Caged Naloxone: Synthesis, Characterization, and Stability of 3-O-(4,5

Subscriber access provided by READING UNIV

Article

Caged Naloxone: Synthesis, Characterization, and Stability of 3-O(4,5-dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (CNV-NLX) Anita H. Lewin, Scott E Fix, Desong Zhong, Louise D Mayer, Jason P. Burgess, Samuel Wayne Mascarella, P. Anantha Reddy, Herbert H. Seltzman, and F. Ivy Carroll ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00378 • Publication Date (Web): 20 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Neuroscience is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Neuroscience

Caged Naloxone: Synthesis, Characterization, and Stability of 3-O-(4,5-dimethoxy-2nitrophenyl)carboxymethyl Naloxone (CNV-NLX)

Anita H. Lewin,* Scott E. Fix, Desong Zhong, Louise D. Mayer, Jason P. Burgess, S. Wayne Mascarella, P. Anantha Reddy, Herbert H. Seltzman, and F. Ivy Carroll Research Triangle Institute, P.O. Box 12194, Research Triangle Park, NC, 27709 United States

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: The photo-labile analog of the broad-spectrum opioid antagonist naloxone, 3-O(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as “caged naloxone”, 3O-(α-carboxy-6-nitroveratryl)naloxone, CNV-NLX), has been found to be a valuable biochemical probe. While the synthesis of CNV-NLX is simple, its characterization is complicated by the fact that it is produced as a mixture of αR,5R 9R,13S,14S and αS,5R,9R,13S,14S diastereomers. Using long range and heteronuclear NMR correlations, the 1H and 13CNMR resonances of both diastereomers have been fully assigned, confirming the structures. Monitoring of solutions of CNV-NLX in saline buffer, in methanol and in DMSO has shown CNV-NLX to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products.

KEYWORDS: photolabile, diastereomers, 3-O-(α-carboxy-6-nitroveratryl)naloxone,

1.

INTRODUCTION Protecting groups have long been used in organic synthesis to allow specific sites to be

modified while preventing modifications from taking place at other sites. For most substituents multiple protecting groups, which vary by stability and by conditions for their removal, have been developed. Among these, photoactivatable protecting groups have found use in cases where exposure of the “protected” substance to chemical reagents would be counter-indicated.1 This concept has been applied not only in chemical synthesis but in biological systems as well. For example, automated DNA or RNA synthesis has benefitted from the use of photolabile protecting groups due to the very mild conditions required for their removal.1 One of the commonly used chromophores has been the 2-nitrophenyl group that defines a family of


Page 2 of 21

Page 3 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


photolabile protecting groups that includes the 2-nitrobenzyl and 4,5-dimethoxy-2-nitrobenzyl groups. Recently photoactivatable protecting groups have been used to achieve high concentrations of signaling molecules at specific neurons. In this case, a ligand in which the functionality essential to its activity has been masked to make it inert is delivered to a specific brain-site, followed by removal of the masking agent to release the neuroactive ligand. Photoactivatable masking groups are uniquely suited to this strategy, since photolysis removes the protecting group rapidly and is unlikely to affect cellular function. This approach was pursued to create so called “caged” neuropeptides in which the hydroxyl group of the tyrosine residue, which was known to have an essential role in receptor activation,2, 3 was protected using a 2-nitrophenyl chromophore that was modified by the addition of a carboxyl functionality to achieve water solubility resulting in a α-carboxynitrobenzyl group that was used to protect the tyrosyl residue as an ether. The thus-protected peptides were reported to be stable at room temperature for >48 hours, unless exposed to illumination, and were determined to be inactive in vitro either as agonists or antagonists in the dark. Laser illumination at 355 nm photo-released the underivatized peptides with high quantum yield (~0.3)4 on the microsecond time scale.5 In an application of this concept Banghart et al6 utilized the 4,5-dimethoxy-2-nitrobenzyl analog ((αcarboxy-6-nitroveratryl), which has been shown to photo release in high quantum yield (~0.2) at a rate 2−3 orders of magnitude larger than the parent compound,7 to derivatize the broadspectrum opioid antagonist naloxone, thereby providing a means to quantitatively measure spatiotemporal dynamics of opioid signaling dynamics and their underlying mechanisms in brain tissue.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Recognizing the importance of this pharmacologic tool, we report our preparation and thorough characterization of 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (CNVNLX). 2.

