Synthesis and antibacterial evaluation of new, unsymmetrical triaryl bisamidine compounds

Article abstract of DOI:10.1016/j.bmcl.2014.05.094

Herein we describe the synthesis and antibacterial evaluation of a new, unsymmetrical triaryl bisamidine compound series, [Am]-[indole]-[linker]-[HetAr/ Ar]-[Am], in which [Am] is an amidine or amino group, [linker] is a benzene, thiophene or pyridine ring, and [HetAr/Ar] is a benzimidazole, imidazopyridine, benzofuran, benzothiophene, pyrimidine or benzene ring. When the [HetAr/Ar] unit is a 5,6-bicyclic heterocycle, it is oriented such that the 5-membered ring portion is connected to the [linker] unit and the 6-membered ring portion is connected to the [Am] unit. Among the 34 compounds in this series, compounds with benzofuran as the [HetAr/Ar] unit showed the highest potencies. Introduction of a fluorine atom or a methyl group to the triaryl core led to the more potent analogs. Bisamidines are more active toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by low CC ₅₀ values). Importantly, we identified compound P12a (MBX 1887) with a relatively narrow spectrum against bacteria and a very high CC₅₀ value. Compound P12a has been scaled up and is currently undergoing further evaluations for therapeutic applications.

Full text of DOI:10.1016/j.bmcl.2014.05.094

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3366–3372
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antibacterial evaluation of new, unsymmetrical triaryl
bisamidine compounds
a
a
a
a
Son T. Nguyen a, , John D. Williams , Michelle M. Butler , Xiaoyuan Ding , Debra M. Mills ,
Tommy F. Tashjian a, Rekha G. Panchal b, Susan K. Weir c, Chaeho Moon d, Hwa-Ok Kim d,
Jeremiah A. Marsden e, Norton P. Peet a, Terry L. Bowlin a
⇑
a Microbiotix, Inc., 1 Innovation Drive, Worcester, MA 01604, USA
b United States Army Medical Research Institute of Infectious Diseases, 1425 Porter Street, Frederick, MD 21702, USA
c Department of Medicine, Boston University School of Medicine, 88 E. Newton Street, Boston, MA 02118, USA
d CreaGen Biosciences, Inc., 23 Rainin Road, Woburn, MA 01801, USA
e Organic Consultants, Inc., 132 E. Broadway, Suite 107, Eugene, OR 97401, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Herein we describe the synthesis and antibacterial evaluation of a new, unsymmetrical triaryl bisamidine
compound series, [Am]-[indole]-[linker]-[HetAr/Ar]-[Am], in which [Am] is an amidine or amino group,
[linker] is a benzene, thiophene or pyridine ring, and [HetAr/Ar] is a benzimidazole, imidazopyridine,
benzofuran, benzothiophene, pyrimidine or benzene ring. When the [HetAr/Ar] unit is a 5,6-bicyclic
heterocycle, it is oriented such that the 5-membered ring portion is connected to the [linker] unit and
the 6-membered ring portion is connected to the [Am] unit. Among the 34 compounds in this series, com-
pounds with benzofuran as the [HetAr/Ar] unit showed the highest potencies. Introduction of a fluorine
atom or a methyl group to the triaryl core led to the more potent analogs. Bisamidines are more active
toward bacteria while the monoamidines are more active toward mammalian cells (as indicated by
low CC50 values). Importantly, we identified compound P12a (MBX 1887) with a relatively narrow spec-
trum against bacteria and a very high CC50 value. Compound P12a has been scaled up and is currently
undergoing further evaluations for therapeutic applications.
Received 14 April 2014
Revised 26 May 2014
Accepted 28 May 2014
Available online 12 June 2014
Ó 2014 Elsevier Ltd. All rights reserved.
