Design, synthesis, and biological characterization of tamibarotene analogs as anticancer agents

Article abstract of DOI:10.1111/cbdd.12778

In our efforts of developing novel compounds as potential anticancer agents, a series of tamibarotene analogs containing Zn²⁺-binding moieties were designed and developed. Biological characterization identified compound 7b as the most potent one with improved antiproliferative activities against multiple cancer cell lines, compared to parent compound tamibarotene. Further characterization also demonstrated that compound 7b exhibited moderate activities as a histone deacetylase inhibitor with IC₅₀ of 1.8?±?0.1?μm, thus suggesting that this could contribute to the improved antiproliferative activities of 7b. Pharmacokinetic studies revealed that compound 7b could release tamibarotene after administration and prolong the circulation time of tamibarotene, and this may also potentially contribute to the improved antiproliferative activities. Collectively, the results demonstrated that compound 7b could serve as a new lead for further development of more potent analogs as potential anticancer agents.

Full text of DOI:10.1111/cbdd.12778

Received: 13 November 2015
DOI: 10.1111/cbdd.12778
Revised: 1 April 2016
Accepted: 18 April 2016
R E S E A R C H A R T I C L E
Design, synthesis, and biological characterization of
tamibarotene analogs as anticancer agents
Yuqi Jiang1
Jingde Wu1
Xiaoyang Li1
Wenfang Xu1
Xue Wang1
Zhonglan Wang1
Jian Zhang2
1Department of Medicinal Chemistry, School of
Pharmaceutical Sciences, Shandong University,
Ji’nan, Shandong, China
2College of Pharmacy, Weifang Medical University,
Wei’fang, Shandong, China
In our efforts of developing novel compounds as potential anticancer agents, a
series of tamibarotene analogs containing Zn2+-binding moieties were designed and
developed. Biological characterization identified compound 7b as the most potent
one with improved antiproliferative activities against multiple cancer cell lines,
compared to parent compound tamibarotene. Further characterization also demon-
strated that compound 7b exhibited moderate activities as a histone deacetylase
Correspondence
Jingde Wu, wujingde70@sdu.edu.cn;
Wenfang Xu, wenfxu@163.com
inhibitor with IC50 of 1.8
0.1 μm, thus suggesting that this could contribute to
the improved antiproliferative activities of 7b. Pharmacokinetic studies revealed
that compound 7b could release tamibarotene after administration and prolong the
circulation time of tamibarotene, and this may also potentially contribute to the
improved antiproliferative activities. Collectively, the results demonstrated that com-
pound 7b could serve as a new lead for further development of more potent analogs
as potential anticancer agents.
K E Y W O R D S
anticancer, histone deacetylases, multitarget drugs, retinoids, tamibarotene
Retinoids have been successfully developed for the treatment of
leukemia, such as all-trans retinoic acid (ATRA, 1) and tamib-
arotene (2, Figure 1A).[1] However, the clinical applications of
these agents are limited by associated toxicities such as muco-
cutaneous dryness, liver toxicity, central nervous system, and
hypertriglyceridemia.[2–4] To address this issue, combination
therapy is practiced to minimize the toxicity and drug resis-
tance problems.[5,6] For example, the combination of retinoic
acid receptor (RAR) agonists with HDAC inhibitors has been
reported to show synergistic effects. [7,8] A phase I trial of the
13-cis-retinoic acid and HDAC inhibitor MS-275 (Entinostat)
combination showed promising results in treating unresect-
able chemotherapy-resistant pancreatic cancer.[9] Furthermore,
prodrugs of ATRA such as compounds 3–5 (Figure 1A) have
been developed and demonstrated improved pharmacological
activities as anticancer agents compared to ATRA itself.[10,11]
Compared to ATRA, tamibarotene exhibited better differen-
tiation induction and lower drug resistance in acute promy-
elocytic leukemia, probably due to its low affinity to cellular
retinoic acid-binding protein.[1,12] However, tamibarotene also
suffers undesirable side-effects such as hypertriglyceridemia,
hypercholesterolemia, and retinoic acid syndrome, among
others.[13] Therefore, it would be of significance to develop
novel tamibarotene analogs for further development.
In our previous studies, tamibarotene derivatives
containing butyric acid moiety, such as 6 (Figure 1A), were
developed and most of them exhibited improved antipro-
liferative activity against human leukemia cells.[14,15] In
continuation of our efforts to develop tamibarotene analogs
with novel pharmacology, herein we report the synthesis
and biological characterization of a new series of analogs
Abbreviations: AM80, tamibarotene; ATRA, all-trans retinoic acid; HDACs,
histone deacetylases; RAR, retinoic acid receptor; ZBG, Zinc-binding group.
© 2016 John Wiley & Sons A/S.
Chem Biol Drug Des 2016; xx: 1–15
wileyonlinelibrary.com/cbdd
1

2
Jiang et al.
FIGURE 1 (A) Chemical structure of 1–6; (B) schematic representation of the design and chemical structure of target compounds
to incorporate a Zn2+-binding group, given the fact that
HDACIs have been added to tamibarotene as combination
therapy and Zn2+ chelating is an essential part of HDACI
design. In the newly designed compounds, the carboxylic
acid moiety of tamibarotene was employed to incorporate
the Zn2+-chelating moiety such as hydroxamic acid and
phenylenediamine via ester or amide linkage (Figure 1B).
It was perceived that these novel compounds will function
as prodrug to improve pharmacokinetic properties of tamib-
arotene. Furthermore, the incorporation of Zn2+-chelating
moieties may potentially introduce inhibitory activities on
HDAC, thus adding another layer of benefits to them as anti-
cancer agents.
1
|
METHODS AND MATERIALS
1.1 | Chemistry
All commercially available starting reagents and solvents
were used without further purification unless otherwise
stated. All reactions were monitored by thin-layer chro-
matography with 0.25-mm silica gel plates (60GF-254).

