Reactions of methotrexate with hypobromous acid and hypochlorous acid

Article abstract of DOI:10.1248/cpb.c19-00602

Methotrexate is a folate antagonist cytotoxic drug employed in the therapy of cancers and rheumatoid arthritis. Hypobromous acid (HOBr) and hypochlorous acid (HOCl) are generated by eosinophils and neutrophils at inflammation sites. The administered metho

Full text of DOI:10.1248/cpb.c19-00602

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Chem. Pharm. Bull. 67, 1250–1254 (2019)
Vol. 67, No. 11
Note
Reactions of Methotrexate with Hypobromous Acid and Hypochlorous Acid
Toshinori Suzuki* and Rie Takeuchi
School of Pharmacy, Shujitsu University; 1–6–1 Nishigawara, Okayama 703–8516, Japan.
Received July 19, 2019; accepted August 20, 2019
Methotrexate is a folate antagonist cytotoxic drug employed in the therapy of cancers and rheumatoid
arthritis. Hypobromous acid (HOBr) and hypochlorous acid (HOCl) are generated by eosinophils and neu-
trophils at inflammation sites. The administered methotrexate may encounter HOBr and HOCl, and react
with them to generate products. When methotrexate was incubated with HOBr or HOCl at pH 7.4 and 37°C
for 30min, a single product was generated almost exclusively in each case, identified as 3ꢀ-bromomethotrexate
for HOBr and 3ꢀ-chloromethotrexate for HOCl. When methotrexate was incubated with HOCl in the pres-
ence of NaBr, the concentration of 3ꢀ-bromomethotrexate increased with decreasing concentration of 3ꢀ-chlo-
romethotrexate in a dose-dependent manner with NaBr, probably due to the formation of HOBr. Free amino
acids suppressed the reactions of methotrexate with HOBr and HOCl. Taurine suppressed the HOCl reaction
but not the HOBr reaction. These results suggest that 3ꢀ-bromomethotrexate and 3ꢀ-chloromethotrexate may
be generated from methotrexate at inflammation sites in humans, although their formation will be sup-
pressed by coexistent amino acids.
Key words methotrexate; hypobromous acid; hypochlorous acid; 3′-bromomethotrexate; 3′-chloromethotrexate
Introduction
Methotrexate (MTX), also known as amethopterin, is one
Results
Reaction with HOBr A solution of 100µM MTX was
of the oldest drugs but is still frequently used.1–3) MTX is incubated with 100µM HOBr in 100mM potassium phosphate
structurally similar to folic acid and acts as an antimetabolite, buffer (pH 7.4) at 37°C for 30min. When the reaction mix-
interfering with the metabolism of folic acid. MTX competi- ture was analyzed by reversed-phase (RP) HPLC, a product
tively inhibits dihydrofolate reductase synthesizing tetrahy- peak appeared in the chromatogram detected at 260nm (Fig.
drofolate that provides single carbon groups for the synthesis 1). The product peak showed a UV spectrum (λmax =260
of precursors of DNA and RNA.4) Thus, MTX can inhibit cell and 372nm) as shown in the inset of Fig. 1. The product was
proliferation. High doses of MTX are prescribed for the treat- isolated by RP-HPLC and identified using MS (electrospray
ment of cancer,5) while low doses MTX are widely prescribed ionization time of flight mass spectrometry, ESI-TOF/MS) and
1
1
for the treatment of rheumatoid arthritis.6,7) These diseases NMR (1H-1d, H–1H correlation spectroscopy (COSY), H–13C
treated with MTX involve inflammation. At sites of inflam- heteronuclear multiple quantum correlation (HMQC), H–13C
1
mation, eosinophils and neutrophils are recruited. Eosinophils, heteronuclear multiple bond correlation (HMBC), 13C-1d). The
one of the white blood cell types, are abundant in blood product showed an ESI-TOF/MS spectrum including peaks
and tissues in various inflammatory disorders.8) Eosinophil of m/z=531 and 533 (1:1) in negative mode. High-resolution
peroxidase, secreted by eosinophils, generates hypobromous (HR) ESI-TOF/MS values of the molecular ion (m/z 531) for
acid (HOBr) using H2O2 and Br−.9,10) Although the plasma the product agreed with the theoretical molecular mass for
concentrations are approx. 60µM for Br− and 100mM for C20H2179BrN8O5− attributable to [MTX+Br–2H]− within
1
Cl−, eosinophil peroxidase uses Br− preferentially to generate 3ppm. A singlet aromatic H signal attributable to the 7-posi-
HOBr.11,12) Meanwhile, neutrophils are a major component of tion of the pteridine moiety was observed. For the benzoyl
white blood cells. Myeloperoxidase, secreted by neutrophils, group, three aromatic protons were observed. Two of them,
generates hypochlorous acid (HOCl) from H2O2 and Cl−,13–15) attributable to 2′ and 6′ protons, showed correlations with
1
which plays a central role in host defense mechanisms against the carbonyl carbon on the H–13C HMBC spectrum. From
infection. HOCl can oxidize Br− to generate HOBr.16,17) Thus, these data, the product was identified as 3′-bromomethotrexate
a portion of the HOCl formed by the myeloperoxidase sys- (3′-Br-MTX) (Fig. 2).
