Reactions of Rebamipide with Hypobromous Acid

Abstract

Rebamipide is a therapeutic agent for gastric ulcers and chronic gastritis. Hypobromous acid (HOBr) is generated not only by eosinophils but also by neutrophils in the presence of bromide ions in the plasma. At inflammation sites, rebamipide may encounter and react with HOBr to generated various products. When rebamipide was incubated with reagent HOBr in potassium phosphate buffer at pH 4.7 and 37°C for 4 h, several products were generated. A major product was identified as 3-bromorebamipide, a novel compound. Rebamipide does not react with hypochlorous acid (HOCl). However, when rebamipide was incubated with HOCl in the presence of NaBr, 3-bromorebamipide was generated in a dose-dependent manner, probably because of formation of HOBr. These results suggest that 3-bromorebamipide may generate from rebamipide at inflammation sites in humans.

Introduction

Rebamipide (RM) was selected from among many synthesized amino acid analogs of 2(1H)-qunilinone as a therapeutic agent for gastric ulcers and chronic gastritis.^1–3) RM increases prostaglandin levels in gastric tissue and protects the gastric mucosa.^4,5) RM is also used as an ophthalmic drug for the treatment of dry eye syndrome, since RM can increase corneal and conjunctival mucin.^6,7) These diseases treated with rebamipide involve inflammation. Eosinophils and neutrophils assemble at sites of inflammation. Eosinophils are a minor component of white blood cells, but are abundant in blood and tissues in various inflammatory disorders.⁸⁾ Eosinophils secrete eosinophil peroxidase, which generates hypobromous acid (HOBr) from H₂O₂ and Br⁻.^9,10) Although the plasma concentrations of Br⁻ and Cl⁻ are 39–84 µM and 100 mM, respectively,¹¹⁾ eosinophil peroxidase uses Br⁻selectively, resulting in HOBr. This HOBr plays an important role in defense mechanisms against parasites and microorganisms. Meanwhile, neutrophils are a major component of white blood cells. Neutrophils secrete myeloperoxidase. Myeloperoxidase generates hypochlorous acid (HOCl) from H₂O₂ and Cl⁻.^12–14) The HOCl formed by myeloperoxidase is of central importance in host defense mechanisms against microorganisms. Since HOCl can react with Br⁻ to generate HOBr, a portion of the HOCl formed by the myeloperoxidase system will react with intra- and intercellular Br⁻, converting to HOBr.^15,16) Under inflammation conditions, RM may encounter HOBr and HOCl, and react with them. However, there is little information about the reaction of RM with HOBr and HOCl. If the reaction occurs, generating products with no physiological activity, it would only cause decrease of concentration of RM administered. If products have some physiological activity, inflammation may influence the effect of RM. In the present study, we examined the reactions of RM with HOBr and HOCl using reversed phase (RP)-HPLC, and found that HOBr reacts with RM to generate brominated products.

