3H-Imidazo[4,5-b]pyridine-6-carboxylic acid derivatives as rexinoids with reduced teratogenicity

Article abstract of DOI:10.1016/j.bmcl.2019.05.050

Several retinoid X receptor (RXR) ligands (rexinoids), such as bexarotene (1), exhibit teratogenicity, which is a serious impediment to their clinical application. We considered that rexinoids with a lower level of maximal RXR transcription activation (i.e., partial agonists) and lower lipid solubility might show weaker adverse side effects. Based on this idea, we modified our previously reported pentamethyltetralin-type RXR partial agonists 5 and 6 to reduce their lipophilicity. Here, we report a new RXR partial agonist, 3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-(trifluoromethyl)-3H-imidazo[4,5-b]pyridine-6-carboxylic acid (8, CATF-PMN), which showed greatly reduced teratogenicity in zebrafish embryos.

Full text of DOI:10.1016/j.bmcl.2019.05.050

Accepted Manuscript
3H-Imidazo[4,5-b]pyridine-6-carboxylic Acid Derivatives as Rexinoids with
Reduced Teratogenicity
Yuta Takamura, Manami Takahashi, Midori Nishii, Osamu Shibahara, Masaki
Watanabe, Michiko Fujihara, Hiroki Kakuta
PII:
S0960-894X(19)30356-7
https://doi.org/10.1016/j.bmcl.2019.05.050
BMCL 26470
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 May 2019
24 May 2019
27 May 2019
Please cite this article as: Takamura, Y., Takahashi, M., Nishii, M., Shibahara, O., Watanabe, M., Fujihara, M.,
Kakuta, H., 3H-Imidazo[4,5-b]pyridine-6-carboxylic Acid Derivatives as Rexinoids with Reduced Teratogenicity,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.05.050
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3H-Imidazo[4,5-b]pyridine-6-carboxylic Acid Derivatives as Rexinoids
with Reduced Teratogenicity
Yuta Takamura,a Manami Takahashi,a Midori Nishii,a,b Osamu Shibahara,a Masaki Watanabe,a
Michiko Fujihara,a,b and Hiroki Kakutaa,*
a Division of Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Dentistry and
Pharmaceutical Sciences, 1-1-1, Tsushima-Naka, Kita-Ku, Okayama 700-8530, Japan.
b AIBIOS Co. Ltd. Tri-Seven Roppongi 8F 7-7-7 Roppongi, Minato-ku, Tokyo 106-0032 Japan.
ABSTRACT: Several retinoid X receptor (RXR) ligands (rexinoids), such as bexarotene (1),
exhibit teratogenicity, which is a serious impediment to their clinical application. We considered
that rexinoids with a lower level of maximal RXR transcription activation (i.e., partial agonists) and
lower lipid solubility might show weaker adverse side effects. Based on this idea, we modified our
previously reported pentamethyltetralin-type RXR partial agonists 5 and 6 to reduce their
lipophilicity.
Here,
we
report
a
new
RXR
partial
agonist,
3-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydronaphthalen-2-yl)-2-(trifluoromethyl)-3H-imidazo[4,5-b]
pyridine-6-carboxylic acid (8, CATF-PMN), which showed greatly reduced teratogenicity in
zebrafish embryos.
KEYWORDS: Nuclear receptor, RXR, rexinoid, teratogenicity, zebrafish
1

Table of Contents Graphic
2

The incidence of birth defects in neonates is about 2–3%,1 and it is reported that about
1–3% of them are caused by medicines.2 A well-known example of teratogenicity as a drug side
effect is provided by thalidomide, which was marketed in 1957 as a remedy for morning sickness in
pregnant women and as a sleeping drug for infants, but was subsequently found to cause birth
defects (thalidomide embryopathy).3,4 Various vitamin A-related compounds (retinoids) also show
teratogenicity via activation of retinoic acid receptors (RARs),5-8 especially RARThus,
assessment of teratogenic potential is an essential part of drug development.
Zebrafish (Danio rerio) are used as a model for prediction of the teratogenic potential of
compounds in humans.10 Zebrafish are useful models for studying various bioprocesses of
vertebrates because of their genetic proximity to humans and the transparency of their eggs and
larvae.11 For example, bexarotene (1, Figure 1), used to treat cutaneous T cell lymphoma (CTCL),12
was found to be teratogenic in zebrafish.13 The molecular targets of 1 are retinoid X receptors
(RXRs), and other RXR activators such as 9-cis retinoic acid (2) and triphenyltin (TPT, 3, Figure 1)
also exhibit teratogenicity.13 It is reported that 2 can activate not only RXR but also RAR.14 For
example, 1 is an RXR agonist that also activates RAR.15 This dual activity results in a synergistic
effect,16 which may account for the teratogenicity of these compounds. Interestingly, the RXR
3