tem in humans should react with Br−, converting to HOBr.
Figure 3A shows the time-dependence of the reaction of
Under inflammation conditions, MTX may encounter HOBr MTX with HOBr when 100µM MTX and 100µM HOBr
and HOCl, and react with them. In the present study, we were incubated in 100mM potassium phosphate buffer at pH
examined the reactions of MTX with HOBr and HOCl, and 7.4 and 37°C for 0–4h. The reaction was fast and completed
report that both HOBr and HOCl react with MTX to generate within 10min. Figure 3B shows the HOBr dose-dependence
monohalogenated products. To the best of our knowledge, this of the reaction of MTX with HOBr when 100µM MTX and
is the first study to examine the reactions of MTX with HOBr 0–100 µM HOBr were incubated in 100mM potassium phos-
and HOCl under neutral conditions, although the reactions of phate buffer at pH 7.4 and 37°C for 30min. With increasing
MTX with Br2 and Cl2 in hydrochloric acid were reported.18)
HOBr concentration, the consumption of MTX and the forma-
tion of 3′-Br-MTX increased. MTX was converted to 3′-Br-
MTX almost exclusively.
*To whom correspondence should be addressed. e-mail: tsuzuki@shujitsu.ac.jp
© 2019 The Pharmaceutical Society of Japan

Vol. 67, No. 11 (2019)
Chem. Pharm. Bull.
1251
Reaction with HOCl A solution of 100µM MTX was 372nm) similar to 3′-Br-MTX. The product was isolated by
incubated with 100µM HOCl in 100mM potassium phos- RP-HPLC and identified using MS and NMR. The product
phate buffer (pH 7.4) at 37°C for 30min. A product peak showed a ESI-TOF/MS spectrum including peaks of m/z=487
appeared in the RP-HPLC chromatogram detected at 260nm and 489 (3:1) in negative mode. HR-ESI-TOF/MS values of
(Fig. 4). The peak showed a UV spectrum (λmax =261 and the molecular ion (m/z 487) for the product agreed with the
theoretical molecular mass for C20H2135ClN8O5− attributable
to [MTX+Cl–2H]− within 3ppm. For NMR, results similar
1
to those of 3′-Br-MTX were obtained. A singlet aromatic H
signal attributable to the 7-position of the pteridine moiety
was observed. For the benzoyl group, three aromatic protons
were observed. Two of them, attributable to 2′ and 6′ protons,
1
showed correlations with the carbonyl carbon on the H–13C
HMBC spectrum. From these data, the product was identified
as 3′-chloromethotrexate (3′-Cl-MTX) (Fig. 5).
Figure 6A shows the time-dependence of the reaction of MTX
with HOCl when 100µM MTX and 100µM HOCl were incu-
bated in 100mM potassium phosphate buffer (pH 7.4) at 37°C
for 0–4h. The reaction was fast and completed within 10min.
Figure 3B shows the HOCl dose-dependence of the reaction of
MTX with HOCl when 100µM MTX and 0–100µM HOCl were
incubated in 100mM potassium phosphate buffer at pH 7.4 and
37°C for 30min. With increasing HOCl concentration, the con-
sumptions of MTX and the formation of 3′-Cl-MTX increased.
MTX was converted to 3′-Cl-MTX almost exclusively.
