Myxopyronin B analogs as inhibitors of RNA polymerase, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmcl.2004.08.045

A series of myxopyronin B analogs has been prepared via a convergent synthetic route and were tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against E. coli and S. aureus. A series of myxopyronin B analogs has been prepared via a convergent synthetic route and were tested for in vitro inhibitory activity against DNA-dependent RNA polymerase and antibacterial activity against E. coli and S. aureus. The parent lead compound proved to be very sensitive to even small changes. Only the achiral desmethyl myxopyronin B (1a) provided enhanced potency.

Full text of DOI:10.1016/j.bmcl.2004.08.045

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5667–5672
Myxopyronin B analogs as inhibitors of RNA
polymerase, synthesis and biological evaluation
Thomas Doundoulakis,a Alan X. Xiang,a Ricardo Lira,a Konstantinos A. Agrios,a
Stephen E. Webber,a Wes Sisson,b Robert M. Aust,b Amit M. Shah,b
Richard E. Showalter,b James R. Applemanb and Klaus B. Simonsena,*
aDepartment of Medicinal Chemistry, Anadys Pharmaceuticals Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
bDepartment of Discovery Biology, Anadys Pharmaceuticals Inc., 3115 Merryfield Row, San Diego, CA 92121, USA
Received 23 June 2004; revised 18 August 2004; accepted 18 August 2004
Available online 15 September 2004
Abstract—A series of myxopyronin B analogs has been prepared via a convergent synthetic route and were tested for in vitro inhibi-
tory activity against DNA-dependent RNA polymerase and antibacterial activity against E. coli and S. aureus. The parent lead com-
pound proved to be very sensitive to even small changes. Only the achiral desmethyl myxopyronin B (1a) provided enhanced
potency.
Ó 2004 Elsevier Ltd. All rights reserved.
Since the discovery of penicillin, natural sources have
become the preferred choice in the search for newanti-
biotics. Today more than half of all approved drugs
from anticancer and antiinfective programs originate
from natural products.1
itors. Myxopyronin B displays: selectivity versus human
RNAP, good cell penetration, reflected in a good corre-
lation between in vitro activity and cell potency, and
potency against rifampicin-resistant S . aureus.4 Although
myxopyronin B was isolated more than 20 years ago, a
total synthesis was reported only recently by Panek and
co-workers.5
Myxopyronin B (1b), a bacterial metabolite isolated
from Myxococcus fulvus Mx 150,2a,b exhibits antibacte-
rial activity against Gram-positive and Gram-negative
bacteria.3 Myxopyronin B belongs to an interesting class
of 3-acyl-4-hydroxy-a-pyronens natural products, which
include the corallopyronins A–C2c and myxopyronin
A.2 Myxopyronin B, inhibits the bacterial DNA-
dependent RNA polymerase (RNAP)3 and represents
a newlead for drug discovery directed at that target.
DNA-dependent RNAP is the principal enzyme of tran-
scription in all living organisms, and a key target in
many regulatory pathways that control gene expression.
Currently, rifampicin is the only known drug that inhib-
its RNAP, but its use has been limited by the rapid
development of resistance.4 Therefore, newleads that in-
hibit RNAP are important. Myxopyronin B represents
an attractive lead for the development of RNAP inhib-
Here, we report the synthesis and biological evaluation
of 15 newmyxopyronin analogs ( Fig. 1 and Table 1) in
which the pyrone core remains constant, and the di-
enone and enecarbamate side chains have been modified.
This convergent route relies on two synthetic differentia-
tions steps, including an alkylation step to install the ene-
carbamate part of the molecule followed by an aldol
condensation to introduce the dienone part (Fig. 1).5
Several features within the structure of myxopyronin B
suggest possible modification in order to modify the
chemical stability of metabolically liable functionalities.
Specifically, the enecarbamate moiety may be prone to
Keywords: RNA polymerase; Antibiotics; Medicinal chemistry.
