A sub-milligram-synthesis protocol for in vitro screening of HDAC11 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2016.03.116

This work demonstrated the high efficiency of a sub-milligram-synthesis based medicinal chemistry method. Totally 72 compounds, consisting a tri-substituted pyrrolidine core, were prepared. Around 0.1 mg of each compound was solid-phase synthesized. Based on the additive property of UV absorptions of unconjugated chromophores of a molecule, these compounds were quantified by UV measurement. A hit, whose IC₅₀ value was 1.2 μM in HDAC11 inhibition assays, highlights the applicability of the approach reported here in future optimization works.

Full text of DOI:10.1016/j.bmcl.2016.03.116

Bioorganic & Medicinal Chemistry Letters 26 (2016) 2434–2437
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A sub-milligram-synthesis protocol for in vitro screening of HDAC11
inhibitors
a,y
a,y
a
a
a
a
a
Yinping Tian , Jin Jin , Congying Wang , Wenhui Lv , Xuewei Li , Xiaona Che , Yanchao Gong ,
a
a
b
a,
⇑
a,
⇑
Yanjun Li , Quanli Li , Jingli Hou , Peng G. Wang , Jie Shen
a
State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071,
People’s Republic of China
Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics, School of Pharmacy, Tianjin Medical University, Tianjin 300070, People’s
b
Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
This work demonstrated the high efficiency of a sub-milligram-synthesis based medicinal chemistry
method. Totally 72 compounds, consisting a tri-substituted pyrrolidine core, were prepared. Around
Received 17 February 2016
Revised 25 March 2016
Accepted 31 March 2016
Available online 1 April 2016
0
.1 mg of each compound was solid-phase synthesized. Based on the additive property of UV absorptions
of unconjugated chromophores of a molecule, these compounds were quantified by UV measurement. A
hit, whose IC50 value was 1.2 M in HDAC11 inhibition assays, highlights the applicability of the
approach reported here in future optimization works.
l
Keywords:
Sub-milligram-synthesis
HDAC11
Ó 2016 Published by Elsevier Ltd.
Solid phase synthesis
UV quantification
Unconjugated chromophore
Constructing a library of derivatives of identified hits or frag-
ments is a tedious task in drug discovery. Whereas a target com-
pound is traditionally synthesized in over ten milligram quantity,
the catalytic pocket and the cap region respectively. From these
fragments, 3 ꢀ 24 = 72 target compounds could be prepared. At
one end of each fr-A–fr-C, there was a hydroxymic acid group,
frequently less than 1
l
g is actually consumed in initial in vitro
2
and this Zn(II)-binding warhead was protected with a NH -tagged
assays. This necessitates the establishment of a sub-milligram-syn-
thesis method whose main challenges come from the synthesis
manipulation and quantity determination. As an example, an ini-
tial SAR study on a new molecular scaffold of HDAC11 inhibitors
was utilized to demonstrate that solid phase synthesis and UV
quantification were suitable to overcome these challenges.
moiety which was linked onto resins and finally cleaved off by TFA
treatment. At the other end, a TMS protected triple bond was
designed to react with the azido group of fr-1–fr-24. For fr-1–fr-
24, a phenyl group and a polar substituent, both being tethered
to a pyrrolidine core, were designed. The phenyl group was fre-
quently seen in class I HDAC inhibitors to contact with loop1 and
loop2. The designed carboxylic acid or hydroxyl group was
expected to interact with HDAC11’s loop3 which contains posi-
tively-charged R206 and H207 residues (Fig. 1).
Taking the sub-milligram synthesis of the 24 compounds in
Group A as an example (Scheme 1), we utilized only 50 mg Merri-
field resins, functionalized with 4-nitrophenyl carbonate, to react
with fr-A to afford Res-A. After removing TMS by TBAF, the resins
were evenly divided into 24 portions to react with fr-1–fr-24
respectively through Cu(I)-catalyzed click reaction. The resins were
then washed with 8-hydroxyquinoline solution in pyridine for
three times to remove the copper catalyst. Then, treatment of the
resins with TFA afforded the crude products which were purified
by semi-preparative HPLC. Finally, 24 stock solutions were
8
Over 5000 lysine residues of human proteins can be reversibly
modified with an acetyl group, which is enzymatically removed
1
–3
by HDACs.
Among the eleven zinc dependent HDACs in human,
4,5
HDAC11 is the only class IV HDAC isozyme. To date, there were
no extensive investigations on HDAC11 inhibitors yet.
Sharing 25–30% sequence identity, HDAC11 is most similar to
class I HDACs, which all have a highly conserved tube-like catalytic
6
,7
pocket. Located at the opening of this pocket is a heterogeneous
cap region which consists of four loops (Fig. 1). Accordingly, frag-
ments fr-A–fr-C and fr-1–fr-24 (Fig. 2) were designed to occupy
⇑
Corresponding authors.
E-mail addresses: pwang@nankai.edu.cn (P.G. Wang), jieshen@nankai.edu.cn
(
J. Shen).
obtained by dissolving each purified compound in 100 lL DMSO.
y
Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2016.03.116
960-894X/Ó 2016 Published by Elsevier Ltd.
0

