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Characterization of cyclodextrin complexes of camostat mesylate by ESI mass spectrometry and NMR spectroscopy
Journal of Molecular Structure 938 (2009) 192–197
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Characterization of cyclodextrin complexes of camostat mesylate by ESI mass spectrometry and NMR spectroscopy Soonho Kwon a,1, Woonhyoung Lee b,1, Hye-Jin Shin a, Sung-il Yoon c, Yun-tae Kim a, Young-Jin Kim a, Kyungruyl Lee a, Sanghoo Lee a,* a b c
Department of Bioanalytical & Mass Spectrometry, Seoul Medical Science Institute, Seoul 152-766, Republic of Korea Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea Department of Bioanalysis, Korean Clinical Research Center, Co., Ltd, Gyeonggi-do 431-836, Republic of Korea
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 9 September 2009 Accepted 16 September 2009 Available online 20 September 2009 Keywords: Camostat mesylate Cyclodextrin Noncovalent complex ESI mass NMR
a b s t r a c t Supramolecular interactions between camostat mesylate, a serine protease inhibitor (1), with a-, b-, and c-cyclodextrin (CD) in water were investigated using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. ESI mass spectral analysis revealed that the 1:1 stoichiometry in all the complexes was formed. The binding constants (Kst) calculated by linear equations constructed from the ESI mass spectra of all the complexes indicated that c-CD was most favorable complexing agent for the binding with 1 among the CDs. Pronounced changes in the 1H chemical shift upon complex formation with c-CD were observed for the protons of the two aromatic rings of 1, with much larger chemical shift changes observed for the protons of the guanidinyl group-linked aromatic ring of 1. These results suggest that the cavity of c-CD rather than that of a- or b-CD is large enough to accommodate the guanidine group of 1. Spatial geometry of 1 within the cavity of c-CD was further identified with two-dimensional rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) experiment. The observed ROESY cross peaks indicated intermolecular dipolar interactions between the two aromatic ring protons of 1 and the protons within the cavity of c-CD. Based on the 1:1 stoichiometry of the complex, ROESY cross peaks suggest that two types of 1:1 complexes of c-CD with 1 exist simultaneously in solution with different geometries. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Active synthetic serine protease inhibitors have been known to play important roles in many biological processes. N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)phenylacetate methanesulphonate (camostat mesylate, 1) (Fig. 1a) is a synthetic proteolytic enzyme inhibitor for trypsin, plasmin, kallikrein, tissue kallikrein, and thrombin [1–3]. The drug has also shown activity in the treatment of pancreatitis [4,5] and reflux esophagitis [6,7], establishing its safety for use in human. Especially, the compound 1 is known to have stronger trypsin inhibitory activity 100–1000 times than other trypsin inhibitors such as aprotinin and gabexate mesylate [8,9]. Metabolic works have also shown that the 1 is rapidly hydrolyzed to 4-(4-guanidinobenzoyloxy)phenylacetic acid
* Corresponding author. Address: Department of Bioanalytical & Mass Spectrometry, Seoul Medical Science Institute & Seoul Clinical Laboratories, #901-1, ACE Technotower V, 197-22, Guro 3-dong, Guro-gu, Seoul 152-766, Republic of Korea. Tel.: +82 70 7115 8697; fax: +82 2 858 2814. E-mail address: [email protected] (S. Lee). 1 These authors contributed equally to this work. 0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.09.025
and 4-guanidinobenzoic acid by esterases during intestinal absorption [10,11]. CDs are a family of cyclic oligosaccharides composed of a-(1,4) linked glucopyranose units. Among them, a-, b- and c-CD are most common cycloamyloses, which have six, seven, and eight glucose units, respectively. Their structures are shown in Fig. 1b. The CDs share a torus-like molecular shape with hydrophobic inner cavity and hydrophilic outer surface and are able to form complexes with various molecules containing apolar groups that can be included partially or completely into their hydrophobic cavities with an internal diameter from 4.7 to 8.3 Å [12,13]. Based on these dimensions, a-CD can typically complex low molecular weight molecules or compounds with aliphatic side chains, b-CD will complex aromatics and heterocycles and c-CD can accommodate larger molecules such as macrocycles and steroids [13,14]. Thus, CDs have been successfully used to control various physico-chemical properties of drugs such as solubility, dissolution, release rates, stability, and bioavailability [12,13,15,24]. Until now, the studies on the drug 1 have been mainly focused on its pharmaceutical activity but no works on noncovalent complexes between CDs and 1 in terms of structural analyses have been reported.
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O
(a) i
f
j
b c
NH
O
e
O
l
O
g
O
N k
h
a H2N
N H
d
(b) OH 4
6
O 5
2
HO 3
OH
1
n : α-CD : β-CD : γ-CD
n=6 n=7 n=8
Fig. 1. Numbered structures of (a) 1 and (b) CDs.
(a)
[β-CD+1+H]+ 1534
(b) [α-CD+H]+
[β-CD+H]+ 1136
973
[α-CD+1+H]+ 1371
(c)
[γ-CD+1+H]+ 1695
[γ-CD+H]+ 1297
Fig. 2. Positive ESI mass spectra of the solutions of 1 with (a) a-CD, (b) b-CD, and (c) c-CD in pure water. Each concentration of the CDs and 1 was 2.5 mM.