RESULTS AND DISCUSSION The synthesis of the “caged naloxone” nicknamed CNV-NLX (6) (Scheme 1) was carried

out with only slight modification of the published methodology.6 Thus, whereas [2(trimethylsilyl)ethoxy]methyl (SEM)-protected carboxynitroveratryl (CNV)-bromide (SEMOCNV-Br) was used as the “caging agent” in the published preparation of CNV-NLX (6), we opted to use the analogous methyl α-bromo-4,5-dimethoxy-2-nitrobenzeneacetate (3). Neither compound has been identified in the Chem Abstracts system. The “caging agent” 3 was prepared Scheme 1. Synthesis of O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (6, CNVNLX)


Page 4 of 21

Page 5 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


by converting commercially procured 4, 5-dimethoxy-2-nitrobenzeneacetic acid (1) to the corresponding methyl ester 2 by acid-promoted esterification in methanol (83% yield), followed by bromination of 2 using N-bromosuccinimide (NBS) and the radical initiator 2,2'-(1,2diazenediyl)bis[2-methylpropanenitrile) (AIBN) in carbon tetrachloride. The chromatographically purified product, methyl α-bromo-4,5-dimethoxy-2-nitrrobenzeneacetate (3), was obtained in 45% yield. Dehydration of naloxone (4) hydrochloride dihydrate using molecular sieves, followed by coupling with 3 in dimethyl formamide in the presence of anhydrous potassium carbonate, gave methyl 3-O-(4,5-dimethoxy-2nitrophenyl)carbomethoxymethyl naloxone (5) in 82% yield after chromatographic purification. Saponification of 5 with lithium hydroxide, essentially as described in the literature, 6 followed by two chromatographic purifications, gave CNV-NLX (6) in 71% yield. Since naloxone (4), which is derived from natural thebaine, has four stereocenters with defined (5R, 9R,13S,14S) stereochemistry, and the ester 3 has one stereocenter and is racemic, the coupling product of 3 and 4 was expected to consist of the diastereomers (αR, 5R, 9R,13S,14S)5 and (αS ,5R, 9R,13S,14S)5, but not necessarily in equal amount, since the coupling reaction may be partially stereoselective. In our hands, HPLC analysis of the coupling product of 3 and 4 (prior to purification) showed the presence of two closely eluting compounds with identical mass spectra, consistent with the formation of the expected diastereomers. Chromatographically purified 5 had a very slight excess of one diastereomer; this was also observed by NMR analysis. Saponification of the diastereomeric mixture 5 gave the target compound, CNV-NLX (6), which appeared as a single component by HPLC, but exhibited NMR spectra consistent with the presence of two components in 2:1 ratio, probably due to stereoselectivity in the chromatographic purification, since saponification is unlikely to have been associated with



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

stereoselectivity. The diastereomeric mixtures 5 and 6 were completely characterized for structural integrity and purity using long range and heteronuclear NMR interactions. The numbering scheme shown in Figure 1 was used in assignment of the proton and carbon

Figure 1. Numbering of 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (6, CNV-NLX) resonances shown in the experimental section. The most striking feature of the 1HNMR spectrum recorded in perdeuterated methanol, is the appearance of the new downfield resonances associated with the aromatic ring protons of the veratryl (cage) component. These aromatic protons (at 3’ and 6’ in Figure 1), which appear as singlets at 7.70 and 7.41 ppm in the 1HNMR spectrum of the bromoester 3, appear as two pairs of singlets at 7.66 (major)/7.70(minor) and 7.42 (major)/7.38 (minor) ppm in the spectrum of CNV-NLX (6) in perdeuterated methanol. On the other hand, the resonances of the methoxy substituents of the veratryl group (at 4’ and 5’ in Figure 1) do not exhibit chemical shift differences between the diastereomers. Chemical shift differences between the diastereomers of 6 are also seen for H-10, where the axial and equatorial protons are observed as separate resonances in the major diastereomer, but are coincident in the minor diastereomer.


