Total synthesis and preliminary antibacterial evaluation of the RNA polymerase inhibitors (±)-myxopyronin A and B

Full text of DOI:10.1021/jo9721610

J . Org. Chem. 1998, 63, 2401-2406
2401
Tota l Syn th esis a n d P r elim in a r y
An tiba cter ia l Eva lu a tion of th e RNA
P olym er a se In h ibitor s (()-Myxop yr on in A
a n d B
†
†
‡
Tao Hu, J ennifer V. Schaus, Kelvin Lam,
‡
‡
Michael G. Palfreyman, Mark Wuonola,
Gary Gustafson, and J ames S. Panek*
‡
,†
Department of Chemistry, Metcalf Center for Science and
Engineering, 590 Commonwealth Avenue, Boston
University, Boston, Massachusetts 02215 and Scriptgen
Pharmaceuticals Inc., 200 Boston Avenue,
Medford, Massachusetts 02155
Received December 9, 1997
In tr od u ction
In the early 1980s H o¨ fle and co-workers reported the
isolation of four novel polyketide derived antibiotics,
myxopyronin A and B (1a and 1b) as well as corallopy-
ronin A and B from the gliding bacteria Myxococcus
fulvus Mx f50 and Corallococcus coralloides, respec-
tively.1 This class of natural products which differ only
by their length of the unsaturated side chain at the C3
position of the R-pyrone nucleus is virtually unexplored.
The absolute stereochemistry of myxopyronin A and B
has been determined by careful degradative and spec-
troscopic methods and was assigned as (R)-configuration
at C7 (Figure 1).1 Those molecules inhibited the growth
of many Gram-positive and several Gram-negative bac-
teria.3 No inhibitory activity was observed for yeasts and
fungi (molds). They were also shown to be selective
F igu r e 1.
,2
chain followed by a titanium(IV)-promoted aldol conden-
sation introducing the (E,E)-dienone of the upper side
chain. Our retrosynthetic analysis of the myxopyronins
is illustrated in Figure 1 with the first disconnection
removing the terminal unsaturated carbamate. This
detachment produced the advanced intermediate 2 bear-
ing an unsaturated carboxylate functionality. The second
disconnection produced the aldol synthons in the form
of the pyrone 3 and the corresponding unsaturated
aldehydes. Further disconnection of 3 produced the
starting 3-propionyl-4-hydroxy-R-pyrone 4.
3
inhibitors of bacterial DNA-dependent RNA polymerase.
The broad spectrum of activity and selectivity for bacte-
rial RNA polymerase over the human polymerase estab-
lished the myxopyronins as promising candidates for
development as antibacterial agents. The exhibition of
activity against rifampicin- or streptolydigin-resistant
bacteria by the myxopyronins suggests that these agents
target a region of RNA polymerase distinct from the one
by rifampicin. In this paper, we report the first total
syntheses of myxopyronin A and B. To the best of our
knowledge, no synthetic investigations have been re-
ported on this class of natural products. Our convergent
synthesis makes use of a 3-propionyl-4-hydroxy-R-pyrone
Resu lts a n d Discu ssion
P r ep a r a tion of th e O1-C14 F r a gm en t. The syn-
thesis of this material relied on a selective alkylation of
the C6 ethyl group of pyrone 4 (Scheme 1). In the
presence of LDA (3.2 equiv), the regioselective alkylation
with primary iodide 55 proceeds through a trianion
6
intermediate to give the alkylated pyrone 6 in good yield.
Intermediate 6 was deprotected under mild acidic condi-
tions to afford the corresponding primary alcohol in 91%
yield. Oxidation of the primary hydroxyl with Dess-
4
as the central building block from which both side
4
chains are introduced. In that regard, an alkylation
strategy was used for the installation of the lower side
7
Martin reagent (2.0 equiv, 0 °C, 2 h) gave aldehyde 7.
Subsequent Horner-Emmons-Wadsworth homologation
using trimethyl phosphonoacetate (2.2 equiv, NaH 2.2
equiv, THF, rt) afforded the R,â-unsaturated ester 8 with
an E:Z isomer ratio greater than 20:1. Silica gel chro-
matography provided geometrically pure 8 in 82% yield.
†
Department of Chemistry, Metcalf Center for Science and Engi-
neering, Boston University, Boston, Massachusetts.
‡
Scriptgen Pharmaceutical Inc., Department of Biology, Medford,
Massachusetts.
(1) (a) Kohl, W.; Irschik, H.; Reichenbach, H.; H o¨ fle, G. Liebigs Ann.
Chem. 1983, 1656-1667. (b) Kohl, W.; Irschik, H.; Reichenbach, H.;
H o¨ fle, G. Liebigs Ann. Chem. 1984, 1088-1093. (c) For clarification,
the natural products have been illustrated with the natural occurring
(5) Alkyl iodide 5 was prepared from 1,3-propanediol by a two-step
reaction sequence: (i) selective protection with TBSCl (1.0 equiv), NaH
(1.0 equiv), 98% yield; (ii) I , PPh , imidazole, 96% yield. See
2 3
(
R)-configuration; however, all materials in this paper were synthesized
in racemic form.
2) J ansen, R.; Irschik, H.; Reichenbach, H.; H o¨ fle, G. Liebigs Ann.
