Macrocyclic compounds as anti-cancer agents: Design and synthesis of multi-acting inhibitors against HDAC, FLT3 and JAK2

Article abstract of DOI:10.1016/j.ejmech.2015.03.034

A novel series of macrocyclic compounds were designed and synthesized as multi-target inhibitors targeting HDAC, FLT3 and JAK2. Some of these compounds exhibited potent HDAC inhibition as well as FLT3 and JAK2 inhibition under both cell-free and cellular conditions. In vitro antiproliferative assay indicated that these compounds were interestingly more cytotoxic to MV4-11 cells bearing FLT3-ITD mutation and HEL cells bearing JAK2^V617F mutation.

Full text of DOI:10.1016/j.ejmech.2015.03.034

European Journal of Medicinal Chemistry 95 (2015) 104e115
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Macrocyclic compounds as anti-cancer agents: Design and synthesis
of multi-acting inhibitors against HDAC, FLT3 and JAK2
Cheng-Qing Ning a, Cheng Lu a, Liang Hu a, Yan-Jing Bi a, Lei Yao a, Yu-Jun He a, Li-Fei Liu a,
,
b
,
, *
Xiao-Yu Liu a **, Nie-Fang Yu a
a School of Pharmaceutical Sciences, Central South University, Changsha 410013, China
b Drug Discovery Department, Hisun Group, Taizhou 317700, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of macrocyclic compounds were designed and synthesized as multi-target inhibitors
targeting HDAC, FLT3 and JAK2. Some of these compounds exhibited potent HDAC inhibition as well as
FLT3 and JAK2 inhibition under both cell-free and cellular conditions. In vitro antiproliferative assay
indicated that these compounds were interestingly more cytotoxic to MV4-11 cells bearing FLT3-ITD
mutation and HEL cells bearing JAK2V617F mutation.
Received 12 November 2014
Received in revised form
22 January 2015
Accepted 13 March 2015
Available online 17 March 2015
© 2015 Elsevier Masson SAS. All rights reserved.
Keywords:
Histone deacetylase
FLT3
JAK2
Macrocyclic
Multi-target inhibitor
1. Introduction
HDACs are a family of enzymes that regulate chromatin
remodeling and gene transcription by catalyzing the deactylation of
Acute myeloid leukemia (AML) is characterized by aberrant
proliferation of myeloid cells in the marrow and an arrest in their
maturation, which typically leads to death within weeks to months
if left untreated [1,2]. The standard treatment for patients with AML
is still chemotherapy regimens including cytarabine and an
anthracycline (such as daunorubicin), which can produce a com-
plete remission in almost 70% of patients. However, most patients
will ultimately relapse and finally die of their disease within 2 years
of achieving first remission [2,3]. Due to the high incidence of
relapse and poor prognosis, new strategies for treatment of AML are
warranted. In recent years, with the growing understanding of the
pathophysiology and molecular biology of AML, the development
of targeted therapies has led to promising results against AML.
Well-explored targets in these therapies include histone deacety-
lases (HDACs), Fms-like tyrosine kinase 3 (FLT3) and Janus kinase 2
(JAK2), in particular their combination [2e6].
the acetyl lysine residue on histone tails [7e9]. Inhibition of HDAC
can impact a variety of cell functions by blocking the deacetylation
of histone or non-histone proteins, such as p53, tubulin, and HSP90,
causing cell cycle arrest, differentiation, and/or apoptosis [10]. FLT3
belongs to the class III of receptor tyrosine kinases, which control
the development of hematopoietic progenitor cells [11]. It has been
reported that more than two-third patients having AML highly
expressed wild-type FLT3 and approximately one-third AML pa-
tients harbor FLT3 mutations [12]. The two major types of muta-
tions that occur are internal tandem duplication (ITD) mutations of
the juxtamembrane region and point mutations in the tyrosine
kinase domain (TKD). Mutant FLT3 results in constitutive activation
of the receptor's tyrosine kinase activity in the absence of ligand
and is associated with poor prognosis of AML patients [2]. The Janus
kinases (JAKs) are a family of non-receptor tyrosine kinases
involved in cytokine signaling via the JAK-STAT pathway [13]. They
play an important role in the pathogenesis of myeloid malignancies
[14]. Most patients with myeloproliferative neoplasms (MPNs)
harbor a somatic JAK2v617f mutation, resulting in constitutively
activation of JAK-STAT pathway [6]. Also, JAK2 mutations have been
found in patients with AML [15].
* Corresponding author.
** Corresponding author. School of Pharmaceutical Sciences, Central South Uni-
versity, Changsha 410013, China.
It has been reported that co-treatment with HDAC inhibitor
E-mail address: niefang_yu@126.com (N.-F. Yu).
http://dx.doi.org/10.1016/j.ejmech.2015.03.034
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
105
Panbinostat (SB939) and JAK2 inhibitor TG101209 could attenuate
JAK2v617f levels and downstream signaling and induce synergistic
apoptosis of MPD cells. It was demonstrated that treatment of
JAK2v617f mutant cells with SB939 disrupted the chaperone asso-
ciation of JAK2v617f with HSP90, consequently promoting protea-
somal degradation of JAK2v617f [16]. More recently, SB939 and FLT3/
JAK2 inhibitor Pacritinib (SB1518) are reported to exert synergistic
cytotoxic effects against cell lines carrying JAK2v617f mutation and
FLT3-ITD mutation. The synergistic effects were attributed to
impaired chaperone function of HSP90 by the HDAC inhibitor, since
both JAK2 and FLT3 have been identified as HSP90 client proteins
[17,18]. In addition, another recent report showed that HDAC in-
hibitors could selectively target FLT3-ITD for degradation in AML
cells [12]. Thus, a combination therapy with an HDAC inhibitor and
a JAK2/FLT3 inhibitor might be a promising approach for the
treatment of AML, including those with FLT3 or JAK2 mutations.
Herein, we report the first exploration of design, synthesis and
evaluation of a single chemical entity that can simultaneously
inhibit HDAC, FLT3, and JAK2.
The study was initiated by understanding the structural re-
quirements of HDAC, FLT3 and JAK2 inhibitors. It has been well
documented that the structural requirements of a typical HDAC
inhibitor should include a zinc binding group to efficiently chelate
the zinc ion, a linker to occupy the hydrophobic tubular channel, a
cap for surface recognition and a unit connect the linker and cap
(Scheme 1). The co-crystal structure of SAHA with HDAC-like pro-
tein (HDLP) reveals that SAHA binds by inserting its six-carbon-
long aliphatic chain into the tube-like pocket through multiple
contacts with hydrophobic residues, the terminal hydroxamic acid
group coordinating the zinc at the bottom of the pocket to disrupt
enzyme activity, the cap group at the other end makes contacts
with residues at the pocket entrance [19]. From our previous
extensive modeling studies [20,21], the cap region of HDLP
appeared large enough to accommodate the macrocyclic skeleton
of SB1317, a FLT3/JAK2 inhibitor now in phase I study for hemato-
logical malignancies (NCT01204164).
centre may be a suitable connecting point to introduce a group
containing a side-chain of a certain length that terminates with a
hydroxamic acid for HDAC binding, without sacrificing FLT3/JAK2
binding affinity at the same time. Thus, series of target compounds
were carefully constructed by combining these structural features
based on macrocycle templates of SB1317 and SB1518 (Scheme 2).
2. Results and discussion
2.1. Chemistry
The preparation of macrocycles with various chain lengths is
shown in Schemes 2 and 3 [21e24]. Scheme 2 describes the syn-
thetic routes for intermediates 6, 7 and 11, and Scheme 3 illustrates
the synthetic routes for macrocycles 30e41.
As shown in Scheme 2, Compounds 4e5 were prepared through
the Suzuki coupling of 2,4-dichloropyrimidine (1) with either
boronic acids 2 or 3. Alkylation of phenol 2 or phenylmethanol 3
with allyl bromide in the presence of base or phase transfer cata-
lyst, gave the allyl ethers 6e7. Coupling of 3-nitrobenzaldehyde (8)
with ally amine in the presence of NaBH(OAc)3 gave the nitroben-
zene 9, which was subsequently reduced by Tin (II) chloride
dehydrate, giving the aniline 10. Then, the secondary amine was
selectively N-Boc protected (11) by using a (Boc)2O/NaOH system.
