Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Article abstract of DOI:10.1038/aps.2013.79

Aim: Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies. Methods: A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System. Results: A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC ₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation. Conclusion: The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.

Full text of DOI:10.1038/aps.2013.79

Acta Pharmacologica Sinica (2013) 34: 1359–1366
© 2013 CPS and SIMM All rights reserved 1671-4083/13 $32.00
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Original Article
Discovery of a retigabine derivative that inhibits
KCNQ2 potassium channels
3,
Hai-ning HU1,2, Ping-zheng ZHOU3, Fei CHEN1,2, Min LI3, Fa-jun NAN1,2, , Zhao-bing GAO
*
*
1State Key Laboratory of Drug Research, 2The National Center for Drug Screening, 3CAS Key Laboratory of Receptor Research,
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
Aim: Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to
explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable
tools for KCNQ channel studies.
Methods: A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2
currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the
recorded cell using an ALA 8 Channel Solution Exchange System.
Results: A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds,
HN38 was the most potent inhibitor of KCNQ2 channels (its IC50 value=0.10±0.05 μmol/L), and was 7-fold more potent than the classi-
cal KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but acceler-
ated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deacti-
vation.
Conclusion: The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinet-
ics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.
Keywords: KCNQ2 channel; retigabine; HN38; XE991; structure modification; CHO cell; whole-cell recording
Acta Pharmacologica Sinica (2013) 34: 1359–1366; doi: 10.1038/aps.2013.79; published online 12 Aug 2013
or from natural products[6, 7]. Both activators and inhibitors
Introduction
The KCNQ family consists of five members termed KCNQ1 are essential as tools for elucidating the mechanism of chan-
to KCNQ5 (or Kv7.1 to Kv7.5). The subthreshold activating nel activity and for developing therapeutic agents. Activators
potassium currents mediated by KCNQ channels play inhi- capable of augmenting KCNQ2 function have proven effective
bitory roles in controlling cellular excitability[1–3]. Expression in animal models of convulsion, pain and neuropsychopa-
of the five isoforms is tissue specific. KCNQ1 is most abun- thy[3, 6, 8]. Retigabine, a KCNQ2-5 activator, was approved for
dantly expressed in cardiac tissue, but its expression in non- adjunctive treatment of partial-onset seizures in adult patients
cardiac tissue should not be ignored[4]. The rest four isoforms in 2011. Linopirdine, an inhibitor of the KCNQ2-3 channels,
(KCNQ2 to KCNQ5) are expressed mainly in the central was studied initially in Alzheimer’s disease due to its enhance-
and peripheral nervous systems, for example, hippocam- ment of acetylcholine release in cholinergic nerve terminals in
pal and dorsal root ganglion[5]. The co-assembly of KCNQ2 the brain. Subsequent clinical trials failed, as treatment with
with KCNQ3 is believed to be the major component of the linopirdine did not improve memory performance in elderly
M-channel[5]. Augmentation of the M-current dampens mem- subjects. However, linopirdine has become a valuable phar-
brane excitability[3]. Recently there is an increasing interest in macological tool compound for KCNQ channel studies[9–11]
.
the discovery of small molecular modulators of Kv channels. Two analogs of linopirdine that exhibit higher potency are
Such compounds can be derived either from synthetic sources XE991 and DMP 543, shown in Figure 1.
During our search for activators of KCNQ2, we found that
certain chemical modifications on the N-2 and N-3 positions of
*
To whom correspondence should be addressed.
retigabine gave rise to an inhibitor of KCNQ2 channels. This
compound (hit 1, Figure 2) inhibits the current of the KCNQ2
channel by 70%. This encouraged us to explore this class of
E-mail fjnan@mail.shcnc.ac.cn (Fa-jun NAN);
zbgao@simm.ac.cn (Zhao-bing GAO)
Received 2013-03-21 Accepted 2013-05-16

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Hu HN et al
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ding to Scheme 1. The procedure for the synthesis of HN311 is
described in the experimental section. Compound 6 was syn-
thesized from commercially available compound 5 and 4-fluo-
robenzaldehyde via reductive amination reaction. Compound
6 was then hydrogenatedly catalyzed by Raney-Ni. Interme-
diate compound 7 was treated with Boc2O to give 8. Reaction
of compound 8 with the corresponding reagents produced
9a–f. Deprotection of 9a–f, followed by further treatment with
the appropriate alkyl chloroformate, gave the final compounds
HN36-HN310 and HN41-HN410. Compound HN21 was syn-
thesized in the same way. Reductive amination using HN21
gave compounds HN31-HN35.
Reagents and conditions
a) 4-Fluorobenzaldehyde, cat. pTsOH, toluene, 120ºC, 77.2%;
b) NaBH4, 1,4-dioxane:MeOH (4:1), rt, 94%; c) H2, Raney-Ni,
MeOH, rt, 100%; d) Boc2O, NaHCO3, H2O:THF (1:2), rt, 85%;
e) R2Br, DIPEA, 60ºC, DMF; f) (1) TFA, DCM, 0ºC; (2) Alkyl
chloroformate, DIPEA, 1,4-dioxane, rt; g) Ethyl chloroformate,
DIPEA, 1,4-dioxane, rt; h) Alkyl aldehyde, AcOH, then sodium
triacetoxyborohydride, DCM, rt.