*
Corresponding author at present address: Department of Medicinal
Chemistry, The Danish University of Pharmaceutical Sciences,
Universitetsparken 2, DK-2100 Copenhagen, Denmark. E-mail:
kbs@dfuni.dk
Figure 1. The naturally occurring enantiomer (R) of Myxopyronin B
(1b). All reported analogs were prepared as racemic mixtures.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.08.045

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T. Doundoulakis et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5667–5672
Table 1. In vitro activity against RNAP (IC50) and antibacterial potency (MIC) of the myxopyronin analogs
Compd
Structure
IC50 (lM)a
MIC (lg/mL)b
Dienone
Enecarbamate
1b
1e
0.92
2.5
>64/2/1
>64/16/16
1d
1c
9
1.04
>64/2/16
70%
<20%
1.26
>64/32/64
>64/>64/>64
>64/4/4
10
8b
1a
<20%
0.34
>64/>64/>64
>64/1/4
13
12
<20%
<20%
>64/>64/>64
>64/>64/>64
18
19
<20%
4.0
>64/>64/>64
>64/8/64
26
<20%
>64/>64/>64
27
11
<20%
4.2
>64/>64/>64
>64/>64/>64
33
<20%
>64/>64/>64
a The IC50 value was not determined for weak inhibitors, instead the percentage of inhibition at 10lM is listed.
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).
hydrolysis. Although pyrones have been used as syn-
thetic scaffold in HIV protease6 and human sputum ela-
stase inhibitors,7 the 2-pyrone moiety and the dienone
could act as possible Michael acceptors.
The syntheses of analogs 1a–d are depicted in Scheme 1.
We will discuss the synthesis of desmethyl myxopyronin
B (1a) as a representative example. Lithiation of 4-hydr-
oxy-6-methyl-3-propionyl-2-pyrone8 (2a) followed by

T. Doundoulakis et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5667–5672
5669
corresponding b-chloro compound that eliminated at
elevated temperature in the presence of DBU to produce
8a. Basic hydrolysis of 8a produced the corresponding
acid, which was converted to the enecarbamate in two
steps by a Curtius rearrangement as reported by Panek
co-workers.5 The elevated temperature necessary to gen-
erate the isocyanate apparently causes isomerization of
the dienone moiety of the molecule. The final products
were isolated as a 3:1 mixture of geometrical isomers.
Therefore, a final HPLC purification was necessary to
produce pure 1a.10
Several other myxopyronin analogs were prepared
through the same reaction sequence utilizing either dif-
ferent pyrones (1a–c) in the alkylation step or aldehydes
(5 or 6) in the aldol condensation step. Racemic myxo-
pyronin B (1b) and its geometrical isomer 1e (see Table
1) was produced from 6-ethyl-4-hydroxy-3-propionyl-2-
pyrone (2b).5,7 Compound 1d, lacking the d-methyl
group, was produced from 2b and trans-2-heptenal 6
and finally compound 1c was produced from 3-acetyl-
6-ethyl-4-hydroxy-2-pyrone (2c), which was prepared
from 6-ethyl-4-hydroxy-2-pyrone11 and acetyl chloride
in the presence of TFA.8 Compound 10, the cis-ene-
carbamate isomer of myxopyronin B, was prepared
from the cis product of the Wittig reaction (see 4b) via
the same protocol. Hydrolysis of intermediate 4b, fur-
nished the corresponding enecarbamate 9 after the Cur-
tius reaction. O-Methylation of myxopyronin B with
MeI produced 11.
Scheme 1. Reagents and conditions: (a) LDA (3.2equiv), HMPA
(3.2equiv), THF, À78°C, 1h; then 3 (1.1equiv), À78°C, 1h, 65–80%;
(b) AcOH/THF/H2O (3:1:1), 23°C, 12h, 85–90%; (c) Dess–Martin
periodinane (2.0equiv), NaHCO3 (5.0equiv), CH2Cl2, 23°C, 2h, 90–
95%; (d) trimethyl phosphonoacetate (2.6equiv), NaH (2.5equiv),
THF, 23°C, 15min; then aldehyde (1.0equiv), THF, 23°C, 3h, 65–
78% (cis/trans ꢀ 1:9); (e) 4a–c (1equiv), TiCl4 (4.0equiv), CH2Cl2,
À78°C, 30min; then DIPEA (5.0equiv), À78°C, 3h; then 5 or 6 (2–
4equiv), CH2Cl2, À78°C, 48h; À78 to 23°C, 1h, 15–35%; (f) MsCl
(2.0equiv), Et3N (3.0equiv), CH2Cl2, 23°C, 4h, 78%; (g) DBU
(3.0equiv), THF, reflux, 2h, 75%; (h) 1M LiOH/THF (1:3), 23°C,
12h, 90–95%; (i) ethyl chloroformate (2.2equiv), DIPEA (2.4equiv),
acetone, 0°C, 90min; then NaN3 (10equiv), H2O, 0°C, 90min; (j)
toluene, 2h, reflux; then MeOH, 18h, 70°C, 50–65% two steps; (k) MeI
(3.0equiv), K2CO3 (10equiv), acetone, reflux, 12h, 55%.