Results and Discussion

Reaction with HOBr

A solution of 100 µM RM was incubated with 100 µM HOBr in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h. When the reaction mixture was analyzed by RP-HPLC, three product peaks, denoted as Peaks 1, 2, and 3, appeared in the chromatogram detected at 230 nm (Fig. 1). Peak 1 showed a UV spectrum (λ_max = 224, 282, and 333 nm) similar to RM (λ_max = 228 and 327 nm), as shown in Fig. 1 insets. The product was isolated by RP-HPLC and identified using MS (electrospray ionization time of flight mass spectrometry (ESI-TOF/MS)) and NMR (¹H-1d, ¹H–¹H correlation spectroscopy (COSY), ¹H–¹³C heteronuclear multiple quantum correlation (HMQC), ¹H–¹³C heteronuclear multiple bond correlation (HMBC), ¹³C-1d). Peak 1 showed a complex ESI-TOF/MS spectrum including peaks of m/z = 447, 449, and 451 in negative mode (Fig. 2A). High-resolution (HR) ESI-TOF/MS values of the molecular ion (m/z 447) for Peak 1 agreed with the theoretical molecular mass for C₁₉H₁₃⁷⁹Br³⁵ClN₂O₄⁻, attributable to [RM + Br − 2H]⁻ within 3 ppm. ¹H-NMR showed two sets of four aromatic protons coupled with each other on the ¹H–¹H COSY spectrum. A proton signal attributable to the 3-position of the quinolinone moiety was not observed. From these data, Peak 1 was identified as a novel compound, 3-bromorebamipide (3-Br-RM) (Fig. 2B). Peak 2 showed an MS spectrum including peaks of m/z = 525, 527, and 529 in negative mode. HR-ESI-TOF/MS values of the molecular ion (m/z 525) for Peak 2 agreed with the theoretical molecular mass for C₁₉H₁₂⁷⁹Br₂³⁵ClN₂O₄⁻, attributable to [RM + 2Br − 3H]⁻ within 3 ppm, suggesting formation of an RM derivative substituted by two bromine atoms. However, ¹H-NMR showed that two compounds included in Peak 2. Thus, we could not identify the structure or determine the yield of the product in Peak 2. Peak 3 showed an MS spectrum including peaks of m/z = 447, 449, and 451 in negative mode. HR-ESI-TOF/MS values of the molecular ion (m/z 447) for Peak 3 agreed with the theoretical molecular mass for C₁₉H₁₃⁷⁹Br³⁵ClN₂O₄⁻, attributable to [RM + Br − 2H]⁻ within 3 ppm, suggesting formation of an RM derivative substituted by a bromine atom. However, ¹H-NMR showed two compounds in the sample. RP-HPLC analysis of the NMR sample showed two peaks, corresponding to Peak 1 and Peak 3. When Peak 3 fractionated by RP-HPLC from the RM/HOBr reaction solution was incubated at 37°C for 1 h, Peak 1 appeared in the PR-HPLC chromatogram, indicating that the product in Peak 3 was converted to 3-Br-RM. Thus, we could not determine the structure and the yield of the product in Peak 3. The concentrations of 3-Br-RM and RM were determined from the integrated peak areas on an RP-HPLC chromatogram detected at 230 nm and by the molecular extinction coefficients at 230 nm. The concentrations were 31.7 ± 2.2 µM for 3-Br-RM with 30.7 ± 3.7 µM of unreacted RM, when a solution of 100 µM RM was incubated with 100 µM HOBr in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h.

Fig. 1. RP-HPLC Chromatogram of a Reaction Mixture of RM with HOBr Detected at 230 nm

A solution of 100 µM RM was incubated with 100 µM HOBr in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h. The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-vis photodiode-array detector (Shimadzu, Kyoto, Japan). For RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 4.6 × 250 mm and particle size 5 µm (GL Sciences, Tokyo) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing acetonitrile. The acetonitrile concentration was increased from 0 to 70% during 30 min in linear gradient mode, and maintained at 70% from 30 to 40 min. The column temperature was 40°C and the flow rate was 1 mL/min.

Fig. 2. (A) Negative Ion ESI-TOF/MS Spectrum of the Product in Peak 1; (B) Structure of 3-Bromorebamipide (3-Br-RM)

Figure 3A shows the time-dependent changes in concentrations of RM and 3-Br-RM when 100 µM RM and 100 µM HOBr were incubated in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 0–4 h, although the reaction was almost complete by 30 min. The yield of 3-Br-RM was about one-half of the consumption of RM. Figure 3B shows the HOBr dose-dependent changes in concentrations of RM and 3-Br-RM when 100 µM RM and 0–200 µM HOBr were incubated in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h. With increasing HOBr concentration, the consumption of RM and the yield of 3-Br-RM increased. Figure 3C shows the pH-dependence of the reaction of RM with HOBr when 100 µM RM and 100 µM HOBr were incubated in 100 mM potassium phosphate buffer at pH 4.3–9.6 and 37°C for 4 h. Under mildly acidic conditions around pH 5.3, the reaction accelerated.