Page 6 of 21

Page 7 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


To determine the light sensitivity of CNV-NLX (6), solutions of 6 trifluoroacetate in aqueous phosphate buffer were exposed to fluorescent laboratory lights, or to low intensity irradiation at λ 405 nm. No change was detected by HPLC or 1HNMR after several days of exposure; methanolic solutions of 6 trifluoroacetate were also stable to these lights. The observed stability of such solutions of 6 trifluoroacetate to fluorescent laboratory lights, or to low intensity irradiation at λ 405 nm, was reassuring, indicating that stock solutions could be prepared and stored. On the other hand, HPLC monitoring (see supplementary material) showed that exposure of such solutions to irradiation with a hand-held UV lamp at λ 365 nm for up to 2.5 hours led to total disappearance of the peak for CNV-NLX (6) and to the appearance of a peak for naloxone (4), confirming the photo-release of intact naloxone (4) from CNV-NLX (6). To allow for the analysis of the photolysis products by 1HNMR and 13CNMR, solutions of 6 trifluoroacetate in dimethyl sulfoxide (DMSO) were likewise irradiated. In particular, it was expected that perdeuterated DMSO would be superior to perdeuterated methanol in enabling detection of potential aldehydic products resulting from the caging group. While the 1HNMR resonances of 6 trifluoroacetate in perdeuterated DMSO were not assigned, the spectrum clearly showed the presence of isomers, just as in perdeuterated methanol. Comparison of the 1HNMR and 13CNMR spectra recorded before and after irradiation demonstrated the appearance of the expected resonances for naloxone (4) as well as several new resonances, presumably associated with the break-down products of the CNV group. Strikingly, the 1HNMR spectrum displays several sharp singlet resonances between δ 3.78 and 3.88 ppm, and between δ 7.1 and 7.4 ppm, suggesting the presence of multiple methoxy-substituted aromatic species. The mechanism for release of CNVlike caging groups has been described8 as proceeding by n—>π* excitation to generate an acinitro intermediate which expels the “uncaged” ligand and affords 4,5-dimethoxy-2-nitroso-α-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

oxobenzeneacetic acid (7, Figure 2) which, in turn, decarboxylates to give 4,5-dimethoxy-2nitrosobenzaldehyde (8). The observed groups of singlet resonances at the chemical shift values associated with methoxy groups and with aromatic protons, in the 1HNMR spectrum of the irradiated solution of 6 trifluoroacetate, are consistent with, but do not prove, the presence of these breakdown products. Both 7 and 8 are strongly UV-absorbing, and thus may serve to

7 8 Figure 2. Breakdown Products of 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl Naloxone (6, CNV-NLX) slow down the photochemical formation of the aci-nitro intermediate thereby slowing down the uncaging process. In fact, it has been pointed out that the decay of their primary quinonoid intermediates does not generally correspond to the rate-determining step of the overall reaction, and the release of the free substrate may be orders of magnitude slower.1 Moreover, both 7 and 8 are potentially toxic, especially the aldehyde 8, and thus photo-release of naloxone (4) from CNV-NLX (6) may not be biocompatible with the system being investigated. The fact that the CNV-NLX (6) consists of a diastereomeric mixture may also be a matter of concern. Thus, the fact that stereochemistry has been shown to play a role in physiologic distribution,9 combined with the likelihood that different preparations (batches), even from the same laboratory, may contain different percentages of each diastereomer, could compromise reproducibility and lead to misleading conclusions. This potential problem could be addressed by using single diastereomers of 6, which would best be separated at the ester precursor stage (5), or by using a single stereoisomer of the caging group, which would then produce a single diastereomer of the caged ligand. For this purpose, methyl α-bromo-4, 5-dimethoxy-2-


Page 8 of 21

Page 9 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nitrobenzeneacetate (3) is probably not a good choice, since chiral integrity at the α-position would be readily compromised due to the lability of the proton α to the carboxyl group. 3.

MATERIALS AND METHODS Instrumentation. 1HNMR spectra were acquired on a Bruker Avance 300 MHz

spectrometer, or a 500 MHz NMR spectrometer. Low-resolution LC-MS data were obtained using a PerkinElmer API 150 EX mass spectrometer outfitted with an ESI (turbospray) source, coupled to a PerkinElmer 200 Series liquid chromatography system. HPLC analyses were performed either on a dual pump system consisting of two HPLC pumps (Varian Prostar 210 solvent system delivery system), a Rheodyne injector and a Varian ProStar 335 DAD UV detector (or a Varian ProStar 320 UV detector) controlled by Varian Star Workstation software or on Agilent HPLC 1100 system (two HPLC pumps, an auto sampler, and a diode-array detector) controlled by ChemStation HPLC software. The water used in HPLC solvent systems was obtained from a Millipore Milli-Q Plus Ultra-Pure Water System. Materials. Naloxone hydrochloride dihydrate was from the NIDA; (4,5-dimethoxy-2nitrophenyl)acetic acid was from Johnson-Matthey; NBS , AIBN, CD3OD and CDCl3 were from Sigma Aldrich; anhydrous DMF, CCl4, and THF were from EM Sciences; all other solvents were from Fisher Scientific. Synthesis of 3-O-(4,5-dimethoxy-2-nitrophenyl)carbomethoxymethyl Naloxone (6, CNV-NLX Methyl 4, 5-dimethoxy-2-nitrobenezeneacetate (2). To a suspension of 4,5-dimethoxy-2nitrobenzeneacetic acid (1) (10 g, 0.0415 mol) in CH3OH (150 mL) under N2 were added a few drops of conc H2SO4. The resulting mixture was heated to 40 °C becoming homogeneous after 2 hours. After overnight at 40 °C the volatiles were removed at reduced pressure. The residual