Chem. 1985, 822-836.
3) Irschik, H.; Gerth, K.; H o¨ fle, G.; Kohl, W.; Reichenbach, H. J .
Antibiot. 1983, 36, 1651-1658.
4) Pyrone was prepared from commercially available ethyl
Supporting Information for details.
(6) Satisfactory spectroscopic data ( H and 13C NMR, IR, CIMS, and
1
(
CIHRMS) were obtained for all new compounds.
(
(7) (a) Dess, D. B.; Martin, J . C. J . Org. Chem. 1983, 48, 4155-
4156. (b) Dess, D. B.; Martin, J . C. J . Am. Chem. Soc. 1991, 113, 7277-
7287. For two improved procedures for the preparation of DMP, see:
(c) Ireland, R. E.; Liu, L. J . Org. Chem. 1993, 58, 2899. (d) Meyer, S.
D.; Schreiber, S. L. J . Org. Chem. 1994, 59, 7549-7552.
(
4
3
1
-oxopropanoate see: Cook, L.; Ternai, B.; Ghosh, P. J . Med. Chem.
987, 30, 1017-1022. See Experimental Section for further details.
S0022-3263(97)02161-0 CCC: $15.00 © 1998 American Chemical Society
Published on Web 03/12/1998

2
402 J . Org. Chem., Vol. 63, No. 7, 1998
Notes
Sch em e 1
Sch em e 2
F igu r e 2. MICs at concentrations comparable to in vitro
bacterial RNAP IC50s.
This sequence completed the preparation of the O1-C14
fragment now set for the subsequent aldol condensation
for the introduction of the C15-C24 and C25 side chains
of myxopyronin A and B.
Syn th esis of th e r,â-Un sa tu r a ted Ald eh yd es 12a
a n d 12b. The synthesis of these subunits relied on
8
Negishi’s carboalumination of the terminal alkynes 9a
and 9b. Treatment of the alkynes with zirconocene
dichloride (1.0 equiv) in the presence of AlMe (2.0 equiv)
3
afforded the (E)-trisubstituted vinyl aluminates, which
were then directly converted to the more reactive alu-
n
minate complex 10 by addition of BuLi (1.0 equiv, THF)
(
Scheme 2). These intermediates were trapped with
F igu r e 3. In vitro transcription assay with Escherichia coli
RNA Polymerase.
excess paraformaldehyde affording the (E)-trisubstituted
allylic alcohol 11 in good overall yield. Oxidation of these
materials with TPAP (0.06 equiv) and NMO (2.0 equiv)
9
densed with freshly prepared aldehyde 12 at -78 °C for
afforded the corresponding aldehydes 12a and 12b,
respectively. These experiments completed the prepara-
tion of the two volatile and labile aldehydes which were
used immediately and without purification in the aldol
condensation for installation of the C15-C24/25 upper
side chain of the myxopyronins.
Tita n iu m (IV)-P r om oted Ald ol Con d en sa tion a n d
Com p letion of th e Syn th eses. The introduction of the
upper side chains of myxopyronin A and B was carried
out utilizing a Ti(IV) tetrachloride-promoted aldol con-
densation between the ethyl ketone 8 and aldehydes 12a
and 12b (Scheme 3). The titanium enolate was gener-
4
8 f 56 h to directly afford after in situ dehydration the
respective (E,E)-dienones 13a and 13b. With both side
chains installed onto the R-pyrone core, completion of the
individual synthesis required the conversion of the R,â-
unsaturated methyl ester to the methyl carbamate. This
2
was initiated by a LiOH (10 equiv, THF/H O 4:1, 35 h)-
promoted hydrolysis of the methyl esters which afforded
the free carboxylic acids 13a and 13b in quantitative
yield. The vinyl carbamate was introduced by a modified
Curtius rearragement,10 employing ethyl chloroformate
and NaN . This sequence completed the assembly of the
3
lower side chain and achieved the individual synthesis
ated at -78 °C by treatment of ethyl ketone 8 with TiCl
4.0 equiv) and DIPEA (4.8 equiv); this excess of Lewis
4
of (()-myxopyronin A and B.
(
An tiba cter ia l Eva lu a tion of (()-Myxop yr on in A
a n d B. The antibacterial activities of myxopyronins
were evaluated with an in vitro transcription assay using
Escherichia coli RNA polymerase (Figures 2 and 3). The
myxopyronins A/B were isolated as a mixture of natural
acid and base was found to be necessary to drive the
reaction to completion. The derived enolate was con-
(8) (a) Negishi, E.; Kondakov, D.; Choueiry, D.; Kasai, K.; Takahashi,
T. J . Am. Chem. Soc. 1996, 118, 9577-9588. (b) Okukado, N.; Negishi,
E. Tetrahedron Lett. 1978, 27, 2357-2360.
(9) (a) Ley, S.; Norman, J .; Griffith, W.; Marsden, S. Synthesis 1994,
6
1
39-666. (b) Griffith, W.; Ley, S. Aldrichimica Acta 1990, 23, 13-
9.
(10) Overman, L.; Taylor, G.; Petty, C.; J essup, P. J . Org. Chem.
1978, 43, 2164-2167.