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In this study, we characterized molecular interactions of the CDs with 1 using ESI mass spectrometry and NMR spectroscopy such as 1H NMR and 2D ROESY. 2. Experimental 2.1. Materials Camostat mesylate was from Ilsung Pharmaceuticals Co., Ltd. a-, b- and c-CD were purchased from Sigma Chemical Co. (St. Louis, MO, USA). D2O (D, 99.96%) as NMR solvent was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). MeOH of analytical grade was purchased from Sigma Chemical Co. and used without further purification. 2.2. Preparation of the complexes of 1 with a-, b-, and c-CD
CDs in the mass spectra. For the complex of a-CD with 1, the ion at m/z 1371 corresponded to [a-CD + 1 + H]+ (Fig. 2a) and the ion at m/z 1534 was also assigned as the peak corresponding to the complex of b-CD with 1 (Fig. 2b). The most intense CD–1 complex ion relative to free CD peak was observed in the spectrum of the complex of c-CD with 1. As shown in Fig. 2c, the ion at m/z 1695 corresponded to the complex of c-CD with 1 ([c-CD + 1 + H]+) and there is no any peak around at m/z 2093, corresponding to the [c-CD + 1 + 1 + H]+ ion. Additionally, the stoichiometry of the complex to make sure whether the complex of c-CD with 1 was 1:1 or 2:1, was determined using the method of continuous variation (Job’s analysis), as shown in Fig. 3. The resulting Job’s plot showed a symmetrical shape and a maximum at 0.5, indicating obviously that the binding stoichiometry between c-CD and 1 was 1:1. Therefore, the 1:1 stoichiometry confirmed from Job’s plot supports that from the ESI-MS analysis.
A stock solution of 1 (2.5 mM) was prepared in water. One mL of the stock solution of 1 in a vial was mixed with each 1 mL of the CDs (2.5 mM) dissolved in water. For evaluation of binding constants of the complexes, the concentration of 1 was constantly kept at 2.5 104 M with increasing the concentrations of the CDs in molar ratios of 1 to the CDs 1:1, 1:2, 1:5, 1:10 and 1:20 in all solutions. The mixtures were stirred for 1 h at room temperature before ESI-MS or NMR analysis. 2.3. ESI-MS analysis All ESI-MS experiments were performed on an API 4000TM triple quadrupole LC/MS/MS System (Applied Biosystems, Foster City, CA, USA) equipped with a turbo electrospray ion source. The sample solutions were directly infused into the ESI source with a flow rate of 10 l min1. Spray voltage, declustering potential, and entrance potential was set to 4.5 keV, 118 eV, and 10 eV in the negative ion mode, respectively. Pressures of curtain gas and ion source gas 1 were 10 and 17 psi, respectively. 2.3.1. 1H NMR and ROESY measurements 1 H NMR experiments were carried out using a Brucker AMX 500 MHz instrument at 298 K in D2O. The chemical shifts were referenced to an internal reference of methanol (d = 3.34 ppm), being a reference without any possible interaction with the CDs. Typical parameters for 1H NMR experiments consisted of spectral width of 7507.50 Hz, pulse width of 9.80 ls, acquisition time of 4.36 s, number of scans of 16, and relaxation delay of 1 s. 2D ROESY experiments for the complex of c-CD with 1 were acquired in the phase-sensitive mode using the same instrument. For ROESY spectrum, the time domain data was zero filled to 2048 points in F2 and 512 points in F1. The ROESY data was acquired with a spin lock power of 21 dB, a mixing time of 200 ms, and a relaxation delay of 2 s.
Fig. 3. Continuous variation plot of the c-CD–1 system ([c-CD] + [1] = 5 mM) in water. Each 1H chemical shift of the Ha,b () and Hc,d (j) resonances of 1 was monitored as the mole fraction of 1 ([1]/([1]+[c-CD])) was varied from 0 to 1.
Table 1 Linear equations and Kst valuesa for 1 with a-, b-, and c-CD.
a-CD b-CD c-CD a
Linear fit equation
r2
Kst (M1)
1/Ir = 4.5 103(1/[a-CD]t) + 1.9369 1/Ir = 3.1 103(1/[b-CD]t) + 2.3269 1/Ir = 1.0 103(1/[c-CD]t) + 3.0084
0.9978 0.9941 0.9960
430.41 ± 9.35 750.71 ± 16.30 3008.42 ± 68.67
All values are average of three measurements (±SD).
3. Results and discussion 3.1. ESI-MS analysis and stoichiometric determination of the complexes of CDs with 1 Relative abundances and stoichiometries of the noncovalent complexes of the CDs with 1 formed in pure water were determined using ESI-MS in the gas phase. The ESI mass spectra were observed for mixtures of uncomplexed CDs and their supramolecular complexes. The 1-CD complexes in the gas phase were stable even when a declustering potential was applied to 118 eV. As shown in Fig. 2, the ions corresponding to the complexes with 1 were clearly observed with those corresponding to uncomplexed
γ
Fig. 4. Linear plot showing the effect of c-CD concentration on the relative intensity (Ir) of c-CD–1 system.
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(d)
(c)
(b) e,f,g,h
a,b
c,d
(a) 8.4
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.5
7.6
7.4
7.3
7.2
7.1
7.0
Chemical Shift (ppm) Fig. 5. Partial 1H NMR spectra of 1 in the (a) absence and presence of (b) a-CD, (c) b-CD, and (d) c-CD. Each concentration of the CDs and 1 was 2.5 mM.
