Small molecule inhibitors of anthrax lethal factor toxin

Article abstract of DOI:10.1016/j.bmc.2013.11.009

This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like compounds.

Full text of DOI:10.1016/j.bmc.2013.11.009

Bioorganic & Medicinal Chemistry 22 (2014) 419–434
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Small molecule inhibitors of anthrax lethal factor toxin
⇑
John D. Williams, Atiyya R. Khan, Steven C. Cardinale, Michelle M. Butler, Terry L. Bowlin, Norton P. Peet
Microbiotix, Inc., Department of Medicinal Chemistry, One Innovation Drive, Worcester, MA 01605, United States
Microbiotix, Inc., Department of Molecular Biology, One Innovation Drive, Worcester, MA 01605, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
This manuscript describes the preparation of new small molecule inhibitors of Bacillus anthracis lethal
factor. Our starting point was the symmetrical, bis-quinolinyl compound 1 (NSC 12155). Optimization
of one half of this molecule led to new LF inhibitors that were desymmetrized to afford more drug-like
compounds.
Received 27 August 2013
Revised 29 October 2013
Accepted 5 November 2013
Available online 13 November 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Bacillus anthracis
Anthrax
Lethal factor
Botulinum neurotoxin A
Light chain
Zinc metalloprotease
Matrix metalloprotease
Quinoline
Hybrid compound
Desymmetrized
1
. Introduction
Aerosolized spores of Bacillus anthracis represent one of the
However, for approximately ten years, the Centers for Disease Con-
trol (CDC) and public health authorities have been investigating
cases of bioterrorism-related anthrax exposure and death in the
4
most serious bioterrorist threats to the security of the United
States. Inhalation of these environmentally stable spores pro-
gresses rapidly to a highly fatal systemic infection. In 2001, this
category A agent was used in several acts of terrorism in the United
States. While ciprofloxacin and doxycycline were effective if
administered immediately after suspected contact with B. anthra-
cis, there is little doubt that capable terrorists will seek to develop
forms of the bacterium resistant to these common antibiotics,
especially since mechanisms of resistance are known and resistant
alleles may be generated readily. New therapeutic agents based on
novel chemical scaffolds are vital to biodefense because they are
likely to be effective against both natural and engineered resistant
forms of B. anthracis, and because there are no pre-existing resis-
tance alleles.
United States. The CDC recognizes B. anthracis as a category A
agent of bioterrorism. It is a serious bioterrorism threat because
its spores are stable under extreme conditions in the environment,
are easily produced and distributed by aerosol (in a powder form),
5
and are highly fatal via inhalation. Penetration of spores via the
skin or gastrointestinal tract is generally less dangerous and results
in mainly localized disease.
Therapy recommended by the CDC includes the use of cipro-
floxacin or doxycycline as well as rifampin, vancomycin, imi-
6
penem, clindamycin or chloramphenicol. Beta-lactamases have
been found in recent clinical isolates, precluding the use of penicil-
6
lins for the treatment of anthrax. In addition, researchers have
been able to generate ciprofloxacin resistant and doxycycline resis-
7
,8
tant strains of B. anthracis in the laboratory, which demonstrates
that unscrupulous scientists could engineer drug resistance into
strains of the species.
Bacillus anthracis is the cause of the acute and often lethal dis-
1
ease called anthrax. In humans, anthrax is a rare infection ac-
quired by inhalation, ingestion or cutaneous contact with the
endospores of B. anthracis.2 Anthrax infection has, until recently,
been observed mostly in farm animals and in humans who have
had direct contact with contaminated animals or animal products.
The B. anthracis bacterium-secreted endotoxin is comprised of
three components: (1) a zinc metallopreotease lethal factor (LF);
(2) a calmodulin-activated edema factor adenylate cyclase (EF);
,3
9
and (3) a protective antigen (PA). Although these proteins are
independently nontoxic, their concerted action disrupts cell signal-
ing events and can lead to cell death. Importantly, lethal factor
combines with protective antigen to form the lethal factor toxin.10
⇑
Corresponding author. Tel.: +1 508 757 2800.
E-mail address: npeet@comcast.net (N.P. Peet).
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.009

4
20
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
When lethal factor is translocated into the cytoplasm of host target
cells, lethal factor cleaves MAP kinase kinases (MAPKKs) and dis-
rupts the signaling pathway mediated by these MAPKKs. NOD-like
receptor (NLR) Nlrp1 protein has recently been identified as a
Peptides and peptide-based analogs have also been shown to
inhibit anthrax toxin. In one approach, polyvalent peptide inhibi-
tors, which block LF or EF association with the PA pre-pore, have
2
4
been shown to protect rats from lethal toxin. A second peptide
approach targeted the LF active site. LF has a deep and long
(40 Å) groove, with an overall negative electrostatic potential, con-
tiguous to the active site, that binds peptide substrates and peptide
1
1–13
physiologically relevant substrate of anthrax lethal toxin,
and interactions of anthrax lethal factor with protective antigen
14
has been defined by site-directed spin labeling studies.
2
5,26
Lethal factor plays a critical role in all stages of anthrax infec-
tion; in the early stages it assists the bacteria by helping evade
the host immune system, and in the later stages, when infection
is systemic, it targets the epithelial cells to cause vascular barrier
inhibitors.
Several peptide inhibitors of LF activity have been
identified on the basis of consensus sequences of the MAP-
2
4,26–29
KKs.
Incorporation of zinc-binding chemical groups into
3
0
these peptides greatly enhanced their potency.
1
5,16
dysfunction.
Zinc metalloprotease enzymes are a well-studied
The LF target is attractive because it is a zinc-dependent metal-
loprotease enzyme, and there is a wealth of information that has
been assembled for the inhibition of this class of enzyme; a crystal
structure for a rhodanine derivative complexed with lethal factor
class of enzymes and many are drug targets for which potent
inhibitors have been designed.17 For these reasons, we chose to ex-
ploit a lethal factor inhibitor that we discovered in a screening
campaign, and to optimize this screening hit for potential use as
an antidote for anthrax intoxication. We specifically chose a non-
hydroxamic acid inhibitor to increase our chances of finding a
drug-like, metabolically stable inhibitor that would allow us to de-
velop a series of compounds with improved pharmacokinetic prop-
erties and fewer undesired interactions.
3
1,32
has been reported.
Several research groups have developed
small molecule LF inhibitor programs based on either structural
information or high-throughput screening hits; a collection of small
molecule inhibitors of anthrax lethal factor is shown in Figure 2.
3
3
The bis-quinoline 1 is the screening hit around which we began
our synthetic efforts of hit and lead optimization. Benzopyranone
3
4
In Figure 1 is shown a cartoon that displays a rudimentary
understanding of binding, assembly, uptake and mechanism of ac-
tion for B. anthracis. A variety of approaches have been taken to
intervene at different points of this process to prevent anthrax tox-
in function. Human antibodies against PA that block receptor bind-
2
was shown to be an inhibitor of Botulinum neurotoxin A in addi-
tion to LF. A class of acylhydrazones represented by compound 3
3
5
also blocked the cleavage of MEK1 in 293T cells. The use of a
hydroxythiopyranone core as a hydroxamic acid replacement led
to the synthesis of compound 4, which represented a class of inhib-
1
8
ing and antibodies specific for lethal factor (LF) that interfere
itor that was more potent than the corresponding hydroxypyro-
19
36,37
with assembly of the toxin components have been shown to pre-
vent lethal toxin induced death in rodents. Soluble TEM8/ATR and
nes.
Furan 5 was one of several compounds discovered in a
HPLC-based high throughput screen in which ‘fragment-based
focusing’ was used to enrich the pool of potential inhibitors.38 Re-
lated compound 6 was designed using an iterative NMR-based
binding assay of LF inhibitors and confirmed by X-ray crystallogra-
CMG2 proteins have also been shown to be effective in cell culture,
preventing anthrax toxin receptor binding.2
0,21
Hexa-D-arginine, a
furin inhibitor that blocks PA cleavage, has been shown to delay
anthrax toxin-mediated cell cytotoxicity in vitro and reduce lethal
toxin mediated lethality in rats.22 Dominant-negative PA mutants
have been generated, which co-assemble with the wild-type PA
protein, preventing translocation of LF or EF across the cell
3
2
phy. Chiral sulfonamide 7 is one of the most potent LF inhibitors
15,16
reported;
a synthetic route using asymmetric hydrogenation
3
9
was also reported for this inhibitor. Hydroxamic acids, which
are known zinc-binding agents that have been widely used for
the inhibition of a variety of metalloproteases, have also been used
2
3
membrane.
Figure 1. Potential drug targets and points of intervention for the prevention of Bacillus anthracis function.

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
421
S
OH
OH
NH
2
NH
2
H
N
H
N
O
S
H
N
H
O N
N
2
N
O
HO
O
O
OH
O
N
N
OH
OH
NSC 12155 (1)
i
DS-998 (3)
i
AM-2S (4)
IC50 = 14 mM
(Lewis, 2006)
ASDI 150016121 (2)
i
K = 0.5 mM
K
= 1.1 mM
K = 0.57 mM
(Panchal, 2004)
(Min, 2004)
(Johnson, 2007)
NHOH
O
F
O
O
O
O O
H
N
O
O
O
MeHN
N
COOH
N
S
N
S
HOHN
S
NH
HO
O O
S
NH
OH
S
Cl
O
ST032629 (5)
K = 3.1 mM
BI-MFM3 (6)
"
K
(
LFI" (7)
= 24 nM
Shoop, 2005)
8
M444264 ( )
i
IC = 1.7 mM
5
0
(Schepetkin, 2006)
i
IC50 = 7 mM
(
Forino, 2006)
(
Chiu, 2009)
Figure 2. Previously reported anthrax lethal factor inhibitors.
for the inhibition of LF. A representative peptidomimetic hydroxa-
mic acid is compound 8. Not shown in Figure 2 are miscellaneous
compound 15 using catalytic hydrogenation gave the correspond-
ing 6-amino compound 16, which we used as a versatile interme-
diate for the synthesis of non symmetrical functionalized
quinolines.
9
additional reported inhibitors of lethal factor protease, including
4
0
41
guanidinylated derivatives of neamine and streptamine, amino-
4
2
43
glycosides, cationic polyamines, and inhibitors from green
The versatile intermediate 16 was subsequently used for the
preparation of amides and ureas as shown in Scheme 2. Target
compounds were prepared with a wide variety of substituents
to determine important features required for good anti-LF activ-
ity. Replacing the second quinoline and forming unsymmetrical
target compounds was an important feature of the newly synthe-
sized inhibitors. Amide intermediates 17a–pp were prepared by
treating 16 with different carboxylic acids, using a standard
amide coupling procedure, followed by deprotection of the
bis-Boc amine with trifluoroacetic acid, to give the amide target
compounds 18a–pp. Likewise, treatment of 16 with isocyanates
followed by the same deprotection procedure afforded the
bis-Boc protected intermediates 19a–i and target ureas 20a–i,
respectively.
Our initial assay results (Table 1) showed that two compounds
in particular, 18m and 18t, were potent inhibitors of LF. Since we
determined that both of these compounds were noncompetitive
inhibitors of LF, and we did not have any knowledge of where these
compounds were binding, we chose to use medicinal chemistry
strategies other than molecular modeling to pursue optimization
of these structures. To further explore the features of these inhib-
itors that provide potent anti-LF activity, two additional series of
compounds were synthesized; one exploring the nature of the
4
4
tea.
In this report we discuss the design and synthesis of additional
quinoline-based inhibitors of LF related to compound 1. Impor-
tantly, we report the synthesis of inhibitor structures that are dif-
ferentiated from the symmetrical bis-quinoline 1, which represent
an expected improvement in pharmacokinetic properties.
2
. Chemistry
To most efficiently probe the different structural requirements
for potent anti-LF activity, we envisioned the preparation of a
key intermediate that would be useful for the synthesis of unsym-
metrical LF inhibitors. The successful preparation of this versatile
diaminoquinoline intermediate, with differentiated amino groups,
which we used for the synthesis of many of the compounds in this
study, is shown in Scheme 1. Condensation of 4-nitroaniline (9)
with ethyl 3-ethoxy-2-butenoic acid (10) gave 6-nitro-2-methyl-
4
5
quinolin-4-one (11). Subsequent conversion of quinolinone 11
to the corresponding 4-chloro compound (12), followed by dis-
placement of the chloro group with azide gave 6-nitro-4-azido-
quinoline (13), which was then reduced using
a standard
Staudinger procedure to provide 4-amino-2-methyl-6-nitroquino-
line (14). Aminoquinoline 14 was converted to the bis-Boc pro-
tected 4-aminoquinoline 15 with Boc anhydride. Reduction of
(
(
quinolinyl)phenyl substituent of 18m, and one exploring the
2-methoxyphenyl)propyl substituent of 18t.
NO
2
O
Cl
N
N3
9
NO2
NO2
NO2
a
b
c
H
2
N
+
OEt
66%
N
H
70%
91%
N
O
11
12
13
1
0
OEt
d
95%
N(Boc)2
N
N(Boc)2
NH
2
NH
2
NO2
NO2
f
e
7
8%
N
74%
N
16
15
14
2 4 3 3
Scheme 1. Synthesis of versatile diaminoquinoline intermediate 16. Reagents and conditions: (a) H SO (cat.), tetrahydronaphthalene, reflux; (b) POCl , reflux; (c) NaN ,
DMF/H O, 100 °C; (d) PPh , THF; (e) Boc O, pyridine, 60 °C; (f) ammonium formate, Pd/C, MeOH.
2
3
2