Coupling of aniline 11 with chloropyrimidines (6 or 7) gave the
corresponding dienes 12e13 as shown in Scheme 3. Compound
14e15 were obtained through ring-closing metathesis (RCM) of the
dienes 12e13 in the presence of Grubbs second generation catalyst
in moderate to good yields. The Boc-group was then removed in the
presence of trifluoroacetic acid, which gave the key intermediates
16e17. The corresponding side chain attached to the macrocycle
was introduced using a coupling reaction. For example, compound
16e17 can be converted to intermediates 18e20 and 24e26 in high
conversion rate by reacting with corresponding aldehydes under
the aforementioned NaBH(OAc)3 conditions, while the amines
16e17 can be also converted to intermediates 21e23 and 27e29 by
coupling with the corresponding carboxylic acids in the presence of
DIC and DMAP in high conversion rate. Finally, the intermediates
were converted to hydroxamates 30e41 in moderate to good
yields, using NH2OH∙HCl/MeONa/MeOH system, a standard pro-
cedure developed in our lab.
Docking studies reveal that SB1317 binds to both FLT3 and JAK2
through the hydrogen bond interactions with residues (Cys694 for
FLT3 and Leu932 for JAK2) at the hinge region (see Fig. 1). The
methyl substituent is solvent exposed and points directly toward a
channel outside of the FLT3 and JAK2 binding pocket. It is pre-
sumably that this methyl could be properly replaced without much
disruption of binding affinity. Interestingly, this observation might
be supported by the fact that a side chain of SB1518 appears in this
nearby region of FLT3/JAK2 (Fig. 2) [22]. Therefore, the amino basic
2.2. In vitro enzymatic inhibitory activities
The enzymatic inhibitory activities of the target compounds
Scheme 1. Design of multi-target inhibitors against HDAC, FLT3 and JAK2.

106
C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
Fig. 1. SB1317 docked into JAK2 (PDB code: 2B7A) and FLT3 (PDB code: 1RJB). A-B), SB1317 docked into JAK2; C-D), SB1317 docked into FLT3. SB1317 is represented as yellow stick;
the structure of hinge region is represented as a cartoon ribbon; H-bond and H- interactions are represented as red and blue dash line, respectively. (For interpretation of the
p
references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. The predicting binding pose of SB1518 (purple red, stick) in the pocket of JAK2 (left) and FLT3 (right). (For interpretation of the references to colour in this figure legend, the
reader is referred to the web version of this article.)
were evaluated in FLT3 and JAK2 kinase activity assays, as well as an
HDAC enzyme assay. The results were summarized in Table 1.
SB1317 and SB1518 are suitable cap groups for HDAC inhibitors. The
enzymatic inhibitory activities of the tested compounds were
marginally affected by the capping group, as indicated by the
comparison between SB1317 hybrids (compounds 30e35) and their
counterparts (SB1518 hybrids, compounds 36e40). In contrast, the
data suggested that the unit Y were critical for HDAC inhibition.
Obviously, stronger HDAC inhibitory activities were observed when
2.2.1. In vitro HDAC inhibition
As shown in Table 1, all the target compounds showed moderate
to potent HDAC inhibitory activities, with IC50 values ranging from
87 to 848 nM. This suggests that both the macrocyclic moiety of

C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
107
Scheme 2. Synthesis of intermediates 6, 7 and 11. Reagents and conditions: (a) Pd(PPh3)4, KF∙2H2O, H2O, dioxane, 90 ꢀC, 4 h; (b) 4-bromo-1-butene, Cs2CO3, DMF, 60 ꢀC, overnight;
(c) 3-bromopropene, KOH, n-Bu4NHSO4, CH3CN, 40 ꢀC, overnight; (d) prop-2-en-1-amine, NaBH(OAc)3, DCM, 0 ꢀC, 1 h; (e) SnCl2∙2H2O, HAc:MeOH ¼ 1:9, 60 ꢀC, 5 h; (f) 4 N NaOH,
(Boc)2O, r.t., overnight.
Scheme 3. Synthesis of target compounds 30e41. Reagents and conditions: (a) Pd(PPh3)4, dppf, Cs2CO3, dioxane, 110 ꢀC, 3 h; (b) Grubbs catalyst II, DCM, r.t., overnight; (c) TFA, DCM,
r.t., 1 h; (d) oxo-alkyl-enoic acid ethyl ester, NaBH(OAc)3, DCM, 0 ꢀC, 0.5~1 h or Alkyl-anedioic acid monomethyl ester, DIC, DMAP, DCM, r.t., 4~5 h; (e) NH2OH∙HCl, MeONa, MeOH,
0
ꢀC, 0.3~1 h.
the unit Y was a methylene group (series A: compounds 30e32 and
36e38) compared to carbonyl group (series B: compounds 33e35
and 39e41). The optimal carbon chain length seems to be five to
six, which gives an IC50 of HDAC inhibition similar to that of SAHA.
Compound 32 exhibited the most potent HDAC inhibition, with an
IC50 value of 87 nM.
2.2.2. In vitro kinase inhibition
The target compounds tested exhibit moderate FLT3 and JAK2
inhibition compared to the reference compound SB1317. The data
indicated that the unit Y was also critical for FLT3 inhibition.
However, it seems not important for JAK2 inhibition. As shown in
Table 1, series A (Y ¼ eCH2e,) showed higher activities against FLT3

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C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
Table 1
Table 2
In vitro enzyme inhibitory activities of target compounds.
In vitro anti-proliferative activities of target compounds.
Compds
GI50
(
m
M)a
HL-60
MV4-11
K-562
HEL
30
31
32
33
34
35
36
37
38
39
40
41
SAHA
SB1317
3.03 0.79
2.62 0.10
1.03 0.15
1.27 0.14
6.73 1.27
1.95 0.06
2.05 0.44
2.30 0.38
0.90 0.11
4.43 1.08
2.87 0.22
1.66 0.13
0.55 0.07
1.13 0.26
0.66 0.13
0.53 0.06
0.27 0.11
1.01 0.11
5.89 0.57
NDb
0.37 0.10
0.52 0.10
0.32 0.04
2.06 0.25
1.41 0.17
0.78 0.03
0.59 0.13
0.66 0.30
2.45 0.22
2.32 0.37
1.02 0.17
1.44 0.27
4.79 1.47
2.02 0.27
1.83 0.93
2.09 0.29
0.94 0.27
4.97 0.13
4.39 1.42
2.64 0.28
0.52 0.16
1.23 0.26
0.51 0.03
2.26 0.82
0.34 0.10
2.11 0.09
5.25 0.75
6.26 1.55
0.49 0.10
0.68 0.04
0.70 0.25
2.94 0.87
5.03 0.15
4.78 0.79
0.76 0.24
0.79 0.25
Compds
X
Y
n
m
IC50 (nM)a
HDACb
FLT3c
JAK2d
293
1044 67
686 28
246
558
1475 186
392
a
30
31
32
33
34
35
36
37
38
39
40
41
SAHA
SB1317
O
O
O
O
O
O
CH2O
CH2O
CH2O
CH2O
CH2O
CH2O
e
CH2
CH2
CH2
CO
CO
CO
CH2
CH2
CH2
CO
CO
CO
e
2
2
2
2
2
2
1
1
1
1
1
1
e
e
1
2
3
2
4
5
1
2
3
2
4
5
e
e
251 59
152 21
87 10
848 118
310 11
355 41
180
108
87 12
556
9
3
3
Anti-proliferative activities against K-562, HL-60, MV4-11 and HEL were
determined using the CellTiter-Glo Assay kit (Promega, Madison, WI, USA). Values
are expressed as means standard deviation of three independent experiments.
b
9
7
3
ND ¼ Not determined.
1256 213
NDe
158
123
159
6
5
9
218
3
4
antiproliferative effect, 32 demonstrated less potent inhibitory ac-
tivities than SB1317 against FLT3 and JAK2. Thus, the greater
cellular potency of 32 against MV4-11 and HEL cells may be
attributed to the synergistic effects resulting from simultaneous
inhibition of HDAC, FLT3 and JAK2.
