The chemistry of pseudomonic acid. 18. Heterocyclic replacement of the α,β-unsaturated ester: Synthesis, molecular modeling, and antibacterial activity

Article abstract of DOI:10.1021/jm960738k

The electronic requirements around the C1-C3 region of pseudomonic acid analogues were investigated. Synthetic routes were developed to access a range of compounds where the α,β-unsaturated ester moiety had been replaced by a 5-membered ring heterocycle.

Full text of DOI:10.1021/jm960738k

J . Med. Chem. 1997, 40, 2563-2570
2563
Th e Ch em istr y of P seu d om on ic Acid .† 18. Heter ocyclic Rep la cem en t of th e
r,â-Un sa tu r a ted Ester : Syn th esis, Molecu la r Mod elin g, a n d An tiba cter ia l
Activity1
Pamela Brown,* Desmond J . Best, Nigel J . P. Broom, Robert Cassels, Peter J . O’Hanlon, Timothy J . Mitchell,
Neal F. Osborne, and J ennifer M. Wilson
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, U.K.
Received October 22, 1996X
The electronic requirements around the C1-C3 region of pseudomonic acid analogues were
investigated. Synthetic routes were developed to access a range of compounds where the R,â-
unsaturated ester moiety had been replaced by a 5-membered ring heterocycle. The inhibition
of isoleucyl tRNA synthetase from Staphylococcus aureus NCTC 6571 was determined as was
the minimum inhibitory concentration (MIC) of the test compounds against that organism.
Compounds possessing a region of electrostatic potential corresponding to that of the carbonyl
group in the R,â-unsaturated ester, and a low-energy unoccupied molecular orbital in the region
corresponding to the double bond, were found to have IC50 values of 0.7-5.3 ng mL-1. However
the MIC values of these compounds were in the range 2.0-8.0 µg mL-1, reflecting their poorer
penetration into the bacterial cell.
In tr od u ction
Pseudomonic acid (1a ), marketed by SmithKline
Beecham as the topical antibacterial agent, Bactroban,
is potent against Gram-positive bacteria and some
Gram-negative organisms such as Haemophilus and
Pasteurella.2 The mechanism of action is the inhibition
of bacterial isoleucyl tRNA synthetase,3a,b for which
pseudomonic acid shows a much greater affinity than
for the corresponding mammalian enzyme.3b However,
in vivo, the ester function of 1a is hydrolyzed to the
antibacterially inactive monic acid 1b. In our search
for metabolically stable analogues, we sought to replace
the R,â-unsaturated ester moiety (C1-C3) with a range
of heterocycles.
In recent years, the structure of several bacterial
tRNA synthetases has been determined.4 However, the
determination of the structure of isoleucyl tRNA syn-
thetase (IRS) has yet to be accomplished, and the precise
interactions of pseudomonic acid in the active site are
not yet known. We sought to build a model of some of
the requirements for activity based on the structure-
activity relationships of various pseudomonic acid ana-
logues.
In this paper we report the synthesis of novel
pseudomonic acid C1-C3 heterocycles, designed to test
a structure-activity hypothesis developed by molecular
modeling of active and inactive derivatives. The bio-
logical activity of the new compounds is reported in
terms of inhibition of the IRS enzyme and whole-cell
antibacterial activity.
Ba ck gr ou n d
We have previously shown that the ester moiety in
pseudomonic acid can be replaced by other esters, for
example ethyl monate 1c,2 and by aryl ketones 21,5 or
heterocycles such as oxazole 3a 6 or oxadiazole 3b,7 while
retaining antibacterial activity. The 2,3-double bond is
solely with the E-geometry. Inactive compounds include
the 2-phenyloxazole 3c7 and the Z-isomers (e.g. 4).6
Reduction of the double bond leads to reduced activity.
The knowledge that only the E-isomers are active, led
an important feature, the antibacterial activity residing
† The approved generic name for pseudomonic acid is mupirocin.
X Abstract published in Advance ACS Abstracts, J uly 1, 1997.
S0022-2623(96)00738-8 CCC: $14.00 © 1997 American Chemical Society

2564 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Brown et al.
Ta ble 1
F igu r e 1.
us toward replacing the C1-C3 region with a cyclic
system. Previous examples of this, the butenolide 5 and
furan 6 showed no activity.5 However, these lacked the
alkyl or aryl substituent incorporated into the other
ester bioisosteric replacements, and as such are not
directly comparable.
Molecu la r Mod elin g
To build up a hypothesis of the requirements for
activity, electronic features of the previously reported
esters and replacements (1, 2, 3a -c) were examined.
Of these, the electrostatic potential appeared to have
the most significance. Using AM-1 potential-derived
charges, electrostatic potential maps around the active
ester bioisosteres (1c, 2, 3a ,b) were calculated. The
intersection of these maps, contoured at -10 kcal mol-1
led to a specific volume common to all active compounds,
shown in Figure 1a superimposed on the ester func-
tionality. The electrostatic potential map of the inactive
3c at this energy level gave no intersection with this
volume.
To test the hypothesis that the electrostatic potential
shown in Figure 1a was a requirement for activity, the
electrostatic potential of a number of 5-membered ring
heterocycles (7b, 8, 14-16, 21, 25, and 26) was calcu-
lated, and the intersection of the individual maps with
the common volume (1a ) was determined. The presence
or absence of an intersection with this volume is given
in Table 1. For example, the isoxazole corresponding
to 7b gave an electrostatic potential map at -10 kcal
mol-1 shown in Figure 1b, the intersection of this with
the common volume 1a being equal to 1a itself.
A second feature believed to be associated with
activity in pseudomonic acid analogues is the C2-C3
double bond, which can be represented by the presence
of a low-energy unoccupied molecular orbital (Figure
2a). To test the hypothesis that this was also a
requirement for activity, the 5-membered ring hetero-
cycles (7b, 8, 14-16, 21, 25, and 26) were examined.
For example the isoxazole corresponding to 8b possesses
a low-energy molecular orbital associated with both
atoms corresponding to the double bond (Figure 2b)
whereas the isoxazole 8 does not (Figure 2c). The
presence or absence of these electronic features of the
5-membered ring heterocycles are given in Table 1.
F igu r e 2.
Ch em istr y
The first route examined for the synthesis of the
3-phenylisoxazole 7b was a 1,3-dipolar cycloaddition of
benzonitrile oxide to the corresponding acetylene (Scheme
1). The readily available ketone 9a 4 was converted via
the kinetic enolate to the enol phosphate 10. However,
attempted â-elimination with base gave no reaction at
-70 °C, while at higher temperatures opening of the
tetrahydropyran ring occurred. Enol acetates have been
shown8 to react with nitrile oxides to form isoxazolines
which eliminate acetic acid on heating to form the
corresponding isoxazoles. We envisaged that a similar
The 5-membered ring heterocycles were then synthe-
sised as described below and tested as inhibitors of
isoleucyl tRNA synthetase and as antibacterial agents.