Table 2 1 H NMR chemical shifts (ppm) of 1 by the interaction with a-, b-, and c-CD. a
a-CD
d1
c
d2
Dd
d2
Dd
8.266 7.275 7.484 3.912 4.910 3.014 2.938
0.001 0.002 0.003 0.017 0.000 0.000 0.001
8.254 7.220 7.504 3.935 4.917 3.018 2.945
0.013 0.053 0.017 0.006 0.007 0.004 0.008
8.138 7.149 7.449 3.910 4.919 3.022 2.950
0.129 0.124 0.038 0.019 0.009 0.008 0.013
d2
H-a,b H-c,d H-e,f,g,h H-i H-j H-k H-l
8.267 7.273 7.487 3.929 4.910 3.014 2.937
c-CD
b-CD
b
Dd
was used to determine the effect of the concentrations of the CDs on relative abundance of the complexes. As reported previously [16–18], Ir is connected with Kst by the following equation:
1=Ir ¼ ð1=kc ½1t K st ½CDt Þ þ ð1=kc ½1t Þ
ð1Þ
3.2. Determination of binding constants of the 1-CD complexes
where kc is a proportionality constant and [1]t and [CD]t are initial concentrations of 1 and CD, respectively. From Eq. (1), Each Kst for the complexes of the CDs with 1 was determined from the corresponding intercepts and slopes of weighted least-squares regression fits of the data to Eq. (1), as presented in Table 1. Moreover, the least-square regression coefficients (r2) of the linear equations were greater than 0.9900 in the three cases. This result indicates a good correlation between the equations and the ESI mass spectral data. The Kst values listed in Table 1 suggest that c-CD is more favorable for noncovalent binding with 1 than a- or b-CD (Table 1 and Fig. 4).
ESI-MS has been used to determine binding constants of the complexes of hosts with guests [16–18]. The 1:1 complexation of 1 to each CD was monitored in the positive ion mode by mixing 1 with up to 20-fold molar excesses of the CDs. Considering that the complexation of a, b, or c-CD to 1 is 1:1 as shown in Figs. 2 and 3, the equilibrium constant, Kst, is defined as Kst = [CD:1]/[CD][1]. A relative intensity Ir, defined as Ir = Ic/(Ic + If) in which If and Ic are defined as the abundances of free 1 and the complex with CD, respectively,
3.2.1. 1H NMR and ROESY experiment of the CD–1 complexes The observed 1H NMR chemical shift changes are also indicative of the noncovalent interactions of the CDs with 1. Fig. 5 shows partial 1H NMR spectra of the ring moieties of 1 in the absence and presence of the CDs. Changes in the chemical shifts of 1 were more pronounced in the presence of c-CD than a- or b-CD (Fig. 5d and Table 2). Generally, upfield chemical shifts of the ring protons of 1 were observed in the complexes with the CDs, indicating that
a b c
Free state. Complexed state. Dd = d2 d1.
Table 3 1 H NMR chemical shifts (ppm) of CDs by the interaction with 1.
a-CD b
c
d1
d2
Dd
d1
d2
Dd
5.050 3.628 3.971 3.584 3.832 3.892
5.046 3.600 3.953 3.587 3.822 3.882
0.004 0.028 0.018 0.003 0.010 0.010
5.068 3.646 3.956 3.583 3.859 3.880
5.061 3.633 3.929 3.572 3.839 3.862
0.007 0.013 0.027 0.011 0.020 0.018
5.108 3.652 3.930 3.589 3.858 3.866
5.093 3.642 3.846 3.574 3.794 3.806
0.015 0.010 0.084 0.015 0.064 0.060
d1
H-1 H-2 H-3 H-4 H-5 H-6 a b c
c-CD
b-CD
a
Free state. Complexed state. Dd = d2 d1.
d2
Dd
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the protons of 1 were surrounded by electron density of the CDs upon the complexation [19,20]. Especially, the resonances of the guanidinyl group-linked aromatic ring protons (H-a,b and H-c,d) in the presence of c-CD were much more shifted upfield than those of the aliphatic protons and the N,N-dimethylcarbamoylmethyl group-linked ring protons, indicating that the guanidinyl grouplinked aromatic ring of 1 was more favorably included into the cavity of c-CD than that of a- or b-CD upon the complexation, while the cavity of a- or b-CD was not large enough to accommodate the guanidine group of 1. Although the H-3 and H-5 resonances of b-CD in the complex with 1 were shifted upfiled, the magnitude of the chemical shifts was much smaller than that of c-CD. Typically, inclusion of aromatic compounds has a pronounced effect on the chemical shift of the protons (H-3 and H-5) within the CD cavities due to ring-current effects [22]. The changes in the chemical shifts of the protons of the CDs upon the complex formation with 1 were also observed (Table 3). As shown in Table 3, the chemical shifts of the c-CD protons were most pronounced
H-i
upon complex formation. Especially, pronounced upfield changes in chemical shift were observed for the resonances of the H-3 and H-5 protons on the inside of the c-CD cavity, resulting from the anisotropic shielding induced by the ring-current effect by the aromatic rings of 1 included into the c-CD cavity [21,22]. The upfield shift of the H-3 resonance of c-CD was the most prominent (Dd = 0.084 ppm), followed by the H-5 proton (Dd = 0.064 ppm) and the H-6 proton (Dd = 0.060 ppm). Combined with 1H chemical shift data of both 1 and c-CD upon complex formation, it is suggested that the guanidinyl group-linked aromatic ring of 1 was preferentially included through the wider rim of c-CD. To obtain further information on the inclusion complexation mode of c-CD with 1, 2D ROESY experiment was carried out. ROE cross peaks were typically observed between the H-3 and H-5 protons on the inside of the c-CD cavity and the aromatic ring protons of 1 (Fig. 6). No cross peaks between the protons within the cavity of c-CD and the aliphatic protons of 1 were observed. As shown in Fig. 6, the protons (H-a,b and H-c,d) of the guanidinyl group-linked
H-6 H-3
H-2
H-4
H-5
7.0
H-e,f,g,h
7.5
8.0
H-a,b
8.5
4.0
4.1
3.9
3.8
3.7
3.6
3.5
F2 Chemical Shift (ppm) Fig. 6. Expansion of ROESY spectrum of the complex of c-CD with 1.
