Selectively guanidinylated derivatives of neamine. Syntheses and inhibition of anthrax lethal factor protease

Article abstract of DOI:10.1016/j.bmcl.2006.07.005

A series of mono-, di-, and tri-guanidinylated derivatives of neamine were prepared via selective guanidinylation of neamine. These molecules represent a novel scaffold as inhibitors of anthrax lethal factor zinc metalloprotease. Methods for the synthesis

Full text of DOI:10.1016/j.bmcl.2006.07.005

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5183–5189
Selectively guanidinylated derivatives of neamine.
Syntheses and inhibition of anthrax lethal factor protease
Guan-Sheng Jiao,a,* Ondrej Simo,a Melissa Nagata,a Sean O’Malley,a
Thomas Hemscheidt,b Lynne Cregar,c Sherri Z. Millis,c
Mark E. Goldmanc, and Cho Tanga,à
aDepartment of Chemistry, Hawaii Biotech, Inc., 99-193 Aiea Heights Dr., Suite 200, Aiea, HI 96701, USA
bDepartment of Chemistry, University of Hawaii at Manoa, Honolulu, HI 96822, USA
cDepartment of Lead Discovery, Hawaii Biotech, Inc., 99-193 Aiea Heights Dr., Suite 200, Aiea, HI 96701, USA
Received 31 May 2006; revised 26 June 2006; accepted 5 July 2006
Available online 25 July 2006
Abstract—A series of mono-, di-, and tri-guanidinylated derivatives of neamine were prepared via selective guanidinylation of
neamine. These molecules represent a novel scaffold as inhibitors of anthrax lethal factor zinc metalloprotease. Methods for the
synthesis of these compounds are described, and structure–activity relationships among the series are analyzed. In addition, initial
findings regarding the mechanism of LF inhibition for these molecules are presented.
Ó 2006 Elsevier Ltd. All rights reserved.
Bacillus anthracis1 is a spore-forming Gram-positive
bacterium that is the causative agent of anthrax infec-
tion.2 Generally, the spores can enter a subject by oral
ingestion, through the skin, or by inhalation.1 The
spores are phagocytized and travel to regional lymph
nodes where they germinate and release toxins3 which
are crucial for the pathogenesis of anthrax.4 Although
the use of vaccines prior to infection is preventive
against anthrax,5 various factors make mass vaccination
impractical. Antibiotics such as ciprofloxacin, penicil-
lins, and tetracyclines may be effective in reducing the
bacterial infection itself at the very early stage,6 but once
the toxins are released, such therapy does not signifi-
cantly arrest the course of the disease because of the
continuing action of the toxins. Given the attractiveness
of anthrax as a biological weapon, the modification of
wildtype B. anthracis to provide antibiotic resistant
strains is a distinct possibility. Therefore, it is important
to provide a post-infection anthrax treatment which is
not prone to antibiotic resistance.
After onset of infection, the toxins are released in the
form of three plasmid-encoded proteins: protective anti-
gen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema
factor (EF, 89 kDa).7 When PA83 (83 kDa)8 specifically
binds to a cell surface anthrax toxin receptor,9 a 20 kDa
fragment is cleaved by a furin-like protease,10 allowing
the remaining PA63 (63 kDa) to heptamerize. After
binding to LF to give lethal toxin (LT) or EF to give
edema toxin (ET), the heptamer behaves as a shuttle
to translocate LF and EF into an intracellular endo-
somal compartment.11 After LT and ET enter the endo-
somes, dissociation occurs, and LF and EF are released
into the cytoplasm to exert their toxic effects.12 Edema
factor is a Ca2+/calmodulin-dependent adenylate cyclase
that triggers the synthesis of cAMP, leading to edema.13
Lethal factor is a Zn2+-dependent metalloprotease that
specifically cleaves mitogen-activated protein kinase ki-
nases (MAPKKs) in macrophages.14 Degradation of
MAPKKs interrupts critical signaling pathways, result-
ing in cell death.15 The dead cells then release cytokines
and NO which cause septic shock and death of the sub-
ject. The fact that EF-deficient B. anthracis strains are
still toxic, while those lacking LF are greatly attenuat-
ed,16 suggests that LF is the dominant virulence factor
of anthrax. Therefore, inhibition of LF should offer an
Keywords: Anthrax; Lethal factor inhibitors; Zn2+-dependent metal-
loprotease; Guanidinylated neamine derivatives.
