Diazeniumdiolated carbamates: A novel class of nitric oxide donors

Abstract

We report an indirect method for synthesis of previously inaccessible diazeniumdiolated carbamates. Synthesis involves use of previously reported triisopropylsilyloxymethylated isopropylamine diazeniumdiolate (TOM-ylated IPA/NO). These novel diazeniumdiolated carbamate prodrugs upon activation release nitric oxide (NO) similar to their secondary amine counterparts. They are also efficient sources of intracellular NO. These prodrugs may have potential applications as therapeutic NO-donors.

1. Introduction

Diazeniumdiolate prodrugs¹ (1, Figure 1) are considered as reliable sources of the potent bioregulatory agent, nitric oxide² (NO). These prodrugs (1), on metabolic activation or hydrolysis, form the parent anion (2), which further decomposes to release up to two moles of NO and the parent amine (Figure 1a). The therapeutic applications of these prodrugs are diverse, and largely depend on the O-2 protecting group (‘R’ in structure 1, Figure 1) and its mechanism of activation. For example, vinyl protected prodrug V-PYRRO/NO (3) is activated by cytochrome P450 to release NO, and shows hepatoprotective properties against a variety of toxins.³ Glutathione (GSH)-activated arylated prodrug JS-K (4) is a lead anticancer agent.⁴ Recently, primary amine diazeniumdiolate prodrug AcOM-IPA/NO (5)⁵ was reported to release nitroxyl (HNO), another potent bioeffector molecule with possible applications in treating heart failure and alcohol abuse.⁶

Figure 1.

(a) Activation of diazeniumdiolate prodrugs to release NO or HNO (b) Structures of, V-PYRRO/NO (3), JS-K (4) and AcOM-IPA/NO (5). (c) Hydrolysis of diazeniumdiolated amides and carbamates (6).

Secondary amine diazeniumdiolate ions are protonated at N-3 (see Figure 1 for numbering) to release NO,¹ whereas, primary amine diazeniumdiolates release nitroxyl⁵ (HNO) on protonation at N-2. Given the difference in product distribution between primary and secondary amine diazeniumdiolate ions seen on hydrolyzing them (mixed NO/HNO donors versus pure NO donors, respectively), we speculated that diazeniumdiolated carbamates (6a) and amides (6b) might hydrolyze with different and potentially advantageous product distributions and/or kinetics (Figure 1c). We have until now had no success in forming diazeniumdiolate ions by reacting amides or other acylated amines directly with NO under basic conditions, so we have resorted to an indirect method for accessing a relevant prototype. Here we describe the synthesis of prodrugs that can be used to probe the chemistry of anionic diazeniumdiolated carbamates as well as to generate analogues that can be used for studies of their biological properties.

2. Results and discussion

2.1. Synthesis

We envisaged that the triisopropylsilyloxymethyl (TOM) protecting group strategy⁷ developed for O-2 substituted diazeniumdiolate prodrugs might be useful for the synthesis of such carbamate or amide derivatives. Thus, IPA/NO (7, Scheme 1) was converted to TOM-ylated IPA/NO (8, Scheme 1) following the reported procedure.⁷ This compound 8 on treatment with a base and Boc-anhydride (Boc₂O) gave carbamate 9. After experimentation with several bases and reaction conditions for this transformation, it was observed that compound 9 is efficiently formed by using lithium tert-butoxide in hexane at 0 °C.

Scheme 1.

Synthesis of carbamate diazeniumdiolate 9.

With compound 9 in hand, it was important to remove the TOM protecting group and introduce a biologically significant protecting group helpful for site-directed delivery of NO. We treated compound 9 with tetra-n-butylammonium fluoride (TBAF) in the presence of a 4-fold excess of 2-bromo-l-[((trifluoromethane)sulfonyl)oxy]ethane (10) and triethylamine (TEA) to obtain the required bromide (Scheme 2). This crude bromide on treatment with Verkade’s superbase (11) gave the vinyl carbamate prodrug 12 in 34% yield (2 steps). Removal of silyl protection in the presence of bromomethyl acetate (13) and TEA gave esterase-labile acetoxymethylated diazeniumdiolate prodrug 14. Similar deprotection in the presence of 2,4-dinitrofluorobenzene (15) gave arylated prodrug 16 in 38% yield. To the best of our knowledge, these are the first examples of diazeniumdiolates with a carbamate functional group at N-3 (for atom numbering system, see structure 1, Figure 1).