Reaction with HOCl in the Presence of NaBr A solu-
Fig. 1. RP-HPLC Chromatogram of a Reaction Mixture of MTX with
HOBr Detected at 260nm
A solution of 100µM MTX was incubated with 100µM HOBr in 100mM
tion of 100µM MTX was incubated with 100µM HOCl in the
presence of 0–100µM NaBr in 100mM potassium phosphate
potassium phosphate buffer at pH 7.4 and 37°C for 30min. The HPLC system
consisted of LC-10ADvp pumps and an SPD-M10Avp UV-vis photodiode-array
detector (Shimadzu). For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column
buffer (pH 7.4) at 37°C for 30min (Fig. 7). As the NaBr dose
was increased up to 100µM, the concentration of 3′-Cl-MTX
decreased, while the concentration of 3′-Br-MTX increased.
Effects of Additives on the Reaction with HOBr To
obtain information about the effect of coexistent compounds
on the reactions of MTX with HOBr, experiments were car-
ried out in the presence of various additives. Table 1 shows
the concentrations of MTX and 3′-Br-MTX when a solution of
100 µM MTX and 1mM additives was incubated with 100µM
HOBr in 100mM potassium phosphate buffer (pH 7.4) at 37°C
for 24h. Ammonium chloride suppressed the reaction. Methyl-
amine and dimethylamine showed little effect, while trimeth-
ylamine and tetramethylammounium showed no effect. Amino
acids suppressed the reaction. Taurine and urea showed no
of 4.6×250mm and particle size 5µm (GL Sciences) was used. The eluent was
20mM ammonium acetate (pH 7.0) containing methanol. The methanol concen-
tration was increased from 0 to 50% during 15min in linear gradient mode, and
maintained at 50% from 15 to 30min. The column temperature was 40°C and the
flow rate was 1mL/min.
Fig. 2. Structure of 3′-Bromomethotrexate (3′-Br-MTX)
Fig. 3. (A) Time Course of the Concentration Changes in MTX and 3′-Br-MTX When a Solution of 100µM MTX Was Incubated with 100µM HOBr
in 100mM Potassium Phosphate Buffer at pH 7.4 and 37°C for 0–4h
(B) HOBr dose-dependence of the concentration changes in MTX and 3′-Br-MTX when a solution of 100µM MTX was incubated with 0–100µM HOBr in 100mM
potassium phosphate buffer at pH 7.4 and 37°C for 30min. MTX (closed circle) and 3′-Br-MTX (open triangle). All the reaction mixtures were analyzed by RP-HPLC.
Means standard deviation (S.D.) (n=3) are presented.

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Chem. Pharm. Bull.
Vol. 67, No. 11 (2019)
effect. Ascorbic acid suppressed the reaction. Glucose and amine strongly suppressed the reaction, while trimethylamine
sodium acetate showed no effect.
and tetramethylammounium showed little effect. Amino acids
Effects of Additives on the Reaction with HOCl To and taurine suppressed the reaction almost completely. Urea,
obtain information about the effect of coexistent compounds glucose, and sodium acetate showed little suppression. Ascor-
on the reactions of MTX with HOCl, experiments were car- bic acid suppressed the formation of 3′-Cl-MTX almost com-
ried out in the presence of various additives. Table 2 shows pletely, although a certain amount of MTX was consumed.
the concentrations of MTX and 3′-Cl-MTX when a solution of
100 µM MTX and 1mM additives was incubated with 100µM
Discussion
HOCl in 100mM potassium phosphate buffer (pH 7.4) at 37°C
3′-Br-MTX and 3′-Cl-MTX were reportedly synthesized
for 24h. Ammonium chloride, methylamine, and dimethyl- from MTX in hydrochloric acid by Br2 and Cl2, respectively.18)
In the present study, we showed that MTX reacted with HOBr
and HOCl to generate 3′-Br-MTX and 3′-Cl-MTX in a neutral
solution, respectively. The reactions were fast and gener-
ated the corresponding halogenated MTX almost exclusively.
Recently, we showed that rebamipide, a therapeutic agent
for gastric ulcers and chronic gastritis, reacted with HOBr
to yield several products including 3-bromorebamipide as a
major product.19) Rebamipide did not react with HOCl. Under
similar conditions, MTX reacted with not only HOBr but also
HOCl to generate the corresponding monohalogenated prod-
ucts. Generally, the reactivity of HOBr to organic compounds
is higher than that of HOCl due to the higher electrophilicity
of HOBr compared to HOCl.20) MTX reacted with HOCl com-
parably to HOBr, especially at a low dose (0.25mM) of the
hypohalous acids as shown in Figs. 3B and 6B. This suggests
that the reactivity of HOCl to MTX is relatively high. In the
presence of NaBr, HOCl generated 3′-Br-MTX from MTX in
addition to 3′-Cl-MTX as shown in Fig. 7. At around serum
Fig. 4. RP-HPLC Chromatogram of a Reaction Mixture of MTX with
HOCl Detected at 260nm
concentration of Br− (approx. 60µM),11,12) the formation of
A solution of 100µM MTX was incubated with 100µM HOCl in 100mM potas-
sium phosphate buffer at pH 7.4 and 37°C for 30min. The HPLC conditions are the
that 3′-Br-MTX, in addition to 3′-Cl-MTX, may also gener-
same as shown in Fig. 1.