*
Corresponding author. Tel: +1 808 792 1367; fax: +1 808 792 1348;
e-mail: gjiao@hibiotech.com
Present address: John A. Burns School of Medicine, University of
Hawaii at Manoa, Honolulu, HI, USA.
à
Present address: Shanghai Hengrui Pharmaceuticals, Shanghai,
China.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.005

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G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5183–5189
efficient therapeutic approach for treating anthrax, par-
ticularly in late stage infection. During the last few
years, progress has been made in the search for specific
inhibitors of LF.17–31
ge density due to the presence of clusters of negatively
charged, acidic Asp and Glu residues.21,22,29,30,37 The elec-
trostatic interactions between the positively charged ami-
no groups of aminoglycosides and the negatively charged
residues of LF have been shown to play a vital role in
determining the inhibitory activity.24,27 We hypothesized
that introduction of highly charged guanidinyl groups
onto the neamine core might result in better potency by
inducing stronger electrostatic interaction with the bind-
ing site.38 To test the hypothesis, tetraguanidinoneamine
4 was synthesized via guanidinylation of neamine with
excess of N,N0-di-(tert-butoxycarbonyl)-N00-triflylguani-
dine 5,39 followed by TFA deprotection, as a TFA salt
(Scheme 1). We were encouraged by the finding that com-
pound 4 showed a Kai pp of 0.7 lM for inhibition of LF,
which is comparable to that of neomycin B.
Our early work in the anthrax project led to the discov-
ery of a series of cationic small molecule LF protease
inhibitors. An intermediate stage of this work was the
identification of the semi-synthetic LF inhibitors derived
from neamine. In this letter, we present the systematic
investigation of these guanidinylated neamine deriva-
tives.32 The significance of this study is twofold. First,
the selective synthesis of guanidinylated neamine deriv-
atives remains a significant challenge in organic synthe-
sis. Efficient and practical methods for mono-, di-, and
tri-guanidinylation of neamine are presented in this let-
ter. Second, these compounds are shown to be potent
inhibitors of anthrax LF protease using an in vitro bio-
chemical assay.33 Based on this work, we further identi-
fied fully synthetic LF inhibitors derived from 2,5-
dideoxystreptamine, which were published earlier.17
Taken together, these studies illustrated the ability to
use a biologically active natural product to identify fully
synthetic, potent small molecule inhibitors of anthrax
lethal factor protease.
The above result strongly suggested the significant effect
of the guanidinyl group on the inhibitory activity
against LF.40 Consequently, our next goal was to deter-
mine the minimum number of guanidinyl groups
required for potent activity as well as to investigate the
structure–activity relationship requirements for the loca-
tion of these guanidinyl groups. Therefore, a series of
mono-, di-, and tri-guanidinylated derivatives of nea-
mine were prepared. In each synthetic strategy described
below, only the key steps are highlighted. The last step
of the synthesis was the same in each case, deprotection
with TFA to give the final products as TFA salts.
The project was initiated by the screening of a focused
library of commercially available cationic compounds.
It was found that some aminoglycosides, commonly
used antibiotics for treatment of Gram-negative and
Gram-positive bacterial infections,34 exhibited strong
inhibitory activity against anthrax LF protease.35 For
example, neomycin B (Fig. 1, 1) showed Kai pp of
0.5 lM for inhibition of LF in our in vitro FRET
assay.32 Because of its structural complexity, neomycin
B does not represent an attractive lead for a medicinal
chemistry program. The decision was then made to
investigate structurally simpler neamine (Fig. 1, 2), a
common pseudodisaccharide present in most naturally
occurring aminoglycosides, for further optimization
because it could be manipulated more easily than neo-
mycin B. Accordingly, we prepared neamine via treat-
ment of commercially available neomycin B with
acetyl chloride in methanol.36 Neamine was found to be-
have as a weak inhibitor of LF with Kai pp of 42.9 lM.
