Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent lethal factor inhibitors with in vivo efficacy

Article abstract of DOI:10.1016/j.bmcl.2010.08.058

Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat lethal toxin model of anthrax disease.

Full text of DOI:10.1016/j.bmcl.2010.08.058

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6850–6853
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent
lethal factor inhibitors with in vivo efficacy
Guan-Sheng Jiao a, Seongjin Kim a, Mahtab Moayeri b, Lynne Cregar-Hernandez a, Linda McKasson a,
Stephen A. Margosiak a, Stephen H. Leppla b, Alan T. Johnson a,
*
a PanThera Biopharma, LLC, Aiea, HI 96701, USA
b Laboratory of Bacterial Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and
Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat
lethal toxin model of anthrax disease.
Received 30 June 2010
Revised 10 August 2010
Accepted 12 August 2010
Available online 15 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Anthrax
Lethal factor
Inhibitor
Small molecule
Three forms of the disease anthrax caused by Bacillus anthracis
are characterized by the route of exposure. Infection of an open
wound leads to cutaneous anthrax, and ingestion of contaminated
food causes gastrointestinal anthrax. While each of these forms can
be fatal, they are less severe compared to inhalation anthrax,
where mortality rates can be very high (>85%).1 Once inhaled, ger-
mination of spores leads to release of bacteria in a vegetative state
and eventual propagation leading to septicemia. To aid in infection
of the host, the bacteria secrete three proteins; edema factor (EF)
and lethal factor (LF), each of which combine with protective anti-
gen (PA) to form two binary toxins that act as virulence factors
possessing potent immunosuppressive activity.2 EF is a Ca+2/cal-
modulin-dependent adenylate cyclase which appears to impair im-
mune function. LF is a Zn2+-dependent metalloproteinase which
disrupts MAPK pathway signaling and leads to suppression of
pro-inflammatory gene expression. PA aids in the translocation of
EF and LF into the target cells.3 If left unchecked, the resulting tox-
emia and sepsis lead to vascular collapse, shock, and the death of
the host within a few days. While EF clearly contributes to the
lethality of anthrax, LF has been shown to be the primary causative
agent leading to death of the host due to toxemia.4 Given the effec-
tiveness of B. anthracis as a weapon of bioterrorism,5 the major role
LF plays in the pathogenesis of anthrax, and validation of LF as a
target for small molecule drug intervention,6 we began our search
for an antidote to LF intoxication. Presented below are the results
from our early discovery phase of a project leading to the identifi-
cation of novel small molecule anthrax LF inhibitors (LFIs).
A number of small molecule inhibitors of anthrax LF are known,
with those shown in Figure 1 being representative of various struc-
tural classes.7 Of these, the sulfonamide-based series represented
by Merck L915 (4) is the best characterized6 and provided a good
starting point for discovery of new lead series.
Using 4 as a design model, we began our investigation with the
goal of identifying novel X–Y linking groups (Fig. 2) using a FRET
based assay to guide the SAR.8 Replacing the NH-group of the sul-
fonamide with a methylene group (X = CH2, Y = SO2) provided
* Corresponding author.
E-mail address: ajohnson@pantherabio.com (A.T. Johnson).
Figure 1. Representative known small molecule inhibitors of anthrax lethal factor.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.058