O O
O O
NH H 2N
N
O
N H
O O
O NH H 2N
N H
O
O
1
N
O
+
O
O NH
γ-CD H 2N
N H
Fig. 7. Proposed geometric model for the inclusion equilibria of c-CD with 1.
O
O
N
F1 Chemical Shift (ppm)
H-c,d
S. Kwon et al. / Journal of Molecular Structure 938 (2009) 192–197
aromatic ring have cross peaks to the H-3, H-5, and H-6 protons of c-CD, indicating the deep insertion of the aromatic ring into the host cavity. Interestingly, additional dipolar correlation was observed between the protons (H-e,f,g,h) of the other aromatic ring and the H-3 proton on the inside of the c-CD cavity, suggesting that the aromatic ring was partially included into the cavity, as opposed to the deep accommodation of the guanidinyl group-linked aromatic ring into the cavity. These results also support the observed 1H chemical shift (Dd) values as presented in Tables 2 and 3. On the basis of the experimental data stated above, two types of 1:1 complexes of c-CD with 1 with different geometry in solution can exist simultaneously in solution. Similar works illustrating the simultaneous presence of two 1:1 complexes have been reported [20,22,23]. Accordingly, hypothetical geometric arrangements of the complex of c-CD with 1 in solution can be proposed as shown in Fig. 7. 4. Conclusions In this study, the noncovalent interactions of 1 with a-, b-, and c-CD were characterized by ESI-MS analysis, 1H NMR and ROESY experiments. ESI mass spectral analysis showed that the complexes of each CD to 1 has a 1:1 stoichiometry and the binding constants for all complexes were determined by linear equations in which the magnitude of the values was in the order of 1-cCD > 1-b-CD > 1-a-CD. 1H NMR spectral analysis also showed that 1 H chemical shifts of 1 were most pronounced in the presence of c-CD, with larger upfield shifts of the guanidinyl group-linked aromatic ring protons of 1. 2D ROESY analysis was showed that the two aromatic rings of 1 were included into the cavity of c-CD upon complex formation, suggesting indicating multimodal molecular encapsulation of the compound 1 by c-CD. This work will be helpful in studying host–guest noncovalent interactions in either the gas phase or the solution phase.
197
Acknowledgment This work was supported by Seoul Medical Science Institute in 2008.
References [1] Y. Tamura, M. Hirado, K. Okamura, Y. Minato, S. Fujii, Biochim. Biophys. Acta 484 (1977) 417. [2] N. Kayama, K. Sakaguchi, S. Fujii, Gendai-Iryo 12 (1980) 1. [3] N. Kayama, K. Sakaguchi, S. Kaneko, S. Fujii, Gendai-Iryo 12 (1980) 11. [4] M. Kitagawa, S. Naruse, H. Ishiguro, T. Hayakawa, Pancreas 16 (1998) 427. [5] M. Sugiyama, Y. Atomi, N. Wada, A. Kuroda, T. Muto, J. Gastroenterol. 32 (1997) 374. [6] I. Sasaki, Y. Suzuki, H. Naito, Y. Funayama, Y. Kamiyama, M. Takahashi, T. Matsuo, K. Fukushima, S. Matsuno, T. Sato, Biomed. Res. 10 (Suppl. 1) (1989) 167. [7] I. Kobayashi, S. Ohwada, T. Ohya, T. Yokomori, H. Iesato, Y. Morishita, Hepatogastroenterology 45 (1998) 558. [8] H. Sarles, Scand. J. Gastroenterol. 6 (1971) 193. [9] H. Sarles, Dig. Dis. Sci. 30 (1985) 573. [10] K. Beckh, B. Goke, R. Muller, R. Arnold, Res. Exp. Med. (Berlin) 187 (1987) 401. [11] T. Nishihata, Y. Saitoh, K. Sakai, Chem. Pharm. Bull. (Tokyo) 36 (1988) 2544. [12] V.J. Stella, Q. He, Toxicol. Pathol. 36 (2008) 30. [13] E.M. Martin Del Valle, Proc. Biochem. 39 (2004) 1033. [14] H. Dodziuk, O.M. Demchuk, W. Schilf, G. Dolgonos, J. Mol. Struct. 693 (2004) 145. [15] T. Yamamoto, T. Kobayashi, K. Yoshikiyo, Y. Matsui, T. Takahashi, Y. Aso, J. Mol. Struct. 920 (2009) 264. [16] Y. Dotsikas, Y.L. Loukas, J. Am. Soc. Mass Spectrom. 14 (2003) 1123. [17] H. Li, J. Zhou, F. Tang, G. Yuan, J. Am. Soc. Mass Spectrom. 17 (2006) 17. [18] Z. Yu, M. Cui, C. Yan, F. Song, Z. Liu, S. Liu, Rapid Commun. Mass Spectrom. 21 (2007) 683. [19] K.H. Kim, Y.H. Park, Int. J. Pharm. 175 (1998) 247. [20] L. Ribeiroa, R.A. Carvalhob, D.C. Ferreirac, F.J.B. Veigaa, Eur. J. Pharm. Sci. 24 (2005) 1. [21] S. Lee, D. Yi, S. Jung, Bull. Korean Chem. Soc. 25 (2004) 216. [22] J.R. Cruz, B.A. Becker, K.F. Morris, C.K. Larive, Magn. Reson. Chem. 46 (2008) 838. [23] H. Achi, T. Niiya, Y. Iwase, M. Yamanoto, J. Pharm. Sci. 90 (2001) 1186. [24] R. Challa, A. Ahuja, J. Ali, R.K. Khar, AAPS PharmSciTech. 6 (2005) E329.
