Heterodimers of histidine and amantadine as inhibitors for wild type and mutant M2 channels of influenza A

Article abstract of DOI:10.1002/cjoc.201090242

Inhibitors bearing the imidazole, adamantane and related structures were synthesized and tested against WT, S31N and S31N-L26I mutant M2 channels. Although amantadine (1) only inhibited WT M2 channel, compound 6 containing the imidazole and adamantane groups showed good inhibitory activity to WT and mutant M2 channels. The stereochemistry and basic pK _a of α-amine are important for the activity of inhibitors and our data showed that derivatives of natural histidine are more active for M2 channels than those of unnatural histidine. The significance of our present results is that we have established a prospective strategy of drug discovery of WT and mutant M2 channels against influenza A.

Full text of DOI:10.1002/cjoc.201090242

FULL PAPER
Heterodimers of Histidine and Amantadine as Inhibitors for
Wild Type and Mutant M2 Channels of Influenza A
Zhang, Wenjuana,b(张文娟)
Jie, Yanlinga(揭艳玲)
Xu, Jinga(徐静)
Liu, Fanga,c(刘芳)
Li, Chufanga(李楚芳)
Chen, Shaopenga(陈少鹏)
Li, Zhiyuana(李志远)
Liu, Jinsonga(刘劲松)
Chen, Linga(陈凌)
Zhou, Guochun*,a,b(周国春)
a Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510663,
China
b Graduate School of Chinese Academy of Sciences, Beijing 100039, China
c School of Life Science, Lanzhou University, Lanzhou, Gansu 730000, China
Inhibitors bearing the imidazole, adamantane and related structures were synthesized and tested against WT,
S31N and S31N-L26I mutant M2 channels. Although amantadine (1) only inhibited WT M2 channel, compound 6
containing the imidazole and adamantane groups showed good inhibitory activity to WT and mutant M2 channels.
The stereochemistry and basic pKa of α-amine are important for the activity of inhibitors and our data showed that
derivatives of natural histidine are more active for M2 channels than those of unnatural histidine. The significance
of our present results is that we have established a prospective strategy of drug discovery of WT and mutant M2
channels against influenza A.
Keywords M2 ion channel, imidazole, histidine, amantadine, inhibitor, amination
Introduction
ing the conformation change of the channel. The influx
of H＋ ions triggers the fusion of viral and endosomal
membranes and then to release the viral genes into the
cytoplasm. Amantadine (Symmetrel, 1) and Riman-
tadine (Flumadine, 2) (Figure 1) are good uncoating
blockers of M2 channel used for the prophylaxis and
therapy of influenza A infections in clinic for decades.4
General assumption for their inhibition3,5 is that entering
1 or bound 1 catches influent proton and inhibits the
proton relay function of imidazole group of His37
or/and the gating function of indole group of Trp41.
Serious concern to avian flu H5N1, which has the
potential to infect human beings to bring out a
life-threatening pandemic of influenza A, promotes in-
ternational actions like worldwide surveillance of the
widespread disease and global cooperative development
of vaccines and drugs. Although several potential drug
targets could be used for drug discovery and develop-
ment to control influenza A virus, current effective drugs
against flu A in clinic are only targeting neuraminidase
(NA) and M2 ion channel.
M2 proton-selective channel is one of the major pro-
teins that are essential for the life cycle of flu virus A
and exists as disulfide-linked homotetramers anchored
in the viral lipid envelope. As an integral membrane
protein containing 97-amino-acids, a small N-terminal
ectodomain, a single transmembrane (TM) domain, and
C-terminal cytoplasmic tail, M2 ion channel opens to
permit proton permeation from its N-terminal (the en-
dosome) to C-terminal when pH is lower than 6.2.1 The
function of M2 ion channel is associated with two cas-
cade events:2,3 bound at the H＋ associative site by its
“ligand” H＋ and then making the channel responsive
sites open for H＋ entering by its conformation changes.
The H＋ associative site is for anchoring and relay of
protons and the channel responsive sites are for deliver-
Figure 1 Structures of M2 channel effectors.
In addition to the occurrence of central nervous sys-
tem (CNS) side effects of 1 and 2, the urgent and serious
challenge for 1 and 2 in clinical use is the rapid emer-
gence of drug resistance and the ready transmissibility
of drug-resistant viruses, especially in H3N2 (higher
than 90% worldwide)6,7 and H5N1 (higher than 90% in
Vietnam and Thailand).8 The mutations of S31N and
L26I-S31N are the most common mutations of M2
*
E-mail: springzhou@yahoo.com
Received January 19, 2010; revised March 22, 2010; accepted May 6, 2010.
