Antidotes to anthrax lethal factor intoxication. Part 2: Structural modifications leading to improved in vivo efficacy

Article abstract of DOI:10.1016/j.bmcl.2011.02.010

New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded analogs with improved efficacy in the rat lethal toxin model.

Full text of DOI:10.1016/j.bmcl.2011.02.010

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2030–2033
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antidotes to anthrax lethal factor intoxication. Part 2: Structural
modifications leading to improved in vivo efficacy
Seongjin Kim a, , Guan-Sheng Jiao a, , Mahtab Moayeri b, Devorah Crown 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:
New anthrax lethal factor inhibitors (LFIs) were designed based upon previously identified potent
inhibitors 1a and 2. Combining the new core structures with modifications to the C2-side chain yielded
analogs with improved efficacy in the rat lethal toxin model.
Received 22 December 2010
Revised 2 February 2011
Accepted 3 February 2011
Available online 18 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Anthrax
Lethal factor
Inhibitor
Small molecule
Anthrax disease results from infection with the gram-positive
bacteria Bacillus anthracis. Inhalation anthrax is especially danger-
ous with a survival rate of <15% if left untreated.1 As the 2001 US
postal service attacks demonstrated, mortality from this disease
approaches 50% even with antibiotic treatment and intensive care.2
While the pathogenesis of anthrax is not fully understood, it is
known that 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; edema toxin and lethal toxin
(ET and LT, respectively). These toxins act as virulence factors
and suppress the immune system of the host.3 The protein EF is
a Ca2+/calmodulin-dependant adenylate cyclase which appears to
impair immune function, while LF acts as a Zn2+-dependent
metalloproteinase and disrupts cell signaling pathways by cleav-
age of MEK proteins. LT is also considered a primary causative
agent leading to death of the host.4 Given the potential for mass
casualties by using anthrax as a weapon of bioterrorism, new
methods of treating patients infected with B. anthracis leading to
improved survival are clearly needed.
a rabbit model of anthrax.5 Since the natural incidence of infection
by B. anthracis is extremely low and lethality can be very high,
phase II and III clinical trials with LFIs are not possible. As a result,
development of an antidote for LF intoxication in humans presents
a special challenge and must rely on the use of the ‘animal rule’ to
demonstrate a potential for efficacy in humans.6 Given this fact, we
chose to incorporate an in vivo toxin model early in our screening
cascade. The rat LT challenge model is attractive from a drug dis-
covery point of view since it is reproducible, has the resolution
needed to rank order compounds being tested in a given study,
and death of the animals results specifically from the action of an-
thrax LF.7 A second benefit of using a pharmacology based model
early in the discovery process is that it provides the potential for
early readouts of Structure Activity Relationships (SAR) as they
One plausible approach is to combine an antibiotic to clear the
active infection and stop further release of toxins, with an LF inhib-
itor (LFI) to block the action of the toxin already present in the
body. Indeed, the first in vivo studies to clearly support the use
of an LFI in this manner were conducted by scientists at Merck in
⇑
Corresponding author.
E-mail address: ajohnson@pantherabio.com (A.T. Johnson).
These authors contributed equally to this work.
Figure 1. Previously identified small molecule LFIs.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.010

S. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2030–2033
2031
death, did provide an increase the Median Survival Time (MST) of
the test animals relative to the vehicle only treated controls. In Part
2 of this series we describe our work toward improving the in vivo
efficacy of these LFIs by exploring further structural modifications
to these two compound classes, guided in part by in vivo data ob-
tained from the rat LT model.