3′-Br-MTX was comparable to that of 3′-Cl-MTX, suggesting
ated in vivo by HOCl formed by myeloperoxidase.
CH3NH2 and (CH3)2NH had little effect on the reactions of
MTX with HOBr, whereas they suppressed the reactions with
HOCl almost completely (Tables 1, 2). CH3NH2 and (CH3)2NH
react with HOBr and HOCl to generate corresponding bro-
mamines and chloramines, respectively. It has been reported
that second-order rate constants for the reactions of phenol
with bromamines of CH3NH2 and (CH3)2NH are several orders
higher than those with chloramines.21) Similarly, MTX would
react with bromamines of CH3NH2 and (CH3)2NH to generate
Fig. 5. Structure of 3′-Chloromethotrexate (3′-Cl-MTX)
Fig. 6. (A) Time Course of the Concentration Changes in MTX and 3′-Cl-MTX When a Solution of 100µM MTX Was Incubated with 100µM HOBr
in 100mM Potassium Phosphate Buffer at pH 7.4 and 37°C for 0–4h
(B) HOCl dose-dependence of the concentration changes in MTX and 3′-Cl-MTX when a solution of 100µM MTX was incubated with 0–100µM HOCl in 100mM
potassium phosphate buffer at pH 7.4 and 37°C for 30min. MTX (closed circle) and 3′-Cl-MTX (open square). All the reaction mixtures were analyzed by RP-HPLC.
Means S.D. (n=3) are presented.

Vol. 67, No. 11 (2019)
Chem. Pharm. Bull.
1253
Table 1. Effects of Additives on the Reactions of MTX with HOBra)
Additives
MTX (µM)
3′-Br-MTX (µM)
None
2.6 0.7
50.4 4.9
10.3 2.9
19.1 3.7
0.2 0.3
0.0 0.0
59.6 1.0
89.2 0.1
95.8 0.1
98.2 0.1
0.9 1.2
0.7 0.6
78.4 0.9
0.7 0.5
4.0 2.5
95.9 1.1
42.5 4.6
87.1 2.6
77.7 3.8
95.8 0.4
95.3 1.9
38.5 0.3
9.0 0.1
NH4Cl
CH3NH2/HCl
(CH3)2NH/HCl
(CH3)3N/HCl
(CH3)4NCl
Gly
Lys
Met
4.1 0.1
Cys
1.6 0.0
Taurine
Urea
95.6 0.7
94.3 1.1
4.9 1.0
Fig. 7. NaBr Dose-Dependence of the Concentration Changes in MTX,
3′-Br-MTX, and 3′-Cl-MTX When a Solution of 100µM MTX Was Incu-
bated with 100µM HOCl in the Presence of 0–100µM NaBr in 100mM
Potassium Phosphate Buffer at pH 7.4 and 37°C for 30min
Ascorbic acid
Glucose
CH3COONa
93.8 4.8
92.7 2.4
MTX (closed circle), 3′-Br-MTX (open triangle), and 3′-Cl-MTX (open square).
All the reaction mixtures were analyzed by RP-HPLC. Means S.D. (n=3) are
presented.
a) Concentrations of MTX and 3′-Br-MTX when a solution of 100µM MTX was
incubated with 100µM HOBr in 100mM potassium phosphate buffer (pH 7.4) at
37°C for 24h in the presence of 1mM of additives. All the reaction mixtures were
analyzed by RP-HPLC. Means S.D. (n=3) are presented.
3′-Br-MTX, whereas MTX would not react with chloramines.
The reactions of both HOBr and HOCl were suppressed by
amino acids, even Gly. It has been reported that α-amino acids
react with both HOBr and HOCl to yield corresponding alde-
hydes and nitriles via monobromamines and dibromamines of
the α-amino acids, respectively.22) The reaction was suppressed
more strongly by Lys, Met, and Cys than by Gly. This implies
that, in addition to the reaction on the α-amino group, a
γ-amino group for Lys, a methyl thiol group for Met, or a thiol
group for Cys in the side chain reacts with HOBr and HOCl.