Contents lists available at ScienceDirect
Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc
Characterization of cyclodextrin complexes of camostat mesylate by ESI mass spectrometry and NMR spectroscopy Soonho Kwon a,1, Woonhyoung Lee b,1, Hye-Jin Shin a, Sung-il Yoon c, Yun-tae Kim a, Young-Jin Kim a, Kyungruyl Lee a, Sanghoo Lee a,* a b c
Department of Bioanalytical & Mass Spectrometry, Seoul Medical Science Institute, Seoul 152-766, Republic of Korea Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea Department of Bioanalysis, Korean Clinical Research Center, Co., Ltd, Gyeonggi-do 431-836, Republic of Korea
a r t i c l e
i n f o
Article history: Received 29 April 2009 Received in revised form 9 September 2009 Accepted 16 September 2009 Available online 20 September 2009 Keywords: Camostat mesylate Cyclodextrin Noncovalent complex ESI mass NMR
a b s t r a c t Supramolecular interactions between camostat mesylate, a serine protease inhibitor (1), with a-, b-, and c-cyclodextrin (CD) in water were investigated using electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectroscopy. ESI mass spectral analysis revealed that the 1:1 stoichiometry in all the complexes was formed. The binding constants (Kst) calculated by linear equations constructed from the ESI mass spectra of all the complexes indicated that c-CD was most favorable complexing agent for the binding with 1 among the CDs. Pronounced changes in the 1H chemical shift upon complex formation with c-CD were observed for the protons of the two aromatic rings of 1, with much larger chemical shift changes observed for the protons of the guanidinyl group-linked aromatic ring of 1. These results suggest that the cavity of c-CD rather than that of a- or b-CD is large enough to accommodate the guanidine group of 1. Spatial geometry of 1 within the cavity of c-CD was further identified with two-dimensional rotating frame nuclear Overhauser effect spectroscopy (2D ROESY) experiment. The observed ROESY cross peaks indicated intermolecular dipolar interactions between the two aromatic ring protons of 1 and the protons within the cavity of c-CD. Based on the 1:1 stoichiometry of the complex, ROESY cross peaks suggest that two types of 1:1 complexes of c-CD with 1 exist simultaneously in solution with different geometries. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Active synthetic serine protease inhibitors have been known to play important roles in many biological processes. N,N-dimethylcarbamoylmethyl 4-(4-guanidinobenzoyloxy)phenylacetate methanesulphonate (camostat mesylate, 1) (Fig. 1a) is a synthetic proteolytic enzyme inhibitor for trypsin, plasmin, kallikrein, tissue kallikrein, and thrombin [1–3]. The drug has also shown activity in the treatment of pancreatitis [4,5] and reflux esophagitis [6,7], establishing its safety for use in human. Especially, the compound 1 is known to have stronger trypsin inhibitory activity 100–1000 times than other trypsin inhibitors such as aprotinin and gabexate mesylate [8,9]. Metabolic works have also shown that the 1 is rapidly hydrolyzed to 4-(4-guanidinobenzoyloxy)phenylacetic acid
* Corresponding author. Address: Department of Bioanalytical & Mass Spectrometry, Seoul Medical Science Institute & Seoul Clinical Laboratories, #901-1, ACE Technotower V, 197-22, Guro 3-dong, Guro-gu, Seoul 152-766, Republic of Korea. Tel.: +82 70 7115 8697; fax: +82 2 858 2814. E-mail address: [email protected] (S. Lee). 1 These authors contributed equally to this work. 0022-2860/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2009.09.025
and 4-guanidinobenzoic acid by esterases during intestinal absorption [10,11]. CDs are a family of cyclic oligosaccharides composed of a-(1,4) linked glucopyranose units. Among them, a-, b- and c-CD are most common cycloamyloses, which have six, seven, and eight glucose units, respectively. Their structures are shown in Fig. 1b. The CDs share a torus-like molecular shape with hydrophobic inner cavity and hydrophilic outer surface and are able to form complexes with various molecules containing apolar groups that can be included partially or completely into their hydrophobic cavities with an internal diameter from 4.7 to 8.3 Å [12,13]. Based on these dimensions, a-CD can typically complex low molecular weight molecules or compounds with aliphatic side chains, b-CD will complex aromatics and heterocycles and c-CD can accommodate larger molecules such as macrocycles and steroids [13,14]. Thus, CDs have been successfully used to control various physico-chemical properties of drugs such as solubility, dissolution, release rates, stability, and bioavailability [12,13,15,24]. Until now, the studies on the drug 1 have been mainly focused on its pharmaceutical activity but no works on noncovalent complexes between CDs and 1 in terms of structural analyses have been reported.
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O
(a) i
f
j
b c
NH
O
e
O
l
O
g
O
N k
h
a H2N
N H
d
(b) OH 4
6
O 5
2
HO 3
OH
1
n : α-CD : β-CD : γ-CD
n=6 n=7 n=8
Fig. 1. Numbered structures of (a) 1 and (b) CDs.
(a)
[β-CD+1+H]+ 1534
(b) [α-CD+H]+
[β-CD+H]+ 1136
973
[α-CD+1+H]+ 1371
(c)
[γ-CD+1+H]+ 1695
[γ-CD+H]+ 1297
Fig. 2. Positive ESI mass spectra of the solutions of 1 with (a) a-CD, (b) b-CD, and (c) c-CD in pure water. Each concentration of the CDs and 1 was 2.5 mM.