Jiang et al.
3
UV light and iodine stain were used to visualize the spots.
Silica gel or C18 silica gel was used for column chroma-
purified by recrystallization with EtOAc and petroleum ether
(ratio 1:4) to achieve a white pure solid 7a (0.13 g, yield: 30%).
mp: 240–241 °C. 1H-NMR (600 MHz DMSO-d ): δ 1.25–1.26
(m, 12H), 1.64–1.67 (m, 4H), 7.26 (s, 2H), 7.31 6(d, J = 8.4 Hz,
1H), 7.44 (S, 1H), 7.62 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H),
7.71 (d, J = 1.8 Hz, 1H), 8.13 (d, J = 8.4 Hz, 2H), 8.19 (d,
J = 7.8 Hz, 2H). 13C-NMR (100 MHz DMSO-d6): δ 165.51,
164.88, 145.04, 140.55, 138.18, 136.91, 136.77, 128.49,
128.09, 127.66, 127.28, 126.93, 118.89, 118.70, 35.06, 34.49,
34.05, 32.15. HRMS (AP-ESI) m/z calculated for C28H31N3O2,
[M + H]+ 442.2489. Found, 442.2489.
1
tography purification. H NMR spectra were recorded on
a Bruker DRX spectrometer at 600 MHz, while the 13C
NMR spectra were recorded at 400 MHz, in parts per
million and J in hertz, using TMS as an internal standard.
High-resolution mass spectra were conducted by Shandong
Analysis and Test Center in Ji’nan, China. ESI-MS spectra
were recorded on an API 4000 spectrometer. Melting
points were determined uncorrected on an electrothermal
melting point apparatus. The tested compounds are >95%
pure by HPLC analysis, performed on Agilent 1100 HPLC
instrument using an ODS HYPERSIL column (5 μm,
4.6 mm × 250 mm), according to the following method
(Figure S1–8). Compounds 7b, 12b, 16a–d, and 18a–c
were eluted with 72% methanol/20% water (containing
0.5% Phosphoric acid)/8% acetonitrile over 30 min, while
the compounds 7a, 12a, and 17a–d were eluted with 74%
methanol/18% water/8% acetonitrile. The absorbance was
measured at 283 nm, the flow rate was 1 mL/min, and the
quantity of injection was 20 μL.
1.2.2
|
N1-hydroxy-N4-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) terephthalamide (7b)
2 (0.7 g, 2.0 mmol) and 4-methylmorpholine (0.24 mL,
2.2 mmol) were dissolved in dry THF (100 mL) under ice
bath followed by the addition of isobutyl chloroformate
(0.3 mL, 2.1 mmol). The mixture was stirred for 30 min at
−10 °C. Hydroxylamine hydrochloride (0.16 g, 2.2 mmol) and
4-methylmorpholine (0.24 mL) dissolved in dry THF (30 mL)
were added into the solution directly. The mixture was stirred
for 12 h at room temperature. THF was evaporated with the
residue being taken up in EtOAc (40 mL). The EtOAc solu-
tion was washed with 1 N aqueous citric acid and brine for
three times, dried over anhydrous sodium sulfate over night,
and evaporated. The residue was purified by column chro-
matography, using DCM–MeOH (20:1) as the mobile phase,
to obtain 7b as pale yellow solid (0.10 g, yield: 14%). mp:
123–124 °C. 1H-NMR (600 MHz DMSO-d ): δ 1.24–1.25 (m,
12H), 1.63–1.66 (m, 4H), 7.30 (d, J = 8.4 6Hz, 1H), 7.59 (dd,
J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.89
(d, J = 8.4 Hz, 2H), 8.02 (d, J = 8.4 Hz, 2H), 9.16 (s, 1H),
10.19 (s, 1H), 11.39 (s, 1H). 13C-NMR (100 MHz DMSO-
d6): δ 164.99, 163.91, 145.02, 140.48, 137.74, 136.93, 135.72,
128.12, 127.36, 126.93, 118.79, 118.56, 35.11, 34.47, 34.03,
32.14. HRMS (AP-ESI) m/z calculated for C22H26N2O3,
[M + H]+ 367.2017. Found, 367.2016.
The compounds 2 were synthesized following reported
procedures.[16]
1.1.1
|
4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalen-2-yl) carbamoyl) benzoic acid (2, Am80)
1
mp: 187–188 °C, H-NMR (600 MHz DMSO-d6): δ 1.24–
1.25 (m, 12H), 1.63–1.66 (m, 4H), 7.30 (d, J = 8.4 Hz, 1H),
7.59 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.68 (d, J = 2.4 Hz,
1H), 8.03–8.08 (m, 4H), 10.26 (s, 1H), 13.26 (s, 1H). 13C-
NMR (100 MHz DMSO-d ): δ 167.24, 164.98, 145.04,
140.56, 139.29, 136.87, 1363.65, 129.72, 128.29, 126.94,
118.82, 118.62, 35.10, 34.47, 34.03, 32.13. ESI-MS m/z:
350.5 [M − H]−.
1.2 | General procedure for the preparation
of 7a and 7b
1.2.1 | N1-(2-aminophenyl)-N4-(5,5,8,8-
tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)
terephthalamide (7a)
A solution of 2 (0.35 g, 1.0 mmol), o-phenylenediamine (0.11 g,
1.0 mmol), DCC (0.22 g, 1.05 mmol), and DMAP (0.03 g,
0.2 mmol) in dichloromethane (DCM) and acetonitrile (ratio
1:1, 20 mL) was stirred at room temperature (rt) for 5 h, diluted
with DCM (20 mL) and filtered. The filtrate was evaporated
with the residue being taken up in ethyl acetate (EtOAc; 40 mL).
The organic phase was washed with saturated NaHCO3 and
brine for three times and dried over anhydrous sodium sulfate
over night. After evaporating the solvent, the crude product was
1.3 | General procedure for the preparation
of 9[17]
1.3.1 | Benzyl-2-hydroxyacetate (9)
8 (4.0 g, 52.0 mmol) and Et3N (8.0 mL, 57.2 mmol) were
dissolved in dry acetone (150 mL). After 5 min of stir-
ring, benzyl bromide (6.0 mL, 46.8 mmol) was added to
the solution. The mixture was refluxed at 60 °C for 12 h.
The solution was filtered and concentrated on vacuo. The
crude material was purified via flash chromatography (PE/
EtOAc = 1:1) to give 9 (5.44 g, yield: 63%). ESI-MS m/z:
167.4 [M + H]+.

