Syntheses of novel myxopyronin B analogs as potential inhibitors of bacterial RNA polymerase

Article abstract of DOI:10.1016/j.bmcl.2007.10.017

Based upon observations from our initial findings, additional myxopyronin B analogs have been prepared and tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against Escherichia coli and Staphylococcus

Full text of DOI:10.1016/j.bmcl.2007.10.017

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6797–6800
Syntheses of novel myxopyronin B analogs as potential inhibitors
of bacterial RNA polymerase
Ricardo Lira, Alan X. Xiang,* Thomas Doundoulakis, William T. Biller,
Konstantinos A. Agrios, Klaus B. Simonsen, Stephen E. Webber,
Wes Sisson, Robert M. Aust, Amit M. Shah, Richard E. Showalter,
Virginia N. Banh, Kevin R. Steffy and James R. Appleman
Anadys Pharmaceuticals, Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
Received 20 September 2007; revised 9 October 2007; accepted 10 October 2007
Available online 17 October 2007
Abstract—Based upon observations from our initial findings, additional myxopyronin B analogs have been prepared and tested
for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against Escherichia coli and
Staphylococcus aureus.
Ó 2007 Elsevier Ltd. All rights reserved.
O
O
OH
O
Although antibiotic resistance has been around for more
than 60 years since the discovery of penicillin, the prob-
lem has grown exponentially in recent years. Factors
contributing to this rapid growth of antibiotic resistance
include the overuse of powerful, broad-spectrum antibi-
otics to treat common infections (e.g., ear infections), as
well as the use of antibiotics in inappropriate situations
(e.g., viral infections like the common cold). Despite im-
proved efforts for the rational use of existing agents,
there remains a strong need for novel antibiotics. Myxo-
pyronin B (Fig. 1, 1), a bacterial metabolite isolated
from Myxococcus fulvus Mx 150, represents such a
candidate.1
H
N
O
O
1 Myxopyronin B
Figure 1. The naturally occurring enantiomer (R) of myxopyronin B
(1). All reported analogs were prepared as racemic mixtures.
Herein we report the synthesis and biological evaluation
of eight new myxopyronin analogs compared to those
presented in our initial report.4 Among them, five com-
pounds are derivatives of desmethyl myxopyronin B
(Fig. 2, 2) since this compound has enhanced potency
and simplified structure. While our initial efforts focused
on the changes to the side chains, our recent studies also
included modifications to the pyrone core (see Schemes
1 and 2).
Myxopyronin B exhibits antibacterial activity against
Gram-positive and Gram-negative bacteria through
selectively inhibiting the bacterial DNA-dependent
RNA polymerase (RNAP).2 As an attractive lead for
the development of RNAP inhibitors, myxopyronin B
displays the following: selectivity versus human RNAP;
good cell penetration that is reflected in a strong corre-
lation between in vitro activity and cell potency; and po-
tency against rifampicin-resistant Staphylococcus
aureus.3
O
O
OH
O
H
N
O
Keywords: Myxopyronin B; Antibiotics; Antibacterial; Curtius rear-
rangement; Ortholithiation.
O
2 Desmethyl myxopyronin B
Figure 2. The synthetic desmethyl myxopyronin B (2).
*
Corresponding author. Tel.: +1 858 530 3633; fax: +1 858 530
3644; e-mail: axiang@anadyspharma.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.017

6798
R. Lira et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6797–6800
O
R1
OH
N
O
O
OH
N
OH
O
O
O
O
R1
O
a
b-c
a
H
O
O
O
O
O
OTBS
R2
R2
7
8
9
3a R2 = Me
3b R2 = H
4a R2 = Me
4b R2 = H
O
d
Cl
10
R1 =
O
O
O
b
e-k
O
R1
OH
O
O
OH
c-d
OTBS
R1
O
11
O
OH
H
N
O
N
N
O
OH
H
R2
O
R2
N
O
6a R2 = Me
6b R2 = H
5a R2 = Me
5b R2 = H
O
12
Scheme 2. Reagents and conditions: (a) MOMCl (1.2 equiv), DIPEA
(1.4 equiv), CH2Cl2, 23 °C, 18 h, 93%; (b) TBSO(CH2)3PPh3+Iꢀ (2.0
equiv), n-BuLi (2.0 equiv), THF, ꢀ78 to 23 °C, 18 h, 82%; (c) H2, 10%
Pd/C (cat.), EtOAc, 23 °C, 18 h, 92%; (d) tert-BuLi (1.1 equiv), Et2O,
0–23 °C, 1 h; then 10 (1.0 equiv), Et2O, ꢀ78 to 23 °C, 18 h, 87%; (e)
TiCl4 (1.1 equiv), CH2Cl2, ꢀ78 °C, 5 min; (f) AcOH/THF/H2O (3:1:1),
23 °C, 2 h, 95% two steps; (g) NMO (2.0 equiv), TPAP (cat.), 4 MS,
CH2Cl2, 0 °C, 45 min; (h) CH3OCOCH2PO(OCH3)2 (2.6 equiv), NaH
(2.5 equiv), THF, 23 °C, 2 h, 50% two steps; (i) 1 M LiOH/THF (1:3),
23 °C, 12 h, 95%; (j) ethyl chloroformate (2.2 equiv), DIPEA (2.4
equiv), acetone, 0 °C, 90 min; then NaN3 (4.8 equiv), H2O, 0 °C,
90 min; (k) toluene, reflux, 2 h; then MeOH, 70 °C, 18 h, 53% two
steps.
