Discovery of a pair of diastereomers as potent HDACs inhibitors: Determination of absolute configuration, biological activity comparison and computational study

Article abstract of DOI:10.1039/c3ra43249a

Histone deacetylase inhibitors (HDACi) are still the focus of epigenetic modulator development due to their effective intervention in many pathological processes. In our previous research, a potent HDACi was designed, synthesized and validated as a promising antitumor candidate named ZYJ-34c. Enlarged scale synthesis of ZYJ-34c for further detailed research was hindered by the occurrence of a by-product, which was identified as an isomer of ZYJ-34c by HRMS and ¹H NMR. Subsequent synthesis route modification and optimization revealed that these two isomers were a pair of epimers and their absolute configurations could be directly determined by our optimized synthesis routes, through which each optically pure epimer could be stereoselectively synthesized, respectively. Based on these results, we concluded that our previously reported absolute configuration of ZYJ-34c was incorrect. It is worth noting that the epimer of ZYJ-34c exhibited more potent HDACs inhibition and both in vitro and in vivo antitumor activities, and moreover, their different HDACs inhibitory activities could be rationalized by computational simulations of their binding modes in HDAC2. The Royal Society of Chemistry 2013.

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ARTICLE TYPE
Discovery of a Pair of Diastereomers as Potent HDACs Inhibitors:
Determination of Absolute Configuration, Biological Activity
Comparison and Computational Study
Yingjie Zhang,a Elizabeth S. Inks,b Mengyuan Zhu,c C. James Chou,b Hao Fang,a Minyong Li,c Yuemao
5
Shen,c Fan Yi,d and Wenfang Xu*a
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Histone deacetylase inhibitors (HDACi) are still the focus of epigenetic modulator development due to
their effective intervention on many pathological processes. In our previous research, a potent HDACi
10 was designed, synthesized and validated as a promising antitumor candidate named as ZYJꢀ34c. Enlarged
scale synthesis of ZYJꢀ34c for further detailed research was hindered by the occurrence of a byꢀproduct,
which was identified as an isomer of ZYJꢀ34c by HRMS and 1H NMR. Subsequent synthesis route
modification and optimization revealed that these two isomers were a pair of epimers and their absolute
configurations could be directly determined by our optimized synthesis routes, through which each
15 optically pure epimer could be stereoselectively synthesized, respectively. Based on these results, we
concluded that our previously reported absolute configuration of ZYJꢀ34c was incorrect. It is worth
noting that the epimer of ZYJꢀ34c exhibited more potent HDACs inhibition and both in vitro and in vivo
antitumor activities, and moreover, their different HDACs inhibitory activities could be rationalized by
computational simulations of their binding modes in HDAC2.
O
O
O
(Boc)2O
1N NaOH, THF
OH
(CH2O)n, 37%HCl
DME, 72-75oC, 18h
I
I
I
20 Introduction
OH
Boc
OH
N
NH2
NH.HCl
HO
HO
HO
1
2
I
I
I
3
An epigenetic trait is a stably heritable phenotype caused by
changes in a chromosome without DNA sequence alterations.1
Aberrant epigenetic covalent modifications of DNA or chromatin
histones will cause disordered gene expression and cellular
25 functions, and consequently many diseases, of which cancer is
the most dreadful.2,3 Hitherto many kinds of epigenetic modifying
enzymes have been revealed as drug intervention targets, such as
histone deacetylases (HDACs), which are responsible for histone
lysine residues deacetylation resulting in chromosomal DNA
30 condensation and gene transcriptional repression.4 Histone
deacetylases inhibitors (HDACi) account for the largest
proportion in epigenetic drug research and development.5
Currently, three HDACi, Vorinostat (SAHA), Romidepsin
(FK228) and Resminostat (4SCꢀ201) have been approved by the
35 FDA as anticancer agents, meanwhile over twenty other HDACi
are in clinical trials.6
O
O
O
O
10%Pd-C, H2
Et3N, MeOH
BrCH2COOCH3, K2CO3
anhydrous DMF
OH H2N
N
H
N
N
DCC, HOBT,
anhydrous THF
HO
Boc
HO
Boc
4
5
O
O
O
O
TFA
DCM
N
H
N
H
O
N
O
NH
O
Boc
O
TBTU, Et3N
THF
O
O
6
7
HO
O
i i ).
