Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation

Article abstract of DOI:10.1016/j.bmcl.2018.10.023

We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and lower levels of metabolic activation than BBR, while maintaining their inhibitory activity toward the uric acid transporter. We propose that these derivatives could serve as effective uricosuric agents that have much better safety profiles than BBR.

Full text of DOI:10.1016/j.bmcl.2018.10.023

Accepted Manuscript
Synthesis of novel benzbromarone derivatives designed to avoid metabolic ac-
tivation
Tomoyuki Ohe, Ryutaro Umezawa, Yumina Kitagawara, Daisuke Yasuda,
Kyoko Takahashi, Shigeo Nakamura, Akiko Abe, Shuichi Sekine, Kousei Ito,
Kentaro Okunushi, Hanae Morio, Tomomi Furihata, Naohiko Anzai, Tadahiko
Mashino
PII:
S0960-894X(18)30826-6
https://doi.org/10.1016/j.bmcl.2018.10.023
BMCL 26082
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 August 2018
9 October 2018
15 October 2018
Please cite this article as: Ohe, T., Umezawa, R., Kitagawara, Y., Yasuda, D., Takahashi, K., Nakamura, S., Abe,
A., Sekine, S., Ito, K., Okunushi, K., Morio, H., Furihata, T., Anzai, N., Mashino, T., Synthesis of novel
benzbromarone derivatives designed to avoid metabolic activation, Bioorganic & Medicinal Chemistry Letters
(2018), doi: https://doi.org/10.1016/j.bmcl.2018.10.023
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Synthesis of novel benzbromarone derivatives designed to avoid metabolic activation
Tomoyuki Ohea,*, Ryutaro Umezawaa, Yumina Kitagawaraa, Daisuke Yasudaa, Kyoko
Takahashia, Shigeo Nakamurab, Akiko Abec, Shuichi Sekinec, Kousei Itoc, Kentaro Okunushid,
Hanae Moriod, Tomomi Furihatad, Naohiko Anzaid, and Tadahiko Mashinoa,*
aFaculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo, Japan
bDepartment of Chemistry, Nippon Medical School, 1-7-1 Kyonan-cho, Musashino-shi, Tokyo,
Japan
cGraduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku,
Chiba, Japan
dGraduate School of Medicine, Chiba University, Inohana 1-8-1, Chuo-ku, Chiba, Japan
* Corresponding authors. Tel.: +81 3 5400 2691; fax +81 3 5400 2691 (T.O.); tel.: +81 3
5400 2694; fax +81 3 5400 2691 (T.M.).
E-mail addresses: ohe-tm@pha.keio.ac.jp (T.O.), mashino-td@pha.keio.ac.jp (T.M.)
Keywords
Benzbromarone; metabolic activation; hepatotoxicity; URAT1
Abbreviations
BBR, benzbromarone; CYP, cytochrome P450; DBBQ, 2,6-dibromo-p-benzoquinone; DBH,
2,6-dibromohydroquinone; DEX, dexamethasone; hURAT1, human uric acid transporter I;
IDR, idiosyncratic adverse drug reactions; LDH, lactate dehydrogenase; MPT, mitochondrial
permeability transition
Abstract
We synthesized six novel BBR derivatives that were designed to avoid metabolic activation via
ipso-substitution and evaluated for their degree of toxicity and hURAT1 inhibition. It was
found that all of the derivatives demonstrate lower cytotoxicity in mouse hepatocytes and
lower levels of metabolic activation than BBR, while maintaining their inhibitory activity
toward the uric acid transporter. We propose that these derivatives could serve as effective
uricosuric agents that have much better safety profiles than BBR.