Scheme 1. Reagents and conditions: (a) tert-BuOCOCH2PO(OCH3)2
(2.7 equiv), NaH (2.4 equiv), THF, 23 °C, 45 min; (b) TFA (10 equiv),
CH2Cl2, 23 °C, 24 h, 79% two steps; (c) ethyl chloroformate (2.2
equiv), DIPEA (2.4 equiv), acetone, 0 °C, 90 min; then NaN3 (4.8
equiv), H2O, 0 °C, 90 min; (d) toluene, reflux, 2 h; then MeOH, 70 °C,
18 h, 60% two steps.
Although pyrones have been used as a synthetic scaffold
in HIV protease5 and human sputum elastase inhibi-
tors,6 the 2-pyrone moiety of myxopyronin B is a poten-
tial electrophile.7 To avoid this problem, we evaluated
the importance of the pyrone core to the activity of
myxopyronin B by preparing analogs 6a,8 6b9 (Scheme
1) and 1210 (Scheme 2). The synthesis of 6a is described
as follows. Compound 3a was prepared as previously
reported11 and subjected to Horner–Emmons–Wadsworth
homologation, followed by acid hydrolysis to afford the
carboxylic acid 5a in 79% yield. It should be noted that
although the ester hydrolysis could also be achieved in
basic media (LiOH, THF/H2O), the pyridone moiety
was simultaneously converted to its corresponding pyr-
one.11 Compound 5a was converted to the enecarbamate
6a in two steps by a Curtius rearrangement as reported
by Panek and co-workers.12
were readily prepared via aldol condensations catalyzed
by piperidine. In the case of 15b, diphenylphosphorazi-
O
OH
O
R
O
O
OCH3
13a R = CH3
13b R = H
a
O
OH
Ar
O
To study the biologic necessity of the enolic pyrone core,
we explored the possibility of replacing it with a simple
phenol. Analog 12 was synthesized from commercial
available 30-hydroxyacetophenone (7) as shown in
Scheme 2. The hydroxyl group of 7 was first protected
as its methoxymethyl ether. A Wittig olefination, fol-
lowed by catalytic hydrogenation, was utilized to intro-
duce the right side chain. For the installation of the left
side chain,13 a directed ortholithiation–acylation strat-
egy was adopted to give rise to compound 11. Com-
pound 11 was then treated with TiCl4 in CH2Cl2,
followed by AcOH/THF/H2O to sequentially remove
the MOM and TBS protecting groups. The resulting
carboxylic acid was converted to the enecarbamate 12
under the same conditions as described for 6a and 6b.
R
O
O
OCH3
14a Ar = 2-Furanyl; R = CH3
14b Ar = 5-Indolyl; R = H
b-d
O
O
OH
Ar
H
N
R
O
O
O
15a Ar = 2-Furanyl; R = CH3
15b Ar = 5-Indolyl; R = H
Scheme 3. Reagents and conditions: (a) ArCHO (1.5 equiv), piperidine
(cat.), CHCl3, 65 °C, 18 h, 75%; (b) 1 M LiOH/THF (1:3), 23 °C, 12 h,
95%; (c) Ar = 2-furanyl: ethyl chloroformate (2.2 equiv), DIPEA (2.4
equiv), acetone, 0 °C, 90 min; then NaN3 (4.8 equiv), H2O, 0 °C,
90 min; Ar = 2-indolyl: (PhO)2PON3 (1.1 equiv), Et3N (3.0 equiv),
toluene, 65 °C, 1 h; (d) toluene, reflux, 2 h; then MeOH, 70 °C, 18 h,
53% for 15a and 60% for 15b two steps.