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

yellow solid was dissolved in CH2Cl2 and the solution was washed with saturated NaHCO3, H2O, and saturated NaCl. Evaporation of the solvent afforded 2 as a pale yellow solid (8.8 g, 83%). 1

HNMR (CDCl3; 300 MHz) δ: 3.73 (s, 3H, OCH3), 3.97 (s, 3H, OCH3) 3.98 (s, 3H, OCH3) 3.99

(m, 2H, CH2), 6.73 (s, 1H, ArH), 7.75 (s, 1H, ArH). Methyl α-bromo-4, 5-dimethoxy-2-nitrobenzeneacetate (3). To a suspension of methyl 4,5dimethoxy-2-nitrobenzeneacetate (2) (6.5 g, 0.025 mol) in CCl4 (260 mL) was added NBS (22.7 g, 0.128 mol) and AIBN (1.8 g, 0.010 mol). After refluxing the suspension overnight under N2 the reaction mixture was filtered, rinsed with CCl4 and concentrated. The residual brown syrup was purified chromatographically (Isco RediSepRf silica Gold, hexanes/EtOAc 95:5) to give 3 as a yellow syrup (3.8 g, 47%). 1HNMR (CDCl3; 500 MHz) δ: 3.79 (s, ,3H, OCH3), 3.93 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 6.24 (s, 1H, CHBr), 7.41 (s, 1H, ArH), 7.70 (s, 1H, ArH). Methyl 3-O-(4,5-dimethoxy-2-nitrophenyl)carbomethoxymethyl naloxone (5). After stirring for 15 min, a mixture of powdered activated molecular sieves (4A, 2.5 µm, 1.25 g) and naloxone (4) hydrochloride dihydrate (1.6 g, 0.004 mol) in anhydrous DMF (6 mL), anhydrous K2CO3 (2.5 g, 0.0192 mol), was added and stirring was continued for 5 min. A solution of methyl α-bromo-4,5-dimethoxy-2-nitrobenzeneacetate (3) (1.6 g, 0.005 mol) in anhydrous DMF (2 mL) was slowly added, the reaction flask was covered with foil, and the mixture was heated overnight at 40 °C under N2. As analysis by TLC (SiO2, hexanes/EtOAc; 2:1) showed the reaction to be complete EtOAc (20 mL) was added and the mixture was washed with saturated NaHCO3 (2 × 20 mL), and then with saturated NaCl (2 × 20 mL). The organic phase was dried over MgSO4, filtered and concentrated to give 5 as a light-yellow syrup (2.25 g), containing excess 3. A portion (1 g) was purified by column chromatography (SiO2, hexane → hexane/EtOAc (1:1)) to give 5 as a light-yellow solid (750 mg, 36%). HPLC: Waters Symmetry