Adverse drug reactions are one of the most important issues that occur during drug
development and the clinical usage of drugs in the postapproval stage. Specifically,
idiosyncratic adverse drug reactions (IDR) with often serious toxicities occur only in a small
percentage of the population and do not show any apparent dose dependency. IDR is a great
problem in terms of safety due to its unpredictability, which presents challenges during drug
development and leads to postmarketing drug withdrawals and black box warnings.1 Although
there are several hypotheses regarding the mechanisms that underlie IDR, it is widely accepted
that IDR is often associated with bioactivation that is mediated by metabolic enzymes, such as
cytochrome P450 (CYP).2
Benzbromarone (BBR) is used as a uricosuric agent due to its inhibition of the renal
reabsorption of uric acid by the human uric acid transporter I (hURAT1). Thus, BBR is
effective in the treatment of hyperuricemia and gout, particularly in cases in which allopurinol,
a xanthine oxidase inhibitor, is ineffective. It has been used in Japan and in parts of Europe for
a long period of time.3 However, BBR is not approved for use in the United States, and it has
been withdrawn from the market in Europe due to being responsible for fatal liver injury.4,5
Although the specific mechanisms responsible for its hepatic toxicity are still unknown,
mitochondrial toxicity and the formation of reactive metabolites have been hypothesized as
potential causes of its toxicity.
In our previous study6, we identified 2,6-dibromohydroquinone (DBH) and mono-
debrominated catechol (CAT), formed via the ipso-substitution of BBR, as novel metabolites
produced within rat and human liver microsomal systems. Considering that the cytotoxicity of
CAT was found to be slightly less in HepG2 cells and that CAT was not produced by CYP2C9
recombinant enzymes, which are supposedly responsible for the toxicity of BBR, it was
previously concluded that hepatocellular toxicity induced by BBR can be attributed to the
formation of DBH or its oxidative metabolite, 2,6-dibromo-p-benzoquinone (DBBQ) (Scheme

1); however, other bioactivation pathways have been proposed by other research groups.7-9
The present study aimed to design and synthesize novel BBR analogues that are not subject to
metabolic activation and, therefore, would not cause hepatotoxicity. In our previous study, we
presented a possible mechanism of ipso-substitution by CYP responsible for metabolic
reactions involving a variety of ortho or para-substituted phenols.10-15 During the first step,
one electron and one proton are abstracted from the phenol moiety, or a hydrogen radical is
abstracted from a hydroxy group within a para or ortho-substituted phenol, by CYP to
produce a phenoxy radical. This radical is delocalized on the aromatic ring and is distributed to
the ipso-position, followed by the rebound of the hydroxyl-radical equivalent on the heme iron
of CYP to produce a semiquinol intermediate, which degrades into hydroquinone (catechol) or
benzoquinone and thereby results in the elimination of the substituent.12 Similarly, in the case
of BBR, a phenoxy radical on the dibromophenol ring is formed during the first step, followed
by the distribution of the radical to the ipso-position of the carbonyl group (para-position of
hydroxy group). Then, the hydroxyl-radical equivalent on the heme iron attacks the ipso-
position to produce an α-hydroxyketone, which is easily degraded into DBH or DBBQ via
ipso-substitution and results in the cleavage of the carbon-carbon bond (Scheme 1). It was
clear that the presence of a hydroxy group at the para-position of the carbonyl group of BBR
is necessary for the ipso-substitution reaction that leads to the metabolic activation of BBR
into DBH or DBBQ. Therefore, we designed multiple BBR derivatives in which the hydroxy
group was transferred to the meta-position of the carbonyl group (Fig. 1), which would
prevent metabolic activation because the radical would not be distributed to the ipso-position
of the carbonyl group and ipso-substitution could not occur at the root of the carbonyl group.