In general, the primary 60-amino group is the most reac-
tive of the four amino groups of neamine with slight dif-
ferences in reactivity among the remaining three
positions (1 > 3 > 20) due to their steric accessibility.41
As
a result, the mono-guanidinylated neamines,
60-guanidinoneamine 7, 1-guanidinoneamine 9, and
3-guanidinoneamine 11, were prepared by selective
guanidinylation of neamine 2, 3,60-di-(tert-butoxycar-
bonyl)-neamine 8,42 and 1,60-di-(tert-butoxycarbonyl)-
neamine 10,43 respectively, employing a limiting amount
of 5 (Schemes 2A–C).44 The 20-guanidinoneamine 13
was prepared from 1,3,60-tri-(tert-butoxycarbonyl)-nea-
mine 1245 with excess of 5 (Scheme 2D).
The synthesis of the di- and tri-guanidinylated neamines
began with either a mono-guanidinylated neamine deriv-
ative or selectively N-protected neamine compounds.
The di-guanidinylated neamines, 1,60-diguanidinone-
amine 14 and 3,60-diguanidinoneamine 15, and tri-gua-
nidinylated analogs, 1,3,60-triguanidinoneamine 16 and
1,20,60-triguanidinoneamine 17, were prepared via a sin-
gle guanidinylation reaction of compound 6 (see Scheme
2A) with 1 equiv of 5 (Scheme 3). The four intermediate
compounds were readily separated from the mixture by
silica gel column chromatography.
The available X-ray crystal structures of LF reveal that
the active site of the protease possesses high negative char-
H2N
O
NH2
OH
NH2
O
H2N
O
NH2
OH
O
2
5
3
4
1
NH2
6
HO
NH2
O
OH
O
6'
OH
5' 1'
OH
H2N
2'
4'
3'
O
HO
NH2
The 1,3-diguanidinoneamine 19 was obtained via selec-
tive guanidinylation of 60-(tert-butoxycarbonyl)-nea-
mine 1846 with 1.9 equiv of 5 (Scheme 4A), and 1,
20-diguanidinoneamine 20 (Scheme 4B), 3,20-diguanidi-
noneamine 21 (Scheme 4C), and 1,3,20-triguanidinone-
amine 23 (Scheme 4E) were prepared from compounds
8,42 10,43 and 18,46 respectively, by using excess of 5.
NH2
OH
OH
1
2
NH2 OH
neomycin B
Figure 1. Structures of neomycin B 1 and neamine 2.
neamine

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5183–5189
5185
HN
HN
NH2
BocN
NHBoc
H
H
NH
NH
H2N
O
NH2
OH
NBoc
NH
N
NH2
NH
HN
N
NHBoc
NBoc
Tf
NH2
N
H2N
BocHN
a
b
O
O
O
OH
O
O
OH
BocHN
NHBoc
OH
OH
OH
5
HO
NH2
HO
NH
HO
NH
NBoc
OH
Guanidinylation Reagent
OH
H2N
OH
BocHN
NH
2
3
4
tetra-guanidinoneamine
Scheme 1. Reagents and conditions: (a) 6 equiv of 5, Et3N, MeOH/H2O, rt; (b) TFA–DCM (1:1), rt.