Chemistry of Pseudomonic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2565
Sch em e 1
Sch em e 2
Sch em e 3
reaction might occur with the enol phosphate 10 en-
abling formation of the isoxazole directly. Indeed,
reaction of benzonitrile oxide with the enol phosphate
10 in refluxing benzene gave a single regioisomer of
isoxazole, albeit in low yield (9%). It is thought that
this low yield is probably due to extensive dimerization
of the nitrile oxide and also to the instability of the
trimethylsilyl (TMS) protecting groups if the reaction
mixture becomes slightly acidic. After cycloaddition/
elimination, the TMS protecting groups were removed
with mild acid. The regiochemistry of cycloaddition was
determined by NOE on the corresponding 4-chloro
analogue 7c, where an NOE effect was noted between
the aromatic ortho H and the isoxazole proton.
The synthesis of the other isoxazole regioisomer 8 was
envisaged via the corresponding diketone 119 (Scheme
2). Reaction of the diketone 119 with hydroxylamine
in ethanol gave the isoxazole 8 in low yield. This was
assigned the structure by comparison with the previ-
ously assigned 3-phenylisoxazole. Also obtained were
the hydroxyisoxazolines 12 and 13 in 36% and 7% yield,
respectively. The outcome of the reaction of hydroxyl-
amine with a diketone is known to depend on many
features,10 and the reaction was not optimized further.
While 12 could be converted to the isoxazole 8 on acid
treatment, this was accompanied by some acid-catalyzed
rearrangement of the pseudomonic acid nucleus.11
For the synthesis of the oxadiazoles 14 and 15, and
the 2-substituted 5-phenyloxazole 16, an activated form
of the “C3-acid” was required. Although we were unable
to isolate this acid directly, a direct synthesis of the
mixed phosphonic anhydride was developed by ozonoly-
sis of the enol phosphate 1012 (Scheme 3).
and 20, respectively. Cyclization of the acylated inter-
mediate under standard conditions,6a,b,13 followed by
mild acid deprotection, gave the required heterocycles
(14-16).
Of the many reported oxazole syntheses,14 the reac-
tion of a carbenoid species with a nitrile appeared an
amenable route to the 2-phenyloxazole (21). The ketone
9a was converted via the kinetic enolate to the diazo
ketone 22 (Scheme 4). Reaction with benzonitrile in the
presence of rhodium(II) acetate gave the oxazole 21 in
moderate yield.
The formation of 5-membered rings linked via a
heteroatom required further manipulation of the
pseudomonic acid skeleton (Scheme 5). Enolisation of
the tris(triethylsilyl) ketone (9b)5 under thermodynamic
conditions, followed by reaction with triisopropylsilyl
triflate gave the triisopropylsilyl enol ether (23), ac-
companied by the kinetic product, together with the
products of epimerisation at C5. However, 23 was
The TMS-protected mixed phosphonic anhydride 17
was not isolated but reacted with the required ami-
doxime, acyl hydrazide, or amino ketone in the presence
of triethylamine to give the acylated derivatives 18, 19,

2566 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Brown et al.
Sch em e 4
carbonyl oxygen/ring nitrogen of previous C1 ester
replacements. It is postulated that this may be a
recognition feature by an H-bond donor in the active
site.
Exp er im en ta l Section
Infrared spectra were determined either in dichloromethane
on a Perkin-Elmer PE 983 spectrophotometer or in KBr on a
Philips PU 9706 spectrophotometer. NMR spectra were
recorded on a Bruker AC-250F spectrometer. Chemical shifts
are expressed in ppm (δ) relative to internal tetramethylsilane.
Mass spectra were obtained on a VG ZAB mass spectrometer.
Merck Kieselgel 60 (<230 mesh ASTM) was used for column
chromatography. Tetrahydrofuran (THF) was dried by distil-
lation from calcium hydride followed by distillation from
sodium benzophenone ketyl.
The purity of all target compounds was confirmed by HPLC,
performed on a Waters Associates instrument using a C18
µ-Bondapak reverse-phase column with pH 4.5 0.05 M am-
monium acetate buffer-methanol solutions as eluant. Detec-
tion was by UV at 240 nm and at the λmax of the test compound.
All target compounds were determined as >95% pure when
examined by this method.
chromatographically stable, enabling its separation from
the byproducts. Ozonolysis of 23 followed by reductive
workup with sodium borohydride gave the alcohol 24.
This was then reacted with 5-phenyltetrazole under
Mitsonobu conditions,15 followed by fluoride deprotec-
tion to give the desired tetrazole 25.
For the synthesis of the thiazole 26, the ketone 9a
was converted to the bromo ketone 27 followed by
reaction with thiobenzamide (Scheme 6). Dehydration
of the hydroxythiazoline 28 via the mesylate gave the
desired product in 29% yield.
6,7,13-O-Tr is(Tr im eth ylsilyl)-1,15-bisn or m on -3-yl Di-
eth yl p h osp h a te (10). A solution of [5(S)-[(2(S),3(S)-epoxy-
5(S)-[(trimethylsilyl)oxy]-4-methylhexyl]-3(R),4(R)-bis[(trime-
thylsilyl)oxy]tetrahydropyran-2(S)-yl]acetone (9a )4 (8.24 g, 16
mmol) in dry THF (40 mL) was added dropwise over a period
of 30 min to a solution of lithium diisopropylamide (17.6 mmol)
in dry THF (40 mL) at -78 °C under an argon atmosphere.
After a further 30 min at -78 °C, the solution was treated
with diethyl chlorophosphate (2.42 mL, 17.6 mmol). The
mixture was allowed to warm to room temperature over 30
min and stirred at this temperature for a further 30 min.
Saturated ammonium chloride (20 mL) was added and the
mixture extracted with ethyl acetate (3 × 50 mL). The organic
phases were combined, washed with brine, and dried over
anhydrous MgSO4. The solvent was evaporated under reduced
pressure and the crude product chromatographed on Kieselgel
60, eluting with 0-50% ethyl acetate in hexane. The product
10 was obtained as a colorless oil (6.51 g, 63%): IR (CH2Cl2)
3671, 1655, 1449, 1372, 1272, and 1249 cm-1; 1H NMR (CDCl3)
δ inter alia 0.91 (3H, d, J ) 7.1 Hz, 17-H3), 1.20 (3H, d, J )
6.4 Hz, 14-H3), 1.36 (6H, dt, J ) 0.73, 6.9 Hz, CH3), 4.16 (4H,
dq, appears as five lines, J ) 7.3 Hz, CH2), and 4.77 (2H, d, J
) 74.5 Hz, dCH2); MS (EI) m/ z 654 (MH+, 1); HRMS calcd
for C28H59O9PSi3 654.3205, found 654.3218.