Combining 1a and 2 into a generic structure (Fig. 2) provided a
starting point for targeting structural modifications and determin-
ing the impact of these changes on intrinsic potency and in vivo
efficacy. These modifications included the synthesis of new core
structures by replacing the X-groups in 1a and 2 with either an
oxygen atom (4, X = O) or methylene group (5, X = CH2), and
exploring alternate substitution patterns on the aryl ring of the
core structure (analog series 6, 7, 8, and 9). Also of interest was
investigating changes to the C2 side chain in terms of overall
length (o and p = 1–3), the role of the Y-group (Y = NH vs O, CH2)
and the position of this group relative to the core structure (e.g.,
3 vs 2). Summarized below are the results of these studies.
Representative synthetic schemes for the preparation of com-
pounds in series 4 and each of the remaining series depicted in Fig-
ure 2 are given in Scheme 1 and the Supplementary Data,9
respectively. Analogs in the phenoxyacetic acid series (4: R2 = Me;
8: R2 = H) were prepared using the asymmetric alkylation route
shown in Scheme 1.10 Preparation of the 3-methyl-4-flouro-phe-
noxyacetic acid from ethyl 2-bromoacetate and conversion to the
oxazolidinone derivative 10 followed standard synthetic methods.
Asymmetric alkylation of 10 (R2 = H) afforded the benzyl protected
allylic alcohol 11 (R2 = H) in moderate yield.11 When the tri-substi-
tuted analog (R2 = Me) was used, the yield for this step dropped
below 20%. A possible explanation for this result came with the
examination of model transition states which suggested the added
Me-group on the aryl ring could sterically hinder access of the
incoming electrophile to the desired re-face of the enolate. Catalytic
hydrogenation to reduce the olefin and deprotect the alcohol was
followed by oxidation to provide the aldyehyde 12 in good overall
yield. Reductive amination to give the secondary amine followed
by treatment of this product with hydroxylamine provide access
to LFIs in 4 and 8 directly from the N-acyloxazolidinone.
Figure 2. Structural changes based upon LFIs 1a and 2.
Scheme 1. Reagents and conditions: (a) LiHMDS, THF, allyl iodide, À70 °C to rt; (b)
H2 (1 atm), Pd–C, EtOH, rt; (c) Dess–Martin periodane, CH2Cl2, rt; (d) ArCH2NH2,
NaBH(OAc)3, dichloroethane, rt; (e) NH2OH, KCN, MeOH, H2O, THF, rt.
relate to in vivo efficacy, pharmacokinetic (PK) properties, and spe-
cific structural features of compound classes being evaluated as
potential candidates for pre-clinical studies.
Table 1 provides Ki values for a set of new LFIs that illustrates
the effect of the planned modifications (Fig. 2) on intrinsic potency
against LF. We were pleased to find that when compared to the
aniline series (1, X = NH), interchanging of X-groups (cf. 1a–d vs
4a–d, and 5a–d) while holding the C2 side chain constant provided
In a previous paper,8 we disclosed the discovery of two com-
pound classes (Fig. 1) with high intrinsic potency and good
in vivo efficacy in the rat LT model of LF intoxication. Compound
1a, representing the one-atom linking series, was found to be a
sub-nanomolar LFI capable of providing 100% protection in the
rat LT model when administered IV at 10 mg/kg. Compound 2, a
member of the two atom linking series, while not preventing
LFIs with similar intrinsic potency (
D
Ki 6 10Â), the one exception
being LFI 1a. In the two-atom link series, analogs represented by
3a–d where the C2-side chain of 2 (o = 1, p = 3) was replaced by
that found in series 1 (o = 3, p = 1; Fig. 1) were also prepared. This
Table 1
Anthrax LF inhibitory data for LFIs represented in Figure 2
LFI
R3
LF (FRET) Ki (nM)
1
3
4
5
6
7
8
9
a
b
c
3-Me,4-FPh
4-FPh
4-ClPh
0.05
0.24
1.0
1.5
0.58
1.2
2.4
1.2
2.0
4.6
0.39
0.13
0.71
9.5
0.62
0.81
0.68
1.3
0.93
1.4
3.0
1.1
0.75
—
1.2
0.25
0.36
d
CH2(4-FPh)
2.1
1.1
—
—
—
Values are means of three experiments; standard deviation is <15%; (—) = not prepared.