Several analogs were made based on modifications to
the left side chain. The syntheses of analogs 15a14 and
15b15 are depicted in Scheme 3. Aromatic side chains

R. Lira et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6797–6800
6799
O
OH
decrease in activity in the RNAP enzymatic assay and
complete loss of potency against all three bacterial
strains. When the pyrone core of compound 12 was
replaced with phenol, it led to weak enzymatic inhibition
and loss of antibacterial activity.
N
H
N
H
O
O
O
O
O
O
O
O
O
16
OH
Attempts to modify the left chain of myxopyronin B and
desmethyl myxopyronin B (Table 1, entries 16, 15a, 15b,
17, and 18) were mostly fruitless as well. Compound 16
displayed mild antibacterial activity against E. coli (Tol
C), but was inactive in the enzymatic assay and against
E. coli and S. aureus.
H
N
O
O
O
17
OH
H
N
In conclusion, the data generated from this continued
study further confirmed that the antibacterial activities
of myxopyronin B were very sensitive to both subtle
and dramatic changes in its structure.
O
O
18
Figure 3. The structures of myxopyronin analogs 16, 17, and 18.
date was utilized for the formation of the acyl azide
intermediate of the Curtius rearrangement.
References and notes
To further investigate the importance of the dienone sys-
tem in the left side chain, and remove a potential electro-
philic species from the molecule, analogs 16,16 17,17 and
1818 (Fig. 3) were synthesized using techniques reported
in our previous publication.4
1. (a) Kohl, W.; Irschik, H.; Reichenbach, H.; Ho¨fle, G.
Liebigs Ann. Chem. 1983, 1656; (b) Kohl, W.; Irschik, H.;
Reichenbach, H.; Ho¨fle, G. Liebigs Ann. Chem. 1984,
1088; (c) Jansen, R.; Irschik, H.; Reichenbach, H.; Ho¨fle,
G. Liebigs Ann. Chem. 1985, 822.
2. Irschik, H.; Gerth, K.; Ho¨fle, G.; Kohl, W.; Reichenbach,
H. J. Antibiot. 1983, 36, 1651.
In vitro inhibitory activity (IC50) against RNAP (Esche-
richia coli) of the myxopyronin B analogs was measured
utilizing an adapted nucleotide coupled NADPH/pyro-
phosphate release assay (Table 1) described in the
literature.19
3. (a) Campbell, E. A.; Korzheva, A.; Mustaev, A.; Mura-
kami, K.; Nair, S.; Goldfarb, A.; Darst, S. Cell 2001, 104,
901; For other inhibitors of RNAP see: (b) Artsimovitch,
I.; Chu, C.; Lynch, A. S.; Landick, R. Science 2003, 302,
650.
4. Doundoulakis, T.; Xiang, A. X.; Lira, R.; Agrios, K. A.;
Webber, S. E.; Sisson, W.; Aust, R. M.; Shah, A. M.;
Showalter, R. E.; Appleman, J. R.; Simonsen, K. B.
Bioorg. Med. Chem. Lett. 2004, 14, 5667.
The antibacterial potency (MIC) of the analogs was
determined in growth inhibition tests against E. coli,
E. coli (Tol C), and S. aureus. All compounds were
tested for cytotoxicity in a T-cell proliferation assay20
and displayed no toxicity up to 40 lM (ꢁ18 lg/mL).
The results are compiled in Table 1.
5. Skulnich, H. I.; Johnson, P. D.; Howe, W. J.; Tomich, P. K.;
Chong, K.-T.; Watenpaugh, K. D.; Janakiraman, M. N.;
Dolak, L. A.; McGrath, J. P.; Lynn, J. C.; Horng, M.-M.;
Hinshaw, R. R.; Zipp, G. L.; Ruwart, M. J.; Schwende, F.
J.; Zhong, W.-Z.; Padbury, G. E.; Dalga, R. J.; Shiou, L.;
Possert, P. L.; Rush, B. D.; Wilkonson, K. F.; Howard, G.