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6850–6853
6851
Figure 2. Design model based upon sulfonamide hydroxamic acids.
sulfone analogs with equivalent potency while the corresponding
amide (X = NH, Y = CO) was inactive. Other two atom linking
groups, such as benzylamines or ethers (X = NH, O, Y = CH2) affor-
ded lower (>10Â) potency analogs. Based on the H-bond impli-
cated in the co-crystal structure between the sulfonyl group pro-
R oxygen of L915 to backbone amide protons (K656 and G657) of
LF,6 we reasoned that a hydroxyl or ether functional group may
provide for a similar interaction. Indeed, use of these two atom
links (X = CH2, Y = CH(OH), CH(OMe)) afforded active analogs, with
the 4-methylether series displaying significantly better potency
compared to the alcohol. We also investigated the corresponding
one atom linking series (X = NH, O, S, and CH2) in the absence of
a Y atom. We were pleased to discover that all of these compounds
were potent inhibitors of anthrax LF, with the aniline and phenyle-
ther series (X = NH, O) being specific (>300Â) for the target metal-
loprotease versus several MMPs (data not shown). These results
led to the further examination of four possible core structures as
novel LFI lead series (Fig. 3).
Scheme 1. Reagents and conditions: (a) 2 equiv of 3,5-dimethyl-4-fluorophenylbo-
ronic acid, 1.1 equiv of Cu(OAc)2, 2 equiv of Et3N, MS 4 Å, DCM, open air, rt; (b) TFA/
DCM (1:1), rt; (c) 1 equiv of R-ZCl (Z = CO, SO2), 5 equiv of Et3N, DCM, rt; or RCHO,
2 equiv of Et3N, 1.4 equiv of NaBH(OAc)3, DCE, rt; (d) KCN (5 mol %), THF/MeOH/50%
NH2OH–H2O (2:2:1), rt.
Our initial goals were to identify the best R1-groups and substi-
tution pattern for the phenyl ring of these core structures, as well
as the preferred stereochemistry at the C2 and C4 positions in each
linking group series. In the one atom series, the aniline derivatives
were selected for further study due to their ease of synthesis
(Scheme 1). Our initial work explored the effect of changing the
size, polarity, and position of the R1-group on inhibitor potency
using racemic compounds and the preparation of various mono-,
di-, and tri-substituted aniline derivatives (Fig. 3; X = NH, R2 = n-
Bu). The resulting SAR led to the identification of the 3,5-di-
methyl-4-fluoroaniline analog as the most potent inhibitor pos-
sessing sub-micromolar inhibitory activity.9 In the two atom
linking series, a similar approach of varying the R1-group in the
Scheme 2. Reagents and conditions: (a) LiHMDS, HMPA, R-CH@CHCH2I, À70 °C to
rt; (b) KOH, H2O, dioxane, rt; (c) MeI, NaH, THF, rt; (d) concd H2SO4, MeOH, rt; (e)
O3, Ph3P, DCM, À70 °C to rt; (f) diphenylamine, NaBH(OAc)3, AcOH, DCE, rt; (g)
NH2–OH, KCN, H2O, MeOH, THF, rt.
racemic 4-methoxy (c-ether) analog series led to the identification
Readily available and optically pure 5-aryl-2-furanones13 provided
access to all four diastereomers. Stereoselective alkylation14 of
furanone 9 using allylic halides led to an easily separable mixture
of the desired major isomer 10 and the minor syn-isomer. Of the
four possible diastereomers, the greatest inhibition of LF activity
was observed with the (2S,4R)-diastereomers (11), while the
(2S,4S)-isomers were found to be >100-fold less potent. The
remaining two diastereomeric series displayed intermediate inhib-
itory activity.
of the 3-methyl-4-fluorophenyl and 4-fluorophenyl analogs as
having the best potency (Fig. 3, R2 = H). This result was consistent
with the two atom sulfonamide linking group found in Merck L915
(4). Determining the preferred stereochemistry in each series re-
quired the development of synthetic schemes capable of providing
optically pure analogs in the aniline10 (Scheme 1) and the 4-meth-
oxy-4-phenylbutanoic acid (c
-ether) series11 (Scheme 2).
In the case of the aniline series, a stereospecific synthesis was
developed following the work by Lam et al.12 which begins with
optically pure amino acid derivatives. Using this synthetic route
with the available (R) and (S) amino acids, a clear preference
(>10 to 100Â) for the (2R) stereochemistry versus (2S) at C2 in
the aniline series was observed.
Having identified the preferred R1-groups and stereochemistry
for the aniline and c-ether series, we began to explore variations
to the C2-side chain in an effort to further increase inhibitor po-
tency. In the aniline series, the primary amine derived from 6
(Scheme 1) provided a convenient branch point for analog synthe-
The presence of two stereogenic centers in the c-ether series led
sis, while in the c-ether series intermediate 11 proved to be good
to the development of the synthetic pathway shown in Scheme 2.
starting point for exploring diversity (Scheme 2, R = H). Tables 1
and 2 provide a representative sample of our findings in each
series.
Collected in Table 1 is a selection of data based on 3,5-dimethyl-
4-fluoroaniline core structure derived from R-lysine which is rep-
resentative of the SAR observed in the one atom linking series.
The synthetic versatility associated with a primary amine led to
an early study on how different nitrogen containing functional
groups would impact intrinsic potency. The primary amine 13
Figure 3. One atom and two atom linking group series selected for study.