A
B
H
NBoc
NH
NH
NH
BocHN
NHBoc
NH2
OH
H2N
O
N
NH2
NH
H2N
O
NH2
OH
H2N
O
NH2
OH
NH2
BocHN
H2N
a
b
c, b
O
O
O
OH
O
O
2
OH
OH
OH
OH
HO
NH2
HO
NH2
HO
NH2
HO
NH2
OH
OH
OH
OH
6
7
8
9
6'-guanidinoneamine
1-guanidinoneamine
C
HN
HN
NH2
D
H2N
NH2
NH2
OH
H2N
NHBoc
OH
BocHN
NHBoc
OH
NH2
NHBoc
O
NHBoc
O
O
O
NH2
c, b
d, b
O
O
OH
O
O
OH
OH
OH
OH
HO
NH
HO
NH2
HO
NH2
OH
H2N
HO
NH2
OH
OH
NH
OH
10
11
12
13
2'-guanidinoneamine
3-guanidinoneamine
Scheme 2. Reagents and conditions: (a) 0.95 equiv of 5, MeOH–H2O (1.3:1), rt; (b) TFA–DCM (1:1), rt; (c) 1 equiv of 5, Et3N, MeOH, rt; (d)
1.5 equiv of 5, Et3N, MeOH, rt.
HN
NH2
NH
HN
HN
NH2
HN
HN
NH2
HN
NH2
NH
NH
NH2
H
NH
NH
NH
NH
NH
NH2
H2N
NH2
OH
N
NH2
NH
H2N
HN
HO
H2N
H2N
a, b
HN
HO
6
O
O
OH
+
+
+
O
O
O
O
OH
O
O
OH
OH
OH
OH
OH
NH
HO
NH2
HO
NH2
NH2
OH
HN
OH
OH
OH
NH2
17
1,2',6'-triguanidinoneamine
14
15
3,6'-diguanidinoneamine
16
1,6'-diguanidinoneamine
1,3,6'-triguanidinoneamine
Scheme 3. Reagents and conditions: (a) 1.0 equiv of 5, Et3N, MeOH, rt; (b) TFA–DCM (1:1), rt.
A highlight of this work was the application of metal-
chelation reactions, which have been used in selective
Cbz- or Boc-protection of neamine,42,43,47 to prepare
20,60-diguanidinoneamine 22 and 3,20,60-triguanidinone-
amine 25.48 Guanidinylation in the presence of nickel
acetate, which ties up the 1- and 3-positions,47 resulted
in 20,60-diguanidinoneamine 22 (Scheme 4D). The inter-
mediate compound 24 was first prepared by using
1 equiv of di-tert-butyl-dicarbonate in the presence of
zinc acetate, which blocks access of 3-position.43 Gua-
nidinylation of compound 24 with 3 equiv of 5 then gave
3,20,60-triguanidinoneamine 25 (Scheme 4F).
results are summarized in Table 1. In general, it was
found that guanidinyl groups added to neamine en-
hanced the potency against LF, but there is no clear lin-
ear relationship between the potency and the number of
guanidinyl groups. Surprisingly, 3,20-diguanidinone-
amine 21 and 1,3,20-triguanidinoneamine 23 exhibited
comparable potency to 1,3,20,60-tetraguanidinoneamine
4 in the sub-micromolar range, which indicates that
two guanidinyl groups are sufficient for potent activity.