Project supported by Guangdong Provincial Natural Science Foundation of China (Team 06200872), National Basic Research Program of China (No.
2005CB523008), National and Guangdong Provincial United Natural Science Foundation of China (No. U0632001).
Chin. J. Chem. 2010, 28, 1417— 1423
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Zhang et al.
FULL PAPER
channel, which can make these mutant viruses 3000-fold
less responsive to 1 and 2 than M2 wild type (WT) vi-
rus.7,9,10 These drawbacks make 1 and 2 almost useless
as single agent in the treatment of avian or human in-
fluenza A virus infections. It is imperative to develop
alternative anti-flu drugs targeting WT and mutant M2
channels for the prophylaxis and treatment of seasonal
or pandemic influenza in the future.
by Raney Ni and generate amines 22 and 23, respec-
tively.
Scheme 1 Synthesis of the compounds 6— 15
The recent data11-13 showed that the binding sites of 1
in the co-crystal and NMR structures are different. Al-
though we cannot conclude right now that two or more
molecules of 1 bind at different binding sites of an intact
M2 channel at the same time, it is true that there are two
or more potential recognition sites for the interaction of
1 with M2 channel. Considering these, we proposed that
it could have a chance to inhibit WT and also mutant
M2 channels by tuning of the basic structure of the
known M2 effectors to make simultaneous bindings of
two or more recognition sites that are the common fea-
tures of M2 channels.
As the first try of our medicinal chemistry efforts to
drug discovery of M2 channel inhibitors, we designed
and synthesized some compounds containing two or
more potential binding groups so that they can interact
with at least two recognition sites of M2 channels at the
same time. Because 1, 2 and their analogues are the ac-
tive inhibitors for WT M2 channel,14 the adamantine
group of 1 and 2 was logically chosen as one class of
potential blocking moiety for the first series of inhibitors
against WT and mutant M2 channels. On the other hand,
imidazole (4, Figure 1) was used as another class of po-
tential M2 channel blocking moiety because it could be
accommodated to interact with M2 channel according to
the observation that imidazole can rescue fully or partly
the M2 channels’ proton-selective activity of single mu-
tations of H37 (H37G, H37S, H37T) and double muta-
tions of H37/W41 (H37G/W41Y, H37G/W41A).15 It is
expected that these compounds with adamantane, imi-
dazole or their related structural units can inhibit WT
and mutant M2 channels by the interaction with M2
channels’ common properties.
Scheme 2 Synthesis of the compounds 16— 23
Results and discussion
The synthetic routes of the compounds are shown in
Schemes 1 and 2. The reductive amination of adaman-
tane aldehyde (5)16 or cyclopropyl aldehyde with L- or
D-amino acid methyl esters produced compounds 6—
917 and 14 by NaCNBH3, and with L- or D-amino acid
gave acid compounds 10—13 through the hydrogena-
tion catalyzed by Pd/C. Amide 15 was formed by the
condensation of L-histidine methyl ester with
1-adamantanecarboxylic acid by the general method.
Amides 16 and 17 were prepared from Boc-protected
L-histidine and D-histidine by the similar method used
for the preparation of 15. After the deprotection of 16
and 17, and the treatment of 18 and 19 with Lawesson’s
reagent, thioamides 20 and 21 were proceeded to reduce
As expected that 1 is a positive compound to WT M2
channel, 1 inhibited WT M2 channel but did not show
obvious inhibitory activity to S31N and S31N-L26I
mutant M2 channels up to 1.0 mmol/L (Figure 2a). Al-
though 1 can not block mutant M2 channels, compounds
6 and 7 bearing adamantane and imidazole groups
showed good inhibitory activity to mutant and WT M2
channels (Figure 2b). The significant activity difference
between the active compounds 6 and 7 and the inactive
compounds 8 and 9 demonstrated that the imidazole
group is a necessary moiety for constructing the block-
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© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Heterodimers of Histidine and Amantadine as Inhibitors
ing activity to WT and mutant M2 channels. Both of
histidine enantiomers were used for the study and natu-
ral histidine derivative 6 possesses 3 times better in-
hibitory activity to WT M2 channel and 10-fold better
inhibitory activity to mutant M2 channel than unnatural
histidine derivative 7; the activity difference of enanti-
omers 18 and 19 follows to that of 6 and 7. These results
demonstrated that M2 channels can distinguish the en-
antiomers and the stereo-configurations of these inhibi-
tors are important for their recognition and inhibition to
M2 channels, especially more distinct for mutant M2
channels with a larger residue of Asn31 than WT M2
channel with a smaller residue of Ser31. Contrary to the
esters 6 and 7, their corresponding acids 10 and 11 ex-
hibited almost no activity to M2 channels; this may be
due to their zwitterion forms that can obstruct 10 and 11
from entering into relatively hydrophobic M2 channels.
