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

Article abstract of DOI:10.1021/acschemneuro.7b00378

The photolabile analogue of the broad-spectrum opioid antagonist naloxone, 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl naloxone (also referred to as "caged naloxone", 3-O-(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 NMR and ¹³C NMR 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.

Full text of DOI:10.1021/acschemneuro.7b00378

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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
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Caged Naloxone: Synthesis, Characterization, and Stability of 3-O-(4,5-dimethoxy-2-
nitrophenyl)carboxymethyl Naloxone (CNV-NLX)
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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
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ABSTRACT: The photoꢀlabile analog of the broadꢀspectrum opioid antagonist naloxone, 3ꢀOꢀ
(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carboxymethyl naloxone (also referred to as “caged naloxone”, 3ꢀ
Oꢀ(αꢀcarboxyꢀ6ꢀnitroveratryl)naloxone, CNVꢀNLX), has been found to be a valuable
biochemical probe. While the synthesis of CNVꢀNLX 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 CNVꢀNLX in saline buffer, in methanol and in DMSO has
shown CNVꢀNLX to be stable for over a week under fluorescent laboratory lights at room
temperature. Exposure of such solutions to λ 365 nm from a handꢀheld UV lamp led to the
formation of naloxone and CNVꢀrelated breakdown products.
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KEYWORDS: photolabile, diastereomers, 3ꢀOꢀ(αꢀcarboxyꢀ6ꢀnitroveratryl)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 counterꢀindicated.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 2ꢀnitrophenyl group that defines a family of
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photolabile protecting groups that includes the 2ꢀnitrobenzyl and 4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl
groups.
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Recently photoactivatable protecting groups have been used to achieve high
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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
brainꢀsite, 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 2ꢀnitrophenyl chromophore that was modified by the addition of a carboxyl functionality to
achieve water solubility resulting in a αꢀcarboxynitrobenzyl group that was used to protect the
tyrosyl residue as an ether. The thusꢀprotected 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 photoꢀreleased
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 4,5ꢀdimethoxyꢀ2ꢀnitrobenzyl analog ((αꢀ
carboxyꢀ6ꢀnitroveratryl), 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 broadꢀ
spectrum opioid antagonist naloxone, thereby providing a means to quantitatively measure
spatiotemporal dynamics of opioid signaling dynamics and their underlying mechanisms in brain
tissue.
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Recognizing the importance of this pharmacologic tool, we report our preparation and
thorough characterization of 3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carboxymethyl naloxone (CNVꢀ
NLX).
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2.
RESULTS AND DISCUSSION
The synthesis of the “caged naloxone” nicknamed CNVꢀNLX (6) (Scheme 1) was carried
out with only slight modification of the published methodology.6 Thus, whereas [2ꢀ
(trimethylsilyl)ethoxy]methyl (SEM)ꢀprotected carboxynitroveratryl (CNV)ꢀbromide (SEMOꢀ
CNVꢀBr) was used as the “caging agent” in the published preparation of CNVꢀNLX (6), we
opted to use the analogous methyl αꢀbromoꢀ4,5ꢀdimethoxyꢀ2ꢀnitrobenzeneacetate (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, CNV-
NLX)
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by converting commercially procured 4, 5ꢀdimethoxyꢀ2ꢀnitrobenzeneacetic acid (1) to the
corresponding methyl ester 2 by acidꢀpromoted esterification in methanol (83% yield), followed
by bromination of 2 using Nꢀbromosuccinimide (NBS) and the radical initiator 2,2'ꢀ(1,2ꢀ
diazenediyl)bis[2ꢀmethylpropanenitrile) (AIBN) in carbon tetrachloride. The
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chromatographically purified product, methyl αꢀbromoꢀ4,5ꢀdimethoxyꢀ2ꢀnitrrobenzeneacetate
(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 3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀ
nitrophenyl)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 CNVꢀNLX (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, CNVꢀNLX (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
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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
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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 CNVꢀNLX (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 Hꢀ10, where the axial and equatorial
protons are observed as separate resonances in the major diastereomer, but are coincident in the
minor diastereomer.