4
Jiang et al.
DCM (40 mL), and filtered, and the filtrate was evaporated
with the residue being taken up in EtOAc (80 mL). The organic
phase was washed with saturated NaHCO3 and brine for three
times and dried over anhydrous sodium sulfate over night.
After evaporating the solvent, the crude product was puri-
fied by column chromatography to afford the compound 12a
1.4 | General procedure for the preparation
of 10
1.4.1 | 2-(benzyloxy)-2-oxoethyl-4-((5,5,8,8-
tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)
carbamoyl) benzoate (10)
1
(0.26 g, yield: 26%). mp: 169–170 °C. H-NMR (600 MHz
2 (0.88 g, 2.5 mmol) was dissolved in the mixture of DCM
and acetonitrile (ratio 2:1, 50 mL) under ice bath followed by
the addition of TBTU (0.9 g, 2.75 mmol), Et3N (0.4 mL) and
stirred for 30 min at room temperature. 9 (0.45 g, 2.75 mmol)
and Et3N (0.4 mL) were added into the solution directly.
The mixture was stirred for 4 h at room temperature. The
solvent was evaporated under vacuum; the crude product was
dissolved with EtOAc, washed by 1 N aqueous citric acid,
saturated NaHCO3, and brine for three times, and dried over
anhydrous sodium sulfate over night, and the solvent was
evaporated under vacuum. The crude product was purified by
column chromatography to afford the compound 10 (0.97 g,
DMSO-d ): δ 1.24–1.25 (m, 12H), 1.64–1.67 (m, 4H), 5.02
(s, 2H), 66.76 (s, 1H), 6.89–7.04 (m, 3H), 7.24 (d, J = 7.2 Hz,
1H), 7.31 (d, J = 8.4 Hz, 1H), 7.60 (d, J = 8.4 Hz, 2H), 7.69
(s, 1H), 8.13–8.19 (m, 4H), 8.98 (s, 1H), 9.69 (s, 1H), 10.34
(s, 1H). 13C-NMR (100 MHz DMSO-d ): δ 165.18, 164.72,
149.04, 145.05, 140.66, 140.14, 136.681, 133.33, 131.33,
130.28, 128.53, 126.95, 125.56, 118.92, 118.74, 115.80,
58.75, 35.09, 34.48, 34.05, 32.14. HRMS (AP-ESI) m/z calcu-
lated for C30H33N3O4, [M + H]+ 500.2546. Found, 500.2544.
1.6.2
|
2-(hydroxyamino)-2-oxoethyl-4-((5,5,8,8-
1
tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)
carbamoyl) benzoate (12b)
yield: 78%). mp: 158–160 °C. H-NMR (600 MHz DMSO-
d ): δ 1.24–1.26 (m, 12H), 1.64–1.66 (m, 4H), 5.06 (s, 2H),
56.23 (s, 2H), 7.31 (d, J = 8.4 Hz, 1H), 7.34–7.38 (m,
1H), 7.39–7.40 (m, 3H), 7.60 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz,
1H), 7.68 (d, J = 2.4 Hz, 1H), 8.09–8.14 (m, 4H), 10.32 (s,
1H). ESI-MS m/z: 499.4 [M + H]+.
11 (0.82 g, 2.0 mmol) and 4-methyl-morpholine (0.24 mL,
2.2 mmol) were dissolved in dry THF (100 mL) under ice bath
followed by the addition of isobutyl chloroformate (0.3 mL,
2.1 mmol) and stirred for 30 min at −10 °C. Hydroxylamine
hydrochloride (0.16 g, 2.2 mmol) and 4-methylmorpholine
(0.24 mL) dissolved in dry THF (30 mL) were added into the
solution directly. The mixture was stirred for 12 h at room
temperature. THF was evaporated with the residue being
taken up in EtOAc (40 mL). The EtOAc solution was washed
with 1 N aqueous citric acid and brine for three times, dried
over anhydrous sodium sulfate over night, and evaporated, and
the residue was chromatographed, using DCM–MeOH (20:1)
as the mobile phase, to obtain 0.10 g of pale yellow solid 12b
1.5 | General procedure for the preparation
of 11
1.5.1 | 2-((4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)
benzoyl) oxy) acetic acid (11)
10 (3.5 g, 15 mmol) was dissolved in mixture of methanol
and THF (ratio 4:1, 200 mL), dry palladium carbon (0.35 g)
was added, and the mixture was stirred under a hydrogen
atmosphere overnight. The palladium carbon was filtered
off over Celite and washed with methanol. After evaporating
the solvent, the residue was dried under vacuum to get white
solid 11 (2.4 g, yield: 79%). ESI-MS m/z: 408.6 [M + H]+.
1
(0.16 g, yield: 19%). mp: 175–176 °C. H-NMR (600 MHz
DMSO-d6): δ 1.24–1.25 (m, 12H), 1.64–1.67 (m, 4H), 4.72
(s, 2H), 7.31 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H),
7.68 (d, J = 1.2 Hz, 1H), 8.08–8.15 (m, 4H), 9.02 (s, 1H),
10.31 (s, 1H), 10.87 (s, 1H). 13C-NMR (100 MHz DMSO-
d ): δ 165.28, 164.80, 163.99, 145.05, 140.63, 139.83, 136.81,
1631.96, 129.96, 128.55, 126.96, 118.88, 118.68, 115.80,
62.34, 35.10, 34.48, 34.05, 32.14. HRMS (AP-ESI) m/z calcu-
lated for C24H28N2O5, [M + H]+ 425.2071. Found, 425.2068.
1.6 | General procedure for the preparation
of 12a and 12b
1.6.1 | 2-((2-aminophenyl) amino)-2-
oxoethyl-4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)
benzoate (12a)
A solution of 11 (0.82 g, 2.0 mmol), o-phenylenediamine
(0.22 g, 2.0 mmol), DCC (0.44 g, 2.1 mmol), and DMAP
(0.06 g) in the mixture of DCM and acetonitrile (ratio 4:1,
40 mL) was stirred at room temperature for 5 h, diluted with
1.7 | General procedure for the preparation
of 14a–d
1.7.1 | Methyl-2-aminoacetate hydrochloride
(14a)
Glycine (9.0 g, 120 mmol) was dissolved in the dry methanol
(MeOH; 200 mL); then, acetyl chloride (25.7 mL, 360 mmol)
was added dropwise at 0 °C; the reaction was refluxed at 75 °C

Jiang et al.
5
for 3 h. The solvent was evaporated under vacuum; the crude
product was purified by recrystallization with ether to achieve
a white pure solid 14a, a white solid (12.41 g, yield: 92%), mp:
176–177 °C (literature reported data[18]: mp: 175–176 °C).
J = 7.0 Hz, 2H), 3.49–4.53 (m, 2H), 3.62 (s, 3H), 7.30 (d,
J = 8.6 Hz, 1H), 7.60 (dd, J1 = 2.1 Hz, J2 = 8.6 Hz, 1H),
7.68 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 8.04 (d,
J = 8.4 Hz, 2H), 8.75 (t, J = 5.4 Hz, 1H), 10.20 (s, 1H).
ESI-MS m/z: 437.4 [M + H]+.
1.7.2
|
Methyl-3-aminopropanoate hydrochloride
(14b)
1.8.3
|
Methyl-4-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl) benzamido)
White solid, 91% yield, mp: 103–105 °C (literature reported
data[19]: mp: 103–104 °C).
butanoate (15c)
White solid, 71% yield. mp: 149–150 °C. 1H-NMR (400 MHz
DMSO-d ): δ 1.24–1.25 (m, 12H), 1.65 (s, 4H), 1.77–1.85 (m,
2H), 2.416(t, J = 7.4 Hz, 2H), 3.28–3.32 (m, 2H), 3.59 (s, 3H),
7.30 (d, J = 8.6 Hz, 1H), 7.60 (dd, J1 = 2.1 Hz, J2 = 8.6 Hz,
1H), 7.69 (d, J = 2.1 Hz, 1H), 7.97 (d, J = 8.4 Hz, 2H), 8.04
(d, J = 8.4 Hz, 2H), 8.66 (t, J = 5.6 Hz, 1H), 10.19 (s, 1H).
ESI-MS m/z: 451.6 [M + H]+.
1.7.3
|
Methyl-4-aminobutanoate hydrochloride (14c)
White solid, 85% yield, mp: 120–122 °C (literature reported
data[19]: mp: 122–124 °C).
1.7.4
|
Methyl-6-aminohexanoate hydrochloride (14d)
White solid, 88% yield, mp: 117–119°C (literature reported
data[20]: mp: 118–122 °C).
1.8.4
|
Methyl-6-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carba moyl) benzamido)
hexanoate (15d)
1.8 | General procedure for the preparation
White solid, 58% yield. mp: 174–176 °C. 1H-NMR (400 MHz
DMSO-d ): δ 1.24–1.25 (m, 12H), 1.32–1.34 (m, 2H), 1.52–
1.59 (m, 64H), 1.65 (s, 4H), 2.34 (t, J = 7.4 Hz, 2H), 3.29
(q, J = 6.8 Hz, 2H), 3.58 (s, 3H), 7.30 (d, J = 8.6 Hz, 1H),
7.60 (dd, J1 = 2.1 Hz, J2 = 8.6 Hz, 1H), 7.69 (d, J = 2.1 Hz,
1H), 7.97 (d, J = 8.4 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H),
8.63 (t, J = 5.6 Hz, 1H), 10.20 (s, 1H). ESI-MS m/z: 479.4
[M + H]+.
of 15a–d
1.8.1 | Methyl-2-(4-((5,5,8,8-tetramethyl-
5,6,7,8-tetrahydronaphthalen-2-yl) carbamoyl)
benzamido) acetate (15a)
2 (0.88 g, 2.5 mmol) was dissolved in the mixture of DCM
and acetonitrile (ratio 4:1, 50 mL) under ice bath followed
by the addition of TBTU (0.9 g, 2.75 mmol), Et3N (0.4 mL)
and stirred for 30 min at room temperature. 14a (0.34 g,
2.75 mmol) and Et3N (0.4 mL) were added into the solution
directly. The mixture was stirred for 4 h at room temperature.
The solvent was evaporated under vacuum; the crude product
was dissolved with EtOAc, washed by 1 N aqueous citric
acid, saturated NaHCO3, and brine for three times, and dried
over anhydrous sodium sulfate over night, and the solvent
was evaporated under vacuum. The crude product was puri-
fied by column chromatography to give the compound 15a
1.9 | General procedure for the preparation
of 16a–d
1.9.1 | 2-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)-
benzamido) acetic acid (16a)
15a (0.42 g, 1.0 mmol) was dissolved in the mixture of meth-
anol (15 mL) and 1.5 mol/L NaOH solution (6 mL). The
mixture was refluxed at 90 °C for 3 h. After removing THF
under vacuum, the solution was acidified to pH 4 with 1 N
HCl solution. The white solid 16a was collected by filtra-
tion, washed with water, and dried (0.33 g, yield: 80%). mp:
1
(0.82 g, yield: 78%). mp: 201–203 °C. H-NMR (600 MHz
DMSO-d6): δ 1.24–1.25 (m, 12H), 1.64–1.67 (m, 4H), 3.67
(s, 3H), 4.02–4.06 (m, 2H), 7.30 (d, J = 9.0 Hz, 1H), 7.60
(dd, J1 = 2.4 Hz, J2 = 9.0 Hz, 1H), 7.69 (d, J = 1.8 Hz, 1H),
8.01 (d, J = 9.0 Hz, 2H), 8.05 (d, J = 9.0 Hz, 2H), 9.13–9.15
(m, 1H), 10.21 (s, 1H). ESI-MS m/z: 423.4 [M + H]+.
1
160–162 °C. H-NMR (600 MHz DMSO-d6): δ 1.23–1.25
(m, 12H), 1.65–1.66 (m, 4H), 3.96–3.97 (m, 2H), 7.30 (d,
J = 8.4 Hz, 1H), 7.60 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 1H),
7.69 (d, J = 1.8 Hz, 1H), 8.01 (d, J = 8.4 Hz, 2H), 8.06 (d,
J = 8.4 Hz, 2H), 9.01–9.03 (m, 1H), 10.22 (s, 1H), 12.80
(s, 1H). 13C-NMR (100 MHz DMSO-d ): δ 171.66, 166.28,
164.97, 145.02, 140.50, 137.92, 136.691, 136.69, 128.14,
127.73, 126.92, 118.86, 118.66, 41.75, 35.05, 34.47, 34.03,
1.8.2
|
Methyl-3-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl) benzamido)
propanoate (15b)
White solid, 63% yield. mp: 182–184°C. 1H-NMR (400 MHz
DMSO-d6): δ 1.24–1.25 (m, 12H), 1.65 (s, 4H), 2.64 (t,