Two analogs, in which a solubilizing group was attached
to the enecarbamate, were prepared from 8b as shown in
Scheme 2.
Further modifications to the enecarbamate of myxopyro-
nin B are depicted in Schemes 3 and 4. Alkylation of 2b
with 1-iodo-4-(tert-butyldimethylsilyloxy)butane (14)12
furnished 16, which was converted to the dienone by
the Ti(IV) mediated aldol condensation with concurrent
silyl deprotection. The resulting alcohol was subse-
quently oxidized to aldehyde 18. The saturated carba-
mate analog 19 was prepared from 2b and 1-iodo-5-
(tert-butyldimethylsilyloxy)pentane (15)13 to furnish
17, which was carried forward as described above to
produce 19.
the addition of 1-iodo-3-(tert-butyldimethylsilyloxy)pro-
pane, 3,5 afforded the regioselective alkylation in excel-
lent yield.
Silyl deprotection followed by Dess–Martin periodinane
oxidation and Horner–Emmons–Wadsworth homologa-
tion afforded 4a as a 9:1 mixture of geometrical isomers
(E:Z), which were separated by chromatography. Addi-
tion of freshly distilled TiCl4 to 4a at À78°C followed by
addition of DIPEA generated the titanium enolate,
which was condensed with E-3-methyl-2-heptenal 5.9
Although TLC indicated complete condensation to the
b-hydroxy ketone 7a within 30min, the dehydration step
was unreliable and we were never able to isolate the de-
sired dehydrated product 8a in more than 15–35% yield
together with a significant amount of b-hydroxy ketone
7a. Fortunately, 7a, could be converted to dienone 8a in
two steps to improve the overall yield. Specifically, treat-
ment of 7a with methanesulfonyl chloride gave rise to
Scheme 2. Reagents and conditions: (a) 1M LiOH/THF (1:3), 23°C,
12h, 95%; (b) ethyl chloroformate (2.2equiv), DIPEA (2.4equiv),
acetone, 0°C, 90min; then NaN3 (10equiv), H2O, 0°C, 90min; (c)
toluene, 2h, reflux; ROH (20equiv), 18h, 70°C, 50–65% two steps.

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T. Doundoulakis et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5667–5672
Scheme 5. Reagents and conditions: (a) phthalimide (1.3equiv), Ph3P
(1.3equiv), DEAD (1.3equiv), THF, 23°C, 6h, 86%; (b) MeNH2
(8.0equiv), EtOH, reflux, 7h; (c) HCl (1.1equiv), MeOH/acetone
(1:14), 0°C, 5min, 99%; (d) COCl2 (10.0equiv), toluene, reflux, 3h,
85%; (e) 31 (1.0equiv), 30 (1.0equiv), Et3N (1.0equiv), xylene, reflux,
4h, 76%; (f) LDA (3.2equiv), HMPA (3.2equiv), THF, À78°C, 1h;
then 3 (1.1equiv), À78°C, 1h; (g) AcOH/THF/H2O (3:1:1), 23°C, 12h;
(h) Dess–Martin periodinane (2.0equiv), NaHCO3 (5.0equiv),
CH2Cl2, 23°C, 2h; (i) trimethyl phosphonoacetate (2.6equiv), NaH
(2.5equiv), THF, 23°C, 15min; then aldehyde (1.0equiv), THF, 23°C,
3h; (j) 1M LiOH/THF (1:3), 23°C, 12h; (k) ethyl chloroformate
(2.2equiv), DIPEA (2.4equiv), acetone, 0°C, 90min; then NaN3
(10equiv), H2O, 0°C, 90min; (l) toluene, 2h, reflux; then MeOH, 18h,
70°C, 16% seven steps.