Fig. 3.  (A) Time Course of the Concentration Changes in RM and 3-Br-RM When a Solution of 100 µM RM Was Incubated with 100 µM HOBr in 100 mM Potassium Phosphate Buffer at pH 4.7 and 37°C for 0–4 h; (B) HOBr dose-Dependence of the Concentration Changes in RM and 3-Br-RM When a Solution of 100 µM RM Was Incubated with 0–200 µM HOBr in 100 mM Potassium Phosphate Buffer at pH 4.7 and 37°C for 4 h; (C) pH-Dependence of the Concentration Changes in RM and 3-Br-RM When a Solution of 100 µM RM Was Incubated with 100 µM HOBr in 100 mM Potassium Phosphate Buffer at pH 4.3–9.6 and 37°C for 4 h

RM (closed circle) and 3-Br-RM (open triangle). All the reaction mixtures were analyzed by RP-HPLC. Means ± standard deviation (S.D.) (n = 3) are presented.

Reaction with N-Bromosuccinimide

As a control, a solution of 100 µM RM was incubated with 100 µM N-bromosuccinimide (NBS), a brominating reagent, in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h. When the reaction mixture was analyzed by RP-HPLC, concentrations were 22.8 ± 1.5 µM for 3-Br-RM with 57.9 ± 2.9 µM of unreacted RM. The result indicates that NBS reacts with RM less effectively than HOBr under this experimental condition.

Reaction with HOCl

A solution of 100 µM RM was incubated with 100 µM HOCl in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 4 h. When the reaction mixture was analyzed by RP-HPLC, no consumption of RM and no product peak were observed. No reaction was observed up to 200 µM HOCl (Fig. 4A). No reaction was observed at pH ranging from 4.3 to 9.6 (Fig. 4B).

Fig. 4. (A) HOCl Dose-Dependence of the Concentration Changes in RM When a Solution of 100 µM RM Was Incubated with 0–200 µM HOCl in 100 mM Potassium Phosphate Buffer at pH 4.7 and 37°C for 4 h; (B) pH-dependence of the Concentration Changes in RM When a Solution of 100 µM RM Was Incubated with 100 µM HOCl in 100 mM Potassium Phosphate Buffer at pH 4.3–9.6 and 37°C for 4 h

All the reaction mixtures were analyzed by RP-HPLC. Means ± S.D. (n = 3) are presented.

Reaction with HOCl in the Presence of NaBr

Figure 5 shows the NaBr dose-dependent changes in concentrations of RM and 3-Br-RM when a solution of 100 µM RM was incubated with 100 µM HOCl in the presence of 0–200 µM NaBr in 100 mM potassium phosphate buffer at pH 4.7 and 37°C for 24 h. With increasing NaBr dose up to 100 µM, the consumption of RM and yield of 3-Br-RM increased. HOCl can oxidize Br⁻ to generate HOBr.¹⁷⁾ The HOBr formed from Br⁻ by HOCl would react with RM, generating 3-Br-RM.

Fig. 5. NaBr Dose-Dependence of the Concentration Changes in RM and 3-Br-RM When a Solution of 100 µM RM Was Incubated with 100 µM HOCl in the Presence of 0–200 µM NaBr in 100 mM Potassium Phosphate Buffer at pH 4.7 and 37°C for 4 h

RM (closed circle) and 3-Br-RM (open triangle). All the reaction mixtures were analyzed by RP-HPLC. Means ± S.D. (n = 3) are presented.