Figure 1. Modulators of KCNQ channels.
Electrophysiology experiment
Cell culture
Chinese hamster ovary (CHO) cells were grown in 50/50
DMEM/F-12 (GibcoTM, Life Technologies, Carlsbad, CA, USA)
with 10% fetal bovine serum (FBS), and 2 mmol/L L-glut-
amine (InvitrogenTM, Life Technologies, Carlsbad, CA, USA).
To express KCNQ2 channels, cells were split for 24 h before
transfection, plated in 60-mm dishes, and transfected with
Lipofectamine 2000TM reagent (InvitrogenTM, Life Technologies,
Carlsbad, CA, USA) according to the manufacturer’s instruc-
tions. At 24 h after transfection, cells were split and replated
onto coverslips coated with poly-L-lysine (Sigma-aldrich, St
Louis, MO, USA). A GFP cDNA (Amaxa, Gaithersburg, MD,
USA) was cotransfected to identify the transfected cells by
fluorescence microscopy.
Figure 2. KCNQ inhibitor hit 1.
compounds further. The most potent derivative (HN38) inhi-
bited the KCNQ2 current by 92% at 10 μmol/L. The IC50 of
HN38 is 0.10±0.05 μmol/L, which is significantly more potent
than either linopirdine or XE991[12]
.
Materials and methods
Chemistry
All the compounds other than HN311 were synthesized accor-
Scheme 1. Synthesis of retigabine derivatives.
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Table 1. Structures of retigabine derivatives and effects on KCNQ2
channels.
Electrophysiological recording
Standard whole-cell recording was used to record KCNQ2
current in CHO cells. Pipettes were pulled from borosilicate
glass capillaries (TW150-4, World Precision Instruments, Sara-
sota, FL, USA). When filled with the intracellular solution,
the pipettes had resistances of 3–5 megaohms. During the
recording, constant perfusion of extracellular solution was
maintained using a BPS perfusion system (ALA Scientific
Instruments). Pipette solution contained (in mmol/L): 145
KCl, 1 MgCl2, 5 EGTA,10 HEPES and 5 MgATP (pH 7.3); extra-
cellular solution contained (in mmol/L): 140 NaCl, 3 KCl, 2
CaCl2, 1.5 MgCl2, 10 HEPES and 10 glucose (pH 7.4). Current
and voltage were recorded using an Axopatch-200B amplifier,
filtered at 2 kHz, and digitized using a DigiData 1440A with
pClamp 10.2 software (Axon Instruments, Sunnyvale, CA,
USA). Series resistance compensation was used and set to
60%–80%.
[a]
[a]
Compound
R2
Me
I/I0
Compound R1
I/I0
HN31
HN32
HN33
HN34
HN35
1.55±0.20
1.17±0.09
0.28±0.10
0.36±0.12
1.01±0.05
HN41
HN42
HN43
HN44
HN45
HN46
hit 1
1.43±0.05
0.16±0.04
0.24±0.10
0.50±0.14
1.24±0.10
1.14±0.20
0.30±0.01
0.27±0.12
0.17±0.07
0.24±0.10
1.08±0.03
Me
nPr
Allyl
iBu
tBu
Me
Et
Et
Compound application
nPr
nPen
nBu
XE991 and HN38 were dissolved in dimethylsulfoxide
(DMSO) to prepare a 20 mmol/L stock solution, from which
the appropriate volumes were added to the external solutions
to produce the desired concentrations. DMSO (less than 0.1%
in the final dilution) elicited no observable effect on the K+ cur-
rents. The testing concentration of each compound in electro-
physiological recording was 10 μmol/L except otherwise sta-
ted. The external solution containing the drugs was delivered
to the recorded cell using ALA 8 Channel Solution Exchange
System (ALA Scientific Instruments Inc, Farmingdale, NY,
USA).
HN36
HN37
HN38
HN39
0.87±0.06
0.66±0.16
0.08±0.02
1.18±0.05
HN47
HN48
HN49
HN410
nPr
Allyl
iPr
iBu
HN310
HN311
0.24±0.02
0.29±0.10
[a] The testing concentration was 10 μmol/L. Each compound was tested
Data analysis
in more than three cells.
Patch clamp data were processed using Clampfit 10.2 (Mole-
cular Devices, Sunnyvale, CA, USA) and then analyzed in
GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).
Voltage-dependent activation curves were fitted with the
Boltzmann equation, G=Gmin+(Gmax–Gmin)/(1+exp(V–V1⁄2)/S),
where Gmax is the maximum conductance, Gmin is the minimum
conductance, V1⁄2 is the voltage for half-maximum conduc-
tance, and S is the slope factor. Dose-response curves were
fitted with the Hill equation, E=Emax/(1–(EC50/C)P), where
EC50 is the drug concentration producing half of the maximum
response, and P is the Hill coefficient. The data are presented
as the mean±SEM. Significance was estimated using Student’s
t test, where P<0.05 was considered significant.