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To determine the light sensitivity of CNVꢀNLX (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 handꢀheld UV lamp at λ 365 nm for up to 2.5 hours led to
total disappearance of the peak for CNVꢀNLX (6) and to the appearance of a peak for naloxone
(4), confirming the photoꢀrelease of intact naloxone (4) from CNVꢀNLX (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
breakꢀdown 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 methoxyꢀsubstituted aromatic species. The mechanism for release of CNVꢀ
like caging groups has been described8 as proceeding by n—>π* excitation to generate an aciꢀ
nitro intermediate which expels the “uncaged” ligand and affords 4,5ꢀdimethoxyꢀ2ꢀnitrosoꢀαꢀ
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oxobenzeneacetic acid (7, Figure 2) which, in turn, decarboxylates to give 4,5ꢀdimethoxyꢀ2ꢀ
nitrosobenzaldehyde (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 UVꢀabsorbing, and thus may serve to
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Figure 2. Breakdown Products of 3-O-(4,5-dimethoxy-2-nitrophenyl)carboxymethyl
Naloxone (6, CNV-NLX)
slow down the photochemical formation of the aciꢀnitro 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 rateꢀdetermining 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 photoꢀrelease of naloxone (4) from
CNVꢀNLX (6) may not be biocompatible with the system being investigated.
The fact that the CNVꢀNLX (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 αꢀbromoꢀ4, 5ꢀdimethoxyꢀ2ꢀ
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nitrobenzeneacetate (3) is probably not a good choice, since chiral integrity at the αꢀposition
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would be readily compromised due to the lability of the proton α to the carboxyl group.
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3.
MATERIALS AND METHODS
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Instrumentation. 1HNMR spectra were acquired on a Bruker Avance 300 MHz
spectrometer, or a 500 MHz NMR spectrometer. Lowꢀresolution LCꢀMS 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 diodeꢀarray
detector) controlled by ChemStation HPLC software. The water used in HPLC solvent systems
was obtained from a Millipore MilliꢀQ Plus UltraꢀPure Water System.
Materials. Naloxone hydrochloride dihydrate was from the NIDA; (4,5ꢀdimethoxyꢀ2ꢀ
nitrophenyl)acetic acid was from JohnsonꢀMatthey; 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, 5ꢀdimethoxyꢀ2ꢀnitrobenezeneacetate (2). To a suspension of 4,5ꢀdimethoxyꢀ2ꢀ
nitrobenzeneacetic 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
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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%).
1HNMR (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).
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Methyl αꢀbromoꢀ4, 5ꢀdimethoxyꢀ2ꢀnitrobenzeneacetate (3). To a suspension of methyl 4,5ꢀ
dimethoxyꢀ2ꢀnitrobenzeneacetate (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 3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carbomethoxymethyl naloxone (5). After