288 10
217 14
221 25
833 24
594 24
613 13
547 10
684 18
835 20
>10,000
170 17
402 12
293
6
334 12
105 16
ND
272
8
>10,000
42 10
To further explore the antitumor effect of these HDAC/FLT3/JAK2
inhibitors, some of the target compounds were selected for evalu-
ation of anti-proliferative effects in solid tumor cells, including
HCT-116 (colon), MCF-7(breast), MDA-MB-435 (melanoma),
NCIeH460 (non-small cell lung) and Ovcar-5 (Ovarian). As shown
in Table 3, the selected compounds all exhibited similar cellular
activities to the positive compound SAHA, with GI50 value ranging
e
e
a
Values are expressed as means
standard deviation of three independent
experiments.
b
HDAC inhibitory activities were determined using the Biomol Flour de Lys
system.
c
FLT3 kinase activities were determined using the LanthaScreen™ Eu kinase
binding assay kit (Invitrogen).
d
JAK2 Kinase activities were determined using the Z0-LYTETM kinase assay kit
from 0.81 to 2.72 mM. It seemed that the cellular potency of these
(Invitrogen).
e
selected compounds against solid tumor cells were more consistent
with their HDAC inhibitory activities. This may be because that FLT3
is expressed in hematopoietic stem/progenitor cells. Thus,
compared to the moderate JAK2 inhibitory activities, the cytotox-
icity of these selected compounds against solid tumor may be
mainly due to their HDAC inhibitory activities. This data also sug-
gested that the target compounds were more cytotoxic to MV4-11
and HEL leukemia cells, bearing FLT3-ITD and JAKV617F mutation,
respectively.
ND ¼ Not determined.
than the series B (Y ¼ eCOe), while the two series showed similar
JAK2 inhibition.
The data also indicated that the carbon chain length appeared to
have some influence on the potency of FLT3 and JAK2 inhibition,
which suggests that the hydroxamic acid at one end of the carbon
chain should have some kind of interaction with the receptors of
FLT3 and JAK2. However, the optimal carbon chain length for HDAC
inhibition seems not favor FLT3 or JAK2 inhibition.
2.4. Western blotting
2.3. Cell growth inhibition
We then explored whether these multi-acting compounds could
inhibit HDAC, and FLT3 and JAK2 phosphorylation in cellular
To explore the antiproliferative effect of the targeted com-
pounds in leukemia cells, HL-60, MV4-11, K-562 and HEL cell lines
were selected for evaluation. Among them, HL-60 is a FLT3 wild
type AML cell line, while MV4-11 is a FLT3 internal tandem dupli-
cation (FLT3-ITD) positive AML cell line. K562 is a JAK2 wild type
CML cell line, while HEL is a JAK2V617F positive erythroleukemia cell
line. The results were summarized in Table 2.
As shown in Table 2, our data suggested that FLT3-ITD bearing
MV4-11 cells and JAK2V617F bearing HEL cells were more sensitive
to the target compounds. This may be because that FLT3-ITD or
JAK2V617F expressing leukemia cells were more addicted to FLT3 or
JAK2 signaling for proliferation and survival. Part of the target
compounds exhibited similar or superior antiproliferative effect to
SAHA and SB1317 in MV4-11 and HEL cells. Compound 32 exhibited
the most potent antiproliferative activities against MV4-11 and HEL
Table 3
In vitro anti-proliferative activities of selected compounds.
Compds GI50 (
HCT-116
m
M)a
MCF-7
MDA-MB-435 NCI-H460
Ovcar-5
30
31
32
33
36
38
SAHA
1.53 0.34 1.13 0.24 2.72 0.03
1.33 0.49 1.32 0.28 2.63 0.57
0.82 0.05 0.95 0.22 1.77 0.08
1.59 0.15 2.09 0.28 2.41 0.48
0.81 0.24 1.07 0.16 1.88 0.37
0.90 0.31 1.13 0.13 1.50 0.54
1.04 0.28 0.61 0.23 1.18 0.21
1.77 0.37 1.22 0.12
1.60 0.43 1.42 0.61
1.07 0.10 1.22 0.19
2.25 0.19 1.52 0.61
1.42 0.10 1.13 0.03
1.19 0.26 1.42 0.27
0.85 0.14 1.78 0.47
a
Cancer cell lines were plated at 3000 cells per well in a 96-well flat-bottomed
plate with varying concentrations of compounds. The cells were incubated with
compounds for 48 h in a humidified atmosphere of 5% CO2 at 37 ꢀC. Growth inhi-
bition was determined by an SRB assay. Values are expressed as means standard
deviation of three independent experiments.
cells, with GI50 value of 0.27 and 0.34
mM, respectively, which was
lower than SAHA and SB1317. In contrary to its superior

C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
109
condition. For this purpose, Leukemia cell lines MV4-11 and HEL
were treated with 32 at 0.1e0.5 M for 24 h. The whole-cell extracts
m
were prepared and cellular proteins were then analyzed by western
blots with antibodies specific for acetylated histone H3 and tubulin,
phosphorylated FLT3 and JAK2. In MV4-11 cell line, compound 32
promoted acetylation of histone H3 and tubulin in a dose-
dependent manner, and effectively decreased the phosphorylated
level of FLT3 (Fig. 3). In HEL cell line, compound 32 treated also
effectively decreased p-JAK2 level.
We also explored the effect of these multi-acting compounds on
downstream pathway, including the phosphorylation of STAT5 and
AKT. As shown in Fig. 4, compound 32 can inhibit the phosphory-
lation of STAT5 and AKT in a dose-dependent manner.
Fig. 4. Compound 32 decreases the expression of phosphorylated STAT5 and AKT. The
MV4-11 cells were plated in the presence of indicated concentration of compound 32.
After 24 h of treatment, total cellular extracts were prepared and analyzed by western
blots with antibodies specific for the indicated proteins.
2.5. Docking studies
The possible binding modes of the macrocyclic compounds on
HDAC, FLT3 and JAK2 were explored with the representative
compounds 32 and 34 using MOE software. As illustrated by Fig. 5,
compounds 32 and 34 binds to HDLP by inserting their aliphatic
chains into the tube-like channel in a way similar as SAHA. The
terminal hydroxamic acid group is coordinated with zinc ion at the
bottom of the pocket. Obviously, the macrocyclic moiety at the
other end of the aliphatic chain is well tolerated. Furthermore, in
comparison to SAHA and compound 34, an additional Hydrogen
bond was observed between the aniline NH of 32 and Tyr264 res-
idue. This may explain the stronger enzymatic inhibitory activity of
compound 32 than that of 34 and SAHA.
FLT3 and JAK2 were conceived, synthesized, and evaluated in vitro.
It was demonstrated that most of these macrocyles exhibited
potent HDAC inhibition as well as FLT3 and JAK2 inhibition under
both cell-free and cellular conditions. In vitro cell growth inhibition
assays indicated that these multi-acting compounds were more
cytotoxic to MV4-11 and HEL cells, bearing FLT3-ITD and JAK2V617F
mutation, respectively. Compound 32 display promise as anticancer
agent with the potential to treat AML patients with FLT3-ITD or
JAK2V617F positive, and has been selected for further study as novel
anticancer agent.
Fig. 6 illustrates the predicted binding modes and detailed in-
teractions of compound 32 and 34 with FLT3 and JAK2, respectively.
Both compounds bind to the ATP binding site through hydrogen
bonding between the amino-pyrimidine and residue at the hinge
region. The side aliphatic chains form additional interactions with
residues in the solvent-exposed area outside of the pocket. For
FLT3, the backbone residue Cys694 participates in hydrogen
bonding with amino-pyrimidine. Ser705 and Asn701 residues in
the solvent-exposed area form electrostatic interactions with
hydroxamate group in one end of the aliphatic chain. For JAK2,
Leu932 residue in the hinge region participates in hydrogen
bonding with amino-pyrimidine. In addition, hydrogen bond was
observed between Leu932 and protonated amino basic central of
compound 32. In the case of compound 34, an additional hydrogen
bond was observed between hydroxamate group and Lys943 res-
idue outside of the pocket. In general, this binding modes support
our hypothesis that the methyl group of SB1317 could be properly
replaced.