Figure 3. Chemical structures of compounds HN21-HN22.
Compound concentration in the assay was 10 μmol/L and
testing voltage was -10 mV, unless otherwise stated. We first
evaluated the effect of substitutions on the N-2 and N-3 posi-
tions of retigabine. Both compounds HN21 and HN22 (Figure
3) lost inhibitory activities (data not shown). These results
indicate that both N-2 and N-3 groups are important for inhi-
bition. Other compounds and their effects on the KCNQ2
channel are shown in Table 1. The effects of substitutions at
N-3 are shown by compounds HN31–HN310. Alkyl chains of
moderate lengths appear to be preferred, as shown in HN33
and HN34, which are nearly equipotent as hit 1. Increasing or
Results
Structure and activity relationship of retigabine derivatives
The electrophysiology experiment was conducted using the
whole-cell patch clamp technique and the effects on the ampli-
tude of outward current (I/I0) were analyzed. I0 is the ampli-
tude of outward current in the absence of a compound. I is the
amplitude of outward current in the presence of a compound.
Compounds leading to I/I0>1 were defined as activators, while
compounds giving with I/I0<1 were defined as inhibitors.
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decreasing the length of the chain had a detrimental effect on
activity (HN31 vs HN33, HN35 vs HN33). Incorporation of a
methyl group at N-3 gave a weak agonist (HN31). Although
saturated alkyl chains did not improve activity, allylic sub-
stitutions gave some promising results (HN36-HN311). The
effect of branching on the allylic group was thus studied. Sub-
stitution with 3-methyl allylic (HN36) and 3,3-dimethyl allylic
(HN37) groups afforded weak inhibitors, while introduction
of a 2-methyl allylic (HN38) side chain caused 92% inhibition.
This compares favorably with 70% inhibition shown by hit
1. A derivative with increased length of the side chain on the
allylic group (HN39) was devoid of activity. A polar branch-
ing group was also investigated, giving rise to a compound
with moderate activity (HN310). One-carbon homologation
of HN39 gave HN311, a compound with attenuated activity.
Compounds HN41-HN45 and HN46-HN410 enabled inves-
tigation of SAR for the N-1 and N-2 positions. The trends
observed were similar to those seen in the two N-3 substituent
series. The nature of the functional groups at N-1 and N-2 was
very important. A critical difference was observed between
the activity of methyl (HN41, HN46) and that of ethyl (HN38,
hit 1) analogues, with HN41 and HN46 behaving as activators
while HN38 and hit 1 as inhibitors. Increasing the length of
the chain led to decreased activity (HN42). Reducing flexibi-
lity at these positions did not improve activity (HN43). Bran-
ching at α- or β-position of the substituents led to attenutated
(HN44) or agonistic (HN45) activity.
Figure 4. Dose-response relationship of HN38 on KCNQ2 channels. (A)
Representative current traces of KCNQ2 in the absence or presence of
HN38 (1, 3, or 10 μmol/L, as indicated). To elicit the currents, holding
potential was set at -80 mV followed by 1500 ms depolarization step to
-10 mV. A hyperpolarization step to -120 mV was used to record the tail
current. (B) Dose-response curve of HN38 on KCNQ2 channels.
Electrophysiological results
Inhibitory effects on KCNQ2 channels by HN38
Compound HN38 was selected to be tested further based
on its potent inhibition of the KCNQ2 channel. Figure 4A
shows that the inhibition of KCNQ2 current by HN38 is con-
centration-dependent. Analysis of the dose-response curves
revealed an IC50 value of 0.10±0.05 μmol/L (Figure 4B). This
value shows that HN38 is 7-fold more potent than XE991 and
activation or deactivation (Figures 5D–5G).
Discussion
XE991 and linopirdine are two classical KCNQ inhibi-
tors and exhibit potent inhibition of both recombinant and
native M-currents[5]. DMP 543 is a derivative of linopirdine.
Although these three compounds have shown potential in
pre-clinical models of memory enhancement, the results of
the clinical trials for Alzheimer’s disease were equivocal. This
result may reflect the suboptimal pharmacokinetics and dis-
tribution properties of these drugs[11]. UCL2077 and ML252
40-fold more potent than linopirdine[12]
.