Scheme 3. Reagents and conditions: (a) LDA (3.2equiv), HMPA
(3.2equiv), THF, À78°C, 1h; then 14 or 15 (1.1equiv), À78°C, 1h, 65–
76%; (b) 16 (1equiv), TiCl4 (4.0equiv), CH2Cl2, À78°C, 30min; then
DIPEA (5.0equiv), À78°C, 3h; then 5 (3.0equiv), CH2Cl2, À78°C,
48h; À78 to 23°C, 1h, 20%; (c) Dess–Martin periodinane (2.0equiv),
NaHCO3 (5.0equiv), CH2Cl2, 23°C, 2h, 80%; (d) AcOH/THF/H2O
(3:1:1), 23°C, 12h, 90%; (e) Dess–Martin periodinane (2.0equiv),
NaHCO3 (5.0equiv), CH2Cl2, 23°C, 2h, 85%; (f) NaClO2 (2equiv),
NaH2PO4 (3equiv), 2-methyl-2-butene (20equiv), THF/t-BuOH/H2O
(2:2:1), 23°C, 2h; (g) TMSCHN2 (1.3equiv), benzene/MeOH (3:1),
23°C, 1h, 65% two steps; (h) 17 (1equiv), TiCl4 (4.0equiv), CH2Cl2,
À78°C, 30min; then DIPEA (5.0equiv), À78°C, 3h; then 5 (3.0equiv),
CH2Cl2, À78°C, 48h; À78 to 23°C, 1h, 15%; (i) 1M LiOH/THF (1:3),
23°C, 12h, 70%; (j) ethyl chloroformate (2.2equiv), DIPEA
(2.4equiv), acetone, 0°C, 90min; then NaN3 (10equiv), H2O, 0°C,
90min; (k) toluene, 2h, reflux; then MeOH, 18h, 70°C, 65% two steps.
prepared (Scheme 4). The alkylation reagents 2014 and
21 were prepared in two steps from 1,3- and 1,4-
bis(hydroxymethyl)benzene,15
respectively.
Hence,
treatment of 1,3- and 1,4-bis(hydroxymethyl)benzene,
with NaH, followed by addition of TBSCl furnished
the mono-protected compounds in good yields, which
were treated with CBr4 and PPh3 to produce 20 and
21, respectively.
To investigate the importance of the dienone moiety
for RNAP-activity and furthermore remove a potential
Michael acceptor moiety from the molecule, the bÀc
unsaturated amide 33 was prepared (Scheme 5). Amine
29 was prepared from alcohol 28,9 following a protocol
developed by Sen and Roach,16 and further converted to
isocyanate 30 under standard conditions.17 Condensa-
tion between 6-ethyl-4-hydroxy-2-pyrone 3111 and iso-
cyanate 30 proceeded in refluxing xylene18 to produce
32 in good yield, which was converted to the final ene-
carbamate 33 in seven steps as described above.
Scheme 4. Reagents and conditions: (a) LDA (3.2equiv), HMPA
(3.2equiv), THF, À78°C, 1h; then 20 or 21 (1.1equiv), À78°C, 1h, 40–
55%; (b) AcOH/THF/H2O (3:1:1), 23°C, 12h, 75–86%; (c) Dess–
Martin periodinane (2.0equiv), NaHCO3 (5.0equiv), CH2Cl2, 23°C,
2h, 92–95%; (d) NaClO2 (2equiv), NaH2PO4 (3equiv), 2-methyl-2-
butene (20equiv), THF/t-BuOH/H2O (2:2:1), 23°C, 2h; (e) TMSCHN2
(1.3equiv), benzene/MeOH (3:1), 23°C, 1h, 60–65% two steps; (f)
TiCl4 (4.0equiv), CH2Cl2, À78°C, 30min; then DIPEA (5.0equiv),
À78°C, 3h; then 5 (3.0equiv), CH2Cl2, À78°C, 48h; À78 to 23°C, 1h,
15–20%; (g) 1M LiOH/THF (1:3), 23°C, 12h; (h) ethyl chloroformate
(2.2equiv), DIPEA (2.4equiv), acetone, 0°C, 90min; then NaN3
(10equiv), H2O, 0°C, 90min; (i) toluene, 2h, reflux; then MeOH, 18h,
70°C, 52–64% two steps.
