Antidotes to anthrax lethal factor intoxication. Part 3: Evaluation of core structures and further modifications to the C2-side chain

Article abstract of DOI:10.1016/j.bmcl.2012.01.095

Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles, and relative efficacy in a rat lethal toxin (LT) m

Full text of DOI:10.1016/j.bmcl.2012.01.095

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2242–2246
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antidotes to anthrax lethal factor intoxication. Part 3: Evaluation of core
structures and further modifications to the C2-side chain
Guan-Sheng Jiao a, , Seongjin Kim , Mahtab Moayeri , Devorah Crown , April Thai ,
a,
b
b
a
a
a
c
a
a
Lynne Cregar-Hernandez , Linda McKasson , Banumathi Sankaran , Axel Lehrer , Teri Wong , Lisa
Johns , Stephen A. Margosiak , Stephen H. Leppla , Alan T. Johnson
a
a
b
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
Advanced Light Source, Lawrence Berkeley Laboratory, Berkeley, CA 94720, USA
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were
evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles,
and relative efficacy in a rat lethal toxin (LT) model of LF intoxication. Poor efficacy in the rat LT model
exhibited by the phenoxyacetic acid series (3) correlated with low rat microsome and plasma stability.
Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine
in the C2-side chain were revealed by X-ray crystallography.
Received 10 November 2011
Revised 24 January 2012
Accepted 24 January 2012
Available online 1 February 2012
Keywords:
Anthrax
Ó 2012 Elsevier Ltd. All rights reserved.
Lethal factor
Inhibitor
In vitro ADME
X-ray crystallography
Anthrax lethal factor (LF) and edema factor (EF) are two pro-
teins secreted by the Gram-positive bacterium Bacillus anthracis
1
to assist in the infection of the host. LF is a zinc metalloproteinase
that disrupts cell signaling pathways by direct cleavage of MEK
2
2+
proteins, while EF is a Ca /calmodulin-dependant adenylate
cyclase that increases cytosolic cAMP and also interferes with cell
3
signaling. When combined with a third protein protective antigen
(
PA), they are known as lethal toxin (LT) and edema toxin (ET). PA
transports EF and LF into cells where they act as potent virulence
4
factors and contribute to the pathogenesis of the disease. The de-
tails of how LF and EF act to suppress the immune system, support
dissemination of the bacteria, and contribute to the lethality of the
disease is beginning to be revealed5 and suggests that inhibiting
the activity of these virulence factors could lead to an increase in
survival of the infected host. Of the three forms of the disease,
death due to inhalation anthrax is significantly higher when com-
pared to fatalities resulting from cutaneous or gastrointestinal
exposure, with fatality rates being >85% without supportive care.
While not contagious, the potential danger from this disease when
used as an of agent bioterrorism was clearly demonstrated by the
Figure 1. Current lead series of anthrax LFIs (1–4).
7
seen even after aggressive treatment with antibiotics. Due to the
relative ease of production and dispersal of anthrax spores, the
potential for mass casualties due to B. anthracis release against
an urban or military population is extremely high. As a result,
new therapeutics are needed to supplement available methods of
treatment and increase the survival rate of patients diagnosed with
inhalation anthrax.
6
2
001 U.S. mail attacks, where fatality rates approaching 50% were
⇑
Corresponding author.
E-mail address: ajohnson@pantherabio.com (A.T. Johnson).
These authors contributed equally to this work.
8
In part 2 of this series we disclosed the identification of four
core structures (Fig. 1) capable of providing anthrax LF inhibitors
0
960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.095

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2242–2246
2243
(
LFIs) with K
i
values of less than 10 nM and in vivo efficacy in a rat
the present structure that the terminal phenyl ring points directly
into the S3 pocket as opposed to the equally accessible S2 subsite12
(see Fig. S2). Of particular interest was the presence of a bridging
water molecule positioned to help stabilize the complex by
H-bonding to both the benzyl amine nitrogen and Glu735. The
clear electron density for the oxygen atom of this water molecule
placed it 2.38 Å from the N-atom of the benzylamine and 2.58 Å
from one of the carboxylate oxygen atoms of Glu735, the other
of which is part of the first coordination sphere of the catalytic zinc
atom. This H-bonding network combined with the SAR discussed
earlier, provides strong support for this interaction as a key con-
tributor to inhibitor potency.