the imidazole group conjugated with very small moiety
of a cyclopropane skeleton was not effective for the
binding to WT and mutant M2 channels but it is possible
to find potent WT and mutant M2 inhibitors by modifi-
cations with relatively bulky skeletons at similar posi-
tion(s). Comparison of 14 (a conjugate of an imidazole
group with a cyclopropane group) with 8 and 9 (conju-
gates of an adamantane group with a phenyl group and
an indole group, respectively), it seems that the imida-
zole group is relatively more important and efficient
than the adamantane group for the binding to M2 chan-
nels. The inhibitory activity of compound 18 is more
obvious to WT M2 channel than to mutant M2 channels.
The inhibitory difference between active amine 6, less
active compound 18 and inactive amide 15 implies that
a basic pKa and a bulky linkage of exact nitrogen con-
figuration are essential for interacting with M2 channels.
In order to know if more amine groups at the aman-
tadine terminus are helpful to binding, we explored to
synthesize compounds 22 and 23 to compare with their
amide forms of 18 and 19. Even though the orientation
of secondary amines with the adamantane group in 6, 19
and 23 is same while that in 7, 18 and 22 is same, the
data showed that only amide 18 with same orientation of
protonable nitrogen as 6 has weak activity while amide
19, diamines 22 and 23 are totally inactive. One reason
is that two protonable amine groups in diamines 22 and
23 possibly made complicated recognition with M2
channels and produced too weak entry ability to rela-
tively hydrophobic M2 channels. In the other aspects,
the suitable bulkiness of the amine and the ideal dis-
tance between the imidazole group and the bulky amine
moiety are indispensable for an inhibitor’s binding ac-
tivity to M2 channels.
Conclusions
In summary, by the tuning of essential molecular
moieties which can potentially interact with different
binding sites in an M2 channel, the first generation of
inhibitors active for WT and mutant M2 channels were
synthesized and identified. From current preliminary
structure-activity relationship (SAR) data, the com-
pounds derived from natural histidine have better activ-
ity against M2 ion channels. Many other factors, in-
cluding suitable blocking moieties and their suitable
distance, feasibly protonated amine, and good entry
ability to M2 channels, may also affect the recognition
and inhibition of inhibitors to M2 channels. This new
line of inhibitors could act in a different mechanism
from amantadine (1) in that they possibly interact with
at least two recognition sites of WT and mutant M2
channels. Although the activity of current inhibitors is
not potent enough yet, the success in the discovery of
active heterodimeric structures of 6 and 7 with the imi-
dazole group and the adamantane group paved a poten-
tial way for drug discovery of WT and mutant M2
channels. Discovering more potent inhibitors is possible
Figure 2 The inhibition of M2 ion channels by amantadine 1 (a)
and compound 6 (b). Membrane currents of 293T-rex cells that
expressed M2-WT, S31N and L26I-S31N with extracellular solu-
tion pH changing from 6.8 to 5.5, holding potential at － 40 mV.
The potency of the compound was estimated by constructing a
dose-response relationship based on electrophysiological re-
cordings (n＝ 4— 6). In all cases, the compounds were pre-applied
for ＞ 30 s before being co-applied with the agonist (pH 5.5). A
logistic curve fit to the data yielded an IC50 which indicated at
Table 1.
The fact that compound 14 expressed only a weak
inhibition to WT and mutant M2 channels showed that
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Zhang et al.
FULL PAPER
after more detailed understanding of the struc-
ture-activity relationship (SAR) is acquired. We are cur-
rently pursuing more compounds for M2 inhibitors
bearing imidazole, adamantane, and other bulky skele-
tons like “cage-like” scaffold moiety14 including the
skeleton of BL-1743 (Figure 1) and related structures.
