DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 2: C-terminal benzimidazoles and derivatives

Article abstract of DOI:10.1016/j.bmcl.2003.12.043

The synthesis and in vitro potency of DNA minor-groove binding antibacterials lacking the C-terminal amide bond are described. The crescent shaped molecules bear the positively charged amino group at an internal pyrrole unit instead of the C-terminus. Thr

Full text of DOI:10.1016/j.bmcl.2003.12.043

Bioorganic & Medicinal Chemistry Letters 14 (2004) 1259–1263
DNA binding ligands targeting drug-resistant Gram-positive
bacteria. Part 2: C-terminal benzimidazoles and derivatives
Roland W. Burli, Peter Jones, Dustin McMinn, Quan Le, Jian-Xin Duan,
Jacob A. Kaizerman, Stacey Difuntorum and Heinz E. Moser*
Genesoft Pharmaceuticals, Inc., 7300 Shoreline Court, South San Francisco, CA 94080, USA
Received 30 October 2003; revised 3 December 2003; accepted 9 December 2003
Abstract—The synthesis and in vitro potency of DNA minor-groove binding antibacterials lacking the C-terminal amide bond are
described. The crescent shaped molecules bear the positively charged amino group at an internal pyrrole unit instead of the C-
terminus. Three structural parameters were investigated: the N-terminal unit, the internal amino group, and the C-terminal ring
system. Several compounds demonstrated good in vitro potency against various Gram-positive bacteria and some molecules were
moderately active against Escherichia coli, a representative Gram-negative strain.
# 2003 Elsevier Ltd. All rights reserved.
In the past decade, there has been a dramatic increase in
the occurrence of bacterial strains that are resistant to
multiple classes of antibiotics;1 for instance, methicillin-
resistant Staphylococcus aureus (MRSA) and vancomy-
cin-resistant Enterococcus faecalis (VRE) are known to
cause problems particularly in hospital settings, while
infections by multidrug-resistant pneumococci occur
also in the community. To combat such drug-resistant
bacterial strains, there is a demand for antibiotics acting
by a novel mechanism and consequently lacking cross-
resistance to existing agents.2 As reported previously,3ꢀ5
we started to optimize analogues of the natural product
distamycin A for antibacterial potency and improved
drug-like properties; in this context, novel pharmaco-
phores have been identified that show excellent in vitro
activity against a wide range of Gram-positive bacterial
strains. These molecules exercise their antibacterial
activity by recognizing and binding the minor-groove of
A/T rich DNA sequences within bacterial genomes. As
a result, they are likely to inhibit DNA dependent pro-
cesses such as DNA replication and RNA transcription.
Our optimization process is schematically illustrated in
Figure 1. In an initial screening, we identified analogues
of distamycin A that consist of three internal N-methyl
pyrrole carboxamides (Py), an N-terminal 4-chloro-
isothiazole (It), and a charged amino group at both ends
of the molecule.3 In a small library approach, we have
prepared analogues with variable substituents at both
termini and have found that the N-terminal charge is
not critical for good antimicrobial activity. Thus, we
rigorously screened for novel N-terminal units and
discovered several promising It-alternatives such as
3-chlorothiophene-2-carboxamide, isoquinoline-3-car-
boxamide, or 2,4-dihalobenzamides, respectively.4
A
compound from this series demonstrated in vivo efficacy
in a mouse peritonitis model against a lethal S. aureus
infection (MSSA).4 In a next step, we started to modify
the internal Py3-element and replaced one Py-carbox-
amide unit by the isosteric benzimidazole group.5 This
formally eliminated one internal amide bond and it was
anticipated that consequently the drug-like properties of
the corresponding molecules should be improved.
Indeed, benzimidazole replacement of the Py unit next
to the C-terminus yielded compounds with comparable
antibacterial activity and—as demonstrated in one
case—improved in vivo efficacy.5 We were interested in
the question whether the drug-like properties of these
molecules could be further increased by removal of an
additional amide bond through internalization of the
C-terminal amino group. Notably, an aliphatic amino
group is at least in part positively charged under physio-
logical conditions and cannot be omitted without ser-
iously compromising the solubility of the polyaromatic
molecules. Herein, we focus on the synthesis and in vitro
antibacterial activity of a small library of compounds in
Keywords: Antibiotics; DNA binder; Minor groove; Lead optimization.