LT model of anthrax. Below we present data from further studies
directed towards evaluation of the current core structures, and
structural modifications which have led to improved in vivo effi-
cacy in the rat LT model.
In a previous report8 we noted the necessity of having a
benzylamine fragment located in the C2 side chain to achieve high
intrinsic potency with this class of LFIs. We also showed that the
replacement of this amine with an oxygen atom (NH to O) to give
the corresponding benzyl ether resulted in a >100-fold loss of
intrinsic potency, and the all carbon chain analogs (NH to CH
played a greater loss (>1000-fold) in potency. This led to the con-
clusion that hydrogen bond or significant electrostatic
interaction was responsible for the observed increase in affinity
of the benzylamine analogs to the LF protein. Since then, we have
obtained a high resolution X-ray crystal structure of LFI 4 bound to
the active site of LF (Fig. 2). Consistent with the LF-inhibitor struc-
2
) dis-
a
a
While clearly important for high binding affinity towards the
target, this benzylamine fragment also fits the classic pharmaco-
1
4
phore model for CYP2D6 inhbitors where the nitrogen is likely
to be H-bonded to Glu216 or Asp301 in the CYP2D6 active site.
Therefore, we sought a way to limit the potential for our LFIs to bind
to this enzyme without sacrificing potency against LF. Since H-
bonding is both distance and angle dependant, one potential way
to disrupt this interaction is to introduce steric hindrance adjacent
to the nitrogen atom. Of the two possible sites for introduction of a
9
ture obtained by the scientists at Merck , the two oxygen atoms of
hydroxamic acid group were found to chelate the catalytic zinc ion.
The orientation and interatomic distances of this group relative to
protein atoms is supportive of H-bonds between the NH-group and
the backbone carbonyl oxygen of Gly657, the carbonyl oxygen with
the hydroxyl group of Tyr728, and the hydroxamate hydroxyl with
the catalytic Glu687 residue. This orientation of the hydroxamic
acid group and the associated H-bonding network is essentially
the same as seen first with hydroxamic acid (HA) based inhibitors
branch point on the C2 side chain, the a-benzyl site appeared to be
most suitable in terms of synthetic accessibility. It should be noted
that at the time these changes in structure were being investigated,
the X-ray crystal structure of 4 discussed above was not yet avail-
able. At the time, consideration of these structural modifications
raised two questions with respect to the potential effect on inhibi-
tor potency; how would the introduction of an R-group at this site,
and the creation of a new stereogenic center, affect the intrinsic
potency and the in vivo efficacy of this class of LFIs?
1
0
thermolysin and later with HA inhibitors bound to the matrix
metalloproteinases (MMPs).11 In contrast to our expectations
based upon the Merck structure we found the C1–C4 axis of the
4
-flourophenyl ring present in the core structure of 4 to be at an
angle of 57° relative to the axis of the 3-methyl-4-fluorophenyl
ring of L915 when bound to LF. This results in a shift of loop
residues Lys673, Gly674, and Val 675 away from the catalytic
zinc-atom and creates a larger S1-prime subsite (see Fig. S1). Early
modeling studies and the structure–activity relationships (SAR) for
these compounds had predicted the core structure of the ligand to
bind on the prime side of the catalytic site, however, we were not
able to identify a single preferred binding mode for the C2-side
chain on the non-prime side with any confidence. It is clear from
To answer these questions our first task was to identify an
efficient synthetic route to stereochemically pure
a-substituted
benzyl amines. Shown in Scheme 1 is the synthesis of the (S)-iso-
butyl-4-fluorobenzyl amine which is representative of the method
used to prepare the needed
a-substituted benzylamines. Com-
pound 5, prepared by reaction of the lithium salt of (S)-isopropyl
oxazolidinone with the acyl chloride derived from 4-fluorophenyl-
acetic acid, was alkylated with methallyl bromide under standard
1
5
conditions. Catalytic hydrogenation using Pd-C provided 6 as a
single diastereomer in good overall yield. Removal of the chiral
auxiliary with lithium hydroperoxide to give carboxylic acid 7
1
6
was followed by a Curtius rearrangement using an aqueous acid
work up to afford the targeted -substituted benzylamine 8. To
a
Scheme 1. Reagents and conditions: (a) (i) LiHMDS, THF, ꢀ78 °C, (ii) methallyl
bromide, ꢀ78 °C to rt, (b) H , Pd-C, EtOH, 3 h at rt, (c) LiOOH, THF/H O, 0 °C, 1 h, (d)
(i) ClCO Et, Et N, acetone, 0 °C, 15 min, (ii) NaN , H O, 0 °C, 30 min, (iii) toluene,
reflux, 45 min, (iv) 8 N HCl, 0 °C to rt.