6
Jiang et al.
32.14. HRMS (AP-ESI) m/z calculated for C24H28N2O4,
[M + H]+ 409.2122. Found, 409.2121.
1.10 | General procedure for the
preparation of 17a–d
1.10.1 | N1-(2-((2-aminophenyl) amino)-
2-oxoethyl)-N4-(5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) terephthalamide
(17a)
1.9.2
|
3-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)-benzamid-o)
propanoic acid (16b)
White solid, 86% yield. mp: 84–85 °C. 1H-NMR (600 MHz
DMSO-d ): δ 1.25–1.29 (m, 12H), 1.63–1.66 (m, 4H), 2.50–
2.56 (m, 62H), 3.47–3.51 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H),
7.60 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz, 1H), 7.68 (d, J = 2.4 Hz,
1H), 7.97 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.73
(t, J = 5.4 Hz, 1H), 10.20 (s, 1H), 12.27 (s, 1H). 13C-NMR
(100 MHz DMSO-d ): δ 173.33, 166.01, 164.99, 145.01,
140.48, 137.68, 1376.25, 136.92, 128.03, 127.63, 126.92,
118.85, 118.64, 36.16, 35.11, 34.47, 34.21, 34.03, 32.14.
HRMS (AP-ESI) m/z calculated for C25H30N2O4, [M + H]+
423.2278. Found, 423.2278.
A solution of 16a (0.82 g, 2.0 mmol), o-phenylenediamine
(0.22 g, 2.0 mmol), DCC (0.44 g, 2.1 mmol), and DMAP
(0.06 g) in the mixture of DCM and acetonitrile (ratio 4:1,
40 mL) was stirred at room temperature for 5 h, diluted
with DCM (40 mL), and filtered, and the filtrate was evap-
orated with the residue being taken up in EtOAc (80 mL).
The organic phase was washed with saturated NaHCO3
and brine for three times and dried over anhydrous
sodium sulfate over night. After evaporating the solvent,
the crude product was purified via flash chromatography
to afford the compound 17a (0.20 g, yield: 21%). mp:
185–186 °C. 1H-NMR (600 MHz DMSO-d6): δ 1.23–1.25
(m, 12H), 1.63–1.65 (m, 4H), 4.10–4.11 (m, 2H), 4.92 (s,
2H), 6.55 (t, J = 7.2 Hz, 1H), 6.72 (d, J = 7.2 Hz, 1H),
6.93 (t, J = 7.2 Hz, 1H), 7.14 (d, J = 7.2 Hz, 1H), 7.30
(d, J = 8.4 Hz, 1H), 7.60 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz,
1H), 7.69 (d, J = 2.4 Hz, 1H), 8.03–8.07 (m, 4H), 9.04
(t, J = 6.0 Hz, 1H), 9.29 (s, 1H), 10.22 (s, 1H). 13C-NMR
(100 MHz DMSO-d ): δ 168.23, 166.53, 164.99, 145.03,
142.94, 140.51, 1376.97, 137.85, 136.92, 128.05, 127.85,
126.92, 126.67, 126.32, 123.29, 118.87, 118.66, 116.52,
116.09, 43.67, 35.07, 34.47, 34.03, 32.14. HRMS (AP-
ESI) m/z calculated for C30H34N4O3, [M + H]+ 499.2704.
Found, 499.2703.
1.9.3
|
4-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)-benzamido)
butanoic acid (16c)
White solid, 81% yield. mp: 175–176 °C. 1H-NMR (600 MHz
DMSO-d ): δ 1.24–1.25 (m, 12H), 1.65 (s, 4H), 1.77–1.78
(m, 2H), 62.31 (t, J = 6.6 Hz, 2H), 3.32 (q, J = 6.6 Hz, 2H),
7.30 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 9.0 Hz, 1H), 7.69 (s,
1H), 7.97 (d, J = 7.8 Hz, 2H), 8.03 (d, J = 7.8 Hz, 2H), 8.67 (t,
J = 5.4 Hz, 1H), 10.20 (s, 1H). 13C-NMR (100 MHz DMSO-
d6): δ 174.70, 166.00, 165.01, 145.01, 140.47, 137.60,
137.47, 136.93, 128.01, 127.63, 126.92, 118.84, 118.63,
39.23, 35.11, 34.47, 34.03, 32.14, 31.64, 24.98. HRMS (AP-
ESI) m/z calculated for C26H32N2O4, [M + H]+ 437.2435.
Found, 437.2437.
1.10.2
|
N1-(3-((2-aminophenyl) amino)-3-oxopropyl)-
N4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-
yl) terephthalamide (17b)
1.9.4
|
6-(4-((5,5,8,8-tetramethyl-5,6,7,8-
tetrahydronaphthalen-2-yl) carbamoyl)-benzamido)
White solid, 26% yield. mp: 112–114 °C. 1H-NMR
(600 MHz DMSO-d ): δ 1.23–1.25 (m, 12H), 1.65–1.66
(m, 4H), 2.66 (t, J = 66.6 Hz, 2H), 3.61 (t, J = 6.6 Hz, 2H),
4.87 (s, 2H), 6.54 (td, J1 = 1.2 Hz, J2 = 7.8 Hz, 1H), 6.71
(dd, J1 = 1.2 Hz, J2 = 7.8 Hz, 1H), 6.91 (td, J1 = 1.8 Hz,
J2 = 8.4 Hz, 1H), 7.16 (dd, J1 = 1.2 Hz, J2 = 7.8 Hz, 1H),
7.30 (d, J = 8.4 Hz, 1H), 7.60 (dd, J1 = 2.4 Hz, J2 = 8.4 Hz,
1H), 7.67–7.68 (m, 1H), 7.96–7.98 (m, 2H), 8.01–8.03 (m,
2H), 8.79 (t, J = 6.0 Hz, 1H), 9.20 (s, 1H), 10.20 (s, 1H).
13C-NMR (100 MHz DMSO-d6): δ 169.91, 166.15, 165.02,
145.03, 142.58, 140.49, 137.66, 137.44, 136.92, 128.02,
127.65, 126.92, 126.38, 126.08, 123.75, 118.85, 118.64,
116.56, 116.27, 36.74, 36.25, 35.12, 34.47, 34.03, 32.14.
HRMS (AP-ESI) m/z calculated for C31H36N4O3, [M + H]+
513.2860. Found, 513.2857.
hexanoic acid (16d)
White solid, 88% yield, mp: 210–211 °C. 1H-NMR
(600 MHz DMSO-d6): δ 1.24–1.26 (m, 12H), 1.33–1.34
(m, 2H), 1.53–1.55 (m, 4H), 1.63–1.65 (m, 4H), 2.23 (t,
J = 7.2 Hz, 2H), 3.27–3.28 (m, 2H), 7.30 (d, J = 8.4 Hz,
1H), 7.59 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 1H), 7.68 (d,
J = 1.8 Hz, 1H), 7.96 (d, J = 8.4 Hz, 2H), 8.03 (d,
J = 8.4 Hz, 2H), 8.60 (s, 1H), 10.18 (s, 1H), 11.99 (s,
1H). 13C-NMR (100 MHz DMSO-d6): δ 174.89, 165.84,
165.01, 145.00, 140.45, 137.58, 137.53, 136.94, 128.01,
127.60, 126.90, 118.85, 118.64, 35.06, 34.47, 34.10,
34.03, 32.14, 29.28, 26.53, 24.74. HRMS (AP-ESI) m/z
calculated for C28H36N2O4, [M + H]+ 465.2748. Found,
465.2748.