4. Experimental section
4.1. General
All moisture-sensitive reactions were performed in an inert at-
mosphere of dry nitrogen. Dichloromethane was distilled from
CaH2. All other reagents and solvents purchased from commercial
sources were used directly without further purification. The prog-
ress of reactions was monitored by LC-MS. All compounds were
purified by recrystallization, column chromatography or reverse
phase chromatography. Column chromatography was performed
on silica gel (200e300 mesh). Reverse phase chromatography was
performed on a Gilson-281 Pre-HPLC equipped with a C-18 column
(Ultimate XB-C18, 5
m
m, 4.6 mm ꢁ 150 mm), using mobile phase A-
acetonitrile and mobile phase B-water containing 0.1% TFA. Frac-
tions containing the desired product were lyophilized or evapo-
rated to dryness under vacuum to provide the dry compound.
1HNMR spectra were recorded at 400 MHz and are reported in parts
3. Conclusion
per million (ppm) on the
d scale relative to tetramethylsilane as an
Novel macroclic compounds simultaneously inhibiting HDAC,
internal standard. High-resolution mass spectrometry (HRMS) was
Fig. 3. Compound 32 induces acetylation of histone H3 and tubulin and downmodulates phosphorylated FLT3 and JAK2. The MV4-11(FLT3-ITD) or HEL (JAK2V617F) cell lines were
plated in the presence of indicated concentration of compound 32. After 24 h of treatment, total cellular extracts were prepared and analyzed by western blots with antibodies
specific for the indicated proteins.

110
C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
Fig. 5. Predicted binding modes of compounds 32 and 34 on HDLP (PDB code: 1C3S) and the detailed protein-inhibitor interactions. A) HDLP is shown as a surface; SAHA is shown
as gray stick; compounds 32 is shown as cyan blue stick; compound 34 is shown as blue stick. B) SAHA, compounds 32 and 34 interacted with the active sites of HDLP. The hydrogen
bond is represented as red dash line; the H-
p interaction is represented as blue dash line. C) Compound 32 interacted with the active sites of HDLP. D) Compound 34 interacted with
the active site of HDLP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
performed on a Bruker MicroTOF QII instrument. The purity of the
synthesized target compounds was determined to be ꢂ95% by
HPLC, conducted on a Waters Xbridge-C18 column (4.6 ꢁ 50 mm,
4.2.3. 4-(3-(but-3-en-1-yloxy)phenyl)-2-chloropyrimidine (6)
A solution of 4 (6 g, 29 mmol) in DMF (100 mL) was treated with
4-bromo-1-butene (11.7 g, 87 mmol) and Cs2CO3 (47.2 g,
145 mmol). The reaction mixture was heated at 60 ꢀC overnight.
After cooling to room temperature, it was filtered and the filtrate
was concentrated under high vacuum. The residue was purified by
column chromatography (Hexane/EtOAc, 15:1) to afford 6 (6.6 g,
5 mm).
4.2. Synthesis
87.3%) as a white solid. 1HNMR (400 MHz, DMSO-d6,
d): 8.82 (d,
4.2.1. 3-(2-Chloropyrimidin-4-yl)phenol (4)
J ¼ 5.2 Hz, 1H), 8.19 (d, J ¼ 5.2 Hz,1H), 7.77 (d, J ¼ 8.0 Hz, 1H), 7.71 (t,
J ¼ 2.0 Hz, 1H), 7.49 (t, J ¼ 8.0 Hz, 1H), 7.19 (dd, J ¼ 8.0 Hz, J ¼ 2.0 Hz,
1H), 5.97e5.87 (m, 1H), 5.22e5.09 (m, 2H), 4.14 (t, J ¼ 6.8 Hz, 2H),
2.55e2.51 (m, 2H); MS m/z: [M þ H]þ 261.
To a degassed (nitrogen) solution of 2,4-dichloropyrimidine
(10 g, 67.1 mmol) and (3-hydroxyphenyl)boronic acid (11.1 g,
80.5 mmol) in dioxane (100 mL) were added KF∙2H2O (25.5 g,
268.4 mmol), Pd(PPh3)4 (5.4 g, 4.7 mmol) and 5 mL of water. The
reaction mixture was heated at 90 ꢀC for 4 h. After cooling to room
temperature, it was filtered and the filtrate was concentrated under
reduced pressure to give the reddish-brown residue, which was
purified by recrystallization from ethyl acetate. Compound 4 was
obtained as a yellowish solid (8.5 g, 61.6%). 1HNMR (400 MHz,
4.2.4. 4-(3-((allyloxy)methyl)phenyl)-2-chloropyrimidine (7)
A mixture of 5 (11 g, 49.9 mmol), KOH (9.8 g, 174.7 mmol), tet-
rabutylammonium hydrogen sulfate (1.2 g, 5 mmol), and 3-
bromopropene (24.1 g, 199.6 mmol) in acetonitrile (150 mL) was
heated at 40 ꢀC overnight. After cooling to room temperature, the
reaction mixture was filtered and concentrated under reduced
pressure. The residue was purified by column chromatography
(Hexane/EtOAc, 7:1) to yield 7 (8.3 g, 63.8%) as a yellow solid.
DMSO-d6,
d
): 9.83 (s, 1H), 8.80 (d, J ¼ 5.2 Hz, 1H), 8.07 (d, J ¼ 5.2 Hz,
1H), 7.63e7.61 (m, 2H), 7.38 (t, J ¼ 8.0 Hz, 1H), 6.99 (ddd, J ¼ 8.0 Hz,
J ¼ 2.4 Hz, J ¼ 1.0 Hz, 1H); MS m/z: [M þ H]þ 207.
1HNMR (400 MHz, CDCl3,
d
): 8.64 (d, J ¼ 7.2 Hz, 1H), 8.08 (s, 1H),
8.03e8.00 (m, 1H), 7.66 (d, J ¼ 7.2 Hz, 1H), 7.56e7.50 (m, 2H),
6.03e5.94 (m, 1H), 5.37e5.22 (m, 2H), 4.61 (s, 2H), 4.09 (d,
J ¼ 7.2 Hz, 2H); MS m/z: [M þ H]þ 261.
4.2.2. (3-(2-chloropyrimidin-4-yl)phenyl)methanol (5)
The title compound was obtained as a yellow solid in 63.1% yield
using a procedure similar to that of compound 4. 1HNMR (400 MHz,
DMSO-d6,
d
): 8.83 (d, J ¼ 5.2 Hz, 1H), 8.17 (br, 1H), 8.14 (d, J ¼ 5.2 Hz,
1H), 8.06 (dt, J ¼ 6.8 Hz, J ¼ 2.0 Hz, 1H), 7.57e7.51 (m, 2H), 5.38 (br,
4.2.5. tert-butyl allyl(3-aminobenzyl)carbamate (11)
1H), 4.61 (s, 2H); MS m/z: [M þ H]þ 221.
A mixture of 3-nitrobenzaldehyde (15.1 g, 0.1 mol) and prop-2-

C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
111
Fig. 6. Compounds 32 and 34 docked into JAK2 (AeB) and FLT3 (CeD). Compound 32, cyan blue stick; Compound 34, blue stick; H-bond, red dash line. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
en-1-amine (8.6 g, 0.15 mol) in dichloromethane (250 mL) was
stirred thoroughly. After cooling to 0 ꢀC with an ice bath, NaB-
H(AcO)3 (63.6 g, 0.3 mol) was added slowly. The resulting mixture
was allowed to stir at room temperature for 1 h. The reaction
mixture was quenched with water (200 mL) and extracted (DCM).
The combined organic layers were dried (Na2SO4), concentrated
under reduced pressure to provide a crude product, which was
used in the next step without further purification.
(3.3 g, 12.7 mmol) in dioxane (30 mL) were added Cs2CO3 (15 g,
46 mmol), Pd(PPh3)4 (1.6 g, 1.4 mmol) and dppf (1.3g, 2.3 mmol).