Page 10 of 21

Page 11 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


C18, 4.6 × 250 mm, Solvent A: (10 g/L NΗ4OAc)/CH3CN (9:1) Solvent B: CH3CN/H2O (9:1), 0-5 min 5% B 5-30 min 5%→80% B, 30-35 min 80% B, 1 mL/min, λ 254 nm) tR 24.30 min and 24.67 min; m/z calcd for C30H32N2O8: 580.21 found (LC/MS, ESI+): 581.21 (M + H) for both retention times. 1HNMR (500 MHz, CD3OD) (major diastereomer) δ: 1.52, 1.85 (ABX, 2H, H15), 1.64, 2.40 ( ABX, 2H, H-7), 2.05, 2.57 (ABX, 2H, H-16), 2.14, , 3.01 (ABX, 2H, H-8), 3.02 (m, 1H, H-H-9), 3.11 (m, 2H,-H-10), 3.15 (m, 2H, H-17), 3.90 (s, 3H, ArOCH3), 3.95 (s, 3H, ArOCH3), 4.76 (s, 1H, H-5), 5.16 (m, 1H, H-19-trans) 5.24 (m, 1H, H-19-cis), 5.87 (m, 1H, H18), 6.65 (d, 8.3, 1H, H-1), 6.69 (2, 1H, CHC(O)OH)), 6.70 (d, 8.3, 1H, H-2), 7.40 (s, 1H, H-6’), 7.69 (s, 1H, H-3’); 13CNMR (125 MHz, CD3OD) (major diastereomer) δ: 23.59 (C-10), 31.35 (C-7), 32.84 (C-15), 36.76 (C-8), 44.33 (C-16), 53.10 (C(O)OCH3), 56.79 (OCH3), 57.00 (OCH3), 58.56 (C-17), 63.25 (C-9), 71.66 (C-14), 79.83 (CHC(O)OH), 92.01 (C-5), 109.38 (C3’), 113.36 (C-6’), 118.21 (C-19), 120.74 (C-1), 121.84 (C-2), 127.63 (C-1’), 129.82 (C-11), 131.81 (C-12), 140.49 (C-3), 141.35 (C-2’), 146.88 (C-4), 150.14 (C-4’), 154.74 (C-5’), 170.67 ((C(O)O), 210.17 (C-6); 1HNMR (500 MHz, CD3OD) (minor diastereomer) δ: 1.52, 1.85 (ABX, 2H, H-15), 1.64, 2.40 ( ABX, 2H, H-7), 2.05, 2.57 (ABX, 2H, H-16), 2.14, , 3.01 (ABX, 2H, H8), 3.02 (m, 1H, H-H-9), 3.11 (m, 2H,-H-10), 3.15 (m, 2H, H-17), 3.89 (s, 3H, ArOCH3), 3.94 (s, 3H, ArOCH3), 4.81 (s, 1H, H-5), 5.16 (m, 1H, H-19-trans) 5.24 (m, 1H, H-19-cis), 5.87 (m, 1H, H-18), 6.61 (d, 8.3, 1H, H-1), 6.64 (2, 1H, CHC(O)OH)), 6.85 (d, 8.3, 1H, H-2), 7.40 (s, 1H, H6’), 7.71 (s, 1H, H-3’); 13CNMR (125 MHz, CD3OD) (major diastereomer) δ: 23.48 (C-10), 31.49 (C-7), 32.72 (C-15), 36.76 (C-8), 44.50 (C-16), 53.13 (C(O)OCH3), 56.79 (OCH3), 57.00 (OCH3), 58.62 (C-17), 63.18 (C-9), 71.51 (C-14), 78.86 (CHC(O)OH), 92.29 (C-5), 109.09 (C3’), 112.02 (C-6’), 118.90 (C-19), 120.26 (C-2), 125.31 (C-1), 127.71 (C-1’), 129.23 (C-11),



