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

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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.

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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