4
22
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
N(Boc)2
N
its entirety. We also incorporated the nitrogen atom of the quino-
NH2
line in the same relative position by appending a pyridine ring to
the 4-position of the 3-phenylpropionic acid. Thus, hybrid com-
pound 35 incorporated the functionalities of both compounds
18m and 18t that we felt might be necessary for the biological
activities of the single molecules.
Synthesis of the hybrid compound 35 is shown in Scheme 5.
This preparation allowed us to make good use of previously pre-
pared building blocks. Treatment of phenylpropionic acid 34b, pre-
pared as shown in Scheme 4, with 3-pyridylboronic acid under
Suzuki coupling conditions, afforded the pyridyl-substituted phen-
ylpropionic acid 36. Coupling of 36 with aminoquinoline 16
(Scheme 1) using HATU provided amide 37, which was then trea-
ted with TFA to remove the amine protecting groups and produce
the hybrid compound 35.
a
c
16
N(Boc)2
N(Boc)2
H
N
H
N
H
N
R
R
O
7a-pp
O
N
N
1
1
9a-i
b
b
NH2
N
NH2
H
N
H
N
H
N
R
R
O
O
N
20a-i
1
8a-pp
3
. Results and discussion
Scheme 2. Synthesis of amide and urea target compounds. Reagents and condi-
tions: (a) HATU, DIPEA, DMF; (b) TFA, CH Cl ; (c) isocyanate, toluene, reflux.
Inhibitory activities of selected compounds against lethal factor
2
2
(
LF), botulinum neurotoxin A (BoNT/A), and matrix metallopro-
teases (MMPs) 1 and 9, as measured in a FRET-based assay are
shown In Table 1. The FRET-bases assay uses a SNAP-based peptide
that is cleaved by LF, which can be measured by a change in fluo-
rescence. BoNT/A, MMP-1 and MMP-9 represent important profil-
ing enzymes used as a gauge of target specificity; the inhibition
of these enzymes was also measured using the corresponding
FRET-tagged peptide substrate. The highly diverse collection of
amides selected for synthesis in Table 1 was chosen to provide a
variety of structures to survey this particular area of the binding
site. Notably, there were two structures that emerged as the high-
est affinity structures; these were then used as templates for the
selection of compounds for Tables 2 and 3. Thus, the (2-methoxy-
phenyl)propionic acid amide 18t displayed good affinity
In the former case, a pair of versatile intermediates (17qq and
7rr) were synthesized and subjected to Suzuki coupling reactions
1
with a variety of quinolinylboronic acids (Scheme 3). Thus, cou-
pling of the 3-iodobenzamide 17qq with quinolinylboronic acids
gave cross-coupled products 21a–d, which were deprotected with
trifluoroacetic acid to produce target compounds 22a–d. In similar
fashion, we prepared the 3-position linked isomers 24a–e, using
the 3-iodobenzamide 17rr in the Suzuki coupling protocol, and
by proceeding through the bis-Boc intermediates 23a–e.
In the case of 18t, we synthesized a range of analogs designed to
test the nature of the linker moiety and the substituents on the
phenyl ring. These were synthesized using commercially available
(IC50 = 3.0
>100 M) as did the very different 4-(6-quinolinyl)benzoic acid
amide 18m, whose IC50 value versus LF was 1.6 M and whose
affinity for BoNT/A was 39 M. Compound 18m became the proto-
lM) for LF and good selectivity versus BoNT/A (IC50
3
-phenylpropionic acids using the coupling reactions found in
l
Scheme 2. Additionally, our initial molecular modeling suggested
that a halogen on the 4- or 5-position of the 2-methoxyphenyl sys-
tem could improve the binding affinity of the inhibitors (results
not shown). The requisite halogenated 3-phenylpropionic acid
starting materials were synthesized using two different methods,
as dictated by the availability of starting materials. Thus, the
l
l
type for the compounds that were prepared in Table 2, and com-
pound 18t was the prototype for the compounds shown in Table 3.
We first turned our attention to prototype compound 18m,
and prepared many isomers of this compound as shown in Ta-
ble 2. Interestingly, all of the isomers of 18m did retain some
inhibitory activity for LF. The most active isomers were com-
pounds 22a, 24a and 24b, wherein the point of attachment to
the quinoline ring is furthest from a bridgehead position. The
remaining isomers, all of which demonstrated less activity than
22a, 24a and 24b, (i.e., double digit micromolar inhibitory in-
stead of single digit micromolar activity), have quinoline attach-
ment points that are adjacent to the bridgehead positions. Thus,
we believe that the protein pocket that accepts the quinoline
ring has a definite preference for the point of attachment, but
that the nitrogen atom in the quinoline nucleus can reside in
a variety of positions.
2
-methoxy-5-halo-3-phenylpropionic acids were prepared using
the first procedure shown in Scheme 4, starting from 2-methoxy-
-halobenzaldehydes. Wittig homologation of 25a and 25b with
5
trimethyl phosphonoacetate gave the corresponding cinnamic es-
ters 26a and 26b, which were catalytically hydrogenated to pro-
vide the saturated phenylpropionic esters 27a and 27b.
Hydrolysis of these esters gave the phenylpropionic acids 28a
and 28b. In the second sequence shown in Scheme 4 2-methoxy-
-substituted benzoic acid methyl esters 29a and 29b were re-
duced with lithium borohydride to give the corresponding benzyl
alcohols 30a and 30b, which were converted to cinnamic esters
2a and 32b in a two-step process. Oxidation of the benzyl alco-
4
3
hols using Swern conditions provided the intermediate corre-
sponding benzaldehydes 31a and 31b, which were then
converted by Wittig homologation to the cinnamic esters. Reduc-
tion of cinnamic esters 32a and 32b, using a rhodium catalyst to
selectively reduce the double bond and not hydrogenate the aryl
halide, gave the 3-phenylpropionic esters 33a and 33b, respec-
tively, which were then hydrolyzed to the corresponding phenyl-
propionic acids 34a and 34b.
Based on the activity of the best bis-quinolinyl amide com-
pound (18m) and the best simple amide compound (18t), we de-
signed a ‘hybrid’ compound by comparing the structures as
shown in Figure 4. In this design, we incorporated amide 18t in
Compounds related to 18t (Table 3) define very distinct and
interesting structure activity relationships (SARs). Remarkably,
when the 2-methoxy group was moved to the 3-position (18v)
or the 4-position (18u), LF inhibitory activity was lost. Addition
of a second methoxy group to the 3-position (18x) or the 5-po-
sition (18w) also led to a complete loss of activity. Addition of
bromo (18ee) or chloro (18ff) to the 5-position also led to inac-
tive compounds, although addition of these substituents to the
4-position (18hh and 18gg, respectively) was tolerated to some
extent, since these compounds had reduced affinities (58
and 78 M, respectively). Benzyloxy (18y), chloro (18z) and
methyl (18aa) groups instead of methoxy in the 2-position were
lM
l

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
423
Table 1
LF and BoNT/A inhibitory activity of selected amides
NH
2
H
N
R
IC50 (lM)
O
N
Compd #
R =
(see Fig. 1)
LFa
3.2
BoNT/Ab
7.2
MMP-1c
31
MMP-9d
1
NSC 12155
100
O
1
8a
70
>100
>100
>100
1
8b
73
>100
n.d.
n.d.
O
1
1
8c
>100
>100
>100
47
n.d.
n.d.
n.d.
n.d.
N
O
8d
1
1
8e
8f
>100
>100
>100
27
n.d.
n.d.
n.d.
n.d.
NMe
2
O
N
N
1
1
8g
8h
52
81
>100
>100
n.d.
n.d.
n.d.
n.d.
N
O
1
1
8i
8j
90
44
>100
35
n.d.
n.d.
n.d.
OMe
HN
>100
1
1
8k
8l
48
43
>100
23
n.d.
n.d.
>100
>100
N
N
1
8m
1.6
38
n.d.
1.7
1
1
1
8n
8o
8p
17
55
33
69
n.d.
>100
n.d.
NMe
OMe
2
>100
>100
n.d.
O
>100
>100
n.d.
O
1
1
8q
8r
>100
>100
>100
>100
n.d.
n.d.
O
O
n.d.
(
continued on next page)

4
24
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
Table 1 (continued)
NH
2
H
N
R
IC50 (lM)
O
N
Compd #
R =
LFa
BoNT/Ab
>100
MMP-1c
n.d.
MMP-9d
n.d.
1
8s
8t
>100
MeO
1
3.0
>100
n.d.
>100
Not determined.
a
Bacillus anthracis lethal factor.
b
c
Clostridium bolulinum neurotoxin A.
Human matrix metalloprotease-1.
Human matrix metalloprotease-9.
d
quinoline
quinoline
I
I
N(Boc)
2
N(Boc)
2
NH
2
H
N
H
N
H
N
a
b
O
O
O
O
O
N
N
N
1
7qq
21a-d
22a-d
N(Boc)
2
N(Boc)
2
NH
2
H
N
H
N
H
N
a
b
quinoline
quinoline
O
N
N
N
17rr
23a-e
24a-e
Scheme 3. Synthesis of (quinolinyl)phenyl amides. Reagents and conditions: (a) (quinlinyl)boronic acid, Pd(PPh
)
3 4
2
, K CO
3 2 2
, DMF 60 °C; (b) TFA, CH Cl .
X
X
CHO
OMe
X
COOMe
X
COOMe
X
COOH
a
b
c
a X = Cl
OMe
OMe
OMe
b X = Br
26a,b
27a,b
2
8a,b
2
5a,b
COOMe
OMe
CH OH
CHO
OMe
COOMe
2
d
e
a
X
OMe
30a,b
X
X
OMe
2
9a,b
31a,b
32a,b
b
COOH
COOMe
c
X
OMe
X
OMe
33a,b
34a,b
Scheme 4. Synthesis of 4-, and 5-halo-2-methoxyphenylpropionic acids. Reagents and conditions: (a) trimethyl phosphonoacetate, t-BuOK, THF; (b) H
c) KOH, MeOH; (d) LiBH , THF, reflux; (e) oxalyl chloride, DMSO, CH Cl
, À78 °C.
2
, Rh/alumina, MeOH;
(
4
2
2
all inactive; surprisingly, a 2-trifluoromethyl group (18bb) affor-
ded moderate activity (IC50 = 39 M). Unsaturation in the propi-
onyl linker (18dd) was consistent with moderate affinity
IC50 = 48 M); however, chain length was intolerant to shorten-
ing (18kk) or lengthening (18mm) by even one methylene unit.
When the 2-methoxy substituent was replaced with 2-ethoxy
Table 3; the 3-pyridyl compound 18nn retained moderate activ-
ity (IC50 = 41 M), whereas the 5-pyridyl isomer 18oo and the
l
l
thienyl compound 18pp were both devoid of activity. Thus, in
spite of the many variations that were made to the prototype
structure 18t in Table 3, compounds with improved affinity
for LF over 18t were not found, and we concluded that a very
narrow but very real range of activity was defined and that
we were dealing with a very specific recognition site in the LF
protein.
(
l
(18ii) or 2-n-propyloxy (18jj) substituents, activity was also to-
tally lost. The phenyl ring itself was also not very tolerant to
modification, as can be seen from the last three compounds in

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
425
N
Table 2
LF and BoNT/A inhibitory activities of quinolinylphenyl amides
MeO
NH
2
NH
2
NH
2
H
N
H
N
H
N
R
IC50 (lM)
O
O
O
N
N
N
18t
1
8m
Compd #
R =
LFa
BoNT/Ab MMP-1c MMP-9d
N
1
NSC 12155 (see Fig. 1)
3.24 7.2
>100
100
N
MeO
MeO
NH2
N
H
N
18m
1.6
3.0
13
38
>100
1.7
O
O
N
N
2
2
2a
2b
>100
65
78
11
NH
2
H
N
N
>100
9.4
N
3
5
Figure 4. Design of ‘hybrid’ amide compound 35.
2
2
2c
10
11
34
65
n.d.
n.d.
17
N
Table 4 contains a group of diverse ureas that were prepared as
described in Scheme 2. Our goal with this series was simply to ex-
plore a different linker functionality. Interestingly, compound 20i,
which has the closest relationship to the prototype amide com-
pound 18t, in that the linker lengths are the same, was devoid of
activity. Although this series of compounds retained a modest de-
gree of inhibitory activity for LF, with benzophenone 20g being the
2d
>100
N
2
2
2
4a
4b
4c
4.6
5.7
21
26
>100
>100
50
>100
16
most active (IC50 = 25
lM), this series as a whole lost selectivity vs.
N
BoNT/A, and several of the compounds displayed greater affinity
for Bont/A than for LF. Thus, we concluded that the propionic
amide linker was important for LF affinity and selectivity.
Given the potent anti-LF activities of compounds 18m and 18t,
we recognized that it might be possible to design a hybrid com-
pound that incorporated elements from both prototype com-
pounds into a single, improved anti-LF agent. In Figure 4 is
shown a simple overlay of these two compounds and also the
structure of a ‘hybrid’ pyridyl compound that we envisioned might
incorporate the important recognition elements from both 18t and
45
N
N
>100
48
N
2
2
4d
4e
33
36
68
79
65
50
1
8m. Synthesis of compound 35 was carried out as described
above, as shown in Scheme 5. The inhibitory activity of hybrid
compound 35 is compared to that of prototype compounds 18t
and 18m in Figure 3. Although the hybrid compound did display
N
>100
>100
moderate inhibitory activity for LF (IC50 = 55 lM), this affinity
was, disappointingly, not additive with respect to the two proto-
type compounds. One possible explanation for the lack of additiv-
ity is that the phenyl ring and the pyridyl ring are accessing two
separate protein pockets, and that the link between these two aro-
matic hydrophobes needs further optimization to increase the
affinity by combining the two aromatic units.
Not determined.
a
Bacillus anthracis lethal factor.
b
c
Clostridium bolulinum neurotoxin A.
Human matrix metalloprotease-1.
Human matrix metalloprotease-9.
d
N
N
N
MeO
MeO
N(Boc)2
N
NH2
H
H
N
MeO
Br
MeO
a
N
c
b
34%
O
O
HOOC
HOOC
37
35
36%
two steps)
N
34b
36
(
Scheme 5. Synthesis of hybrid amide compound 35. Reagents and conditions: (a) 3-pyridylboronic acid, Pd(PPh
CH Cl
3 4 2 3
) , K CO , DMF, 60 °C; (g) 16, HATU, DIPEA, DMF; (h) TFA,
2
2
.