The in vitro inhibitory activity (IC50) against RNAP (E.
coli) of the myxopyronin analogs was evaluated in a
nucleotide coupled NADPH/pyrophosphate release
assay (Table 1) based upon a scheme described
previously.19
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 assay
and displayed no toxicity up to 40lM (ꢀ18lg/mL).
The results are compiled in Table 1.
To introduce rigidity and chemical stability to the ene-
carbamate, two analogs, 26 and 27, in which the double
bond was incorporated into an aromatic system were

T. Doundoulakis et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5667–5672
5671
Changes to the dienone region of myxopyronin B
References and notes
revealed several interesting features required for the
activity of this class of compounds (Table 1, entries
2–5 and 16). The geometrical isomer of myxopyronin
B, 1e, showed a threefold decrease in activity in the
RNAP enzymatic assay and a 10-fold decrease in
potency in the growth inhibition test.
1. Cragg, G. M.; Newman, D. J.; Snader, K. M. J. Nat. Prod.
1997, 60, 52–60.
2. (a) Kohl, W.; Irschik, H.; Reichenback, H.; Ho¨fle, G.
Liebigs Ann. Chem. 1983, 1656–1667; (b) Kohl, W.;
Irschik, H.; Reichenback, H.; Ho¨fle, G. Liebigs Ann.
Chem. 1984, 1088–1093; (c) Jansen, R.; Irschik, H.;
Reichenback, H.; Ho¨fle, G. Liebigs Ann. Chem. 1985,
822–836.
Although compound 1d, lacking the methyl d to the carb-
onyl group, showed similar activity against RNAP in
the biochemical assay and toward E. coli (Tol C), the
activity against S. aureus decreased significantly.
Removing the methyl adjacent to the carbonyl group
(1c) resulted in an almost complete loss of antibacterial
activity. Replacement of the dienone with an amide
functionality as in 33 or complete absence of the diene
moiety, as in 9, resulted in a loss of antibacterial
potency, as well as the enzymatic activity. The enecarba-
mate component of myxopyronin B was even more sen-
sitive toward changes (Table 1, entries 6–14). Extending
the carbamate with solubilizing groups (12 and 13),
replacement of the carbamate with an ester (8b) and
the incorporation of the carbamate alkene into an aro-
matic moiety (26–27) reduced the enzymatic activity
and resulted in a complete loss of potency against all
three bacterial strains. Aldehyde 18, which is a potential
degradation product of myxopyronin B, lacked potency
and was therefore dismissed as an RNAP inhibitor.
3. Irschik, H.; Gerth, K.; Ho¨fle, G.; Kohl, W.; Reichenback,
H. J. Antibiot. 1983, 36, 1651–1658.
4. (a) Campbell, E. A.; Korzheva, A.; Mustaev, A.; Mura-
kami, K.; Nair, S.; Goldfarb, A.; Darst, S. Cell 2001, 104,
901–912; (b) For other inhibitors of RNAP see: Artsi-
movitch, I.; Chu, C.; Lynch, A. S.; Landick, R. Science
2003, 302, 650–654.
5. Hu, T.; Schaus, V.; Lam, K.; Palfreyman, M. G.;
Wuonola, M.; Gustafso, G.; Panek, J. S. J. Org. Chem.
1998, 63, 2401–2406.
6. 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, 4968–4971.
7. Cook, L.; Ternai, B.; Ghosh, P. J. Med. Chem. 1987, 30,
1017–1023.