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In this study, we characterized molecular interactions of the CDs with 1 using ESI mass spectrometry and NMR spectroscopy such as 1H NMR and 2D ROESY. 2. Experimental 2.1. Materials Camostat mesylate was from Ilsung Pharmaceuticals Co., Ltd. a-, b- and c-CD were purchased from Sigma Chemical Co. (St. Louis, MO, USA). D2O (D, 99.96%) as NMR solvent was purchased from Cambridge Isotope Laboratories, Inc. (Andover, MA, USA). MeOH of analytical grade was purchased from Sigma Chemical Co. and used without further purification. 2.2. Preparation of the complexes of 1 with a-, b-, and c-CD
CDs in the mass spectra. For the complex of a-CD with 1, the ion at m/z 1371 corresponded to [a-CD + 1 + H]+ (Fig. 2a) and the ion at m/z 1534 was also assigned as the peak corresponding to the complex of b-CD with 1 (Fig. 2b). The most intense CD–1 complex ion relative to free CD peak was observed in the spectrum of the complex of c-CD with 1. As shown in Fig. 2c, the ion at m/z 1695 corresponded to the complex of c-CD with 1 ([c-CD + 1 + H]+) and there is no any peak around at m/z 2093, corresponding to the [c-CD + 1 + 1 + H]+ ion. Additionally, the stoichiometry of the complex to make sure whether the complex of c-CD with 1 was 1:1 or 2:1, was determined using the method of continuous variation (Job’s analysis), as shown in Fig. 3. The resulting Job’s plot showed a symmetrical shape and a maximum at 0.5, indicating obviously that the binding stoichiometry between c-CD and 1 was 1:1. Therefore, the 1:1 stoichiometry confirmed from Job’s plot supports that from the ESI-MS analysis.
A stock solution of 1 (2.5 mM) was prepared in water. One mL of the stock solution of 1 in a vial was mixed with each 1 mL of the CDs (2.5 mM) dissolved in water. For evaluation of binding constants of the complexes, the concentration of 1 was constantly kept at 2.5 104 M with increasing the concentrations of the CDs in molar ratios of 1 to the CDs 1:1, 1:2, 1:5, 1:10 and 1:20 in all solutions. The mixtures were stirred for 1 h at room temperature before ESI-MS or NMR analysis. 2.3. ESI-MS analysis All ESI-MS experiments were performed on an API 4000TM triple quadrupole LC/MS/MS System (Applied Biosystems, Foster City, CA, USA) equipped with a turbo electrospray ion source. The sample solutions were directly infused into the ESI source with a flow rate of 10 l min1. Spray voltage, declustering potential, and entrance potential was set to 4.5 keV, 118 eV, and 10 eV in the negative ion mode, respectively. Pressures of curtain gas and ion source gas 1 were 10 and 17 psi, respectively. 2.3.1. 1H NMR and ROESY measurements 1 H NMR experiments were carried out using a Brucker AMX 500 MHz instrument at 298 K in D2O. The chemical shifts were referenced to an internal reference of methanol (d = 3.34 ppm), being a reference without any possible interaction with the CDs. Typical parameters for 1H NMR experiments consisted of spectral width of 7507.50 Hz, pulse width of 9.80 ls, acquisition time of 4.36 s, number of scans of 16, and relaxation delay of 1 s. 2D ROESY experiments for the complex of c-CD with 1 were acquired in the phase-sensitive mode using the same instrument. For ROESY spectrum, the time domain data was zero filled to 2048 points in F2 and 512 points in F1. The ROESY data was acquired with a spin lock power of 21 dB, a mixing time of 200 ms, and a relaxation delay of 2 s.
Fig. 3. Continuous variation plot of the c-CD–1 system ([c-CD] + [1] = 5 mM) in water. Each 1H chemical shift of the Ha,b () and Hc,d (j) resonances of 1 was monitored as the mole fraction of 1 ([1]/([1]+[c-CD])) was varied from 0 to 1.
Table 1 Linear equations and Kst valuesa for 1 with a-, b-, and c-CD.
a-CD b-CD c-CD a
Linear fit equation
r2
Kst (M1)
1/Ir = 4.5 103(1/[a-CD]t) + 1.9369 1/Ir = 3.1 103(1/[b-CD]t) + 2.3269 1/Ir = 1.0 103(1/[c-CD]t) + 3.0084
0.9978 0.9941 0.9960
430.41 ± 9.35 750.71 ± 16.30 3008.42 ± 68.67
All values are average of three measurements (±SD).
3. Results and discussion 3.1. ESI-MS analysis and stoichiometric determination of the complexes of CDs with 1 Relative abundances and stoichiometries of the noncovalent complexes of the CDs with 1 formed in pure water were determined using ESI-MS in the gas phase. The ESI mass spectra were observed for mixtures of uncomplexed CDs and their supramolecular complexes. The 1-CD complexes in the gas phase were stable even when a declustering potential was applied to 118 eV. As shown in Fig. 2, the ions corresponding to the complexes with 1 were clearly observed with those corresponding to uncomplexed
γ
Fig. 4. Linear plot showing the effect of c-CD concentration on the relative intensity (Ir) of c-CD–1 system.
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(d)
(c)
(b) e,f,g,h
a,b
c,d
(a) 8.4
8.3
8.2
8.1
8.0
7.9
7.8
7.7
7.5
7.6
7.4
7.3
7.2
7.1
7.0
Chemical Shift (ppm) Fig. 5. Partial 1H NMR spectra of 1 in the (a) absence and presence of (b) a-CD, (c) b-CD, and (d) c-CD. Each concentration of the CDs and 1 was 2.5 mM.