O
O
i ). HCl, CH3OH;
OH
isobutyl chloroformate, NMM
THF
OH
HN
9
H2N
i i i ). 2M NaOH, C2H5OH
O
8
O
O
O
O
NH2OK
CH3OH
N
H
H
N
H
H
N
N
O
N
H
HO
O
O
N
O
N
O
O
O
O
O
α
α
Previously reported
structure of ZYJ-34c
10
Scheme 1 Previously reported synthesis route and structure of ZYJꢀ34c
Through our previous several rounds of structural optimization
and
activity
evaluation,7ꢀ9
we
obtained
a
potent
However, enlarged scale synthesis of ZYJꢀ34c for further
detailed research was hindered by the occurrence of a byꢀproduct.
50 In fact, this impurity has already been detected in our milligram
scale synthesis. According to the peak areas (Fig. 1a), the ratio of
the two components is about 3:1. At that time, we took it for
granted that the major component at retention time (RT) 6.4 min
was our desired compound ZYJꢀ34c and that the minor
55 component at RT 7.2 min was some useless byꢀproduct. We tried
tetrahydroisoquinolineꢀbased HDACi, ZYJꢀ34c with marker in
40 vitro and in vivo antitumor potency.9 Because ZYJꢀ34c was
initially synthesized according to the methods described in
1
Scheme 1 and its H NMR (Fig. S1†) and HRMS data (Fig. S2†)
appeared reasonable, we took it for granted that the structure of
ZYJꢀ34c should be the one shown in Scheme 1 as previously
45 reported.9
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recrystallization using almost all common laboratory solvents and
mixed solvent but it did not work. Because the RT of the byꢀ
saponification, which might cause racemization of 9. Due to no
available reported specific rotation of 9, we derivatized our
product was too close to that of our main product (Fig. 1a), we 30 synthesized
9 by condensation with other amines having
could only collect the main product by preparative C18 column for
further activity evaluation. This dramatically hindered the further
research and development of ZYJꢀ34c.
ultraviolet absorption so that we could easily use HPLC to detect
the optical purity of 9. The HPLC analysis results of these
condensation products (Fig. S6†) indirectly demonstrated that
intermediate 9 obtained in Scheme 1 was optical pure. Above
35 mentioned information further confirmed our hypothesis that the
racemization of Cα of ZYJꢀ34c occurred during the amide bond
formation between 7 and 9. So we took it for granted that the
structures of ZYJꢀ34c and its epimer should be the ones shown in
Fig. 1a.
5
40
Subsequently, we tried to eliminate the racemization in the
condensation of 7 and 9 by controlling reaction temperature and
using some other coupling reagents such as DCC and DEPBT,
however, no satisfying results were obtained according to the
HPLC analysis results (Fig. S7†).
45
Considering the most important mechanism of racemization
involving the oxazolone intermediate formation (Scheme S1†),
which is not so facile when the acyl substituent on the αꢀamine
group is an alkoxycarbonyl protecting group such as tertꢀ
butoxycarbonyl (Boc) group,10,11 we established a modified
50 synthesis route (Scheme 2) in which compound 7 was coupled
with BocꢀLꢀisoleucine 11. Then Boc group cleavage of 12 and
subsequent coupling with 3,3ꢀdimethylbutyric acid afforded the
intermediate 10, which was finally transformed into the
corresponding hydroxamic acid. HPLC analysis result revealed
55 that this product was optically pure (Fig. 1b), however, its RT
was 7.312 min, which seemed close to that of the ZYJꢀ34c
epimer (7.157 min, Fig. 1a). NMR spectrums confirmed that the
target compound synthesized in Scheme 2 was exactly ZYJꢀ34c
epimer separated from the crude product of Scheme 1. This result
60 indicated that our previously reported structure of ZYJꢀ34c was
incorrect.