Effects of Additives on the Reaction with HOBr

To obtain information about the effect of coexistent compounds on the reactions of RM with HOBr, experiments were carried out in the presence of various additives. Table 1 shows the concentrations of RM and 3-Br-RM when a solution of 100 µM RM and 1 mM additives was incubated with 100 µM HOBr in 100 mM potassium phosphate buffer (pH 4.7) at 37°C for 24 h. Ammonium chloride, methylamine, and dimethylamine suppressed the reaction. Trimethylamine and tetramethylammonium had no effect for consumption of RM, although the yield of 3-Br-RM was increased. Amino acids, even glycine (Gly), suppressed the reaction almost completely. Taurine suppressed the reaction. Urea slightly suppressed the consumption of RM, although the yield of 3-Br-RM was comparable. Glucose and sodium acetate showed no effect for consumption of RM, although the yield of 3-Br-RM was increased. In the presence of trimethylamine, tetramethylammonium, glucose, and sodium acetate, the integrated peak areas of Peak 2 on the RP-HPLC chromatograms of the reaction solutions decreased. These compounds may suppress the generation of a dibromo derivative of RM, resulting in the increase of 3-Br-RM, although the mechanism is unclear. In human plasma, total concentration of free amino acids is several mM, whereas taurine concentration is several dozen µM.^18,19) The reaction of RM with HOBr in plasma should be strongly suppressed by free amino acids.

Table 1. Effects of Additives on the Reactions of RM with HOBr^a)

Additives	RM (µM)	3-Br-RM (µM)
None	30.7 ± 3.7	31.7 ± 2.2
NH4Cl	87.3 ± 2.6	7.3 ± 0.6
CH3NH2/HCl	91.3 ± 0.6	4.8 ± 0.2
(CH3)2NH/HCl	76.1 ± 6.4	15.3 ± 3.3
(CH3)3N/HCl	29.3 ± 3.6	45.1 ± 2.6
(CH3)4NCl	37.3 ± 4.3	38.9 ± 1.8
Gly	98.7 ± 3.7	0.4 ± 0.3
Lys	100.6 ± 1.2	0.5 ± 0.4
Met	99.4 ± 1.7	0.2 ± 0.3
Cys	97.5 ± 0.6	0.0 ± 0.0
Taurine	77.8 ± 3.3	12.3 ± 2.0
Urea	49.2 ± 2.6	30.4 ± 1.4
Glucose	28.9 ± 6.6	43.3 ± 3.7
CH3COONa	27.8 ± 2.3	45.4 ± 1.6

a) Concentrations of RM and 3-Br-RM when a solution of 100 µM RM was incubated with 100 µM HOBr in 100 mM potassium phosphate buffer (pH 4.7) at 37°C for 24 h in the presence of 1 mM each of various additives. All the reaction mixtures were analyzed by RP-HPLC. Means ± S.D. (n = 3) are presented.

Effects of Additives on the Reaction with HOCl

To obtain information about the effect of coexistent compounds on the reactions of RM with HOCl, experiments were carried out in the presence of the same additives. A solution of 100 µM RM and 1 mM additives was incubated with 100 µM HOCl in 100 mM potassium phosphate buffer (pH 4.7) at 37°C for 24 h. No consumption of RM was observed in the presence of any additives (data not shown).

Conclusion

The present study showed that RM reacts with HOBr to generate 3-Br-RM as the major product, along with and other brominated derivatives of RM. Also, in the presence of Br⁻ at serum concentrations, HOCl reacted with RM to generate 3-Br-RM. Free amino acids effectively suppressed the reactions of RM with HOBr. These results suggest that 3-Br-RM may be generated from RM at inflammation sites in humans, although the reaction is considerably suppressed by coexistent free amino acids. No information about physiological activity and metabolism is available for 3-Br-RM. Further study of 3-Br-RM may be necessary in the future.

Experimental

Materials

RM was purchased from ChemCruz (TX, U.S.A.). NaBr (>99.99%) was purchased from Sigma-Aldrich (MO, U.S.A.). Other chemicals were obtained from Nacalai Tesque (Kyoto, Japan) or TCI (Tokyo, Japan). Bromide-free HOBr was prepared by the addition of silver nitrate and subsequent distillation, as previously reported.^16,20) The concentration of HOBr was determined spectrophotometrically at 331 nm in 10 mM NaOH using a molar extinction coefficient of 315 M⁻¹ cm⁻¹.²⁰⁾ Chloride-free sodium hypochlorite (NaOCl) was prepared by the method previously reported.²¹⁾ The concentration of NaOCl was determined spectrophotometrically at 290 nm using a molar extinction coefficient of 350 M⁻¹ cm⁻¹.²²⁾ Water was purified with a Millipore Milli-Q deionizer.

