Synthesis and evaluation of novel neamine derivatives effectively targeting to RNA

Article abstract of DOI:10.1016/j.bmcl.2009.03.021

Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA

Full text of DOI:10.1016/j.bmcl.2009.03.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2103–2106
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of novel neamine derivatives effectively
targeting to RNA
*
Yanli Xu, Hongwei Jin, Zhenjun Yang, Liangren Zhang, Qi Wang, Manning Li, Lihe Zhang
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine
or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of
these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of nea-
mine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some anti-
bacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may
provide a promising way for the development of potential candidates effectively targeting to RNAs.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 26 December 2008
Revised 18 February 2009
Accepted 9 March 2009
Available online 13 March 2009
Keywords:
Neamine derivative
TAR RNA
16S RNA
Molecular modeling
RNA has become an attractive target for drug design due to its
structural complexity and diversity and the important roles played
in the process of life.1,2 The recognition between mRNA and tRNA is
an important step in the process of protein synthesis, and in bacte-
ria the recognition occurs at the A site decoding region of 16S
rRNA.3 Neomycin B (Fig. 1), an effective aminoglycoside antibiotic,
can bind potentially to the A site of 16S RNA and induce miscoding
or inhibition of the bacterial protein synthesis, thus leading to bac-
terial cell death in the end.4 Besides binding to 16S RNA, neomycin
B can also potentially target to some other RNAs including HIV-1
trans-activation response element (TAR) RNA.5 The interaction be-
tween TAR RNA and Tat protein has been known the key step in the
transcription of HIV-1 genome by activating and stabilizing the
synthesis of HIV-1 mRNA.6 Neomycin B can prevent the binding
of Tat protein to TAR RNA thus inhibiting the replication of HIV-1
virus.7 Unfortunately, neomycin B can not be directly used in clinic
mainly because of its nonspecific electrostatic interaction to RNA
in a large part which causes the high toxicity.8 In addition, it is rel-
atively unstable and prone to be modified by aminoglycoside mod-
ifying enzymes (AME) which can lead to drug resistance.9
amino acid modified aminoglycosides has been proved to be an
effective strategy.11,12 Arginine-rich peptide region of HIV Tat
and Rev proteins is the important binding site in the recognition
of the target RNAs.13,14 Many aminoglycoside-arginine conjugates
(AACs) and arginine peptide-aminoglycoside conjugates (APACs)
have been successfully synthesized, including arginine conjugates
of neomycin B, paromomycin, gentamicin and kanamycin A.15–26
These AACs and APACs all display a high binding affinities to HIV
virus RNAs and show potent antiviral activities. Lysine-rich protein
is another one of the HIV RNA binding proteins.13 The mono-lysine
neamine conjugate and hexa-lysine neomycin conjugate also both
showed higher bioactivities compared with their mother com-
pound neamine and neomycin, respectively.27,28,17 Up to now, all
of the reported aminoglycoside conjugates were designed connect-
ing neamine with arginine, arginine peptide or lysine directly to
one of amino groups on the aminosugar moiety. Here, we reported
the synthesis of neamine derivatives, 3 (NE), 6 (NEA) and 9 (NEL),
6'
II
NH2
5'
4'
O
HO
HO
Neamine (Fig. 1), a simplified neomycin B mimic, keeps the
same targeting site as neomycin B and indicates the lower toxicity.
However, compared with neomycin B, it shows only poor antibiotic
activity and can not also be clinically used.10 It appears a promising
way to keep the minimal structural motif and optimize the struc-
ture of neamine. Many efforts have been made to prepare new nea-
mine derivatives potentially targeting to RNAs, among which
H2N
4
1'
2
2'
6'
II
3
NH2
5'
3'
4'
NH2
H2N
O
1
O
I
5
HO
HO
HO
O
5''
H2N
4
6
1'
O
III
OH
2
2'
3
1''
3'
4''
NH2
H2N
O
1
I
2''
3''
6'''
HO
O
6
NH2
OH
5
OH
1'''
5'''
OH
O
IV
3'''
H2N
4'''
HO
2'''
Neomycin B
Neamine
* Corresponding author. Tel.: +86 10 82801700; fax: +86 10 82802724.