Jiang et al.
7
1.10.3
|
N1-(4-((2-aminophenyl) amino)-4-oxobutyl)-
1 mmol) was dissolved in the NH2OK solution and stirred
at room temperature for 6 h. After removing MeOH under
vacuum, the residue was acidified with 1 N HCl to a pH 3 and
then extracted with EtOAc. The organic phase was washed
with brine for three times and dried over anhydrous sodium
sulfate over night. After evaporating the solvent, the crude
product was purified via flash chromatography to afford the
N4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-
yl) terephthalamide (17c)
White solid, 23% yield. mp: 124–126 °C. 1H-NMR (600 MHz
DMSO-d6): δ 1.24–1.25 (m, 12H), 1.65 (s, 4H), 1.84–1.88
(m, 3H), 1.99 (s, 1H), 2.42 (t, J = 7.2 Hz, 2H), 4.88 (s, 2H),
6.54 (t, J = 7.2 Hz, 1H), 6.72 (d, J = 7.8 Hz, 1H), 6.90 (t,
J = 7.8 Hz, 1H), 7.16 (d, J = 7.2 Hz, 1H), 7.30 (d, J = 8.4 Hz,
1H), 7.60 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.98–8.04 (m,
4H), 8.71 (s, 1H), 9.15 (s, 1H), 10.21 (s, 1H). 13C-NMR
(100 MHz DMSO-d ): δ 171.32, 166.03, 165.00, 145.02,
142.50, 140.48, 1376.59, 137.53, 136.93, 128.00, 127.65,
126.91, 126.22, 125.92, 123.93, 118.86, 118.65, 116.55,
116.27, 47.99, 35.07, 34.47, 34.03, 32.14, 25.81, 24.93.
HRMS (AP-ESI) m/z calculated for C32H38N4O3, [M + H]+
527.3017. Found, 527.3016.
1
compound 18a (0.16 g, yield: 38%). mp: 198–200 °C. H-
NMR (600 MHz DMSO-d6): δ 1.24–1.25 (m, 12H), 1.63–
1.67 (m, 4H), 3.81–3.82 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H),
7.60 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 8.01 (d,
J = 8.4 Hz, 2H), 8.05 (d, J = 8.4 Hz, 2H), 8.84 (s, 1H), 8.93
(d, J = 5.4 Hz, 1H), 10.21 (s, 1H), 10.65 (s, 1H). 13C-NMR
(100 MHz DMSO-d ): δ 171.75, 166.27, 165.00, 145.02,
140.50, 137.92, 1366.92, 136.73, 128.13, 128.00, 127.84,
127.72, 126.92, 118.86, 118.67, 41.81, 35.12, 34.47, 34.03,
32.14. HRMS (AP-ESI) m/z calculated for C24H29N3O4,
[M + H]+ 424.2231. Found, 424.2232.
1.10.4
|
N1-(6-((2-aminophenyl) amino)-6-oxohexyl)-
N4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-
yl) terephthalamide (17d)
1.11.2
|
N1-(3-(hydroxyamino)-3-oxopropyl)-N4-
(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)
White solid, 19% yield. mp: 104–105°C. 1H-NMR
(600 MHz DMSO-d6): δ 1.24–1.25 (m, 12H), 1.37–1.39
(m, 2H), 1.57–1.62 (m, 2H), 1.65–1.66 (m, 6H), 2.34 (t,
J = 6.6 Hz, 2H), 3.31 (q, J = 6.6 Hz, 2H), 4.82 (s, 2H), 6.54
(td, J1 = 1.2 Hz, J2 = 8.4 Hz, 1H), 6.72 (dd, J1 = 1.2 Hz,
J2 = 8.4 Hz, 1H), 6.90 (td, J1 = 1.2 Hz, J2 = 8.4 Hz,
1H), 7.15 (dd, J1 = 1.2 Hz, J2 = 8.4 Hz, 1H), 7.30 (d,
J = 8.4 Hz, 1H), 7.59 (dd, J1 = 2.4 Hz, J2 = 9.0 Hz, 1H),
7.68 (d, J = 2.4 Hz, 1H), 7.97 (d, J = 8.4 Hz, 2H), 8.03
(d, J = 8.4 Hz, 2H), 8.65 (t, J = 6.0 Hz, 1H), 9.10 (s, 1H),
10.19 (s, 1H). 13C-NMR (100 MHz DMSO-d6): δ 171.58,
165.87, 165.03, 145.02, 142.33, 140.48, 137.61, 137.56,
136.93, 128.00, 127.59, 126.91, 126.14, 125.75, 124.09,
118.85, 118.64, 116.65, 116.37, 36.22, 35.08, 34.48,
34.04, 32.14, 29.39, 26.70, 25.57. HRMS (AP-ESI) m/z
calculated for C34H42N4O3, [M + H]+ 555.3333. Found,
555.3335.
terephthalamide (18b)
White solid, 26% yield. mp: 123–125 °C. 1H-NMR (600 MHz
DMSO-d ): δ 1.24–1.25 (m, 12H), 1.65–1.66 (m, 4H), 2.30 (t,
J = 7.2 H6z, 2H), 3.46–3.49 (m, 2H), 7.30 (d, J = 8.4 Hz, 1H),
7.59 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz, 1H), 7.68 (d, J = 1.8 Hz,
1H), 7.96 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 8.4 Hz, 2H), 8.73
(t, J = 6.0 Hz, 1H), 8.77 (d, J = 1.2 Hz, 1H), 10.20 (s, 1H),
10.48 (s, 1H). 13C-NMR (100 MHz DMSO-d6): δ 167.76,
165.99, 165.00, 145.03, 140.50, 137.67, 137.32, 136.91,
128.02, 127.61, 126.91, 118.85, 118.65, 36.61, 35.12, 34.47,
34.03, 32.84, 32.14. HRMS (AP-ESI) m/z calculated for
C25H31N3O4, [M + H]+ 438.2387. Found, 438.2386.
1.11.3
|
N1-(4-(hydroxyamino)-4-oxobutyl)-N4-
(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)
terephthalamide (18c)
White solid, 31% yield. mp: 205–207 °C. 1H-NMR (600 MHz
DMSO): δ 1.24–1.25 (m, 12H), 1.63–1.66 (m, 4H), 1.76–1.78
(m, 2H), 2.05 (t, J = 7.8 Hz, 2H), 3.29 (q, J = 6.6 Hz, 2H),
7.30 (d, J = 8.4 Hz, 1H), 7.60 (dd, J1 = 1.8 Hz, J2 = 8.4 Hz,
1H), 7.69 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 2H),
8.04 (d, J = 8.4 Hz, 2H), 8.70 (t, J = 6.0 Hz, 1H), 8.73 (d,
J = 1.8 Hz 1H), 10.22 (s, 1H), 10.43 (s, 1H). 13C-NMR
(100 MHz DMSO-d ): δ 169.38, 165.96, 165.01, 145.00,
140.45, 137.55, 1376.46, 136.95, 128.02, 127.65, 126.88,
118.88, 118.70, 39.48, 35.07, 34.47, 34.02, 32.14, 30.50,
25.72. HRMS (AP-ESI) m/z calculated for C26H33N3O4,
[M + H]+ 452.2544. Found, 452.2543.
1.11 | General procedure for the
preparation of 18a–c
1.11.1 | N1-(2-(hydroxyamino)-2-oxoethyl)-
N4-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaph
thalen-2-yl) terephthalamide (18a)
KOH (28 g, 509 mmol) and NH2OH·HCl (23.35 g,
343 mmol) were dissolved, respectively, in 70 and 120 mL
of MeOH to get solution A and solution B. Next, solution A
was added dropwise to solution B. After filtering the precip-
itate (KCl), a NH2OK solution was obtained. 15a (0.42 g,