* Corresponding author. Tel.:+1-650-837-1820; fax:+1-650-827-0476;
e-mail: hmoser@genesoft.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.12.043

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¨
which the amino group is attached at the ring nitrogen
of an internal Py moiety.
benzimidazole building block 10 and its aza-analogues
11–13 were prepared in two steps from the nitro pyrrole
4.8 Coupling of 4 under standard conditions (HBTU
activation)4 to the corresponding 1,2-diamines provided
the amino amides 6–9. Heating these amides under
acidic conditions led to the corresponding fused ring
systems 10–13. Notably, the trichloromethyl ketone 59
can be used instead of the acid 4; details for the synth-
esis of benzimidazole 10 starting from ketone 5 are
described.10 Hydrogenolytic reduction of the nitro
dimers 10–13 followed by in situ coupling to the corres-
ponding N-terminal dimers gave the final compounds.
All products were purified by preparative HPLC and
The synthesis of the antibacterial compounds 17–35
(Table 1) is described in Scheme 1. Alkylation of the
known pyrrole 16 and subsequent hydrogenation yiel-
ded the amino ester 3.7 Acylation of amine 3 with either
an acyl chloride or an activated carboxylic acid, fol-
lowed by saponification of the resulting dimeric esters
gave the N-terminal dimers; analogous chemistry has
been described in detail.4,5 The C-terminal pyrrole–
1
analyzed by H NMR and mass spectroscopy (general
procedure below).11
The compounds 36–42 (Table 2) were synthesized from
the common tetrameric intermediate 16 (Scheme 2).
Coupling of activated 4-chloro-2-fluorobenzoic acid
(HBTU) to the amino ester 14,12 followed by saponifi-
cation of the dimeric intermediate, provided the
hydroxy alcohol 15 in good yields. Reaction of 15 and
the in situ generated C-terminal benzamide (obtained
from 10, by hydrogenation) gave the tetramer 16.
Mesylation of the alcohol 16 and treatment with excess
of the desired secondary amines led to the final
compounds that were purified by HPLC; a general
procedure is documented.13
In a ‘matrix-type’ fashion, we first investigated the
influence of the nature of the terminal heterocycles on
antibacterial activity in compounds that carry a basic
diaminopropyl group linked to the ring nitrogen of an
internal Py module. It has previously been shown that
the DNA binding properties of similar minor-groove
binding ligands are not diminished by similar
substituents at this position.12,14 In the case of short
linkers, an electrostatic interaction between the pending
Figure 1. Schematic representation of optimization of distamycin A
analogues. Optimized element is underlined (—). Empty circle: N-
methyl-pyrrole-2-carboxamide. It: 4-chloroisothiazole-2-carboxamide.
Aryl: uncharged aromatic/heteroaromatic N-terminus. BI: benzimida-
zole. (+: alkyl amine.
Table 1. Antibacterial activity of compounds 17–35 (Scheme 1)
!
R
3
#R2
MRSAa PISP VSEF
MRSA PISP VSEF
MRSA PISP VSEF
MRSA PISP VSEF
17
18
2
8
0.5
8
1
8
22
23
4
2
1
4
2
—
31
32
16
2
16
2
0.5
27
28
29
30
1
0.25
1
2
0.03
0.03
19
20
21
1
0.5
1
0.25
0.13
0.5
1
0.25
2
24
25
26
2
0.5
2
1
0.25
1
1
0.25
2
4
4
33
34
35
16
2
4
8
4
1
0.5
0.5
0.5
0.25
0.5
0.5
0.13
0.25
1
a MIC values in (mg/mL).15 MRSA, methicillin-resistant S. aureus (ATCC 27660). PISP, penicillin-intermediate S. pneumoniae (ATCC 49619).
VSEF, vancomycin-susceptible E. faecalis (ATCC 29212).

R. W. Burli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1259–1263
¨
1261
Scheme 2. Synthesis of final compounds (Table 2): (a) 4-chloro-2-
fluorobenzoic acid (1 equiv), HBTU (1 equiv), 14 (1 equiv), DMF,
iPr2EtN (3 equiv), 60 ꢁC, 12 h, 74%; then, NaOH (3 equiv), EtOH/
H2O (1:2), 60 ꢁC, 16 h, 88%; (h) 15 (1.1 equiv), HBTU (1.05 equiv), 10
(1 equiv), DMF/ Pr2EtN (10:1), 60 ꢁC, 12 h, crude; (c) MeSO2Cl (4.8
i
equiv), Pr2EtN (3 equiv), DMF, 40 ꢁC, 1 h; then, amine (10 equiv),
i
60 ꢁC, 12 h; then, prep. HPLC.
while the purine derivative 34 was somewhat less potent.
In the isoquinoline group, no dramatic change in activ-
ity was observed upon introduction of ring nitrogens in
the C-terminal unit; the imidazo[4,5-c]pyridine 30 was
slightly more potent compared to the other derivatives
and showed excellent activity against all strains (MIC
values: 0.25–0.5 mg/mL).