stirring for 15 min, a mixture of powdered activated molecular sieves (4A, 2.5 ꢁm, 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 αꢀbromoꢀ4,5ꢀdimethoxyꢀ2ꢀnitrobenzeneacetate (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 lightꢀyellow 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 lightꢀyellow solid (750 mg, 36%). HPLC: Waters Symmetry
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C18, 4.6 × 250 mm, Solvent A: (10 g/L NΗ4OAc)/CH3CN (9:1) Solvent B: CH3CN/H2O (9:1),
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0ꢀ5 min 5% B 5ꢀ30 min 5%→80% B, 30ꢀ35 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, Hꢀ
15), 1.64, 2.40 ( ABX, 2H, Hꢀ7), 2.05, 2.57 (ABX, 2H, Hꢀ16), 2.14, , 3.01 (ABX, 2H, Hꢀ8), 3.02
(m, 1H, HꢀHꢀ9), 3.11 (m, 2H,ꢀHꢀ10), 3.15 (m, 2H, Hꢀ17), 3.90 (s, 3H, ArOCH3), 3.95 (s, 3H,
ArOCH3), 4.76 (s, 1H, Hꢀ5), 5.16 (m, 1H, Hꢀ19ꢀtrans) 5.24 (m, 1H, Hꢀ19ꢀcis), 5.87 (m, 1H, Hꢀ
18), 6.65 (d, 8.3, 1H, Hꢀ1), 6.69 (2, 1H, CHC(O)OH)), 6.70 (d, 8.3, 1H, Hꢀ2), 7.40 (s, 1H, Hꢀ6’),
7.69 (s, 1H, Hꢀ3’); 13CNMR (125 MHz, CD3OD) (major diastereomer) δ: 23.59 (Cꢀ10), 31.35
(Cꢀ7), 32.84 (Cꢀ15), 36.76 (Cꢀ8), 44.33 (Cꢀ16), 53.10 (C(O)OCH3), 56.79 (OCH3), 57.00
(OCH3), 58.56 (Cꢀ17), 63.25 (Cꢀ9), 71.66 (Cꢀ14), 79.83 (CHC(O)OH), 92.01 (Cꢀ5), 109.38 (Cꢀ
3’), 113.36 (Cꢀ6’), 118.21 (Cꢀ19), 120.74 (Cꢀ1), 121.84 (Cꢀ2), 127.63 (Cꢀ1’), 129.82 (Cꢀ11),
131.81 (Cꢀ12), 140.49 (Cꢀ3), 141.35 (Cꢀ2’), 146.88 (Cꢀ4), 150.14 (Cꢀ4’), 154.74 (Cꢀ5’), 170.67
((C(O)O), 210.17 (Cꢀ6); 1HNMR (500 MHz, CD3OD) (minor diastereomer) δ: 1.52, 1.85 (ABX,
2H, Hꢀ15), 1.64, 2.40 ( ABX, 2H, Hꢀ7), 2.05, 2.57 (ABX, 2H, Hꢀ16), 2.14, , 3.01 (ABX, 2H, Hꢀ
8), 3.02 (m, 1H, HꢀHꢀ9), 3.11 (m, 2H,ꢀHꢀ10), 3.15 (m, 2H, Hꢀ17), 3.89 (s, 3H, ArOCH3), 3.94 (s,
3H, ArOCH3), 4.81 (s, 1H, Hꢀ5), 5.16 (m, 1H, Hꢀ19ꢀtrans) 5.24 (m, 1H, Hꢀ19ꢀcis), 5.87 (m, 1H,
Hꢀ18), 6.61 (d, 8.3, 1H, Hꢀ1), 6.64 (2, 1H, CHC(O)OH)), 6.85 (d, 8.3, 1H, Hꢀ2), 7.40 (s, 1H, Hꢀ
6’), 7.71 (s, 1H, Hꢀ3’); 13CNMR (125 MHz, CD3OD) (major diastereomer) δ: 23.48 (Cꢀ10),
31.49 (Cꢀ7), 32.72 (Cꢀ15), 36.76 (Cꢀ8), 44.50 (Cꢀ16), 53.13 (C(O)OCH3), 56.79 (OCH3), 57.00
(OCH3), 58.62 (Cꢀ17), 63.18 (Cꢀ9), 71.51 (Cꢀ14), 78.86 (CHC(O)OH), 92.29 (Cꢀ5), 109.09 (Cꢀ
3’), 112.02 (Cꢀ6’), 118.90 (Cꢀ19), 120.26 (Cꢀ2), 125.31 (Cꢀ1), 127.71 (Cꢀ1’), 129.23 (Cꢀ11),
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131.6 (Cꢀ12), 140.71 (Cꢀ3), 141.41 (Cꢀ2’), 146.54 (Cꢀ4), 150.14 (Cꢀ4’), 155.07 (Cꢀ5’), 170.76
((C(O)O), 210.21 (Cꢀ6). m/z calcd for C30H32N2O10: 580.2; found: 581.8 (M + H).
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3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carbomethoxymethyl naloxone (6, CNV-NLX)
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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 3ꢀOꢀ(4,5ꢀ
dimethoxyꢀ2ꢀnitrophenyl)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 XꢀBridge C18 5 ꢁM; 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, Hꢀ7), 1.69 (m, 1H, Hꢀ8), 1.99 (m, 1H, Hꢀ7), 2.22
(m, 1H, Hꢀ15), 2.72 (m, 1H, Hꢀ16), 2.80 (m, 1H, Hꢀ8), 3.00 (m, 1H, Hꢀ15),) 3.04 (m, 1H, Hꢀ10),