Comparison of HN38 with XE991
We next compared the effects of HN38 and XE991 on the
KCNQ2 channel. Homomeric KCNQ2 mediates a character-
istic outward current with slow activation and deactivation
(Figures 5A and 5B). In the presence of 3 μmol/L HN38 or
XE991, steady-state outward currents were significantly inhib-
ited at all applied depolarizing voltages. Figure 5C shows
the current-voltage relationships of the KCNQ2 channel in
the absence and presence of 3 μmol/L HN38 or 3 μmol/L
XE991. Then the effects of HN38 and XE991 on the activation
and deactivation kinetics of the KCNQ2 channel were ana-
lyzed. HN38 at 3 μmol/L had no detectable effect on channel
activation (Figures 5D and 5F), while it accelerated deactiva-
tion in hyperpolarizing voltages (Figures 5E and 5G). After
application of HN38, the time constant of deactivation was
accelerated from 38.3±6.6 ms to 17.2±4.2 ms (n=3, P<0.05). In
contrast, XE991 did not affect the kinetics of either channel
are KCNQ2 inhibitors that have been reported recently[13, 14]
.
Of all the KCNQ2 inhibitors reported to date, only ML252,
a synthetic compound discovered from a high-throughput
screen of 300000 compounds, shows potency comparable to
HN38. The IC50 of ML252 against KCNQ2 is approximately 70
nmol/L[14]. Detailed kinetics studies suggest the compound
HN38 might belong to a different class of inhibitor of KCNQ2
channels from XE991. It accelerated the deactivation phase of
KCNQ2 channels significantly while XE991 exhibited negli-
gible effects on the deactivation. As a derivative of retigabine,
an approved anti-epileptic drug, compound HN38 with its
potent efficiency might be a good leading compound for clini-
cal development of KCNQ inhibitors.
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Figure 5. Effects of HN38 and XE991 on KCNQ2 channels. Representative traces of KCNQ2, before and after application of 3 μmol/L HN38 (A) or 3
μmol/L XE991 (B). The holding potential was -80 mV, followed by a series of 1500 ms depolarization steps from -70 to +50 mV in 10 mV increments. A
1000 ms hypopolarization step to -120 mV was then applied to record the tail current. (C) I–V curves in the absence (open circle) and presence (filled
circle) of 3 μmol/L HN38 or presence (filled triangle) of 3 μmol/L XE991 as indicated. (D) Normalized activation phase from full traces in the absence
and presence of 3 μmol/L HN38 (left) or 3 μmol/L XE991 (right). The testing depolarization was -10 mV. (E) Normalized deactivation phase from full
traces in the absence and presence of 3 μmol/L HN38 (left) or 3 μmol/L XE991 (right). The testing depolarization was -120 mV. (F) Histogram summa-
rized the effects of 3 μmol/L HN38 and 3 μmol/L XE991 on activation of KCNQ2 channels. (G) Histogram summarized the effects of 3 μmol/L HN38 or
3 μmol/L XE991 on deactivation of KCNQ2 channels (n>3, bP<0.05 vs control).
vatives, exhibits an IC50 value of 0.10±0.05 μmol/L, which is
Conclusion
In summary, we have discovered a series of KCNQ2 inhibitors 7-fold potent than XE991 and 40-fold potent than linopirdine.
based on SAR studies of retigabine, an approved anti-epileptic Our work provides a new strategy for the design and develop-
drug (Figure 1). HN38, the most potent one among the dire- ment of potent KCNQ2 channel inhibitors.
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Experimental section
Representative procedure for the synthesis of compounds HN21, 1,4-dioxane (2.0 mL), and the mixture was stirred at room tem-
hit 1, HN36-HN10, and HN41- HN410
perture for 1 h. The mixture was extracted with EtOAc (3×10
mate (18 mg, 0.17 mmol) was added dropwise as a solution in
To a solution of 2-nitro-1,4-phenylenediamine 5 (7.65 g, 0.05 mL). The combined organic phases were washed with water
mol) and PTSA (0.25 g) in toluene (200 mL), was added 4-fluo- and brine, dried over sodium sulfate and concentrated in in
robenzaldehyde (6.8 g, 55.0 mmol). The stirred reaction mix- vacuo. Purification (4:1 to 2:1, petroleum ether: EtOAc, v/v) by
ture was heated to reflux under a water sepatator for 3 h. The silica gel column chromatography gave HN22 (33.6 mg, yield:
mixture was filtered quickly through a Büchner funnel packed 58%). 1H NMR (300 MHz, CDCl3): δ 7.18 (t, J=8.1 Hz, 2H), 6.99
with silica gel. The filtrate was stirred and allowed to cool to (t, J=8.4 Hz, 2H), 6.91 (d, J=8.7 Hz, 1H), 6.12 (d, J=8.7Hz, 2H),
room temperature over 6 h. The orange solid was filtered and 6.06 (d, J=2.1 Hz, 1H), 5.80-5.86 (m, 1H), 5.19 (s, 1H), 5.14 (d,
washed with petroleum ether (3×15 mL) to give an orange J=3.0 Hz, 1H), 4.44 (s, 2H), 4.20 (q, J=6.9 Hz, 2H), 3.91 (d, J=4.5
solid (10.0 g, yield: 77.2%). The resulting solid was suspended Hz, 2H), 3.59 (brs, 2H), 1.28 (t, J=6.9 Hz, 3H); LC-MS m/z (%):
in a mixture of 1,4-dioxane (40 mL) and methanol (10 mL). To 366.2 (100) [M+Na]+.