The reaction mixture was heated at 110 ꢀC for 3 h. After cooling to
room temperature, it was filtered and the filtrate was concentrated
under reduced pressure. The residue was purified by column
chromatography (Hexane/EtOAc, 5:1) to yield 12 as orange-yellow
oil (3.3 g, 58.9%). 1HNMR (400 MHz, DMSO-d6,
d): 9.71 (s, 1H), 8.53
(d, J ¼ 5.2 Hz, 1H), 7.72e7.67 (m, 4H), 7.46e7.40 (m, 2H), 7.27 (t,
J ¼ 7.6 Hz, 1H), 7.13 (dd, J ¼ 7.2 Hz, J ¼ 2.0 Hz, 1H), 6.83 (d, J ¼ 7.6 Hz,
1H), 5.91 (m, 1H), 5.76 (m, 1H), 5.15 (m, 4H), 4.34 (br, 2H), 4.13 (t,
J ¼ 6.4 Hz, 2H), 3.80 (br, 1H), 3.74 (br, 1H), 2.54 (m, 2H), 1.39 (s, 9H);
MS m/z: [M þ H]þ 488.
The residue obtained above was dissolved in MeOH (252 mL).
HAc (28 mL) and SnCl2∙2H2O (67.7 g, 0.3 mol) was added. The
resulting reaction mixture was stirred at 70 ꢀC overnight, and then
KOH/MeOH solution was added to adjust the pH z 9. The resulting
mixture was filtered, washed with DCM. The filtrate was condensed
under reduced pressure, and then 150 mL DCM and 4 M NaOH
aqueous solution (200 mL) were added. After cooling to 5 ꢀC with
an ice bath, (Boc)2O (0.08 mol) was added dropwise. The reaction
mixture was allowed to stir at room temperature for 2 h. Then, it
was extracted with DCM, dried over Na2SO4, filtered and concen-
trated under reduced pressure. The crude product was purified by
column chromatography on silica gel using hexane/EtOAc (5:1) as
eluent to afford the title compound as reddish-brown oil (14.9 g,
4.2.7. tert-butyl allyl(3-((4-(3-((allyloxy)methyl)phenyl)pyrimidin-
2-yl)amino)benzyl)carb eamate (13)
The title compound was obtained as orange-yellow oil in 51%
yield using a procedure similar to that of compound 12. 1HNMR
(400 MHz, DMSO-d6,
d
): 8.46 (d, J ¼ 7.2 Hz, 1H), 8.01 (m, 2H), 7.99
(br,1H), 7.54e7.47 (m, 4H), 7.33e7.28 (m,1H), 7.17 (d, J ¼ 7.2 Hz,1H),
6.93 (br, 1H), 6.04e5.91 (m, 1H), 5.77 (br, 1H), 5.37e5.08 (m, 4H),
4.61 (s, 2H), 4.45 (s, 2H), 4.08 (d, J ¼ 6.4 Hz, 2H), 3.88 (br, 1H), 3.78
(br, 1H), 1.47 (s, 9H); MS m/z: [M þ H]þ 488.
56.8%). 1HNMR (400 MHz, DMSO-d6,
d
): 6.95 (t, J ¼ 8.0 Hz, 1H),
6.45e6.42 (m, 2H), 6.34 (d, J ¼ 8.0 Hz, 1H), 5.78e5.68 (m, 1H),
5.12e5.04 (m, 4H), 4.19 (s, 2H), 3.72 (br, 1H), 3.67 (br, 1H), 1.41 (s,
9H); MS m/z: [M þ Na]þ 285.
4.2.8. (16E)-14-N-Boc-20-oxa-5,7,14,26-tetraazatetracyclo
[19.3.1.1(2,6)0.1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-
decaene (14)
4.2.6. tert-butyl allyl(3-((4-(3-(but-3-en-1-yloxy)phenyl)
pyrimidin-2-yl)amino)benz-yl)carb -amate (12)
A degassed (nitrogen) solution of 12 (3 g, 6.2 mmol) in dry DCM
(450 mL) was heated to 45 ꢀC with stirring. Then, the second
generation Grubbs catalyst (764 mg, 0.9 mmol), predissolved in dry
To a degassed (nitrogen) solution of 6 (3 g, 11.5 mmol) and 11

112
C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
DCM (15 mL), was added to the reaction mixture in three portions.
The reaction mixture was stirred at reflux overnight. The solvent
was removed under reduced pressure and subsequent purified by
column chromatography (Hexane/EtOAc, 5:1) to yield 14 as a
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 515.
4.2.15. Methyl 6-(7-oxa-macrocycle-1-yl)-6-oxohexanoate (21)
yellowish solid (2.2 g, 79%). 1HNMR (400 MHz, DMSO-d6,
d): 9.77 (s,
To a solution of 6-methoxy-6-oxohexanoic acid (39 mg,
1H), 8.56 (d, J ¼ 5.2 Hz, 1H), 8.51 (s, 1H), 7.88 (s, 1H), 7.65 (d,
J ¼ 7.6 Hz, 1H), 7.46 (t, J ¼ 7.6 Hz, 1H), 7.41 (d, J ¼ 5.2 Hz, 1H),
7.29e7.23 (m, 2H), 7.09 (d, J ¼ 8.0 Hz, 1H), 6.76 (d, J ¼ 7.6 Hz, 1H),
5.62e5.55 (m,1H), 5.41e5.35 (m,1H), 4.89 (s, 2H), 4.58 (br, 2H), 4.15
(t, J ¼ 5.2 Hz, 2H), 2.43 (m, 2H), 1.44 (s, 9H); MS m/z: [M þ H]þ 459.
0.25 mmol) in DCM (5 mL) was added DIC (33 mg, 0.26 mmol) and
stirred at room temperature for 10 min. Then, DMAP (13 mg,
0.11 mmol) and 16 (80 mg, 0.22 mmol) were added. After stirring at
room temperature for 4 h, water (5 mL) was added and extracted
(DCM). The combined organic layer was dried (Na2SO4), filtered,
and concentrated under reduced pressure to afford the crude
product, which was used in next step without further purification.
LC/MS (ESI positive mode) m/z [M þ H]þ 501.
4.2.9. (16E)-14-N-Boc-19-oxa-5,7,14,26-tetraazatetracyclo
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-
decaene (15)
The title compound was obtained as a yellowish solid in 71%
4.2.16. Methyl 8-(7-oxa-macrocycle-1-yl)-8-oxooctanoate (22)
The title compound was prepared from 16 and 8-methoxy-8-
oxooctanoic acid using a procedure similar to that of compound
21. The obtained crude product was used in next step without
further purification. LC/MS (ESI positive mode) m/z [M þ H]þ 529.
yield using a procedure similar to that of compound 14. 1HNMR
(400 MHz, CDCl3, d): 8.75 (s, 1H), 8.47e8.43 (m, 2H), 7.85e7.81 (m,
1H), 7.52e7.47 (m, 2H), 7.33e7.20 (m, 3H), 6.84e6.81 (m, 2H),
5.80e5.70 (m, 2H), 4.62 (s, 2H), 4.59 (s, 2H), 4.07 (d, J ¼ 6.8 Hz, 2H),
3.91 (br, 2H), 1.53 (s, 9H); MS m/z: [M þ H]þ 459.
4.2.17. Methyl 9-(7-oxa-macrocycle-1-yl)-9-oxononanoate (23)
The title compound was prepared from 16 and 9-methoxy-9-
oxononanoic acid using a procedure similar to that of compound
21. The obtained crude product was used in next step without
further purification. LC/MS (ESI positive mode) m/z [M þ H]þ 543.
4.2.10. (16E)-20-oxa-5,7,14,26-tetraazatetracyclo
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-
decaene (16)
A solution of 14 (2 g, 4.4 mmol) in DCM (20 mL) was added TFA
(2 mL). The reaction mixture was stirred at room temperature for
1 h. The solvent was evaporated and the residue was dissolved in
DCM (30 mL), washed by Na2CO3 aqueous solution, dried over
Na2SO4, and concentrated under reduced pressure to afford com-
pound 16 as a yellowish solid (1.5 g. 97%). 1HNMR (400 MHz,
4.2.18. Ethyl 5-(6-oxa-macrocycle-1-yl)pentanoate (24)
The title compound was prepared from 17 and ethyl 5-
oxopentanoate using a procedure similar to that of compound 9.