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

131.6 (C-12), 140.71 (C-3), 141.41 (C-2’), 146.54 (C-4), 150.14 (C-4’), 155.07 (C-5’), 170.76 ((C(O)O), 210.21 (C-6). m/z calcd for C30H32N2O10: 580.2; found: 581.8 (M + H). 3-O-(4,5-dimethoxy-2-nitrophenyl)carbomethoxymethyl naloxone (6, CNV-NLX) Trifluoroacetate. The following operations were carried out under dim red lights. A solution of LiOH (47 mg, 1.12 mmol) in H2O (10 mL) was added to a solution of methyl 3-O-(4,5dimethoxy-2-nitrophenyl)carbomethoxymethyl naloxone (5) (216 mg, 0.372 mmol) in THF/MeOH (1:1, 20 mL) and the reaction mixture was stirred overnight at room temperature. The solvents were removed under reduced pressure and the resulting residue was purified chromatographically (ISCO C18 (43 g), H2O/CH3CN (95:5) + 0.1% TFA in H2O/CH3CN (20:80)). A second purification was carried out by TLC (C18 0.1% TFA in H2O/CH3CN (65:35). These purifications afforded 6, as the TFA salt, (150 mg, 71%) with purity > 95% (HPLC, Waters X-Bridge C18 5 µM; 4.6 mm × 100 mm; gradient from 0.1% TFA in H2O/CH3CN (95:5) to 0.1% TFA in H2O/CH3CN (20:80), 1 mL/min; λ 240 nm, (tR= 8.28 min). 1HNMR (500 MHz, CD3OD) (major diastereomer) δ: 1.66 (m, 1H, H-7), 1.69 (m, 1H, H-8), 1.99 (m, 1H, H-7), 2.22 (m, 1H, H-15), 2.72 (m, 1H, H-16), 2.80 (m, 1H, H-8), 3.00 (m, 1H, H-15),) 3.04 (m, 1H, H-10), 3.23 (m, 1H, H-16), 3.42 (m, 1H, H-10), 3.71 (d, 6.41 Hz, 1H, H-9), 3.90 (m, 3H, OCH3), 3.95 (m, 3H, OCH3), 3,97 (m, 2H, H-18), 4.91 (s, 1H, H-5), 5.66 (m, 2H, H-20), 5.94 (m, 1H, H-19), 6.63 (s, 1H, ArCHO), 6.79 (d, 8.18 Hz, 1H, H-1), 6.85 (d, 8.18 Hz, 1H- H-2), 7.42 (s, 1H, H-6’), 7.66 (s, 1H, H-3’); (minor diastereomer) (500 MHZ, CD3OD) δ: 1.66 (m, 1H, H-7), 1.69 (m, 1H, H-8), 1.99 (m, 1H, H-7), 2.22 (m, 1H, H-15), 2.72 (m, 1H, H-16), 2.80 (m, 1H, H-8), 3.00 (m, 1H, H-15), 3.05 (m, 1H, H-10), 3.23 (m, 1H, H--16), 3.42 (m, 2H, H-10), 3.71 (d, 6.41 Hz, 1H, H-9), 3.90 (m, 3H, OCH3), 3.95 (m, 3H, OCH3), 3,97 (m, 2H, H-18), 4.96 (s, 1H, H-5), 5.66 (m, 2H, H-20), 5.94 (m, 1H, H-19), 6.70 (s, 1H, ArCHO), 6.79 (d, 8.18 Hz, 1H, H-1), 6.85 (d, 8.18


Page 12 of 21

Page 13 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Hz, 1H- H-2), 7.38 (s, 1H, H-6’), 7.70 (s, 1H, H-3’); 13CNMR (major diastereomer) (125 MHz, CD3OD) δ: 24.06 (C-15), 28.72 (C-8), 32.16 (C-7), 35.61 (C-15), 47.53 (C-16), 50.50 (C-5a), 56.80 (C-18), 56.86 (OCH3), 57.03 (OCH3), 63.93 (C-15), 71.27 (C-14), 78.10 (ArCHO), 91.35 (C-5), 109.14 (C-3’), 113.16 (C-6’), 121.12 (C-2), 121.87 (C-1), 125.6 (C-1a), 126.76 (C-20), 127.70 (C-1’), 127.78 (C-17), 129.59 (C-4a), 141.89 (C-2’), 142.2 (C-3), 146.92 (C-4), 150.26 (C-4’), 155.05 (C-5’), 171.59 (C=O), 208.01 (C-6); minor diastereomer) (125 MHz, CD3OD) δ: 24.06 (C-15), 28.72 (C-8), 32.28 (C-7), 35.61 (C-15), 47.53 (C-16), 50.50 (C-5a), 56.80 (C-18), 56.86 (OCH3), 57.03 (OCH3), 63.86 (C-15), 71.02 (C-14), 79.58 (ArCHO), 91.06 (C-5), 109.46 (C-3’), 111.64 (C-6’), 121.12 (C-2), 121.997 (C-1), 126.16(C-1a), 126.76 (C-20), 127.70 (C-1’), 127.78 (C-17), 129.84 (C-4a), 141.66 (C-2’), 142.12 (C-3), 147.02 (C-4), 150.19 (C-4’), 154.72 (C-5’), 171.83 (C=O), 207.96 (C-6). Irradiation of 3-O-(4,5-dimethoxy-2-nitrophenyl)carbomethoxymethyl naloxone (6, CNV-NLX). Solutions of 6•TFA were analyzed by HPLC under the following conditions: Phenomenex Gemini-NX (150 × 4.6 mm, 3 µm), solvent A: [5 mM Na2HPO4), solvent B: CH3CN, 0-20 min 20→80% B, 20-30 min 80% B, 0.8 mL/min, λ1: 210 nm, λ2: 350 nm. tR for 6: 10.9 min; for 4: 14.9 min. 6•TFA in Methanol D4—Low Energy 405 nm. A solution of CNV-NLX (6) trifluoroacetate in CD3OD was irradiated at 405 nm (by the UV detector) for up to 2.5 hours. No change in composition was observed by 1HNMR and HPLC. 6•TFA in Physiologic Medium—Low Energy 405 nm and Tungsten. A solution of CNV-NLX (6) trifluoroacetate (0.5 mM) in phosphate-buffered brine (25 mM Na2HPO4-H2O, pH7.2, containing NaCl) in a HPLC cuvette was irradiated at 405 nm (by the UV detector) for up to 2 hours. No