5-[[5(S)-[2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
3-p h en ylisoxa zole (7b). The enol phosphate 10 (1.3g, 2
mmol) was dissolved in dry benzene (10 mL) and treated with
benzohydroximinoyl chloride16 (343 mg, 2.2 mmol) and tri-
ethylamine (0.3 mL). The mixture was heated to reflux under
an argon atmosphere for 24 h and then cooled to room
temperature and the solvent evaporated under reduced pres-
sure. The crude product was chromatographed on Kieselgel
60 eluting with 0-30% ethyl acetate in hexane to give the
protected isoxazole as a colorless oil (110 mg, 9%): IR (CH2-
Cl2) 2950, 2890, 1602, and 1580 cm -1; 1H NMR (CDCl3) δ 0.10
(9H, s, SiCH3), 0.17 (9H, s, SiCH3), 0.18 (9H, s, SiCH3), 0.90
(3H, d, J ) 7.1 Hz, 17-H3), 1.20 (3H, d, J ) 6.3 Hz, 14-H3),
2.67-2.73 (2H, m, 10-H and 11-H), 2.98 (1H, dd, J ) 9.6, 15.7
Hz, 4-H), 3.15 (1H, dd, J ) 2.5, 15.7 Hz, 4′-H), 6.45 (1H, s,
isoxazole-H), 7.42-7.45 (3H, m, Ar-H), and 7.79-7.84 (2H, m,
Ar-H); MS (EI, m/ z) 625 (M+, 1), 117 (100).
Resu lts a n d Discu ssion
Table 1 shows the heterocycles synthesized, together
with pseudomonic acid and ethyl monate for compari-
son, the presence or absence of the electrostatic potential
and low-energy unoccupied molecular orbitals, the
inhibition of isoleucyl tRNA synthetase from Staphylo-
coccus aureus NCTC 6571, and the minimum inhibitory
concentration of the test compounds against that organ-
ism.
The most potent compounds (14, 15, and 21) have a
level of inhibition in the region of that seen with
pseudomonic acid and ethyl monate and a reasonable
level of whole-cell activity. All of these compounds
possess both the electrostatic potential and unoccupied
molecular orbital features. The isoxazole 7b, tetrazole
25, and thiazole 26 are slightly weaker inhibitors. Of
these, the thiazole 26 lacks the unoccupied molecular
orbital feature. The isoxazole 8 which also lacks the
unoccupied molecular orbital feature has substantially
reduced inhibition of IRS. The oxazole 16 lacking the
electrostatic potential feature, does not inhibit the
isolated enzyme, confirming that this is an important
feature for recognition.
The electrostatic potential may correspond to a re-
quirement for interaction with a hydrogen-bond donor
in the active site. The role of the low-energy molecular
orbital is less clear, but may indicate a site for charge-
transfer interaction within the active site.
While the level of inhibition approaches that seen
with pseudomonic acid (and is equipotent in the case of
the oxadiazole 14), none of the compounds synthesized
have whole-cell activity approaching that of pseudomon-
ic acid. It is believed that this is due to poorer
penetration of these lipophilic compounds into the
bacterial cell.
This material (100 mg, 0.16 mmol) was dissolved in a
mixture of THF (3 mL) and water (0.75 mL) and treated with
5 M HCl (2 drops). After 5 min, the reaction was quenched
with saturated aqueous NaHCO3 solution (5 mL), and the
mixture was extracted with ethyl acetate. The organic phases
were combined and dried over anhydrous MgSO4, the solvent
was evaporated, and the crude product was chromatographed
on Kieselgel 60 eluting with 5% methanol in dichloromethane
to give 7b as a white foam (56 mg, 87%): IR (KBr) 3465, 3318,
1610, and1580 cm-1; UV (EtOH) λmax 240.5 nm (ꢀm 15079); 1H
It is concluded that bioisosteric replacement of the
R,â-unsaturated ester by aryl heterocycles has been
successfully achieved by 5-membered ring heterocycles
possessing electrostatic potential in the region of the

Chemistry of Pseudomonic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2567
Sch em e 5
4(S)-methylhexyl)tetrahydropyran (11)9 (100 mg, 0.25 mmol)
was dissolved in ethanol (1 mL) and treated with triethylamine
(0.038 mL, 0.27 mmol) and hydroxylamine hydrochloride (19
mg, 0.27 mmol). The reaction mixture was stirred at room
temperature for 3 days. Further triethylamine (0.27 mmol)
and hydroxylamine hydrochloride (0.27 mmol) were added, and
the mixture was stirred for a further 6 h. The solution was
diluted with ethyl acetate and washed with water and brine.
The organic phase was dried over anhydrous MgSO4 and
evaporated under reduced pressure. The crude product was
purified by chromatography on Keiselgel 60, eluting with
0-10% methanol in dichloromethane, to give 8 as a colorless
oil (4 mg, 4%): IR (CH2Cl2) 3602, 3430, 1643, 1613, 1574, and
Sch em e 6
1
1451 cm-1; UV (MeOH) λmax 259 nm; H NMR (CDCl3) δ 0.92
(3H, d, J ) 7.0 Hz, 17-H3), 1.21 (3H, d, J ) 6.3 Hz, 14-H3),
2.65 (1H, dd, J ) 2.0, 8.0 Hz, 11-H), 2.79 (1H, dd, J ) 2.0, 5.7
Hz, 10-H), 3.00 (1H, dd, J ) 6.6, 15.1 Hz, 4-H), 3.15 (1H, dd,
J ) 4.2, 15.1 Hz, 4′-H), 6.55 (1H, s, isoxazole-H), 7.42-7.48
(3H, m, Ar-H), and 7.76-7.80 (2H, m, Ar-H); MS (EI) m/ z 403
(M+, 2), 188 (45), 159 (100). HRMS calcd for C22H29NO6
403.1995, found 403.2017.
NMR (CDCl3) δ 0.92 (3H, d, J ) 7.0 Hz, 17-H3), 1.21 (3H, d, J
) 6.3 Hz, 14-H3), 2.69 (1H, dd, J ) 2.0, 7.8 Hz, 11-H), 2.80
(1H, dt, J ) 2.0, 5.5 Hz, 10-H), 3.01 (1H, dd, J ) 8.0, 15.6 Hz,
4-H), 3.29 (1H, dd, J ) 3.1, 15.6 Hz, 4′-H), 6.47 (1H, s,
isoxazole-H), 7.43-7.47 (3H, m, Ar-H), 7.77-7.83 (2H, m, Ar-
H);13C NMR (CD3OD) δ 12.1 (C-17), 20.2 (C-14), 30.4 and 32.9
(C-4) and (C-9), 41.7 (C-8), 43.6 (C-12), 56.7(C-10), 61.2 (C-
11), 66.3 (C-16), 69.4 (C-6), 70.6 (C-7), 71.4 (C-13), 75.9 (C-5),
101.5 (C-2), 127.6 (2 × Ar), 129.9 (2 × Ar), 130.4 (Ar), 130.9
(Ar), 163.8 (CdN), and 173.2 (C-O); MS m/ z 403 (M+, 1), 188
(40), and 159 (100); found M+ 403.2002, C22H29NO6 requires
M 403.1995.