8
Jiang et al.
1.12 | In vitro antiproliferative assay
1.15 | APN inhibition assay
Cells were maintained in RPMI1640 medium containing 10%
FBS at 37 °C in a 5% CO2 humidified incubator. Cell prolifer-
ation was determined by the MTT method. Briefly, cells were
seeded into a 96-well cell plates, allowed to grow for 12 h
and then treated with different concentrations of compounds
for 48 h. A 0.5% MTT solution was added to each well. After
incubation for another 4 h, formazan formed was extracted
by adding 150 μL of DMSO. Absorbance was then recorded
at 570 nm.
The inhibitory activity of target compounds toward APN was
determined using microsomal aminopeptidase from porcine
kidney microsomes (Sigma) as the enzyme.[24] Briefly, the
assay was performed in 96-well plates in 50 mm PBS, pH 7.2
as the assay buffer, at 37 °C. The APN solution was mixed with
compounds at various concentrations and incubated at 37 °C
for 5 min. Subsequently, the substrate (l-leucine-p-nitroanilide)
was added and the mixture was incubated for 30 min at 37°C.
Finally, the hydrolysis of the substrate was measured by a
microplate reader (Thermo Fisher, Shanghai, China) at 405 nm.
1.13 | In vitro HDACs inhibition
fluorescence assay
1.16 | Stability of representative compound
in human plasma, fresh rat plasma, and rat
liver homogenate
In vitro HDACs inhibition assays were conducted as previ-
ously described.[21] In brief, 10 μL of enzyme solution
(HeLa nuclear extract contain HDAC1, 2) was mixed with
compound at various concentrations (50 μL). The mixture
was incubated at 37 °C for 5 min, then 40 μL of fluorogenic
substrate Boc-Lys(acetyl)-AMC (300 μm) was added. After
incubation at 37 °C for 1 h, the reaction was stopped by the
addition of 100 μL of developer containing trypsin (10 μg/
mL) and TSA (2 μm). Fluorescence intensity was measured
by a microplate reader at excitation and emission wave-
lengths of 390 and 460 nm, respectively. The IC50 values
were calculated using a regression analysis of the concentra-
tion/inhibition data.
Preparation of human plasma, fresh rat plasma, and rat liver
homogenate was conducted as previously described.[25]
Human plasma, fresh rat plasma, and rat liver homogenate
were added to the stock solution of 7b (4 mg/mL in meth-
anol) and incubated at 37 °C for 48 h. At scheduled times,
sample aliquots were collected and the enzymatic reaction
was quenched by adding acetonitrile. Human plasma and
fresh rat plasma samples underwent extraction using 300 μL
of acetonitrile, while rat liver homogenate samples under-
went extraction using 400 μL of acetonitrile. The samples
were filtered (0.22 μm) after shocking 30 seconds and centrif-
ugation at 12 000 rpm for 10 min. Analytical HPLC was
performed on Agilent 1200 HPLC instrument using a Alltima
C18 column (5 μm, 4.6 mm × 250 mm), and the compound
was eluted with 72% methanol/20% water (containing 0.5%
Phosphoric acid)/8% acetonitrile over 15 min. The absor-
bance was measured at 283 nm, the flow rate was 1 mL/min,
and the quantity of injection was 20 μL. Retention times
(min) for 7b and tamibarotene were as follows: 8.064 (7b)
and 11.845 (tamibarotene).
1.14 | In vitro HDAC8 assay
The HDAC8 was expressed and purified as previously
described.[22] Briefly, HDAC8 gene was amplified by using
a polymerase chain reaction, the products were subcloned
into pGEX-6p-1 plasmid. Subsequently, the constructs
were used to transform Escherichia coli cells. The human
HDAC8 proteins were purified using a GST column. The
purity of hHDAC8 was above 95%, and the specific activity
of hHDAC8 was 0.73 0.23 U/mg. In vitro HDAC8 inhi-
bition assays were conducted as previously described.[23]
IC50 values against HDAC8 were determined using HDAC8
expressed in Escherichia coli as the enzyme. Briefly, the
HDAC8 enzyme was diluted 20 times. Then, 10 μL of
HDAC8 solution was mixed with compounds at various
concentrations (50 μL). Then, 40 μL of the substrate [Boc-
Lys-(acetyl)-AMC, 300 μm] was added, and the mixture was
incubated at 37 °C for 30 min. Subsequently, the reaction was
stopped by the addition of 100 μL of developer containing
trypsin (10 μg/mL) and TSA (2 μm). Fluorescence intensity
was measured by a microplate reader at excitation and emis-
sion wavelengths of 390 and 460 nm, respectively. The IC50
values were calculated using a regression analysis of the
concentration/inhibition data.
1.17 | In vivo pharmacokinetics
For pharmacokinetic studies, six Wistar rats, weight
200 20 g, were randomly divided into two groups (injected
through tail vein with 20 mg/kg 7b and tamibarotene). The
rats were fasted for 12 h before the experiment, free drinking
water. For test and control groups, 0.3 mL of blood sample
from the carotid sinus before the administration of 0 h and
after the administration of 0.05, 0.083, 0.167, 0.333, 0.667, l,
2, 4, 6, 10, and 24 h, respectively, was added in the centrifuge
tube handled by heparin sodium, centrifuged at 4000 rpm for
l0 min, and stored at −80 °C until sample extraction. The test
samples were prepared for quantification by protein precipi-
tation and evaporation. 7b and tamibarotene were quantified
by HPLC analysis.