The reaction of MTX with HOBr and HOCl in plasma should
be suppressed by free amino acids, since the total concentra-
tion of free amino acids in human plasma is several mM.23)
Taurine suppressed the HOCl reaction almost completely,
whereas taurine did not suppress the HOBr reaction. Taurine
reacts with HOBr and HOCl resulting in taurine bromamine
(Tau-NHBr) and taurine cholamine (Tau-NHCl), respectively.
It has been reported that the reactivity of Tau-NHBr to amino
acids is higher than that of Tau-NHCl.24) Tau-NHBr may react
with MTX to generate 3′-Br-MTX, whereas Tau-NHCl could
not react with MTX. Although the taurine concentration is
low in plasma (38µM), it is high in neutrophils (19mM) and
eosinophils (15mM).25) The reaction of MTX with HOCl gen-
erated by myeloperoxidase might be suppressed by taurine,
Table 2. Effects of Additives on the Reactions of MTX with HOCla)
Additives
MTX (µM)
3′-Cl-MTX (µM)
None
19.1 3.4
84.0 2.3
98.5 0.7
97.3 0.7
29.6 3.0
23.1 2.2
99.1 0.5
98.4 0.3
100.1 0.4
99.8 0.3
98.8 0.3
21.5 8.2
81.4 0.2
27.9 3.9
26.3 9.8
64.6 2.5
3.0 0.2
1.0 0.1
2.1 0.2
64.5 3.4
63.5 2.2
0.9 0.1
0.7 0.0
0.7 0.0
0.7 0.1
0.8 0.0
62.4 6.6
0.6 0.1
58.9 3.4
59.7 8.3
NH4Cl
CH3NH2/HCl
(CH3)2NH/HCl
(CH3)3N/HCl
(CH3)4NCl
Gly
Lys
Met
Cys
Taurine
Urea
Ascorbic acid
Glucose
CH3COONa
a) Concentrations of MTX and 3′-Cl-MTX when a solution of 100µM MTX was
incubated with 100µM HOCl in 100mM potassium phosphate buffer (pH 7.4) at
37°C for 24h in the presence of 1mM of additives. All the reaction mixtures were
analyzed by RP-HPLC. Means S.D. (n=3) are presented.
whereas the reaction with HOBr by eosinophil peroxidase may rats. Treatment of the retransplanted tumors with the alkyl-
not be suppressed. ating agent reduced tumor successfully. This suggests that
Several studies have examined the concerning biological ac- 3′-Br-MTX can work as an antitumor reagent as well as or
tivities of 3′-Br-MTX and 3′-Cl-MTX. 3′-Br-MTX, as well as stronger than MTX under certain conditions. 3′-Cl-MTX in-
MTX, suppressed uptake of tritium-labeled MTX (MTX-3H) hibited both cultured human leukemic lymphoblasts and the
by the choroid plexus of a rabbit brain in vitro.26) Bromine- corresponding MTX-resistant subline at a similar efficiency
77-labeled 3′-Br-MTX (3′-77Br-MTX) injected into the femoral to MTX,29) suggesting that 3′-Cl-MTX works as an antitumor
artery of a tumor bearing rat leg together with MTX showed reagent as well as MTX.
high initial retention activity by the tumor.27) These reports
The present study showed that MTX reacts with HOBr and
indicate that 3′-Br-MTX can be taken up in organs or cells HOCl to generate 3′-Br-MTX and 3′-Cl-MTX, respectively.
comparable to MTX. A Yoshida lymphosarcoma line resistant Free amino acids suppressed both the HOBr and HOCl reac-
to an alkylating agent showed collateral sensitivity against tions, while taurine suppressed the HOCl reaction but not the
3′-Br-MTX.28) The original Yoshida sarcoma was transplanted HOBr reaction. These results suggest that 3′-Br-MTX and
into rats, which were then treated with 3′-Br-MTX. Initially, 3′-Cl-MTX may be generated from MTX at inflammation
the tumor volume reduced, but tumor regrowth commenced, sites, although the reactions will be suppressed by coexistent
and less response was seen to the second dose. Some of the amino acids. If these halogenations occur on MTX adminis-
3′-Br-MTX treated tumors were then transplanted into other tered to humans with inflammation, they may affect the medi-

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Chem. Pharm. Bull.
Vol. 67, No. 11 (2019)
cal efficacy of MTX.
