, thus providing the possibility of donor/acceptor interactions leading to formation of diastereomeric complexes having different binding energies [16,17]. The compounds in our series meet this condition and these CSPs were screened extensively. The four Pirkle-type chiral stationary phases examined were 1-(3,5-dinitrobenzamido)1,2,3,4,-tetrahydrophenanthrene based Regis WHELK-O 1, a 3,5-dinitrobenzyol derivative of diphenylethylenediamine (Regis ULMO), a 3-(3,5-dinitrobenzamido)-4-phenyl--lactam (Regis PIRKLE 1-J), and a 3,5-dinitrobenzoyl derivative of leucine (Regis LEUCINE). As can be seen in Table 1, the Pirkle-type CSPs were almost completely ineffective in chiral recognition of the study compounds. Only compound 2, 1,5-methano-3-methyl-6-oxo-
1,2,3,4,5,6-hexahydro-3-benzazocine, was shown to be optically resolved on the Pirkle-type phases investigated. 3.3. Cyclodextrin CSPs The primary mode of chiral interaction with cyclodextrin based CSPs is thought to involve formation of an inclusion complex wherein a portion of the solute is accommodated inside the cavity of the cyclodextrin. Also, it is believed that these phases perform best when the enantiomeric pairs contain chiral centers juxtaposed between systems [18]. While the use of cyclodextrin based CSP’s is often cited in enantiomeric separations employing capillary electrophoresis, we still make it a practice to screen for HPLC separations using cyclodextrin based columns. Derivatized cyclodextrins can provide modes of interaction that do not require the formation of an inclusion complex between the solute and stationary phase and have been shown to exhibit optical resolving power in both polar-organic and normal-phase modes of
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Fig. 2. Enantioseparation of compounds 2–5 on Chiralcel OD. Mobile phase conditions: compound 2, 99.5/0.5 Hex/IPA with 0.2% TEA (v/v); 3 80/20 Hex/EtOH with 0.2% TEA (v/v); 4 95/5 Hex/IPA with 0.1% TEA (v/v); 5 90/10 Hex/EtOH with 0.1% TEA (v/v).
Fig. 3. Enantioseparation of compounds 2–5 on Chiralpak AD. Mobile phase conditions: compound 2, 98/2 Hex/EtOH with 0.2% TEA (v/v); 3 85/15 Hex/EtOH with 0.2% TEA (v/v); 4 85/15 Hex/EtOH with 0.2% TEA (v/v); 5 90/10 Hex/IPA with 0.2% TEA (v/v).
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Fig. 4. Enantioseparation of 2,6-methanobenzazocines 3–5 on Cyclobond I 2000. Mobile phase conditions: compound 3, 50/50 pH 4.1 NH4 OAcbuffer/MeOH; 4 and 5, 65/35 pH 7 NH4 OAcbuffer/MeOH.
chromatography [12,19,20]. The utility of preparative chromatography to produce enantiomerically pure material is frequently a foremost consideration when we set out to develop chromatographic methods for chiral molecules and since the majority of chiral separations described using cyclodextrin based CSPs occur in reverse-phase, our laboratories generally examine cyclodextrin phases after the polysaccharide and Pirkle-type phases have been evaluated. The native -CD column first screened, Cyclobond I 2000, did not produce chiral separation of 1,5-methano-3-benzazocines (1, 2) in any chromatographic mode. However, the 2,6 methano-3benzazocines (3–5) all demonstrated chiral interaction with this CSP under reverse-phase conditions (Fig. 4). Similarly when the 3,5dimethylphenyl carbamate derivative of -CD (Cyclobond I 2000 DMP) was utilized, chiral recognition of analytes occurred only in reverse-phase conditions (compounds 3–5). The polar-organic and normal-phase modes screened on the Cyclobond I 2000 DMP column did not produce enantiomeric separation of any compounds. Use of Cyclobond I 2000 DNP, a 3-trifluromethyl-1,5-dinitrophenyl derived -CD, under reverse-phase conditions yielded the best results, exhibiting chiral resolution with 4 out of the 5 compounds (1, 3–5) (Fig. 5). As observed with the other -CD CSPs investigated, use of the polar-organic or normal-phase modes did not resolve any of the mixtures. Interestingly, none of the -CD CSPs were able to resolve compound 2. This finding supports the assertion that resolution of enantiomeric mixtures on these CSPs is most probable when the chiral center is juxtaposed between two aromatic moieties.
Fig. 5. Separation of enantiomers of compounds 1, 3, 4, and 5 on Cyclobond I 2000 DNP. Peak identities are confirmed by mass spectrometry. Mobile phase conditions: 1, 65/35 pH 7 NH4 OAcbuffer/ACN; 2, 65/35 pH 7 NH4 OAcbuffer/MeOH; 3, 55/45 pH 5 NH4 OAcbuffer/MeOH; 4 and 5, 65/35 pH 5 NH4 OAcbuffer/ACN.