2032
S. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2030–2033
Table 2
in the ligand binding site. Given that the LF protease is selective
Effect of the Y-group on intrinsic potency
for substrates having Arg or Lys rich sequences on the nonprime
side of the catalytic site,12 the preference for a basic amine group
in the C2-side chain suggests a similar interaction occurs with this
class of LFI during the binding process. While the ether analogs
(Y = O) display intrinsic potencies in a range suitable for small mol-
ecule drugs,13 the Ki values for the all carbon C2-side chain series
(Y = CH2) fall outside this range and were no longer considered tar-
gets for analog synthesis.
LFI
X
Y
LF (FRET)
After exploring a number of modifications to the basic core
structure of our LFIs (Fig. 2) and discovering that most led to only
slight differences in intrinsic potency versus LF, we investigated
whether these changes would affect the ability of these LFIs to pro-
tect animals in the rat LT challenge model. As discussed above, this
model provides a quick read out on the ability of an LFI to block the
action of LF in vivo and also relative metabolic stability and
distribution properties as it relates to the observed efficacy. All
new LFIs were dosed initially at 5.0 mg/kg and those found to
provide complete protection at this dose, re-tested at the lower
dose of 2.5 mg/kg. Given that inter-experimental variability exists
for in vivo models, we normalized the data for each in vivo study
(Tables 3 and 4) to the Median Survival Time (MST) of the control
animals in each experiment. Parameters used to evaluate LFI effi-
cacy were MST, percent survival, and the presence or absence of
morbidity observed in the survivors. In order compare data be-
tween tables, we included a standard compound in each study.
In the first experiment (Table 3), the survival curves9 for all of
the LFIs tested were found to be statistically different (P <0.05) rel-
ative to the vehicle control group. Since a previous study had
shown that LFI 1a was capable of providing 100% protection at
10, 5, and 2.5 mg/kg in the rat LT model,8 LFI 1a served as the stan-
dard to which the new analog data were compared. In this exper-
iment, LFI 1a and two other compounds demonstrated complete
protection when dosed at 5.0 mg/kg, although 1a was unable to
provide full protection the at the 2.5 mg/kg dose. Interestingly,
the removal of the methyl group from the meta-position of the
terminal phenyl ring (R3 = Me to H) to give 1b led to a significant
increase in efficacy providing 100% protection without morbidity
at both the 5 and 2.5 mg/kg doses even though the intrinsic po-
tency of 1b is five-fold lower relative to 1a. This same trend ap-
pears to be present in the two atom linking group series where
3b provides qualitatively better protection relative to the methyl
Ki (nM)
1a
13
14
5a
15
16
NH
NH
NH
CH2
CH2
CH2
NH
O
CH2
NH
O
0.05
30.4
273
0.39
25
CH2
577
Values from three experiments; standard deviation <15%.
change resulted in a 10-fold increase in intrinsic potency against LF
(cf. 3a vs 2) and was our first indication that the position of the
amino-group of the C2-side chain was important for inhibitor po-
tency. Modifications to the aryl ring of each core structure by re-
moval of a methyl group (6a–c, 8a–c, and 9a–c) or the addition
of a methyl group (7a–c) had no significant effect on the intrinsic
potency. Finally, the extension of the C2 side chain below the
N-atom (p = 2, d-series) appeared to slightly decrease potency of
these compounds. Taken together, the similar inhibitory activity
of these LFIs seen with simple changes in the core structure pro-
vides a potential way to modulate the physicochemical (PC) and
PK properties of these compounds without sacrificing intrinsic po-
tency against the target.