The cis analog, 10, displayed comparable IC50 value as
the parent trans compound with a small decrease in
activity toward E. coli and S. aureus. The saturated
carbamate analog 19 displayed comparable activity
against RNAP, but a significant decrease in MIC was
observed. Methylation of the pyrone skeleton (11)
resulted in fivefold decrease toward RNAP and com-
plete loss of cellular activity, indicating the importance
of the free hydroxyl group for biological activity.
8. Groutas, W. C.; Stanga, M. A.; Brubaker, M. J.; Huang,
T. L.; Moi, M. K.; Carroll, R. T. J. Med. Chem. 1985, 28,
1106–1109.
9. (a) Aldehyde 5 was prepared from methyl 2-butynoate
immediately prior to addition, in a three-step reaction
sequence: (i) n-BuLi, CuI, methyl 2-butynoate; (ii) LiAlH4;
(iii) TPAP, NMO. See: Anderson, R. J.; Corbin, V. L.;
Cotterrell, G.; Cox, G. R.; Henrick, C. A.; Schaub, F.;
Siddall, J. B. J. Am. Chem. Soc. 1975, 97, 1197–1204; (b)
Goura, K.; Nishino, T.; Koyama, T.; Seto, S. J. Am.
Chem. Soc. 1970, 92, 1197–1204.
The desmethyl analog, 1a, is the most active analog
synthesized in this series. The activity against RNAP
was improved threefold, without any increase in the cel-
lular potency. This result shows that it is possible to
remove the methyl group and hence the chirality from
this class of compounds without diminishing the biolog-
ical activity. Thus, the preparation of the next genera-
tion of myxopyronin B analogs, was designed without
the chiral center. The synthesis and biological evaluation
on the second-generation analogs will be reported
elsewhere.
10. Satisfactory spectroscopic data were obtained for all
newcompounds and all final analogs were purified by
reverse phase HPLC and characterized by 1H NMR and LC–
MS. HPLC (MeOH/H2O/AcOH = 70:30:4, flowrate =
40mL/min) tr = 2.6min (1a, minor), tr = 3.9min (1a, major).
11. 6-Ethyl-4-hydroxy-2-pyrone was prepared from 3-oxo-
pentanoic acid and meldrumÕs acid using similar condi-
tions as described for the preparation of other 2-pyrones,
see: Lokot, I. P.; Pashkovsky, F. S.; Lakhvich, F. A.
Tetrahedron 1999, 55, 4783–4792.
12. Poleschner, H.; Heydenreich, M.; Dietemr, M. Synthesis
1991, 1231–1235.
13. Hu, T. Q.; Weiler, L. Can. J. Chem. 1994, 72, 1500–1511.
14. Cumming, J. N.; Wang, D.; Park, S. B.; Shapiro, T. A.;
Posner, G. H. J. Med. Chem. 1998, 41, 952–964.
15. Gorins, G.; Kuhnert, L.; Johnson, C. R.; Marnett, L. J.
J. Med. Chem. 1996, 39, 4871–4878.
16. Sen, S. E.; Roach, S. L. J. Org. Chem. 1998, 61, 6646–
6650.
17. Molho, D. U.S. Patent 3,122,557, 1964.
18. Lee, B. H.; Clothier, M. F.; Dutton, F. E.; Conder, G. A.;
Johnson, S. S. Bioorg. Med. Chem. Lett. 1998, 8, 3317–
3320.
19. Johnson, J. C.; Shanoff, M.; Boezi, J. A.; Hansen, R. G.
Anal. Biochem. 1968, 26, 137–145. The cited assay was
adapted to 96-well plates to obtain reasonable throughput.
In conclusion, the SAR generated from this study
showed that minor structural changes to myxopyronin
B led to loss of biological activity. At the present time
natural product within this class do not represent viable
lead candidates for antibacterial therapy until a better
understanding of the exact mode of action is
determined.
Acknowledgement
We thank Dr. K. Steffy for the cytotoxicity studies.

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T. Doundoulakis et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5667–5672
NADPH formation typically was monitored by fluores-
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.
cence (excitation wavelength, ꢀ340nm; emission wave-
length, ꢀ440nm) rather than absorbance at 340nm as in
the original assay to improve sensitivity. Although com-
pounds with strong absorbance at 340nm can in principle