E-mail address: zdszlh@bjmu.edu.cn (L. Zhang).
Figure 1. Structures of neomycin B and neamine.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.021

2104
Y. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2103–2106
NH2
N3
O
O
HO
HO
HO
HO
H2N
HN
N3
N3
O
NH2
N3
H2N
O
AcO
AcO
N3
O
N3
N3
OH
N3
O
OH
HO
HN
OAc
1
c
e
a
NH
NH
O
O
H
H
NHR1
N3
R2HN
d
N
NH2
H2N
N
O
HO
HO
N3
NH
NEA
NH
6
N3
4, R1=Fmoc, R2=Pbf
5, R1=H, R2=Pbf
N3
O
OH
2
HN
NH2
O
N3
O
b
HO
HO
HO
N3
H2N
HO
NH2
N3
NH2
H2N
O
N3
O
NH2
O
OH
OH
h
f
HO
HO
H2N
HN
HN
HN
NH2
H2N
O
OH
3
NH
NH
NH2
NE
O
O
NHR3
R4HN
7, R3=Boc, R4=Fmoc
8, R3=H, R4=Fmoc
H2N
NEL
NH2
9
g
Scheme 1. Synthesis of neamine derivatives 3 (NE), 6 (NEA) and 9 (NEL). Reagents and conditions: (a) (i) Tf2O, Py, CH2Cl2; (ii) H2N(CH2)2NH2, rt; (b) H2S, Py/Et3N/H2O (4/3/2),
rt; (c) Fmoc-Arg(Pbf)-OH, HOBt, DCC, DMF, 0 °C to rt; (d) Et2NH/DMF (1:9), rt; (e) (i) CF3COOH/H2O/PhSCH3 (94/3/3); (ii) H2S, Py/Et3N/H2O (4/3/2), rt; (f) Boc-Lys(Fmoc)-OH,
HOBt, DCC, DMF, 0 °C to rt; (g) CF3COOH/CH2Cl2 (1:4), rt; (h) (i) Et2NH/DMF (1:9); (ii) H2S, Py/Et3N/H2O (4/3/2), rt.
in which amino acid modified on the 5-hydroxyl of neamine
G
G
C
G
U
C
A
C
C
C
G
C
U
G
A
G
U
G
G
G 15
(Scheme 1). The amino groups on neamine play an important role
in their binding to the target RNAs. Keeping the amino group free
in the neamine molecule and modification of other positions of
neamine may lead the derivatives contributing a various interac-
tion to RNA. In this report, compounds 3 (NE), 6 (NEA) and 9
(NEL) show the interesting binding properties to the A site of 16S
RNA and TAR RNA and the binding data were discussed by molec-
ular modeling. In addition, compound 9 (NEL) exhibits some anti-
25
5
20
A
A
bacterial activities against Staphylococcus aureus (MIC = 25
and Escherichia coli (MIC = 50 M).
lM)
10
C
C
U
l
Compound 1 and 2 were obtained by the published proce-
dures.29,30 As shown in Scheme 1, the reduction of all of four azido-
groups on compound 2 can directly afford the desired product 3
(NE) in 83% yield. For the synthesis of compounds 6 (NEA) and 9
(NEL), Fmoc-Arg(Pbf)-OH or Boc-Lys(Fmoc)-OH was condensed
with compound 2 in the presence of HOBt/DCC in DMF at room
temperature to obtain the intermediate 4 or 7 in 79% or 74% yield,
respectively. After removal of the protected groups on arginine/ly-
sine moiety, compound 5 and 8 were obtained in 85% and 76%
yields, respectively. As shown in Scheme 1, Fmoc group on arginine
U C
Figure 2. Structure of A site of 16S RNA.