Based on the observation that analogs in the 5-amino series
were consistently more potent compared to the 3-amino series
(e.g., 1a vs 2), we were interested in knowing if the secondary
amine was essential for LFI activity (Fig. 2, Y = NH vs O, CH2). The
data in Table 2 show a clear preference for the secondary amine
at the 5-position of the C2-side chain, followed by oxygen and then
a methylene group. Comparing 1a and 5a with their carbon analogs
(Y = CH2, 14, 16), reveals a >1000-fold loss in binding affinity
(
DDG >4.5 Kcal/mol) and suggests the amine may participate in a
directed hydrogen-bond, electrostatic, or specific ionic interaction
Table 3
Rat LT model survival data for LFIs, experiment 1
LFI
Ki (nM)
X
R1
R2
R3
5.0 mg/kg
Survivala
2.5 mg/kg
Survivala
rMSTb
rMSTb
1a
1b
5a
0.05
0.24
0.39
NH
NH
CH2
Me
Me
Me
Me
Me
Me
Me
H
Me
3/3
3/3
2/3
—
—
7.8
0/3
3/3
nt
4.2
—
—
6a
17
8a
0.62
3.8
3.0
NH
NH
O
Me
Cl
Me
H
H
H
Me
H
Me
0/3
0/3
0/3
5.8
1.4
1.3
nt
nt
nt
—
—
—
3a
3b
1.5
0.58
CH2CH(OMe)
CH2CH(OMe)
H
H
H
H
Me
H
1/3
3/3
5.0
—
nt
0/3
—
5.0
Fischer 344 rats were dosed IV with LFIs followed 20–30 min later by 10
l
g of LT (10
l
g PA + 10
l
g LF) dosed IV.
a
Number of survivors per group. Survival curves for all LFIs versus controls showed statistical significance (P <0.05). When comparing curves for individual LFIs at 5.0 mg/
kg, significance (P <0.05) was observed for 1a versus 6a, 6a versus 17, and 6a versus 8a; but was not considered significant for 1a versus 5a, or 3a versus 3b.
b
rMST = MST(LFI)/MST(controls) where vehicle treated controls (MST = 82.3 9.6 min, n = 9); when less than 50% deaths occurred the average survival time was used:
rMST = aveST(LFI)/MST(controls); nt = not tested, (—) = not applicable.

S. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2030–2033
2033
Table 4
ence of survivors for 18 make this trend less convincing, comparing
the data for 6a versus 8a (Table 3, P = 0.025) and subsequent exper-
iments testing this hypothesis (data not shown) provide additional
support for this conclusion. The data in Table 4 also suggest that
an increase in chain length between the secondary amine nitrogen
and the terminal aryl ring does not have a significant impact on
in vivo efficacy (cf. 1b, p = 1–19, p = 3; P = 0.130). In contrast, adding
an additional methylene to the C2 side chain to give 6-amino analogs
(o = 4) provided LFIs with good intrinsic potency (20–23) but no
ability to protect against LF intoxication. This effect is best observed
by comparing 20 and 21 with compound 3b in Table 3 where com-
plete protection by the latter compound was observed at the same
dose of LFI. Further, the two- to three-fold increase in survival time
when comparing 22 and 23 with 4b, while modest (4b vs 22,
P = 0.004) is also supportive of this positional effect. This lack of
in vivo efficacy without a commensurate loss of intrinsic potency
implies a metabolic liability is associated with the secondary amine
at position 6 of the C2 side chain that is absent in the 5-amino series.
In summary, we have used the SAR from in vitro and in vivo
experiments to identify structural features in new LFIs with
improved efficacy relative to 1a and 2 in the rat LT challenge mod-
el. Part 3 of this series will present a closer look into how variation
in the X-group and additional changes to the C2-side chain can
affect in vivo efficacy in the rat LT challenge model.