this suspension, was added three separate portions of NaBH4
(1.2 g, 31.6 mmol) at 15-min intervals. The reaction was allo- Similar methods were used to synthesize compounds hit 1,
wed to stir at room temperature for 2 h, and quenched with HN36-HN310, and HN41-HN410
1
water (200 mL). The resulting solid was collected by filtration, hit 1: H NMR (300 MHz, CDCl3): δ 7.18 (dd, J=8.4, 5.4 Hz,
washed with water, and dried under vacuum to give com- 2H), 7.09 (brs, 1H), 6.99 (t, J=8.4 Hz, 2H), 6.60 (m, 2H), 5.96 (m,
pound 6 (4.91 g, yield: 94%). 1H NMR (300 MHz, CDCl3): δ 2H), 5.67 (m, 1H), 5.47 (m, 1H), 5.35 (d, J=17.4 Hz, 2H) 5.26
7.31 (t, J=8.1 Hz, 3H), 7.03 (t, J=8.7 Hz, 2H), 6.84 (dd, J=8.7, 2.7 (d, J=12.0 Hz, 2H), 4.65 (d, J=5.7 Hz, 4H), 4.48 (s, 2H), 3.95 (d,
Hz, 1H), 6.69 (d, J=9.0 Hz, 1H), 5.74 (s, 2H), 4.25 (d, J=5.1 Hz, J=5.7 Hz, 2H), 1.67 (d, J=6.3 Hz, 6H); LC-MS m/z (%): 438.2 (100)
2H); LC-MS m/z (%): 284.1 (100) [M+Na]+.
[M+Na]+.
Compound 6 (2.61 g, 10.0 mmol) was hydrogenated under
1
atmospheric pressure of hydrogen for 3 h in MeOH (30 mL) HN36: H NMR (300 MHz, CDCl3): δ 7.18 (dd, J=7.5, 5.7 Hz,
catalysed by Raney-Ni (10%, 261 mg). The catalyst was filte- 2H), 7.06 (d, J=8.7 Hz, 1H), 6.99 (t, J=8.7 Hz, 2H), 6.42 (s, 1H),
red off and the filtrate was concentrated. The product, com- 6.42 (d, J=8.7 Hz, 2H), 5.48–5.62 (m, 2H), 4.45 (s, 2H), 4.20 (m,
pound 7, should be used immediately in the next reaction. To 4H), 3.88 (d, J=4.5 Hz, 2H), 1.57 (d, J=6.0 Hz, 2H), 1.24–1.32 (m,
a solution of 7 (1.0 g, 4.32 mmol) in THF: H2O (40 ml, 2:1, v/v), 6H); LC-MS m/z (%): 452.2 (100) [M+Na]+.
was added Boc2O (2.83 g, 12.96 mmol) and NaHCO3 (1.16 g,
1
12.96 mmol). The mixture was stirred at room temperature HN37: H NMR (300 MHz, CDCl3): δ 7.19 (dd, J=8.4, 5.7 Hz,
overnight. The mixture was extracted with EtOAc (3×30 mL). 2H), 7.07 (d, J=8.7 Hz, 1H), 6.98 (t, J=8.4 Hz, 3H), 6.41 (s, 1H),
The combined organic phases were dried over sodium sulfate 6.41 (dd, J=8.7, 2.4 Hz, 2H), 5.23 (t, J=6.3 Hz, 2H), 4.43 (s, 2H),
and concentrated in vacuo. Purification (5:1 petroleum ether: 4.12–4.24 (m, 4H), 3.90 (d, J=6.0 Hz, 2H), 1.71 (s, 3H), 1.62 (s,
EtOAc, v/v) by silica gel column chromatography gave com- 3H), 1.28 (t, J=7.2 Hz, 6H); LC-MS m/z (%): 466.2 (100) [M+Na]+.
pound 8 (1.67 g, yield: 89.5%). 1H NMR (300 MHz, CDCl3): δ
1
7.28 (t, J=8.7 Hz, 2H), 7.00 (t, J=8.7 Hz, 3H), 6.89 (s, 1H), 6.26 (d, HN38: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.7, 5.4 Hz,
J=8.7 Hz, 1H), 4.25 (s, 2H), 1.48 (s, 18H); LC-MS m/z (%): 454.2 2H), 7.06 (d, J=9.0 Hz, 1H), 6.99 (t, J=8.7 Hz, 2H), 6.94 (s, 1H),
(100) [M+Na]+.
6.38 (s, 1H), 6.38 (dd, J=8.7, 2.4 Hz, 2H), 4.97 (m, J=6.3 Hz, 2H),
To a solution of 8 (150 mg, 0.348 mmol) in DMF (3 mL), was 4.88 (s, 1H), 4.81 (s, 1H), 4.50 (s, 2H), 4.11–4.23 (m, 4H), 3.84 (s,
added allyl bromide (39.0 μL, 0.452 mmol) and DIPEA (99.0 2H), 1.73 (s, 3H), 1.23–1.31 (m, 6H); LC-MS m/z (%): 452.2 (100)
mg, 0.766 mmol). The mixture was stirred at 60 °C for 2 h. [M+Na]+.