5-[[5(S)-[2(S),3(S)-Epoxy-5(S)-[(tr im eth ylsilyl)oxy]-4(S)-
m eth ylh exyl]-3(R),4(R)-bis-[(tr im eth ylsilyl)oxy]tetr a h y-
d r op yr a n -2(S)-yl]m eth yl}ben za m id e Oxim e (18). 6,7,13-
O-Tris(trimethylsilyl)-1,15-bisnormon-3-yl diethyl phosphate
(10) (300 mg 0.45 mmol) was dissolved in dry dichloromethane
(30 mL), the mixture was cooled to -70 °C, and ozone bubbled
through until a blue color was observed. Argon was bubbled
through to remove excess ozone, and the solution was treated
with dimethyl sulfide (0.36 mL, 0.5 mmol) and warmed to -30
°C. After 30 min the reaction mixture was treated with
triethylamine (0.068 mL, 0.5 mmol) followed by benzami-
doxime (68 mg, 0.5 mmol) and allowed to warm to room
temperature. After 20 min the reaction mixture was washed
with water and brine, dried over anhydrous MgSO4, and
evaporated under reduced pressure. The crude product was
chromatographed on Kieselgel 60, eluting with 10-25% ethyl
acetate in hexane, to give 18 as a colorless oil (222 mg, 76%):
IR (CH2Cl2) 3513, 3414, 3356, 1750, and 1636 cm -1; 1H NMR
(CDCl3) δ 0.91 (3H, d, J ) 7.0 Hz, 17-H3), 1.19 (3H, d, J ) 6.3
Hz, 14-H3), 2.58 (1H, dd, J ) 7.7,14.4 Hz, 4-H), 2.62-2.70 (2H,
m, 10-H and 11-H), 2.84 (1H, dd, J ) 4.4, 14.4 Hz, 4′-H), 5.44
(2H, br s, OH and NH), 7.38-7.50 (3H, m, Ar-H), and 7.73
(2H, d, J ) 6.7 Hz, Ar-H); MS (EI) m/ z 639 (MH+, 8); HRMS
calcd for C30H55N2O7Si3 639.3317, found 639.3317.
5-[[5(S)-[2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
3-(4-ch lor op h en yl)isoxa zole (7c). The enol phosphate 10
(654 mg, 1 mmol) was reacted with chlorobenzohydroximinoyl
chloride (266 mg,1.4 mmol) as described in the preparation of
7b. After deprotection as previously described, the isoxazole
7c was obtained as a white foam in 9% overall yield: IR (KBr)
3421, 1731, 1606, 1568, 1509, 1454, and 1430 cm-1; UV (EtOH)
1
λmax 247 nm (ꢀm 16 908); H NMR (CDCl3) δ 0.93 (3H, d, J )
7.0 Hz, 17-H3), 1.22 (3H, d, J ) 6.3 Hz, 14-H3), 2.49 (1H, dd,
J ) 2.1, 7.9 Hz, 11-H), 2.79 (1H, dt, J ) 2.1, 6.4 Hz, 10-H),
3.00 (1H, dd, J ) 8.2, 15.7 Hz, 4-H), 3.28 (1H, dd, J ) 3.1,
15.7 Hz, 4′-H), 6.43 (1H, s, isoxazole-H), 7.43 and 7.73 (4H, 2
× d, Ar-H); 13C NMR (CDCl3) δ 12.3 (C-17), 20.3 (C-14), 30.4
and 32.9 (C-4 and C-9), 41.8 (C-8), 43.7 (C-12), 56.8 (C-10),
61.2 (C-11), 66.4 (C-16), 69.4 (C-6), 70.7 (C-7), 71.5 (C-13), 75.9
(C-5), 101.5 (C-2), 129.2, (2 × Ar), 130.2 (2 × Ar), 136.9 (Ar-
Cl), 162.8 (CdN), and 173.6 (C-O); MS (EI) m/ z 438 (MH+,
10), 222 (50), 193 (95); HRMS calcd for C22H29NO6Cl 438.1683,
found 438.1689.
5-[[5(S)-[2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl)-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
3-p h en yl-1,2,4-oxa d ia zole (14). The oxime 18 (200 mg) was
dissolved in diglyme (5 mL) and heated to 120 °C for 4 h. The
solution was cooled to room temperature, diluted with ethyl
acetate (20 mL), and washed with water (10 mL) and brine
(10 mL). The organic phase was dried over anhydrous MgSO4,
the solvent evaporated, and the crude product chromato-
3-[[5S-(2(S),3(S)-Epoxy-5(S)-h ydr oxy-4(S)-m eth ylh exyl)-
3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-5-
p h en ylisoxa zole (8). 3(R),4(R)-Dihydroxy-2(S)-[4-(phenyl-
2,4-dioxo-4-phenylbut-1-yl]-5(S)-(2(S),3(S)-epoxy-5(S)-hydroxy-

2568 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Brown et al.
graphed on Kieselgel 60 eluting with 10% ethyl acetate in
hexane to give the protected oxadiazole as a colorless oil (89
(1H, dt, J ) 2.2, 5.5 Hz, 10-H), 3.12 (1H, d, J ) 15.5, 8.6 Hz,
4-H), 3.41 (1H, dd, J ) 15.5, 3.6 Hz, 4-H), 3.77 (1H, dq, J )
6.3, 7.4 Hz, 13-H), 4.04 (1H, dt, J ) 3.6,8.8 Hz, 5-H), 7.55-
7.62 (3H, m, Ar-H), and 8.02-8.06 (2H, m, Ar-H); MS (EI) m/ z
404 (M+, 5), 160 (100); HRMS calcd for C21H28N2O6 404.1947,
found 404.1956.
1
mg, 45%): IR (CH2Cl2) 1595, 1571, and 1446 cm-1; H NMR
(CDCl3) δ 0.90 (3H, d, J ) 7.1 Hz, 17-H3), 1.18 (3H, d, J ) 6.3
Hz, 14-H3), 2.65-2.68 (2H, m, 10-H and 11-H), 2.99 (1H, dd,
J ) 8.9, 15.1 Hz, 4-H), 3.31 (1H, dd, J ) 8.9, 15.1 Hz, 4′-H),
4.04 (1H, dt, J ) 3.4, 9.1 Hz, 5-H), 7.45-7.48 (3H, m, Ar-H),
and 8.07-8.10 (2H, m, Ar-H); MS (EI) m/ z 621 (MH+, 1), 117
(100).
