Microwave-Assisted Synthesis of Nucleoside Acyl-sulfamate Backbone HINT1 Inhibitors
Andrew Zhou, Rachit Shah, & Carston R. Wagner
University of Minnesota, Department of Medicinal Chemistry
12/12/2014
Abstract
Synthetic inhibitors of histidine triad nucleotide binding protein 1 (HINT1) have shown promise
as potential therapeutic adjuvants to morphine. HINT1 plays a regulatory role in opioid signaling
pathways.1 Preparation of HINT1 inhibitors involves a multistep synthesis, with the rate-limiting
coupling step traditionally performed under ambient conditions requiring overnight incubation.
Microwave chemistry offers a means of shortening the overnight reaction to a span of minutes.
The coupling step was attempted via microwave assisted synthesis on the HINT1 inhibitor
intermediates with acyl-sulfamate backbones: 5’-O-(N-(Indole) sulfamoyl)2’,3’-isopropylidine
guanosine triethylammonium salt (IndGst) and 5’-O-(N-(butyrate) sulfamoyl)2’,3’-
isopropylidene guanosine triethylammonium salt (butyGSt). Yields of 39 % and 29 % were
obtained for IndGstKp and butyGStKp, respectively, in reaction times of roughly 10 minutes.
The formation of side products evidenced by flash chromatography and 1H NMR spectroscopy in
conjunction with the meager yields indicate that the microwave adapted syntheses led to a
significant reduction in reaction efficiency. Parameters such as solvents, temperatures, and time
points could be modified in successive attempts to assess whether higher yields may be obtained.
Introduction
The recently discovered link between histidine triad nucleotide-binding protein 1 (HINT1) and
opioid tolerance offers a promising strategy for optimizing the therapeutic use of opioids.1
Opioid based pain medications like morphine are commonly administered to postoperative
patients and others experiencing moderate to severe pain. However, the clinical use of opioids is
limited by the onset of acute opioid tolerance following initial dosing. Dosing periods are often
shortened and dosages are increased to maintain adequate pain control. This can lead to a variety
of adverse side effects, such as nausea, vomiting, and respiratory insufficiency.2 We have
recently shown that HINT1 plays a critical role in the development of acute opioid tolerance.1
Like many other opioids, morphine induced analgesia is produced through activation of the μ-
opioid receptor (MOR) in the central nervous system.3 The analgesic effect is then terminated by
subsequent activation of the nearby N-methyl D-aspartate receptor (NMDAR), which negatively
regulates MOR signaling.3 HINT1, which is widely expressed in the central nervous system,
facilitates NMDAR activation by interacting with the C terminus of both MOR and NMDAR.4
The importance of HINT1 in NMDAR activation has recently been demonstrated in HINT1
knockout mice, which display enhanced morphine analgesia and a failure to develop NMDAR
mediated opioid tolerance.1
The relationship between HINT1 and opioid tolerance was further explored by synthesizing two
series of HINT1 inhibitors, designed by replacing the hydrolysable backbone of HINT1 substrate
adenosine 5’-indole-3-propionic adenylate (AIPA) with a non-hydrolyzsable backbone (Figure
1). The first series was designed with a carbamate backbone, while the second series was
designed with a sulfamate backbone. As substrate mimics, the HINT1 inhibitors act via
competitive inhibition.5 Intravenous co-administration of morphine along with guanosine-5’-
tryptamine carbamate (TrpGc), an inhibitor from the carbamate series, has shown effective in a
mouse model at both enhancing morphine analgesia and preventing the onset of acute opioid
tolerance.1 Animal studies have yet to be performed using the sulfamate inhibitors, although they
possess the added advantage of complete water solubility and they more closely mimic the
structure of AIPA.
The preparation of HINT1 inhibitors involves a multistep synthesis, with the rate-limiting step
being a coupling reaction (Figures 2 & 3). The coupling step has previously been performed
under ambient conditions requiring overnight incubation. However, the advent of microwave
chemistry introduces a potential means of condensing this overnight process into a reaction that
can be completed in the course of minutes. Microwave chemistry involves the excitation of polar
molecules with microwave radiation to drive chemical reactions at astonishing rates.6 Molecules
realign their dipoles with the high frequency electric field produced by the microwave radiation.