M.; Toth, L. N.; Williams, M. G.; Kakuk, T. J.; Cole, S. L.;
Zaya, R. M.; Lovasz, K. D.; Morris, J. K.; Romines, K. R.;
Thaisrivomgs, S.; Aristoff, P. A. J. Med. Chem. 1995, 38,
4968.
When the pyrone core of myxopyronin B was replaced
with a more stable N-methyl pyridone, the analogs
(Table 1, entries 6a and 6b) displayed substantial
Table 1. In vitro activity against RNAP (IC50) and antibacterial
potency (MIC) of the myxopyronin analogs based on myxopyronin B
(1) and desmethyl myxopyronin B (2)
6. Cook, L.; Ternai, B.; Ghosh, P. J. Med. Chem. 1987, 30,
1017.
7. For examples, see: (a) Kiang, A. K.; Tan, S. F.; Wong, W.
S. J. Chem. Soc. C 1971, 2721; (b) Castillo, S.; Ouadahi,
H.; Herault, V. Bull. Soc. Chim. Fr. 1982, 2, 257.
8. Satisfactory spectroscopic data were obtained for all new
compounds. All final analogs were purified by flash
MICb (lg/mL)
a
Compound
IC50 (lM)
1
0.92
20.5
>64/2/1
6a
12
16
>64/>64/>64
>64/>64/>64
>64/16/>64
38%
<20%
1
column chromatography and characterized by H NMR
and LC–MS. 6a: 1H NMR (400 MHz, CD3OD) d: 0.86 (t,
J = 7.2 Hz, 3H), 1.20 (d, J = 6.8 Hz, 3H), 1.20–1.29 (m,
2H), 1.34–1.41 (m, 2H), 1.49–1.56 (m, 2H), 1.68–1.76 (m,
2H), 1.79 (s, 3H), 1.97–2.06 (m, 2H), 2.03 (s, 3H), 2.47–
2.57 (m, 1H), 3.08 (s, 3H), 3.66 (s, 3H), 5.01–5.08 (m, 1H),
5.49–5.52 (m, 1H), 5.74 (s, 1H), 6.36–6.41 (m, 2H).
2
0.34
15
>64/1/4
6b
15a
15b
17
18
>64/>64/>64
>64/>64/>64
>64/>64/>64
>64/>64/>64
>64/>64/>64
<20%
<20%
<20%
<20%
1
9. 6b: H NMR (400 MHz, CD3OD) d: 0.95 (t, J = 7.2 Hz,
a The IC50 value was not determined for weak inhibitors, instead the
percentage of inhibition at 10 lM is listed.
3H), 1.32–1.38 (m, 2H), 1.44–1.50 (m, 2H), 1.66–1.72 (m,
2H), 1.77 (s, 3H), 1.98 (s, 3H), 2.02–2.07 (m, 2H), 2.17 (t,
J = 7.2 Hz, 2H), 2.40 (t, J = 7.6 Hz, 2H), 3.13 (s, 3H), 3.66
(s, 3H), 5.04–5.11 (m, 1H), 5.73 (s, 1H), 6.12–6.20 (m, 2H),
6.35–6.44 (m, 1H).
b MIC: minimum inhibitory concentration (lg/mL), determined as
average of triplicate measurements in serial dilution against E. coli
(first value), E. coli Tol C (second value), and S. aureus (third value).

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R. Lira et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6797–6800
1
10. 12: H NMR (400 MHz, CD3OD) d: 0.95 (t, J = 7.2 Hz,
3H), 1.24 (d, J = 6.8 Hz, 3H), 1.30–1.40 (m, 2H), 1.43–1.55
(m, 2H), 1.60–1.69 (m, 2H), 1.77 (s, 3H), 1.83–1.95 (m,
2H), 2.02 (s, 3H), 2.23 (t, J = 7.6 Hz, 2H), 2.73 (q,
J = 7.2 Hz, 1H), 3.66 (s, 3H), 5.02–5.09 (m, 1H), 6.28–6.34
(m, 2H), 6.76–6.79 (m, 2H), 6.95 (dd, J = 1.6, 11.6 Hz,
1H), 7.47 (d, J = 8.0 Hz, 1H).
16. 16: 1H NMR (400 MHz, CD3OD) d: 1.60 (s, 3 H), 1.66 (s,
3H), 1.73 (s, 3H), 1.69–1.77 (m, 2H), 2.00–2.15 (m, 8H),
2.54 (t, J = 8.4 Hz, 2H), 3.66 (s, 3H), 3.96 (d, J = 8.0 Hz,
2H), 5.07–5.10 (m, 1H), 5.25–5.28 (m, 1H), 6.14 (s, 1H),
6.42 (d, J = 14.4 Hz, 1H).