New therapies are urgently needed for treatment of Gram-
negative bacterial infections. Due to the emergence of bacterial
strains resistant to all classes of b-lactam antibiotics (penicillins,
cephalosporins, and carbapenems), aminoglycosides, and quino-
lones we have reached a crisis in the availability of effective ther-
apy.1 Among the five novel antibiotics introduced since 2000
(linezolid, daptomycin, retapamulin, fidaxomicin and bedaquiline),
none are effective against Gram-negative infections.2 All of the
newly approved antibiotics for Gram-negative infections are
analogs of known drugs, for example, b-lactams, fluoroquinolones,
tetracyclines and macrolides.2 To manage infections complicated
by the continued development of resistance, new therapeutic
approaches and rapid development of novel antibiotics are
essential.
N
N
N
N
N
H
N
H
NH
NH
HN
HN
MBX 1066
(original hit compound)
linker
N
H
N
H
previous work: symmetrical scaffold
aromatic
linker
Am
ring
N
Am: amidine or amine
NH
In collaboration with the U.S. Army Medical Research Institute
of Infectious Diseases (USAMRIID), we have developed a series of
bisamidine compounds that show potent activities against preva-
lent Gram-negative and Gram-positive pathogens.3 Details on the
this work: unsymmetrical scaffold
Figure 1. Unsymmetrical and symmetrical scaffolds.
synthesis and structure–activity relationship studies of com-
pounds in the symmetrical head-to-head bisindole series (Fig. 1)
have been published elsewhere.4 In this Letter we describe the
⇑
Corresponding author. Tel.: +1 508 757 2800; fax: +1 508 757 1999.
E-mail address: snguyen@microbiotix.com (S.T. Nguyen).
http://dx.doi.org/10.1016/j.bmcl.2014.05.094
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
3367
synthesis and biological evaluation of a new, unsymmetrical series,
in which one of the indole rings in the triaryl core has been
replaced with another aromatic unit (benzimidazole, imidazopyri-
dine, benzofuran, benzothiophene, pyrimidine or benzene) and the
central linker unit contains benzene, thiophene, or pyridine rings.
Our initial synthetic efforts focused on the synthesis of
unsymmetrical compounds containing an indole, a phenyl linker,
and a non-indole 5:6 heterocyclic system. These compounds were
prepared from the following three indole derivatives:
N-Boc-2-(4-bromophenyl)indole-6-carbonitrile (5), (4-(N-Boc-6-
cyanoindol-2-yl)phenyl)boronic acid pinacol ester (6), and
(N-Boc-6-cyanoindol-2-yl)boronic acid (7) following path A, B or
C as illustrated in Scheme 1. Piperidine-catalyzed condensation
of 4-methyl-3-nitrobenzonitrile and 4-bromobenzaldehyde pro-
vided a mixture of cis- and trans-stilbenes 3. Treatment of 3 with
triethylphosphite led to reduction of the nitro group and formation
of the indole 4.5 Protection of the indole nitrogen with a Boc group
provided the intermediate 5, which could then be directly
converted to the intermediate nitrile 9 by conducting a Suzuki–
Miyaura cross-coupling6 with an aryl pinacolboronate (path A).
Alternatively, borylation of the bromide 5 with bis(pinacola-
to)diboron in the presence of a palladium catalyst gave intermedi-
ate boronic acid ester 67 which could also be converted to 9 using
palladium-catalyzed coupling methodology (path B). Intermediate
9 could also be synthesized by condensation of commercially
available boronic acid 7 with aryl bromide 8 (path C). Subsequent
treatment of compounds 9 with diamines in the presence of phos-
phorous pentasulfide provided the desired amidine products 10.8
Preparation of benzimidazole and imidazopyridine containing
3,4-diaminobenzonitrile (11) and 4-bromobenzaldehyde (2) at
reflux in the presence of sodium metabisulfite provided benzimid-
azole 12.9 Treatment of 2-amino-5-cyanopyridine (13) and
2-amino-4-cyanopyridine (14) with 2,40-dibromoacetophenone
(15) provided the corresponding imidazopyridines 16 and 17,
respectively.10 Coupling of the diarylbromides 12, 16 and 17 with
(N-Boc-6-cyanoindol-2-yl)boronic acid (7), followed by amidine
formation provided products P1a–P3b.