Scheme 2.

Synthesis of carbamate diazeniumdiolate prodrugs 12, 14 and 16.

2.2. NO-release

It was important to identify and quantify the NO and/or HNO released on activation of these prodrugs. Chemiluminescence assay was used to determine NO. After NO evolution has ceased, the residual solution was further used for Griess’s assay in the presence of air (oxygen). The Griess’s assay was used to determine amount of nitrite (NO₂⁻) present in the solution. Nitrite is the oxidation product of NO in aerobic aqueous solution. Quantification of HNO was carried out using quantification of nitrous oxide (N₂O), a dimerization product of HNO, by gas chromatography. The results are summarized in Table 1. Compound 14 on activation by porcine liver esterase under physiological conditions for 5 days gave 1.25 mol (63%) of NO, 0.06 mol (3%) of nitrite and an undetectable amount of N₂O. The decreased amount of NO measured on hydrolysis of 14 may be attributed to one or more effects. Nitrosative inactivation of the enzyme could occur, ceasing hydrolysis and resulting in protein nitrosation products not detectable by Griess assay. Formation of N-nitrosocarbamate might also be expected. This product could further rearrange to the O-isopropyl carbamate with expulsion of N₂, a difficult product to measure. Similarly, compound 16 on activation by GSH in PBS under physiological conditions gave about 1.35 mol of NO, 0.32 mol of nitrite and trace amount of N₂O. Prodrug 16 also shows less than the calculated amount of NO, probably due to similar reasons as speculated for compound 14. Thus, these N-diazeniumdiolated carbamates release NO via a pathway similar to that of their secondary amine counterparts as shown in Figure 2.

Table 1.

Quantification of NO, NO₂⁻ and N₂O for prodrugs 14 and 16.

prodrug	activation conditions	NO mol/mol of prodrug (% yield)a	NO2− mol/mol of prodrug (% yield)a	N2O mol/mol of prodrug
14	Porcine liver esterase in PBS at 37 °C	1.25 (63%) over 5 days	0.06 (3%)	N.D. b
16	GSH (4 mM) in PBS at 37 °C	1.35 (68%) over 400 min	0.32 (16%)	0.02

^a2 mol/mol prodrug = 100% yield;

^bN.D.= not detected.

Figure 2.

Proposed path for activation of diazeniumdiolated carbamate 17 to release NO, the predominant hydrolysis product.

2.3. Intracellular NO-release

Next, we studied the intracellular NO release and cell permeability of these new compounds. The intracellular NO was estimated by using the nitric oxide-sensitive and commercially available dye, 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM diacetate).⁸ Normal human skin fibroblast BJ-5ta cells and U937 human leukemia cells were selected, and JS-K (4) was taken as a positive control for the experiments. Both the cell lines were pre-loaded with DAF-FM diacetate, followed by treatment with DMSO solutions of prodrugs 14, 16 and JS-K. The fluorescence measurements after 90 min provided estimates of the levels of intracellular NO. It was observed that at equal concentrations, prodrug 16 showed significantly higher levels of NO as compared to JS-K, while 14 showed a comparable fluorescence level as that of JS-K for BJ-5ta and U937 cells (Figure 3). A reviewer has pointed out that the DAF-FM assay is subject to false positive readings under certain experimental settings. Nevertheless, we conclude that the present results are consistent with the view that the prodrugs 14 and 16 are taken up by the cells and are activated therein to release NO.

Figure 3.

Levels of intracellular NO formation upon treatment of (a) BJ-5ta and (b) U-937 cells with compounds (10 µM final concentration) and DMSO (control) as determined by DAF-FM diacetate fluorescence study.