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 487.
DMSO-d6, d): 10.00 (s, 1H), 9.16 (br, 1H), 9.01 (s, 1H), 8.60 (d,
J ¼ 5.2 Hz,1H), 7.94 (s, 1H), 7.64 (d, J ¼ 8.0 Hz,1H), 7.51 (d, J ¼ 8.0 Hz,
1H), 7.48 (d, J ¼ 5.2 Hz,1H), 7.38 (t, J ¼ 8.0 Hz,1H), 7.28 (d, J ¼ 8.0 Hz,
1H), 7.26 (td, J ¼ 8.0 Hz, J ¼ 2.4 Hz, 1H), 7.12 (d, J ¼ 8.0 Hz, 1H),
6.10e6.03 (m, 1H), 5.71e5.64 (m, 1H), 4.23 (t, J ¼ 5.2 Hz, 2H),
4.12e4.09 (m, 2H), 3.73 (br, 2H), 2.58e2.54 (m, 2H); MS m/z:
[M þ H]þ 359.
4.2.19. Ethyl 6-(6-oxa-macrocycle-1-yl)hexanoate (25)
The title compound was prepared from 17 and ethyl 6-
oxohexanoate using a procedure similar to that of compound 9.
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 501.
4.2.11. (16E)-19-oxa-5,7,14,26-tetraazatetracyclo
[19.3.1.1(2,6).1(8,12)]heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-
decaene (17)
4.2.20. Ethyl 6-(6-oxa-macrocycle-1-yl)heptanoate (26)
The title compound was prepared from 17 and ethyl 7-
oxoheptanoate using a procedure similar to that of compound 9.
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 515.
The title compound was obtained as a yellowish solid in 94%
yield using a procedure similar to that of compound 16. 1HNMR
(400 MHz, CDCl3,
d
): 8.81 (s, 1H), 8.45 (d, J ¼ 4.8 Hz, 1H), 7.83 (d,
J ¼ 7.6 Hz, 1H), 7.59 (d, J ¼ 7.6 Hz, 1H), 7.51e7.48 (m, 1H), 7.30e7.26
(m, 2H), 7.21 (d, J ¼ 5.2 Hz, 1H), 7.08 (d, J ¼ 7.2 Hz, 1H), 6.81 (d,
J ¼ 8.0 Hz, 1H), 5.79e5.76 (m, 2H), 4.64 (s, 2H), 4.08 (d, J ¼ 4.0 Hz,
2H), 3.86 (s, 2H), 3.41 (d, J ¼ 4.4 Hz, 2H), 1.82 (br, 1H); MS m/z:
[M þ H]þ 359.
4.2.21. Methyl 6-(6-oxa-macrocycle-1-yl)-6-oxohexanoate (27)
The title compound was prepared from 17 and 6-methoxy-6-
oxohexanoic acid using a procedure similar to that of compound
21. The obtained crude product was used in next step without
further purification. LC/MS (ESI positive mode) m/z [M þ H]þ 501.
4.2.12. Ethyl 5-(7-oxa-macrocycle-1-yl)pentanoate (18)
4.2.22. Methyl 8-(7-oxa-macrocycle-1-yl)-8-oxooctanoate (28)
The title compound was prepared from 17 and 8-methoxy-8-
oxooctanoic acid using a procedure similar to that of compound
21. The obtained crude product was used in next step without
further purification. LC/MS (ESI positive mode) m/z [M þ H]þ 530.
The title compound was prepared from 16 and ethyl 5-
oxopentanoate using a procedure similar to that of compound 9.
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 487.
4.2.13. Ethyl 6-(7-oxa-macrocycle-1-yl)hexanoate (19)
4.2.23. Methyl 9-(7-oxa-macrocycle-1-yl)-9-oxononanoate (29)
The title compound was prepared from 17 and 9-methoxy-9-
oxononanoic acid using a procedure similar to that of compound
21. The obtained crude product was used in next step without
further purification. LC/MS (ESI positive mode) m/z [M þ H]þ 543.
The title compound was prepared from 16 and ethyl 6-
oxohexanoate using a procedure similar to that of compound 9.
The obtained crude product was used in next step without further
purification. LC/MS (ESI positive mode) m/z [M þ H]þ 501.
4.2.14. Ethyl 6-(7-oxa-macrocycle-1-yl)heptanoate (20)
The title compound was prepared from 16 and ethyl 7-
oxoheptanoate using a procedure similar to that of compound 9.
4.2.24. General procedure for the synthesis of the hydroxamic acids
30e41
A solution of methyl esters or ethyl esters (0.2 mmol) in MeOH

C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
113
(1 mL) was added NH2OH∙HCl (5.6 mmol). After cooling to 0 ꢀC
with an ice bath, NaOMe solution in MeOH (25%, 6 mmol) was
poured into the reaction mixture. The progress of reaction was
monitored by LC-MS (15e60 min). The reaction was quenched by
cold 6 N HCl, and the resulting mixture was centrifuged, and
sentenced to prep-HPLC chromatography. The target compounds
were obtained as yellow solid with yield from 30% to 65%.
4.2.24.6. 9-(7-Oxa-macrocycle-1-yl)-N-hydroxy-9-oxononanamide
(35). 1HNMR (400 MHz, DMSO-d6,
): 10.34 (br, 1H), 9.78 (s, 1H),
d
8.58 (s, 1H), 8.56 (d, J ¼ 5.2 Hz,1H), 7.87 (t, J ¼ 2.0 Hz,1H), 7.68e7.65
(m, 1H), 7.47 (t, J ¼ 8.0 Hz, 1H), 7.42 (t, J ¼ 5.6 Hz, 1H), 7.31e7.22 (m,
2H), 7.08 (d, J ¼ 8.0 Hz, 1H), 6.69e6.66 (m, 1H), 5.60e5.53 (m, 1H),
5.40e5.32 (m, 1H), 4.66 (s, 1H), 4.60 (s, 1H), 4.19e4.16 (m, 2H),
3.97e3.96 (m, 1H), 3.85e3.84 (m, 1H), 2.44e2.40 (m, 4H), 1.96e1.89
(m, 2H), 1.56e1.45 (m, 4H), 1.28e1.22 (m, 6H); MS m/z: [M þ H]þ
544; HRMS (m/z): [M þ H]þ calcd for C31H38N5O4, 544.2918; found,
544.2900.
4.2.24.1. 5-(7-Oxa-macrocycle-1-yl)-N-hydroxypentanamide
(30).
1HNMR (400 MHz, DMSO-d6,
d
): 10.29 (br, 1H), 9.99 (s, 1H), 9.77 (br,
1H), 8.91 (s, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 7.90 (s, 1H), 7.65 (d,
J ¼ 7.6 Hz, 1H), 7.51e7.47 (m, 2H), 7.40 (t, J ¼ 8.0 Hz, 1H), 7.32 (d,
J ¼ 8.0 Hz, 1H), 7.25 (dd, J ¼ 8.0 Hz, J ¼ 2.4 Hz, 1H), 7.16 (d, J ¼ 7.6 Hz,
1H), 6.20e6.13 (m, 1H), 5.82e5.74 (m, 1H), 4.59e4.56 (m, 1H),
4.26e4.23 (m, 2H), 4.05e3.99 (m, 2H), 3.85e3.82 (m, 1H),
2.93e2.83 (m, 2H), 2.59e2.56 (m, 2H), 1.80 (t, J ¼ 7.2 Hz, 2H),
1.70e1.68 (m, 1H), 1.41e1.36 (m, 1H), 1.27e1.15 (m, 2H); MS m/z:
[M þ H]þ 474; HRMS (m/z): [M þ H]þ calcd for C27H32N5O3,
474.2500; found, 474.2487.
4.2.24.7. 5-(6-Oxa-macrocycle-1-yl)-N-hydroxypentanamide (36).