2-[5(S)-[2(S),3(S)-Ep oxy-5(S)-[(tr im eth ylsilyl)oxy]-4(S)-
m eth ylh exyl]-3(R),4(R)-bis[(tr im eth ylsilyl)oxy]tetr a h y-
dr opyr an -2(S)-yl]-N-(2-ph en yl-2-oxoeth yl)acetam ide (20).
6,7,13-O-Tris(Trimethylsilyl)-1,15-bisnormon-3-yl diethyl phos-
phate (10) (327 mg 0.5 mmol) was dissolved in dry dichlo-
romethane (30 mL) and cooled to -70 °C, and ozone was
bubbled through until a blue color was observed. Argon was
bubbled through to remove excess ozone, and the solution was
treated with dimethyl sulfide (0.40 mL, 0.55 mmol) and
warmed to -30 °C. After 30 min the reaction mixture was
treated with triethylamine (0.15 mL, 1.1 mmol) followed by
2-aminoacetophenone hydrochloride (102 mg, 0.6 mmol) and
allowed to warm to room temperature. After 20 min the
reaction mixture was washed with water and brine, dried over
anhydrous MgSO4, and evaporated under reduced pressure.
The crude product was chromatographed on Kieselgel 60,
eluting with 25% ethyl acetate in hexane, to give 20 as a
colorless oil (241 mg, 76): ΙR (CH2Cl2) 3416, 3368, 1732, and
1696 cm -1;1H NMR (CDCl3) δ 0.93 (3H, d, J ) 7.1 Hz, 17-H3),
1.21 (3H, d, J ) 6.2 Hz, 14-H3), 2.34 (1H, dd, J ) 9.8, 15.6 Hz,
4-H), 2.66-2.75 (23H, m, 10-H, 11-H, and 4′-H),4.78 (2H, dd,
CH2NH), 7.48-7.56 (3H, m, Ar-H), and 7.98-8.02 (2H, m, Ar-
H); MS (EI) m/ z 639 (MH+, 8); HRMS calcd for C30H55N2O7-
Si3 639.3317, found 639.3317.
This material (67 mg) was dissolved in a mixture of THF (3
mL) and water (0.75 mL) and treated with 5 M HCl (2 drops).
After 5 min, the reaction was quenched with saturated
aqueous NaHCO3 solution (5 mL), and the mixture was
extracted with ethyl acetate. The organic phases were com-
bined and dried over anhydrous MgSO4, the solvent was
evaporated, and the crude product was chromatographed on
Kieselgel 60, eluting with 5% methanol in dichloromethane,
to give 14 as a white solid (42 mg, 95%): IR (KBr) 3426, 1625,
1596, and 1447 cm-1; UV (EtOH) λmax 238 nm (ꢀm 15 731); 1H
NMR (CDCl3) δ 0.94 (3H, d, J ) 7.0 Hz, 17-H3), 1.21 (3H, d, J
) 6.3 Hz, 14-H3), 2.68 (1H, dd, J ) 2.2, 8.0 Hz, 11-H), 2.80
(1H, dt, J ) 2.2, 5.6 Hz, 10-H), 3.24 (1H, dd, J ) 7.0, 15.5 Hz,
4-H), 3.39 (1H, dd J ) 4.8, 15.5 Hz, 4′-H), 7.44 -7.50 (3H, m,
Ar-H), and 8.05-8.09 (2H, m, Ar-H); 13C NMR (CD3OD) δ 10.7
(C-17), 16.7 (C-14), 29.2 and 31.3 (C-4) and (C-9), 40.2 (C-8),
42.1 (C-12), 55.2 (C-10), 59.3 (C-11), 64.9 (C-16), 67.8 (C-6),
69.1 (C-7), 69.8 (C-13), 74.0 (C-5), 126.5 (Ar), 126.7 (2xAr),
128.4 (2 × Ar), 130.7 (Ar), 167.8 (C)N), and 178.4 (C-O); MS
(EI) m/ z 405 (MH+, 90), 227 (100), 119 (100); HRMS calcd for
C21H29N2O6 405.2026, found 405.2029.
N′-[2-[5(S)-[2(S),3(S)-E p oxy-5S-[(t r im et h ylsilyl oxy]-
4(S)-m eth ylh exyl]-3(R),4(R)-bis[(tr im eth ylsilyl)oxy]tet-
r a h yd r op yr a n -2(S)-yl]a cetylben zoic h yd r a zid e (19). 6,7,
13-O-Tristrimethylsilyl)-1,15-bisnormon-3-yl diethyl phosphate
(10) (327 mg, 0.5 mmol) was dissolved in dry dichloromethane
(30 mL), and cooled to -70 °C, and ozone was bubbled through
until a blue color was observed. Argon was bubbled through
to remove excess ozone, and the solution was treated with
dimethyl sulfide (0.040 mL, 0.55 mmol) and warmed to -30
°C. After 30 min the reaction mixture was treated with
triethylamine (0.075 mL, 0.56 mmol) followed by benzoylhy-
drazine (0.150 g, 0.56 mmol) and allowed to warm to room
temperature. After 1 h the reaction mixture was washed with
water and brine, dried over anhydrous MgSO4, and evaporated
under reduced pressure. The crude product was chromato-
graphed on Kieselgel 60, eluting with 25% ethyl acetate in
hexane, to give 19 as a colorless oil (220 mg, 69%): IR (CH2-
Cl2) 3393, 3303, 1716, 1678, and 1637 cm-1; 1H NMR (CDCl3)
δ 0.92 (3H, d, J ) 7.1 Hz, 17-H3), 1.20 (3H, d, J ) 6.4 Hz,
14-H3), 2.40 (1H, dd, J ) 9.4, 15.8 Hz, 4-H), 2.68-2.88 (3H,
m, 10-H, 11-H and 4′-H), 7.42-7.57 (3H, m, Ar-H), 7.80-7.84
(2H, m, Ar-H), 8.88 (1H, d, J ) 6.1 Hz, N-H), and 9.49 (1-H,
d, J ) 6.1 Hz, N-H); MS (EI) m/ z 639 (MH+, 0.5); HRMS calcd
for C30H55N2O7Si3 639.3317, found 639.3317.