Preparation of benzofuran- and benzothiophene-containing
analogs followed path A (Scheme 3). The preparation of (6-cya-
nobenzofuran-2-yl)boronic acid pinacol ester (24) has been opti-
mized and scaled up to hundreds of grams and involves no
chromatographic purifications.11 The synthesis began with iodin-
ation of 3-hydroxybenzoic acid (18) to provide the iodide 19 in
51% yield.12 Conversion of the carboxylic group of 19 to the corre-
sponding nitrile was accomplished in 3 steps, and involved chlori-
nation, amination and dehydration to provide the nitrile 20 in 51%
overall yield.13 Sonogashira coupling of 20 with TMS-acetylene
gave arylacetylene 21 in 89% yield.11 Treatment of 21 with CuI
and triethylamine provided benzofuran-6-carbonitrile (22) in 68%
yield.11 Iridium-catalyzed borylation of 22 and the commercially
available benzofuran-5-carbonitrile (23) occurred selectively at
the 2-position to provide (6-cyanobenzofuran-2-yl)boronic acid
pinacol ester (24) and (5-cyanobenzofuran-2-yl)boronic acid pina-
col ester (25) in 74% and 83% yields, respectively.11 Cross-coupling
of 24, 25 and the commercially available (6-cyanobenzothiophen-
2-yl)boronic acid (26) with the intermediate 5, followed by
amidine formation as described above, provided products P4–P6f.
Minimum inhibitory concentration (MIC) values for compounds
P1a–P6f against a range of clinically important Gram-positive and
analogs followed path
C
(Scheme 2). Condensation of
Br
CH3
b
c
O
H
a
+
Br
Br
N
H
NC
NO2
NC
NC
NO2
1
2
4
3
d
Br
Bpin
N
N
Boc
NC
NC
NC
Boc
6
path A
5
ArBpin
path B
Br-Ar-X
path C
e
Ar Am
B(OH)2
Ar
X
HN
N
H
N
N
Boc
NC
Br
Ar
X
Boc
N
10
[Am = amidine or amino]
7
9
8
[X = CN or amino]
Scheme 1. General methods for compound preparation. Reagents and conditions: (a) piperidine, 125 °C; (b) P(OEt)3, reflux; (c) Boc2O, DMAP, THF; (d) PdCl2(dppf), B2pin2,
NaOAc, dioxane, 80 °C; (e) P2S5, diamine, 120–130 °C.
N
NH2
O
H
N
b, c, d
a
e
H
N
H
N
+
Br
Br
Br
Br
N
H
N
H
N
H
NC
NH2
NC
N
N
N
11
n
2
n
P1a
12
: n = 1
P1b: n = 2
H
N
4
NC
5
5
6
O
N
b, c, d
5
N
H
N
+
NC
N
N
N
H
n
Br
6
N
N
NH2
P2a
P2b
P3a
P3b
: amidine at C(5), n = 1
: amidine at C(5), n = 2
: amidine at C(6), n = 1
: amidine at C(6), n = 2
15
n
13
14
: CN at C(5)
: CN at C(4)
16
: CN at C(5)
: CN at C(6)
17
Scheme 2. Preparation of benzimidazole and imidazopyridine containing analogs. Reagents and conditions: (a) Na2S2O5, EtOH, DMSO, H2O, 100 °C; (b) 7, Pd(Ph3P)4 or
Pd(OAc)2/2-(Biphenyl)t-Bu2P, K2CO3, EtOH, toluene, 70 °C; (c) CF3CO2H, MeOH, CH2Cl2; (d) P2S5, NH2CH2(CH2)nNH2, 120 °C; (e) EtOH, reflux.