Hz, 1H), 7.16—7.20 (m, 1H), 7.10—7.14 (m, 1H), 7.04
(s, 1H), 3.63 (s, 3H), 3.56 (t, J＝6.4 Hz, 1H), 3.10—
3.20 (m, 1H), 2.31 (d, J＝11.2 Hz, 1H), 2.07—2.18 (m,
1H), 1.88—1.95 (m, 3H), 1.59—1.74 (m, 6H), 1.43—
1.48 (m, 6H); 13C NMR (CDCl3, 100 MHz) δ: 175.8,
136.2, 127.6, 122.8, 121.9, 119.3, 118.9, 111.6, 111.1,
63.4, 61.0, 51.5, 40.7 (3C), 37.2 (3C), 33.6, 29.2, 28.5
(3C); ESIMS m/z (%): 367.0 ([M＋H]＋, 100).
Experimental
N-(1-Adamantylmethyl)-L-phenylalanine methyl
ester17 (9) Yield 25%; 1H NMR (CDCl3, 400 MHz) δ:
7.21—7.32 (m, 5H), 3.66 (s, 3H), 3.44 (t, J＝6.8 Hz,
1H), 2.90—2.98 (m, 2H), 2.29 (d, J＝11.2 Hz, 1H), 2.07
(d, J＝11.2 Hz, 1H), 1.92—1.98 (m, 3H), 1.60—1.74 (m,
6H), 1.44—1.49 (m, 6H); 13C NMR (CDCl3, 100 MHz)
δ: 175.4, 137.7, 129.2 (2C), 128.3 (2C), 126.6, 64.2,
60.8, 51.5, 40.6 (3C), 39.6, 37.2＋(3C), 33.55, 28.4 (3C);
ESIMS m/z (%): 328.2 ([M＋H] , 100).
Materials and instruments
1H and 13C NMR spectra were recorded on a Bruker
AV-400 spectrometer at 400 and 100 MHz. Coupling
constants (J) are expressed in hertz (Hz). Chemical
shifts (δ) of NMR are reported in parts per million units
relative to the solvent. The low resolution of EIMS and
high resolution of FABMS were recorded on a VG
ZAB-HS mass spectrometer and a MAT 95XP (Thermo)
mass spectrometer, respectively. The low resolution of
ESIMS was recorded on an Agilent 1200 HPLC-MSD
mass spectrometer. All the reagents were of analytical
reagent grade.
General procedures of the reductive amination of
aldehyde 5 and amino acids by Pd/C hydrogenation
The mixture of 0.5 mmol of amino acid hydrochloric
salt and 0.6 mmol of 1-adamantadine aldehyde 516 in 5
mL of MeOH was treated with 1.0 equiv. of Et3N and
then stirred at r.t. for 5 h. After the careful evacuation of
air and the addition of catalytic amount of Pd/C, the re-
action mixture was charged with H2 at one atmosphere.
The reaction progress was checked by TLC and the re-
action was generally completed after stirred at room
temperature for 8 h. After filtered and evaporated in
vacuum, the residue was purified by silica gel column
chromatography (MeOH in DCM, 5% to 15% in volume)
to give the title compound.
General procedures of the reductive amination of an
aldehyde and amino acid methyl esters by NaCNBH3
After the mixture of 0.53 mmol of methyl ester of an
amino acid and 0.65 mmol of an aldehyde in 5 mL of
MeOH was stirred at room temperature for 1.5 h, 0.07 g
(1.11 mmol) of NaCNBH3 was added to the mixture at 0
℃. The reaction progress was checked by TLC and the
completed reaction was treated with sat. NaHCO3, ex-
tracted with CH2Cl2 and then dried over anhydrous
MgSO4. After evaporated in vacuum and purified by
silica gel column chromatography (MeOH in CH2Cl2,
0% to 2% in volume), the title compound was prepared.
N-(1-Adamantylmethyl)-L-histidine methyl ester
(6) Yield 31%; 1H NMR (CDCl3, 400 MHz) δ: 7.50 (s,
1H), 6.78 (s, 1H), 3.65 (s, 3H), 3.37—3.41 (m, 1H),
2.96 (dd, J＝5.8, 15.0 Hz, 1H), 2.82 (dd, J＝8.4, 6.4 Hz,
1H), 2.24—2.28 (m, 1H), 2.03—2.07 (m, 1H), 1.90—
1.97 (m, 3H), 1.55—1.70 (m, 6H), 1.40—1.50 (m, 6H);
13C NMR (CDCl3, 100 MHz) δ: 174.9, 134.6, 131.6,
120.2, 62.4, 60.84, 51.8, 40.7 (3C), 37.1 (3C), 33.4, 29.5,
28.4 (3C); HRFABMS calcd for C18H28O2N3 318.2176,
found 318.2166 [M＋H]＋ (100).