The mixture was extracted with EtOAc (3×10 mL). The combi-
ned organic phases were washed with water and brine, dried HN39: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.4, 5.4 Hz,
1
over sodium sulfate and then concentrated in vacuo. Purifica- 4H), 7.06 (d, J=9.3 Hz, 2H), 6.99 (t, J=8.7 Hz, 2H), 6.88 (s, 1H),
tion (4:1, petroleum ether: EtOAc, v/v) by silica gel column 6.38 (dd, J=9.0, 2.7 Hz, 1H), 6.38 (s, 1H), 4.87 (s, 1H), 4.82 (s,
chromatography gave product 9a (143 mg, yield: 87.2%). 1H 1H), 4.50 (s, 2H), 4.13–4.22 (m, 4H), 3.86 (s, 2H), 2.00 (t, J=7.2
NMR (300 MHz, CDCl3): δ 7.18 (t, J=6.9 Hz, 2H), 6.90–7.06 (m, Hz, 2H), 1.43–1.48 (m, 2H), 1.23–1.35 (m, 10H), 0.89 (t, J=7.5
4H), 6.49 (s, 1H), 6.48 (d, J=8.7 Hz, 1H), 5.79–5.88 (m, 1H), 5.18 Hz, 3H); LC-MS m/z (%): 550.3 (100) [M+Na]+.
(s, 1H), 5.13 (s, 1H), 4.44 (s, 2H), 3.92 (d, J=3.6Hz, 2H), 1.49 (s,
1
18H); 13C NMR (75 MHz, CDCl3): δ 163.5, 160.3, 154.9, 153.5, HN310: H NMR (300 MHz, CDCl3): δ 7.17 (t, J=8.7 Hz, 2H),
147.5, 134.4, 134.3, 133.4, 128.4, 128.3, 127.0, 116.7, 115.5, 115.2, 7.08 (d, J=9.0 Hz, 1H), 6.99 (t, J=8.4 Hz, 2H), 6.91 (s, 1H), 6.73
80.4, 53.4, 53.1, 28.3; LC-MS m/z (%): 494.3 (100) [M+Na]+.
(s, 1H), 6.32–6.40 (m, 2H), 6.30 (s, 1H), 5.63 (s, 1H), 4.50 (s, 2H),
To a solution of 9a (80 mg, 0.17 mmol) in DCM (0.2 mL), was 4.18–4.23 (m, 6H), 3.83 (s, 3H), 1.85–1.91 (m, 6H); LC-MS m/z
added TFA (1.0 mL), and the mixture was stirred at room tem- (%): 496.2 (100) [M+Na]+.
perature for 1 h. The mixture was concentrated in vacuo. The
residue was taken up in 1,4-dioxane (10 mL) and to this solu- HN41:
tion was added DIPEA (66.0 mg, 0.51 mmol). Ethyl chlorofor- 1H NMR (300 MHz, CDCl3): δ 7.21 (dd, J=8.7, 5.7 Hz, 2H),
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7.09 (d, J=9.0 Hz, 1H), 7.03 (t, J=8.7 Hz, 2H), 6.41 (s, 1H), 6.41 Hz, 1H), 4.46 (s, 2H), 3.94 (d, J=4.5 Hz, 2H), 1.46 (s, 18H); LC-
(d, J=8.7 Hz, 2H), 4.92 (s, 1H), 4.85 (s, 1H), 3.88 (s, 2H), 3.78 MS m/z (%): 494.3 (100) [M+Na]+.
(s, 3H), 3.77 (s, 3H), 1.77 (s, 3H); LC-MS m/z (%): 424.2 (100)
[M+Na]+.
Preparation of HN21
To a solution of 7 (1.0 g, 4.32 mmol) in 1,4-dioxane (30 mL)
1
HN42: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.7, 5.4 Hz, cooled in an ice bath, was added DIPEA (836 mg, 6.48 mmol).
2H), 7.06 (d, J=9.0 Hz, 1H), 6.99 (t, J=8.7 Hz, 2H), 6.95 (s, 1H), Ethyl chloroformate (703 mg, 6.48 mmol) was added dropwise
6.36–6.41 (m, 2H), 4.88 (s, 1H), 4.81 (s, 1H), 4.50 (s, 2H), 4.04–4.12 as a solution in 1,4-dioxane (20 mL) and the mixture was stir-
(m, 4H), 3.84 (s, 2H), 1.73 (s, 3H), 1.63–1.69 (m, 4H), 0.91–0.99 red for 1 h. The mixture was extracted with EtOAc (3×30 mL).
(m, 6H); m/z (%): 480.2 (100) [M+Na]+.