of the Scatchand plot.31,32 The SPR results for neamine and its three
derivatives 3, 6 and 9 binding to the A site of 16S RNA are summa-
rized in Table 1. The KD value of neamine was determined as
M. After modification of an ethylenediamine at 5-hydroxyl of
neamine, compound 3 shows 7
potential in comparison with neamine. Interestingly, compounds 6
and 9 further increase their potential in binding to the A site of 16S
RNA and pose 2 lM and 1 lM of KD values which indicate 3.5 and 7
19
l
l
M of KD value which is 2.7 times
a
-amino group and lysine
Et2NH/DMF (1:9) and the deprotections of Pbf group on arginine
d-guanidino group and the Boc group on lysine -amino group
e-amino group can be removed using
a
can be completed using CF3COOH/H2O/PhSCH3 (94/3/3) and
CF3COOH/CH2Cl2 (1:4), respectively. Compounds 6 (NEA) and 9
(NEL) were obtained in 64% and 71% yield, respectively, by the
reduction of intermediate 5 and 8 by H2S in a mixture of pyridine
and triethylamine. All the new neamine derivatives 3, 6 and 9 were
purified by reversed phase chromatography on C18 column (1‰
CF3COOH in H2O) and obtained as the appropriate trifluoroacetic
salts in the end. The intermediates and final products were charac-
terized by 1H NMR, 13C NMR, HRMS and optical rotations.
times potential in comparison with derivative 3, respectively.
The binding properties of neamine and its derivatives 3, 6 and 9
to TAR RNA (Fig. 3) were also determined by SPR.31,32 The results
Table 1
The KD values of neamine and its derivatives 3, 6 and 9 binding to A site of 16S RNA
Compd
16S RNA (lM)
Neamine
3 (NE)
6 (NEA)
9 (NEL)
19
7
2
The binding properties of three neamine derivatives 3, 6 and 9
to the A site of 16S RNA (Fig. 2) were evaluated by SPR (Surface
Plasmon Resonance) using Biacore 3000 instrument, the dissocia-
1
tion constants (KD value in
lM) were calculated from the slope

Y. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2103–2106
2105
G
G
C
C
A
G
C
C
30
G
G
5 U
C
25
A
U
U
U
C
C
U
G
A 20
G
C
C
C
G
10
A
U
15
G
G
G
Figure 3. Structure of HIV-1 TAR RNA.
are summarized in Table 2. The KD value of neamine is 24
Three neamine derivatives 3, 6 and 9 show more potential binding
activities to TAR RNA in comparison with neamine, the KD value is
l
M.
terminal amino group of ethylenediamine and two novel neamine
derivatives 6 and 9 were synthesized and showed the higher bind-
ing affinities to RNA compared with compound 3. The KD data of
compounds 6 and 9 indicate more interactions between these
compounds with RNA in existence. It is unexpected that the lysine
conjugate 9 shows the stronger binding activity (Table 1 and 2)
compared with the arginine conjugate 6. To obtain more informa-
tion about the interactions between the neamine derivatives 6 and
9 and A site of 16S RNA, molecular modeling study was per-
formed.33 The structure of A site of 16S RNA fragment was ex-
tracted from the protein databank (PDB code 1PBR) and the
software AUTODOCK 3.0 was used for the molecular docking calcula-
tion of three neamine derivatives. By computer calculation, the fi-
nal configuration (Fig. 4a, b, and c) with the lowest docking energy
was obtained and used for the analysis.
14
l
M, 3
lM and 2 lM, respectively.