6852
G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6850–6853
Table 1
Shown in Table 2 are data for a representative set of LF inhibi-
tors based upon the -ether core structure. Simple symmetric dial-
LF inhibitory data for selected aniline derivatives
c
kyl and cyclic amine derivatives (29–32) afforded LF inhibitors
with moderate activity. The 3-substituted piperidine derivatives
33 and 34 displayed slightly higher potency relative to the unsub-
stituted compounds, with the individual (R) and (S) isomers in each
case having Ki values essentially unchanged from the mixture
shown. Examination of 4-substituted piperidines showed a similar
profile having Ki values in the high double digit nanomolar range
(data not shown).
Compound
Z
R
LF (FRET)
Kia (nM)
Preparation and testing of 4-substituted piperazine analogs
(35–38) resulted in inhibitors with Ki values in the 100 nM range.
Somewhat surprisingly, the size of the 4-substitutent had no effect
on LF inhibitory activity. In contrast, preparation of unsymmetrical
tertiary amines where one R-group was methyl (39–41) provided
analogs with improved potency. Of the many analogs made, those
having a total C2 length equivalent to the N-substituted lysine
derivatives discussed above (Table 1) exhibited the highest poten-
cies, with compound 40 displaying a Ki of 11 nM.
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
—
CO
SO2
–H
8.2
7.3
3.1
0.05
9.3
0.69
0.85
0.24
1.1
0.41
0.44
0.47
0.30
1.4
3-Me-4-F-Ph
3-Me-4-F-Ph
3-Me-4-F-Ph
3-Me-4-F-Ph
2-F-Ph
3-F-Ph
4-F-Ph
4-Cl-Ph
4-Me-Ph
4-OMe-Ph
4-CF3-Ph
4-NO2-Ph
4-t-Bu-Ph
4-F-Ph
CH2
C(O)NH
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
(CH2)2
(CH2)3
Kinetic studies conducted with representatives of both the ani-
line and c-ether series showed them to be slow tight-binding com-
petitive inhibitors (Fig. 4); a result in line with the finding of the
2.1
1.3
Merck scientists and 4.6a
4-F-Ph
After identifying potent LFIs in two unique structural series, our
next goal was to demonstrate their efficacy in an in vivo Lethal
Toxin (LT) model of anthrax.16 Based upon selection criteria in
our LFI screening cascade which included intrinsic potency (Ki
<100 nM), selectivity for the target (>500Â vs MMP-1, 3, 9, 12,
14) drug-like properties (E log D <3, Sk >5 mg/mL 50% DMSO/PBS)
a
Values are means of three experiments; standard deviation is <10%.
Table 2
LF inhibitory data for selected c-ether derivatives
Compound
R
LF (FRET)
Kia (nM)
29
30
31
32
33
34
35
36
37
38
39
40
41
–N(Me)2
Pyrrolydin-1-yl
Piperidin-1-yl
Morpholin-4-yl
(R,S)-3-Hydroxymethyl-piperidin-1-yl
(R,S)-3-(4-F-Ph)CH2-piperidin-1-yl
Piperazin-1-yl
96
122
230
350
99
33
90
151
115
118
42
4-Ph-piperazin-1-yl
4-PhCH2-piperazin-1-yl
4-Ph(CH2)2-piperazin-1-yl
–N(Me)–(CH2)3Ph
–N(Me)(CH2)3-3Me-4-F-Ph
–N(Me)CH2-4(3-Me-4-F-Ph)-Ph
11
135
Figure 4. Kinetic inhibition data for 40 versus anthrax LF. Global non-linear fitting
(GraFit v5.0) to multiple inhibition models and statistical results indicate compet-
itive behavior.
a
Values are means of three experiments; standard deviation is <10%.
was found to be a potent inhibitor of LF activity with a Ki = 8.2 nM.
The corresponding amide 14, sulfonamide 15 and urea 17 derived
from this amine and bearing the same R-group were potent inhib-
itors but less active compared to the sub-nanomolar benzyl amine
derivative 16 (Ki = 0.05 nM), a compound >500Â more potent than
4 (Ki = 46 nM). As a result, our focus was directed towards further
exploration of the benzylamine series.15 Positioning one or more
small groups anywhere on the phenyl ring (18–20) afforded potent
LFIs. Further exploration at the C4-position was investigated with
the preparation of inhibitors 21–26. Little or no effect was seen
with variation in the nature of the R-group at C4 with all of the
compounds displaying single digit to sub-nanomolar inhibition of
LF activity. This suggested that these R-groups either extend into
a large cavity or that they are directed into the solvent. Finally,
extension of the chain length to give the phenethyl (27) or phen-
propyl (28) analogs lead to a slight decrease in potency relative
to the benzyl analogs.
Figure 5. Survival curves for Fischer 344 rats (n = 4) dosed (iv) with LFIs followed
by LT (10 lg PA + 10 lg LF) dosed iv.