The kinetic energy from this realignment along with the electric field excitation results in the
highly efficient superheating of the molecules, effectively shortening reaction times from days to
minutes and hours to seconds. In some cases, microwave adapted synthesis has even been shown
to increase yields and reduce the formation of side products.6
While the first reported use of microwave chemistry was over 25 years ago, 7 only during the past
decade has it evolved into an established technique widely used to assist in organic synthesis.8
Over the first decade following its introduction, microwave assisted syntheses were largely
attempted in traditional microwaves such as those found in kitchens. The sudden rise in interest
in microwave synthesis is attributed to the development of sophisticated microwave instruments
specialized for performing organic synthesis, which have become increasingly available over the
last ten years.6 These instruments are programmable with precise temperature, pressure, and time
controls, allowing for reaction conditions to be optimized for a specific reaction. The shift to
specialized instruments has also significantly reduced the risks associated with the superheating
of highly flammable organic solvents.6
We have previously performed the microwave assisted synthesis of HINT1 inhibitors from the
carbamate series, with the conditions optimized through successive experiments until similar
yields could be achieved (unpublished results). The synthesis of HINT1 inhibitors belonging to
the sulfamate series has yet to be attempted under microwave conditions. The objective of this
work is to apply microwave chemistry to the coupling step in the synthesis of two inhibitors
belonging to the sulfamate series, 5’-O-(N-(Indole) sulfamoyl)2’,3’-isopropylidine guanosine
triethylammonium salt (IndGst) and 5’-O-(N-(butyrate) sulfamoyl)2’,3’-isopropylidene
guanosine triethylammonium salt (butyGSt). The overall synthetic scheme for each inhibitor
involves four steps: 1. Protection of guanosine, 2. Attachment of a sulfamate group, 3. Coupling
with side chain, 4. Deprotection (Figures 2 & 3). As IndGSt and butyGSt differ only in the
identity of the side chain, the product of the second step could be used to proceed to the third
coupling step in the synthesis of each inhibitor. The first, second, and final steps are performed
under ambient conditions, as they all involve reaction times of two hours or less. The rate-
limiting coupling step, typically requiring overnight incubation, is completed through microwave
assisted synthesis to form IndGStKp and butyGStKp (Kp denotes the ketal protected 2’ and 3’
hydroxyl groups on the ribose unit of guanosine). Based on the yields obtained for IndGStKp and
butyGStKp and their purities determined through mass spectrometry and 1H NMR spectrometry,
an assessment can be made on whether the rate limiting step in the synthesis of nucleoside-acyl
sulfamate HINT1 inhibitors can be optimized by controlled microwave heating.
Materials & Methods
Materials: 2,2-dimethoxypropane, argon, p-toluenesulfonic acid monohydrate, perchloric acid,
ammonium hydroxide, acetone, methanol, dichloromethane, triethylamine, chloroform, 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU), and dimethylformamide (DMF) were purchased from
Sigma-Aldrich. Guanosine was purchased from Acros Organics. The synthetic chemistry was
performed in 9-foot chemical fume hoods equipped for multi-step organic synthesis. Reactions
were monitored by thin layer chromatography (TLC) on silica plates. Microwave assisted
synthesis was performed using the Explorer®-12 Hybrid (CEM corporation). A standard rotary
evaporator was used to remove excess solvents. Purification was performed using a Combliflash
chromatography system (Teledyne-Isco). Products were freeze-dried using a lyophilizer
(Labconco). Product characterization was performed using a mass spectrometer (Agilent ESI)
and a 5400 MHz NMR spectrometer (Bruker).