Scheme 1. Synthesis of final compounds (Table 1): (a) 3-(dimethyl-
amino)propyl chloride (1.3 equiv), K2CO3 (2.2 equiv), NaI (1.0 equiv),
DMF, 75 ꢁC, 16 h, 53%; (b) Pd–C, H2 (1 atm), AcOEt/MeOH (1:1),
25 ꢁC, 12 h, 91%; (c) RCOOH (1.2 equiv), HBTU (1.14 equiv), 3 (1.0
equiv), DMF/ iPr2EtN (10:1), 25 ꢁC, 12 h; (d) 4 (1.0 equiv), HBTU (1.0
equiv), diamine (1.1 equiv), DMF/iPr2EtN (10:1), 60 ꢁC, 15 h, 76% for
6, 84% for 7, 51% for 8, 35% for 9; (e) AcOH, 60 ꢁC, 2 h, 52% for 10;
AcOH, 60 ꢁC, 16 h, 61% for 11, 51% for 13; aqueous HCl (conc.),
reflux, 16 h, 59% for 12; (f) 10–13, Pd–C, H2 (1 atm), DMF, 25 ꢁC,
We next studied the influence of the amino group on
antibacterial potency (Table 2). It has previously been
shown that the nature of the positively charged group
can substantially impact the in vitro potency as well as
i
4 h, crude; then, acid (1.2 equiv), HBTU (1.15 equiv), DMF/ Pr2EtN
(10:1), 25 ꢁC, 12 h, prep. HPLC.
Table 2. Antibacterial activity of compounds 36–42 (Scheme 2)
positively charged amine and the phosphodiester back-
bone of DNA might stabilize the DNA–ligand complex.
For the N-terminus, we selected five end caps that
previously have shown good activity in the context of
the internal Py3 scaffold,4 whereas the C-terminus con-
sisted of either benzimidazole or an aza-analogue with
one or two additional ring nitrogens at various posi-
tions. The antibacterial activities against MRSA, PISP,
and VSEF, are summarized in Table 1.15 In the benzi-
midazole series, the benzothiophene 18 was only mod-
erately potent, whereas the thiophene 17, the
benzamides 19 and 20, and the isoquinoline 21 showed
MIC (minimum inhibitory concentration) values close
to 1 mg/mL or lower. The aza-analogues of the thio-
phene 17—namely the imidazo[4,5-b]pyridine 22 and the
purine 31—were significantly less potent. Interestingly,
the opposite structure–activity relationship was
observed for the benzothiophene series; addition of ring
nitrogens at the C-terminus resulted in improved in
vitro potency (! 23, 27, 32). In fact, the purine 32
showed excellent MIC values of 0.03 mg/mL against S.
aureus and S. pneumoniae, and good activity against E.
faecalis (2 mg/mL). The 2,4-difluorobenzamides (19, 24,
28, 33) were consistently less potent than their corres-
ponding 4-chloro-analogues (20, 25, 29, 34). The 4-
chloro-2-fluorobenzamides 20, 25, and 29 were active at
MIC values well below 1 mg/mL against all strains,
R
MRSAa
27660
PISP
49619
VSEF
29212
E. coli
25922
36
37
0.13
0.13
0.06
0.25
0.5
0.5
>32
>32
38
39
40
41
42
0.13
0.5
2
0.25
1
1
0.5
4
16
>32
32
1
2
0.5
4
2
>32
>32
4
2
a MIC values in (mg/mL) against ATCC strains.15 MRSA, methicillin-
resistant S. aureus. VSEF, vancomycin-susceptible E. faecalis. PISP,
penicillin-intermediate S. pneumoniae.

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R. W. Burli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1259–1263
¨
other pharmacologically important parameters. For
this, we concentrated on analogues of the 4-chloro-2-
fluorobenzamide 20, as this compound showed the most
promising in vitro profile against all Gram-positive
strains. The morpholine 36, the thiomorpholine 37, and
the unsubstituted piperidine 38 exhibited similar
potency (MIC values of 0.06–1 mg/mL against all Gram-
positive strains), while the 4,4-difluoropiperidine 39, the
4-hydroxypiperidine 40 and the piperazine 41 were
slightly less active. The bis(hydroxyethyl)amine 42 was
significantly less potent. Possibly, the hydroxy groups
might be highly solvated in aqueous medium and thus
reduce cellular penetration. Interestingly, out of this set
of compounds, three molecules showed moderate activ-
ity against E. coli, a representative Gram-negative bac-
terial strain (16 mg/mL for 20, 16 mg/mL for 38, 32 mg/
mL for 40). As outlined in the preceding publication,5
some benzimidazole-containing antibacterials exhibited
activity against E. coli, whereas the corresponding Py3
derivatives did not.4 Removal of the C-terminal amide
bond, however, did not improve E. coli potency.