Notes
J . Org. Chem., Vol. 63, No. 7, 1998 2403
Sch em e 3
inhibition toward streptolygidin-resistant bacteria S.
aureus than toward rifampicin-resistant strain (Figure
4
).
Con clu sion
We have described an efficient and highly convergent
synthesis of myxopyronin A and B. Biological evaluation
of these agents against RNA polymerase for a variety of
bacteria including mammalian culture cells holds con-
siderable promise as potential antibacterial agents. The
asymmetric synthesis and SAR programs are currently
underway in our laboratories and will be reported at the
appropriate time.
Exp er im en ta l Section
1H and 13C NMR spectra were taken in CDCl
at 400 and 75
3
F igu r e 4. Myxopyronin A/B against rifampicin- and strep-
tolydigin-resistant Staphylococcus aureus.
MHz, respectively, unless specified otherwise. Chemical shifts
are reported in parts per million using the solvent resonance
1
internal standard (chloroform, 7.24 and 77.0 ppm for H and
1
3
products containing a 9:1 ratio of A and B. The pyronins
exhibited comparable micromolar inhibitory activity in
enzymatic assays against a panel of seven different
bacteria, in accordance with their previously established
activity. These seven pathogens represent both Gram-
positive and Gram-negative bacterial and illustrate the
broad spectrum activity of the myxopyronins.
C NMR, respectively, unless specified otherwise). NMR data
are reported as follows: chemical shift, multiplicity (app )
apparent, par obsc ) partially obscured, ovrlp ) overlapping, s
)
singlet, d ) doublet, t ) triplet, q ) quartet, m ) multiplet,
br ) broad, abq ) ab quartet), coupling constant, and integra-
tion. Infrared resonance (IR) spectra were recorded on a FTIR
spectrophotometer. High-resolution mass spectra were ob-
stained on a Finnegan spectrometer in the Boston University
Mass Spectrometry Laboratory. Reversed phase preparative
HPLC was conducted with a diode array detector, using a 22 ×
2
As a point of comparison, the synthetic myxopyronin
A and B were tested against E. coli RNA polymerase
separately and myxopyronin B is shown to be an ap-
proximately 4 times more potent molecule than A (Figure
250 mm C18 column (218TP1022). Methylene chloride (CH -
2 6 6
Cl ), methanol (MeOH), benzene (C H ), toluene, and hexane
were distilled from calcium hydride, and tetrahydrofuran (THF)
and hexamethylphosphoramide (HMPA) were distilled from
sodium and benzophenone prior to use. Titanium tetrachloride
3
). As a validation of the in vitro transcription assay, a
known transcription inhibitor, rifampicin, was assayed
with the natural product mixture (myxopyronin A/B)
against rifampicin and streptolydigin-resistant S. aureus
(
TiCl
pressure before each use. Anhydrous 1,2-dichloroethane (ClCH
CH Cl), trimethylaluminum (AlMe3, 2.0 M solution in hexanes),
and zirconocene dichloride (Cp ZrCl ) was purchased from
4
) was freshly distilled from copper powder under reduced
2
-
2
(Figure 4).
2
2
Aldrich Chemical Co. Inc. All other reagents were used as
supplied. All reactions were carried out in oven-dried glassware
under argon atmosphere unless otherwise noted. Analytical
thin-layer chromatography was performed on 0.25 mm silica gel
Table 1 summarizes in vitro IC50 and MIC values
obtained from the cell-based evaluation of the myxopy-
ronins using both Gram-positive and Gram-negative
bacteria. The data show that myxopyronins have in vivo
cell-based activities against rifampicin-resistant bacte-
ria.11 In complement to the in vitro transcription activi-
ties, (()-myxopyronin B is also shown to be at least 30-
fold more potent in cell-based activities for the S. aureus
Gram-positive strain than (()-myxopyronin A (Table 1).
Table 1 summarizes the in vitro IC50 and MIC values for
myxopyronin A and B. The pyronins exhibited similar
(
11) In vitro transcription reactions were performed as described
32
in the Supporting Information. [R- P] UTP-incorporated RNA was
synthesized in 50 µL reaction volumes containing transcription buffer
(
2
50 mM Tris-HCl, pH 8.0, 200 mM KCl, 10 mM MgCl , 10 mM DTT,
and 1.5 µM BSA), 1 µg of DNA template, 4 µM UTP containing 5 µCi
3
2
of [R- P] UTP, 400 µM each of ATP, GTP, and CTP. After incubation
for 60 min at 25 °C, the reaction is terminated with 100 µL of 10%
TCA, which also precipitates the newly transcribed RNA. See Sup-
porting Information for details.

2
404 J . Org. Chem., Vol. 63, No. 7, 1998
Notes
Ta ble 1
in vitro
transcription
(
E. coli RNAP)
IC50 (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
E. colia
MIC (µg/mL)
S. aureusb
MIC (µg/mL)
b
compound
E. coli
S. aureus
(
R)-Myxo A/B
natural mixture
()-Myxo B
8
200
5
4
5
(
(
5
20
>30
>30
2
4-8
0.5
15
0.5
8
()-Myxo A
a
Mutant strain that has a permeabilized cell wall. b Mutant strain is rifampicin-resistant bacteria. See Supporting Information for
details.