The finding that the most potent derivatives bear guan-
idinyl groups on both the 3- and 20-positions suggests
that the spatial location of these groups on neamine is
preferred for good inhibitory activity against LF.49
Once the mono-, di-, and tri-guanidinylated neamine
derivatives were prepared, their inhibition of LF was
evaluated using an in vitro biochemical assay.33 The
Finally, to explore the significance of the OH groups
on the inhibition of LF activity by neamine, the

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G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5183–5189
A
B
HN
HN
NH2
H
H2N
O
N
NH2
H2N
O
NH2
OH
H
N
NH2
NH
NH2
NHBoc
O
NH
OH
a, b
NH2
c, b
O
8
O
O
OH
OH
OH
OH
HO
NH
HO
NH2
HO
NH2
OH
OH
HN
NH2
20
OH
18
19
1,3-diguanidinoneamine
1,2'-diguanidinoneamine
C
D
HN
HN
NH2
NH
H2N
NH2
OH
NH2
OH
H2N
NH
NH2
c, b
d, b
O
O
O
O
10
6
OH
OH
HO
NH
HO
NH
OH
H2N
OH
H2N
NH
NH
21
22
3,2'-diguanidinoneamine
2',6'-diguanidinoneamine
E
F
HN
HN
NH2
HN
HN
NH2
H
N
NHBoc
NH
NH2
NH
H2N
O
NHBoc
OH
NH2
NH2
OH
BocN
NH2
HN
e, b
f
c, b
NH
OH
O
O
O
18
6
O
O
OH
OH
OH
HO
NH2
HO
NH
HO
NH
OH
OH
OH
NH
23
1,3,2'-triguanidinoneamine
NH2
H2N
HN
24
25
3,2',6'-triguanidinoneamine
Scheme 4. Reagents and conditions: (a) 1.9 equiv of 5, Et3N, MeOH, rt; (b) TFA–DCM (1:1), rt; (c) 3 equiv of 5, Et3N, MeOH, rt; (d) 1 equiv of 5,
Ni(OAc)2, MeOH, rt; (e) 4.5 equiv of 5, Et3N, MeOH, rt; (f) 1 equiv of Boc2O, Cu(OAc)2, MeOH, rt.
Table 1. Inhibition constant (Kai pp) values for guanidinylated neamine derivatives against lethal factor
R2
R1
3
1
R4
R1, R2, R3, R4 = NH2
O
O
OH
6'
NH
or
OH
2'
R3
N
NH2
HO
H
OH
Compound
Trivial name
No. of guanidinyl group
Kai pp (lM)a
1
2
Neomycin B
Neamine
1,3,20,60-Tetraguanidinoneamine
1-Guanidinoneamine
0
0
4
1
1
1
1
2
2
2
2
2
2
3
3
3
3
4
0.5 0.1
42.9 6.3
0.7 0.1
5.0 0.7
5.2 0.7
10.9 1.5
24.3 0.6
5.6 0.7
5.3 1.1
10.2 3.2
0.7 0.1
8.8 2.4
7.7 1.1
0.5 0.1
8.7 1.0
3.1 0.8
9.2 1.1
1.5 0.2
4
9
11
13
7
3-Guanidinoneamine
20-Guanidinoneamine
60-Guanidinoneamine
19
20
14
21
15
22
23
16
17
25
29
1,3-Diguanidinoneamine
1,20-Diguanidinoneamine
1,60-Diguanidinoneamine
3,20-Diguanidinoneamine
3,60-Diguanidinoneamine
20,60-Diguanidinoneamine
1,3,20-Triguanidinoneamine
1,3,60-Triguanidinoneamine
1,20,60-Triguanidinoneamine
3,20,60-Triguanidinoneamine
1,3,20,60-Tetraguanidino-5,6,30,4-tetramethoxylneamine
a The values are means of three experiments.

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5183–5189
5187
HN
HN
NH2
H
NH2
NH
N3
O
N3
N3
O
N3
H2N
O
NH2
N
NH
NH2
N3
N3
NH2
HN
a
b
c,d
O
OH
O
OMe
O
OMe
O
O
OMe
OH
OMe
OMe
OMe
HO
N3
MeO
N3
MeO
NH2
MeO
NH
OH
MeO
MeO
MeO
NH2
HN
26
27
28
29
1,3,2',6'-tetraguanidino-
5,6,3',4'-tetramethoxylneamine
Scheme 5. Reagents and conditions: (a) MeI, NaH, THF, rt; (b) H2, Pd/C, MeOH, rt; (c) 6 equiv of 5, Et3N, MeOH, rt; (d) TFA-DCM (1:1), rt.