Step 1. Protection of hydroxyl groups: The 2’ and 3’ hydroxyl groups of the ribose were first
protected in preparation for the selective reaction with sulfamoyl chloride in the second step of
the synthesis. Guanosine (5.0 g, 18 mmol) was added to a 500 mL round bottom flask and dried
under vacuum overnight. Guanosine was then suspended in 300 mL acetone, and perchloric acid
(1.25 mL) was added dropwise while under ice-bath. The reaction was allowed to run at room
temperature for 90 minutes during which the suspension became clear. TLC analysis was
performed using a 20 % methanol / 80 % chloroform / 0.1 % ammonium hydroxide solvent
system. TLC indicated major product formation at this time, but with some starting material
remaining. After an additional 20 minutes, TLC indicated total consumption of starting
materials. Ammonium hydroxide (2.75 mL) was added dropwise while under ice-bath to
neutralize the perchloric acid, during which the product began precipitating out of the reaction
mixture. The solution was placed under a rotary evaporator to remove the solvent. 200 mL of
chilled DI water was added, and the solution was stirred for 30 minutes. The insoluble product
was collected through vacuum filtration, then transferred to a 50 mL flask and put under vacuum
to dry overnight. The product was characterized by mass spectrometry (Figure 4) and 1H NMR
spectroscopy (Figure 5).
Step 2. Attachment of sulfamate group: The second step involves deprotonation of the hydroxyl
on ribose followed by nucleophilic attack of the sulfamoyl ion. The resulting 2’,3’-
isopropylidine-5’-O-sulfamoyl nucleoside was previously synthesized by Rachit Shah in the
Wagner Lab.
Step 3. Synthesis of 5’-O-(N-(Indole) sulfamoyl)2’,3’-isopropylidine guanosine
triethylammonium salt (IndGstKp): The coupling of 2’,3’-isopropylidine-5’-O-sulfamoyl
nucleoside and indole 3-propionic hydroxysuccinimide ester was performed via microwave.
Indole 3-propionic hydroxysuccinimide ester was previously synthesized by Rachit Shah. In a 5
mL microwave tube, DBU (1.1 equiv, 20.5 ul, 0.138 mmol) was added to a solution of 2’,3’-
isopropylidine-5’-O-sulfamoyl nucleoside (1.0 equiv, 50 mg, 0.13 mmol) and indole 3-propionic
hydroxysuccinimide ester (1.5 equiv, 53.5 mg, 0.188 mmol) in DMF (0.8 mL). The reaction
mixture was purged with an argon line. The mixture was microwaved at room temperature and
200 mV for 5 minutes. TLC analysis was performed using a 20 % methanol / 80 %
dichloromethane / 0.1 % ammonium hydroxide solvent system. TLC indicated mostly starting
material was present. In an attempt to accelerate the rate of product formation, the reaction
mixture was microwaved for an additional 5 minutes at 50 oC. TLC indicated some product
formation, but with additional side products and some starting material present. The solution was
microwaved for an additional 2 minutes at 50 oC. TLC indicated complete consumption of the
starting material. The solution was placed under a rotary evaporator to remove the solvent.
IndGstKp was purified from the reaction mixture by flash chromatography using a Combiflash
RF200 (Figure 6). Product was confirmed in fractions 36-42 by mass spectrometry (Figure 7).
Fractions 36-42 were combined and placed under a rotary evaporator to remove the solvent. A
solid carbon dioxide / acetone bath was used to freeze the product, and the product was
lyophilized overnight. The product was characterized by 1H NMR spectroscopy (Figure 8).
Step 3. Synthesis of 5’-O-(N-(butyrate)sulfamoyl)2’,3’-Isopropylidene Guanosine
triethylammonium salt (butyGStKp): The coupling of 2’,3’-isopropylidine-5’-O-sulfamoyl
nucleoside and butyrate hydroxysuccinimide ester was performed via microwave. Butyrate
hydroxysuccinimide ester was previously synthesized by Rachit Shah. In a 5 mL microwave
tube, DBU (1.1 equiv, 20.5 ul, 0.138 mmol) was added to a solution of 2’,3’-isopropylidine-5’-
O-sulfamoyl nucleoside (1.0 equiv, 50 mg, 0.125 mmol) and butyrate hydroxysuccinimide ester
(1.5 equiv, 34.5 mg, 0.188 mmol) in DMF (0.8 mL). The reaction mixture was purged with an
argon line. The mixture was microwaved at 50 oC and 200 mV for 5 min. TLC analysis was
performed using a 20 % methanol / 80 % dichloromethane / 0.1 % ammonium hydroxide solvent
system. TLC indicated product formation with some side products. To complete the consumption
of the starting material, the solution was microwaved for an additional 2 min at 50 oC and 200
mV. TLC indicated little starting material remained. The solution was placed under a rotary
evaporator to remove the solvent. Combiflash was performed (Figure 9), and the product was
confirmed in fractions 24-26 by mass spectrometry (Figure 10). Fractions 24-26 were combined
and placed under a rotary evaporator. A solid carbon dioxide / acetone bath was used to freeze
dry the product, and the product was lyophilized overnight. The product was characterized by 1H
NMR spectroscopy (Figure 11).