3. Conclusions

In summary, we have synthesized diazeniumdiolated carbamate prodrugs with established, biologically significant protecting groups starting from TOM-ylated diazeniumdiolate. To the best of our knowledge, this is the first report of diazeniumdiolated carbamate prodrugs. We have studied their activation mechanism and measured the amount of NO released. These new prodrugs release NO by a mechanism similar to their secondary amine counterparts. We have also demonstrated their ability to release NO inside cells. The compounds synthesized have led to a new class of NO-donors with potential biological applications.

4. Experimental

4.1. Synthesis

4.1.1. General

Starting materials were purchased from Aldrich Chemical Co. (Milwaukee, WI) unless otherwise indicated. NMR spectra were recorded on a 400 MHz Varian UNITY INOVA spectrometer; chemical shifts (δ) are reported in parts per million (ppm) downfield from tetramethylsilane. Ultraviolet (UV) spectra were recorded on an Agilent Model 8453 or a Hewlett-Packard model 8451A diode array spectrophotometer. High resolution mass spectra (HRMS) were recorded on Agilent 6250 series Accurate-Mass Q-TOF LC/MS by electrospray ionization (ESI). Elemental analyses were performed by Midwest Microlab (Indianapolis, IN). Chromatography was performed on a Biotage SP1 Flash Purification System. Prepacked silica gel flash chromatography columns were purchased from Yamazen Science Inc. (San Bruno, CA). Compounds 8⁷ and 10⁹ were prepared by using the reported procedures. Diethyl ether was used to dissolve and transfer all the liquid compounds from rotary evaporator flask to smaller containers and sample vials.

4.1.2. Procedures and analytical data

Compound 9. Under N₂, a 1 M solution of LiO^t Bu in hexane (3.8 mL, 3.84 mmol) was added to an ice-cold solution of compound 8 (1.06 g, 3.49 mmol) in hexane (10 mL). After 20 min, a solution of Boc₂O in hexane was added to this ice-cold reaction mixture. After 20 min of stirring, the reaction was diluted with distilled water (10 mL) and hexane (10 mL). The organic layer was washed with brine, dried over anhydrous Na₂SO₄ and evaporated. The crude product was purified by flash column chromatography (9:1 hexane/ethyl acetate) to give compound 9 as an oil (1.0 g, 71%). UV (ethanol) λ_max (ε) 235 nm (5.9 mM⁻¹cm⁻¹); ¹H NMR (CDCl₃, 400 MHz) δ 1.05–1.17 (m, 3H), 1.08 (d, J = 6.1 Hz, 18H), 1.32 (d, J = 6.7 Hz, 6H), 1.49 (s, 9H), 4.36 (septet, J = 6.7 Hz, 1H), 5.53 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 151.42, 91.58, 83.53, 51.44, 27.96, 19.86, 17.64, 11.81. HRMS (ESI) m/z calculated for C₁₈H₄₃N₄O₅Si [M+NH₄]⁺ 423.2997, found 423.2993. Anal. Calcd for C₁₈H₃₉N₃O₅Si·0.5Et₂O: C, 54.27; H, 10.02; N, 9.49, Found: C, 54.36; H, 9.79; N, 9.66.

Compound 12. To a solution of 9 (140 mg, 0.35 mmol), triethylamine (TEA) (0.2 mL, 1.4 mmol) and 2-bromo-l-[((trifluoromethane)sulfonyl)oxy]ethane (10) (360 mg, 1.4 mmol) in CH₂Cl₂ (3 mL) was added tetrabutylammonium fluoride trihydrate (TBAF) (133 mg, 0.42 mmol) in CH₂Cl₂ (2 mL) at 0 °C. Reaction was allowed to attain rt over 2 h, then the reaction was diluted with 5% sodium bicarbonate, and extracted with CH₂Cl₂. The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄ and evaporated. The crude product was used for the next step without further purification. This crude product was dissolved in acetonitrile (2 mL) and a solution of Verkade’s superbase 11 (151 mg, 1.4 mmol) in acetonitrile (1 mL) was added at rt. After 60 min at rt, volatiles were evaporated and the crude product was purified by flash column chromatography (9:1 hexane/ethyl acetate) to give compound 12 as a colorless oil (29 mg, 34% for 2 steps). UV (ethanol) λ_max (ε) 258 nm (5.73 mM⁻¹cm⁻¹); ¹H NMR (CDCl₃, 400 MHz) δ 1.33 (d, J = 6.7 Hz, 6H), 1.50 (s, 9H), 4.37 (septet, J = 6.7 Hz, 1H), 4.53 (dd, J = 6.6, 2.7 Hz, 1H), 4.97 (dd, J = 14.0, 2.7 Hz, 1H), 6.90 (dd, J = 14.0, 6.6 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 151.25, 148.27, 94.08, 83.98, 51.66, 27.94, 19.85. Anal. Calcd for C₁₀H₁₉N₃O₄: C, 48.97; H, 7.81; N, 17.13, Found: C, 48.92; H, 7.73; N, 17.16.