4
26
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
Table 3
Table 3 (continued)
LF and BoNT/A inhibitory activity of (methoxyphenyl)propionic amides and related
compounds
NH
2
H
N
R
NH
2
IC50 (lM)
H
N
R
O
N
N
IC50 (lM)
O
LFa
BoNT/Ab
MMP-1c
MMP-9d
Compd #
R =
N
LFa
BoNT/Ab MMP-1c MMP-9d
OMe
Compd #
R =
1
NSC 12155 (see Fig. 1)
3.24 7.2
>100
100
18mm
>100 >100
n.d.
n.d.
MeO
1
8t
3.0
>100
>100
>100
MeO
1
1
8nn
8oo
41
>100
>100
>100
n.d.
>100
n.d.
OMe
1
1
8u
8v
>100 >100
>100 >100
n.d.
n.d.
n.d.
n.d.
MeO
>50
N
MeO
OMe
OMe
1
8pp
>100 >100
n.d.
n.d.
MeO
S
1
8w
>100 >100
n.d.
n.d.
Not determined.
a
Bacillus anthracis lethal factor.
OMe
b
c
Clostridium bolulinum neurotoxin A.
Human matrix metalloprotease-1.
Human matrix metalloprotease-9.
MeO
BzO
Cl
1
1
8x
8y
>100 >100
>100 >100
n.d.
n.d.
n.d.
n.d.
d
4. Conclusion
1
1
1
8z
>100 >100
>100 >100
n.d.
n.d.
In this study we successfully optimized one half of the bis-
quinolinyl, symmetrical screening hit 1 and produced desym-
metrized compounds that are selective inhibitors of Bacillus
anthracis lethal factor. Compounds 18m and 18t are two of
the best compounds in terms of potency and selectivity versus
BoNT/A, MMP-1 and MMP-9 that were produced in this syn-
thetic campaign, and provide new starting points with more
drug-like features than compound 1. The present work repre-
sents a midpoint in the total optimization of compound 1, and
represents a significant amount of structure activity relationship
development that has mapped a portion of a lethal factor bind-
ing site that we think resides at or close to the active site of the
enzyme.
8aa
8bb
n.d.
n.d.
3
F C
38
>100
>100
>100
F
1
1
8cc
>100 >100
n.d.
n.d.
MeO
MeO
8dd
46
>100
>100
>100
1
1
8ee
8ff
>100 >100
>100 >100
n.d.
n.d.
n.d.
n.d.
Br
MeO
5
5
. Experimental section
Cl
Cl
.1. General methods
MeO
MeO
O
1
1
1
1
8gg
8hh
8ii
78
58
>100
>100
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1H NMR spectra were recorded on a Bruker AV 300 MHz
spectrometer in the solvent indicated. Chemical shifts are re-
ported in d (ppm) units relative to internal standard tetrameth-
ylsilane (TMS). Mass spectra were recorded on
Br
a Thermo
Scientific LCQ Fleet LC/MS instrument. Elemental analyses were
performed by Columbia Analytical Services, 19408 Park Row
Dr., Suite 320, Houston, TX 19408. Thin-layer chromatography
was carried out with silica gel plates (Analtech, Newark, DE)
with visualization by UV light (254 nm). Melting points were re-
corded with an EZ-Melt Apparatus (Stanford Research Systems,
Stanford, CA). Column chromatography was carried out on silica
gel 60 (Merck 230–400 mesh). All non-aqueous reactions were
conducted in oven-dried glassware under a positive pressure
of argon, using commercially available dry solvents. The synthe-
sis of protected aminoquinoline 16 is found in the Supplemen-
tary data section; all other reagents were obtained from
commercial sources.
>100 >100
>100 >100
O
8jj
OMe
Cl
1
1
8kk
8ll
>100 >100
>100 >100
n.d.
n.d.
n.d.
n.d.

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
427
Table 4
Na
CO
(1 Â 150 mL), brine (1 Â 150 mL), dried (MgSO
), and evap-
2
3
4
LF and BoNT/A inhibitory activities of ureas
orated to provide a brown solid. The solid was re-crystallized from
CH CN to provide 2.93 g (61%) of the final product as a fine tan-col-
ored powder: R 0.54 (25% EtOAc/Hex); mp 196–256 °C decomp.;
H NMR (DMSO-d ) d 10.63 (s, 1H), 8.38 (d, 1H), 8.13 (dd, 1H),
NH
2
3
H
N
H
N
f
R
IC50
(
l
M)
1
6
O
N
7.97 (d, 1H), 7.94 (d, 2H), 7.79 (d, 1H), 7.76 (d, 1H), 7.38 (s, 1H),
2.65 (s, 3H), 1.34 (s, 18H).
LFa
BoNT/Ab MMP-1c MMP-9d
Compd #
R =
1
NSC 12155 (see Fig. 1)
3.24
7.2
>100
n.d.e
100
5
6
.2.2. N-(4-[Bis{tert-butoxycarbonyl}amino]-2-methylquinolin-
-yl)-3-iodobenzamide (17rr)
A
O
O
2
0a
>100 41
n.d.
mixture of 4-(bis[tert-butoxycarbonyl]amino)-6-amino-2-
methylquinoline (3.00 g, 8.0 mmol), 3-iodobenzoic acid (3.98 g,
6 mmol, 2.0 equiv); 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU; 7.60 g,
0 mmol, 2.5 equiv), DIEA (15 mL, 86 mmol, 10 equiv) in DMF
100 mL) was stirred at room temperature for 48 h. The mixture
was poured into 200 mL of 5% aq Na CO and extracted with EtOAc
(2 Â 200 mL). The combined organic extracts were washed with 5%
aq Na CO ), and
(1 Â 150 mL), brine (1 Â 150 mL), dried (MgSO
evaporated to provide a brown solid. The solid was re-crystallized
from CH CN to provide 2.75 g (58%) of product as a brown granular
solid: mp 199–253 °C decomp.; H NMR (DMSO-d
8.39 (d, 1H), 8.32 (dd, 1H), 8.12 (dd, 1H), 8.01–7.96 (m, 3H), 7.38 (t,
1
2
0b
79
79
n.d.
n.d.
2
(
2
2
2
0c
0a
0e
>100 41
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
2
3
OMe
COOEt
F
2
3
4
87
95
51
32
3
1
6
) d 10.66 (s, 1H),
2
H), 2.89 (s, 3H), 1.34 (s, 18H).
2
2
0f
83
62
n.d.
n.d.
5
.2.3. General synthesis of compounds 18a–pp
Mixtures of 4-(di-tert-butoxycarbonylamino)-6-amino-2-meth-
ylquinoline (100 mg, 0.27 mmol), the respective carboxylic acid
(0.53 mmol, 2 equiv), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU; 190 mg,
0g
26
61
67
>100
>100
O
0
.500 mmol, 2.0 equiv) and di-iso-propylethylamine (0.5 mL,
Cl
2
.9 mmol, 10 equiv) in DMF (5 mL) were stirred at room tempera-
2
2
0h
0i
>100
n.d.
n.d.
n.d.
n.d.
ture for 16 h. The mixtures were poured into 50 mL of 5% aq Na2-
CO
and extracted with EtOAc (3 Â 50 mL). The combined organic
layers were washed with 5% aq Na CO
(1 Â 50 mL), brine
), and evaporated. The residual materials
MeO
3
>100 >100
2
3
(
1 Â 50 mL), dried (MgSO
4
a
b
c
Bacillus anthracis lethal factor.
Clostridium bolulinum neurotoxin A.
Human matrix metalloprotease-1.
Human matrix metalloprotease-9.
Not determined.
were co-evaporated with diethyl ether and treated without further
purification with 1:1 TFA/DCM (5 mL) for 30 min–2 h. Removal of
solvents gave crude materials as solids or oils that were triturated
d
e
with diethyl ether and recrystallized from CH
the final product as a TFA salt or purified using prep TLC
(80:18:2 CHCl /MeOH/aq CH NH ) to provide the free base. Some
3
CN/MeOH) to yield
5
.2. Synthesis
3
3
2
of the free bases were further reacted with a slight excess of
p-toluenesulfonic acid in diethyl ether to provide the correspond-
ing p-TsOH salts.
5
6
.2.1. N-(4-[Bis{tert-butoxycarbonyl}amino]-2-methylquinolin-
-yl)-4-iodobenzamide (17qq)
A
mixture of 4-(bis[tert-butoxycarbonyl]amino)-6-amino-2-
methylquinoline (3.00 g, 8.0 mmol), 4-iodobenzoic acid (3.98 g,
6 mmol, 2.0 equiv); 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HATU; 7.60 g, 20
mmol, 2.5 equiv), DIEA (15 mL, 86 mmol, 10 equiv) in DMF
5.2.3.1. N-(4-Amino-2-methylquinolin-6-yl)-4-phenoxybenza-
1
mide trifluoroacetate (18a).
Compound 18a was obtained
0.33 (80:18:2 CHCl /MeOH/aq CH3-
); mp 202–216 °C; H NMR (DMSO-d ) d 13.02 (b, 1H), 10.60
as a light yellow powder: R
f
3
1
NH
2
6
(
100 mL) was stirred at room temperature for 48 h. The mixture
was poured into 200 mL of 5% aq Na CO and extracted with EtOAc
2200 mL). The combined organic extracts were washed with 5% aq
(b, 1H), 8.80 (b, 1H), 8.60 (b, 1H), 8.06–8.09 (m, 3H), 7.82 (d, 1H),
7.48 (t, 2H), 7.27–7.12 (m, 5H), 6.60 (s, 1H), 2.58 (s, 3H); m/z ex-
2
3
+
(
pected 369.1, found 370.5 ([M+H] ).
N
N
MeO
MeO
NH
N
2
NH
2
NH2
N
H
N
H
N
H
N
O
O
O
N
1
8m
18t
35
IC50 = 3.0 µM
IC50 = 1.5 µM
IC50 = 55 µM
Figure 3. Activity of hybrid amide compound and corresponding parent compounds.