3368
S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
TMS
I
I
b, c, d
a
e
f
HO2C
OH
HO2C
OH
NC
OH
O
NC
NC
OH
18
19
20
22
21
g
5
NC
6
5
NC
6
Bpin
H
N
O
O
R
5
6
h
H
22: CN at C(6)
24: CN at C(6)
N
N
n
Z
N
H
23
25
: CN at C(5)
: CN at C(5)
R
i
N
n
h
5
P4: amidine at C(6), Z = S, R = H, n = 2
B(OH)2
NC
P5
: amidine at C(5), Z = O, R = H, n = 2
6
S
Z
N
P6a
P6b
: amidine at C(6), Z = O, R = H, n = 1
: amidine at C(6), Z = O, R = CH3, n = 1
NC
CN
j
Boc
26
27: CN at C(6), Z = O
28
29
: CN at C(5), Z = O
: CN at C(6), Z = S
H
N
H
N
O
N
H
N
N
R'
R'
P6c: R' = H
P6d
: R' = OH
P6e: R' = OMe
P6f
: R' = F
Scheme 3. Preparation of benzofuran and benzothiophene containing compounds. Reagents and conditions: (a) ICl, AcOH, 45 °C; (b) EtCO2Cl, Et3N, THF; (c) NH4OH; (d)
(CF3CO)2O, pyridine, CH2Cl2; (e) TMS-acetylene, Pd(PPh3)2Cl2, CuI, Et3N, THF, 40 °C; (f) CuI, Et3N, EtOH, reflux; (g) B2pin2, [Ir(OMe)COD]2, hexanes; (h) 5, Pd(PPh3)4, Na2CO3,
EtOH, toluene; (i) P2S5, NH2CHR(CH2)nNH2, 120 °C; (j) P2S5, NH2CH2CHR0CH2NH2, 120 °C.
Table 1
MIC and CC50 values for compounds P1a–P6f
d
Cpd #
Structures
5,6-HetAr
MIC (μg/mL)a,b,c
CC50
Am
E.col+ E.col- P.aer P.ae- K.pne B.sub MRSA VRE B.anth HeLa
1066
1.25
0.16
7.5
2.5
0.24
0.12
0.16
0.16
0.31
32.5
P1a
P1b
P2a
P2b
P3a
10
0.31
0.31
0.63
0.31
0.31
20
40
20
0.16
0.08
0.31
0.08
2.5
1.25
0.31
80
0.63
0.31
20
5
8.6
27.6
-
,,
,,
2.5
40
2.5
>80
5.0
40
2.5
>80
5.0
>80
5.0
80
0.31
40
5.0
2.5
5.0
5.0
0.63
>80
0.63
>80
1.25
>80
19.7
2.5
,,
P3b
P4
2.5
0.31
0.31
0.39
40
10
2.5
>80
10
5.0
0.16
0.10
0.08
2.5
1.25
0.31
0.08
1.25
0.16
0.08
20.9
5.9
0.63
0.63
1.25
1.25
0.31
0.31
P5
2.5
8.5
P6a
P6c
P6b
0.47
0.63
1.09
0.24
0.16
0.63
10.0
0.63
80.0
15.0
0.47
80.0
0.94
0.55
5.0
0.16
0.14
0.35
0.16
0.10
0.55
0.16
0.39
0.63
0.31
5.0
9.7
26
,,
,,
,,
,,
,,
,,
0.63
7.6
P6b-
iso
0.63
0.63
60.0
80.0
2.5
0.20
0.31
0.39
0.31
5.7
P6d
P6e
P6f
2.5
0.16
0.24
0.27
6.3
>80
5.0
15.0
>80
2.5
1.09
1.25
0.63
0.08
0.08
0.16
0.31
0.31
0.31
0.31
0.63
0.31
0.55
0.31
0.31
11.9
6.0
2.5
1.25
47.5
a
MIC values obtained by the broth dilution method.16
Bacterial strains: Ecol+ = Escherichia coli 700 TolC+, Ecolꢀ = E. coli TolCꢀ, P.aer = Pseudomonas aeruginosa PAO 1, P.aeꢀ = P. aeruginosa PAO 1
b
D
(mexABꢀoprM), K.