6
2
0-A plates. Flash chromatography was performed on silica gel
30-400 mesh. Due to the similarities of the two natural
in hexanes as the eluant) to afford the primary iodide 5 (14.4 g,
96% yield) as a pale yellow oil: 1H NMR (400 MHz, CDCl
): δ
3.64 (t, J ) 5.7 Hz, 2H), 3.25 (t, J ) 6.6 Hz, 2H), 1.96 (m, 2H),
0.87 (s, 9H), 0.05 (s, 6H); 13C NMR (67.5 MHz, CDCl
) δ 62.3,
36.1, 25.9, 8.3, 3.6, -5.3; IR (neat) νmax: 2929, 1472, 1257, 1101
3
products, detailed experimental procedures are provided only
for myxopyronin B; see Supporting Information.
3
3
-(1-P r op ion yl)-4-h yd r oxy-6-eth yl-2-p yr on e (4). Ethyl
-
1
+
3
-oxopentanoate (5.0 g, 35 mmol) was dissolved in 30 mL of 1.5
cm ; CIHRMS (NH
3 9 2
gas) calcd for C H22SiO I (M + H )
M solution NaOH and stirred at rt for 30 h. The reaction was
cooled to 0 °C, and 3 M HCl was slowly added until the reaction
system reached a pH ∼2, and then solid KCl was added to
saturate the solution. The reaction was extracted with EtOAc
301.0485, found: 301.0470.
3-(1-P r op ion yl)-4-h yd r oxy-6-[1-m eth yl-4-[(ter t-bu tyld im -
eth ylsilyl)oxy]bu ta n yl]-2-p yr on e (6). To a stirred solution
of diisopropylamine (3.34 mL, 24 mmol) in 25 mL of THF at -78
°C under an argon atmosphere was added n-butyllithium (9.45
mL, 2.5 M solution in hexanes, 23.6 mmol). The mixture was
allowed to warm to 0 °C for 30 min and then recooled to -78
°C. The resulting solution was treated with a solution of pyrone
4 (1.47 g, 7.5 mmol) in 10 mL of THF, and stirred for 1.0 h at
-78 °C. The derived trianion was treated with iodide 5 (2.5 g,
8.25 mmol) in 10 mL of THF, followed by the addition of HMPA
(4.0 mL, 23.2 mmol). The reaction mixture was allowed to stir
and CHCl
Na SO , filtered, and concentrated in vacuo to afford the crude
-oxopentanoic acid (3.1 g, 76% yield) as a white solid. The
material needed no further purification and was used directly
3
, and the combined organic layers were dried with
2
4
3
1
in the next step: H NMR (300 MHz, CDCl
3
) δ 3.52 (s, 2H), 2.60
1
3
(
q, J ) 7.3 Hz, 2H), 1.10 (t, J ) 7.3 Hz, 3H); C NMR (75 MHz,
CDCl
3
) δ 204.3, 172.3, 47.9, 36.6, 7.5; IR (neat) νmax: 3400, 1708,
-
1
1
620 cm
.
To a stirring solution of the above 3-oxopentanoic acid (3.1 g,
6.7 mmol) in 50 mL of THF was added carbonyldiimidazole (5.6
g, 34.7 mmol). The reaction was left stirring for 18 h before
4
for 30 min at -78 °C, before being diluted with saturated NH -
2
Cl aqueous solution. The organic layer was separated, and the
aqueous layer was extracted with ether. The organic layers were
being quenched with 2% HCl aq solution (pH measured 2∼3).
4
combined, dried (MgSO ), filtered, and concentrated in vacuo.
It was extracted with EtOAc, dried (Na
2
SO
4
), filtered, and then
The resulting crude oil was purified by flash chromatography
(20% EtOAc in hexanes as the eluant) to afford the pure
concentrated in vacuo. The product needed no further purifica-
tion as long as it was left on a vacuum pump for 2 h in order to
remove any remaining starting acid. This protocol afforded
1
alkylation product 6 (2.4 g, 87% yield) as a yellow oil: H NMR
(400 MHz, CDCl
(q, J ) 7.3 Hz, 2H), 2.56 (m, 1H), 1.43-1.73 (m, 4H), 1.22 (d, J
3
) δ 5.90 (s, 1H), 3.57 (t, J ) 6.0 Hz, 2H), 3.08
1
compound 4 (2.22 g, 86%) as a pale yellow solid: H NMR (400
1
3
MHz, CDCl
3
) δ 5.91 (s, 1H); 3.10 (q, J ) 7.3 Hz, 2H); 2.53 (q, J
) 7.3 Hz, 3H), 1.13 (t, 3H), 0.86 (s, 9H), 0.01 (s, 6H); C NMR
(67.5 MHz, CDCl ) δ 207.8, 180.6, 175.6, 160.6, 99.3, 62.2, 38.3,
34.8, 30.0, 29.7, 25.4, 17.8, 17.5, 7.3, -5.8; IR (neat) νmax: 2936,
)
7.2 Hz, 2H); 1.24 (t, J ) 7.5 Hz, 3H); 1.15 (t, J ) 7.2 Hz, 3H).
C NMR (75 MHz, CDCl
3
1
3
3
) δ 208.4, 181.2, 173.5, 161.2, 99.9,
1
-
-1
9
9.6; 35.4, 27.5, 10.5, 7.8; IR (neat) νmax: 3420, 1731, 1640 cm
;
3 6
1743, 1636, 1100 cm ; CIHRMS (NH gas) calcd for C19H33SiO
+
+
CIHRMS (NH
found: 197.0818.