The combined organic phases were dried over sodium sulfate
and concentrated in vacuo. Purification (5:1 petroleum ether:
1
HN43: H NMR (300 MHz, CDCl3): δ 7.16 (dd, J=8.7, 5.4 Hz, EtOAc, v/v) by silica gel column chromatography gave HN21
2H), 7.07 (d, J=9.0 Hz, 1H), 6.99 (t, J=8.7 Hz, 2H), 6.39 (s, 1H), (1.13 g, yield: 68%). 1H NMR (300 MHz, CDCl3): δ 7.31 (dd,
6.39 (dd, J=8.7, 2.4 Hz, 2H), 5.90–5.97 ( m, 2H), 5.34 (d, J=17.1 J=8.1, 5.7 Hz, 2H), 7.08 (brs, 2H), 6.95 (t, J=8.7 Hz, 2H), 6.44
Hz, 2H), 5.25 (d, J=10.2 Hz, 2H), 4.88 (s, 1H), 4.81 (s, 1H), (brs, 1H), 6.28 (d, J=6.6 Hz, 1H), 4.24 (s, 2H), 4.06–4.16 (m, 5H),
4.61–4.68 (m, 4H), 4.51 (s, 2H), 3.84 (s, 2H), 1.73 (s, 3H); LC-MS 0.87–0.97 (m, 6H); LC-MS m/z (%): 398.2 (100) [M+Na]+.
m/z (%): 476.2 (100) [M+Na]+.
Preparation of HN31-HN35
1
HN44: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.4, 5.7 Hz, To a solution of HN21 (75 mg, 0.20 mmol) in DCM (2 mL) was
2H), 7.06 (d, J=9.0 Hz, 1H), 6.99 (t, J=8.7 Hz, 2H), 6.86 (s, 1H), added formaldehyde (75 μL, 40% in water, w/w), one drop of
6.38 (s, 1H), 6.38 (dd, J=8.7, 2.4 Hz, 2H), 4.94–4.99 (m, J=6.3 Hz, acetic acid and sodium triacetoxyborohydride (43 mg, 0.20
2H), 4.88 (s, 1H), 4.81 (s, 1H), 4.51 (s, 2H), 3.88–3.93 (m, 6H), 3.84 mmol). The mixture was stirred at room temperature for 10 h,
(s, 2H), 1.91–1.97 (m, 2H), 0.93 (d, J=6.9 Hz, 6H); LC-MS m/z (%): then quenched with saturated aqueous K2CO3 (3 mL). The
508.3 (100) [M+Na]+.
mixture was extracted with EtOAc (3×10 mL). The combined
organic phases were washed with water and brine, dried over
1
HN45: H NMR (300 MHz, CDCl3): δ 7.16 (dd, J=7.5, 5.7 Hz, sodium sulfate and concentrated in vacuo. Purification (4:1
2H), 7.06 (d, J=8.7 Hz, 1H), 6.98 (t, J=8.4 Hz, 2H), 6.91 (brs, 1H), to 2:1 petroleum ether: EtOAc, v/v) by silica gel column chro-
6.74 (s, 1H), 6.37 (s, 1H), 6.37 (d, J=8.7 Hz, 2H), 4.87 (s, 1H), 4.80 matography gave HN31 (47.6 mg, yield: 61%). 1H NMR (300
(s, 1H), 4.49 (s, 2H), 3.83 (s, 2H), 1.72 (s, 3H), 1.48 (s, 18H); LC- MHz, CDCl3): δ 7.18 (dd, J=8.1, 5.4 Hz, 2H), 7.08 (t, J=8.4 Hz,
MS m/z (%): 508.3 (100) [M+Na]+.
1H), 6.99 (d, J=8.7 Hz, 2H), 6.45 (d, J=8.7 Hz, 1H), 6.40 (brs,
1H), 4.47 (s, 2H), 4.17–4.24 (m, 4H), 3.98 (s, 3H), 3.59 (brs, 2H),
HN46: H NMR (300 MHz, CDCl3): δ 8.69 (brs, 2H), 7.58 (s, 1.28 (t, J=6.9 Hz, 6H); LC-MS m/z (%): 412.2 (100) [M+Na]+.
1H), 7.46 (d, 1H), 7.40 (d, J=9.0, 8.7 Hz, 1H), 7.18 (dd, J=8.1, 5.4
1
Hz, 2H), 5.74–5.83 (m, 1H), 5.37 (d, J=5.7 Hz, 1H), 5.33 (s, 1H), Similar methods were used to synthesize of compounds HN32-
4.58 (s, 2H), 4.15 (d, J=5.7 Hz, 2H), 3.79 (s, 6H); LC-MS m/z (%): HN35
1
410.2 (100) [M+Na]+.