3. Burli, R. W.; Ge, Y.; White, S.; Baird, E. E.; Touami,
S. M.; Taylor, M.; Kaizerman, J. A.; Moser, H. E. Bioorg.
Med. Chem. Lett. 2002, 12, 2591.
4. Kaizerman, J. A.; Gross, M. I.; Ge, Y.; White, S.; Hu, W.;
Duan, J. X.; Baird, E. E.; Johnson, K. W.; Tanaka, R. D.;
Moser, H. E.; Burli, R. W. J. Med. Chem. 2003, 46, 3914.
5. Burli, R.; McMinn, D.; Kaizerman, J.; Hu, W.; Pack, Q.;
Le, Q.; Ge, Y.; Gross, M.; Difuntorum, S.; Moser, H.
Bioorg. Med. Chem. Lett., submitted.
6. Bremer, R. E.; Szewczyk, J. W.; Baird, E. E.; Dervan,
P. B. Bioorg. Med. Chem. 2000, 8, 1947.
7. Synthesis of 3. A mixture of pyrrole 1 (10.0 g, 54.3 mmol),
3-(dimethylamino)propyl chloride (HCl salt, 10.74 g, 67.9
mmol), NaI (8.14 g, 54.3 mmol), and K2CO3 (16.51 g,
119.4 mmol) in DMF (150 mL) was stirred at 75 ꢁC for 16
h, cooled to 25 ꢁC, poured into 1 M aqueous HCl (500
mL), and washed with AcOEt (2ꢂ300 mL). The mixture
was neutralized by addition of Na2CO3 and extracted
with AcOEt (3ꢂ300 mL). The combined organic layers
were dried (Na2SO4) and evaporated to give nitro pyrrole
2 (7.77 g, 53%). A suspension of 2 (7.65 g, 28.4 mmol)
and PdC (10%, 0.38 g) in AcOEt/MeOH (1:1, 150 mL)
was stirred under H2 (1 atm) at 25 ꢁC for 12 h, and filtered
through Celite. The filtrate was evaporated and the resi-
due dissolved in AcOEt (200 mL), and treated with HCl
gas. Evaporation of solvents gave amino pyrrole 3 (8.05 g,
91%).
8. Dudouit, F.; Goossens, J.-F.; Houssin, R.; Henichart, J.-P.;
Colson, P.; Houssier, C.; Gelus, N.; Bailly, C. Bioorg.
Med. Chem. Lett. 2000, 10, 553.
9. Baird, E. E.; Dervan, P. B. J. Am. Chem. Soc. 1996, 118,
6141.
10. Synthesis of 10. A mixture of ketone 5 (25.1 g, 92.5 mmol)
and 1,2-phenylenediamine (10.0 g, 92.47 mmol) in THF
(300 mL) and iPr2EtN (30 mL) was stirred at 60 ꢁC for 15
h and added dropwise into H2O (ca. 1 L). The resulting
precipitate was collected by filtration and dried to give
amide 6 (16.05 g, 67%). A solution of 6 (16.05 g, 61.67
mmol) in glacial AcOH (200 mL) was heated at 60 ꢁC for
2 h and concentrated in vacuo. Addition of Et2O resulted
in precipitation of 10, which was collected by filtration
and dried (!7.82 g, 52%).
The goal of this study was to investigate whether the
C-terminal amide bond could be eliminated by inter-
nalization of the charged function without loss of anti-
bacterial potency. Three structural parameters were
varied: the N-terminal unit, the internal amino group,
and the C-terminal ring system, respectively. The N-
termini and the internal amines were selected based on
previous results, whereas the C-terminus consisted of a
benzimidazole-type structure. We also studied the effect
of one or two additional ring nitrogens in the benzimi-
dazole unit and found that, depending on the nature of
the N-terminus, extra ring nitrogens will enhance or
reduce antibacterial potency of the molecules. In sum-
mary, some compounds bearing the charged group at an
internal unit demonstrated good in vitro potency
against various Gram-positive bacteria and are structu-
rally quite different from the original natural product
distamycin A and earlier analogues thereof. Internaliz-
ing the amino group clearly opens a new avenue for
structural optimization DNA minor-groove binding
ligands, since the nature of the C-terminal aryl group
has largely remained unexplored. The in vivo behavior
of this new subclass of DNA minor-groove binding
antibacterial agents remains to be assessed.