1HNMR (400 MHz, DMSO-d6,
d): 10.40 (br, 1H), 9.98 (s, 1H), 9.68 (br,
1H), 8.78 (s, 1H), 8.61 (d, J ¼ 5.2 Hz, 1H), 8.33 (s, 1H), 8.09e8.07 (m,
1H), 7.61e7.58 (m, 2H), 7.53 (d, J ¼ 5.2 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz,
1H), 7.32 (d, J ¼ 8.0 Hz, 1H), 7.16 (d, J ¼ 7.2 Hz, 1H), 6.22e6.16 (m,
1H), 5.93e5.82 (m, 1H), 4.46e4.11 (m, 4H), 3.94e3.93 (m, 1H),
3.83e3.82 (m, 1H), 3.01e2.95 (m, 2H), 1.95 (t, J ¼ 7.2 Hz, 2H),
1.77e1.41 (m, 4H); MS m/z: [M þ H]þ 474; HRMS (m/z): [M þ H]þ
calcd for C27H32N5O3, 474.2500; found, 474.2487.
4.2.24.2. 6-(7-Oxa-macrocycle-1-yl)-N-hydroxyhexanamide
(31).
4.2.24.8. 6-(6-Oxa-macrocycle-1-yl)-N-hydroxyhexanamide
(37).
1HNMR (400 MHz, DMSO-d6,
d): 10.28 (br, 1H), 9.99 (s, 1H), 9.81 (br,
1HNMR (400 MHz, DMSO-d6,
d
): 10.36 (br, 1H), 9.98 (s, 1H), 9.65 (br,
1H), 8.90 (s, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 7.89 (s, 1H), 7.64 (d,
J ¼ 8.0 Hz, 1H), 7.51e7.46 (m, 2H), 7.39 (t, J ¼ 8.0 Hz, 1H), 7.31 (d,
J ¼ 8.0 Hz, 1H), 7.24 (dd, J ¼ 8.0 Hz, J ¼ 2.4 Hz, 1H), 7.16 (d, J ¼ 7.6 Hz,
1H), 6.20e6.13 (m, 1H), 5.83e5.76 (m, 1H), 4.60e4.57 (m, 1H),
4.27e4.18 (m, 2H), 4.07e4.00 (m, 2H), 3.88e3.81 (m, 1H), 2.91e2.79
(m, 2H), 2.59e2.58 (m, 2H), 1.75 (t, J ¼ 7.2 Hz, 2H), 1.32e1.23 (m,
4H), 0.99e0.87 (m, 2H); MS m/z: [M þ H]þ 488; HRMS (m/z):
[M þ H]þ calcd for C28H34N5O3, 488.2656; found, 488.2664.
1H), 8.78 (s, 1H), 8.61 (d, J ¼ 5.2 Hz, 1H), 8.32 (s, 1H), 8.09e8.07 (m,
1H), 7.60e7.58 (m, 2H), 7.53 (d, J ¼ 5.2 Hz, 1H), 7.40 (t, J ¼ 8.0 Hz,
1H), 7.31 (d, J ¼ 8.0 Hz, 1H), 7.16 (d, J ¼ 7.2 Hz, 1H), 6.22e6.16 (m,
1H), 5.93e5.85 (m, 1H), 4.62e4.56 (m, 2H), 4.46e4.36 (m, 2H),
4.14e4.11 (m, 2H), 3.95e3.94 (m, 1H), 3.81e3.80 (m, 1H), 2.98e2.91
(m, 2H), 1.92 (t, J ¼ 7.2 Hz, 2H), 1.81e1.42 (m, 4H), 1.23e1.18 (m, 2H);
MS m/z: [M þ H]þ 488; HRMS (m/z): [M þ H]þ calcd for C28H32N5O3,
488.2656; found, 488.2662.
4.2.24.9. 7-(6-Oxa-macrocycle-1-yl)-N-hydroxyheptanamide (38).
4.2.24.3. 7-(7-Oxa-macrocycle-1-yl)-N-hydroxyheptanamide (32).
1HNMR (400 MHz, DMSO-d6,
d): 10.34 (br, 1H), 9.97 (s, 1H), 9.69 (br,
1HNMR (400 MHz, DMSO-d6,
d): 10.26 (br, 1H), 9.99 (s, 1H), 9.74 (br,
1H), 8.78 (s, 1H), 8.61 (d, J ¼ 5.2 Hz, 1H), 8.33 (s, 1H), 8.09e8.07 (m,
1H), 7.60e7.58 (m, 2H), 7.53 (d, J ¼ 5.2 Hz, 1H), 7.41 (t, J ¼ 8.0 Hz,
1H), 7.31 (d, J ¼ 8.0 Hz, 1H), 7.16 (d, J ¼ 7.2 Hz, 1H), 6.21e6.16 (m,
1H), 5.92e5.89 (m, 1H), 4.61e4.58 (m, 2H), 4.45e4.11 (m, 4H),
3.95e3.94 (m, 1H), 3.82e3.81 (m, 1H), 2.99e2.92 (m, 2H), 1.91 (t,
J ¼ 7.2 Hz, 2H), 1.76e1.43 (m, 4H), 1.19 (br, 4H); MS m/z: [M þ H]þ
502; HRMS (m/z): [M þ H]þ calcd for C29H36N5O3, 502.2813; found,
502.2791.
1H), 8.90 (s, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 7.90 (t, J ¼ 2.0 Hz, 1H), 7.64
(d, J ¼ 7.6 Hz, 1H), 7.49 (t, J ¼ 8.0 Hz,1H), 7.51e7.46 (m, 2H), 7.31 (dd,
J ¼ 8.0 Hz, J ¼ 2.4 Hz,1H), 7.23 (dd, J ¼ 8.0 Hz, J ¼ 2.4 Hz,1H), 7.15 (d,
J ¼ 7.6 Hz, 1H), 6.19e6.12 (m, 1H), 5.84e5.77 (m, 1H), 4.60e4.58 (m,
1H), 4.27e4.18 (m, 2H), 4.07e4.00 (m, 2H), 3.89e3.86 (m, 1H),
2.92e2.82 (m, 2H), 2.59e2.56 (m, 2H), 1.74 (t, J ¼ 7.2 Hz, 2H),
1.30e1.21 (m, 4H), 0.97e0.89 (m, 4H); MS m/z: [M þ H]þ 502;
HRMS (m/z): [M þ H]þ calcd for C29H36N5O3, 502.2813; found,
502.2808.
4.2.24.10. 6-(6-Oxa-macrocycle-1-yl)-N-hydroxy-6-oxohexanamide
(39). 1HNMR (400 MHz, Acetone-d6,
d): 9.80 (s, 1H), 8.68 (br, 1H),
4.2.24.4. 6-(7-Oxa-macrocycle-1-yl)-N-hydroxy-6-oxohexanamide
8.61 (s, 1H), 8.50 (d, J ¼ 5.2 Hz,1H), 8.44 (s, 1H), 8.03 (s, 1H), 7.80 (br,
1H), 7.60e7.52 (m, 2H), 7.43 (d, J ¼ 5.2 Hz, 1H), 7.25 (t, J ¼ 7.6 Hz,
1H), 7.13 (d, J ¼ 8.0 Hz, 1H), 6.99 (d, J ¼ 6.8 Hz, 1H), 5.84e5.68 (m,
2H), 4.61 (s, 2H), 3.96 (d, J ¼ 4.8 Hz, 2H), 3.64 (s, 1H), 3.16 (d,
J ¼ 6.0 Hz, 2H), 2.39 (t, J ¼ 7.2 Hz, 2H), 2.04 (t, J ¼ 7.2 Hz, 2H),
1.50e1.43 (m, 3H); MS m/z: [M þ H]þ 502; HRMS (m/z): [M þ H]þ
calcd for C28H32N5O4, 502.2449; found, 502.2431.