2
2
Figure 2. Representation13 showing the crystallographic water bridging the amine
of LFI 4 to Glu735 in the catalytic site of LF.
2
3
3
2

2
244
G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2242–2246
Table 1
interactions essential for high affinity binding (see Fig. S3). A sec-
Anthrax LF inhibitory data for LFIs bearing a-benzyl substituents on the C2-side chain
ond finding was that with the exception of R = methyl, the (R)-iso-
mers were more potent by three to fivefold relative to analogs of
the (S)-configuration. When considering both of these parameters,
a change in potency of 50-fold can be observed (cf. 11 vs 16). Per-
haps most important, it should be possible to modify the drug-like
properties of a given LFI by introduction of various R-groups to this
site with minimal concern for size or absolute stereochemistry.
Having identified structural features which provided sub
LFI
(⁄)
R
i
LF (FRET) (K nM)
-
nanomolar LFIs in four core structures, we set out to see if one
9
R
S
R
S
R
S
R
S
Me
Me
Et
Et
iso-Butyl
iso-Butyl
–CH
–CH
0.30
0.49
0.04
0.11
0.23
1.30
0.60
2.00
or more of these structural classes would be preferred for the lead
optimization phase of this work. Towards this end we chose to
compare in vitro potency, toxicity, physicochemical (PC) proper-
ties, and in vivo efficacy of matched pairs of LFIs representing each
10
11
12
13
14
15
16
core structure bearing an unsubstituted or
amine on the C2-side chain. Based on the results presented in Table
, and to ease the burden of synthesis, we selected the racemic
-ethylbenzylamine analogs for our comparison of C2-side chain
effects. The data collected from these studies is provided in Tables
a-substituted benzyl-
2
Ph
Ph
2
1
a
Values are the average of two experiments; sd <15%
2
and 3 below.
investigate this modification in the C2-side chain, we selected the
phenylpropionic acid core series (2) due to their ease of synthesis.
These LFIs were then prepared following our previously published
procedure involving reductive amination of the benzylamine re-
agent with the C2-side chain aldehyde connected to the phenyl-
The in vitro potency, potential for toxicity, PC properties, and
the activity of these compounds versus possible anti-targets
(MMPs, CYP2D6, and hERG) is provided in Table 2. In terms of
intrinsic potency, the analogs based upon the phenoxyacetic acid
core structure are the least potent, though they still possess K val-
ues <2 nM. Cytoxicity is very low for all of the analogs with the ob-
i
8
propionic acid core.