4
28
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
5
.2.3.2.
mide trifluoroacetate (18b).
as a light brown powder: R
N-(4-Amino-2-methylquinolin-6-yl)-3-benzoylbenza-
Compound 18b was obtained
0.37 (80:18:2 CHCl /MeOH/aq CH3-
); mp 209–218 °C; H NMR (DMSO-d ) d 13.45 (b, 1H), 10.87
obtained as a yellow microcrystalline solid: R
f
0.37 (80:18:2 CHCl
3
/
1
3
MeOH/aq CH NH
2
); mp 205–206 °C; H NMR (DMSO-d ) d 13.53 (s,
6
f
3
1H), 11.63 (s, 1H), 10.62 (s, 1H), 8.94 (s, 3H), 8.13 (d, 1H), 7.97 (d,
1H), 7.40 (s, 2H), 7.16 (s, 1H), 6.91 (d, 1H), 6.60 (s, 1H), 3.83 (s, 3H),
2.56 (s, 3H); m/z expected 346.1, found 347.4 ([M+H] ).
1
NH
2
6
+
(
(
(
s, 1H), 8.77 (s, 1H), 8.68 (b, 2H), 8.38–8.31 (m, 2H), 8.06–7.97
m, 2H), 7.85–7.63 (m, 5H), 7.71–7.58 (m, 2H), 6.59 (s, 1H), 2.58
s, 3H); m/z expected 381.1, found 382.6 ([M+H] ).
+
5.2.3.11. N-(4-Amino-2-methylquinolin-6-yl)biphenyl-4-carbox
amide trifluoroacetate (18k).
as a light yellow powder: R 0.35 (80:18:2 CHCl
); mp 263–267; H NMR (DMSO-d ) d 13.40 (b, 1H), 10.74 (s,
Compound 18k was obtained
5
.2.3.3. N-(4-Amino-6-methylquinolin-6-yl)nicotinamide tri-
fluoroacetate (18c). Compound 18c was obtained as a yellow
powder: R 0.19 (80:18:2 CHCl /MeOH/aq CH NH ); mp 175–
80 °C; H NMR (DMSO-d ) d 13.46 (b, 1H), 10.88 (s, 1H), 9.18 (d,
f
3
/MeOH/aq CH3-
1
NH
2
6
f
3
3
2
1H), 8.80 (s, 1H), 8.60 (s, 2H), 8.16–8.06 (m, 4H), 7.79–7.77 (m,
1
1
1
1
6
4H), 7.55–7.44 (m, 3H), 6.56 (s, 1H), 2.58 (s, 3H); m/z expected
+
H), 8.82–8.78 (m, 3H), 8.70 (b, 2H), 8.38–8.34 (m, 1H), 8.03 (d,
H), 7.84 (d, 1H), 6.60 (s, 1H), 2.60 (s, 3H); m/z expected 278.1,
323.2, found 354.6 ([M+H] ).
+
found 279.4 ([M+H] ).
5.2.3.12. N-(4-Amino-2-methylquinolin-6-yl)-4-(pyridin-3-yl)
benzamide trifluoroacetate (18l).
tained as a tan powder: R 0.27 (80:18:2 CHCl
mp 227–228 °C; H NMR (DMSO-d ) d 13.45 (s, 1H), 10.84 (s, 1H),
Compound 18l was ob-
5
.2.3.4. N-(4-Amino-2-methylquinolin-6-yl)furan-2-carboxam-
f
3
/MeOH/aq CH NH );
3
2
1
ide trifluoroacetate (18d).
a yellow-orange powder: R
NH
Compound 18d was obtained as
0.15 (80:18:2 CHCl /MeOH/aq CH3-
) d 13.46 (s, 1H), 10.64
6
f
3
9.06 (d, 1H), 8.84 (d, 1H), 8.68 (d, 3H), 8.28 (d, 1H), 8.18 (d, 2H),
1
2
); mp 192–207 °C; H NMR (DMSO-d
6
8.07 (dd, 1H), 7.99 (d, 2H), 7.85 (d, 1H), 7.64–7.60 (m, 1H), 6.60
+
(
6
s, 1H), 8.69 (b, 3H), 8.10–7.99 (m, 2H), 7.82 (d, 1H), 7.24 (d, 1H),
.76–6.72 (m, 1H), 6.59 (s, 1H), 2.58 (s, 3H); m/z expected 267.1,
(s, 1H), 2.59 (s, 3H); m/z expected 354.1, found 355.2 ([M+H] ).
+
found 268.2 ([M+H] ).
5.2.3.13.
yl)benzamide trifluoroacetate (18m).
obtained as a tan powder: R 0.29 (80:18:2 CHCl
); mp 214–216 °C; H NMR (DMSO-d ) d 13.49 (s, 1H), 10.80
N-(4-Amino-2-methylquinolin-6-yl)-4-(quinoline-6-
Compound 18m was
/MeOH/aq CH3-
5
.2.3.5. N-(4-Amino-2-methylquinolin-6-yl)-3-(dimethylamino)
benzamide trifluoroacetate (18e). Compound 18e was ob-
tained as a tan powder: R 0.43 (80:18:2 CHCl /MeOH/aq CH NH );
mp 210–212 °C; H NMR (DMSO-d ) d 13.53 (s, 1H), 10.57 (s, 1H),
.77 (s, 1H), 8.76 (s, 2H), 8.06 (d, 1H), 7.83 (d, 1H), 7.65 (b, 1H),
f
3
1
NH
2
6
f
3
3
2
(s, 1H), 8.98 (s, 1H), 8.82 (s, 1H), 8.71 (b, 2H), 8.53 (d, 2H), 8.47
1
6
(s, 1H), 8.21 (d, 4H), 8.09 (s, 3H), 7.86 (d, 1H), 7.64 (s, 1H), 6.60
+
8
7
3
(s, 1H), 2.60 (s, 3H); m/z expected 404.2, found 405.5 ([M+H] ).
.61–7.56 (m, 3H), 6.96 (s, 1H), 6.59 (s, 1H), 2.98 (s, 6H), 2.55 (s,
H); m/z expected 320.2, found 321.5 ([M+H] ).
+
5.2.3.14. N-(4-Amino-2-methylquinolin-6-yl)-3-(4-dimethylam-
inophenyl) benzamide (18n).
as an off-white powder: R 0.28 (80:18:2 CHCl
mp 206–213 °C; H NMR (DMSO-d ) d 10.64 (s, 1H), 8.70 (s, 1H),
Compound 18n was obtained
5
.2.3.6. N-(4-Amino-2-methylquinolin-6-yl)-4-(1-morpholino)
f
3
/MeOH/aq CH NH );
3
2
1
benzamide trifluoroacetate (18f).
tained as a tan powder: R 0.48 (80:18:2 CHCl
mp 109–111 °C; H NMR (DMSO-d ) d 13.48 (s, 1H), 10.42 (s, 1H),
.77 (s, 1H), 8.76 (b, 2H), 8.07 (m, 1H), 8.04 (d, 2H), 7.81 (d, 1H),
.06 (d, 2H), 6.58 (s, 1H), 3.76 (t, 4H), 3.28 (t, 4H), 2.58 (s, 3H);
Compound 18f was ob-
6
f
3
/MeOH/aq CH NH );
3
2
8.40 (s, 1H), 8.38 (b, 2H), 8.03 (d, 1H), 7.84 (m, 3H), 7.65–7.51
1
6
(m, 3H), 6.84 (d, 2H), 6.57 (s, 1H), 2.89 (s, 6H), 2.56 (s, 3H); m/z ex-
+
8
7
pected 396.2, found 397.5 ([M+H] ).
+
m/z expected 362.2, found 363.5 ([M+H] ).
5.2.3.15.
cinamamide trifluoroacetate (18o).
tained as pink powder: 0.41 (80:18:2 CHCl
CH NH ); mp >300 °C; H NMR (DMSO-d ) d 13.4 (b, 1H), 10.53
(E)-N-(4-Amino-2-methylquinolin-6-yl)-4-methoxy-
Compound 18o was ob-
/MeOH/aq
5
.2.3.7. N-(4-Amino-2-methylquinolin-6-yl)quinoline-2-carbox-
amide trifluoroacetate (18g). Compound 18f was obtained
as a pale yellow powder: R 0.45 (90:9:1 CHCl /MeOH/aq CH NH );
mp 224–227 °C; H NMR (DMSO-d ) d 13.40 (s, 1H), 10.91 (s, 1H)
.86 (s, 1H), 8.66 (b, 2H), 8.56 (d, 1H), 8.42 (d, 1H), 8.48 (dd, 1H),
a
R
f
3
1
3
2
6
f
3
3
2
(s, 1H), 8.66 (s, 1H), 8.48 (b, 2H), 7.93 (d, 1H), 7.80 (d, 1H), 7.62–
1
6
7.58 (m, 3H), 7.03 (d, 2H), 6.75 (d, 1H), 6.56 (s, 1H), 3.81 (s, 3H),
+
8
8
2
2.56 (s, 3H); m/z expected 333.1, found 334.4 ([M+H] ).
.08–8.03 (m, 2H), 7.92–7.84 (m, 2H), 7.74 (t, 1H), 6.61 (s, 1H),
.56 (s, 3H); m/z expected 328.1, found 329.4 ([M+H] ).
+
5.2.3.16. N-(4-Amino-2-methylquinolin-6-yl)-2-[(4-benzyloxy)
phenoxy]acetamide trifluoroacetate (18p).
was obtained as a light yellow powder: R
Compound 18p
0.23 (80:18:2 CHCl
); mp 208–210 °C; H NMR (DMSO-d ) d 13.53
5
.2.3.8. N-(4-Amino-2-methylquinolin-6-yl)isoquinoline-3-car-
f
3
/
1
boxamide trifluoroacetate (18h).
tained as a yellow powder: R 0.40 (80:18:2 CHCl
); mp 288–290 °C; H NMR (DMSO-d ) d 14.49 (s, 1H),
Compound 18h was ob-
3
MeOH/aq CH NH
2
6
f
3
/MeOH/aq
(b, 1H), 10.55 (b, 1H), 8.90 (d, 3H), 7.41 (m, 5H), 6.97 (s, 4H),
1
CH
3
NH
2
6
6.57 (s, 1H), 5.08 (d, 2H), 4.70 (d, 2H), 2.57 (s, 3H); m/z expected
+
1
8
2
1.05 (s, 1H), 9.52 (s, 1H), 8.82 (s, 1H), 8.75 (s, 1H), 8.75 (s, 2H),
.73–8.72 (m, 3H), 8.12 (d, 1H), 7.94–7.88 (m, 2H), 6.68 (s, 1H),
.62 (s, 3H); m/z expected 328.1, found 329.4 ([M+H] ).
413.2, found 414.4 ([M+H] ).
+
5.2.3.17.
amide (18q).
N-(4-Amino-2-methylquinolin-6-yl)-2-phenylacet-
Compound 18q was obtained as a pink crystal-
5
.2.3.9. N-(4-Amino-2-methylquinolin-6-yl)benzofuran-2-car-
line solid: /MeOH/aq CH
R
f
0.27 (80:18:2 CHCl
3
3 2
NH ); mp
1
boxamide trifluoroacetate (18i).
tained as a yellow microcrystalline solid: R
MeOH/aq CH
Compound 18i was ob-
0.42 (80:18:2 CHCl
); mp 218–219 °C; H NMR (DMSO-d ) d 13.48
(sublimed 160 °C); H NMR (DMSO-d ) d 10.67 (s, 1H), 8.61 (d,
6
f
3
/
1H), 8.55 (s, 2H), 7.90 (dd, 1H), 7.88 (d, 1H), 7.40–7.24 (m, 5H),
1
3
NH
2
6
6.56 (s, 1H), 3.73 (s, 2H), 2.55 (s, 3H); m/z expected 291.4, found
+
(
(
(
s, 1H), 10.99 (s, 1H), 8.77 (s, 1H), 8.71 (b, 2H), 8.12 (d, 1H), 7.86
dd, 3H), 7.75 (d, 1H), 7.50 (t, 1H), 7.40 (t, 1H), 6.61 (s, 1H), 2.59
s, 3H); m/z expected 317.1, found 318.5 ([M+H] ).
292.9 ([M+H] ).
+
5.2.3.18. N-(4-Amino-2-methylquinolin-6-yl)-2-(benzo[d][1,3]-
dioxol-5-yl)acetamide trifluoroacetate (18r).
Compound
5
2
.2.3.10. N-(4-Amino-2-methylquinolin-6-yl)-5-methoxyindole-
-carboxamide trifluoroacetate (18j). Compound 18j was
18r was obtained as a brown crystalline solid: R
f
0.30 (90:9:1
1
CHCl
3
3
/MeOH/aq CH NH
2
); mp >160 °C (subl.);
H
NMR