pne = Klebsiella pneumonia 13882, B. sub = Bacillus subtilis BD54, MRSA = methicillin-resistant Staphylococcus aureus 1094, VRE = vancomycin-resistant Enterococcus faecalis
ATCC 51575, B. anth = Bacillus anthracis Sterne.
c
MIC 61: green; 1 < MIC 6 5: blue; 5 < MIC 6 20: yellow; MIC >20: pink.
d
Cytotoxicity determined by using the vital stain MTS,17 units in
lg/mL.

S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
3369
Gram-negative pathogens, including those proficient and deficient
in efflux mechanisms, as well as cytotoxic concentration (CC50
indole-benzofuran containing scaffold. Again, the tetrahydropyr-
imidine amidine imparts the greatest activity; methylimidazoline,
hydroxyl- or methoxytetrahydropyrimidine amidines all exhibit
significantly lower potency against P. aeruginosa. The fluorotetra-
hydropyrimidine amidine makes compound P6f nearly as active
as the corresponding norfluoro analog P6c, but less cytotoxic.
Lastly, we noted that the position of the amidine on the imidazo-
pyridine does not affect the activity but on the benzofuran it has
a moderate effect, with the 6-position being generally more pre-
ferred. An exception is that P5 is more potent than P6c against B.
anthracis.
)
values against human HeLa cells, are shown in Table 1. Compounds
containing benzimidazole and imidazopyridine heterocyclic
systems are more susceptible to efflux by Escherichia coli than
those containing benzothiophene and benzofuran systems, as the
MIC values against the efflux deficient (TolCꢀ) strains are substan-
tially lower than those against the wild-type (TolC+) strains.
Against the efflux-deficient (TolCꢀ) strain, all compounds are very
active (MIC <1 lg/mL). The MIC values against Klebsiella pneumonia
closely resemble those against the E. coli wild type strain
(differences within 2-fold). In contrast, their MIC values against
To determine whether both amidine groups are required for
antibacterial activity, we prepared a number of analogs that have
different amine-containing groups. Preparation of these analogs
followed paths A and B as shown in Scheme 4. Coupling of interme-
diate 6 with commercially available building blocks 30, 31 and
intermediate 5 with (commercially available) 34 followed by ami-
dine formation provided products P7a–P9b.
MIC and CC50 values for P7a to P9b are shown in Table 2.
Overall, these compounds possess significantly lower antibacterial
activities compared to the bisamidine compounds, and higher
cytotoxicity against HeLa cells. Notably, P7a and P8a are potent
against the efflux deficient strain of E. coli but are not active against
the wild type strain, suggesting that they are strongly susceptible
to efflux. Compounds P7a, P8a, P9a and P9b are active against
Pseudomonas aeruginosa varied widely, from <1
>80 g/mL (P2a, P6e). These values remained essentially the same
against the efflux deficient strain PAO1
(mexAB ꢀ oprM), indicat-
lg/mL (P6c) to
l
D
ing that the reduced potency against Pseudomonas for some com-
pounds was not due to efflux by the mexAB ꢀ oprM pump. Most
of the compounds are broadly active against Gram-positive patho-
gens with the exception of compounds P2a and P3a. Notably, the
tetrahydropyrimidine amidine homologs P2b and P3b are still very
potent against MRSA, VRE and Bacillus anthracis, following a trend
that we found for the bisindoles series.4 The differences in MIC
values caused by a simple change from a 5-membered ring to
6-membered ring amidine in these analogs are striking. Effects of
the amidine group on activity were further examined in the
N
N
N
N
a
b
N
H
N
N
Br
N
R
R
N
H
R
N
N
Boc
N
CN
R'
N
P7a
: R = OCH2, R' = CH2CH2
30
32
: R = OCH2
31: R = CH2
: R = OCH2
P7b: R = OCH2, R' =CHCH3
P8a: R = CH2, R' = CH2CH2
33: R = CH2
P8b
: R = CH2, R' = CHCH3
c, d
N
H
N
N
Bpin
N
H
N
34
P9a
n
: n = 1
P9b: n = 2
Scheme 4. Preparation of monoamidine compounds. Reagents and conditions: (a) 6, Pd(PPh3)4, Na2CO3, DME, H2O, 75 °C; (b) P2S5, NH2-R0-CH2NH2, 120 °C; (c) 5, Pd(PPh3)4,
Na2CO3, EtOH, toluene, 75 °C; (d) P2S5, NH2CH2(CH2)nNH2, 120 °C.