5-[[5(S)-[2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
2-p h en yl-1,3,4-oxa d ia zole (15). The acyl hydrazide 19 (150
mg, 0.23 mmol) was dissolved in dry acetonitrile (1.2 mL) and
treated with triethylamine (0.1 mL), tetrachloromethane (0.14
mL), and triphenylphosphine (0.18 g). The reaction mixture
was stirred at room temperature for 30 min, diluted with ethyl
acetate (20 mL), and washed with saturated aqueous NaHCO3
(15 mL), water (10 mL), and brine (10 mL). The solution was
dried over anhydrous MgSO4 and evaporated under reduced
pressure. The crude product was dissolved in a mixture of
THF (6 mL) and water (1.5 mL) and treated with 5 M HCl (4
drops). After 5 min, the reaction was quenched with saturated
aqueous NaHCO3 solution (5 mL), and the mixture was
extracted with ethyl acetate. The organic phases were com-
bined and dried over anhydrous MgSO4, the solvent was
evaporated, and the crude product was chromatographed on
Kieselgel 60 eluting with 5% methanol in dichloromethane to
give 15 as a white solid (63 mg, 68%): IR (KBr) 3458, 3346,
1571, and 1558 cm-1; UV (EtOH) λmax 251 nm (ꢀm 18 058); 1H
NMR (CD3OD) δ 0.95 (3H, d, J ) 7.1 Hz, 17-H3), 1.19 (3H, d,
J ) 6.3 Hz, 14-H3), 2.70 (1H, dd, J ) 2.3, 7.6 Hz, 11-H), 2.80
2-[[5(S)-[2(S),3(S)-Epoxy-5(S)-h ydr oxy-4S-m eth ylh exyl]-
3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-5-
p h en yloxa zole (16). The keto amide 20 (150 mg, 0.23 mmol)
was dissolved in dry dichloromethane (3 mL) and treated with
pyridine (76 µL, 4 equiv) and trichloroacetyl chloride (52 µL,
2 equiv). The reaction mixture was stirred at room temper-
ature for 30 min, diluted with dichloromethane (10 mL),
washed with NaHCO3, 10% citric acid, water, and brine, dried
(MgSO4), and evaporated. The crude product was chromato-
graphed on Kieselgel 60 eluting with 10-20% ethyl acetate
in hexane to give the protected oxazole (46 mg, 31%). This
was deprotected under the conditions described for compound
18 to give the oxazole 16 as a white amorphous solid (16 mg,
55%): IR (KBr) 3464, 1760, 1629, 1558, and 1450 cm-1; UV
(EtOH) λmax 273 (ꢀm 17 566), 266 nm (17 782); 1H NMR (CDCl3)
δ 0.95 (3H, d, J ) 7.0 Hz, 17-H3), 1.21 (3H, d, J ) 6.3 Hz,
14-H3), 2.69 (1H, dd, J ) 2.2, 8.0 Hz, 11-H), 2.79 (1H, dt, J )
2.2, 5.6 Hz, 10-H), 3.22 (2H, d, J ) 6.0 Hz, 4-H2),7.25 (1H,s,
oxazole-H), 7.31-7.46 (3H, m, Ar-H), and 7.51-7.64 (2H, m,
Ar-H); MS (EI) m/ z 403 (M+, 2); HRMS calcd for C22H29NO6
403.1995, found 403.2002.
5-[[5(S)-(2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
2-p h en yloxa zole (21). A solution of 6,7,13-O-tris-(trimeth-
ylsilyl)monone (9a ) (518 mg, 1 mmol) in dry THF (5 mL) was
added dropwise to a solution of lithium bis(trimethylsilyl)-
amide (1 mmol) in dry THF (3 mL) at -78 °C under an argon
atmosphere. After 30 min at -78 °C the solution was treated
with p-toluenesulfonyl azide (1.2 mmol) in dry THF (2 mL).
After 30 min, the mixture was quenched with saturated
ammonium chloride, allowed to warm to room temperature,
and extracted with ethyl acetate. The organic phases were
combined, washed with brine, and dried over anhydrous
MgSO4. The solvent was evaporated under reduced pressure
and the crude product chromatographed on Kieselgel 60,
eluting with 10-15% ethyl acetate in hexane, to give the diazo
ketone 22 as a colorless oil (107 mg, 20%): IR (CH2Cl2) 3417,
2959, 2898, 2107, 1640, and 1452 cm-1;1H NMR (CDCl3) δ 0.91
(3H, d, J ) 6.9 Hz, 17-H3), 1.20 (3H, d, J ) 6.4 Hz, 14-H3),
2.28 (1H, d, J ) 10.3, 14.6 Hz, 4-H), 2.62-2.70 (3H, m, 10-H,
11-H and 4′-H), 3.42 (1H, dd, J ) 2.4, 9.1 Hz, 6-H), 3.55 (1H,
d, J ) 11.3 Hz, 16- H), 5.40 (1H, br s, CHN2); MS (FAB,
3-NOBA-Na) m/ z 567 (MNa+, 45), 539 (MNa+ - N2, 30).
The diazo ketone 22 (74 mg 0.14 mmol) was dissolved in
benzonitrile (2 mL), tetrakis(acetato)dirhodium(II) (7 mg) was

Chemistry of Pseudomonic Acids
J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16 2569
added, and the mixture was heated to 100 °C under an argon
atmosphere for 40 min. The solution was cooled to room
temperature, diluted with ethyl acetate, and washed with
saturated aqueous NaHCO3, water, and brine. The organic
phase was dried over anhydrous MgSO4 and the solvent
evaporated under reduced pressure. The crude product was
chromatographed on Kieselgel 60 eluting with 0-10% ethyl
acetate in hexane to give a colorless oil (22 mg, 25%). This
was dissolved in a mixture of THF (1.5 mL) and water (0.4
mL) and treated with 5 M HCl (1 drop). After 5 min, the
reaction was quenched with saturated aqueous NaHCO3
solution (5 mL), and the mixture was extracted with ethyl
acetate. The organic phases were combined and dried over
anhydrous MgSO4, the solvent was evaporated, and the crude
product was chromatographed on Kieselgel 60 eluting with 5%
methanol in dichloromethane to give 21 as a white solid (11
mg, 87%): IR (KBr) 3458, 1640, 1606, 1549, 1484, and1450
matographed on Kieselgel 60 eluting with 5% ethyl acetate in
hexane to give the protected product as a colorless oil (95 mg,
78%): 1H NMR (CDCl3) δ 1.19 (3H, d, J ) 6.3 Hz, 14- Hz),
2.64-2.66 (2H, m, 10-H and 11-H), 3.54 (1H, d, J ) 11.3 Hz,
16-H), 3.69 (1H, dd, J ) 9.3, 2.2 Hz, 6-H), 4.33 (1H, dt, J )
9.4, 2.7 Hz, 5-H), 4.65 (1H, dd, J ) 9.2, 13.6 Hz, 4-H), 4.90
(1H, dd, J ) 2.8, 13.5 Hz, 4′-H), 7.45-7.53 (3H, m, Ar-H),
8.15-8.21 (2H, m, Ar-H); MS (FAB, thioglycerol) m/ z 747
(MH+, 20), 159 (100). This material (84 mg, 0.11 mmol) was
dissolved in THF (4.5 mL) and treated with a solution of tetra-
n-butylammonium fluoride (1 M solution in THF, 0.44 mL, 4
equiv). After the mixture was stirred for 1 h at room
temperature the solvent was evaporated and the residue
chromatographed on Kieselgel 60 eluting with 5% methanol
in dichloromethane. The product 25 was obtained as a white
foam (34 mg, 77%): IR (KBr) 3413, 1639, 1529, 1466, 1450
cm-1;UV (EtOH) λmax 239nm (ꢀm 16 021); 1H NMR (CD3OD) δ
0.92 (3H, d, J ) 7.1 Hz, 17-H3), 1.18 (3H, d, J ) 6.4 Hz, 14-
H3), 1.34-1.43 (1H, m, 12-H), 1.58-1.73 (2H, m, 9-H), 1.92-
1.97 (1H, m, 8-H), 2.68 (1H, dd, J ) 2.2, 7.6 Hz, 11-H), 2.97
(1H, dt, J ) 2.2, 5.6 Hz, 10-H), 3.55 (1H, d, J ) 11.5 Hz, 16-
H), 3.64 (1H, dd, J ) 3.1, 5.8 Hz, 6-H), 3.72-3.78 (1H, m, 13-
H), 3.86 (1H, dd, J ) 2.5, 11.5 Hz, 16′-H), 3.74 (1H, appears
as t, J ) 3.0 Hz, 7-H), 4.7 (1H, dt, J ) 2.8, 8.2 Hz, 5-H), 4.83
(1H, dd, J ) 5.6, 13.8 Hz, 4-H), 4.98 (1H, dd, J ) 2.8, 14.0 Hz,
4′-H), 7.43-7.53 (3H, m, Ar-H), 8.03-8.12 (2H, m, Ar-H); MS
(EI) m/z 404 (M+), 131 (100); HRMS calcd for C20H29N4O5
405.2138, found 405.2142.