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
429
(
7
1
DMSO-d
6
) d 13.52 (b, 1H), 10.62 (s, 1H), 8.61 (s, 2H), 8.58 (d, 1H),
.91 (dd, 1H), 7.77 (d, 1H), 6.95 (d, 1H), 6.89–6.81 (m, 2H), 6.55 (s,
H), 5.99 (s, 2H), 3.62 (s, 2H), 2.56 (s, 3H); m/z expected 335.1,
5.2.3.26.
phenyl)propionamide trifluoroacetate (18z).
18z was obtained as a white powder: R 0.42 (90:9:1 CHCl
); mp 214–216 °C; H NMR (DMSO-d ) d 13.38
s, 1H), 10.38 (s, 1H), 8.60 (b, 3H), 7.84 (dd, 1H), 7.77 (d, 1H),
N-(4-Amino-2-methylquinolin-6-yl)-3-(2-chloro-
Compound
f
3
/
+
1
found 336.4 ([M+H] ).
MeOH/aq CH
(
3
NH
2
6
7
2
.46–7.39 (m, 2H), 7.32–7.25 (m, 2H), 6.56 (s, 1H), 3.07 (t, 2H),
.74 (t, 2H), 2.56 (s, 3H); m/z expected 339.1, found 340.5 ([M+H] ).
5
.2.3.19. N-(4-Amino-2-methylquinolin-6-yl)-3-phenylpropiona
mide trifluoroacetate (18s). Compound 18s was obtained as
a yellow powder: R 0.18 (80:18:2 CHCl /MeOH/aq CH NH ); mp
07–209 °C; H NMR (DMSO-d ) d 13.41 (b, 1H), 10.39 (s, 1H),
+
f
3
3
2
5
.2.3.27.
N-(4-Amino-2-methylquinolin-6-yl)-3-(2-methyl-
Compound
0.44 (90:9:1 CHCl
); mp 239–240 °C; H NMR (DMSO-d ) d 13.40
b, 1H), 10.39 (s, 1H), 8.59 (s, 3H), 7.85 (dd, 1H), 7.76 (d, 1H),
1
2
8
6
3
6
phenyl)propionamide trifluoroacetate (18aa).
8aa was obtained as a tan powder: R
.50 (s, 3H), 7.81 (dd, 2H), 7.33–7.28 (m, 3H), 7.22–7.17 (m, 2H),
.58 (s, 1H), 2.96 (t, 2H), 2.72 (t, 2H), 2.56 (s, 3H); m/z expected
05.2, found 306.5 ([M+H] ).
1
f
3
/
1
MeOH/aq CH
3
NH
2
6
+
(
7
3
.21–7.07 (m, 4H), 6.56 (s, 1H), 2.94 (t, 2H), 2.67 (t, 2H), 2.56 (s,
H), 2.32 (s, 3H); m/z expected 319.2, found 320.5 ([M+H] ).
5
.2.3.20.
N-(4-Amino-2-methylquinolin-6-yl)-3-(2-methoxy-
Compound
0.27 (90:9:1 CHCl /MeOH/
); mp 240–242 °C; H NMR (DMSO-d ) d 13.30 (b, 1H),
0.33 (s, 1H), 8.60 (b, 3H), 7.80 (d, 2H), 7.19 (d, 2H), 6.97 (d, 1H),
+
phenyl)propionamide trifluoroacetate (18t).
8t was obtained as a tan powder: R
1
f
3
5
.2.3.28.
N-(4-Amino-2-methylquinolin-6-yl)-3-[2-(trifluoro-
1
aq CH
3
NH
2
6
methyl)phenyl]propionamide trifluoroacetate (18bb).
pound 18bb was obtained as a pale green crystalline solid: R
Com-
0.29
NMR
1
6
2
f
.88 (d, 1H), 6.56 (s, 1H), 3.80 (s, 3H), 2.90 (t, 2H), 2.66 (t, 2H),
.56 (s, 3H); m/z expected 335.2, found 336.5 ([M+H] ).
1
(90:9:1 CHCl
(DMSO-d
(m, 5H), 7.49 (t, 1H), 6.56 (s, 1H), 3.13 (t, 2H), 2.75 (t, 2H), 2.56 (s,
3 3 2
/MeOH/aq CH NH ); mp 220–225 °C;
H
+
6
) d 13.42 (s, 1H), 10.45 (s, 1H), 8.64 (br s, 3H), 7.82–7.55
+
5
.2.3.21.
N-(4-Amino-2-methylquinolin-6-yl)-3-(4-methoxy-
Com-
0.40 (90:9:1
); mp 234–236 °C; H NMR (DMSO-d ) d
3H); m/z expected 373.1, found 374.5 ([M+H] ).
phenyl)propionamide p-toluenesulfonate (18u).
pound 18u was obtained as a white powder: R
CHCl
5.2.3.29.
N-(4-Amino-2-methylquinolin-6-yl)-3-(4-fluoro-
f
1
3
/MeOH/aq CH
3
NH
2
6
phenyl)propionamide p-toluenesulfonate (18cc).
Com-
0.32 (90:9:1
); mp 222–225 °C; H NMR (DMSO-d ) d
13.33 (s, 1H), 10.34 (s, 1H), 8.58 (b, 3H), 7.82–7.75 (m, 2H), 7.47
d, 2H), 7.33–7.28 (m, 2H), 7.14–7.08 (m, 4H), 6.59 (s, 1H), 2.94
1
7
2
3.33 (s, 1H), 10.33 (s, 1H), 8.6 (b, 3H), 7.78 (q, 2H), 7.48 (d, 2H),
.14 (dd, 4H), 6.85 (d, 2H), 6.56 (s, 1H), 3.71 (s, 3H), 2.89 (t, 2H),
.67 (t, 2H), 2.56 (s, 3H), 2.28 (s, 3H).
pound 18cc was obtained as a white powder: R
f
1
CHCl
3
/MeOH/aq CH
3
NH
2
6
(
5
.2.3.22.
N-(4-Amino-2-methylquinolin-6-yl)-3-(3-methoxy-
Compound
0.38 (90:9:1 CHCl
); mp 234–236 °C; H NMR (DMSO-d ) d 13.40
b, 1H), 10.37 (s, 1H), 8.58 (s, 3H), 7.86–7.71 (m, 2H), 7.20 (t,
(t, 2H), 2.70 (t, 2H), 2.56 (s, 3H), 2.28 (s, 3H); m/z expected 323.1,
found 324.4 ([M+H] ).
+
phenyl)propionamide trifluoroacetate (18v).
8v was obtained as a white powder: R
1
f
3
/
1
MeOH/aq CH
(
3
NH
2
6
5.2.3.30. (E)-N-(4-Amino-2-methylquinolin-6-yl)-2-methoxyci-
nanamide trifluoroacetate (18dd).
Compound 18dd was ob-
0.32 (90:9:1 CHCl /MeOH/aq
); mp 218–220 °C; H NMR (DMSO-d ) d 13.74 (b, 1H),
10.66 (s, 1H), 8.65 (s, 1H), 8.57 (b, 2H), 7.99–7.81 (m, 3H), 7.60
d, 1H), 7.43 (t, 1H), 7.16 (d, 1H), 7.04 (t, 1H), 6.97–6.92 (m, 1H),
1
2
H), 6.83 (s, 2H), 6.76 (d, 1H), 6.56 (s, 1H), 3.72 (s, 3H), 2.93 (t,
H), 2.71 (t, 2H), 2.56 (s, 3H); m/z expected 335.2, found 336.5
tained as a yellow powder: R
f
3
1
3
CH NH
2
6
+
(
[M+H] ).
(
6
3
.58 (s, 1H), 3.91 (s, 3H), 2.57 (s, 3H); m/z expected 333.1, found
34.4 ([M+H] ).
5
.2.3.23. N-(4-Amino-2-methylquinolin-6-yl)-3-(2,5-dimethoxy-
phenyl)propionamide p-toluenesulfonate (18w). Compound
8w was obtained as a white powder: R 0.43 (90:9:1 CHCl /MeOH/
); mp 240–244 °C; H NMR (DMSO-d ) d 13.33 (s, 1H),
+
1
f
3
5
.2.3.31.
N-(4-Amino-2-methylquinolin-6-yl)-3-(5-bromo-2-
1
aq CH
NH
3 2
6
methoxyphenyl)propionamide trifluoroacetate (18ee).
pound 18ee was obtained as an off-white crystalline solid: R
Com-
0.52
NMR
1
6
3
3
0.33 (s, 1H), 8.61 (s, 3H), 7.82 (m, 2H), 7.47 (d, 2H), 7.11 (d, 2H),
.88 (d, 1H), 6.79–6.76 (m, 2H), 6.56 (s, 1H), 3.75 (s, 3H), 3.66 (s,
H), 2.88 (t, 2H), 2.65 (t, 2H), 2.56 (s, 3H), 2.28 (s, 3H); m/z expected
f
1
(
(
2
(
90:9:1 CHCl
DMSO-d
H), 7.37–7.35 (m, 2H), 6.94 (d, 1H), 6.56 (s, 1H), 3.80 (s, 3H), 2.89
t, 2H), 2.67 (t, 2H), 2.56 (s, 3H); m/z expected 413.1, found 414.5
3 3 2
/MeOH/aq CH NH ); mp 135–139 °C;
H
6
) d 13.42 (s, 1H), 10.35 (s, 1H), 8.61 (b, 3H), 7.82–7.76 (m,
+
65.2, found 366.4 ([M+H] ).
+
5.2.3.24. N-(4-Amino-2-methylquinolin-6-yl)-3-(2,3-dimethoxy-
([M+H] ).
phenyl)propionamide p-toluenesulfonate (18x).
8x was obtained as an off-white powder: R 0.48 (90:9:1 CHCl
); mp 204–206 °C; H NMR (DMSO-d ) d 13.33 (s,
Compound
1
f
3
/
5.2.3.32.
N-(4-Amino-2-methylquinolin-6-yl)-3-(5-chloro-2-
Com-
pound 18ff was obtained as a white powder: R 0.33 (90:9:1 CHCl /
1
MeOH/aq CH
3
NH
2
6
methoxyphenyl)propionamide trifluoroacetate (18ff).
1
2
H), 10.31 (d, 1H), 8.81 (s, 1H), 8.65 (b, 2H), 7.79 (q, 2H), 7.47 (d,
H), 7.11 (d, 2H), 6.96–6.77 (m, 3H), 6.55 (s, 1H), 3.81 (s, 3H), 3.75
f
3
1
MeOH/aq CH NH ); mp 186–189 °C; H NMR (DMSO-d ) d 13.31 (s,
1H), 10.24 (s, 1H), 8.48 (b, 3H), 7.68 (d, 2H), 7.11 (s, 2H), 6.87 (d,
1H), 6.44 (s, 1H), 3.68 (s, 3H), 2.77 (m, 2H), 2.55 (m, 2H), 2.44 (s,
3
2
6
(
3
s, 3H), 2.93 (t, 2H), 2.66 (t, 2H), 2.55 (s, 3H), 2.28 (s, 3H); m/z expected
65.2, found 366.5 ([M+H] ).
+
+
3H); m/z expected 369.1, found 370.4 ([M+H] ).
5
.2.3.25. N-(4-Amino-2-methylquinolin-6-yl)-3-(2-benzoyloxy-
phenyl)propionamide trifluoroacetate (18y).
8y was obtained as a white powder: R 0.46 (90:9:1 CHCl
); mp 176–180 °C; H NMR (DMSO-d ) d 13.37
s, 1H), 10.32 (s, 1H), 8.57 (b, 3H), 8.18 (d, 2H), 7.81–7.73 (m,
Compound
5.2.3.33.
methoxyphenyl)propionamide trifluoroacetate (18gg).
pound 18gg was obtained as a white powder: R 0.31 (90:9:1 CHCl /
MeOH/aq CH NH ); mp 141–143 °C; H NMR (DMSO-d ) d 13.30 (s,
3 2 6
1H), 10.36 (s, 1H), 8.61 (b, 3H), 7.80 (t, 2H), 7.25 (t, 1H), 7.05 (s, 1H),
N-(4-Amino-2-methylquinolin-6-yl)-3-(4-chloro-2-
1
f
3
/
Com-
1
MeOH/aq CH
(
3
NH
2
6
f
3
1
3
1
H), 7.61 (t, 2H), 7.45–7.43 (m, 1H), 7.36–7.25 (m, 3H), 6.55 (s,
H), 2.90 (t, 2H), 2.72 (t, 2H), 2.55 (s, 3H); m/z expected 425.2,
6.95 (dd, 1H), 6.55 (s, 1H), 3.87 (s, 3H), 2.70 (t, 2H), 2.86 (t, 2H), 2.56
+
+
found 426.5 ([M+H] ).
(s, 3H); m/z expected 369.1, found 370.4 ([M+H] ).