HN32: H NMR (300 MHz, CDCl3): δ 7.18 (dd, J=8.7, 5.7 Hz,
2H), 7.11 (s, 1H), 7.02 (d, J=8.4 Hz, 2H), 6.99 (t, J=8.7 Hz, 2H),
1
HN47: H NMR (300 MHz, CDCl3): δ 7.19 (dd, J=8.4, 5.4 Hz, 6.38 (dd, J=9.0, 2.7 Hz, 1H), 6.37 (brs, 1H), 4.45 (s, 2H), 4.16–4.23
2H), 6.95–7.08 (m, 5H), 6.39–6.43 (m, 2H), 5.79–5.91 (m, 1H), 5.20 (m, 4H), 3.42 (q, J=7.2 Hz, 2H), 1.28 (t, J=6.9 Hz, 6H), 1.18 (t,
(s, 1H), 5.14 (d, J=3.0 Hz, 1H), 4.48 (s, 2H), 4.08–4.13 (m, 4H), J=7.2 Hz, 3H); LC-MS m/z (%): 426.2 (100) [M+Na]+.
3.96 (d, J=4.5 Hz, 2H), 1.65–1.72 (m, 4H), 0.95 (t, J=7.5 Hz, 6H);
1
LC-MS m/z (%): 466.2 (100) [M+Na]+.
HN33: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.1, 5.4 Hz,
2H), 7.02–7.05 (m, 2H), 6.95 (t, J=8.4 Hz, 2H), 6.34–6.38 (m, 2H),
1
HN48: H NMR (300 MHz, CDCl3): δ 7.34 (brs, 2H), 6.80–7.24 4.48 (s, 2H), 4.15–4.23 (m, 4H), 3.31 (t, J=7.2 Hz, 2H), 1.65 (m,
(m, 6H), 6.87 (d, J=8.7 Hz, 1H), 5.85–5.98 (m, 3H), 5.10–5.38 (m, 2H), 1.28 (t, J=7.2 Hz, 6H), 0.92 (t, J=7.2 Hz, 3H); LC-MS m/z (%):
6H), 4.45–4.71 (m, 4H), 4.50 (s, 2H), 4.03 (d, J=4.2 Hz, 2H); LC- 440.2 (100) [M+Na]+.
MS m/z (%): 462.2 (100) [M+Na]+.
1
HN34: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.4, 5.4 Hz,
1
HN49: H NMR (300 MHz, CDCl3): δ 7.28 (brs, 2H), 7.18 (dd, 2H), 6.94–7.06 (m, 5H), 6.35–6.38 (m, 2H), 4.47 (s, 2H), 4.15–4.23
J=8.4, 5.4 Hz, 2H), 7.10 (s, 1H), 6.62 (s, 1H), 6.63 (d, J=7.2 Hz, (m, 4H), 3.42 (t, J=7.2Hz, 2H), 1.56–1.66 (m, 2H), 1.33–1.45 (m,
1H), 5.79–5.83 (m, 1H), 5.26 (s, 1H), 5.22 (d, J=3.0 Hz, 1H), 4.52 8H), 0.93 (t, J=7.2 Hz, 3H); LC-MS m/z (%): 454.2 (100) [M+Na]+.
(s, 2H), 4.04 (d, J=5.1 Hz, 2H), 3.90–3.96 (m, 4H), 1.93–1.99 (m,
1
2H), 0.85–0.89 (m, 12H); LC-MS m/z (%): 466.2 (100) [M+Na]+.
HN35: H NMR (300 MHz, CDCl3): δ 7.17 (dd, J=8.7, 5.4 Hz,
2H), 6.94–7.06 (m, 5H), 6.35–6.39 (m, 2H), 4.47 (s, 2H), 4.15–4.23
1
HN410: H NMR (300 MHz, CDCl3): δ 7.18 (dd, J=8.1, 5.7 Hz, (m, 4H), 3.33 (t, J=7.2 Hz, 2H), 1.57–1.68 (m, 2H), 1.28–1.37
2H), 7.07 (d, J=8.7 Hz, 1H), 6.98 (t, J=8.7 Hz, 2H), 6.73 (s, 1H), (m, 10H), 0.89 (t, J=7.2 Hz, 3H); LC-MS m/z (%): 468.2 (100)
6.40 (d, J=6.6 Hz, 1H), 6.29 (brs, 1H), 5.18 (s, 1H), 5.13 (d, J=5.4 [M+Na]+.
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Scheme 2. Synthesis of HN311.
Preparation of HN311 (Scheme 2)
research; Hai-ning HU, Fei CHEN, and Ping-zheng ZHOU
Reagents and conditions: (i) Methyl vinyl ketone, Et3N, MeOH, performed the research; all authors analyzed data; Hai-ning
reflux; (j) Methyltriphenylphosphonium bromide, n-BuLi, HU and Ping-zheng ZHOU wrote the paper.
THF, -78°C;
To a solution of HN21 (150 mg, 0.40 mmol) in MeOH (5 mL),
was added methyl vinyl ketone (MVK) (49 μL, 0.6 mmol) and
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Acknowledgements
The project was supported by National Science and Techno-
logy Major Project on “Key New Drug Creation and Manuf-
acturing Program” (2013ZX09103001-016), National Key Basic
Research Program of China (2013CB910604), and National
Natural Science Foundation of China for Excellent Key Labo-
ratory (81123004).
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Author contribution
Fa-jun NAN, Zhao-bing GAO, and Min LI designed the
Acta Pharmacologica Sinica