-Iod o-3-[(ter t-bu tyld im eth ylsilyl)oxy]p r op a n e (5). To a
3
gas) calcd for C10
H
13
O
4
(M + H ) 197.0814,
(M + H ) 369.2097, found: 369.2085.
3-(1-P r op ion yl)-4-h yd r oxy-6-(1-m eth yl-4-h yd r oxybu tyl)-
2-p yr on e (7′). A solution of TBS ether 6 (1.66 g, 4.5 mmol) in
1
suspension of NaH (4.0 g, 100 mmol, 60% dispersion in mineral
oil) in THF (100 mL) at rt was added 1,3-propanediol (7.6 g, 100
mmol, dissolved in 50 mL of THF) via a cannula. The resulting
mixture was stirred at rt for 45 min, before a solution of TBSCl
2
50 mL of AcOH/THF/H O (3:1:1) was stirred at rt for 22 h. The
reaction was diluted with water and extracted with EtOAc. The
combined organic layers were washed with a saturated solution
of NaHCO
layer was back extracted with EtOAc. The combined organic
layers were dried (MgSO ), filtered, and concentrated in vacuo.
3
. The organic layer was separated, and the aqueous
(
15.0 g, 100 mmol) in 50 mL of THF was added into the reaction
mixture by a cannula. This resulting reaction was stirred for
.0 h at rt. The reaction was quenched with saturated aqueous
NaHCO solution and extracted with ether. The combined
organic layers were dried (MgSO ), filtered, and concentrated
4
1
The crude product was purified by flash chromatography (40%
EtOAc in hexanes eluant) to afford diol 7′ (1.04 g, 91% yield) as
3
1
4
a yellow oil: H NMR (400 MHz, CDCl
3
) δ 5.90 (s, 1H), 3.63 (br
in vacuo. The crude product was purified by flash chromatog-
raphy (20% EtOAc in petroleum ether eluant) to afford the pure
monoprotected alcohol (18.1 g, 98% yield) as a clear oil: 1H NMR
t, 2H), 3.08 (q, J ) 7.3 Hz, 2H), 2.58 (m, 1H), 1.75 (m, 1H), 1.50-
1.62 (m, 3H), 1.33 (br s, 1H), 1.23 (d, J ) 7.3 Hz, 3H), 1.13 (t, J
13
) 7.3 Hz, 3H); C NMR (67.5 MHz, CDCl
3
) δ 180.5, 175.2, 160.6,
(
400 MHz, CDCl
3
) δ 3.73-3.80 (m, 4H); 2.71 (br s, 1H); 1.74 (m,
H); 0.86 (s, 9H); 0.04 (s, 6H); 13C NMR (67.5 MHz, CDCl
) δ
2.7, 62.2, 34.2, 25.9, 25.8, 25.8, 18.1, -5.6; IR (neat) νmax: 3371,
99.3, 99.0, 61.6, 38.1, 34.7, 29.8, 29.5, 17.3, 7.1, 1.51; IR (neat)
-1
2
6
2
3
ν
max: 3425, 2940, 1734, 1636 cm ; CIHRMS (NH
3
gas) calcd
+
for C13
H
19
O
5
(M + H ) 255.1232, found: 255.1252.
-1
929, 1473, 1257, 1099 cm ; CIHRMS (NH
3
gas) calcd for C
9
H
23
-
3-(1-P r op ion yl)-4-h yd r oxy-6-(1-m eth yl-4-for m ylbu tyl)-2-
p yr on e (7). To a solution of alcohol 7′ (483 mg, 1.9 mmol) in
+
SiO
To a solution of imidazole (10.2 g, 150 mmol) and triph-
enylphosphine (14.4 g, 55 mmol) in CH Cl (200 mL) at 0 °C
was added I (14.0 g, 55 mmol). After 10 min, a solution of the
above monoprotected alcohol (9.5 g, 50 mmol) in CH Cl (100
2
(M + H ) 191.1467, found: 191.1472.