4
30
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
5
.2.3.34.
methoxyphenyl)propionamide trifluoroacetate (18hh).
pound 18hh was obtained as a white powder: R 0.34 (90:9:1 CHCl
); mp 122–126 °C; H NMR (DMSO-d ) d 13.44 (s,
H), 10.36 (s, 1H), 8.60 (b, 3H), 7.79 (m, 2H), 7.15–7.05 (m, 3H), 6.56
s, 1H), 3.83 (s, 3H), 2.87 (t, 2H), 2.65 (t, 2H), 2.56 (s, 3H); m/z expected
N-(4-Amino-2-methylquinolin-6-yl)-3-(4-bromo-2-
(d, 1H), 6.52 (s, 1H), 3.87 (s, 3H), 2.90 (t, 2H), 2.67 (t, 2H), 2.51
(s, 3H); m/z expected 336.2, found 337.4 ([M+H] ).
+
Com-
f
3
/
1
MeOH/aq CH
1
(
3
NH
2
6
5.2.3.42. N-(4-Amino-2-methylquinolin-6-yl)-3-(3-methoxythi-
ophene-2-yl)propionamide trifluoroacetate (18pp).
pound 18pp was obtained as a cream-colored powder: R
Com-
0.29
NMR
f
+
1
413.1, found 414.5 ([M+H] ).
(90:9:1 CHCl
DMSO-d
3
/MeOH/aq CH
) d 13.43 (b, 1H), 10.40 (s, 1H), 8.61 (b, 3H), 7.87–7.70
3
NH
2
); mp 229–231 °C;
H
(
6
5
.2.3.35.
N-(4-Amino-2-methylquinolin-6-yl)-3-(2-ethoxy-
Compound
0.34 (90:9:1 CHCl
); mp 122–126 °C; H NMR (DMSO-d ) d 13.38
(m, 2H), 7.24 (d, 1H), 6.97 (d, 1H), 6.56 (s, 1H), 3.78 (s, 3H), 2.98
phenyl)propionamide trifluoroacetate (18ii).
8ii was obtained as a white powder: R
(t, 2H), 2.66 (t, 2H), 2.56 (s, 3H); m/z expected 341.1, found 342.3
+
1
f
3
/
([M+H] ).
1
MeOH/aq CH
3
NH
2
6
(
(
2
s, 1H), 10.33 (s, 1H), 8.62 (b, 3H), 7.84–7.75 (m, 2H), 7.20–7.15
m, 2H), 6.95 (d, 1H), 6.87–6.83 (m, 1H), 6.56 (s, 1H), 4.05 (q,
5.2.4. General synthesis of compounds 20a–i
Mixtures of 4-(di-tert-butoxycarbonylamino)-6-amino-2-meth-
ylquinoline (100 mg, 0.27 mmol) and the corresponding isocya-
nates (0.29 mmol, 1.1 equiv) in dichloromethane (3 mL) were
stirred at room temperature for 16 h. The residues obtained after
removing the solvents were treated with 1:1 TFA/DCM until depro-
tection was complete (4 h). Removal of the TFA solutions gave
essentially pure solids that were washed with ether and dried to
give the final compounds as TFA salts.
H), 2.92 (t, 2H), 2.67 (t, 2H), 2.56 (s, 3H), 1.36 (t, 3H); m/z expected
+
3
49.2, found 350.4 ([M+H] ).
5
.2.3.36. N-(4-Amino-2-methylquinolin-6-yl)-3-[2-(1-propoxy)
phenyl]propionamide trifluoroacetate (18jj).
8jj was obtained as a white powder: R 0.47 (90:9:1 CHCl
); mp 125–127 °C; H NMR (DMSO-d ) d 13.36
b, 1H), 10.32 (s, 1H), 8.62 (s, 1H), 8.57 (b, 2H), 7.84–7.75 (m,
Compound
1
f
3
/
1
MeOH/aq CH
3
NH
2
6
(
2
3
1
H), 7.20–7.15 (m, 2H), 6.94 (d, 1H), 6.85 (t, 1H), 6.56 (s, 1H),
.95 (t, 2H), 2.93 (t, 2H), 2.70 (t, 2H), 2.56 (s, 3H), 1.76 (q, 2H),
.02 (t,3H); m/z expected 363.2, found 364.4 ([M+H] ).
5.2.4.1.
dioxol-5-yl)urea trifluoroacetate (20a).
was obtained as a yellow microcrystalline solid: R
1-(4-Amino-2-methylquinolin-6-yl)-3-(benzo[d][1,3]
Compound 20a
0.30 (90:9:1
); mp 209–210 °C; H NMR (DMSO-d ) d
+
f
1
CHCl
3
3
/MeOH/aq CH NH
2
6
5
.2.3.37.
N-(4-Amino-2-methylquinolin-6-yl)-2-(2-methoxy-
13.36 (s, 1H), 9.18 (s, 1H), 8.98 (s, 1H), 8.59 (b, 2H), 8.33 (d, 1H),
7.89 (dd, 1H), 7.78 (d, 1H), 7.25 (d, 1H), 6.83 (m, 2H), 6.55 (s,
1H), 5.98 (s, 2H), 2.56 (s, 3H); m/z expected 336.1, found 337.3
phenyl)acetamide
trifluoroacetate
(18kk).
0.48 (80:18:2 CHCl
) d 13.45
Compound
1
8kk was obtained as a white powder: R
f
3
/
1
+
MeOH/aq CH
3
NH
2
); mp 178–180 °C; H NMR (DMSO-d
6
([M+H] ).
(
s, 1H), 10.51 (s, 1H), 8.65 (d, 1H), 8.60 (b, 2H), 7.87 (dd, 1H),
7
1
.80 (d, 1H), 7.28–7.24 (m, 2H), 6.99 (d, 1H), 6.92 (t, 1H), 6.55 (s,
5.2.4.2. 1-(4-Amino-2-methylquinolin-6-yl)-3-(biphenyl-4-yl)
H), 3.77 (s, 3H), 3.71 (s, 2H), 2.56 (s, 3H); m/z expected 321.1,
urea trifluoroacetate (20b).
Compound 20b was obtained
0.30 (90:9:1 CHCl /MeOH/aq CH3-
) d 13.37 (s, 1H), 9.32
+
found 322.4 ([M+H] ).
as a cream-colored powder: R
f
3
1
NH
2
); mp 211–212 °C; H NMR (DMSO-d
6
5
.2.3.38.
phenyl)acetamide trifluoroacetate (18ll).
was obtained as a white powder: R 0.18 (90:9:1 CHCl
); mp 215–216 °C; H NMR (DMSO-d ) d 13.42 (s, 1H),
N-(4-Amino-2-methylquinolin-6-yl)-2-(2-chloro-
Compound 18ll
/MeOH/aq
(s, 1H), 9.24 (s, 1H), 8.55 (b, 2H), 8.39 (d, 1H), 7.92 (dd, 1H), 7.76
(d, 1H), 7.63 (m, 6H), 7.45 (t, 2H), 7.32 (t, 1H), 6.56 (s, 1H), 2.57
+
f
3
(s, 3H); m/z expected 368.2, found 369.4 ([M+H] ).
1
CH
3
NH
2
6
1
0.69 (s, 1H), 8.63 (b, 3H), 7.86 (dd, 2H), 7.48–7.45 (m, 2H),
5.2.4.3.
1-(4-Amino-2-methylquinolin-6-yl)-3-(4-methoxy-
7
.33–7.29 (m, 2H), 6.55 (s, 1H), 3.93 (s, 2H), 2.56 (s, 3H); m/z ex-
phenyl)urea trifluoroacetate (20c).
Compound 20c was ob-
0.29 (90:9:1 CHCl /MeOH/
) d 13.37 (s, 1H),
+
pected 325.1, found 326.5 ([M+H] ).
tained as a cream-colored powder: R
f
3
1
3
aq CH NH
2
); mp 170–171 °C; H NMR (DMSO-d
6
5
.2.3.39.
phenyl)butyramide trifluoroacetate (18mm).
8mm was obtained as a white powder: R 0.38 (90:9:1 CHCl
); mp 199–210 °C; H NMR (DMSO-d ) d 13.34
b, 1H), 10.29 (s, 1H), 8.65 (b, 3H), 7.83–7.72 (m, 2H), 7.14 (d,
N-(4-Amimo-2-methylquinolin-6-yl)-4-(4-methoxy-
9.19 (s, 1H), 8.93 (s, 1H), 8.58 (b, 2H), 8.35 (d, 1H), 7.89 (dd, 1H),
Compound
7.76 (d, 1H), 7.41 (d, 2H), 6.89 (d, 2H), 6.55 (s, 1H), 3.72 (s, 3H),
+
1
f
3
/
2.56 (s, 3H); m/z expected 322.1, found 323.3 ([M+H] ).
1
MeOH/aq CH
(
3
NH
2
6
5.2.4.4. 1-(4-Amino-2-methylquinolin-6-yl)-3-(4-ethoxycarbon-
2
2
H), 6.86 (d, 2H), 6.56 (s, 1H), 3.77 (s, 3H), 2.69–2.60 (m, 5H),
.41 (t, 2H), 1.93 (t, 2H); m/z expected 349.2, found 350.5 ([M+H] ).
ylphenyl)urea trifluoroacetate (20d).
obtained as a light gray powder: R 0.30 (90:9:1 CHCl
); mp 202–204 °C; H NMR (DMSO-d ) d 13.42 (s, 1H),
Compound 20d was
+
f
3
/MeOH/aq
1
3
CH NH
2
6
9
7
1
.76 (s, 1H), 9.65 (s, 1H), 8.62 (b, 2H), 8.43 (s, 1H), 7.90 (t, 3H),
.79 (d, 1H), 7.66 (d, 2H), 6.56 (s, 1H), 4.28 (q, 2H), 2.56 (s, 3H),
.31 (t, 3H); m/z expected 364.2, found 365.4 ([M+H] ).
5
.2.3.40. N-(4-Amino-2-methylquinoline-6-yl)-3-(2-methoxy-
pyridin-3-yl)propionamide trifluoroacetate (18nn).
pound 18nn was obtained as a white powder: R 0.29 (90:9:1
); mp 224–226 °C; H NMR (DMSO-d ) d
Com-
+
f
1
CHCl
3
/MeOH/aq CH
3
NH
2
6
5
.2.4.5. 1-(4-Amino-2-methylquinolin-6-yl)-3-(4-fluorophenyl)
1
2
2
3.83 (b, 1H), 10.49 (s, 1H), 8.59 (b, 3H), 8.02 (t, 1H), 7.85 (dd,
H), 7.58 (d, 1H), 6.92 (dd, 1H), 6.58 (s, 1H), 3.90 (s, 3H), 2.89 (t,
H), 2.71 (t, 2H), 2.57 (s, 3H); m/z expected 336.2, found 337.4
urea trifluoroacetate (20e).
a light yellow powder: R 0.32 (90:9:1 CHCl
mp 206–207 °C; H NMR (DMSO-d ) d 13.39 (s, 1H), 9.38 (s, 1H),
.26 (s, 1H), 8.37 (b, 2H), 8.15 (s, 1H), 7.90 (d, 1H), 7.77 (d, 1H),
.52 (dd, 2H), 7.15 (t, 2H), 6.55 (s, 1H), 2.56 (s, 3H); m/z expected
Compound 20e was obtained as
f
3
/MeOH/aq CH NH );
3
2
1
+
6
(
[M+H] ).
9
7
+
5
.2.3.41.
pyridin-3-yl)propionamide (18oo).
obtained as a tan powder: R 0.21 (90:9:1 CHCl
); mp 205–207 °C; H NMR (DMSO-d ) d 10.25 (s, 1H), 8.49
s, 1H), 8.33 (d, 1H), 8.26 (s, 1H), 8.03 (b, 2H), 7.74 (s, 2H), 7.02
N-(4-Amino-2-methylquinolin-6-yl)-3-(4-methoxy-
Compound 18oo was
/MeOH/aq CH3-
310.1, found 311.3 ([M+H] ).
f
3
5.2.4.6. 1-(4-Amino-2-methylquinolin-6-yl)-3-(4-isopropylphe-
nyl)urea trifluoroacetate (20f).
as a cream-colored powder: R 0.27 (90:9:1 CHCl /MeOH/aq CH
1
NH
(
2
6
Compound 20f was obtained
f
3
3-