N-(1-Adamantylmethyl)-L-histidine (10)
Yield
53%; 1H NMR (CD3OD, 400 MHz) δ: 7.66 (s, 1H), 7.00
(s, 1H), 3.59—3.63 (m, 1H), 3.20—3.30 (m, 1H), 2.29
—3.07 (m, 1H), 2.83 (d, J＝12.4 Hz, 1H), 2.62 (d, J＝
12.4 Hz, 1H), 1.95—2.06 (m, 3H), 1.67—1.75 (m, 9H);
13C NMR (CD3OD, 100 MHz) δ: 171.3, 135.7, 134.9,
114.2, 64.4, 58.6, 39.3 (3C), 36.2 (3C＋), 32.1, 28.1 (3C),
26.5; ESIMS m/z (%): 304.0 ([M＋H] , 100).
N-(1-Adamantylmethyl)-D-histidine (11)
Yield
50%; 1H NMR (CD3OD, 400 MHz) δ: 7.68 (s, 1H), 7.02
(s, 1H), 3.60—3.66 (m, 1H), 2.29 (dd, J＝3.6, 12.0 Hz,
1H), 3.05 (dd, J＝6.0, 9.6 Hz, 1H), 2.86 (d, J＝12.4 Hz,
1H), 2.65 (d, J＝12.4 Hz, 1H), 2.06—2.10 (m, 3H), 1.78
—1.93 (m, 6H), 1.69—1.73 (m, 6H); 13C NMR (CD3OD,
100 MHz) δ: 171.2, 135.7, 134.9, 114.1, 64.4, 58.6, 39.3
(3C), 36.2 (3C), 32.1, 28.1 (3C), 26.5; ESIMS m/z (%):
304.0 ([M＋H]＋, 100).
N-(1-Adamantylmethyl)-D-histidine methyl ester
(7) Yield 40%; 1H NMR (CDCl3, 400 MHz) δ: 7.53 (s,
1H), 6.82 (s, 1H), 3.71 (s, 3H), 3.39 (dd, J＝4.0, 4.4 Hz,
1H), 3.01 (dd, J＝4.0, 10.8 Hz, 1H), 2.80 (dd, J＝6.8,
8.4 Hz, 1H), 2.32 (d, J＝11.2 Hz, 1H), 2.10 (d, J＝11.2
Hz, 1H), 1.95—1.99 (m, 3H), 1.65—1.75 (m, 3H), 1.62
—1.68 (m, 3H), 1.49—1.60 (m, 6H); 13C NMR (CDCl3,
100 MHz) δ: 174.6, 134.4, 130.3, 122.1, 62.1, 60.9, 52.0,
40.8 (3C), 37.1 (3C), 33.4, 28.7, 28.4 (3C); HREIMS
calcd for C18H27O2N3 317.2098, found 317.2098 [M]＋
(100).
N-(1-Adamantylmethyl)-L-tryptophan
(12)
1
Yield 51%; H NMR (CD3OD, 400 MHz) δ: 7.73 (d, J
＝8.0 Hz, 1H), 7.43 (d, J＝8.0 Hz, 1H), 7.29 (s, 1H),
7.17—7.21 (m, 1H), 7.10—7.14 (m, 1H), 3.81—3.85 (m,
1H), 3.63 (dd, J＝5.2, 10.0 Hz, 1H), 3.27 (dd, J＝6.0,
9.2 Hz, 1H), 2.67 (d, J＝12.4 Hz, 1H), 2.42 (d, J＝12.4
Hz, 1H), 1.92—1.96 (m, 3H), 1.72—1.77 (m, 3H), 1.58
— 1.65 (m, 3H), 1.39 — 1.414 (m, 6H); 13C NMR
(CD3OD＋CDCl3, 100 MHz) δ: 171.2, 136.8, 126.6,
N-(1-Adamantylmethyl)-L-tryptophan
methyl
1
ester (8) Yield 30%; H NMR (CDCl3, 400 MHz) δ:
8.32 (brs, 1H), 7.64 (d, J＝7.6 Hz, 1H), 7.33 (d, J＝8.0
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Heterodimers of Histidine and Amantadine as Inhibitors
124.1, 122.2, 119.6, 117.9, 111.7, 108.0, 64.2, 59.2, 39.0
(3C), 36.1 (3C), 32.0, 27.6 (3C), 26.2; ESIMS m/z (%):
353.2 ([M＋H]＋, 100).