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

change in composition was observed by HPLC. The solution was irradiated with a 300W tungsten lamp for 30 min; HPLC analysis indicated no change in composition. 6•TFA in Physiologic Medium—365 nm. A solution of CNV-NLX (6) trifluoroacetate (0.8 mg, 0.001 mmol) in phosphate-buffered brine (25 mM Na2HPO4-H2O, pH7.2, containing NaCl) (1 mL) was irradiated with a hand-held UV lamp set at long wavelength (365 nm) for 30 min, 1.5 hours, and 2.5 hours. After 2.5-hour irradiation a light brown solution formed. HPLC analysis showed only 4; no 6 was detected. 6•TFA in Methanol—365 nm. A solution CNV-NLX (6) trifluoroacetate in MeOH was irradiated with a hand-held UV lamp set at long wavelength (365 nm) for 30 min, 2.5 hours, and 3.5 hours. After 3.5-hour a light brown solution formed. HPLC analysis showed the presence of 4 and a trace amount of 6. 6•TFA in Dimethyl Sulfoxide (DMSO)—365 nm. A colorless solution of CNV-NLX (6) trifluoroacetate (1 mg, 0.002 mmol) in DMSO (1 mL) in a UV cuvette (1 mL) was irradiated with a hand-held UV lamp set at long wavelength (365 nm) for 2.5 hours to give a light brown solution. HPLC analysis showed only 4 (no 6). 6•TFA in DMSO-D6—365 nm. A colorless solution of CNV-NLX (6) trifluoroacetate (18 mg, 0.03 mmol) in DMSO-D6 (0.75 mL) in a UV curvette (1 mL) was irradiated with a hand-held UV lamp set at long wavelength (365 nm) for 45 min, 2.5 hours, 5 hours, and 10 hours and the photolysis reaction was monitored by HPLC. After 10-hour irradiation a light brown solution formed which contained 4 and no 6. 1HNMR (300 MHz, DMSO-d6) δ: 1.51 (m, 2H), 1.97 (d, J = 12.2 Hz, 1H), 2.11 (m, 1H), 2.62 (m, 1H), 2.92 (m, 2H), 3.13 (m, 1H), 3.58 (m. 1H), 3.76 (m. 1H), 3.72–3.89 (m, 4H), 3.94 (m, 1H), 4.95 (s, 1H), 5.52 (d, J = 10.3 Hz, 1H), 5.61 (d, J = 17.1 Hz, 1H), 5.84 (m, 1H), 6.63 (d, J = 8.3 Hz, 1H), 6.68 (d, J = 7.8 Hz, 1H), 6.75–7.50 (m, 2H),


Page 14 of 21

Page 15 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.40 ( s., 1H), 9.52 (s, 1H). 13C NMR (75 MHz, DMSO-d6) ppm: 207.6, 143.5, 140.2, 127.8, 127.7, 124.7, 120.5, 119.8, 118.1, 88.5, 69.8, 61.3, 55.1, 48.6, 46.0, 34.9, 30.6, 27.1, 22.4. 4.

CONCLUSIONS The synthesis of 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (caged