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
431
1
); mp 220–223 °C; 1H NMR (DMSO-d
NH
2
); mp 203–204 °C; H NMR (DMSO-d
6
) d 13.37 (b, 1H), 9.21 (s,
3
MeOH/aq CH NH
2
6
) d 13.47
1
7
H), 9.00 (s, 1H), 8.60 (b, 2H), 8.35 (s, 1H), 7.91 (d, 1H), 7.77 (d, 1H),
(s, 1H), 10.81 (s, 1H), 8.99 (d, 1H), 8.85 (s, 1H), 8.72 (b, 2H),
8.26–8.21 (m, 3H), 8.15–8.07 (m, 2H), 7.92 (d, 1H), 7.86 (d, 1H),
7.73 (d, 2H), 7.66 (d, 1H), 7.60 (dd, 1H), 6.61 (s, 1H), 2.60 (s, 3H);
.42 (d, 2H), 7.17 (d, 2H), 6.56 (s, 1H), 2.84 (t, 1H), 2.56 (s, 3H), 1.18
+
(
d, 6H); m/z expected 334.2, found 335.4 ([M+H] ).
+
m/z expected 404.2, found 405.6 ([M+H] ).
5
.2.4.7. 1-(4-Amino-2-methylquinolin-6-yl)-3-(4-benzoylphe-
nyl)urea trifluoroacetate (20g).
Compound 20g was ob-
0.25 (90:9:1 CHCl /MeOH/
) d 13.39 (s, 1H),
5
.2.5.4. N-(4-Amino-2-methylquinolin-6-yl)-4-(quinolin-8-yl)
tained as a cream-colored powder: R
f
3
benzamide trifluoroacetate (22d).
tained as a cream-colored powder: R
MeOH/aq CH
Compound 22d was ob-
0.2–0.4 (80:18:2 CHCl
) d 13.43
1
aq CH
3
NH
2
); mp 193–195 °C; H NMR (DMSO-d
6
f
3
/
9
7
.65 (s, 1H), 9.47 (s, 1H), 4.64 (b, 2H), 8.42 (s, 1H), 7.92 (d, 1H),
.81–7.60 (m, 8H), 7.55 (d, 2H), 6.57 (s, 1H), 2.57 (s, 3H); m/z ex-
1
3
NH
2
); mp 230–232 °C; H NMR (DMSO-d
6
(
s, 1H), 10.76 (s, 1H), 8.93 (dd, 1H), 8.85 (d, 1H), 8.68 (b, 2H),
+
pected 396.2, found 397.4 ([M+H] ).
8
7
4
.46 (dd, 1H), 8.14–8.06 (m, 4H), 7.88–7.84 (m, 4H), 7.74 (t, 1H),
.62 (dd, 1H), 6.61 (s, 1H), 2.59 (s, 3H); m/z expected 404.2, found
05.5 ([M+H] ).
5
.2.4.8. 1-(4-Amino-2-methylquinolin-6-yl)-3-(2-chlorobenzyl)
urea trifluoroacetate (20h). Compound 20h was obtained
as a white solid: R 0.19 (90:9:1 CHCl /MeOH/aq CH NH ); mp
94–198 °C; H NMR (DMSO-d ) d 13.34 (s, 1H), 9.18 (s, 1H),
.51 (br s, 2H), 8.30 (s, 1H), 7.85 (d, 1H), 7.73 (d, 1H), 7.44–7.33
+
f
3
3
2
1
5
.2.6. General synthesis of compounds 24a–e
1
8
6
Mixtures of N-(4-amino-2-methylquinolin-6-yl)-4-iodobenza-
mide (17rr; 100 mg, 0.17 mmol), the corresponding quinolinyl
boronic acid (0.17 mmol, 1.1 equiv), Pd(OAc)
(
m, 4H), 7.06 (s, 1H), 6.52 (s, 1H), 4.40 (br s, 2H), 2.54 (s, 3H); m/
+
2
(1.0 mg,
z expected 340.1, found 341.2 ([M+H] ).
0
0
.005 mmol), PPh
.198 mmol) in 2:1 i-PrOH/H
3
(3.9 mg, 0.015 mmol) and Na
2
CO
3
(21.0 mg,
2
O (10 mL) were heated to 85 °C in a
5
.2.4.9. 1-(4-Amino-2-methylquinolin-6-yl)-3-(2-methoxyben-
zyl)urea trifluoroacetate (20i). Compound 20i was obtained
as a white solid: R 0.15 (90:9:1 CHCl /MeOH/aq CH NH ); mp
28–131 °C; H NMR (DMSO-d ) d 13.36 (s, 1H), 10.33 (s, 1H),
sealed tube under an argon atmosphere. After 45 min the mixtures
were cooled to room temperature and filtered through a plug of
celite. The filtrates were dissolved in EtOAc (150 mL), washed with
f
3
3
2
1
1
8
1
6
brine (3 Â 30 mL), dried (MgSO
4
), and concentrated to yield crude
.47 (br s, 3H), 7.85–7.72 (m, 2H), 7.25–7.13 (m, 2H), 6.99 (d,
H), 6.56 (s, 1H), 3.8 (s, 3H), 2.9 (t, 2H), 2.69 (t, 2H), 2.56 (s, 3H);
product that was co-evaporated with diethyl ether. The solids thus
obtained were dissolved in 1:1 TFA/DCM (20 mL) and stirred at
room temperature for 4 h. The solutions were evaporated in vacuo
to give crude products which were purified by recrystallization
+
m/z expected 336.2, found 337.3 ([M+H] ).
from MeOH/CHCl
3
.
5
.2.5. General synthesis of compounds 22a–d
Mixtures of N-(4-amino-2-methylquinolin-6-yl)-4-iodobenza-
mide (17qq) (100 mg, 0.17 mmol), the corresponding quinolinyl
5.2.6.1. N-(4-Amino-2-methylquinolin-6-yl)-3-(quinoline-6-yl)
boronic acid (0.17 mmol, 1.1 equiv), Pd(OAc)
2
(1.1 mg,
(21.0 mg,
benzamide trifluoroacetate (24a).
Compound 24a was ob-
0.38 (80:18:2 CHCl /MeOH/
) d 13.46 (s, 1H),
0
.005 mmol), PPh
3
(3.9 mg, 0.015 mmol) and Na
2
CO
3
tained as a cream-colored powder: R
f
3
1
0
2
.198 mmol) in 2:1 i-PrOH/H O (10 mL) were heated to 85 °C in a
3
aq CH NH
2
); mp 212–214 °C; H NMR (DMSO-d
6
sealed tube under an argon atmosphere. After 45 min the mixtures
were cooled to room temperature and filtered through a plug of
celite. The filtrates were dissolved in EtOAc (150 mL), washed with
10.85 (s, 1H), 8.97 (d, 1H), 8.80 (s, 1H), 8.71 (b, 2H), 8.50 (dd,
3H), 8.22 (q, 3H), 8.09 (q, 3H), 7.85 (d, 1H), 7.75 (t, 1H), 7.67–
7.72 (m, 1H), 6.60 (s, 1H), 2.56 (s, 3H); m/z expected 404.2, found
+
brine (3 Â 30 mL), dried (MgSO
4
), and concentrated to yield crude
405.5 ([M+H] ).
product that was co-evaporated with diethyl ether. The solids thus
obtained were dissolved in 1:1 TFA/DCM (20 mL) and stirred at
room temperature for 4 h. The solutions were evaporated in vacuo
to give crude products which were purified by recrystallization
5.2.6.2. N-(4-Amino-2-methylquinolin-6-yl)-3-(quinolin-3-yl)
benzamide trifluoroacetate (24b).
tained as a cream-colored powder: R
Compound 24b was ob-
0.2–0.4 (80:18:2 CHCl
) d 13.49
f
3
/
1
from MeOH/CHCl
3
.
3
MeOH/aq CH NH
2
); mp 238–239 °C; H NMR (DMSO-d
6
(
s, 1H), 10.85 (s, 1H), 9.39 (d, 1H), 8.79 (s, 2H), 8.72 (b, 2H), 8.54
5
.2.5.1. N-(4-Amino-2-methylquinolin-6-yl)-4-(quinolin-3-yl)
(s, 1H), 8.18 (d, 1H), 8.10 (d, 4H), 7.88–7.69 (m, 4H), 6.61 (s, 1H),
2.59 (s, 3H); m/z expected 404.2, found 405.5 ([M+H] ).
+
benzamide trifluoroacetate (22a).
tained as a cream-colored powder: R
MeOH/aq CH
Compound 22a was ob-
0.2–0.4 (80:18:2 CHCl
) d 10.81
f
3
/
1
3
NH
2
); mp 268–270 °C; H NMR (DMSO-d
6
5.2.6.3. N-(4-Amino-2-methylquinolin-6-yl)-3-(isoquinolin-4-
(
(
s, 1H), 9.36 (s, 1H), 8.79 (s, 2H), 8.70 (b, 2H), 8.22 (d, 2H), 8.12
yl)benzamide trifluoroacetate (24c).
obtained as a white powder: R 0.2–0.4 (80:18:2 CHCl
); mp 213–215 °C; H NMR (DMSO-d ) d 13.47 (s, 1H),
0.77 (s, 1H), 9.42 (s, 1H), 8.78 (s, 1H), 8.69 (b, 2H), 8.56 (s, 1H),
Compound 24c was
t, 5H), 7.85 (dd, 2H), 7.70 (t, 1H), 6.62 (s, 1H), 2.59 (s, 3H); m/z ex-
f
3
/MeOH/aq
+
1
pected 404.2, found 405.6 ([M+H] ).
CH
1
3
NH
2
6
5
.2.5.2. N-(4-Amino-2-methylquinolin-6-yl)-4-(isoquinolin-4-
yl)benzamide trifluoroacetate (22b). Compound 22b was
obtained as a tan powder: R 0.2–0.4 (80:18:2 CHCl /MeOH/aq CH3-
); mp 205–207 °C; H NMR (DMSO-d ) d 13.53 (s, 1H), 10.84 (s,
H), 9.41 (s, 1H), 8.85 (s, 1H), 8.73 (b, 2H), 8.53 (s, 1H), 8.29–8.17
m, 3H), 8.11 (d, 1H), 7.86–7.77 (m, 6H), 6.62 (s, 1H), 2.60 (s,
8.29–8.17 (m, 3H), 8.06 (d, 1H), 7.87–7.79 (m, 6H), 6.59 (s, 1H),
2.59 (s, 3H); m/z expected 404.2, found 405.5 ([M+H] ).
+
f
3
1
NH
1
(
2
6
5.2.6.4. N-(4-Amino-2-methylquinolin-6-yl)-3-(quinolin-5-yl)
benzamide trifluoroacetate (24d).
tained as a beige powder: R 0.2–0.4 (80:18:2 CHCl
); mp 244–247 °C; H NMR (DMSO-d ) d 13.47 (s, 1H), 10.76 (s,
H), 8.99 (d, 1H), 8.78 (s, 1H), 8.68 (b, 2H), 8.24 (d, 1H), 8.18–8.13
Compound 24d was ob-
f
3
/MeOH/aq CH3-
+
1
3
H); m/z expected 404.2, found 405.5 ([M+H] ).
NH
2
6
1
5
.2.5.3. N-(4-Amino-2-methylquinolin-6-yl)-4-(quinolin-5-yl)
(m, 3H), 8.06 (d, 1H), 7.91 (t, 1H), 7.85–7.79 (m, 3H), 7.69 (d, 1H),
benzamide trifluoroacetate (22c).
tained as a cream-colored powder: R
Compound 22c was ob-
0.2–0.4 (80:18:2 CHCl
7.59 (dd, 1H), 6.59 (s, 1H), 2.58 (s, 3H); m/z expected 404.2, found
405.5 ([M+H] ).
+
f
3
/

4
32
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
5
.2.6.5.
yl)benzamide trifluoroacetate (24e).
obtained as an off-white powder: R 0.38 (80:18:2 CHCl
); mp 247–248 °C; H NMR (DMSO-d ) d 13.46 (s, 1H),
0.73 (s, 1H), 8.93 (dd, 1H), 8.80 (s, 1H), 8.70 (b, 2H), 8.48 (dd,
N-(4-Amino-2-methylquinolin-6-yl)-3-(quinolin-8-
Compound 22e was
/MeOH/
(45 mL, 45 mmol, 5 equiv), and the combined solution was heated
at reflux overnight. The solvent was evaporated, and the resulting
solid was dissolved in water (15 mL). The pH was adjusted to 3
with 10% aq HCl, and the precipitate formed was filtered and
washed with water. The solid was dissolved in diethyl ether and
f
3
1
aq CH
3
NH
2
6
1
1
1
H), 8.32 (s, 1H), 8.09–8.05 (m, 3H), 7.95–7.66 (m, 5H), 7.61 (dd,
H), 6.59 (s, 1H), 2.58 (s, 3H); m/z expected 404.2, found 405.5
the solution was dried (MgSO
4
), filtered, and evaporated to give
0.36 (25% EtOAc/hex-
) d 12.14 (s, 1H), 7.25–7.20
m, 2 H), 6.96 (d, 1H), 3.78 (s, 3 H), 2.75 (t, 2H), 2.47 (t, 2H).
2.70 g (79%) of product as a white powder: R
f
+
1
(
[M+H] ).
ane); mp 92–94 °C; H NMR (DMSO-d
6
(
5
.2.7. (E)-Methyl 3-(5-chloro-2-methoxyphenyl)acrylate (26a)
To suspension of trimethyl phosphonoacetate (6.66 g,
a
5.2.12. 3-(5-Bromo-2-methoxyphenyl)propionic acid (28b)
Methyl 3-(5-bromo-2-methoxyphenyl)propionate (27a; 1.19 g,
4.0 mmol) was dissolved in a 1.0 M solution of KOH in MeOH
(20 mL, 20 mmol, 5 equiv), and the combined solution was heated
at reflux overnight. The solvent was evaporated, and the resulting
solid was dissolved in water (15 mL). The pH was adjusted to 3
with 10% aq HCl, and the precipitate formed was filtered and
washed with water. The solid was dissolved in diethyl ether and
3
6.6 mmol, 1.2 equiv) and potassium tert-butoxide (5.13 g,
4
5.7 mmol, 1.5 equiv) in anhydrous THF (150 mL) at 0 °C was
added a solution of 5-chlorobenzaldehyde (5.20 g, 30.5 mmol) in
THF (10 mL) over 10–15 min. The resulting suspension was stirred
from 0 °C to room temperature overnight. The solvent was evapo-
rated under reduced pressure, and the residue was diluted with
water (50 mL). The aqueous layer was extracted with EtOAc
(
2 Â 100 mL), and the combined organic layers were washed with
brine (1 Â 30 mL), dried (MgSO ) and concentrated to yield a crude
product as an oil. Recrystallization from 10% EtOAc/hexanes gave
.77 g (55%) of a yellow crystalline solid: R 0.17 (5% EtOAc/hexane;
) d 7.9 (d, 1H), 7.45 (s, 1H), 7.3–7.26
m, 1H), 6.84 (d, 1H), 6.49 (d, 1H), 3.87 (s, 3H), 3.80 (s, 3H).
the solution was dried (MgSO
4
), filtered, and evaporated to give
0.33 (1:1 hexane/
) d 12.12 (br s, 1H),
2
= 9 Hz, 1H), 6.92 (d, 1H), 3.78 (s, 3H), 2.75 (t, 2H),
4
0.99 g (95%) of product as a white powder: R
f
1
EtOAc); mp 114–116 °C; H NMR (DMSO-d
7.35 (dd, J = J
2.46 (t, 2H).
6
3
f
1
1
mp 56–58 °C; H NMR (CDCl
(
3
5
.2.13. 4-Bromo-2-methoxybenzyl alcohol (30b)
5
.2.8. (E)-Methyl 3-(5-bromo-2-methoxyphenyl)acrylate (26b)
To suspension of trimethyl phosphonoacetate (11.6 g,
To a solution of methyl 4-bromo-2-methoxybenzoate (7.14 g,
29 mmol) in THF (35 mL) was added a 2 M solution of LiBH in
4
a
2
3
8 mmol, 1.2 equiv) and potassium tert-butoxide (3.91 g,
4.9 mmol, 1.5 equiv) in anhydrous THF (100 mL) at 0 °C was
THF (35 mL, 70 mmol, 2.4 equiv). The solution was heated at
60 °C for 16 h. The reaction was then cooled to room temperature,
and the solvent was removed under vacuum. The residue was stir-
red in a 5% aqueous acetic acid solution (pH 6) for 15 min. The
aqueous layer was extracted with EtOAc (2 Â 50 mL), then the
added a solution of 5-bromobenzaldehyde (5.0 g, 23.3 mmol) in
THF (100 mL) over 15 min. The resulting suspension was stirred
from 0 °C to room temperature overnight. The solvent was evapo-
rated under reduced pressure, and the residue was diluted with
water (50 mL). The aqueous layer was extracted with EtOAc
4
combined organic extracts were washed with brine, dried (MgSO ),
filtered, and concentrated to yield the crude product as a tan oil.
The oil was purified by flash chromatography using 5% EtOAc in
(
2 Â 100 mL), and the combined organic layers were washed with
brine (1 Â 30 mL), dried (MgSO ) and concentrated to yield a crude
product as an oil. Recrystallization from 10% EtOAc/hexanes gave
.55 g (25%) of a yellow crystalline solid: R 0.50 (2:1 hexane/
) d 7.89 (d, Hz, 1H), 7.59 (d, 1H), 7.42 (dd,
H), 6.78 (d, 1H), 6.49 (d, 1H), 3.87 (s, 3H), 3.80 (s, 3H).
4
hexanes to provide 5.96 g (94%) of the product as a white solid:
1
R
f
0.30 (10% EtOAc/hexane); H NMR (CDCl
3
) d 7.30 (d, 1H), 7.14
1
f
(d, 1H), 7.11 (s, 1H), 5.09 (t, 1H), 4.42 (d, 2H,), 3.77 (s, 3H).
1
EtOAc); H NMR (CDCl
2
3
5.2.14. 4-Bromo-2-methoxybenzaldehyde (31b)
A solution of anhydrous DMSO (5.1 mL, 73 mmol, 3.0 equiv) in
anhydrous dichloromethane (50 mL) was cooled to À78 °C in a
dry ice/i-PrOH bath. Oxalyl chloride (6.4 mL, 73 mmol, 3.0 equiv)
was added dropwise over 45 min. The mixture was stirred for
5
.2.9. Methyl 3-(5-chloro-2-methoxyphenyl)propionate (27a)
To a solution of (E)-methyl 3-(5-chloro-2-methoxyphenyl)acry-
late (26a; 3.70 g, 16 mmol) in MeOH (100 mL) was added 5% Rh/
alumina (0.2 g). The reaction flask was evacuated, flushed with
hydrogen (3Â), and the mixture was stirred under 1 atm of hydro-
gen for 6 h. The mixture was filtered through celite, and the filtrate
was concentrated under vacuum to give a clear oil that was dried
15 min and
a solution of 4-bromo-2-methoxybenzyl alcohol
(30b; 5.30 g, 24 mmol) in dichloromethane (10 mL) was added
dropwise. The resulting mixture was stirred for 30 min at À78 °C.
Triethylamine (12.4 g, 120 mmol, 5.0 equiv) was then added, and
the mixture was allowed to warm to rt over 16 h. The mixture
was quenched with water (100 mL) and extracted with chloroform
(3 Â 200 mL). The combined organic extracts were washed with
under high vacuum to give 3.45 g (93%) of the product as a clear
oil; H NMR (CDCl
1
3
) d 7.15–7.1 (m, 1H), 6.74 (d, 2H), 3.79 (s, 3H),
3
.66 (s, 3H), 2.89 (t, 2H), 2.58 (t, 2H).
water (1 Â 150 mL), brine (1 Â 150 mL), dried (MgSO
4
), filtered,
5
.2.10. Methyl 3-(5-bromo-2-methoxyphenyl)propionate (27b)
and evaporated to yield the crude product as dark residue. The res-
idue was purified by flash chromatography using chloroform to ob-
tain an off-white solid that was recrystallized from hexanes to
To a solution of (E)-methyl 3-(5-chloro-2-methoxyphenyl)acry-
late (26b; 1.42 g, 5.2 mmol) in MeOH (40 mL) was added 5% Rh/
alumina (70 mg). The reaction flask was evacuated, flushed with
hydrogen (3Â), and the mixture was stirred under a 1 atm of
hydrogen for 6 h. The mixture was filtered through celite, and
the filtrate was concentrated under vacuum to give a clear oil that
yield 3.73 g (71%) of the product as a white solid: R
f
0.18 (5%
1
EtOAc/hexane); mp 65–67 °C; H NMR (CDCl
3
) d 10.39 (s, 1H),
7.68 (d, 1H), 7.2–7.16 (m, 2H), 3.94 (s, 3H).
was dried under high vacuum to give 1.08 g (76%) of the product
5.2.15. (E)-Methyl 3-(4-bromo-2-methoxyphenyl)acrylate (32b)
To a suspension of potassium tert-butoxide (7.83 g, 70 mmol,
3.0 equiv) in THF (300 mL) at 0 °C was added trimethyl phosphono-
acetate (10.6 g, 58 mmol, 2.5 equiv). The resulting clear yellow
solution was stirred for 15 min at 0 °C, then a solution of (4-bro-
mo-2-methoxy)benzaldehyde (31b; 5.00 g, 23 mmol) in THF
(20 mL) was added dropwise. The resulting mixture was warmed
1
3
as; H NMR (CDCl ) d 7.3–7.24 (m, 2H), 6.69 (d, 1H), 3.79 (s, 3H),
3
5
1
.67 (s, 3H), 2.88 (t, 2H), 2.58 (t, 2H).
.2.11. 3-(5-Chloro-2-methoxyphenyl)propionic acid (28a)
Methyl 3-(5-chloro-2-methoxyphenyl)propionate (27a; 3.40 g,
5 mmol) was dissolved in a 1.0 M solution of KOH in MeOH