Rat LT model survival data for LFIs, experiment 2
LFI Ki (nM)
X
R1
R2
R3
o
p
5.0 mg/kg
Survivala rMSTb
1b
5b
4b
18
19
0.24
0.13
1.2
2.0
2.2
NH
CH2
O
O
NH
Me Me
Me Me
Me Me
Me Me Cl
Me Me
F
F
F
3
3
3
3
3
1
1
1
1
3
2/4
1/5
0/5
3/6
1/5
17.0
13.6
2.9
1.3
2.0
F
20
21
22
23
4.6
5.0
3.1
3.3
CH2CH(OMe)
CH2CH(OMe)
O
O
H
H
H
H
F
F
F
F
4
4
4
4
1
2
1
2
0/4
0/4
0/3
0/3
1.4
1.3
1.4
1.1
Me Me
Me Me
Fischer 344 rats were dosed IV with LFIs followed 20–30 min later by 10
(10 g PA + 10 g LF) dosed IV.
Number of survivors per group. Survival curves for all LFIs except 23 versus
lg of LT
l
l
a
controls showed statistical significance (P <0.05). When comparing curves for
individual LFIs at 5.0 mg/kg, significance (P <0.05) was observed for 1b versus 4b,
4b versus 5b, and 4b versus 22; but was not considered significant for 1b versus 19,
or 4b versus 18.
b
Acknowledgements
rMST = MST(LFI)/MST(controls) where vehicle treated controls (MST = 70.7
5.1 min, n = 9); when less than 50% deaths occurred the average survival time was
used: rMST = aveST(LFI)/MST(controls).
We thank the National Institutes of Health for their support of
this work with grants R44 AI052587 and U01 AI078067. Animal
studies were supported by the Intramural Research Program of
the NIH, National Institute of Allergy and Infectious Diseases. The
content is solely the responsibility of the authors and does not nec-
essarily represent the official views of the NIAID or the NIH.
homolog 3a (P = 0.12) suggesting that the R3 methyl group poses a
metabolic liability with respect to in vivo efficacy. In support of
this hypothesis, the C2-side chain present in these compounds fits
the classic pharmacophore model supporting CYP2D6 activity; an
alkylarylamine with a site of oxidation, in this case the benzylic
methyl group, located 5–7 Å from the amine nitrogen atom.14 In
regard to these experiments, the rat possesses six CYP2D isoforms,
four of which are similar to human CYP2D6 in their ability to
metabolize xenobiotic molecules.15 This finding alerted us to the
possibility that the lower efficacy observed in the a-series relative
to the b-series may be due to CYP2D metabolism and should be
considered in future inhibitor design. Comparing LFI 5a with 1a
suggests that the change in the X-group from NH to CH2 had no
measurable effect on activity (5a vs 1a, P = 0.34). What came as a
surprise was the finding that removal of a methyl group (R2) from
the core structure aryl ring of 1a to give 6a, while having only a
minor effect on intrinsic potency (Table 3), resulted in a dramatic
loss of in vivo efficacy (P = 0.026). While this result appears coun-
terintuitive to the metabolic liability expected for decreasing the
number of benzylic methyl groups in a molecule, the data for 8a
and 17 also support this finding.
In a second experiment using LFI 1b as the standard (Table 4),
we were interested in determining the effect of changing the
X-group and variations in chain length on in vivo efficacy. With
the exception of LFI 23, the survival curves9 for each LFI tested
were found to be statistically different (P <0.05) relative to the con-
trol group, though in this case, none of the compounds provided
100% protection. Unlike in the previous experiment where LFI 1b
was fully protective at both doses tested, only 50% protection
was observed in this study at the 5.0 mg/kg dose. Even with this
difference, the data obtained from this experiment still supports
certain trends.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.02.010.
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For example, when comparing a change in the X-group on effi-
cacy, the data for LFIs 1b, 4b, and 5b suggest that the phenoxyacetic
acid derivatives (4, X = O) are less effective in this model than the
aniline (1b vs 4b, P = 0.011) or carbon series (5b vs 4b, P = 0.011).
While the limited data available from this experiment and the pres-
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