HPLC and MS Conditions

The HPLC system consisted of LC-10ADvp pumps and an SPD-M10Avp UV-vis photodiode-array detector (Shimadzu, Kyoto, Japan). For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of 4.6 × 250 mm and particle size 5 µm (GL Sciences, Tokyo, Japan) was used. The eluent was 20 mM ammonium acetate (pH 7.0) containing acetonitrile. The acetonitrile concentration was increased from 0 to 70% during 30 min in linear gradient mode, and maintained at 70% from 30 to 40 min. The column temperature was 40°C and the flow rate was 1 mL/min. ESI-TOF/MS measurements were performed on a MicroTOF spectrometer (Bruker, Bremen, Germany) in negative mode. The sample isolated by RP-HPLC was directly infused into the MS system by a syringe pump without a column.

Spectrometric Data

Peak 1 (3-bromorebamipide, 3-Br-RM). ESI-TOF/MS (negative mode): m/z 447, 449, 451. HR-ESI-TOF/MS (negative mode): 446.973933 obsd, 446.975271 calcd for C₁₉H₁₃⁷⁹Br³⁵ClN₂O₄⁻. UV: λ_max = 224, 282, and 333 nm (pH 7.0). ¹H-NMR (500 MHz, dimethyl sulfoxide (DMSO)-d₆): δ (ppm/tetramethylsilane (TMS)) 8.37 (br, 1H, α-NH), 8.13 (d, J = 8.1 Hz, 1H, H-5), 7.70 (d, J = 8.0 Hz, 2H, H-2′ and 6′), 7.50 (d, J = 7.5 Hz, 1H, H-7), 7.47 (d, J = 8.6 Hz, 2H, H-3′ and 5′), 7.30 (d, J = 8.0 Hz, 1H, H-8), 7.21 (dd, J = 8.1, 7.5 Hz, 1H, H-6), 4.58 (m, 1H, H-α), 3.74 (dd, J = 13.5, 3.7 Hz, 1H, C-β), 3.50 (dd, J = 13.2, 10.3 Hz, 1H, H-β), ¹³C-NMR (125 MHz, DMSO-d₆): δ (ppm/TMS) 171.8 (COOH), 164.3 (CONH), 157.2 (C-2), 148.3 (C-4), 137.1 (C-8a), 135.7 (C-4′), 133.1 (C-1′), 130.1 (C-7), 128.9 (C-2′ and 6′), 128.2 (C-3′ and 5′), 125.4 (C-5), 122.0 (C-6), 120.0 (C-3), 119.1 (C-4a), 115.4 (C-8), 53.0 (C-α), 37.5 (C-β).

Peak 2. ESI-TOF/MS (negative mode): m/z 525, 527, 529. HR-ESI-TOF/MS (negative mode): 524.884958 obsd, 524.885783 calcd for C₁₉H₁₂⁷⁹Br₂³⁵ClN₂O₄⁻. UV: λ_max = 233, 282, and 337 nm (pH 7.0).

Peak 3. ESI-TOF/MS (negative mode): m/z 447, 449, 451. HR-ESI-TOF/MS (negative mode): 446.975219 obsd, 446.975271 calcd for C₁₉H₁₃⁷⁹Br³⁵ClN₂O₄⁻. UV: λ_max = 235 and 292 nm (pH 7.0).

Quantitative Procedures

The concentrations of compounds were evaluated from the integrated peak areas on RP-HPLC chromatograms detected at 230 nm and by the molecular extinction coefficient at 230 nm (ε_230 nm). The ε_230 nm value of 34400 M⁻¹ cm⁻¹ was used for RM. The ε_230 nm value of 3-Br-RM was determined from the integration of proton signals of NMR and the HPLC peak area detected at 230 nm relative to that of RM in the mixed solution. The estimated ε_230 nm value for 3-Br-RM was 33900 M⁻¹ cm⁻¹.

Conflict of Interest

The authors declare no conflict of interest.

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