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6850–6853
6853
and low cytotoxicity (CC50 >10
for an in vivo study. Fischer 344 rats were dosed (iv) with three
aniline and two -ether analogs at 10 mg/kg in DMSO/PBS (1:1,
v/v) followed 20–30 min later with a 10 g dose (iv) of LT (10
PA + 10 g LF). Using the extension of Median Survival Time
(MST) and percent survival as endpoints, the results in Figure 5
show that with the exception of compound 14, all of the com-
pounds extended the MST relative to the control group, and one
compound, the aniline 16, was capable of providing 100% protec-
tion at this dose. Repeating this experiment with lower doses of
16 indicated that this compound was fully protective at both 5.0
and 2.5 mg/kg, although at the lowest dose the animals became
ill approximately 3 h after treatment with LT but appeared fully
recovered by 24 h post exposure (data not shown).
lM), five compounds were selected
References and notes
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Chem. Lett. 2006, 16, 964.
c
l
lg
l
7. (a) Turk, B. E. Curr. Pharm. Biotech. 2008, 9, 24; (b) Schepetkin, I. A.; Khlebnikov,
A. I.; Kirpotina, L. N.; Quinn, M. T. J. Med. Chem. 2006, 49, 5232.
8. LF proteolytic assay: A fluorescence resonance energy transfer (FRET) assay was
used to determine the inhibition kinetics and Ki values using MCA-
KKVYPYPME-Dap(Dnp)-NH2 as the peptide substrate. Cleavage was measured
In summary, we have identified sub-nanomolar inhibitors of
anthrax lethal factor with potent in vivo efficacy. Part 2 of this
work will describe the initial steps taken to optimize these com-
pounds with the goal of developing LFIs to treat anthrax in
humans.
as an increase in fluorescence with kex = 324 nm, kem = 395 nm. The 100
reaction contained 0.5 nM LF (List Biologicals) in 20 mM HEPES pH 7.4, 0.1%
Tween-20, 0.5 mg/mL BSA and 40 M peptide substrate. The reaction was
lL
l
initiated with enzyme addition and incubated 4 h (23 °C) to achieve a steady
state rate of product formation. Steady state rates were used to determine Ki
values by fitting to the classical tight-binding equation.
9. Relative potency: mono- < di- ꢀ tri-substitution; C2 << C3 ꢀ C4; R1 = F <
Cl ꢀ Me > Et > i-Pr, Ph.
Acknowledgments
10. Jiao, G.-S.; Johnson, A. T. WO 2008/094592 A1.
We thank Dr. Sherri Millis, Dr. Petr Kuzmic, and Devorah Crown
for help with preliminary experiments. We also thank the National
Institutes of Health for their support of this work with grant R44
AI052587. Animal studies were supported by the Intramural Re-
search Program of the NIH, National Institute of Allergy and Infec-
tious Diseases. The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the NIAID or the NIH.
11. Johnson, A. T.; Kim, S. WO 2009/008920 A2.
12. Lam, P. Y. S.; Bonne, D.; Vincent, G.; Clark, C. G.; Combs, A. P. Tetrahedron Lett.
2003, 44, 1691.
13. Ghosh, A. K.; Swanson, L. J. Org. Chem. 2003, 68, 9823.
14. Tomioka, K.; Cho, Y.-S.; Sato, F.; Koga, K. J. Org. Chem. 1988, 53, 4094.
15. Relative
potency:
Z = CH2,
R = Ph > heteroaryl ꢀ cycloalkyl ꢀ
heterocycloalkyl P alkyl.
16. Gupta, P. K.; Moayeri, M.; Crown, D.; Fattah, R. J.; Leppla, S. H. PLoS ONE 2008, 3,
e3130.