J＝6.0, 8.8 Hz, 1H), 1.97—2.05 (m, 3H), 1.85—1.89 (m,
6H), 1.58—1.60 (m, 6H), 1.43 (s, 9H).
Deprotection of 16 and 17
N-(1-Adamantylmethyl)-L-phenylalanine
(13)
Yield 57%; 1H NMR (CD3OD, 400 MHz) δ: 7.28—7.38
(m, 5H), 3.71 (t, J＝7.2 Hz, 1H), 3.34—3.38 (m, 1H),
3.12 (dd, J＝6.8, 7.4 Hz, 1H), 2.58 (dd, J＝12.4, 27.2
Hz, 2H), 1.98—2.05 (m, 3H), 1.68—1.86 (m, 6H), 1.53
—1.60 (m, 6H); 13C NMR (CD3OD＋CDCl3, 100 MHz)
δ: 170.7, 135.9, 129.1 (2C), 129.0 (2C), 65.2, 59.4, 39.2
(3C), 36.2 (3C), 35.7, 32.2, 27.7 (3C); ESIMS m/z (%):
314.2 ([M＋H]＋, 100).
The solution of 0.9 g (1.84 mmol) of compound 16
or 17 in 20 mL of DCM was treated with 1.0 mL of
trifluoroacetic acid (TFA) from 0 ℃ to room tempera-
ture for 5 h. After the removal of volatile components,
the residue was purified by silica gel column chroma-
tography (MeOH in DCM, 6.3% in volume with 1%
concentrated NH4OH) to provide the compound 18 or
19.
N-(1-Adamantyl)-L-histidine amide (18) Yield
93%; 1H NMR (CD3OD, 400 MHz) δ: 7.57 (s, 1H), 6.82
(s, 1H), 3.43 (dd, J＝1.6, 5.6 Hz, 1H), 2.88 (dd, J＝5.6,
8.8 Hz, 1H), 2.71 (dd, J＝6.8, 7.6 Hz, 1H), 1.90—1.98
(m, 3H), 1.85—1.94 (m, 6H), 1.58—1.68 (m, 6H); 13C
NMR (CDCl3, 100 MHz): δ: 174.2, 134.9, 133.4, 117.8,
55.3, 51.1, 43.5, 40.9, 36.1, 35.7, 32.7, 29.5; HRFABMS
calcd for C16H25O1N4 289.2023, found 289.2014 [M]＋
(100).
N-(1-Cyclopropylmethyl)-L-histidine methyl ester
(14)
14 was got by similar procedure as the
1
preparation of compounds 6—9: Yield 35%; H NMR
(CDCl3, 400 MHz) δ: 7.54 (s, 1H), 6.83 (s, 1H), 3.71 (s,
3H), 3.53—3.56 (m, 1H), 3.02 (dd, J＝4.4, 10.8 Hz, 1H),
2.84 (dd, J＝6.8, 8.4 Hz, 1H), 2.53 (dd, J＝5.6, 6.4 Hz,
1H), 2.36 (dd, J＝4.4, 7.2 Hz, 1H), 0.89—0.95 (m, 1H),
0.47—0.50 (m, 2H), 0.11—0.16 (m, 2H); 13C NMR
(CDCl3, 100 MHz) δ: 174.7, 134.7, 131.6, 119.9, 61.1,
53.2, 51.9, 29.7, 11.1, 3.52, 3.38; HRFABMS calcd for
C11H18O2N3 224.1394, found 224.1392 [M]＋ (100).
N-(1-Adamantyl)-D-histidine amide (19) Yield
90%; 1H NMR (CD3OD, 400 MHz) δ: 7.54 (s, 1H), 7.21
(s, 1H), 6.80 (s, 1H), 4.66 (brs, 1H), 3.48—3.55 (m, 1H),
2.97 (dd, J＝4.4, 10.4 Hz, 1H), 2.84 (dd, J＝6.8, 7.6 Hz,
1H), 1.98—2.06 (m, 3H), 1.90—1.95 (m, 6H), 1.58—
1.65 (m, 6H); 13C NMR (CDCl3, 100 MHz) δ: 173.7,
135.2, 132.4, 119.5, 55.4, 51.3, 41.4 (3C), 36.＋3 (3C),
32.0, 29.4 (3C); ESIMS m/z (%): 289.4 ([M＋H] , 100).