Table 2
MIC and CC50 values for compounds P7a–P9ba
Cpd #
Structures
CC50
MIC (μg/mL)
K.pne B. sub MRSA
E.col+ E.col-
P.aer
>80
VRE
B. anth
0.55
HeLa
P7a
35
0.63
70
0.78
7.5
3.75
2.0
P7b
P8a
P8b
>80
>80
>80
60.0
0.63
40.0
>80
>80
>80
>80
>80
>80
10.0
0.63
40.0
>80
2.5
>80
5.0
22.5
0.63
40.0
1.3
5.7
1.1
40.0
>80
P9a
P9b
>80
>80
>80
>80
>80
>80
>80
>80
0.63
0.63
1.04
1.09
3.3
3.1
2.1
≤0.63
≤0.63
0.47
a
Bacterial strains: Ecol+ = E. coli 700 TolC+, Ecolꢀ = E. coli TolCꢀ, P.aer = P. aeruginosa PAO 1, K. pne = K. pneumonia 13882, B. sub = B. subtilis BD54, MRSA = methicillin-
resistant S. aureus 1094, VRE = vancomycin-resistant E. faecalis ATCC 51575, B. anth = B. anthracis Sterne; MIC 61: green; 1 < MIC 6 5: blue; 5 < MIC 6 20: yellow; MIC >20:
pink; CC50 units in g/mL
l

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S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
Gram-positive pathogens. These activities sharply decline in the
methylimidazoline isomers, P7b and P8b. Lastly, compounds P9a
and P9b are highly cytotoxic. Thus, the data suggests that the
bisamidine is important for activity against bacteria, especially
Gram-negative bacteria, while monoamidines have more inhibi-
tory effects on the growth of mammalian cells.
Because the benzofuran-containing analog P6c has the most
potent antibacterial activity of this series and an acceptable level
of cytotoxicity, we decided to explore the indole-aryl-benzofuran
scaffold further by varying the central linker unit and the amidine
substitutions. Preparation of these compounds followed a route
similar to path A, as shown in Scheme 5. Syntheses of
N-Boc-2-(4-bromo-3-fluorophenyl)indole-6-carbonitrile (37) and
N-Boc-2-(5-bromothiophen-2-yl)indole-6-carbonitrile (40) were
based on the route used previously for the preparation of 5. Thus,
condensation of 4-bromo-3-fluorobenzaldehyde (35) and 5-bro-
mothiophene-2-carboxaldehyde with 4-methyl-3-nitrobenzonitri-
le (1), followed by treatment with triethylphosphite, and
subsequent protection of the indole nitrogen with a Boc group,
provided 6-cyanoindoles 37 and 40, respectively. Coupling of 37,
Br
CH3
O
H
b, c
d
a
+
Br
Br
O
F
N
Boc
NC
NO2
NC
NC
e
NO2
36
F
F
1
35
37
H
N
H
N
N
H
O
N
NC
CN
R1
F
Boc
N
N
P10: R1 = F
38
P11: R1 = NH(CH2)3NH2
CH3
NO2
O
H
a', b, c
f
S
+
N
Boc
O
N
Boc
40
S
NC
Br
NC
CN
NC
S
Br
41
1
39
g
h
S
H
N