antagonist UVI3003 (4, Figure 1) is also teratogenic.17 Therefore, RXR activation itself is unlikely
to be involved in teratogenicity. In fact, the teratogenicity of 4 was reported to be caused by
PPAR(peroxisome proliferator-activated receptor ) activation.17 On the other hand, pioglitazone
hydrochloride, a well-known PPAR agonist, is not teratogenic in zebrafish.18 Since pioglitazone is
used as pioglitazone hydrochloride, we speculated that its high molecular polarity might account for
the lack of teratogenicity. Actually, it is reported that the solubilities of pioglitazone and pioglitazone
hydrochloride in water are 0.039 mM and 0.70 mM, respectively.19 Therefore, we hypothesized that
RXR agonists with reduced teratogenicity might be obtained by increasing their molecular polarity
or hydrophilicity. Here, we report the successful application of this idea to obtain novel RXR
ligands with reduced teratogenicity.
4

Figure 1. Chemical structures of 1–8.
Based on the fact that highly lipophilic full RXR agonists, such as 1, show teratogenicity,
we first examined the teratogenicity of our previously reported RXR partial agonists CBt-PMN20 (5,
Figure 1) and CBTF-PMN21 (6, Figure 1) in zebrafish. We focused on 5 and 6 because they show a
lower blood triglyceride-elevating effect than bexarotene, so we considered that they might also show
lower teratogenicity. These compounds have a similar pentamethyltetralin structure to 1, but have
lower lipophilicity (Table 1).
Table 1 shows the hatching rate, survival rate, and malformation rate in zebrafish after
5

exposure of the eggs to 1, 5 or 6 for 120 hours. The group treated with 1 at 100 nM showed 67%
hatching rate versus the untreated control, and the malformation rate was 100%. Malformations
included spine curvature and an increased proportion of the head in the whole body (Figure 2). The
groups treated with 5 or 6 at the same concentration showed hatching rates of 75% and 83%,
respectively, but notably the malformation rate was 0% for both compounds. However, when the
eggs were exposed to a higher concentration (1,000 nM) of 5 or 6, the malformation rate increased
to 100% and 17%, respectively. As regards RXR transcriptional activation, 6 shows a higher Emax
value than 5 and the EC50 value of 6 is much larger than that of 5. To examine whether the triazole
structure of 5 might be associated with the teratogenicity and to examine the influence of increased
molecular polarity, we therefore synthesized 7 and 8 (Figure 1) and evaluated their teratogenicity.
6

100 nM
Normal
1
100 nM
1000 nM
5
100 nM
1000 nM
6
Figure 2. Representative photos of juvenile zebrafish exposed to RXR full agonist 1, and partial
agonists 5 and 6. Compound 1 at 100 nM induced malformations such as spine curvature and head
enlargement. Compounds 5 and 6 showed no effect at 100 nM, but at 1,000 nM they induced
similar, though less marked, changes.
7

Table 1. Teratogenicity of RXR full agonist 1 and partial agonists 5 and 6 in zebrafish.
Exposure to 5 and 6 was associated with higher hatching rates and lower malformation rates,
compared with 1.
RXR transcriptional
activity
EC50
Emax
Conc.
[nM]
Hatching Survival
[%] [%]
Teratogenicity
[%]
Solubility
[µM]
[nM]
[%]
Normal
1
–
100
100
100
–
–
–
–
100
67
99
75
82
100
20
100
0.25
ref 12
287
100
99
100
100
100
100
100
100
0
44
100
0
5
6
300
143
15
75
85
305
1,000
100
100
83
300
92
0
12.8
1,000
100
17
N = 9－12.
Scheme 1 illustrates the syntheses of 7 and 8. The common precursor 9 for 5 and 6 was
synthesized as reported previously (ref 16). Compound 9 was reacted with methyl
6-chloro-5-nitronicotinate (10) to afford 11. After catalytic reduction of the nitro group of 11, ring
closure under appropriate conditions afforded 12, which has a triazole skeleton, or 13, which has an
imidazole skeleton. Hydrolysis of the ester gave the target compounds 7 and 8, respectively.
8