1
cm-1; UV (EtOH) λmax 272 nm; H NMR (CD3OD) δ 0.94 (3H,
d, J ) 7.1 Hz, 17-H3), 1.19 (3H, d, J ) 6.4 Hz, 14-H3), 1.39
(1H,dq, J ) 7.1, 4.9 Hz,12-H), 1.93-2.01 (1H,m, 8-H), 2.68 (1H,
dd, J ) 2.1, 7.9 Hz, 11-H), 2.80 (1H, dt, J ) 2.1, 5.6 Hz, 10-H),
2.91 (1H, dd, J ) 8.4, 15.8 Hz, 4-H), 3.24 (1H, dd, J ) 2.3,
15.8 Hz, 4′-H), 3.47 (1H, dd, J ) 3.1, 9.2 Hz, 6-H), 3.61 (1H, d,
J ) 11.3 Hz, 16-H), 3.77 (1H, dq, J ) 6.4, 4.9 Hz, 13-H), 3.82-
3.95 (3H, m, 5, 16′, and 7-H), 7.00 (1H, s, oxazole-H), 7.45-
7.49 (3H, m, Ar-H), and 7.94-7.98 (2H, m, Ar-H); MS (EI) m/ z
403 (M+, 5), 159 (100). HRMS calcd for C22H29NO6 403.1995,
found 403.1994.
2-[[5(S)-(2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
5-p h en yltetr a zole (25). A solution of [5(S)-[2(S),3(S)-epoxy-
5(S)-[(triethylsilyl)oxy]-4-methylhexyl]-3(R),4(R)-bis[(tri-
ethylsilyl)oxy]tetrahydropyran-2(S)-yl]acetone (9b) (8.0g, 12.42
mmol) in dry tetrahydrofuran (20 mL) was added to a solution
of lithium diisopropylamide (10 mmol) in dry tetrahydrofuran
(20 mL) at -70 °C under an argon atmosphere. The cooling
bath was removed and the mixture warmed to room temper-
ature over a period of 15 min, and stirred for a further 30 min.
Triisopropylsilyl trifluoromethanesulfonate (2.55 mL, 10 mmol)
was then added, and the mixture stirred for an additional 1
h. The solvent was evaporated under reduced pressure, the
residue taken up in dry pentane (10 mL), and the mixture
filtered. The filtrate was evaporated under reduced pressure
and the residue chromatographed on Kieselgel 60 eluting with
2-20% ethyl acetate in hexane to give the triisopropylsilyl enol
ether 23 as a colorless oil (3.41 g, 43%), contaminated with
10% of the 5-epimer: 1H NMR (CDCl3) δ 0.91 (3H, d, J ) 7.1
Hz, 17-H3), 1.20 (3H, d, J ) 6.4 Hz, 14-H3), 1.80 (3H, s, CH3),
2.64-2.68 (2H, m, 10-H and 11-H), 3.40 (1H, dd, J ) 2.3, 8.3
Hz, 6-H), 3.50 (1H, d, J ) 11.4 Hz, 16-H), 3.81 (1H, appears
as br s, 7-H), 3.81-3.91 (1H, m, 13-H), 3.97 (1H, d, J ) 11.4
Hz, 16′-H), 4.38 (1H, d, J ) 9.2 Hz, 4-H), 4.60 (1H, appears as
t, J ) 9.1 Hz, 5-H).
4-[[5(S)-(2(S),3(S)-Ep oxy-5(S)-h yd r oxy-4(S)-m eth ylh ex-
yl]-3(R),4(R)-d ih yd r oxytetr a h yd r op yr a n -2(S)-yl]m eth yl]-
2-p h en ylth ia zole (26). A solution of 6,7,13-O-tris(trimeth-
ylsilyl)monone (9a ) (540 mg, 1.04 mmol) in dry dichloromethane
(10 mL) was cooled to 0 °C and treated with triethylamine
(0.29 mL, 2.0 equiv) followed by (trimethylsilyl)trifluo-
romethanesulfonate (0.2 mL, 1.0 equiv). The mixture was
stirred for 30 min. N-Bromosuccinimide (0.2 g, 1.1 equiv) was
added and the mixture stirred at room temperature for 30 min.
Triethylamine (0.29 mL, 2.0 equiv) and thiobenzamide (0.16
g, 1.1 equiv) were added, and the mixture was stirred for a
further 2 h. The solvent was evaporated under reduced
pressure and the residue chromatographed on Kieselgel 60,
eluting with 10-20% ethyl acetate in hexane, to give the
4-hydroxythiazoline (352 mg) as a mixture of diastereomers.