Table 2 1 H NMR chemical shifts (ppm) of 1 by the interaction with a-, b-, and c-CD. a
a-CD
d1
c
d2
Dd
d2
Dd
8.266 7.275 7.484 3.912 4.910 3.014 2.938
0.001 0.002 0.003 0.017 0.000 0.000 0.001
8.254 7.220 7.504 3.935 4.917 3.018 2.945
0.013 0.053 0.017 0.006 0.007 0.004 0.008
8.138 7.149 7.449 3.910 4.919 3.022 2.950
0.129 0.124 0.038 0.019 0.009 0.008 0.013
d2
H-a,b H-c,d H-e,f,g,h H-i H-j H-k H-l
8.267 7.273 7.487 3.929 4.910 3.014 2.937
c-CD
b-CD
b
Dd
was used to determine the effect of the concentrations of the CDs on relative abundance of the complexes. As reported previously [16–18], Ir is connected with Kst by the following equation:
1=Ir ¼ ð1=kc ½1t K st ½CDt Þ þ ð1=kc ½1t Þ
ð1Þ
3.2. Determination of binding constants of the 1-CD complexes
where kc is a proportionality constant and [1]t and [CD]t are initial concentrations of 1 and CD, respectively. From Eq. (1), Each Kst for the complexes of the CDs with 1 was determined from the corresponding intercepts and slopes of weighted least-squares regression fits of the data to Eq. (1), as presented in Table 1. Moreover, the least-square regression coefficients (r2) of the linear equations were greater than 0.9900 in the three cases. This result indicates a good correlation between the equations and the ESI mass spectral data. The Kst values listed in Table 1 suggest that c-CD is more favorable for noncovalent binding with 1 than a- or b-CD (Table 1 and Fig. 4).
ESI-MS has been used to determine binding constants of the complexes of hosts with guests [16–18]. The 1:1 complexation of 1 to each CD was monitored in the positive ion mode by mixing 1 with up to 20-fold molar excesses of the CDs. Considering that the complexation of a, b, or c-CD to 1 is 1:1 as shown in Figs. 2 and 3, the equilibrium constant, Kst, is defined as Kst = [CD:1]/[CD][1]. A relative intensity Ir, defined as Ir = Ic/(Ic + If) in which If and Ic are defined as the abundances of free 1 and the complex with CD, respectively,
3.2.1. 1H NMR and ROESY experiment of the CD–1 complexes The observed 1H NMR chemical shift changes are also indicative of the noncovalent interactions of the CDs with 1. Fig. 5 shows partial 1H NMR spectra of the ring moieties of 1 in the absence and presence of the CDs. Changes in the chemical shifts of 1 were more pronounced in the presence of c-CD than a- or b-CD (Fig. 5d and Table 2). Generally, upfield chemical shifts of the ring protons of 1 were observed in the complexes with the CDs, indicating that
a b c
Free state. Complexed state. Dd = d2 d1.
Table 3 1 H NMR chemical shifts (ppm) of CDs by the interaction with 1.
a-CD b
c
d1
d2
Dd
d1
d2
Dd
5.050 3.628 3.971 3.584 3.832 3.892
5.046 3.600 3.953 3.587 3.822 3.882
0.004 0.028 0.018 0.003 0.010 0.010
5.068 3.646 3.956 3.583 3.859 3.880
5.061 3.633 3.929 3.572 3.839 3.862
0.007 0.013 0.027 0.011 0.020 0.018
5.108 3.652 3.930 3.589 3.858 3.866
5.093 3.642 3.846 3.574 3.794 3.806
0.015 0.010 0.084 0.015 0.064 0.060
d1
H-1 H-2 H-3 H-4 H-5 H-6 a b c
c-CD
b-CD
a
Free state. Complexed state. Dd = d2 d1.
d2
Dd
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the protons of 1 were surrounded by electron density of the CDs upon the complexation [19,20]. Especially, the resonances of the guanidinyl group-linked aromatic ring protons (H-a,b and H-c,d) in the presence of c-CD were much more shifted upfield than those of the aliphatic protons and the N,N-dimethylcarbamoylmethyl group-linked ring protons, indicating that the guanidinyl grouplinked aromatic ring of 1 was more favorably included into the cavity of c-CD than that of a- or b-CD upon the complexation, while the cavity of a- or b-CD was not large enough to accommodate the guanidine group of 1. Although the H-3 and H-5 resonances of b-CD in the complex with 1 were shifted upfiled, the magnitude of the chemical shifts was much smaller than that of c-CD. Typically, inclusion of aromatic compounds has a pronounced effect on the chemical shift of the protons (H-3 and H-5) within the CD cavities due to ring-current effects [22]. The changes in the chemical shifts of the protons of the CDs upon the complex formation with 1 were also observed (Table 3). As shown in Table 3, the chemical shifts of the c-CD protons were most pronounced
H-i
upon complex formation. Especially, pronounced upfield changes in chemical shift were observed for the resonances of the H-3 and H-5 protons on the inside of the c-CD cavity, resulting from the anisotropic shielding induced by the ring-current effect by the aromatic rings of 1 included into the c-CD cavity [21,22]. The upfield shift of the H-3 resonance of c-CD was the most prominent (Dd = 0.084 ppm), followed by the H-5 proton (Dd = 0.064 ppm) and the H-6 proton (Dd = 0.060 ppm). Combined with 1H chemical shift data of both 1 and c-CD upon complex formation, it is suggested that the guanidinyl group-linked aromatic ring of 1 was preferentially included through the wider rim of c-CD. To obtain further information on the inclusion complexation mode of c-CD with 1, 2D ROESY experiment was carried out. ROE cross peaks were typically observed between the H-3 and H-5 protons on the inside of the c-CD cavity and the aromatic ring protons of 1 (Fig. 6). No cross peaks between the protons within the cavity of c-CD and the aliphatic protons of 1 were observed. As shown in Fig. 6, the protons (H-a,b and H-c,d) of the guanidinyl group-linked
H-6 H-3
H-2
H-4
H-5
7.0
H-e,f,g,h
7.5
8.0
H-a,b
8.5
4.0
4.1
3.9
3.8
3.7
3.6
3.5
F2 Chemical Shift (ppm) Fig. 6. Expansion of ROESY spectrum of the complex of c-CD with 1.