(33). 1HNMR (400 MHz, DMSO-d6,
d): 10.34 (br, 1H), 9.79 (d,
J ¼ 4.0 Hz, 1H), 8.58 (s, 1H), 8.56 (d, J ¼ 5.2 Hz, 1H), 7.87 (br, 1H),
7.68e7.65 (m, 1H), 7.47 (t, J ¼ 8.0 Hz, 1H), 7.44e7.41 (m, 1H),
7.31e7.22 (m, 2H), 7.08 (d, J ¼ 8.0 Hz, 1H), 6.68 (d, J ¼ 7.6 Hz, 1H),
5.60e5.53 (m, 1H), 5.46e5.41 (m, 1H), 4.66 (s, 1H), 4.60 (s, 1H),
4.19e4.16 (m, 2H), 3.97e3.95 (m, 1H), 3.85e3.83 (m, 1H), 2.45e2.43
(m, 4H), 1.98e1.95 (m, 2H), 1.55e1.51 (m, 4H); MS m/z: [M þ H]þ
502; HRMS (m/z): [M þ H]þ calcd for C28H32N5O4, 502.2449; found,
502.2450.
4.2.24.11. 8-(6-Oxa-macrocycle-1-yl)-N-hydroxy-8-oxooctanamide
(40). 1HNMR (400 MHz, DMSO-d6,
d): 10.43 (s, 2H), 10.01 (d,
J ¼ 3.2 Hz, 1H), 8.61 (s, 1H), 8.60 (d, J ¼ 5.2 Hz, 1H), 8.31 (d,
J ¼ 8.0 Hz, 1H), 8.08 (br, 1H), 7.55e7.52 (m, 3H), 7.35e7.31 (m, 1H),
7.17 (t, J ¼ 7.6 Hz, 1H), 6.71 (d, J ¼ 7.6 Hz, 1H), 5.69e5.62 (m, 2H),
4.65e4.49 (m, 4H), 3.99 (m, 3H), 3.89 (m, 1H), 2.48e2.42 (m, 2H),
1.98e1.93 (m, 2H), 1.57e1.45 (m, 4H), 1.29e1.24 (m, 4H); MS m/z:
[M þ H]þ 530; HRMS (m/z): [M þ H]þ calcd for C30H36N5O4,
530.2761; found, 530.2765.
4.2.24.5. 8-(7-Oxa-macrocycle-1-yl)-N-hydroxy-8-oxooctanamide
(34). 1HNMR (400 MHz, DMSO-d6,
d): 10.34 (br, 1H), 9.79 (s, 1H),
8.58 (s, 1H), 8.56 (d, J ¼ 5.2 Hz, 1H), 7.87 (br, 1H), 7.68e7.65 (m, 1H),
7.47 (t, J ¼ 8.0 Hz, 1H), 7.44e7.41 (m, 1H), 7.31e7.23 (m, 2H), 7.08 (d,
J ¼ 8.0 Hz,1H), 6.69e6.67 (m,1H), 5.60e5.54 (m,1H), 5.44e5.37 (m,
1H), 4.66 (s, 1H), 4.60 (s, 1H), 4.19e4.16 (m, 2H), 3.97e3.95 (m, 1H),
3.85e3.83 (m, 1H), 2.45e2.39 (m, 4H), 1.96e1.91 (m, 2H), 1.56e1.46
(m, 4H), 1.28e1.23 (m, 4H); MS m/z: [M þ H]þ 530; HRMS (m/z):
[M þ H]þ calcd for C30H36N5O4, 530.2762; found, 530.2743.
4.2.24.12. 9-(6-Oxa-macrocycle-1-yl)-N-hydroxy-9-oxononanamide
(41). 1HNMR (400 MHz, DMSO-d6,
d): 10.35 (br, 1H), 9.85 (s, 1H),

114
C.-Q. Ning et al. / European Journal of Medicinal Chemistry 95 (2015) 104e115
8.66 (br, 1H), 8.58 (dd, J ¼ 5.2 Hz, J ¼ 1.6 Hz, 1H), 8.32 (d, J ¼ 7.6 Hz,
1H), 8.07e8.05 (m, 1H), 7.54e7.45 (m, 3H), 7.30e7.27 (m, 1H), 7.15
(t, J ¼ 7.2 Hz, 1H), 6.89 (d, J ¼ 7.2 Hz, 1H), 5.76e5.67 (m, 1H),
5.63e5.61 (m, 1H), 4.64 (br, 2H), 4.51e4.87 (m, 2H), 3.99e3.98 (m,
3H), 3.88e3.87 (m, 1H), 2.48e2.42 (m, 2H), 1.96e1.91 (m, 2H),
1.58e1.45 (m, 4H),1.29e1.24 (m, 6H); MS m/z: [M þ H]þ 544; HRMS
(m/z): [M þ H]þ calcd for C31H38N5O4, 544.2918; found, 544.2899.
whole cell lysis buffer (Beyotime, #p0013). SDS-polyacrylamide gel
electrophoresis and western blotting were performed according to
the standard procedures using total cellular extracts. Antibodies
against acetylated histone H3 (#06-599) were obtained from Mil-
lipore, antibodies against acetylated tubulin (T7451) and tubulin
(T6199) were obtained from SigmaeAldrich, and antibodies against
phosphor-FLT3 Y591 (#3461), phosphor-JAK2 Y1007/1008 (#3776),
phosphor-STAT5 Y694 (#3939) and phosphor-AKT T308 (#4056)
were obtained from Cell Signaling Technologies. Detection was
performed using horse-radish peroxidase-labeled secondary anti-
bodies (Santa Cruz).
4.3. HDAC inhibition assay
The HDAC inhibitory activities were assessed using the Biomol
Fluor de Lys assay kit (AK-500, Enzo Life Sciences, Inc.), as previously
described by us [20,21]. Briefly, HeLa cell nuclear extracts were
incubated with test compounds, and HDAC reaction was initiated
by addition of Fluor de Lys substrate. The resulting mixture was
incubated for 2 h at room temperature, followed by adding devel-
oper to stop the reaction. Fluorescence was measured by a micro-
plate reader (SpectraMax M5) with excitation at 360 nm and
emission at 460 nm.
4.8. Molecular modeling
Protein complexes were obtained directly from protein data
bank (PDB). For HDLP (PDB code: 1C3S) and JAK2 (PDB code: 2B7A),
all waters were eliminated and one chain was retained. Hydrogen
and partial charges were added through protonate 3D program of
MOE. Default parameter settings generated by the program dock of
MOE were used for docking. For FLT3, the FLT3 X-ray structure (PDB
code: 1RJB) obtained from PDB is in the DFG-out conformation that
prevents our inhibitors from being docked into the ATP-binding
site. Thus, an X-ray/homology model of DFG-in conformation of
FLT3 was constructed based on FGFR2 X-ray structure (PDB code:
1OEC) according to the method described by Poulsen using ho-
mology model program of MOE [25]. Then, the protein preparation
and ligand-docking were accomplished as previously described.
4.4. FLT3 kinase assay
FLT3 kinase activities were measured using LanthaScreen™
assay platform (Invitrogen) according to the manufacturer's in-
structions. Briefly, the time-resolved fluorescence energy transfer
(TR-FRET) assay was performed in white, low-volume 384-well
plates (Corning Part#3673). The recombinant enzyme FLT3 (cata-
log no.PV3182, Invitrogen) was incubated with LanthaScreen™ Eu-
anti-His antibody, Alexa Fluor 647-labeled kinase tracer 236 and
various concentrations of tested compounds for 1 h at room tem-
perature. Then, the single was measured at 665/615 nm emission
ration on a microplate reader (SpectraMax M5).
Acknowledgments
We thank the National Natural Science Foundation of China for
financial support (Project Number: 30873137, 30772645 and
81172927).
4.5. JAK2 kinase assay
Appendix A. Supplementary data
JAK2 kinase activities were determined using Z0-LYTETM kinase
assay platform (Invitrogen). The assay was carried out in black, low-
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.034.
volume 384-well plates (Corning Part#3676). Briefly, in a 10
kinase reaction, each well contains 30 ng/mL JAK2 (catalog
no.PV4210, Invitrogen), 4 mM
M Z0-LYTE Try6 peptide substrate, 25
mL
m
References
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reaction was performed at room temperature for 1 h. Then,
development reagent was added into each well. After additional 1 h
incubation at room temperature, stop reagent was added to halt the
development reaction. The single was measured at 445/520 nm
emission ration on a microplate reader (SpectraMax M5e).
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