Compound 14. To a solution of 9 (400 mg, 0.98 mmol), triethylamine (TEA) (0.6 mL, 3.92 mmol) and bromomethyl acetate (13) (0.4 mL, 3.92 mmol) in CH₂Cl₂ (9 mL) was added tetrabutylammonium fluoride trihydrate (TBAF) (372 mg, 1.18 mmol) in CH₂Cl₂ (4 mL) at 0 °C. Reaction was allowed to attain rt over 2 h, then the reaction was diluted with 5% sodium bicarbonate and extracted with CH₂Cl₂. The combined organic layer was washed with brine, dried over anhydrous Na₂SO₄ and evaporated. The crude product was purified by flash column chromatography (9:1 hexane/ethyl acetate) to give compound 14 as colorless oil (89 mg, 31%). UV (ethanol) λ_max (ε) 247 nm (5.52 mM⁻¹cm⁻¹); ¹H NMR (CDCl₃, 400 MHz) δ 1.32 (d, J = 6.7 Hz, 6H), 1.50 (s, 9H), 2.12 (s, 3H), 4.35 (septet, J = 6.7 Hz, 1H), 5.86 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 168.99, 151.14, 87.24, 83.86, 51.61, 27.93, 20.64, 19.78. HRMS (ESI) m/z calculated for C₁₁H₂₂N₃O₆ [M+H]⁺ 292.1503, found 292.1500.

Compound 16. To a solution of 9 (360 mg, 0.9 mmol) and 2,4-dinitrofluorobenzene (15) (170 mg, 0.9 mmol) in CH₂Cl₂ (9 mL) was added tetrabutylammonium fluoride trihydrate (TBAF) (347 mg, 1.10 mmol) in CH₂Cl₂ (4 mL) at 0 °C. Reaction was allowed to attain rt over 2 h, then the volatiles were evaporated and the crude product was purified by flash column chromatography (5:1 hexane/ethyl acetate) to give compound 16 as a yellow solid (130 mg, 38%). UV (ethanol) λ_max (ε) 248 nm (6.01 mM⁻¹cm⁻¹) and 286 nm (7.29 mM⁻¹cm⁻¹); ¹H NMR (CDCl₃, 400 MHz) δ 1.39 (d, J = 6.8 Hz, 6H), 1.53 (s, 9H), 4.43 (septet, J = 6.8 Hz, 1H), 7.73 (d, J = 9.2 Hz, 1H), 8.49 (ddd, J = 9.2, 2.7, 0.6 Hz, 1H), 8.90 (d, J = 2.7 Hz, 1H); ¹³C NMR (CDCl₃, 100 MHz) δ 152.94, 150.94, 143.15, 129.00, 121.98, 118.79, 84.95, 52.50, 27.95, 19.86. HRMS (ESI) m/z calculated for C₁₄H₂₃N₆O₈ [M+NH₄]⁺ 403.1572, found 403.1578.

4.2. NO-release

Chemiluminescence

Calibration of the Sievers Nitric Oxide Analyzer (NOA), model 280i (Instruments Business Group, Boulder, CO) was performed by injecting various volumes of known concentrations of NO in helium (50 ppm, 500 ppm and 5%) certified standards into the reaction chamber and recording the peak areas. Samples and reaction chambers were incubated at 37 °C. The gas was sparged with argon and swept into the chemiluminescence detector. Data were recorded using Agilent Chemstation software and processed using Microsoft Excel.