Scheme 1 Proposed mechanism for metabolic activation of BBR via ipso-substitution.
Fig. 1 Chemical structure of BBR derivatives.
In the present study, we synthesized six novel BBR derivatives (1-6), designed to avoid
metabolic activation via ipso-substitution, that were evaluated for their degree of toxicity and
hURAT1 inhibition. The purpose of this study was to transform benzbromarone into
compounds that have a reduced risk of hepatotoxicity while retaining efficacy.
Synthesis of BBR derivatives. We designed and chemically synthesized six BBR derivatives
(1-6) that are shown in Fig. 1. The hydroxy group was moved to the meta-position of the
carbonyl group in all of the compounds in order to avoid metabolic activation. The bromine
atoms were retained within the molecules to minimize any changes in their physicochemical

properties, with the aim of producing levels of hURAT1 inhibition and pharmacokinetics that
were similar to those of BBR. The bromine atom may be subject to ipso-substitution that could
produce catechol metabolites; however, as described above, it is hypothesized that these
metabolites would not contribute greatly to toxicity. Compounds 1-4 are structural isomers
that have a bromine atom at different sites on the phenol ring, while compounds 5-6 contain
two bromine atoms.
For the synthesis of the BBR derivatives, six different (di)bromomethoxybenzoic acids (1a-6a)
were synthetically prepared as shown in Scheme 2, with the exception of 2-bromo-3-
methoxybenzoic acid (1a) and 4-bromo-3-methoxybenzoic acid (2a), which were commercially
available. With respect to compound 3, 3-bromo-5-methoxybenzoic acid (3a) was prepared by
the oxidation of 1-bromo-3-methoxy-5-methylbenzene with KMnO4. In the case of compound
4, 2-bromo-5-methoxybenzoic acid (4a) was prepared via the Pinnick oxidation of 2-bromo-5-
methoxybenzaldehyde. For the synthesis of compound 5, 3-hydroxybenzoic acid was used as
the starting material. The methylation of 3-hydroxybenzoic acid after selective dibromination at
the position ortho to the hydroxy group of 3-hydroxybenzoic acid with N-bromosuccinimide
(NBS) produced 2,4-dibromo-3-methoxybenzoic acid (5a). Regarding compound 6,
bromination with NaBr and Oxone® followed by purification using silica gel column
chromatography produced 2,4-dibromo-5-methoxybenzaldehyde, which was further oxidized
via Pinnick oxidation to produce 2,4-dibromo-5-methoxybenzoic acid (6a).
After the treatment of these six benzoic acid derivatives (1a-6a) with SOCl2, Friedel-Crafts
acylation with 2-ethylbenzofuran was directed to the 3-position of the benzofuran ring.
Subsequent demethylation of the methoxy groups with BBr3 produced compounds 1-6.

Scheme 2 Synthesis of BBR derivatives. (a) KMnO4, pyridine/H2O (2:5), reflux, 48 hr; (b)
NaClO2, NaH2PO4, tert-butyl alcohol, 2-methyl-2-butene, THF, H2O, rt, 1 hr; (c) NBS, conc.
H2SO4, 50 °C, 1 hr; (d) CH3I, DMF, rt, 1 hr, then, NaOH, CH3OH, H2O, rt, 4 hr; (e) Oxone®,
NaBr, CH3OH/H2O (1:1), rt, 1.5 hr; (f) NaClO2, NaH2PO4, tert-butyl alcohol, 2-methyl-2-
butene, THF, H2O, rt, 45 min; (g) SOCl2, reflux, 2-6 hr, then, 2-ethylbenzofuran, SnCl4.
CH2Cl2, rt, 2.5-24 hr; (h) BBr3, CH2Cl2, rt, 1-3 hr. More details are described in Supplemental
data.
Cytotoxicity of BBR derivatives in mouse hepatocytes. The cytotoxicity of BBR and


compounds 1-6 at concentrations of 1 M or 5 M was assessed by measuring the lactate
dehydrogenase (LDH) activity after 16 hr of drug exposure in hepatocytes that were prepared
from mice treated with dexamethasone (DEX) to induce the production of CYP enzymes.


BBR caused a dose-related LDH release of 27% and 52% at 1 M and 5 M, while compounds

1-6 showed less than 16% even at 5 M. Although there was no concentration dependence in
some compounds, it is clear that the cellular toxicities of compounds 1-6 are much lower than
that of BBR, which indicates that 1-6 are much safer for metabolism by hepatocytes, as was
expected (Fig. 2).