3.23 (m, 1H, Hꢀ16), 3.42 (m, 1H, Hꢀ10), 3.71 (d, 6.41 Hz, 1H, Hꢀ9), 3.90 (m, 3H, OCH3), 3.95
(m, 3H, OCH3), 3,97 (m, 2H, Hꢀ18), 4.91 (s, 1H, Hꢀ5), 5.66 (m, 2H, Hꢀ20), 5.94 (m, 1H, Hꢀ19),
6.63 (s, 1H, ArCHO), 6.79 (d, 8.18 Hz, 1H, Hꢀ1), 6.85 (d, 8.18 Hz, 1Hꢀ Hꢀ2), 7.42 (s, 1H, Hꢀ6’),
7.66 (s, 1H, Hꢀ3’); (minor diastereomer) (500 MHZ, CD3OD) δ: 1.66 (m, 1H, Hꢀ7), 1.69 (m, 1H,
Hꢀ8), 1.99 (m, 1H, Hꢀ7), 2.22 (m, 1H, Hꢀ15), 2.72 (m, 1H, Hꢀ16), 2.80 (m, 1H, Hꢀ8), 3.00 (m,
1H, Hꢀ15), 3.05 (m, 1H, Hꢀ10), 3.23 (m, 1H, Hꢀꢀ16), 3.42 (m, 2H, Hꢀ10), 3.71 (d, 6.41 Hz, 1H,
Hꢀ9), 3.90 (m, 3H, OCH3), 3.95 (m, 3H, OCH3), 3,97 (m, 2H, Hꢀ18), 4.96 (s, 1H, Hꢀ5), 5.66 (m,
2H, Hꢀ20), 5.94 (m, 1H, Hꢀ19), 6.70 (s, 1H, ArCHO), 6.79 (d, 8.18 Hz, 1H, Hꢀ1), 6.85 (d, 8.18
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Hz, 1Hꢀ Hꢀ2), 7.38 (s, 1H, Hꢀ6’), 7.70 (s, 1H, Hꢀ3’); 13CNMR (major diastereomer) (125 MHz,
CD3OD) δ: 24.06 (Cꢀ15), 28.72 (Cꢀ8), 32.16 (Cꢀ7), 35.61 (Cꢀ15), 47.53 (Cꢀ16), 50.50 (Cꢀ5a),
56.80 (Cꢀ18), 56.86 (OCH3), 57.03 (OCH3), 63.93 (Cꢀ15), 71.27 (Cꢀ14), 78.10 (ArCHO), 91.35
(Cꢀ5), 109.14 (Cꢀ3’), 113.16 (Cꢀ6’), 121.12 (Cꢀ2), 121.87 (Cꢀ1), 125.6 (Cꢀ1a), 126.76 (Cꢀ20),
127.70 (Cꢀ1’), 127.78 (Cꢀ17), 129.59 (Cꢀ4a), 141.89 (Cꢀ2’), 142.2 (Cꢀ3), 146.92 (Cꢀ4), 150.26
(Cꢀ4’), 155.05 (Cꢀ5’), 171.59 (C=O), 208.01 (Cꢀ6); minor diastereomer) (125 MHz, CD3OD) δ:
24.06 (Cꢀ15), 28.72 (Cꢀ8), 32.28 (Cꢀ7), 35.61 (Cꢀ15), 47.53 (Cꢀ16), 50.50 (Cꢀ5a), 56.80 (Cꢀ18),
56.86 (OCH3), 57.03 (OCH3), 63.86 (Cꢀ15), 71.02 (Cꢀ14), 79.58 (ArCHO), 91.06 (Cꢀ5), 109.46
(Cꢀ3’), 111.64 (Cꢀ6’), 121.12 (Cꢀ2), 121.997 (Cꢀ1), 126.16(Cꢀ1a), 126.76 (Cꢀ20), 127.70 (Cꢀ1’),
127.78 (Cꢀ17), 129.84 (Cꢀ4a), 141.66 (Cꢀ2’), 142.12 (Cꢀ3), 147.02 (Cꢀ4), 150.19 (Cꢀ4’), 154.72
(Cꢀ5’), 171.83 (C=O), 207.96 (Cꢀ6).
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Irradiation of 3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carbomethoxymethyl naloxone (6, CNV-NLX).
Solutions of 6•TFA were analyzed by HPLC under the following conditions: Phenomenex
GeminiꢀNX (150 × 4.6 mm, 3 µm), solvent A: [5 mM Na2HPO4), solvent B: CH3CN, 0ꢀ20 min
20→80% B, 20ꢀ30 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 CNVꢀNLX (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 CNVꢀNLX (6)
trifluoroacetate (0.5 mM) in phosphateꢀbuffered brine (25 mM Na2HPO4ꢀH2O, pH7.2, containing
NaCl) in a HPLC cuvette was irradiated at 405 nm (by the UV detector) for up to 2 hours. No
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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 CNVꢀNLX (6) trifluoroacetate (0.8 mg,
0.001 mmol) in phosphateꢀbuffered brine (25 mM Na2HPO4ꢀH2O, pH7.2, containing NaCl) (1
mL) was irradiated with a handꢀheld UV lamp set at long wavelength (365 nm) for 30 min, 1.5
hours, and 2.5 hours. After 2.5ꢀhour irradiation a light brown solution formed. HPLC analysis
showed only 4; no 6 was detected.
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6•TFA in Methanol—365 nm. A solution CNVꢀNLX (6) trifluoroacetate in MeOH was irradiated
with a handꢀheld UV lamp set at long wavelength (365 nm) for 30 min, 2.5 hours, and 3.5 hours.