H
N
S
NH
O
H
N
H
NH
O
N
R2
R2
N
N
P12c: R3 = H
N
N
P12a: R2 = H
P12b
R3
R3
: R3 = OH
: R2 = CH3
P12d
P12e
3
: R = F
d, h
H
N
H
Br
N
N
H
O
N
N
Boc
N
NC
N
N
R3
R3
P13a: R3 = H
42
P13b: R3 = CH3
P13c: R3 = OH
: R3 = F
P13d
Scheme 5. Preparation of compounds with the indole-aryl-benzofuran scaffold. Reagents and conditions: (a) piperidine, sulfolane, 145 °C; (a0) piperidine, 110 °C; (b) P(OEt)3,
130 °C; (c) Boc2O, DMAP, THF; (d) 24,Pd(PPh3)4, Na2CO3, EtOH, toluene, 70 °C; (e) P2S5, NH2(CH2)3NH2, 120 °C; (f) 24, Pd(dppf)Cl2, Na2CO3, MeCN, 70 °C; (g) P2S5,
NH2CHR2CH2NH2, 120 °C; (h) P2S5, NH2CH2CHR3CH2NH2, 120 °C.
CH3
CH3
.
4
4
NHNH2 HCl
O
b
c
a
NC
6
NC
6
Br
Br
Br
+
N
Boc
N
H
CN
44
45: CN at C(4)
46: CN at C(6)
43
47: CN at C(4)
(not separated)
(not separated)
48: CN at C(6)
CN
CH3
CH3
N
+
N
Boc
O
O
NC
CN
CN
Boc
50
49
(isomers separated)
d
d
CN
CH3
CH3
H
N
N
N
N
O
N
H
O
H
N
NH
HN
P14
P15
Scheme 6. Synthesis of 3-methylindole containing compounds. Reagents and conditions: (a) p-TsOH.H2O, toluene, reflux; (b) Boc2O, DMAP, DMF; (c) Pd(PPh3)4, Na2CO3, DME,
H2O, 85 °C; (d) P2S5, NH2(CH2)3NH2, 120 °C.

S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
3371
Table 3
MIC and CC50 values for compounds P10–P15a
Cpd #
linker
Structures
Am
MIC (μg/mL)
CC50
HeLa
E.col+ E.col- P.aer K.pne B.sub MRSA VRE
B. anth
0.08
P10
0.63
0.31
2.5
0.63
1.25
0.16
0.16
0.08
14.3
4.6
P11
2.5
0.63
2.5
0.63
1.25
0.63
0.24
P12a
2.80
1.25
0.27
0.63
66.7
26.7
60.0
20.0
0.94
1.04
1.56
0.63
1.09
0.52
0.16
0.31
182.2
14.5
,,
P12b
,,
P12c
0.63
5.0
0.31
0.47
0.31
0.16
0.27
0.63
0.31
0.31
10.0
10.0
40.0
53.3
1.25
5.0
2.5
0.31
1.88
0.63
0.31
0.31
2.5
0.31
2.5
0.16
1.25
0.31
0.16
0.63
1.09
0.63
0.08
2.5
0.08
0.43
0.16
0.08
0.08
3.75
0.16
0.08
2.5
8.7
12.7
22.5
15.6
2.2
,,
P12d
15.0
2.5
,,
P12e
1.25
1.25
1.88
80
0.63
0.39
0.63
5.0
P13a
1.56
2.5
P13b
‘’
,,
P13c
25
8.8
16.7
19.6
1.5
P13d
8.3
8.3
2.5
0.83
0.16
5.0
0.63
0.16
5.0
,,
P14
P15
See scheme 6
See scheme 6
0.31
20.0
1.25
40.0
0.31
20.0
-
a
Bacterial strains: Ecol+ = E. coli 700 TolC+, Ecolꢀ = E. coli TolCꢀ, P. aer = P. aeruginosa PAO 1, K. pne = K. pneumonia 13882, B. sub = B. subtilis BD54, MRSA = methicillin-
resistant S. aureus 1094, VRE = vancomycin-resistant E. faecalis ATCC 51575, B. anth = B. anthracis Sterne; MIC 61: green; 1 < MIC 6 5: blue; 5 < MIC 6 20: yellow; MIC >20:
pink; CC50 units in g/mL.