Fig. 2 Cytotoxicity of BBR derivatives in DEX-treated mouse hepatocytes. Cell toxicity was
assessed by measuring LDH release after 18 hr of drug exposure. The data represent the
average of measurements made in duplicate experiments.
Induction of NADPH-dependent mitochondrial permeability transition (MPT) by BBR
derivatives in rat liver microsomes. Next, we evaluated the mitochondrial toxicity of the
synthesized compounds compared to that of BBR; the hepatic toxicity of BBR has been
hypothesized to be associated with its mitochondrial toxicity and metabolic activation.16 The
induction of MPT is one of the mechanisms that underlies drug-induced mitochondrial toxicity
and is caused by many toxic drugs, including anticancer drugs, nonsteroidal anti-inflammatory
drugs, and some thiazolidinones. The loss of mitochondrial membrane impermeability is
associated with pathways involved in cell death, and MPT is thought to be a key event in this
process.16 In the present study, we investigated MPT induction after the incubation of liver
microsomes with BBR derivatives according to a previously reported method16 to evaluate the
mitochondrial toxicity of the derivatives and their metabolites.


Initially, either BBR or compounds 1-6 (50 M) were incubated with DEX-treated rat liver
microsomes in the presence or absence of NADPH. After incubation for 30 min, the isolated
mitochondrial fraction was added and the mitochondrial swelling was measured to determine
the level of MPT induction. To evaluate the induction of MPT, we plotted the change in
absorbance (540 nm) over time, then calculated the integral area over the curve (AOC). In the
absence of NADPH, no significant differences were observed in the levels of MPT induced by
any of the compounds, including BBR. On the other hand, the greatest level of NADPH-
dependent, drug-induced MPT was observed for BBR, while no NADPH-dependent MPT
induction was observed for compounds 1-6 (Fig. 3). These results suggest that compounds 1-6
are not subject to metabolic activation and have a lower potential to cause metabolite-induced
mitochondrial toxicity than BBR.
As discussed above, the exact mechanisms underlying the hepatocellular toxicity of BBR are
still unknown; multiple bioactivation pathways have been proposed by a number of research
groups.7-9 In our previous study, we demonstrated that metabolites formed via the ipso-
substitution of BBR by CYP2C9 are cytotoxic (Scheme 1).6 The fact that mitochondrial
toxicity in hepatocytes treated with compounds 1-6, which were designed to avoid ipso-
substitution, was reduced clearly indicates that the metabolism of BBR via ipso-substitution is
a major contributor to its hepatocellular toxicity.

400
350
300
250
200
150
100
50
NADPH(-)
NADPH(+)
*
0
BBR
1
2
3
4
5
6
Fig. 3 NADPH-dependent MPT induction by BBR derivatives. The compounds (50 μM) were
incubated with dexamethasone-treated rat liver microsomes at 37 °C in the absence or
presence of NADPH for 30 min, and the isolated mitochondria were added at the indicated
time point. Data represent the mean ± SD (n = 3). *: p < 0.05 vs. NADPH (-).
Inhibitory effects of the BBR derivatives on uric acid uptake by hURAT1-expressing
HEK cells. To examine whether the inhibition of hURAT1 by BBR was retained in
compounds 1-6, we evaluated uric acid uptake in the presence of BBR or the synthesized

compounds in HEK cells that overexpressed hURAT1 (10 M), using a previously reported
method.17 As shown in Fig. 4, BBR strongly inhibited the uptake of uric acid to a level that
was less than 10% of that of the control. While slightly weaker than BBR, compounds 1-6 also
produced significant inhibition of uric acid uptake. There were no significant differences in the
inhibitory potencies of the compounds.