Jiang et al.
9
To make analogs 16a–d, 17a–d, and 18a–c, coupling reac-
tions of 2 with various amino esters 14a–d produced 15a–d.
Reactions of 15a–d with NH2OK in dry methanol directly
delivered hydroxamic acids 18a–c. Hydrolysis of 15a–d
followed by coupling reactions with o-phenylenediamine
gave 17a–d (Scheme 3).
1.18 | Determination of 7b in whole blood,
plasma, and blood cells
Three Wistar rats were injected through tail vein with 20 mg/
kg 7b. Before the experiment, the rats were forbidden to eat
anything but drink water. We got 0.5 mL of blood sample
from the jugular sinus, after the time of administration of
5 min and 10 min, and added the blood sample in the centri-
fuge tube handled by heparin sodium. Preparation of whole
blood samples: 0.2 mL of acetonitrile was added into the
0.1 mL of rat blood. The samples were filtered (0.22 μm)
after shocking 30 seconds and centrifugation at 12 000 rpm
for 10 min. Preparation of plasma and blood cells samples:
0.35 mL of rat blood was centrifuged at 4000 rpm for 10 min,
and then, the plasma and blood cells were prepared. About
0.2 mL of acetonitrile was added into the 0.15 mL of each of
plasma and blood cells. The samples were filtered (0.22 μm)
after shocking 30 seconds and centrifugation at 12 000 rpm
for 10 min, and then, we obtained the cell samples. To deter-
mine the concentration of compound 7b in 20 μL of each of
samples of whole plasma and blood cell samples at 5 and
10 min, the amount of drug extracted from whole blood was
taken as the full, then the plasma and the distribution of drug
in blood cells were calculated.
2.2 | Antiproliferative activities of the
designed analogs
To evaluate whether the incorporation of Zn2+-chelating
moiety into tamibarotene will improve anticancer activi-
ties, we examined the antiproliferative activities of these
compounds in human leukemia K562 cells, which was most
frequently used in evaluating tamibarotene derivatives.[14]
Initially, a single concentration of 30 μm was chose for the
screening to identify compounds with reasonable activity. As
shown in Table 1, in general, the analogs with hydroxamic
acid moiety exhibited better antiproliferative activities at the
tested concentration. Analogs with carboxylic acid moiety
(16a–d) lost antiproliferative activities compared to tamib-
arotene or SAHA. This may suggest that the Zn2+-chelating
moiety is important for the biological activities within the
designed analogs. Based on the initial screening results,
compounds 7a, 7b, and 18a were selected for further dose–
response studies to obtain potency against six cancer cells
lines including solid and hematological tumor cells, which
were most frequently used in evaluating tamibarotene deriva-
tives and HDACIs (Table 2). Overall, compounds 7a, 7b, and
18a showed improved antiproliferation potency against six
cancer cell lines, compared to tamibarotene. When compared
to SAHA, they exhibited comparable potencies against K562,
HL60, and ES-2 cell lines. Among these three compounds,
7b displayed an overall optimal potency than the other two
analogs, specifically for HL60, MDA-MB-231, HCT116, and
ES-2 cancer cells. This may suggest that the observed activity
of 7b could be due to both of the tamibarotene moiety and
the hydroxamic acid through interactions with retinoid recep-
tors and HDAC. Additionally, cytotoxicity of 7b against
human liver cell HL-7702 was tested in our lab, and the IC50
value was 108.9 15.4 μm, which revealed the selectivity
of 7b between non-transformed cells and tumor cells. The
compound 7b has been reported as an HDACI, but no further
2
|
RESULTS AND DISCUSSION
2.1 | Chemistry
To prepare the target compounds, key intermediate 2 (tamib-
arotene/Am80) was synthesized following reported proce-
dures.[16] Coupling reactions of 2 with hydroxylamine
or o-phenylenediamine accomplished the synthesis of
compounds 7a and 7b, respectively, following the conditions
outlined in Scheme 1.
The synthesis of target compounds 12a–b was shown
in Scheme 2. Briefly, reaction of 8 with benzyl bromide in
acetone in the presence of triethylamine (Et3N) yielded
9, which was followed by reaction with 2 to give interme-
diate 10 in the presence of O-(benzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium tetrafluoroborate (TBTU). Deprotection
of 10 followed by coupling reactions with hydroxylamine or
o-phenylenediamine finally gave target compounds 12a–b.
O
O
O
N
H
O
H
N
H
N
H
H
O
H
N
NH2
H
N
b
a
O
O
O
a
7
2
7
b
SCHEME 1 Synthesis of 7a–b. Reagents and conditions: (a) DCC/DMAP, DCM; (b) isobutyl chloroformate, THF, hydroxylamine hydrochloride

10
Jiang et al.
O
O
H
O
O
O
H
N
O
H
N
O
H
O
O
O
c
a
2
H
O
H
O
O
11
b
O
O
8
O
9
10
NH2
O
O
H
N
H
N
O
H
O
O
H
N
H
N
e
d
O
O
11
O
O
12b
12a
SCHEME 2 Synthesis of 12a–b. Reagents and conditions: (a) benzyl bromide, acetone, Et3N, 60 °C; (b) TBTU/Et3N; (c) Pd/C, H2; (d) DCC/DMAP,
DCM; (e) isobutyl chloroformate, THF, hydroxylamine hydrochloride
O
O
O
OH
N
H
N
H
n
n
HCl
H2N
14a–14d
H
N
H
N
O
O
c
2
b
OH
O
a
H2N
n
n
O
O
O
O
16a–16d
15a–15d
13a–13d
e
d
O
NH2
O
H
N
H
N
N
H
n
N
OH
n
H
H
H
16a–18a: n = 1;
16b–18b: n = 2;
16c–18c: n = 3;
16d–18d: n = 5;
N
O
N
O
O
O
17a–17d
18a–18c
SCHEME 3 Synthesis of 16a–d, 17a–d and 18a–c. Reagents and conditions: (a) acetyl chloride, MeOH, 70 °C; (b) TBTU/Et3N; (c) MeOH/NaOH,
90 °C; (d) DCC/DMAP, DCM; (e) NH2OK/MeOH, THF
characterization and biological results available from the
early report.[26]
on HDACs and no inhibitory effects were observed for
compounds 16, consistent with the antiproliferative activ-
ities (Table 1). This again may suggest the important roles
of the Zn2+-chelating moieties on the newly designed
analogs. Overall, compounds containing hydroxamic acid
(18a–c) were more potent than compounds with benzamide
moiety (17a–d) under the current testing conditions.
Notably, compound 7b exhibited the most potent inhi-
bition on HDACs with single-digit micromolar potency
2.3 | HeLa cell extract inhibition by the
target compounds
Given the fact that the hydroxamic acid moiety is essen-
tial for HDACIs as a Zn2+-chelating group, we next exam-
ined the inhibitory effects of these compounds on HDAC.
To this end, HeLa cell nuclear extract (which contains
primarily HDAC1 and HDAC2) was used as the HDACs
enzyme source.[25] As shown in Table 3, compounds
12, 17, and 18 exhibited moderate inhibitory activities
(IC50: 1.8
0.1 μm). Although the inhibitory potency
on HDAC is lower than SAHA, the combination of dual
functions from 7b, namely RAR agonist and HDACI, may
explain the overall improved cytotoxic effects compared
TABLE 1 Growth inhibition rates toward K562 cells at concentration of 30 μm
Compound
Inhibition (%)a
Compound
Inhibition (%)a
Compound
Inhibition (%)a
7a
77.0
67.2
41.8
58.4
35.8
16c
16d
17a
17b
17c
8.1
32.3
49.1
56.7
45.2
18a
66.4
58.4
60.7
49.4
47.1
7b
18b
12a
12b
16a
18c
SAHA
Tamibarotene
16b
38.3
17d
60.1
aAssays were performed in replicate (n ≥ 2); the SD values are <20% of the mean.