The SPR data indicate that the ethylenediamine modified nea-
mine 3 (NE) could increase the binding affinity to RNA in compar-
ison with neamine, ethylenediamine in NE plays not only a linker
but a functional group for the binding to RNA. Based on the struc-
ture of compound 3, an arginine or lysine was conjugated to the
Table 2
The KD values of neamine and its derivatives 3, 6 and 9 binding to TAR RNA
Compd
TAR RNA (
l
M)
Neamine
3 (NE)
6 (NEA)
9 (NEL)
24
14
3
The molecular docking study suggests that except the hydrogen
bonds and electrostatic interactions which are formed between the
neamine moiety of compound 3 and 16S RNA, the NH2 group on
2
a
b
c
O18 PO2
NH3
U18 PO2
G17 PO2
G17 PO2
G19
N7
G4 O2P
HO
G17 O2P
G19 PO2
U18 O1P
NH3
O
NH3
O
O
U5 PO2
HO
H3N
HO
HO
HO
G17 PO2
NH3
H3N
H3N
HO
NH3
H3N
O
NH3
H3N
G4 PO2
O
H3N
O
OH
U5 PO2
HN
OH
OH
HN
HN
G4 PO2
NH3
A7 O2P
U5 O2P
NH
NH
NH3
G4 PO2
A
C6 PO2
U5 PO2
O
O
H
NH3
H3N
N
H3N
C6 O2P
NH
C
B
Figure. 4. Molecular modeling for the interactions between compounds 3 (NE) (a, A), 6 (NEA) (b, B), 9 (NEL) (c, C) and A site of 16S RNA. a, b and c show the hydrogen bonds
(dotted line) only and A, B and C indicate the interactions including all the hydrogen bonds and electrostatic interactions.

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Y. Xu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2103–2106
Table 3
Supplementary data
The MIC values (lM/L) of neamine and its three derivatives 3, 6 and 9
Compd
E. coli
S. aureus
P. aeruginos
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.021.
Neamine
3 (NE)
6 (NEA)
9 (NEL)
50
100
100
50
50
>100
50
>100
>100
>100
>100
25
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ethylenediamine side chain of compound 3 can also form an elec-
trostatic interaction with POÀ2 of G4 on the A site (Fig. 4a and A).
After conjugation of an arginine or lysine to compound 3, more
interactions including hydrogen bonds and electrostatic interac-
tions are observed between 6 or 9 and the A site of 16S RNA
(Fig. 4b, B, c, and C). Comparing with the molecular modeling of
compound 6 or 9, it shows clearly that the arginine residue in com-
pound 6 contributes only one NH of d-guanidino group and one a-
NH2 group for the interaction with RNA and the ethylenediamine
moiety loses its interaction with RNA. However, in the case of com-
pound 9, the
x-NH2 and a-NH2 of lysine residue and the NH of eth-
ylenediamine part contribute significantly for the interaction with
the A site of 16S RNA. These results are in agreement with the
experimental data and could explain the difference of binding
affinity between compound 6 and 9.
The antibacterial activities of neamine and its three neamine
derivatives against E. coli ATCC 25922, S. aureus ATCC 29213 and
P. aeruginosa (Pseudomonas aeruginosa) ATCC 27853 were deter-
mined, respectively,34 the results are summarized in Table 3. Com-
pound 9 shows some activities against S. aureus (MIC = 25
E. coli (MIC = 50 M). This result exhibits that compound 9 could
be used as a lead for the further structural optimization.
lM) and
l
In summary, three new neamine derivatives 3, 6 and 9 were
synthesized by modifying 5-hydroxyl of neamine. Their binding
affinities to 16S and TAR RNA indicate that the modification to
5-hydroxyl of neamine by amino acid can enhance the binding
affinity of neamine. Compound 9 (NEL) shows more potential of
binding affinity compared with its arginine derivative and exhibits
some antibacterial activities against S. aureus (MIC = 25
lM) and
E. coli (MIC = 50 M). These results demonstrate that lysine mod-
l
ifying at 5-position of neamine may provide a promising way for
the development of potential candidates effectively targeting to
RNAs.
Acknowledgment
This study was supported by the National Natural Science
Foundation of China (20332010) and The Ministry of Science and
Technology of China (2004cb518904).
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