The ε260nm value of 22700M−1 cm−1 was used for MTX.
The ε260nm values of 3′-Br-MTX and 3′-Cl-MTX were deter-
mined from the integration of proton signals of NMR and
Experimental
Materials L-(+)-Amethopterin (MTX) was purchased the HPLC peak area detected at 260nm relative to that of
from Nacalai Tesque (Kyoto, Japan). NaBr (>99.99%) was MTX in the mixed solution. The estimated ε260nm values were
purchased from Sigma-Aldrich (MO, U.S.A.). Other chemicals 26800M−1 cm−1 for 3′-Br-MTX and 26500M−1 cm−1 for 3′-Cl-
were obtained from Nacalai Tesque or TCI (Tokyo, Japan). MTX.
Bromide-free hypobromous acid (HOBr) was prepared by
the addition of silver nitrate and subsequent distillation, as
Conflict of Interest The authors declare no conflict of
previously reported.17,29) The concentration of HOBr was de- interest.
termined spectrophotometrically at 331nm in 10mM NaOH
using a molar extinction coefficient of 315M−1 cm−1.30) Chlo-
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Spectrometric Data 3′-Bromomethotrexate (3′-Br-MTX).
ESI-TOF-MS: m/z 531, 533 (1:1). HR-ESI-TOF-MS: m/z
531.074154 obsd, 531.074552 calcd for C20H2179BrN8O5−.
UV: λmax =260, 372nm (pH 7.0). ε260nm =26800M−1 cm−1.
1H-NMR (500MHz, dimethyl sulfoxide (DMSO)-d6):
δ
(ppm/tetramethylsilane (TMS)) 8.77 (s, 1H, H-7), 8.30 (d,
J=6.9Hz, 1H, NH), 8.12 (d, J=2.3Hz, 1H, H-2′), 7.81 (dd,
J=8.6, 1.7Hz, 1H, H-6′), 7.28 (d, J=8.6Hz, 1H, H-5′),
4.44 (s, 2H, H-9), 4.25 (m, 1H, H-α), 2.78 (s, 3H, CH3),
2.25 (m, 2H, H-γ), 1.92 (m, 2H, H-β). 13C-NMR (125MHz,
DMSO-d6): δ (ppm/TMS) 174.8 (γ-COOH), 172.3 (α-COOH),
163.5 (C=O), 162.8, 162.7, 155.1 (C-8a), 152.3 (C-4′), 149.8
(C-7), 145.2 (C-6), 132.5 (C-2′), 130.2 (C-1′),127.3 (C-6′),
121.7 (C-5′), 121.1, 117.7 (C-3′), 58.2 (C-9), 53.2 (C-α), 40.8
(CH3), 32.2 (C-γ), 27.7 (C-β). 3′-Chloromethotrexate (3′-Cl-
MTX). ESI-TOF-MS: m/z 487, 489 (3:1). HR-ESI-TOF-MS:
487.125732 obsd, 487.125067 calcd for C20H2135ClN8O5−. UV:
1
λmax =261, 372nm (pH 7.0). ε260nm =26500M−1 cm−1. H-NMR
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(500MHz, DMSO-d6): δ (ppm/TMS) 8.74 (s, 1H, H-7), 8.28
(d, J=6.9Hz, 1H, NH), 7.93 (s, 1H, H-2′), 7.75 (d, J=8.3Hz,
1H, H-6′), 7.26 (d, J=8.6Hz, 1H, H-5′), 4.46 (s, 2H, H-9),
4.22 (m, 1H, H-α), 2.81 (s, 3H, CH3), 2.22 (m, 2H, H-γ), 1.92
(m, 2H, H-β). 13C-NMR (125MHz, DMSO-d6): δ (ppm/TMS)
175.0 (γ-COOH), 173.7 (α-COOH), 163.6 (C=O), 162.8, 162.7,
155.1 (C-8a), 150.8 (C-4′), 149.6 (C-7), 145.2 (C-6), 129.4
(C-2′), 129.3 (C-3′), 126.6 (C-6′), 126.3 (C-1′), 121.1 (C-5′),
121.0, 57.8 (C-9), 53.4 (C-α), 40.3 (CH3), 32.5 (C-γ), 27.8 (C-β).
Quantitative Procedures The concentrations of the
products were evaluated according to integrated peak areas
on RP-HPLC chromatograms detected at 260nm and by the
molecular extinction coefficients at 260nm (ε260nm).
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