3.3.1. Evaluation of ˇ-CD interactions: influence of pH upon reverse-phase separations Concentration and the ionic strength of the buffer are known to have significant influence upon retention and separation of chiral analytes tested on cyclodextrin CSPs [14]. The charge state of analyte may also effect its affinity for the hydrophobic cavity of a neutral cyclodextrin and alter the equilibrium constant for inclusion into the cyclodextrin cavity [21]. The pH of the mobile phase buffer was considered when evaluating reverse-phase separations with these CSPs because of its effect on the polarity and charge of solutes. Variation of the buffer concentration to levels above 50 mM was not considered because of issues this might cause with the inlet of the mass detector. While preparing sample solutions it was observed that, as neutral species, these solutes exhibited poor aqueous solubility. For compounds incorporating amine functionalities, solubility in 50/50 water/acetonitrile was markedly improved upon the addition of a small quantity of glacial acetic acid. For a few compounds issues with analyte solubility appeared to produce deleterious results, particularly in the case of methanol as the organic modifier of the mobile phase. In these cases poor peak efficiency and reduced UV detection were observed. As the pH of the buffer component used to prepare the mobile phase is lowered from ∼7 (neutral buffer) to 4.1, the compounds with an amine function change from predominately neutral to ionized species and become less hydrophobic. This change produced significant chromatographic changes in the retention, peak shape and enantioseparation
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Fig. 6. (Left) Retention and chiral resolution of compound 5 decreases as pH is lowered. Mobile phase conditions A, 65/35 pH 7 NH4 OAcbuffer/MeOH; B, 65/35 pH 5 NH4 OAcbuffer/MeOH; C, 65/35 pH 4.1NH4 OAcbuffer/MeOH. (Right) compound 3, a neutral analyte is not significantly influenced by mobile phase pH. Mobile phase conditions A, 50/50 pH 7 NH4 OAcbuffer/MeOH; B, 50/50 pH 5 NH4 OAcbuffer/MeOH; C, 50/50 pH 4.1 NH4 OAcbuffer/MeOH.
for ionizable compounds in both the 1,5-methano-3-benzazocine and 2,6-methano-3-benzazocine series on each type of -CD. An illustration of the influence that buffer pH and the resulting charge state of the solute has upon the interactions between the -CD CSP and an ionizable compound 5 in comparison to the chromatographs of a non-ionizable compound 3 is shown in Fig. 6. As expected, compound 3, a neutral molecule whose charge, polarity, or solubility should not be altered by changes in mobile phase pH, is observed to retain relatively constant peak shape, retention factor, and enantiomeric resolution factor. Identification of optimized mobile phase conditions was challenged by the need to balance the solubility of ionizable compounds in our series and their interaction with the cyclodextrin cavity for a given charge state brought about by pH of the mobile phase. 3.3.2. Evaluation of ˇ-CD interactions: influence of organic solvent selection in reverse-phase conditions The polarity of solvents used to prepare the mobile phase may change the enantioselectivity of cyclodextrins by altering interactions (e.g., non-polar, electrostatic, and dipolar) between the cyclodextrin and its guest [21]. Additionally, organic modifiers are known to compete with solutes for residence in the cyclodextrin cavity and therefore interfere in the formation of an inclusion complex [22]. Change of the organic modifier from methanol to acetonitrile sometimes produced dramatic effects upon the enantioseparation of solutes by the -CD phases, while in other cases enantioseparation was maintained. The interaction between compound 4 and the native -CD changes dramatically when the organic content of the mobile phase is altered from 35% MeOH to 35% ACN. Under the methanol condition compound 4 is well retained and resolved. Replacement of methanol with acetonitrile leads to a decrease in retention of the solutes and resolution of the enantiomers collapses. These effects are also observed on the derivatized -CD CSPs 4. Conclusions We had hoped to identify generic chromatographic conditions to support any further medicinal chemistry efforts in this series, however none of the columns surveyed was able to achieve enantio-separation for all five analytes. Columns with a carbohydrate backbone performed better than silica based CSPs containing