Inhibitory data for these LFIs against anthrax LF are provided in
Table 1.1 As a first observation, the size of the R-group can have a
significant effect on the intrinsic potency of the LFI, with the most
potent inhibitor bearing an ethyl group at the benzylic position. Gi-
ven that this work was conducted prior to obtaining the X-ray
structure of 4 bound to LF, we were somewhat surprised to see that
introduction of a large substituent such as benzyl (15) can still af-
ford sub-nanomolar inhibitors of LF. By examination of the crystal
served CC50 values being 20K-fold greater than their respective Ki
values. The PC properties for all of the core structures are very sim-
ilar. Molecular mass is well below 500 Da for each series and dif-
fers by <50 mass units. ClogP values are on the high side of the
preferred range with all being >3.0 but still less than 5.0 while
the calculated polar surface areas are at the lower end of the pre-
ferred range of 75 Å < PSA < 125 Å. Determination of relative per-
2
1
7
meability using a hexadecane based PAMPA assay show all of
1
2
structure of 4, it can be easily seen how a large R-group attached
the LFIs to have fair to good permeability with logP values being
e
to the
a-position can be accommodated without disrupting the
>ꢀ6.0. While no apparent trend emerges with respect to the
Table 2
The in vitro potency, potential for toxicity, and PC properties for match pairs of LFIs representing each of the four core structures
a
CC50a
ClogPb
PSAc (Å2)
PAMPAd (logP
M)a
ꢀ12
>50
>50
>50
>50
>50
>50
2.3
CYP2D6a
hERGa
(lM)
LFI
1
MW
K
i
(nM)
(l
M)
e
)
MMP K
i
(l
(l
M)
ꢀ
1
ꢀ3
ꢀ9
ꢀ14
391.2
419.2
390.2
418.2
392.2
420.2
406.2
434.3
0.24
0.17
0.13
0.31
1.20
1.00
0.58
0.27
61
32
16
15
26
21
55
35
3.7
4.6
4.0
4.8
3.8
4.7
3.2
4.0
92.6
88.1
88.9
74.6
86.9
83.9
81.9
80.1
ꢀ5.9
ꢀ5.1
ꢀ4.8
ꢀ4.1
ꢀ5.2
ꢀ4.4
ꢀ5.8
ꢀ5.1
>50
>50
>50
>50
>50
>50
33
>50
>50
>50
>50
>50
>50
>50
31
>50
>50
>50
>50
>50
>50
21
>50
>50
>50
>50
>50
>50
>50
>50
1.35
0.24
0.02
0.08
0.23
0.34
1.26
0.16
1.23
0.95
4.65
13.3
0.79
2.7
17
2
18
3
19
4
20
2.0
10.0
47
11
12
a
b
c
See Supplementary data for experimental procedures; IC50 values given for CYP2D6 and hERG.
ClogP calculated with ChemDraw v12.0.
PSA calculated using PCModel v. 8.5.
d
17
PAMPA determined by the method of Wohnsland using Mllipore multiscreen 96-well plates

G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2242–2246
2245
Table 3
The in vitro ADME and rat LT challenge assay data for match pairs of LFIs representing each of the four core structures
a
CC50a
ClogPb
ElogDc
In vitro ADMEd
P
5.0 mg/kge
surv rMST
2.5 mg/kge
1.25 mg/kge
surv rMST
LFI
K
i
(nM)
(lM)
M
U
surv
rMST
1
0.24
0.17
0.13
0.31
1.20
1.00
0.58
0.27
61
32
16
15
26
21
55
35
3.7
4.6
4.0
4.8
3.8
4.7
3.2
4.0
2.3
3.3
2.2
2.9
3.1
3.3
2.4
2.9
60
75
18
55
3
7
91
75
90
84
100
95
61
38
38
74
79
100
76
75
3/3
3/3
3/3
3/3
0/3
1/3
2/3
3/3
—
—
—
—
16.0
16.0
16.0
—
3/5
3/3
1/3
3/3
nt
nt
nt
2/5
16.8
—
19.3
—
—
—
nt
0/3
nt
5/6
nt
nt
—
2.4
—
4.2
—
—
1
2
1
3
1
4
2
7
8
9
0
98
94
99
100
—
16.8
nt
nt
—
—
a
b
c
See Supplementary data for experimental procedure.
ClogP calculated with ChemDraw v12.0.
ElogD measure using the method of Lombardo.
15
d
e
Values given indicate percent of compound remaining at t = 30 min for M = miocrosome stability, U = UGT assays and the percent remaining at 3 h for P = plasma stability.