l
40 and 4214 with (6-cyanobenzofuran-2-yl)boronic acid pinacol
ester (24) followed by amidine formation provided products
P10–P13d.
P12a could be useful for narrow-spectrum treatment of select
pathogens. For example, we found that P12a is very active against
Francisella tularensis (MIC 0.063–2.5 lg/mL across 13 strains
Compounds with a 3-methyl substituent on the indole were pre-
pared as shown in Scheme 6, using the Fischer indole synthesis.
Condensation of 3-cyanophenylhydrazine (43)15 with 40-bromo-
propiophenone (44) provided a mixture of regioisomers 45 and
46, which were carried on to the next step. Protection of the indole
nitrogen with a Boc group followed by coupling of the product mix-
ture with (6-cyanobenzofuran-2-yl)boronic acid pinacol ester (24)
provided triaryl compounds 49 and 50, which were separated by
silica gel chromatography. Amidination of 49 provided the bisami-
dine P14 as expected. Under the same conditions, only one nitrile
group in 50 was converted to the corresponding amidine, leading
to isolation of the monoamidine product P15. We speculate that
the 4-cyanoindole functionality in bis-nitrile 50 is hindered with
respect to the 6-cyanoindole functionality in bis-nitrile 49, which
is the reason for the production of monoamidated compound P15.
MIC and CC50 values for the indole-benzofuran analogs with
various linkers are summarized in Table 3. Introduction of methyl
and fluoro groups to the core provided potent compounds (P14,
P10), while the more polar diaminopropane functionality was less
effective (P11). This pattern is consistent with what we observed in
the first series (Table 1): addition of nitrogen atoms to the core
produced compounds with higher levels of efflux. Replacing the
phenyl linker with pyridine or thiophene led to compounds that
were more susceptible to efflux and less active against Gram-
negative bacteria. Notably, thiophene-linked compound P12a
showed very low cytotoxicity. This unique feature suggests that
tested). We have subsequently optimized the synthesis of P12a
to the hundred-gram scale. Further evaluation of this compound
is pending and will be reported in the future.
In summary, we have prepared 34 new unsymmetrical triaryl
bisamidine and monoamidine compounds, featuring 15 new triaryl
core structures, and evaluated them for antibacterial activities and
cytotoxicity. We found that benzofuran can replace one indole ring
from the original hit compound (MBX 1066) to deliver broad-
spectrum compounds with improved potency such as P5, P6c
and P6f. Introduction of a fluorine atom or a methyl group to the
triaryl core led to the more potent analogs P10 and P14, respec-
tively. Bisamidines are more active toward bacteria while the
monoamidines are more active toward mammalian cells (as
indicated by low CC50 values). Importantly, we have identified
compound P12a with a relatively narrow spectrum of activity
against bacteria and a very high CC50 value. The synthesis of com-
pound P12a has been optimized and scaled up, and the compound
is currently under further evaluations for therapeutic applications.
Acknowledgments
We thank Ms. Atiyya Khan for providing some quantities of the
intermediates 40 and 42.
Research reported in this article was generously supported by
the National Institute of Allergy and Infectious Disease of the
National Institutes of Health under award numbers U01AI082052

3372
S. T. Nguyen et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3366–3372
4. Williams, J. D.; Nguyen, S. T.; Gu, S.; Ding, X.; Butler, M. M.; Tashjian, T. F.;
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authors and does not necessarily represent the official views of
the National Institutes of Health.
Supplementary data
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