chiral appendages. The polysaccharide Chiralcel OD and Chiralpak AD columns baseline separated 4 analytes (compounds 2–5) and 3 analytes (compounds 3–5), respectively. The modified -CD based Cyclobond I 2000 DNP column also provided separation for 4 of the analytes and was the only column tested demonstrating chiral recognition of compound 1. This emphasizes the necessity for the formation of an inclusion complex to achieve baseline separation for this molecule. Interestingly, compound 2 was not resolved by any -CD CSP, but separation was observed on 4 out of 5 polysaccharide columns examined. In grouping the analogous phases and reviewing the performance of each under equivalent chromatographic conditions we find support for the notion that no single chiral stationary phase will resolve every solute and that even minor changes within a chemical series can dramatically alter the chromatographic conditions necessary for optical resolution of enantiomers. References [1] [2] [3] [4] [5] [6] [7]
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A.A. Patchett, R.P. Nargund, Ann. Rep. Med. Chem. 35 (2000) 289. D.A. Horton, G.T. Bourne, M.L. Smythe, Chem. Rev. 103 (2003) 893. L.A. Walter, W.K. Chang, R.I. Taber, J. Med. Chem. 14 (10) (1971) 1011. F.B. Block, F.H. Clarke Jr., J. Med. Chem. 12 (5) (1969) 845. T. Kometani, S. Shiotani, K. Mitsuhashi, Chem. Pharm. Bull. 24 (3) (1976) 541. J. Boivin, J. Pothier, S.Z. Zard, Tetrahedron Lett. 40 (19) (1999) 3701. C.G. Bashore, M.G. Vetelino, M.C. Wirtz, P.R. Brooks, H.N. Frost, R.E. McDermott, D.C. Whritenour, J.A. Ragan, J.L. Rutherford, T.W. Makowski, S.J. Brenek, J.W. Coe, Org. Lett. 8 (26) (2006) 5947. N. Yokoyama, P.I. Almaula, F.B. Block, F.R. Granat, N. Gottfried, R.T. Hill, E.H. McMahon, W.F. Munch, H. Rachlin, et al., J. Med. Chem. 22 (5) (1979) 537. A.M. Krstulovic, J. Chromatogr.: Biomed. Appl. 488 (1) (1989) 53. M.C. Nunez, M.E. Garcia-Rubino, A. Conejo-Garcia, O. Curz-Lopez, M. Kimatrai, M.A. Gallo, A. Espinosa, J.M. Campos, Curr. Med. Chem. 16 (16) (2009) 2064. NMR data (400 MHz): compound 1: 1 H NMR (DMSO), ı 1.81 (dd, J = 12 Hz and 1 Hz, 1H), 1.88 (m, 1H), 1.91 (s, 3H), 2.01 (m, 2H), 2.23 (dd, J = 12 Hz and 1 Hz, 1H), 2.29 (d, J = 12, 1H), 2.74 (d, J = 12 Hz, 1H), 2.95 (br s, 1H), 4.35 (d, J = 8 Hz, 1H), 6.67 (d, J = 8 Hz, 1H), 7.03 (m, 3H), 7.22 (m, 2H), 7.29 (br s, 4H). Compound 2: 1 H NMR (DMSO), ı 1.87 (dt, J = 13 Hz and 3 Hz, 1H), 1.99 (s, 3H), 2.21 (d, J = 13 Hz, 1H), 2.27 (dd, J = 12 Hz and 3 Hz, 1H), 2.35 (dd, J = 12 Hz and 3 Hz, 1H), 2.57 (m, 1H), 2.67 (d, J = 12 Hz, 1H), 2.94 (d, J = 12 Hz, 1H), 3.15 (m, 1H), 7.36 (m, 2H), 7.53 (dt, J = 8 Hz and 2 Hz, 1H), 7.80 (m, J = 8 Hz and 2 Hz, 1H). Compound 3: 1 H NMR (DMSO), ı 1.65 (dd, J = 13 Hz and 3 Hz, 1H), 1.83 (dd, J = 12 Hz and 1 Hz, 1H), 2.12 (dt, J = 13 Hz and 5 Hz, 1H), 2.29 (d, J = 13 Hz, 1H), 2.45 (m, 1H), 2.64 (d, J = 17 Hz, 1H), 3.10 (dd, J = 17 Hz and 5 Hz, 1H), 3.78 (br s, 1H), 4.50 (br s, 1H), 5.86 (d, J = 2 Hz, 1H), 5.96 (s, 2H), 6.50 (dd, J = 8 Hz and 3 Hz, 1H), 6.91 (d, J = 8 Hz, 1H), 7.25 (m, 1H), 7.36 (m, 4H), 8.91 (s, 1H). Compound 4: 1 H NMR (DMSO), ı 1.74 (m, 2H), 2.04 (m, 2H), 2.23 (m, 2H), 2.31 (s, 3H), 2.56 (m, 1H), 2.70 (dd, J = 17 Hz and 6 Hz, 1H), 3.03 (d, J = 18 Hz, 1H), 5.79 (d, J = 3 Hz, 1H), 6.47 (dd, J = 8 Hz and 3 Hz, 1H), 6.92 (d, J = 8 Hz, 1H), 7.26 (m, 1H), 7.34 (m, 4H), 8.83 (br s, 1H). Compound 5: 1 H NMR (DMSO), ␦ 2.03 (d, J = 13 Hz, 1H), 2.13 (d,
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