O O
O O
NH H 2N
N
O
N H
O O
O NH H 2N
N H
O
O
1
N
O
+
O
O NH
γ-CD H 2N
N H
Fig. 7. Proposed geometric model for the inclusion equilibria of c-CD with 1.
O
O
N
F1 Chemical Shift (ppm)
H-c,d
S. Kwon et al. / Journal of Molecular Structure 938 (2009) 192–197
aromatic ring have cross peaks to the H-3, H-5, and H-6 protons of c-CD, indicating the deep insertion of the aromatic ring into the host cavity. Interestingly, additional dipolar correlation was observed between the protons (H-e,f,g,h) of the other aromatic ring and the H-3 proton on the inside of the c-CD cavity, suggesting that the aromatic ring was partially included into the cavity, as opposed to the deep accommodation of the guanidinyl group-linked aromatic ring into the cavity. These results also support the observed 1H chemical shift (Dd) values as presented in Tables 2 and 3. On the basis of the experimental data stated above, two types of 1:1 complexes of c-CD with 1 with different geometry in solution can exist simultaneously in solution. Similar works illustrating the simultaneous presence of two 1:1 complexes have been reported [20,22,23]. Accordingly, hypothetical geometric arrangements of the complex of c-CD with 1 in solution can be proposed as shown in Fig. 7. 4. Conclusions In this study, the noncovalent interactions of 1 with a-, b-, and c-CD were characterized by ESI-MS analysis, 1H NMR and ROESY experiments. ESI mass spectral analysis showed that the complexes of each CD to 1 has a 1:1 stoichiometry and the binding constants for all complexes were determined by linear equations in which the magnitude of the values was in the order of 1-cCD > 1-b-CD > 1-a-CD. 1H NMR spectral analysis also showed that 1 H chemical shifts of 1 were most pronounced in the presence of c-CD, with larger upfield shifts of the guanidinyl group-linked aromatic ring protons of 1. 2D ROESY analysis was showed that the two aromatic rings of 1 were included into the cavity of c-CD upon complex formation, suggesting indicating multimodal molecular encapsulation of the compound 1 by c-CD. This work will be helpful in studying host–guest noncovalent interactions in either the gas phase or the solution phase.
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Acknowledgment This work was supported by Seoul Medical Science Institute in 2008.
References [1] Y. Tamura, M. Hirado, K. Okamura, Y. Minato, S. Fujii, Biochim. Biophys. Acta 484 (1977) 417. [2] N. Kayama, K. Sakaguchi, S. Fujii, Gendai-Iryo 12 (1980) 1. [3] N. Kayama, K. Sakaguchi, S. Kaneko, S. Fujii, Gendai-Iryo 12 (1980) 11. [4] M. Kitagawa, S. Naruse, H. Ishiguro, T. Hayakawa, Pancreas 16 (1998) 427. [5] M. Sugiyama, Y. Atomi, N. Wada, A. Kuroda, T. Muto, J. Gastroenterol. 32 (1997) 374. [6] I. Sasaki, Y. Suzuki, H. Naito, Y. Funayama, Y. Kamiyama, M. Takahashi, T. Matsuo, K. Fukushima, S. Matsuno, T. Sato, Biomed. Res. 10 (Suppl. 1) (1989) 167. [7] I. Kobayashi, S. Ohwada, T. Ohya, T. Yokomori, H. Iesato, Y. Morishita, Hepatogastroenterology 45 (1998) 558. [8] H. Sarles, Scand. J. Gastroenterol. 6 (1971) 193. [9] H. Sarles, Dig. Dis. Sci. 30 (1985) 573. [10] K. Beckh, B. Goke, R. Muller, R. Arnold, Res. Exp. Med. (Berlin) 187 (1987) 401. [11] T. Nishihata, Y. Saitoh, K. Sakai, Chem. Pharm. Bull. (Tokyo) 36 (1988) 2544. [12] V.J. Stella, Q. He, Toxicol. Pathol. 36 (2008) 30. [13] E.M. Martin Del Valle, Proc. Biochem. 39 (2004) 1033. [14] H. Dodziuk, O.M. Demchuk, W. Schilf, G. Dolgonos, J. Mol. Struct. 693 (2004) 145. [15] T. Yamamoto, T. Kobayashi, K. Yoshikiyo, Y. Matsui, T. Takahashi, Y. Aso, J. Mol. Struct. 920 (2009) 264. [16] Y. Dotsikas, Y.L. Loukas, J. Am. Soc. Mass Spectrom. 14 (2003) 1123. [17] H. Li, J. Zhou, F. Tang, G. Yuan, J. Am. Soc. Mass Spectrom. 17 (2006) 17. [18] Z. Yu, M. Cui, C. Yan, F. Song, Z. Liu, S. Liu, Rapid Commun. Mass Spectrom. 21 (2007) 683. [19] K.H. Kim, Y.H. Park, Int. J. Pharm. 175 (1998) 247. [20] L. Ribeiroa, R.A. Carvalhob, D.C. Ferreirac, F.J.B. Veigaa, Eur. J. Pharm. Sci. 24 (2005) 1. [21] S. Lee, D. Yi, S. Jung, Bull. Korean Chem. Soc. 25 (2004) 216. [22] J.R. Cruz, B.A. Becker, K.F. Morris, C.K. Larive, Magn. Reson. Chem. 46 (2008) 838. [23] H. Achi, T. Niiya, Y. Iwase, M. Yamanoto, J. Pharm. Sci. 90 (2001) 1186. [24] R. Challa, A. Ahuja, J. Ali, R.K. Khar, AAPS PharmSciTech. 6 (2005) E329.