J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
433
from 0 °C to room temperature over 16 h. The solvent was removed
under vacuum, then the crude mixture was diluted with water
tan-colored residue. The reside was co-evaporated with diethyl
ether and treated without further purification with 1:1 TFA/DCM
(5 mL) for 2 h. The solvent was evaporated and the crude product
(
50 mL) and extracted with EtOAc (2 Â 100 mL). The combined or-
ganic extracts were washed with water (1 Â 50 mL), brine
), filtered, and concentrated to provide
was purified by preparative TLC using 80:18:2 CHCl
CH NH . The solids thus obtained were recrystallized from CH3-
CN/MeOH to yield 61 mg (36%) of product as an off-white powder:
3
/MeOH/aq
(
1 Â 50 mL), dried (MgSO
4
3
2
a dark residue. The residue was purified by flash chromatography
1
using 5% EtOAc in hexanes to obtain 3.53 g (56%) of product as a
R
f
0.19 (80:18:2 CHCl
3 3 2
/MeOH/aq CH NH ); mp 219–221 °C; H
1
white solid: R
f
0.19 (5% EtOAc/hexane; H NMR (CDCl
3
) d 8.02 (d,
NMR (DMSO-d ) d 13.30 (b, 1H), 10.36 (s, 1H), 8.93 (s, 1H), 8.61
6
1
6
H), 7.47 (dd, 1H), 7.26–7.22 (m, 1H), 6.93 (t, 1H), 6.84 (d, 1H),
.62 (d, 1H), 6.2 (s, 1H), 3.83 (s, 3H).
(s, 2H), 8.57 (b, 2H), 8.11–8.08 (d, 1H), 7.86–7.76 (m, 2H), 7.50–
7.46 (m, 1H), 7.30 (m, 1H), 7.23 (d, 2H), 6.56 (s, 1H), 3.92 (s, 3H),
2
.97 (t, 2H), 2.71 (t, 2H), 2.56 (s, 3H); m/z expected 412.2, found
+
5
.2.16. Methyl 3-(4-bromo-2-methoxyphenyl)propionate (33b)
413.3 ([M+H] ).
To a solution of (E)-methyl 3-(4-bromo-2-methoxyphenyl)acry-
late (32b; 1.43 g, 5.6 mmol) in MeOH (20 mL) was added 5% Rh/
alumina (200 mg). The reaction flask was evacuated, flushed with
hydrogen (3Â), and the mixture was shaken under 20 psi of hydro-
gen for 3 h. The mixture was filtered through celite, and the filtrate
was concentrated under vacuum to give a clear oil that was dried
5
5
.3. Biological assays
.3.1. BoNT/A LC FRET assay: 96-well format for IC50
determinations
Hit confirmations, IC50 determinations, and kinetic analysis for
inhibitor mechanism of action were carried out utilizing a 96-well
FRET assay microplate format. For hit confirmation, compound
under high vacuum to give 1.38 g (97%) of the product as a color-
1
less oil; H NMR (CDCl
3
) d 7.0–6.96 (m, 3H), 3.82 (s, 3H), 3.66 (s,
3
5
5
H), 2.87 (t, 2H), 2.57 (t, 2H).
(
25
lM final), 20 lM SNAP-25 substrate (MOCAc-Lys-Lys-Val-
4
6
Tyr-Pro-Tyr-Pro-Met-Glu-Lys(Dnp)-NH
were incubated at 37 °C for 40 min, which was the initial linear
rate phase of catalytic activity, in the presence of buffer (50 mM
HEPES 0.05% Tween [pH 7.4]) in a volume of 100lL. The reactions
were stopped by the addition of acetic acid to 0.5% prior to measur-
ing the fluorescence of the cleaved substrate at 485 nm following
excitation at 398 nm in a Molecular Devices (Sunnyvale, CA) plate
reader.
2
), and 2 nM BoNT/A LC)
.2.17. 3-(4-Bromo-2-methoxyphenyl)propionic acid (34b)
Methyl 3-(4-bromo-2-methoxyphenyl)propionate (1.38 g,
.1 mmol) was dissolved in a 1.0 M solution of KOH in MeOH
(
25 mL, 25 mmol, 5 equiv), and the combined solution was heated
at 60 °C overnight. The solvent was evaporated, and the resulting
solid was dissolved in water (15 mL). The aqueous solution was
washed with CHCl
was adjusted to 3 with 3 M aqueous HCl. The precipitate that
formed was filtered and washed with water. The solid was dis-
3
(2 Â 20 mL), and the pH of the aqueous portion
5
.3.2. Bacillus anthracis lethal factor (BaLF) FRET assay
A BaLF FRET assay was performed using 20 M peptide sub-
solved in diethyl ether and the solution was dried (MgSO
4
), filtered,
l
and evaporated to give 1.12 g (86%) of product as a white powder:
1
strate (MCA-KKVYPYPME[dnp]K amide), 20 mM HEPES–0.05%
Tween [pH 8.2] and 5.55 nM BaLF and incubating at 37 °C for
R
f
0.31 (25% EtOAc/hexane; mp 112 °C (subl.); H NMR (DMSO-d
6
)
d 12.1 (b, 1H), 6.9–6.83 (m, 3H), 3.58 (s, 3H), 2.51 (t, 2H), 2.22 (t,
3
3
3
0 min as described previously. Inactivation of the enzyme was
2
H).
achieved by addition of acetic acid to 0.5% and the fluorescent sig-
nal of the cleaved substrate was measured at 395 nm after excita-
tion at 324 nm.
5
.2.18. 3-(2-Methoxy-4-(pyridin-3-yl)phenyl)propionic acid (36)
A mixture of 3-(4-bromo-2-methoxyphenyl)propionic acid (34;
20 mg, 2.0 mmol), pyridine-3-boronic acid pinacol ester (470 mg,
.11 mmol, 1.05 equiv), palladium acetate (14 mg, 0.06 mmol, 3%),
5
2
5.3.3. Human matrix metalloproteinase (MMP) 1 and 9 assays
MMP 1 and 9 FRET assays were performed to assess specificity
of the compounds. An assay mixture consisting of 0.05 % NP-40,
triphenylphosphine (48 mg, 0.18 mmol, 9%), and sodium carbonate
254 mg, 2.40 mmol, 1.2 equiv) in 10% i-PrOH/water (20 mL) was
(
heated to 80 °C in a sealed vessel under argon for 4 h. The mixture
was then cooled and filtered through celite. The filtrate was diluted
with EtOAc (100 mL) and washed with water (45 mL). The organic
50 mM MOPS (pH 6.0), 50 lM fluorogenic substrate (Enzo Life Sci-
ences Catalog Number P-128), and either MMP-1 (38 nM) or MMP-
9 (13 nM) was incubated at 37 °C for 60 min (assay linear range
90 min). The reactions were then stopped with the addition of ace-
tic acid (0.5% [final]) and fluorescence read at 440 nm after excita-
tion at 340 nm.
layer was dried (MgSO
was purified by preparative TLC using 1% AcOH in EtOAc to provide
17 mg (34%) of product as a white solid: R 0.11 (25% EtOAc/hex-
) d 12.12 (br s, 1H), 8.91 (d,
H), 8.55 (d, 1H), 8.1–8.06 (m, 1H), 7.47 (dd, 1H), 7.28–7.20 (m,
4
), filtered, and evaporated. The crude solid
1
f
1
ane; mp >300 °C; H NMR (DMSO-d
6
1
5.3.4. Kinetic analysis of BaLF inhibition
3
H), 3.90 (s, 3H), 2.82 (t, 2H), 2.52 (t, 2H, partially under DMSO).
The kinetics of BaLF inhibition by compounds 18t and 18m
were examined in detail utilizing the FRET assay results. Eadie-
Hofstee transformations of the assay results (Atkins and Nimmo,
1975) indicate these compounds exert a noncompetitive mecha-
nism of inhibition on BaLF activity (see Supplementary data) as
evidenced by a decrease in Vmax (y intercept) and no change in
5
.2.19. N-(4-Amino-2-methylquinolin-6-yl)-3-(2-methoxy-4-
(
pyridin-3-yl)phenyl)propanamide (35)
mixture of 4-(di-tert-butoxycarbonylamino)-6-amino-2-
methylquinoline (16; 128 mg, 0.342 mmol), 3-(2-methoxy-4-
pyridin-3-yl)phenyl)propionic acid (36; 176 mg, 0.680 mmol,
.0 equiv), 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl-
uronium hexafluorophosphate (HATU; 258 mg, 0.68 mmol,
A
(
2
m
K (line slope) with increasing inhibitor concentrations. The ki-
netic assay data was also analyzed utilizing a Michaelis-Menten
plot (GraphPad PrismÒ Software). The analyzed data revealed that
the inhibitor IC50 value did not change significantly as a function of
substrate concentration and, the substrate K value did not change
m
significantly as a function of inhibitor concentration; both observa-
tions are indicative of a noncompetitive inhibition mechanism.
2
1
2
.0 equiv), and N,N-diisopropylethylamine (0.7 mL, 4.0 mmol,
0 equiv) in DMF (5 mL) was stirred at room temperature for
0 h. The mixture was then poured into 5% aqueous Na
2
CO
3
(
50 mL), and extracted with EtOAc (3 Â 50 mL). The combined or-
ganic extracts were washed with 5% aqueous Na CO (50 mL),
brine (50 mL), dried (MgSO ), and evaporated produce
Compound 18t had a calculated K
i
value of 2.9
M.
lM while Com-
2
3
a
pound 18m had a calculated K value of 1.1 l
i
4

4
34
J. D. Williams et al. / Bioorg. Med. Chem. 22 (2014) 419–434
1
1
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Research reported in this article was generously supported by
the National Institute of Allergy and Infectious Disease of the Na-
tional Institutes of Health under award number R43 AI066476.
The content is solely the responsibility of the authors and does
not necessarily represent the official views of the National Insti-
tutes of Health.
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