2 2
CH Cl (20 mL) was added pyridine (845 µL, 10.45 mmol),
2
2
followed with Dess-Martin periodinate reagent (2.8 mg, 6.65
2
mmol) in one portion. The resulting reaction mixture was stirred
2
2
at rt for 2.0 h before being quenched with saturated NaHCO
aqueous solution. The reaction mixture was extracted with CH
Cl , dried (MgSO
3
mL) was added over 5 min. The mixture was warmed to rt,
covered in aluminum foil, and stirred for an additional 15 h in
the dark. The reaction was then diluted with 2.0 mL of
2
-
2
4
), filtered, and concentrated in vacuo. The
crude product was flash chromatographed (15% EtOAc in
saturated Na
water. The organic layer was separated, and the aqueous layer
was back extracted with CH Cl . The combined organic layers
were dried (MgSO ), filtered, and concentrated in vacuo. The
crude product was purified by flash chromatography (2% EtOAc
2
S
2
O
4
aqueous solution before further dilution with
hexanes eluant) to afford aldehyde 11 (426 mg, 89% yield) as
1
yellow oil: H NMR (400 MHz, CDCl
1H), 3.07 (q, J ) 7.3 Hz, 2H), 2.59 (m, 1H), 2.46 (t, J ) 7.3 Hz,
2H), 1.83-2.02 (m, 2H), 1.23 (d, J ) 7.3 Hz, 3H), 1.12 (t, J )
3
) δ 9.73 (s, 1Η), 5.90 (s,
2
2
4
7.3 Hz, 3H); 13C NMR (67.5 MHz, CDCl
) δ 200.7, 180.7, 174.4,
3

Notes
J . Org. Chem., Vol. 63, No. 7, 1998 2405
1
ν
60.6, 99.9, 99.5, 40.8, 37.8, 35.0, 25.9, 17.5, 7.4, 1.8; IR (neat)
reaction turned to an yellow slurry mixture immediately. After
stirring for 20 min at -78 °C, DIPEA (483 µL, 2.77 mmol) was
added, and the reaction became a red-dark reaction mixture.
This reaction mixture was allowed to stir at -78 °C for 3.0 h,
and then aldehyde 12b (254 mg, 2.0 mmol) dissolved in 2.0 mL
CH Cl was added via a cannula. This dark-red reaction mixture
2 2
was stirred at -78 °C for 48 h and then warmed to 0 °C for 5
min, before it was quenched with distilled water. The reaction
-
1
max: 2978, 1727, 1636, 1560 cm ; CIHRMS (NH
3
gas) calcd
+
for C13
H
17
O
6
(M + H ) 253.1076, found: 253.1076.
3
-(1-P r op ion yl)-4-h yd r oxy-6-[5-(m e t h oxyca r b on yl)-1-
m eth yl-4-p en ten yl]-2-p yr on e (8). To a solution of trimethyl
phosphonoacetate (1.7 g, 9.3 mmol) in THF (20 mL) at room
temperature was added NaH (360 mg, 8.95 mmol, 60% dispen-
sion in mineral oil). The resulting reaction was stirred at rt for
2 2 2 4
was extracted with CH Cl , dried (Na SO ), filtered, and con-
centrated in vacuo. The crude product was flash chromato-
1
5 min, before aldehyde 7 (906 mg, 3.58 mmol) in 10 mL of THF
was added, via cannula. The reaction was allowed to stir at rt
for 3.0 h before being diluted with aqueous NH Cl solution. The
mixture was extracted with EtOAc, dried (MgSO ), filtered, and
graphed (25% EtOAc in hexanes as the eluant) to provide diene
4
1
1
3b (147 mg, 61% yield) as a sticky yellow oil: H NMR (400
4
MHz, CDCl ): δ 6.97 (d, J ) 11.6 Hz, 1H), 6.88 (dt, J ) 6.7,
3
concentrated in vacuo. The crude product was flash chromato-
graphed (20% EtOAc in hexanes as the eluant) to afford
1
5.3 Hz, 1H), 6.13 (d, J ) 11.6 Hz, 1H), 5.91 (s, 1H), 5.81 (d, J
1
) 15.3 Hz, 1H), 3.70 (s, 3H), 2.58 (m, 1H), 2.20 (m, 2H), 2.14 (t,
J ) 7.3 Hz, 2H), 1.98 (s, 3H), 1.88 (m, 1H), 1.82 (s, 3H), 1.66 (m,
1
compound 8 (904 mg, 82% yield) as a yellow oil: H NMR (400
MHz, CDCl
3
) δ 6.79 (dt, J ) 6.8, 15.6 Hz, 1H), 5.83 (s, 1H), 5.70
d, J ) 15.6 Hz, 1H), 3.57 (s, 3H), 2.97 (q, J ) 7.1 Hz, 2H), 2.49
m, 1H), 2.11 (m, 2H); 1.78 (m, 1H), 1.58 (m, 1H), 1.14 (d, J )
H), 1.45 (m, 2H), 1.30 (m 2H), 1.24 (d, J ) 6.7 Hz, 3 H), 0.89 (t,
(
(
6
13
J ) 7.3 Hz, 3H); C NMR (75 MHz, CDCl
166.7, 160.3, 149.4, 147.5, 133.6, 132.9, 121.9, 120.4, 100.0, 99.1,
1.5, 40.5, 38.2, 32.2, 30.0, 29.6, 22.4, 17.7, 17.3, 14.0, 13.5; IR
3
) δ 201.6, 180.7, 174.7,
.7 Hz, 3H), 1.02 (t, J ) 7.1 Hz, 3H); 1 C NMR (75 MHz, CDCl
3
3
)
5
δ 208.2, 180.8, 174.8, 166.6, 160.8, 147.4, 121.8, 100.0, 99.6, 51.4,
-
1
3
(neat) νmax: 2932, 1726, 1636, 1546 cm ; CIHRMS (NH gas)
3
1
3
8.2, 35.2, 32.1, 29.5, 17.7, 7.6; IR (neat) νmax: 2980, 1726, 1637,
+
-
1
+
calcd for C24
32 6
H O (M ) 416.2199, found: 416.2208.