Step 4. Deprotection: The deprotection of the 2’ and 3’ hydroxyl groups on ribose was
previously performed by Rachit Shah to obtain final product.
Results
Protection of hydroxyl groups: Protection of the 2’ and 3’ hydroxyl groups of the ribose unit of
guanosine was carried out to achieve selective reactivity of the 1’ hydroxyl group. The purity of
the product is demonstrated by the molecular ion peak in the mass spectrum at 324 m/z, with the
peak at 152 m/z being a fragment produced upon ionization (Figure 4). The product purity is
further confirmed by the single set of peaks on the 1H NMR spectrum for Guanosine Kp (Figure
5).
Microwave synthesis of 5’-O-(N-(Indole) sulfamoyl)2’,3’-isopropylidine guanosine
triethylammonium salt (IndGstKp): The coupling step in the synthesis of IndGSt was performed
under microwave conditions to see if reaction completion could be achieved in a significantly
reduced time period. Product formation was confirmed in the mass spectrum (Figure 7), with the
peak at 574.0 m/z corresponding to the molecular ion peak for IndGstKp. The microwave
assisted coupling of 2’,3’-isopropylidine-5’-O-sulfamoyl nucleoside and indole 3-propionic
hydroxysuccinimide ester yielded 27.7 mg (0.048 mmol, 39 % yield) of pure IndGStKp product
(Table 1). The purity was evaluated through 1H NMR spectroscopy (Figure 8). The single set of
peaks in the 1H NMR spectrum indicates that a clean product was obtained.
Microwave synthesis of 5’-O-(N-(butyrate)sulfamoyl)2’,3’-Isopropylidene Guanosine
triethylammonium salt (butyGStKp): The coupling step in the synthesis of butyGSt was
performed under microwave conditions to see if reaction completion could be achieved in a
significantly reduced time period. ButyGStKp formation was confirmed by the peak at 473.0 m/z
in the mass spectrum, however the presence of several other minor peaks in the spectrum
indicates the presence of side products (Figure 10). The microwave assisted coupling of 2’,3’-
isopropylidine-5’-O-sulfamoyl nucleoside and butyrate hydroxysuccinimide ester yielded 15.0
mg (0.026 mmol, 29 % yield) of butyGStKp product (Table 1). The purity was evaluated through
1H NMR spectroscopy (Figure 11). Minor peaks in the 1H NMR spectrum reveal the presence of
a side product with similar structure to butyGStKp.
Discussion
The product formation evidenced by mass spectrometry and 1H NMR analysis confirms that the
rate-limiting coupling step in the synthesis of nucleoside-acyl sulfamate HINT1 inhibitors can be
completed under microwave conditions. The meager yields of 39 % and 29 % obtained for
IndGStKp and butyGStKp, respectively, demonstrate a significant reduction in reaction
efficiency when compared to the typical yields of 60 – 80 % obtained through overnight
incubation at room temperature (Table 1). The reduced yields are likely resultant of the
formation of unwanted side products during the microwave excitation process, which are
indicated by the additional peaks in the Combiflash data for IndGStKp (Figure 6) and
butyGStKp (Figure 9). Mass spectra were obtained for the non-product containing fractions
obtained from Combiflash in an attempt to identify some of the side products, although the
identification was unsuccessful. The several minor peaks present in the mass spectrum for
butyGStKp also indicate the presence of side products. These findings suggest that under the
microwave conditions performed, the desired coupling reactions are in competition with other
reactions that result in the formation of unwanted side products.