After 3.5ꢀhour 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 CNVꢀNLX (6)
trifluoroacetate (1 mg, 0.002 mmol) in DMSO (1 mL) in a UV cuvette (1 mL) was irradiated
with a handꢀheld 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 DMSOꢀD6—365 nm. A colorless solution of CNVꢀNLX (6) trifluoroacetate (18 mg,
0.03 mmol) in DMSOꢀD6 (0.75 mL) in a UV curvette (1 mL) was irradiated with a handꢀheld
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 10ꢀhour irradiation a light brown solution
formed which contained 4 and no 6. 1HNMR (300 MHz, DMSOꢀd6) 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),
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9.40 ( s., 1H), 9.52 (s, 1H). 13C NMR (75 MHz, DMSOꢀd6) 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.
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CONCLUSIONS
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The synthesis of 3ꢀOꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)carboxymethyl naloxone (caged
naloxone, CNVꢀNLX (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 CNVꢀNLX 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
handꢀheld UV lamp led to the formation of naloxone and CNVꢀrelated breakdown products.
AUTHOR INFORMATION
Corresponding Author
Telephone: 1ꢀ919ꢀ541ꢀ6691
Fax: 1ꢀ919ꢀ541ꢀ8868
Email: ahl.emeritus@rti.org
ORCID
Anita H. Lewin: orcid.org/0000ꢀ0001ꢀ6020ꢀ322X
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.
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ABBREVIATIONS
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CNVꢀNLX, carboxynitroveratryl naloxone; NMR, nuclear magnetic resonance; DMSO,
dimethyl sulfoxide; THF, tetrahydrofuran; SEMOꢀCNVꢀBr, [2ꢀ(trimethylsilyl)ethoxy]methyl
protected carboxynitroveratryl bromide; DNA, Deoxyribonucleic acid; RNA, ribonucleic acid;
NBS, Nꢀbromosuccinimice; 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.
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SUPPORTING INFORMATION: HPLC traces showing the monitoring of the phototylic
decomposition of CNVꢀNLX 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, 119ꢀ191.
[2] Morley, J. S. (1980) Structureꢀactivity relationships of enkephalinꢀlike peptides, Annu Rev
Pharmacol Toxicol 20, 81ꢀ110.
[3] Chavkin, C., and Goldstein, A. (1981) Specific receptor for the opioid peptide dynorphin:
structureꢀꢀactivity relationships, Proc Natl Acad Sci U S A 78, 6543ꢀ6547.
[4] Sreekumar, R., Ikebe, M., Fay, F. S., and Walker, J. W. (1998) Biologically active peptides
caged on tyrosine, Methods Enzymol 291, 78ꢀ94.
[5] Tatsu, Y., Shigeri, Y., Sogabe, S., Yumoto, N., and Yoshikawa, S. (1996) Solidꢀphase
synthesis of caged peptides using tyrosine modified with a photocleavable protecting
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group: application to the synthesis of caged neuropeptide Y, Biochem Biophys Res
Commun 227, 688ꢀ693.
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[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, 687ꢀ
695.
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[7] Russell, A. G., Ragoussi, M. E., Ramalho, R., Wharton, C. W., Carteau, D., Bassani, D. M.,
and Snaith, J. S. (2010) Alphaꢀcarboxyꢀ6ꢀnitroveratryl: a photolabile protecting group for
carboxylic acids, J. Org. Chem. 75, 4648ꢀ4651.
[8] Woodrell, C. D., Kehayova, P. D., and Jain, A. (1999) Photochemicallyꢀtriggered
decarboxylation/deamination of oꢀnitrodimethoxyphenylglycine, Org Lett 1, 619ꢀ621.
[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, 635ꢀ638.
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OMe
OMe
OMe
MeO
MeO
MeO
O
O
O
H2SO4
MeOH
NBS/AIBN
CCl4
OH
OMe
OMe
NO2
NO2
NO2 Br
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2
3
HO
DMF
K2CO3, 4Å
molecular
sieves
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N
OH
O
OMe
OMe
4
MeO
MeO
O
O
OMe
OH
LiOH
NO2
O
NO2
O
O
O
N
N
OH
OH
O
O
5
6
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OMe
5'
MeO
4'
6'
1'
O
3'
OH
2
2'
NO2
O
3
1
4
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16
O
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20
9
N
5
14
19
OH
6
8
O
7
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OMe
OMe
MeO
MeO
O
H
OH
N
O
N
O
O
O
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52
53
54
55
56
57
58
59
60
OMe
NO2
OMe
MeO
MeO
O
O
(S)
(R)
OH
OH
O
NO2
O
4
O
O
(S)
(S)
(R)
(R)
(R)
N
(R)
N
(S)
(S)
OH
OH
O
O
ACS Paragon Plus Environment