Fischer 344 rats were dosed IV with LFIs follwed 20–30 min later by 10 g of LT (10 g of PA + 10 g of LF); surv. = Number of survivors per group. rMST = MST(LFI)/
l
l
l
MST(controls) where vehicle treated controls (MST = 74.8 ± 7.2 min (n = 11; when less than 50% deaths occurred the average survival time was used: rMST = aveST(LFI)/
MST(controls); nt = not tested, (ꢀ) = not applicable. Survival curves for all LFIs at each dose showed statistical significance (P <0.05) relative to controls.
Table 4
PK data for LFI 20 after single dose of 10 mg/kg (SC; DMSO:PEG400:pH6 citrate buffer,
served in the rat LT challenge experiments and aid in selection of
2
:10:88) in NZW rabbits.
future candidates for in vivo studies. The results of this work are
provided in Table 3. We also determined the ElogD values for these
compounds which show good correlation with logD7.4 values when
K
i
0.27 nM
Dose (SC)
10 mg/kg
0.3 h
561.3 ng/mL (1.3
5.3 h
2637.6 ng h/mL
79 nM
T
max
max
1/2(b)
AUC
20]12 h
19
applied to neutral or basic compounds. In this case, the data
C
lM)
shows these LFIs to have the potential for better drug-like proper-
ties at physiologic pH when compared to evaluation of the ClogP
values alone.
t
1
[
As in our previously studies, the rat LT challenge study was con-
ducted by dosing Fischer 344 rats (IV) with the LFI followed 20–
3
0 min later by a 10 lg dose (IV) of LT (10 lg PA + 10 lg LF). When
specific core structures, better permeability is seen with the
ethyl analogs 17 to 20. Selectivity for LF versus various MMPs
was excellent, being >50K-fold for the one atom linking group ser-
a
-
administered at a dose of 5.0 mg/kg (Table 3), five of the eight LFIs
provided 100% protection, one animal was lost in the group treated
with LFI 4, but only a single animal survived from the two groups
treated with the LFIs based on the phenoxy acetic acid core struc-
ture (3 and 19). This finding was qualitatively consistent with the
observed in vitro ADME data where 3 and 19 displayed the lowest
stability in the microsome and plasma stability assays among all of
the compounds tested. Of particular significance was the low met-
abolic stability for these compounds in the rat liver microsome as-
say where <10% of either compound remained after incubation for
30 min. This observation combined with the fact that the animals
were challenged with lethal toxin 20–30 min after receiving the
LFI is consistent with the inability of 3 and 19 to provide significant
protection from the lethal effects of LF in this experiment. In an at-
tempt to probe further into possible differences in efficacy due to
the core structure, the LFIs which provided full protection at
5.0 mg/kg were then tested at 2.5 mg/kg. In this case, two of the
five compounds tested (17 and 18) were still able to protect all
of the animals in their respective groups. The finding that the LFIs
ies (X = NH, CH
sented by LFI
2
, O) and >4000-fold for the
c-ether series repre-
4
and 20. Consistent with the current
pharmacophore models for binding to CYP2D6, many of these com-
pounds were found to be strong inhibitors of this enzyme. Further,
when comparing analogs where R = H versus R = ethyl, introduc-
tion of steric hindrance by a simple alkyl group at the benzylic po-
sition did not decrease CYP2D6 inhibitory activity. Activity against
hERG was considerably lower compared to CYP2D6, but still signif-
icant (<10 lM) for all but two analogs. Taken together, these data
did not show a bias towards any one core structure in terms their
suitability for the lead optimization process, however, the potential
for each series to significantly inhibit CYP2D6 and hERG was made
clear.