Based on the insubstantial yields obtained for IndGStKp and butyGStKp under the executed
microwave conditions, a trade off clearly exists between the convenience offered by the
significant reduction in reaction time and the poor yields obtained. Further experiments should
be performed to determine whether the microwave assisted synthesis of nucleoside-acyl
sulfamate HINT1 inhibitors can be optimized for better yields and higher purity. This can be
done by testing the use of different solvents, temperature settings, and time points in the reaction.
One possible explanation for the unwanted side products is that the reaction mixture was
overheated, causing some of the newly formed product to degrade into unwanted side products.
The microwave syntheses could be repeated using shorter time periods and at lower temperatures
to see if side product formation still occurs to a similar extent. The second step involving
attachment of the sulfamate group could also be tested under microwave conditions to see if this
two hour reaction can be reduced to minutes or seconds.
Literature Cited
1. Garzon, J., Herrero-Labrador, R., Rodriguez-Munoz, M., Shah, R., Vicente-Sanchez, A., Wagner. C. R., Sanchez-Blazquez, P., 2015. HINT1 protein: a new therapeutic target to enhance opioid antinociception and block mechanical allodynia. Neuropharmacology. 89, 412-413.
2. Zhao, S. Z., Chung, F., Hanna, D. B., Raymundo, A. L., Cheung, R. Y., Chen, C., 2004. Dose response relationship between opioid use and adverse effects after ambulatory surgery. J Pain Symptom Manage. 28, 35-46.
3. Rodriquez-Munoz, M., Sanchez-Blazquez, P., Vicente-Sanchez, A., Berrocoso, E., Garzon, J., 2012. The mu-opioid receptor and the NMDA receptor associate in PAG neurons: implications in pain control. Neuropsychopharmacology. 37, 338-349.
4. Guang, W., Wang, H., Su, T., Weinstein, I.B., Wang, J.B., 2004. Role of mPKCI, a novel mu-opioid receptor interactive protein, in receptor desensitization, phosphorylation, and morphine-induced analgesia. Mol. Pharmacol. 66, 1285e1292.
5. Bardaweel, S.K., Ghosh, B., Wagner C.R., 2012. Synthesis and evaluation of potential inhibitors of human and Escherichia coli histidine triad nucleotide binding proteins. Bioorganic & Medicinal Chemistry Letters. 22, 558-560.
6. Kappe, C.O., 2004. Controlled microwave heating in modern organic synthesis. Angewandte Chemie International Edition. 43, 6520-6284.
7. Barbier, E., Wang, J.B., 2009. Anti-depressant and anxiolytic like behaviors in PKCI/ HINT1 knockout mice associated with elevated plasma corticosterone level. BMC Neurosci. 10, 132.
8. Ajit, S.K., Ramineni, S., Edris, W., Hunt, R.A., Hum, W.T., Hepler, J.R., et al., 2007. RGSZ1 interacts with protein kinase C interacting protein PKCI-1 and modulates mu opioid receptor signaling. Cell. Signal. 19, 723e730.
Appendix
Table 1: Comparison of Time Points & Yields for Standard & Microwave Assisted Synthesis
Standard Conditions MicrowaveTime Point Temp % Yield Time Point Temp % Yield
Coupling Step for IndGSt
Overnight 21 oC ≈ 70 12 min 50 oC 39
Coupling Step for butyGSt
Overnight 21 oC ≈ 70 7 min 50 oC 29
Figure 1: structures of AIPA, TrpGc, and IndGSt
Figure 2: Synthetic Scheme for IndGSt
Figure 3: Synthetic Scheme for butyGSt
Figure 4: Mass Spectrum for Guanosine Kp
Figure 5: 1H NMR Spectrum (300MHz, DMSO) for Guanosine Kp
Figure 6: Combiflash Data for IndGStKp
Figure 7: Mass Spectrum for IndGStKp
Figure 8: 1H NMR Spectrum (300MHz, DMSO) for IndGStKp
Figure 9: Combiflash Data for butyGStKp
Figure 10: Mass Spectrum for butyGStKp
Figure 11: 1H NMR Spectrum (300MHz, DMSO) for butyGStKp
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