O
O
O
O
N
H
NH
O
N
TBTU, Et3N
THF
O
H
7
O
N
O
H
N
O
O
O
O
Fig.1 a) HPLC chromatogram of Scheme 1 crude product and the
hypothetic structures of ZYJꢀ34c and its epimer. b) HPLC chromatogram
O
O
O
α
O
(Boc)2O
1M NaOH
OH
O
OH
12
HN
11
10 of Scheme 2 product and the actual structure of ZYJꢀ34c epimer. c)
HPLC chromatogram of Scheme 3 product and the actual structure of
ZYJꢀ34c.
H2
N
8
O
O
O
O
NH2OK
CH3OH
N
N
H
N
H
H
i ). TFA, DCM
N
O
N
H
N
H
HO
O
O
O
HO
i i ).
N
O
Results and Discussion
O
O
O
O
O
α
α
TBTU, Et3N, THF
10
In order to synthesize ZYJꢀ34c without formation of this impurity
15 by optimizing reaction conditions or synthesis route, we firstly
collected this impurity using preparative HPLC to analyze what
Scheme 2 Modified synthesis route
O
O
1
exactly it was. H NMR (Fig. S3†) and HRMS data (Fig. S4†)
O
O
N
H
revealed that this byꢀproduct was an isomer of ZYJꢀ34c. Based
on the analysis of our synthesis route shown in Scheme 1 we
20 hypothesized that the isomer should be an epimer of ZYJꢀ34c and
the racemization most probably happened in the Cα of ZYJꢀ34c
during the condensation of intermediates 7 and 9. So we
performed HPLC analysis of the methyl ester 10 and the result
that intermediate 10 contained two adjacent peaks (Fig. S5†)
25 confirmed our hypothesis. There was another possibility that
intermediate 9 was obtained as a mixture of two epimers because
its synthesis methods involved esterification, condensation and
O
NH
N
TBTU, Et3N
THF
O
H
7
O
N
O
H
N
O
O
O
O
O
O
O
α
O
(Boc)2O
OH
O
OH
1M NaOH
15
HN
14
H2
N
13
O
O
O
O
NH2OK
CH3OH
N
N
H
N
H
H
i ). TFA, DCM
N
O
N
H
N
H
HO
O
O
O
HO
i i ).
N
O
O
O
O
O
O
α
α
TBTU, Et3N, THF
16
65 Scheme 3 Modified synthesis route
2
| Journal Name, [year], [vol], 00–00
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In order to determine the real structure of ZYJꢀ34c, we used
30
the same reaction conditions of Scheme 2 to establish Scheme 3,
in which Dꢀalloisoleucine 13 was substituted for Lꢀisoleucine 8 in
Scheme 2. As expected, HPLC analysis result revealed that the
product of Scheme 3 was also optically pure (Fig. 1c) and its RT
To further compare their antiproliferative activities, this pair of
diastereomers was evaluated against several tumor cell lines.
Results in Table 2 showed that ZYJꢀ34c epimer exhibited more
potent in vitro antitumor activities than ZYJꢀ34c and SAHA
5
(6.446 min) and NMR spectrums all demonstrated that it was 35 against all tested tumor cell lines. Meanwhile, it was notable that
exactly ZYJꢀ34c published in our previous work.9
ZYJꢀ34c epimer and ZYJꢀ34c possessed lower toxicity to normal
human lung fibroblast cell line (WI38) compared with SAHA.
Encouraged by its excellent in vitro activity, ZYJꢀ34c epimer
was progressed to an in vivo experiment. We applied the same
Compound ZYJꢀ34c was validated as a promising antitumor
candidate with superior in vivo antitumor potency compared with
10 the approved drug SAHA.9 Through above mentioned Scheme 3,
we could obtain optically pure ZYJꢀ34c on a large scale for 40 MDAꢀMBꢀ231 xenograft mouse model as in our previous
further preclinical research. However, the starting material Dꢀ
alloisoleucine 13 is a very expensive unnatural amino acid, which
makes the production cost of ZYJꢀ34c unacceptable. Therefore,
15 we focused our attention on ZYJꢀ34c epimer because of its much
more available starting material Lꢀisoleucine 11.