560 cm ; CIHRMS (NH
08.1260, found: 308.1234.
E)-3-Meth ylh ep t-2-en -1-ol (11b). To a white slurry solu-
tion of zirconocene dichloride (7.3 g, 25 mmol) in 60 mL of (CH
Cl was added AlMe (25 mL, 2.0 M in hexanes, 50 mmol) at 0
C, stirred for 30 min, and then warmed to rt for 1.0 h. To this
lemon-yellow solution was added 1-hexyne (9b; 2.05 g, 25 mmol,
dissolved in 20 mL of (CH Cl ) at rt. The reaction was allowed
3 20 6
gas) calcd for C16H O (M + H )
3
-[(E,E)-2,5-Dim et h yl-2,4-n on a d ien oyl]-4-h yd r oxy-6-(5-
ca r boxy-1-m eth yl-4-p en ten yl)-2-p yr on e (2b). To a stirred
solution of 13b (42 mg, 0.10 mmol) in THF (6.0 mL) was added
LiOH aqueous solution (1.5 mL, 1.0 M aq, 1.5 mmol) at rt, and
(
2 2
) -
2
3
the resulting reaction mixture (THF/H
stir at rt for 35 h before it was diluted with EtOAc and then
quenched by saturated NH Cl aq solution. The reaction was
further acidified to pH ) 2 by slow addition of 5% HCl. It was
extracted with EtOAc, dried (Na SO ), filtered through a short
2
O ) 4:1) was allowed to
°
)
2
2
4
2
to stir at rt for 16 h. The volatile components were evaporated
under reduced pressure (maximum 50 °C, 0.3 mmHg, 1-2 h).
The remaining orange-yellow organic residue was extracted with
dry hexanes (4 × 30 mL), and the yellow extract was transferred
to a 500 mL round-bottom flask via a cannula. To this was
2
4
pad of silica gel, and then concentrated in vacuo to afford the
crude acid 2b (40 mg, 100% yield) as a sticky yellow oil. This
material was sufficiently pure and used without further
n
purification: H NMR (270 MHz, CDCl ): δ 7.05-6.94 (m, 2H),
1
added BuLi (10 mL, 2.5 M in hexanes, 25 mmol) at 0 °C. This
3
orange-yellow slurry solution was stirred from 0 °C to rt for 1.5
h, and then THF (50 mL) was added to dissolve the precipitate.
The resulting solution (homogeneous, brown-yellow color) was
cannulated to a suspension of paraformaldehyde (3.75 g, 125
mmol) in THF (50 mL) under a argon atmosphere. This orange-
yellow suspension was allowed to stir at rt for 20 h before it
was cooled to 0 °C (ice-water bath). Ice was added to dilute
6.14 (d, J ) 11.5 Hz, 1H), 5.92 (s, 1H), 5.81 (d, J ) 15.4 Hz,
1H), 2.59 (m, 1H), 2.24 (m, 2H), 2.14 (t, J ) 7.3 Hz, 2H), 1.97 (s,
3H), 1.89 (m, 1H), 1.82 (s, 3H), 1.68 (m, 1H), 1.43 (m, 2H), 1.28
1
3
(m 2H), 1.24 (d, J ) 6.8 Hz, 3 H), 0.89 (t, J ) 7.3 Hz, 3H);
C
NMR (75 MHz, CDCl
3
) δ 201.6, 180.7, 174.5, 171.4, 160.4, 150.2,
149.1, 133.6, 132.9, 128.3, 121.6, 120.6, 100.1, 99.1, 42.8, 38.2,
32.0, 29.7, 20.9, 17.7, 17.2, 13.8, 13.4; IR (neat) νmax: 2960, 1724,
+
the reaction, and then saturated NH
4
Cl was added. The ice bath
1636, 1561; CIHRMS (NH
403.2121, found: 403.2136.
3
gas) calcd for C23
H
31
O
6
(M + H )
was removed, and the reaction was further acidified with 3 M
HCl until the reaction turned to a clear yellow (homogeneous)
solution. At this time, the reaction pH was measured 2-3. The
organic layer was separated, and the aqueous layer was ex-
tracted with ether. The organic extracts were combined, washed
(()-Myxop yr on in B (1b). To a stirred solution of acid 2b
(22 mg, 0.0547 mmol) in dry acetone (1.0 mL) were sequentially
added DIPEA (23 µL, 0.13 mmol) and ethyl chloroformate (11
µL, 0.12 mmol) at 0 °C. The reaction was stirred at 0 °C for 1.5
with a saturated solution NaHCO
3 2
, and then dried with Na -
h, and then NaN
3
(17 mg, 0.263 mmol, dissolved in 300 µL of
SO , filtered, and concentrated under reduced pressure to
4
distilled H O) was added via a syringe. The resulting reaction
2
provide crude allylic alcohol 11b. The crude product was
purified by flash chromatography (20% EtOAc in hexanes as the
eluant) to afford pure 11b (2.46 g, 77% yield) as a colorless oil:
mixture was stirred at 0 °C for 70 min before being quenched
by ice-water, and extracted with distilled toluene, dried (Mg-
4
SO ), filtered, and concentrated in vacuo. The organic residue
was taken up by dry toluene (6 mL) and refluxed for 2.0 h before
fresh distilled MeOH (3.0 mL) was added to trap the isocyanate
intermediate. The resulting reaction solution was further
refluxed for 12 h and then concentrated in vacuo to provide crude
1
H NMR (400 MHz, CDCl ) δ 5.37 (t, J ) 7.1 Hz, 1H), 4.11 (d,
3
J ) 7.1 Hz, 2H), 1.98 (t, J ) 7.5 Hz, 2H), 1.64 (s, 3H), 1.37 (m,
1
3
2
H), 1.28 (m, 2H), 0.87 (t, J ) 7.3 Hz, 3H); C NMR (67.5 MHz,
CDCl ) δ 139.9, 123.1, 59.2, 39.2, 29.8, 22.3, 16.1, 13.9; IR (neat)
3
-
1
ν
3
max: 3330, 2958, 1670, 1467 cm ; CIHRMS (NH gas) calcd
(
()-myxopyronin B (1b) as a yellow oil. This crude material was
purified by preparative reverse phase HPLC (70:30:4 MeOH:
O:AcOH) to provide pure (()-myxopyronin B (1b, 15.6 mg,
+
for C
8
H
16
O
1
(M ) 128.1201, found: 128.1199.