naloxone, CNV-NLX (6)), on a practical laboratory scale and using only common reagents, has been carried out and the product, which is a mixture of αR,5R 9R,13S,14S and αS,5R,9R,13S,14S diastereomers has been thoroughly characterized. The 1H and 13CNMR resonances of both diastereomers have been fully assigned, confirming the structures. Solutions of CNV-NLX in saline buffer, in methanol and in DMSO have been found to be stable for over a week under fluorescent laboratory lights at room temperature. Exposure of such solutions to λ 365 nm from a hand-held UV lamp led to the formation of naloxone and CNV-related breakdown products. AUTHOR INFORMATION Corresponding Author Telephone: 1-919-541-6691 Fax: 1-919-541-8868 Email: [email protected] ORCID Anita H. Lewin: orcid.org/0000-0001-6020-322X Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding This research was supported by National Institute on Drug Abuse Contract No. HHSN2712013000007C.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABBREVIATIONS CNV-NLX, carboxynitroveratryl naloxone; NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; SEMO-CNV-Br, [2-(trimethylsilyl)ethoxy]methyl protected carboxynitroveratryl bromide; DNA, Deoxyribonucleic acid; RNA, ribonucleic acid; NBS, N-bromosuccinimice; AIBN, azobisisobutyronitrile; TLC, thin layer chromatography; HPLC. High pressure liquid chromatography; UV, ultraviolet; TFA, trifluoroacetic acid; tR, retention time; mL, milliliter; DMF, dimethyl formamide; CCl4, carbon tetrachloride; CH3OH, methanol; CD3OD, perdeuterated methanol. SUPPORTING INFORMATION: HPLC traces showing the monitoring of the phototylic decomposition of CNV-NLX in aqueous sodium phosphate/NaCl (pH 7.2) buffer. This information is available free of charge via the Internet at http://pubs.acs.org. REFERENCES [1]

Klan, P., Solomek, T., Bochet, C. G., Blanc, A., Givens, R., Rubina, M., Popik, V., Kostikov, A., and Wirz, J. (2013) Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy, Chem Rev 113, 119-191.

[2]

Morley, J. S. (1980) Structure-activity relationships of enkephalin-like peptides, Annu Rev Pharmacol Toxicol 20, 81-110.

[3]

Chavkin, C., and Goldstein, A. (1981) Specific receptor for the opioid peptide dynorphin: structure--activity relationships, Proc Natl Acad Sci U S A 78, 6543-6547.

[4]

Sreekumar, R., Ikebe, M., Fay, F. S., and Walker, J. W. (1998) Biologically active peptides caged on tyrosine, Methods Enzymol 291, 78-94.

[5]

Tatsu, Y., Shigeri, Y., Sogabe, S., Yumoto, N., and Yoshikawa, S. (1996) Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting


Page 16 of 21

Page 17 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


group: application to the synthesis of caged neuropeptide Y, Biochem Biophys Res Commun 227, 688-693. [6]

Banghart, M. R., Williams, J. T., Shah, R. C., Lavis, L. D., and Sabatini, B. L. (2013) Caged naloxone reveals opioid signaling deactivation kinetics, Mol Pharmacol 84, 687695.

[7]

Russell, A. G., Ragoussi, M. E., Ramalho, R., Wharton, C. W., Carteau, D., Bassani, D. M., and Snaith, J. S. (2010) Alpha-carboxy-6-nitroveratryl: a photolabile protecting group for carboxylic acids, J. Org. Chem. 75, 4648-4651.

[8]

Woodrell, C. D., Kehayova, P. D., and Jain, A. (1999) Photochemically-triggered decarboxylation/deamination of o-nitrodimethoxyphenylglycine, Org Lett 1, 619-621.

[9]

Valentova, J., Bauerova, K., Farah, L., and Devinsky, F. (2010) Does stereochemistry influence transdermal permeation of flurbiprofen through the rat skin?, Arch Dermatol Res 302, 635-638.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OMe MeO

Page 18 of 21

OMe O

MeO

H2SO4 OH

OMe MeO

O

MeOH

OMe

NO2

NO2

1

2

O

NBS/AIBN CCl4

OMe NO2 Br 3 HO DMF K2CO3, 4Å molecular sieves

O N OH O

OMe

OMe

4

MeO

MeO

O

O

OMe

OH LiOH

NO2 O

NO2 O

O

O

N

N OH O

OH O 5

6


Page 19 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55

OMe 5'

MeO

4'

6'

O

1'

3'

OH

2'

2

NO2 O

3

1

4

11 16

15 9

13

5

14 6

O

10

12

O

OH

N

18

20 19

8 7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

OMe

Page 20 of 21

OMe

MeO

MeO

O

H

OH O

N

O

O

N


O

Page 21 of 21

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


55

OMe

OMe

MeO

MeO

O (S)

O (R)

OH

OH

NO2 O

NO2 O 4

O (R)

O

(S)

O

(R) (S)

N

(R)

OH O


(S)

(R) (S)

N OH

Caged Naloxone: Synthesis, Characterization, and Stability of 3-O-(4,5

Recommend Documents