Coupling reaction for the preparation of 15, 16 and
17
To the suspension of 8.54 mmol of hydrochloride
salt of an amine in 100 mL of dichloromethylene (DCM)
with ice-water cooling bath, 3.0 mL of trimethylamine,
8.45 mmol of an acid, 2.72 g (8.47 mmol) of
Formation of thio-amides 20 and 21 by Lawesson’s
reagent
O-benzotriazole-N,N,N',N'-tetramethyluronium
tetra-
fluoroborate (TBTU) and 1.0 mL of N-methyl mor-
pholine (NMM) were added orderly. After the addition,
the reaction was stirred at room temperature for 18 h
and then treated with sat. NaHCO3, extracted by CH2Cl2,
dried over Na2SO4, evaporated in vacuum and silica gel
column chromatographed (MeOH in CH2Cl2, 1% to 3%
plus 1% concentrated NH4OH in volume for 15, MeOH
in CH2Cl2, 3.3% in volume for 16 or 17) to give the
compound 15, 16 or 17.
0.1 g (0.35 mmol) of compound 18 or 19 and 0.35 g
(0.87 mmol) of Lawesson’s reagent were dissolved in 20
mL of dried benzene. Compound 18 or 19 was almost
converted to thio-amide 20 or 21 after the reaction mix-
ture was refluxed for 20 h. The reaction mixture was
evaporated and purified by silica gel column chroma-
tography to yield the mixture of compounds 18 and 20
or 19 and 21.
N-Adamantylcarboxy-L-histidine methyl ester (15)
Yield 68%; 1H NMR (CDCl3, 400 MHz) δ: 7.49 (s, 1H),
6.72 (s, 1H), 4.62—4.65 (m, 1H), 3.56 (s, 3H), 2.96—
3.10 (m, 2H), 1.93—2.00 (m, 3H), 1.72—1.82 (m, 6H),
1.58—1.68 (m, 6H); 13C NMR (CDCl3, 100 MHz) δ:
178.7, 172.2, 135.4, 134.6, 115.3, 52.5, 52.1, 40.5, 38.9
(3C), 36.＋4 (3C), 29.0, 28.0 (3C); ESIMS m/z (%): 332.0
([M＋H] , 100).
Reduction of thio-amides 20 and 21
To the solution of the mixture 18 and 20 or 19 and 21
from the last step (about 0.35 mmol) in 20 mL of 80%
tert-butanol, 1.0 g of Raney Ni was added and then the
reaction was refluxed for 8 h. After filtered and evapo-
rated, the residue was silica gel column chromatogra-
phed (MeOH in DCM, 20% in volume) to provide the
compound 22 or 23.
N,N'-Di(tert-butylcarbamate)-adamantylamino-
L-histidine amide (16) Yield 87%; HNMR (CDCl3,
1
(2S)-1-[N-(1-Adamantyl)]-3-[(1H-imidazol-4-yl)-
propane-1,2-diamine (22)
Yield 76%; 1H NMR
400 MHz) δ: 8.00 (s, 1H), 7.15 (s, 1H), 6.25 (s, 1H),
5.71 (s, 1H), 4.18 (s, 1H), 2.92—3.00 (m, 1H), 2.72—
2.82 (m, 1H), 2.65—2.70 (m, 1H), 1.92—1.97 (m, 3H),
1.78—1.85 (m, 6H), 1.50—1.60 (m, 6H), 1.40 (s, 9H).
N,N'-Di(tert-butylcarbamate)-adamantylamino-
(CD3OD, 400 MHz) δ: 7.56 (s, 1H), 6.83 (s, 1H), 2.92—
2.99 (m, 1H), 2.53—2.70 (m, 3H), 2.35—2.42 (m, 1H),
1.96—2.06 (m, 3H), 1.57—1.70 (m, 12H); 13C NMR
(CD3OD, 100 MHz) δ: 134.8, 133.6, 117.8, 51.4, 50.3,
45.6, 41.7 (3C), 36.4 (3C), 32.9, 29.62 (3C); HRFABMS
calcd for C16H27N4 275.2230, found 275.2213 [M]＋
(100).