It was exciting that ZYJꢀ34c epimer exhibited more potent
inhibitory activities than both ZYJꢀ34c and SAHA against
HDAC1, HDAC2 and HDAC3. Although ZYJꢀ34c epimer was
20 inferior to SAHA against HDAC6, it was still superior to ZYJꢀ
34c. All tested compounds exhibited no obvious inhibition
against class IIa HDACs using MDAꢀMBꢀ231 cell lysate as
enzyme source (Table 1).
research8,9 with ZYJꢀ34c and SAHA as positive control. The final
dissected tumor volume, tumor growth inhibition (TGI) and
relative increment ration (T/C) shown in Fig. 2 all indicated that
ZYJꢀ34c epimer was the most potent compound, which was in
45 line with its HDACs inhibitory activities and in vitro
antiproliferative activities.
Table 1. HDACs inhibitory activities of tested compounds
IC50 (nM) α
Compds
Class I
Class IIa
Class IIb
HDAC1 HDAC2 HDAC3 Cell lysate HDAC6
Fig. 2 In vivo antitumor activity comparison of tested compounds
ZYJꢀ34c
epimer
NAβ
8.1
117.1
11.0
570
NAβ
NAβ
ZYJꢀ34c
39.3
75.6
428.4
256.4
58.3
28.4
1320
118
SAHA
25 α Values are the means of at least two independent experiments.
β NA: Not active at 10 ꢁM.
Table 2. IC50 values (ꢁM) α of tested compounds in MTT assays
ZYJꢀ34c
ZYJꢀ34c SAHA
epimer
MDAꢀMBꢀ231 2.29
50 Fig. 3 Proposed binding modes of ZYJꢀ34c (blue stick, a) and its epimer
(cyan stick, b) in HDAC2. Protein is represented in ribbon diagrams with
amino acid residues labeled in purple sticks. The Zn2+ is shown as a gray
sphere.
4.67
6.10
4.14
3.46
3.02
4.09
3.33
5.73
6.58
3.15
6.00
9.27
3.41
2.54
1.62
52.0
4.70
3.74
1.81
3.35
1.20
1.45
1.25
1.86
1.88
4.20
3.00
3.22
1.70
1.25
1.23
4.59
MDAꢀMBꢀ468 1.70
MDAꢀMBꢀ435 1.00
Breast Cancer
Colon Cancer
BT549
BT474
HCT116
HT29
0.96
0.50
0.95
0.86
1.52
1.66
0.91
1.25
2.24
0.87
0.84
0.46
16.0
The proposed binding modes of ZYJꢀ34c epimer and ZYJꢀ34c
55 in the active site of HDAC2 were respectively navigated by
molecular dynamic (MD) simulations to probe the reason why
ZYJꢀ34c epimer was more potent than its diastereomer. We chose
HDAC2 for the following three reasons. First, all Zn2+
dependant HDACs, especially isoforms belonging to the same
60 class bear a highly conserved active site. Second, Class I HDACs,
especially HDAC1, HDAC2 and HDAC3 are the most tumorꢀ
related HDACs isoforms.12 Third, the HDAC2 crystal structure
has been reported (PDB ID: 3MAX). After 200 ps of simulation,
both the complexes had converged and reached equilibrium (Fig.
65 S8†). After MD simulation, MMꢀGBSA method was used to
calculate the Gibbs free energy associated with the binding of
inhibitors to HDAC2. The total binding energy (ꢀGb) of ZYJꢀ34c
epimer (ꢀ63.44 kJ/mol) was slightly lower than that of ZYJꢀ34c (ꢀ
SW620
SKꢀOVꢀ3
PCꢀ3
Ovarian Cancer
Prostate Cancer
Lung Cancer
A549
Cervical Cancer HeLa
Melanoma
Leukemia
SKꢀMELꢀ28
K562
U937
WI38
Lymphoma
Normal Cell
α Values are the means of three independent experiments.