(
E)-3-Meth ylh ep t-2-en -1-a l (12b). To a suspension solution
of alcohol 11b (739 mg, 5.7 mmol), 4 Å molecular sieves (2.9 g,
activated), and NMO (1.33 g, 11.4 mmol) in CH Cl (12 mL) at
°C was added TPAP (120 mg, 0.34 mmol) in one portion. The
resulting dark reaction mixture was allowed to stir at 0 °C for
0 min, before being diluted with CH Cl , and then filtered
H
6
2
1
6%) as a yellow sticky oil: H NMR (400 MHz, CD
3
OD, 3.27
2
2
ppm) δ 7.15 (d, J ) 11.6 Hz, 1H), 6.36 (d, J ) 14.0 Hz, 1H), 6.23
0
(
1
2
d, J ) 11.6 Hz, 1H), 6.01 (s, 1H), 5.02 (dt, J ) 7.3, 14.0 Hz,
H), 3.62 (s, 3H), 2.61 (m, 1H), 2.18 (t, J ) 7.3 Hz, 2H), 1.97 (m,
3
2
2
H), 1.90 (s, 3H), 1.78 (s, 3H), 1.72 (m, 1H), 1.55 (m, 1H), 1.49
through a short pad of silica gel. The filtrate was concentrated
(
m, 2H), 1.31 (m, 2H), 1.21 (d, J ) 7.3 Hz, 3H), 0.90 (t, J ) 7.0
in vacuo to afford aldehyde 12b (704 mg, 98% yield) as a colorless
oil. The aldehyde was sufficiently pure and used immediately
13
Hz, 3H); C NMR (75 MHz, CD
3
OD, 49.15 ppm) δ 199.1, 175.2,
73.2, 165.0, 156.9, 151.6, 138.4, 135.2, 126.1, 122.3, 110.7, 102.6,
01.9, 52.8, 41.6, 39.2, 35.9, 31.3, 29.7, 23.6, 18.6, 17.4, 14.4,
1
1
1
1
without further purification: H NMR (400 MHz, CDCl
(
3
) δ 9.94
d, J ) 8.1 Hz, 1H), 5.82 (d, J ) 8.1 Hz, 1H), 2.16 (t, J ) 7.6 Hz,
H), 2.11 (s, 3H), 1.44 (m, 2H), 1.28 (m, 2H), 0.87 (t, J ) 7.3 Hz,
-
1
1.9; IR (neat) νmax: 3315, 2931, 1734, 1681 cm ; UV (metha-
2
3
2
nol): λmax (log ꢀ) ) 213, 298 nm; CIHRMS (NH
C
3
gas) calcd for
(M + H ) 432.2386, found: 432.2377.
13
H); C NMR (75 MHz, CDCl
2.3, 17.4, 13.8; IR (neat) νmax: 2959, 1676 cm
-[(E,E)-2,5-Dim et h yl-2,4-n on a d ien oyl]-4-h yd r oxy-6-[5-
m eth oxyca r bon yl)-1-m eth yl-4-p en ten yl]-2-p yr on e (13b).
Compound 8 (178 mg, 0.578 mmol) was dissolved in CH Cl (10
mL) and stirred at -78 °C under argon atmosphere. To this
was added freshly distilled TiCl (254 µL, 2.31 mmol), and the
3
) δ 191.3, 164.5, 127.2, 40.3, 29.2,
+
24
H
34
N
1
O
6
-
1
.
3
Ack n ow led gm en t. This work has been financially
(
supported by Scriptgen Pharmaceuticals, Inc. We are
grateful to Mr. Brian Mendelsohn for technical as-
sistance.
2
2
4

2
406 J . Org. Chem., Vol. 63, No. 7, 1998
Notes
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
is contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
cedures and characterization for compounds 1a , 2a , 11a , 12a ,
1
13
and 13a reported herein; copies of H and C NMR spectra
for all reaction products. In addition, details for microdilution
minimal inhibitory concentration (MIC) and minimum bacte-
ricidal concentration (MBC) assays (39 pages). This material
J O9721610