Y. Tian et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2434–2437
2435
Similar cases were observed for Group B and Group C. We envi-
sioned that for each group, we might use the UV standard curve
of one compound to quantify the other 23 compounds. To verify
the assumption, over ten milligram B1 and B13 were synthesized.
Then, on one side, the concentration of B13 solution was precisely
determined to be 15.0 lg/mL by using analytical balance and vol-
umetric flask. On the other side, by using the UV standard curve
of B1 at 254 nm, the concentration of the same B13 solution was
calculated to be 15.7 lg/mL. The deviation of these two data was
approximately 5% (see Supporting information), which could well
satisfy the accuracy requirement in initial assays. In this regard,
over ten milligram of A2, B1 and C1 were synthesized, and their
2
54 nm standard UV curves were employed to determine the con-
centrations of the stock solutions of Group A, B and C respectively.
His-tagged human HDAC11 was expressed in SF-9 cells by bac-
ulovirus, and was applied in an established fluorogenic assay
1
6
method. In the initial assay screenings, the HDAC11 inhibition
capacities of these compounds at 0.5 M were measured (Fig. 4).
Figure 1. HDAC11 inhibitor design.
l
Among them, A13 was the most active. Then, over 10 mg A13
was obtained by using traditional liquid phase synthesis strategies
(see Supporting information). Its chemical structure was confirmed
by NMR and HRMS, and its IC50 value in HDAC11 inhibition assay
Similarly, stock solutions of the other 48 targets were also
obtained.
For
a molecule containing multiple unconjugated chro-
mophores, its UV absorption is additively constituted of the
Consistent with this
UV additive property, the normalized UV spectra of the 24 com-
was 1.2 lM.
absorptions of all chromophores (Fig. 3).9
–15
Preliminary SAR studies on HDAC11 inhibitor were investi-
gated. Of the three zinc-binding fragments, fr-A and fr-B are better
than fr-C, implying the catalytic pocket of HDAC11 might prefer
pounds of Group A are almost identical from 240 nm to 300 nm.
Figure 2. Target molecules and key fragments.

2
436
Y. Tian et al. / Bioorg. Med. Chem. Lett. 26 (2016) 2434–2437
Scheme 1. Solid-phase synthesis of Group Aa. aReagents and conditions: (a) PPh
, THF/H O, 40 °C, 6 h; (b) (i) 4-nitrophenyl carbonate Merrifield resin, DMF, 50 °C, 24 h, (ii)
3 2
1
.0 M TBAF in THF, rt, 30 min; (c) (i) fr-1–fr-24, CuIꢁP(OEt)
3
, DIEA, DMF, 50 °C, 48 h; (ii) 0.5 M 8-hydroxyquinoline in pyridine, rt, 6 h, repeated for three times; (d) 50% TFA,
TIS, DCM, rt, 30 min.
More importantly, this work is the proof-of-concept to establish
a sub-milligram-synthesis method, which requires dramatically
less labor, less time and fewer materials than the traditional
over-ten-milligram-synthesis based method for in vitro screening
studies. Solid phase synthesis and UV quantification are the two
key features. Besides simplifying the purification processes, resins
utilized in solid phase synthesis act as a ‘mass-amplifier’ to facili-
tate sub-milligram-synthesis. As for the UV quantification, two
points should be addressed. Firstly, considering the UV detection
limit can reach micromolar level, this UV method can theoretically
quantify compounds in micrograms. In case of screening for tight
i
binding inhibitors whose K value is less than 10 nM, compounds
in microgram quantity are enough for in vitro screening. In such
cases, it might be necessary to furtherly develop a microgram-syn-
thesis based method. Secondly, although the UV quantification
method is not as accurate as the traditional approaches, it can still
satisfy the requirement of initial screenings unless there is a strong
interaction between chromophores. Counterexamples in publica-
tions could possibly be attributed to: (a) a short linker, such as
Figure 3. UV quantification.
1
2
two methylene units,
chromophores forced by a rigid scaffold, or (c) two large size
or (b) the close contact of the two
1
3
1
4,15
chromophores linked by a flexible linker.
Considering the cen-
tral role of this UV quantification method in this newly described
sub-milligram synthesis protocol, factors that might significantly
affect the intramolecular UV additive property should be exten-
sively investigated in the future.
Acknowledgements
This study was supported by National Natural Science
Foundation of China (No. 21272127).
Supplementary data
Figure 4. Initial one-dose (0.5 lM) inhibition assay of target molecules against
HDAC11.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.03.
1
16.
ligands with a straight ‘arm’. The two chiral centers surely take
important roles since A16, A19 and A22, stereoisomers of hit com-
pound A13, are all weak inhibitors. The expected interaction
between the designed polar substituent and HDAC11’s positively
charged loop3 should not occur in these 72 compounds, since A1
is less potent than A13 which has a carboxylic group. Elongation
of the chain between the polar functionality and the pyrrolidine
core might be necessary. The identified hit compound A13, a mod-
erate HDAC11 inhibitor bearing a new molecular scaffold, is a
launch pad for the future hit-to-lead studies.
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