Scheme 1a
aReagents and conditions: (a) methyl 6-chloro-5-nitronicotinate (10), MeOH, r.t., 4 h. (b) 1) H2,
Pd/C, EtOAc, r.t., 16 h. 2) NaNO2, dil. H2SO4, THF, 5 °C, 1 h. (c) 1) H2, Pd/C, EtOAc, r.t., 16 h. 2)
trifluoroacetic anhydride, trifluoroacetic acid, r.t., 1 h. (d) 1) 2 N NaOH, MeOH, THF, 60 °C , 3 h.
2) 2 N HCl. (e) 1) 2 N NaOH, MeOH, 60 °C, 1.5 h. 2) 2 N HCl.
Table 2 summarizes the results of RXR transcriptional activation assay, water solubility
measurement, and teratogenicity evaluation of 7 and 8. Conversion of the benzoic acid derivatives 5
and 6 to nicotinic acid derivatives 7 and 8 increased the EC50 values, while the Emax values were
little changed. The water solubility of 7 and 8 was significantly increased compared to that of 5 or 6,
respectively. Even when zebrafish embryos were exposed to the high concentration of 1,000 nM, the
9

malformation rate was 25% for 7 and 10% for 8, respectively. At this concentration, individuals
with marked spinal curvature were observed in the case of 7, but not in the case of 8 (Figure 2).
Since the results for 5 and 7 are rather similar, the teratogenicity may be related to the triazole
skeleton. Furthermore, 6 and 8 showed similarly low rates of malformation at 1,000 nM. By
analogy with pioglitazone hydrochloride, this suggests that high molecular polarity may reduce
teratogenicity, possibly by decreasing the permeability of the compound across the zebrafish egg
membrane, resulting in lower intra-egg exposure.
10

Table 2. Teratogenicity of nicotinic acid derivatives 7 and 8 in zebrafish. Compound 7 or 8
induced lower malformation rates than benzoic acid derivatives 5 and 6.
RXR transcriptional
activity
EC50
Emax
Conc.
[nM]
Hatching
[%]
Survival
[%]
Teratogenicity
[%]
Solubility
[µM]
[nM]
[%]
1
5
6
100
100
100
100
100
100
100
92
100
100
100
100
100
100
100
100
100
100
100
17
0
20
143
15
100
75
0.25
305
1,000
1,000
100
85
12.8
7
8
300
0
275
27
89
86
670
1,000
100
25
0
300
100
83
0
222
1,000
10
N = 9－12.
11

100 nM
1000 nM
7
8
100 nM
1000 nM
Figure 3. Representative photos of juvenile zebrafish exposed to nicotinic acid derivatives 7 and 8.
Compound 7 induced spinal curvature at the high concentration of 1,000 nM, but 8 did not induce
any major malformation at this concentration.
In this study, we aimed to develop RXR agonists with reduced teratogenicity by modifying
our previously reported RXR partial agonists CBt-PMN (5) and CBTF-PMN (6) to reduce their
lipophilicity. We first established that 5 and 6 are less teratogenic than the full agonist bexarotene 1,
although 5 exhibited moderate teratogenicity. Conversion of the benzoic acid moiety of 5 and 6 to
nicotinic acid to increase the molecular polarity and reduce the lipophilicity of these compounds
12

afforded 7 and 8, which showed reduced teratogenicity, although 7 was still moderately teratogenic
at high concentration. Compound 8 was identified as a new RXR partial agonist with greatly
reduced teratogenicity, supporting the usefulness of our strategy of focusing on partial RXR
agonists with reduced lipophilicity. More detailed teratogenicity evaluation using rodents or other
models should be the next step to further validate this approach.
Associated contents
Supporting information
HPLC charts and NMR charts for compounds (PDF). The Supporting Information is available free
of charge on the website.
Author infoirmation
Corresponding Author
* Hiroki Kakuta (kakuta-h@okayama-u.ac.jp; phone: +81-(0)86-251-7963)
ORCID
Hiroki Kakuta: 0000-0002-3633-8121
13

Author Contributions
H.K. conceived and designed the project. Y.T. and O.S. synthesized compounds. Y.T., M.T., M.N.,
M.W. performed zebrafish experiments. Y.T., M.W., and H.K. performed LRMS and HRMS. The
manuscript was written by Y.T., M.W., and H.K. All authors analyzed and discussed the data.
Funding Sources
This work was partially supported by The Tokyo Biochemical Research Foundation (TBRF) (to
H.K.).
Notes
This research was partially performed in collaboration with AIBIOS Co., Ltd.. Michiko Fujihara is
an employee of AIBOS. No other author reports any potential conflict of interest relevant to this
article.
Acknowlegement
The authors are grateful to the Division of Instrumental Analysis, Okayama University for the NMR
and MS measurements.
14

Abbreviations
CTCL, cutaneous T cell lymphoma; EC50, half-maximal effective concentration; Emax, efficacy
maximal response; PPAR, peroxisome proliferator-activated receptor; RAR, retinoic acid receptor;
RXR, retinoid X receptor; TPT, triphenyltin.
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