11. General procedure for the synthesis of 17–35. A solution
of nitro compound 10–13 (150 mmol) and PdC (10%, 30
mg) in DMF (0.75 mL) was stirred under H2 (1 atm) at
25 ꢁC for 4 h, and filtered through Celite. The filtrate was
treated with a mixture of preactivated acid (1.2 equiv acid,
1.15 equiv HBTU in DMF/iPr2EtN=10:1, 25 ꢁC, 30 min),
stirred at 37 ꢁC for 12 h, diluted with 50% aqueous AcOH
to a volume of 15 mL, and purified by preparative HPLC
(Hamilton PRP-1 column, 250ꢂ21.5 mm, A: 0.5% AcOH
in H2O, B: CH3CN, 0–60% B in 60 min, 20 mL/min, UV
detection at 310 nm). Analytical data for 17: 1H NMR
(DMSO-d6) d 12.51 (br. s, 1H), 10.22 (s, 1H), 10.06 (s,
1H), 7.87 (d, J=5.2 Hz, 1H), 7.62–7.55 (m, 1H), 7.48–7.41
(m, 1H), 7.34 (d, J=1.3 Hz, 1H), 7.32 (d, J=1.5 Hz, 1H),
7.17 (d, J=5.3 Hz, 1H), 7.16–7.13 (m, 2H), 7.06 (d, J=1.5
Hz, 1H), 7.03 (d, J=1.5 Hz, 1H), 4.34 (t, J=7.0 Hz, 2H),
4.08 (s, 3H), 2.17 (t, J=6.8 Hz, 2H), 2.13 (s, 6H), 1.86–1.80
(m, 2H). ESI MS 552.1 (40%), 550.2 (100%, [M+H]+).
Purity (analyt. HPLC,4 310 nm) 95%. All compounds
were characterized by 1H NMR and mass spectrometry
and showed purity of at least 90%. The isolated yields
after HPLC purification ranged from ca. 10–30%.
12. Bremer, R. E.; Wurtz, N. R.; Szewczyk, J. W.; Dervan,
P. B. Bioorg. Med. Chem. 2001, 9, 2093.
Acknowledgements
This work was supported in part by the Defense
Advanced Research Projects Agency (DARPA Grant
Number N65236-99-1-5427). The authors would like to
thank Vernon Jiang, Zhijun Ye, and Hsiu Chen for
analytical support.
References and notes
1. McDevitt, D.; Rosenberg, M. TRENDS in Microbiology
2001, 9, 611 and references cited therein.
2. Projan, S. J.; Youngman, P. J. Curr. Opin. Microbiology
2002, 5, 463.
13. General procedure for the synthesis of 36–42. A solution
of alcohol 16 (25 mg, 47 mmol) in DMF (1 mL) and
iPr2EtN (0.1 mL) was treated with MeSO2Cl (18 mL, 227

R. W. Burli et al. / Bioorg. Med. Chem. Lett. 14 (2004) 1259–1263
¨
1263
mmol), stirred at 40 ꢁC for 1 h, treated with amine (470
mmol), and stirred at 60 ꢁC for 12 h. The mixture was
diluted with 50% aqueous AcOH to a volume of 15 mL
and purified by preparative HPLC (as above). Data for
2H), 2.21 (t, J=7.0 Hz, 2H), 1.88–1.86 (m, 2H). ESI MS
606.2 (40%), 604.3 (100%, [M+H]+). Purity (analyt.
HPLC, 310 nm) 95%.
14. Bruice, T. C.; Mei, H.-Y.; He, G.-X.; Lopez, V. Proc.
Natl. Acad. Sci. U.S.A. 1992, 89, 1700.
1
36: H NMR (DMSO-d6) d 12.51 (s, 1H), 10.44 (s, 1H),
10.06 (s, 1H), 7.68 (t, J=8.1, 1H), 7.61 (br. d, J=8.0 Hz,
1H), 7.59 (br. t, J=8.0 Hz, 1H), 7.42 (br. d, J=7.4 Hz,
2H), 7.37 (br. s, 1H), 7.32 (br. s, 1H), 7.15–7.13 (m, 2H),
7.02 (br. s, 2H), 4.36 (t, J=6.6 Hz, 2H), 4.07 (s, 3H), 3.65
(t, J=4.8 Hz, 2H), 3.58–3.56 (m, 4H), 3.07 (t, J=4.8 Hz,
15. National Committee for Clinical Laboratory Standards.
Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria That Grow Aerobically, 5th ed; Approved Stan-
dard (M7A5); National Committee for Clinical Labora-
tory Standards, Wayne, PA, 2000.