Continuing our investigation of the four core structures, the
same set of eight LFIs were tested in various in vitro ADME assays
and finally in the rat LT challenge assay.18 A concurrent goal of
these studies was to determine if the data obtained from the
in vitro ADME assays would correlate with survival outcomes ob-
which perform best were
a
-substituted benzylamine analogs,
-ether analogs 4
combined with a similar trend seen with the
c

2
246
G.-S. Jiao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2242–2246
(
R = H) and 20 (R = Et), indicates that
a-substitution appears to
LT challenge studies were supported by the Intramural Research
benefit survival in this model of anthrax LT intoxication. In a final
experiment LFIs 17 and 18 were tested at 1.25 mg/kg. At this dose,
neither LFI was able to fully protect the animals from death due to
LF, however, the phenylpropionic acid derivative 18 appeared to be
more effective (5/6 survivors) compared to the aniline based ana-
log 17 (0/3 survivors).
Program of the NIH, National Institute of Allergy and Infectious
Diseases. The PK study was conducted by Covance Laboratories,
Inc. (Madison, WI). The content is solely the responsibility of the
authors and does not necessarily represent the official views of
the NIAID or the NIH.
The pathogenesis of inhalation anthrax observed in the rabbit is
considered to be a good representation of the disease course in hu-
Supplementary data
2
0
mans. In preparation for these studies we determined the PK pro-
file of LFI 20 in NZW rabbits. Since the current protocol call for the
administration of the drug over several days, and may require mul-
tiple doses of the test compound per day, we selected the subcuta-
neous (SC) route of administration for this initial study. The PK
data for LFI 20 shown in Table 4 indicates that this compound is
rapidly absorbed when dosed at 10 mg/kg via the SC route and re-
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.095.
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value which
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6
In summary, evaluation of four core structures capable of pro-
viding sub-nanomolar inhibitors of anthrax lethal factor was
accomplished by comparing the potential for toxicity, PC proper-
ties, in vitro ADME profiles, and relative efficacy in a rat LT model
of LF intoxication. Based upon these results, the phenoxyacetic acid
core structure appears to be inferior to the other three tested. All of
the core structures provided analogs which had significant inhibi-
tory activity against CYP2D6 and hERG indicating that new analogs
should be tested against these anti-targets as part of the lead can-
didate selection process. Addition of an R-group at the benzylic po-
sition of the C2-side chain was well tolerated with respect to
intrinsic potency, was consistent with the X-ray structure of LFI
7. Jernigan, J. A.; Stephens, D. S.; Ashford, D. A.; Omenaca, C.; Topiel, M. S.;
Galbraith, M., et al Emerg. Infect Dis. 2001, 7, 933.
8.
Kim, S.; Jiao, G.-S.; Moayeri, M.; Crown, D.; Cregar-Hernandez, L.; McKasson, L.;
Margosiak, S. S.; Leppla, S. H.; Johnson, A. T. Bioorg. Med. Chem. Lett. 2011, 21,
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Shoop, W. L.; Xiong, Y.; Wiltsie, J.; Woods, A., et al Proc. Natl. Acad. Sci. U.S.A.
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12. See Supplementary data for details on experimental procedures and additional
figures from the crystallographic study.
1
3. This image was made with VMD which was developed with NIH support by the
Theoretical and Computational Biophysics group at the Beckman Institute,
University of Illinois at Urbana-Champaign. http://www.ks.uiuc.edu/Research/
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1
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bound to the catalytic site of LF, but did not decrease inhibitor
potency against CYP2D6. In addition, this modification appeared
to provide analogs with improved protection profiles in the rat
LT challenge model. Compound 20 was also found to have a good
PK profile when administered SC to NZW rabbits and supports
the potential of these inhibitors to provide protection in a rabbit
spore challenge model of inhalation anthrax.
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e3130. Note: this assay was not designed to model inhalation anthrax in
humans but to demonstrate that a small molecule LFI can prevent death due to
anthrax lethal factor in an in vivo model. The protocol used provides for a
reproducible assay with the resolution necessary to rank order the in vivo
efficacy of LFIs in an animal model where death results specifically from the
action of anthrax LF.
Acknowledgments
19. Lombardo, F.; Shalaeva, M. Y.; Tupper, K. A.; Gao, F. J. Med. Chem. 2001, 44,
We thank the National Institutes of Health for their support of
this work with Grants R44 AI052587 and U01 AI078067. The rat
2490.
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