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6
7
http://www.clinicaltrials.gov/
61.58 kJ/mol), which was in accordance with their HDACs
inhibitory activity. In order to investigate the influence of
different chirality on proteinꢀligand interaction, MMꢀGBSA
decomposition calculation was performed. Calculation results of
two key residues (PROꢀ23 and ASPꢀ93, Table S1†), which
interacted with the chiral side chains of the two epimers, and the
binding modes in HDAC2 (Fig. 3) indicated that compared with
ZYJꢀ34c, its epimer could not only form an additional ꢀ0.503
kcal/mol of hydrophobic interaction with PROꢀ23 (Fig. 3b) but
60
65
Y. Zhang, J. Feng, C. Liu, L. Zhang, J. Jiao, H. Fang, L. Su, X.
Zhang, J. Zhang, M. Li, B. Wang, W. Xu, Bioorg. Med. Chem.
2010, 18, 1761ꢀ1772.
Y. Zhang, J. Feng, Y. Jia, X. Wang, L. Zhang, C. Liu, H. Fang, W.
Xu, J. Med. Chem. 2011, 54, 2823ꢀ2838.
8
9
5
Y. Zhang, H. Fang, J. Feng, Y. Jia, X. Wang, W. Xu, J. Med. Chem.
2011, 54, 5532ꢀ5539.
10 J. Jones, Amino Acid and Peptide Synthesis; Oxford University
Press: New York, 2002; Chapter 5.
11 M. M. Joullié, K. M. Lassen, Arkivoc 2010, 8, 189ꢀ250.
70 12 B. Venugopal, T. R. Evans, Curr. Med. Chem. 2011, 18, 1658ꢀ1671.
10 also reduce 3.579 kcal/mol of repulsive force against ASPꢀ93
(Fig. 3a).
Conclusions
In conclusion, we successfully determined the exact absolute
configurations of the previous HDACi ZYJꢀ34c and its newly
15 discovered epimer by a facile asymmetric synthetic method. It is
interesting that ZYJꢀ34c epimer exhibited more potent HDACs
inhibition and antitumor activities than ZYJꢀ34c. More
importantly, both diastereomers could be obtained on large scale
using our asymmetric synthetic method, which laid a solid
20 foundation for further research and development of ZYJꢀ34c
epimer as a promising antitumor candidate. Moreover, the
different HDACs inhibitory activities of the two epimers could be
rationalized by computational study, validating MD simulations
and MMꢀGBSA as reliable methods for HDACi discovery, at
25 least for rational design and screening of our
tetrahydroisoquinolineꢀbased HDACi.
Acknowledgements
This work was supported by National Scientific and
Technological Major Project of Ministry of Science and
30 Technology of China (Grant No.2011ZX09401ꢀ015), National
Natural Science Foundation of China (Grant No.21302111, Grant
No.21172134), Independent Innovation Foundation of Shandong
University, IIFSDU (Grant No.2013GN013) and National Cancer
Institute of the National Institute of Health (Award
35 No.R01CA163452).
Notes and references
a Department of Medicinal Chemistry, School of Pharmaceutical
Sciences, Shandong University, Ji'nan, Shandong, 250012, P. R. China
b Department of Drug Discovery and Biomedical Sciences, South
40 Carolina College of Pharmacy, Medical University of South Carolina,
Charleston, South Carolina, 29425, United States.
.
c Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, Ji'nan, Shandong,
250012, P. R. China.
45 d Department of Pharmacology, School of Medicine, Shandong
University, Ji'nan, Shandong, 250012, P. R. China.
* Email: wenfxu@gmail.com; Fax/Tel: +86ꢁ531ꢁ88382264
† Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
50 DOI: 10.1039/b000000x/
1
S. L. Berger, T. Kouzarides, R. Shiekhattar, A. Shilarifard, Genes
Dev. 2009, 23, 781ꢀ783
2
3
P. A. Jones, S. B. Baylin, Cell 2007, 128, 683ꢀ692.
N. Tsankova, W. Renthal, A. Kumar, E. J. Nestler, Nat. Rev.
Neurosci. 2007, 8, 355ꢀ367
55
4
5
A. P. Wolffe, Science 1996, 272, 371ꢀ372.
J. D. Best, N. Carey, Drug Discov. Today. 2010, 15, 65ꢀ70.
4
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Graphical Abstract