1
D-histidine amide (17) Yield 90%; H NMR (CDCl3,
400 MHz) δ: 8.02 (s, 1H), 7.16 (s, 1H), 6.21 (s, 1H),
5.83 (s, 1H), 4.27 (s, 1H), 2.99—3.05 (m, 1H), 2.87 (dd,
Chin. J. Chem. 2010, 28, 1417— 1423
© 2010 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
1421

Zhang et al.
FULL PAPER
(2R)-1-[N-(1-Adamantyl)]-3-[(1H-imidazol-4-yl)-
propane-1,2-diamine (23)
performed using a commercially available automated
fast solution exchange system (RSC-200 Rapid solution
changer). All data are reported for n number of cells.
Differences in the compound’s inhibition were deter-
mined from statistical tests using IC50 value and a com-
parison between two groups was made using Student’s t
test (Table 1).
Yield 75%; 1H NMR
(CDCl3, 400 MHz) δ: 7.49 (s, 1H), 6.76 (s, 1H), 3.66
(brs, 2H), 2.95—3.06 (m, 1H), 2.75 (dd, J＝4.8, 10.0 Hz,
1H), 2.65 (dd, J＝4.4, 7.2 Hz, 1H), 2.52—2.57 (m, 1H),
2.40—2.45 (m, 1H), 2.00—2.07 (m, 3H), 1.55—1.66 (m,
12H); 13C NMR (CDCl3, 100 MHz) δ: 134.8, 133.1,
119.4, 51.9, 50.6, 46.4, 42.6 (3C), 36.6 (3C), 32.9, 29.5
(3C); ESIMS m/z (%): 275 ([M]＋, 100).
Cytotoxicity of the compounds to the three trans-
formed cell lines
Electrophysiological recordings by patch clamp
The cytotoxicity (CC50) of these compounds to three
transformed cell lines was detected by the general MTT
method to quantify the number of viable cells without
the induction 3 d after the addition of the compound or
the carrier. Briefly, 1000 cells/well were seeded in
96-well plates and allowed to attach for 20 h before the
treatment with a compound in 0.1% DMSO or the same
volume of the carrier. Every concentration of each
compound or the carrier is in triplicate. After incubated
for 72 h, cells were treated with MTT reagents and rela-
tive cell viability of each well was calculated. The data
of CC50 are shown in Table 1.
The stable cell lines of transformed 293T-rex ex-
pressed WT, S31N-L26I and S31N mutant M2 channels
of avian H5N1 were successfully established according
to the literature.18 M2-transformed 293T-rex cells were
used 24—48 h after induction with 1 µg/mL tetracycline.
Perforated whole-cell voltage-clamp recordings were
carried out at room temperature (23—25 ℃) using an
Axopatch 200B amplifier (Axon Instruments Inc., Union
City, CA) as described previously19 (Li et al., 2004).
Drug applications and extracellular pH changes were
Table 1 The IC50 (µmol/L) for compounds estimated on patch-clamp recording the 293T-rex cells expressed M2 WT, mutants of S31N
and L26I/S31N and the CC50 (µmol/L) for compounds detected by general MTT method
WT-M2
S31N-M2
S31N/L26I-M2
IC50 CC50
Compd.
IC50
4.18
5.84
18.9
NA
CC50
200
IC50
NAa
10.96
NDb
ND
CC50
200
1
＞
＞
NA
9.77
112.1
ND
＞
200
400.4
500
6
332.5
463.1
63.9
419.0
500
300.2
399.7
62.6
372.2
500
7
＞
8
59.3
500
9
NA
ND
ND
＞
＞
＞
10
11
12
13
14
15
18
19
22
23
NA
＞
＞
ND
＞
＞
ND
500
NA
500
ND
500
ND
500
NA
336.1
500
ND
371.1
96.1
500
ND
482.5
500
NA
＞
＞
ND
ND
＞
＞
319.0
NA
500
465.0
ND
＞
399.0
ND
500
70.3
428.1
500
80.6
352.0
500
64.5
500
137.2
NA
ND
NA
＞
＞
＞
＞
＞
＞
＞
ND
＞
＞
＞
ND
500
NA
500
NA
500
NA
500
NA
500
NA
500
ND
500
a NA means “IC50＞ 500 µmol/L”; b ND means “not determined yet”.
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www.cjc.wiley-vch.de
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