120
100
80
60
40
20
0
**
***
***
**
***
***
***
Control
BBR
1
2
3
4
5
6
Fig. 4 Inhibitory effects of the BBR derivatives (10 μM) on uric acid uptake by hURAT1-
expressing HEK cells. The uric acid uptake values were expressed as percentage relative to
that obtained from the control (0.5% DMSO with no inhibitor). The data represent the mean ±
SD (n = 4). **: p < 0.01 vs. control, ***: p< 0.001 vs. control.
In summary, we synthesized a novel set of BBR derivatives that were designed to avoid
metabolic activation via ipso-substitution and found that they exhibited lower cytotoxicity in
mouse hepatocytes; we also found that their metabolites exhibited lower mitochondrial toxicity
than that of BBR. The BBR derivatives also showed significant hURAT1 inhibitory activity,
while slightly weaker than BBR. Although the weaker pharmacological activities are
unfavorable profiles for the alternative drugs aiming to mitigate the hepatotoxicity of BBR
with retaining efficacy, we propose that these derivatives might serve as lead compounds that
could be investigated for use as effective uricosuric agents with much better safety profiles
than BBR, with the aim of enabling more rapid dose escalation in vivo and overcoming the
attenuation of potency in vitro. The in vivo pharmacology and safety studies of the derivatives
are currently underway.

Acknowledgements
This work was supported by the Platform for Drug Discovery, Informatics, and Structural Life
Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT),
the Japan Agency for Medical Research and Development (AMED; 2012-2016), and the
Platform Project for Supporting Drug Discovery and Life Science Research, AMED (2017-
2018). This work was also supported by JSPS KAKENHI (Grant Number 16K08379) and a
special grant generously provided by the Hoansha Foundation.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at
https://doi.org/***
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Figure captions
Scheme 1 Proposed mechanism for metabolic activation of BBR via ipso-substitution.
Fig. 1 Chemical structure of BBR derivatives.
Scheme 2 Synthesis of BBR derivatives. (a) KMnO4, pyridine/H2O (2:5), reflux, 48 hr; (b)
NaClO2, NaH2PO4, tert-butyl alcohol, 2-methyl-2-butene, THF, H2O, rt, 1 hr; (c) NBS, conc.
H2SO4, 50 °C, 1 hr; (d) CH3I, DMF, rt, 1 hr, then, NaOH, CH3OH, H2O, rt, 4 hr; (e) Oxone®,
NaBr, CH3OH/H2O (1:1), rt, 1.5 hr; (f) NaClO2, NaH2PO4, tert-butyl alcohol, 2-methyl-2-
butene, THF, H2O, rt, 45 min; (g) SOCl2, reflux, 2-6 hr, then, 2-ethylbenzofuran, SnCl4.
CH2Cl2, rt, 2.5-24 hr; (h) BBr3, CH2Cl2, rt, 1-3 hr. More details are described in Supplemental
data.
Fig. 2 Cytotoxicity of BBR derivatives in DEX-treated mouse hepatocytes. Cell toxicity was
assessed by measuring LDH release after 18 hr of drug exposure. The data represent the
average of measurements made in duplicate experiments.
Fig. 3 NADPH-dependent MPT induction by BBR derivatives. The compounds (50 μM) were
incubated with dexamethasone-treated rat liver microsomes at 37 °C in the absence or

presence of NADPH for 30 min, and the isolated mitochondria were added at the indicated
time point. Data represent the mean ± SD (n = 3). *: p < 0.05 vs. NADPH (-).
Fig. 4 Inhibitory effects of the BBR derivatives (10 μM) on uric acid uptake by hURAT1-
expressing HEK cells. The uric acid uptake values were expressed as percentage relative to
that obtained from the control (0.5% DMSO with no inhibitor). The data represent the mean ±
SD (n = 4). **: p < 0.01 vs. control, ***: p < 0.001 vs. control.




Synthesis of a novel set of benzbromarone derivatives
Reduced cytotoxicity in mouse hepatocytes
Attenuated mitochondrial toxicity with metabolic activation
Maintained activity of inhibition toward uric acid transporter