1
17. 17: H NMR (400 MHz, CD3OD) d: 0.89 (t, J = 7.2 Hz,
3H), 1.28–1.36 (m, 10H), 1.74–1.82 (m, 4H), 2.07–2.13 (m,
2H), 2.65 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H), 3.66
(s, 3H), 5.04–5.11 (m, 1H), 6.42 (d, J = 12.8 Hz, 1H), 6.76
(s, 1H).
11. Lira, R.; Agrios, K. A.; Doundoulakis, T.; Simonsen, K. B.;
Webber, S. E.; Xiang, A. X. Heterocycles 2006, 68, 1099.
12. Hu, T.; Schaus, J. V.; Lam, K.; Palfreyman, M. G.;
Wuonola, M.; Gustafson, G.; Panek, J. S. J. Org. Chem.
1998, 63, 2401.
1
18. 18: H NMR (400 MHz, CD3OD) d: 0.90–0.93 (m, 3H),
1.35–1.40 (m, 4H), 1.74–1.82 (m, 4H), 2.07–2.12 (m, 2H),
2.65 (t, J = 7.6 Hz, 2H), 2.89 (t, J = 7.6 Hz, 2H), 3.66 (s,
3H), 5.03–5.11 (m, 1H), 6.42 (d, J = 12.8 Hz, 1H), 6.76 (s,
1H).
13. Compound 10 was synthesized based on the following
route:
1. n-BuLi, CuI, THF
O
O
19. Johnson, J. C.; Shanoff, M.; Boezi, J. A.; Hansen, R. G.
Anal. Biochem. 1968, 26, 137, The cited assay was adapted
to 96-well plates to obtain reasonable throughput.
NADPH formation typically was monitored by fluores-
cence (excitation wavelength, ꢁ340 nm; emission wave-
length, ꢁ440 nm) rather than absorbance at 340 nm as in
the original assay to improve sensitivity. Although com-
pounds with strong absorbance at 340 nm can in principle
influence the assay, we found it to be acceptable for all
compounds discussed herein. Inhibitory potencies of
selected compounds were also evaluated by effects on
incorporation of radiolabeled nucleotide into RNA, and
comparable results were obtained.
20. Cell toxicity assays for CC50 determination were
conducted on CEM-SS T lymphocyte cells. Cellular
toxicity was measured using XTT (Sigma) and detected
with a fluorometer at 450 nm/650 nm using Genios/
Tecan (Xfluor). CEM-SS cells were incubated in 96-well
microtiter plates for 3 days. At the end of that time,
the cells were treated with an XTT solution, measure-
ments were recorded and CC50 determinations were
made.
2. LiAlH4, Et2O
Bu
H
O
3. TPAP, NMO, CH2Cl2
82%
1. CH3C(PPh3)COOEt
CH2Cl2
2. 1M NaOH, EtOH
92%
O
(COCl)2, DMF, CH2Cl2
100%
10
Bu
OH
.
14. 15a: 1H NMR (400 MHz, DMSO-d6) d: 1.57–1.64 (m,
2H), 1.98–2.03 (m, 2H), 2.09 (s, 3H), 2.44–2.50 (m, 2H),
3.58 (s, 3H), 5.00–5.07 (m, 1H), 6.05 (s, 1H), 6.33–6.39 (m,
1H), 6.67 (s, 1H), 6.94 (d, J = 3.2 Hz, 1H), 7.14 (s, 1H),
7.91 (s, 1H), 9.23 (d, J = 9.6 Hz, 1H).
15. 15b: 1H NMR (400 MHz, DMSO-d6) d: 1.59–1.67 (m,
2H), 1.98–2.05 (m, 2H), 2.48–2.55 (m, 2H), 3.57 (s, 3H),
4.98–5.05 (m, 1H), 6.22 (s, 1H), 6.32–6.38 (m, 1H), 6.55 (s,
1H), 7.42–7.44 (m, 1H), 7.48–7.53 (m, 1H), 7.96 (s, 1H),
8.12 (dd, J = 30.4, 16.0 Hz, 2H), 9.22 (d, J = 8.8 Hz, 1H),
11.45 (s, 1H).