Esterase-activated

3.0 mL of pH 7.4 buffer containing porcine liver esterase (ammonium sulfate suspension, Sigma, 5KU) (0.55 µL) and diethylenetriaminepentaacetic acid (DTPA, 50 µM) was placed into the reaction chamber of the NOA and then sparged for several minutes with argon. A DMSO solution (10 mM) of the prodrug 14 (60 µL) was injected into the reaction chamber and nitric oxide release was recorded. Total amount of NO released was determined by integrating the area under the curve and applying a calibration curve. The NO release is an average of three independent experiments.

Glutathione (GSH)-activated

3.0 mL of pH 7.4 buffer containing GSH (3.6–3.9 mM) and DTPA (50 µM) was placed into the reaction chamber of the NOA and then sparged for several minutes with argon. A DMSO solution (10 mM) of the prodrug 16 (100 µL) was injected into the reaction chamber and nitric oxide release was recorded by Sievers NOA. Total amount of NO released was determined by integrating the area under the curve and applying a calibration curve. The NO release is an average of three independent experiments.

Griess assay

Sample volume was measured after the chemiluminescence experiment in the presence of air (oxygen). 100 µL of the Griess reagent, 300 µL of sample and 2.6 mL of deionized water were added in a spectrophotometer cuvette. The mixture was incubated for 30 minutes at rt. Photometric reference sample was prepared by mixing 100 µL of the Griess reagent and 2.9 mL of deionized water. The absorbance of the nitrite-containing sample at 548 nm was measured relative to the reference sample. Then absorbance readings were converted to the nitrite concentrations according to calibration curve.

Gas Chromatography for N₂O

• Instrument: Shimadzu GC- 2014

• Detector: ECD (Electron capture Detector)

• Column: Restek ShinCarbon 80/100 Packed Column

• Length = 2 m; ID = 2.0 mm;

• Test condition: Injector temp = 250 °C, Detector temp = 250 °C; Initial column temp = 90 °C; Final temp = 200 °C; Rate = 20 °C/min; Final temp Hold = 1.1 min; Total program time = 6.7 min; Carrier: He (UHP) = 30 mL/min.

4.3. Cell culture and intracellular NO-release

BJ-5ta

Human hTERT immortalized BJ-5ta skin fibroblasts were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen, Carlsbad, CA):199 medium (Sigma) (4:1) supplemented with 10% fetal calf serum (Gemini Bio-Products, Sacramento, CA), 100 U/mL penicillin and 2 mM glutamine. The intracellular level of nitric oxide after diazeniumdiolate prodrug treatment was quantified using the NO-sensitive fluorophore 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate; Invitrogen, Carlsbad, CA). BJ-5ta cells growing on 96-well plates were loaded with 2.5 µM DAF-FM diacetate in Hanks' balanced salt solution (HBSS) at 37 °C and 5% CO₂. After 30 min of incubation the cells were rinsed with HBSS to remove excess probe, and fresh HBSS was added to the wells. The compounds were added to the cells at 10 µM final concentration. After 90-min incubation the fluorescence of the benzotriazole derivative formed on DAF-FM’s reaction with aerobic NO was analyzed using a PerSeptive Biosystems CytoFluor 4000 microplate reader with the excitation source at 485 nm and emission at 525 nm. The mean value of 16 independent experiments is reported (see Table S1 in the Supporting Information).

U937

U937 cell lines were obtained from ATCC, Manassas, VA. Cells were maintained in RPMI 1640 medium (Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (Gemini Bio-Products, Sacramento, CA), 100 U/mL penicillin and 2 mM glutamine, at 37 °C and 5% CO₂. U937 cells were loaded with 2.5 µM DAF-FM diacetate in HBSS at 37 °C and 5 % CO₂. After 30 min of incubation the cells were collected by centrifugation, rinsed with HBSS to remove excess probe, and resuspended in fresh HBSS. The compounds were added to the cells at 10 µM final concentration. After 90-min incubation the fluorescence of the benzotriazole derivative formed on DAF-FM’s reaction with aerobic NO was analyzed using a PerSeptive Biosystems CytoFluor 4000 microplate reader with the excitation source at 485 nm and emission at 525 nm. The mean value of 16 independent experiments is reported (see Table S2 in the Supporting Information).