Jiang et al.
11
a
TABLE 2 Antiproliferative activities of 7a, 7b, 18a, tamibarotene, and SAHA against six tumor cell lines (IC50 in μm )
IC50
Compound
HCT116
ES-2
K562
HL60
PC-3
MDA-MB-231
7a
26.9
7.8
12.8
81.3
0.3
17.5
12.9
26.8
ND
4.6
34.6
31.1
42.4
40.3
14.7
4.0
10.7
16.1
5.7
>100
13.7
5.2
98.6
>100
0.7
7b
11.0
18a
6.5
79.4
21.4
Tamibarotene
SAHA
13.4
ND, Not determined.
aAssays were performed in replicate (n ≥ 2); the SD values are <20% of the mean.
TABLE 3 HDAC inhibition activity of compounds 7a–b, 12a–b, 16a–d, 17a–d, 18a–c, tamibarotene, and SAHA
O
N
H
R2
X
R1
O
Compound
X
R1
R2
IC50 against HeLa extract (μm)a
7a
–
–
o-H2NPhNH
>100
7b
–
–
NHOH
1.8 0.1
51.6 2.4
15.1 0.5
>100
12a
12b
16a
16b
16c
16d
17a
17b
17c
17d
18a
18b
18c
SAHA
O
O
CH2CO
CH2CO
CH2CO
(CH2)2CO
(CH2)3CO
(CH2)5CO
CH2CO
(CH2)2CO
(CH2)3CO
(CH2)5CO
CH2CO
(CH2)2CO
(CH2)3CO
o-H2NPhNH
NHOH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
OH
OH
>100
OH
>100
OH
>100
o-H2NPhNH
o-H2NPhNH
o-H2NPhNH
o-H2NPhNH
NHOH
75.2 4.8
46.1 2.1
57.8 3.6
32.4 1.3
9.3 0.4
21.4 0.5
12.1 0.3
0.1 0.0
NHOH
NHOH
O
H
N
OH
N
H
O
Tamibarotene
OH
–
–
>100
aAssays were performed in replicate (n ≥ 2); values are shown as mean SD.
TABLE 4 In vitro inhibition of HDAC-8 and APN of 7ba
the positive control to APN. The result in Table 4 showed
that compound 7b exhibited low micromolar IC50 value
against HDAC8, which was similar to its inhibitory activity
toward HeLa cell nuclear extract (which contains primarily
HDAC1 and HDAC2) and no inhibitory effects were
observed for APN. These results validated our strategy for
rational designing of potential HDACIs.
Compound
IC50 (μm)a 7b
SAHA
Tamibarotene
Bestatin
HDAC-8
APN
4.3 0.1
>100
1.1 0.0
>100
>100
>100
ND
2.5 0.3
aAssays were performed in replicate (n ≥ 2); values are shown as mean SD.
2.4 | Stability of compound 7b in human
plasma, fresh rat plasma, and rat liver
homogenate
Given the improved antiproliferative activities of 7b, we next
set out to assess its in vivo pharmacokinetic profiles. To this
end, in vitro stability of 7b in human plasma, fresh rat plasma,
to tamibarotene or SAHA alone. This further supports our
design rationale of such tamibarotene analogs.
Compound 7b was further evaluated for its inhibitory
activity against HDAC8 and APN. Similar to HDACs, APN
is zinc-dependent metalloproteinase as well and associ-
ated closely with of tumorigenesis. Bestatin was used as

12
Jiang et al.
FIGURE 2 (A–B) Stability of compound 7b
in human plasma and fresh rat plasma; Points were
achieved after 4, 8, 12, 24, and 48 h, respectively.
(C) Stability of compound 7b in rat liver homogenate;
Points were achieved after 1, 2, 4, 8, 24, and 48 h,
respectively
FIGURE 3 (A) Concentration–time curve after
intravenous injection of 7b (20 mg/kg) in rats. (B)
Concentration–time curve after intravenous injection
of tamibarotene (20 mg/kg) in rats
TABLE 5 Main pharmacokinetic parameters of 7b and tamibarotene after i.v. administration (20 mg/kg) in rat (mean SD, n = 3)
7b administration
Tamibarotene administration
Pharmacokinetic parameters
7b
Tamibarotene
Tamibarotene
t1/2α (h)
0.08 0.05
2.28 1.03
2.21 0.77
15.70 20.31
16.58 14.87
0.06 0.02
6.70 3.61
1.38 1.39
3.88 1.76
1.11 1.40
18.09 14.05
92.16 79.64
4.00 2.83
3.89 0.02
0.80 1.23
2.22 38.20
0.409 0.11
4.04 1.04
t1/2β (h)
V1 (L/kg)
MRT (h)
AUC0–∞ (mg/L*h)
Tmax (h)
136.67 9.41
0.08 0.01
Cmax (mg/L)
42.40 10.29
CL (L/h/kg)
3.65 3.91
0.35 0.30
0.15 0.01
t1/2α, absorption half-life; t1/2β, elimination half-life; V1, apparent volume of distribution; MRT, mean residence time; AUC0–∞, area under curve; Tmax, peak time;
Cmax, maximum plasma drug concentration; CL, clearance.
and rat liver homogenate was studied using a HPLC method.
As shown in Figure 2, 7b was partially cleaved in human
plasma, fresh rat plasma, and rat liver homogenate within 48 h.
Data showed that compound 7b released significant levels of
tamibarotene in vitro. To evaluate whether 7b will function as
a prodrug to release the parent compound tamibarotene upon
metabolism, we also determined the level of tamibarotene
under these experimental conditions. As shown in Figure 2B
and C, there is a steady increase of tamibarotene accompanied
FIGURE 4 The distribution of 7b in plasma and blood cells

Jiang et al.
13
with the degradation of 7b, thus suggesting that 7b gradually
release the parent compound tamibarotene upon metabolism.
This is consistent with our initial design rationale.
compound for development of more potent analogs as treat-
ment agents for leukemic patients.
ACKNOWLEDGMENTS
2.5 | The pharmacokinetic study of 7b
This work was supported by National Major Scientific and
Technological Special Project for ‘Significant New Drugs
Development’ of China (Grant no. 2012ZX09103101-015)
and National Natural Science Foundation of China (Grant no.
21302111, 81373282).
in vivo
Given the promising results of 7b from the in vitro studies,
we decided to study the in vivo pharmacokinetic properties
of 7b that will help prepare further preclinical studies of
7b as well as guide development of more potent analogs
of 7b.
CONFLICT OF INTEREST
As shown in Figure 3A and Table 5, after the intravenous
administration of 7b, at 20 mg/kg dose, tamibarotene was
detected 5 min later, and it reached the peak concentration at
4 h postadministration of 7b. This clearly indicated that 7b
was metabolized to release tamibarotene in vivo. Compared
to the profile of tamibarotene, administration of 7b signifi-
cantly improved the half-life of tamibarotene (from 2.22 to
3.88 h), mean residence time (MRT; from 4.44 to 18.09 h),
and peak time (Tmax; from 0.08 to 4 h; Table 5). Collectively,
the results suggest that compound 7b could provide more
beneficial properties as a potential anticancer agent compared
to tamibarotene by exhibiting the properties such as RAR
agonist and HDAC inhibitor and extending the residence time
of tamibarotene.
The authors confirmed that this article content has no conflict
of interest.
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3
|
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could potentially contribute to the improved antiproliferative
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Figure S2. HPLC analysis chromatogram of 7b.
Figure S3. HPLC analysis chromatogram of 16a.
Figure S4. HPLC analysis chromatogram of 16b.
Figure S5. HPLC analysis chromatogram of 17a.
Figure S6. HPLC analysis chromatogram of 17b.
Figure S7. HPLC analysis chromatogram of 18b.
Figure S8. HPLC analysis chromatogram of 18c.
[26] B. Leblond, E. Beausoleil, EP: 1541549 A1 2005.
SUPPORTING INFORMATION
Additional Supporting Information may be found online in
the supporting information tab for this article:
Figure S1. HPLC analysis chromatogram of 7a.