4.0
1,3,20,60-tetraguanidino-5,6,30,40-tetramethoxylneamine
29 was prepared from 1,3,20,60-tetraazidoneamine 2650
via methylation with methyl iodide, followed by reduc-
tion, guanidinylation, and deprotection (Scheme 5). It
3.5
[I] = 0.00
[I] = 0.50
[I] = 1.00
[I] = 2.00
3.0
2.5
2.0
1.5
1.0
0.5
0
was found that the OH groups in the guanidinylated
neamine derivatives had negligible influence on the
potency (29 vs. 4, Table 1). This result implies that sub-
stitution or replacement of the OH groups might pro-
vide an opportunity to modify neamine as a lead
structure to improve bioavailability and other pharma-
cological properties without loss of potency.
Mechanistic studies suggest that the guanidinylated nea-
mine derivatives are mixed-type inhibitors of LF.51 As
illustrated in the double reciprocal Lineweaver–Burk
plot of compound 21 (Fig. 2), the primary plot of inhi-
bition gave straight lines with a point of intersection in
the second quadrant. Both the slope and the vertical axis
intercepts increased with increasing inhibitor concentra-
tion, indicating a mixed-type inhibition. It was also
observed that the addition of NaCl (up to 40 mM) to
the assay buffer solution resulted in dramatic increase
of Kai pp values for these compounds (ca. 10-fold, data
not shown), thus supporting the idea that the interaction
between LF and the guanidinylated neamine derivatives
is predominantly electrostatic. Since aminoglycosides
have been shown to chelate to zinc,43 we briefly investi-
gated the effect of the nonspecific Zn-chelation on the
inhibition of LF. As an example, compound 21 was
assayed at various concentrations of ZnCl2 to determine
if the added zinc ions would compete with the catalytic
zinc of LF for binding to the inhibitor. The results
showed that addition of ZnCl2 at concentrations below
16 lM does not significantly affect the Kai pp values,52
indicating that the nonspecific Zn-chelation is unlikely
to govern the inhibition of LF. We also conducted
counterscreens of the most potent compounds 4, 21,
23, and 29 against several relevant zinc-dependent
metalloproteases such as MMP-1, MMP-3. MMP-9,
MMP-12, and MMP-14. Very weak or no activity
ðKai pp > 300 lMÞ was observed against these enzymes,
suggesting that the guanidinylated neamine derivatives
are selective inhibitors of LF.
-0.5
-0.30
-0.25
-0.20
-0.15
-0.10
-0.05
0
0.05
0.10
0.15
1/[S]
Figure 2. Lineweaver–Burk plot of compound 21.
idinoneamine, and 1,3,20,60-tetraguanidinoneamine
exhibited the most potent activities in the sub-micromo-
lar range. Our work with this series demonstrates that
multiple cationic groups are important for the inhibition
of LF, and the full complex neomycin B structure is not
needed for potency.
Acknowledgments
We thank Dr. Alan T. Johnson for editorial comment
on the manuscript, Dr. Dominique Nguyen for helpful
discussion, and Ms. Linda McKasson for analytical
assistance. One reviewer is acknowledged for the com-
ment on the mechanism of inhibition. This work was
funded by the US Department of Defense, US Army
Medical Research and Materials Command, Ft. Det-
rick, MD, and administered by the Pacific Telehealth
and Technology Hui, Honolulu, HI, Contract #
V549P-6073. The views expressed in this article are those
of the authors and do not reflect the official policy or po-
sition of the Department of Defense, US Department of
the Army, or the US Government. The appearance of
name-brand products in this article does not constitute
endorsement by the US Department of the Army,
Department of Defense, or the US Government of the
information, products or services contained therein.
In summary, a series of novel guanidinylated neamine
derivatives have been synthesized via selective mono-,
di-, and tri-guanidinylation of neamine. These molecules
were shown to be potent, selective inhibitors of LF.
Among them, 3,20-diguanidinoneamine, 1,3,20-triguan-

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27. Lee, L. V.; Bower, K. E.; Liang, F.-S.; Shi, J.; Wu, D.;
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32. Part of this letter has been presented in ACS National
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diate metal chelates are formed between the proximal
52. When the concentration of ZnCl2 is below 16 lM, the
effect on ionic strength by ZnCl2 is negligible.