This was dissolved in dry dichloromethane (10 mL), cooled to
0 °C, and treated with triethylamine (0.15 mL, 2.0 equiv),
methanesulfonyl chloride (0.042 mL, 1.0 equiv), and 4-(di-
methylamino)pyridine (5.0 mg). The mixture was stirred at
0 °C for 1 h, the solvent evaporated under reduced pressure,
and the residue chromatographed on Kieselgel 60 eluting with
5-15% ethyl acetate in hexane to give the protected product
1
(190 mg, 29%): IR (CH2Cl2) 1519, 1459, 1376 cm-1; H NMR
(CD3OD) δ 0.91 (3H, d, J ) 7.1 Hz, 17-H3), 1.21 (3H, d, J )
6.4 Hz, 14-H3), 2.71-2.85 (3H, m, 10, 11, and 4-H), 3.22 (1H,
dd, J ) 2.8, 15.0 Hz, 4′-H), 4.05 (1H, dt, J ) 2.8, 9.1 Hz, 5-H),
7.22 (1H, s, thiazole-H), 7.41-7.48 (3H, m, Ar-H), 7.91-7.94
(2H, m, Ar-H); MS (EI) m/z 636 (M+).
The silyl enol ether 23 (1.6 g, 2.0 mL) was dissolved in dry
dichloromethane (56 mL) and methanol (32 mL) and cooled
to -70 °C and ozone passed through until a blue color was
observed. Argon was passed through to remove excess ozone
and the solution treated with sodium borohydride (92 mg, 2.0
mmol). The mixture was stirred at -70 °C for 1 h, a further
quantity of sodium borohydride (92 mg) added, and the
mixture allowed to warm to room temperature. After 1 h the
reaction mixture was evaporated, and the residue was dis-
solved in ethyl acetate (80 mL), washed with saturated
NaHCO3, water, and brine, dried over anhydrous MgSO4, and
evaporated. The residue was chromatographed on Kieselgel
60 eluting with 5-20% ethyl acetate in hexane to give 24 as
a white foam (0.335 g, 27%); IR (CH2Cl2) 3582, 1459 cm-1; 1H
NMR (CDCl3) δ 1.20 (3H, d, J ) 6.4 Hz, 14-H3), 2.00 (1H, t, J
This material (176 mg, 0.28 mmol) was deprotected under
the conditions described for 7b. After workup, the crude
product was crystallized from acetone-ether to give 26 as a
white solid (68 mg, 60%): IR (KBr) 3463, 1518, 1501, 1460,
1436 cm-1; UV (EtOH) λmax 295 nm (ꢀm 13 000); 1 H NMR (CD3-
OD) δ 0.95 (3H, d, J ) 7.1 Hz, 17-H3), 1.21 (3H, d, J ) 6.4 Hz,
14-H3), 2.71 (1H, dd, J ) 2.2, 7.6 Hz, 11-H), 2.81 (1H, dt, J )
2.2, 5.8 Hz, 10-H), 2.93 (1H, dd, J ) 8.7, 15.1 Hz, 4-H), 3.52
(1H, dd, J ) 3.1, 8.7 Hz, 16-H), 7.26 (1H, s, thiazole-H), 7.42-
7.48 (3H, m, Ar-H), 7.90-7.95 (2H, m, Ar-H); 13C NMR (CD3-
OD) δ 12.2 (C-17), 20.2 (C-14), 33.0 (C-4), 34.6 (C-9), 41.5 (C-
8), 43.7 (C-12), 56.9 (C-10), 61.3 (C-11), 66.3 (C-16), 69.9 (C-
6), 70.8 (C-13), 71.6 (C-7), 77.3 (C-5), 118.4 (thiazole C-H),
127.4, 130.1 and 131.1 (Ar-CH), 134.8 (Ar-C), 158.3 (thiazole-
C), 168.9 (thiazole-C); MS (EI) m/z 419 (M+), 204 (100); HRMS
calcd for C22H29NO5S 419.1766, found 419.1769.
+
) 6.5 Hz, OH), 2.65-2.71 (2H, m, 10-H and 11-H); MS (NH3
DCI) m/z 619 (MH+, 50), 633 (MNH4+).
5-Phenyltetrazole (35 mg, 0.24 mmol) was dissolved in dry
THF (2 mL) and treated with triphenylphosphine (63 mg, 0.24
mmol) and diethyl azodicarboxylate (38 µL, 0.24 mmol),
followed by 24 (100 mg, 0.16 mmol) in dry THF (2 mL). The
reaction mixture was stirred at room temperature under argon
for 2 h. The solvent was evaporated and the residue chro-
Electr osta tic P oten tia l Ca lcu la tion s. Model fragments,
containing the equivalents of the atoms shown in Figure 1, of
compounds 1c, 7b, 8, 14-16, 21, 25, and 26 were built using
the program Chem-X17. Models of the ketone 2 and the

2570 J ournal of Medicinal Chemistry, 1997, Vol. 40, No. 16
Brown et al.
heterocyclic compounds 3a ,c were generated from the model
of 1c. The geometries of these compounds were fully optimized
using the AM1 Hamiltonian within the AMPAC semiempirical
molecular orbital program.18 Electrostatic potential derived
charges were calculated by the method of Ferenczy et al.19
The optimized structures of the ketone 2 and the heterocy-
clic compounds 3a ,c were overlaid onto the ester 1c by a least
squares fitting of the carbonyl oxygen (or equivalent nitrogen
in the case of 3a ,c) and carbon atoms 1,2,3 and 15. Similarly,
the heterocyclic model compounds 7b, 8, 14-16, 21, 25, and
26 were overlaid onto 1c by a least squares fitting the
corresponding atoms of the 5-membered heterocyclic ring to
the carbonyl oxygen and carbon atoms 1, 2, 3, and 15 of
compound 1c.
The common electrostatic potential map of the active
compounds 1c, 2, 3a , and 3c was generated from the individual
maps of the potentials of the region around the carbonyl
oxygen (or equivalent nitrogen in the case of 3a and 3c). A
logical AND operation on these potential maps was performed
to generate the map, shown in Figure 1a , of the electrostatic
features common to these compounds. The potential maps of
compounds 1c, 7b, 8, 14-16, 21, 25, and 26 were individually
AND-ed to the common potential map. Each resultant inter-
section map gave a specific representation of the electrostatic
potential of the test molecule at -10 kcal mol-1 in the region
identified as common to the active compounds 1c, 2, 3a , and
3b.
Molecu la r Or bita l Ca lcu la tion s. Calculations were per-
formed with a STO 6-31G* basis set using AM-1-optimized
geometries, using the Spartan electronic structure program.20
The orbitals shown in Figure 2 were contoured at 0.09 e/au3.
Deter m in a tion of Min im u m In h ibitor y Con cen tr a -
tion s (MICs). Compounds were serially diluted in blood agar
base (Oxoid) containing 5% chocolated horse blood. Organisms
were grown overnight in nutrient broth media. One milliliter
spots were inoculated on to the surface of the agar plates giving
an inoculum of ca. 106 colony-forming units per spot. Plates
were incubated for 18-24 h at 37 °C. The minimum inhibitory
concentration was determined as the lowest concentration fully
inhibiting bacterial growth.
Refer en ces
(1) Part 17: Broom, N. J . P.; Cassels, R.; Cheng, H.-Y.; Elder, J . S.;
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J . M. The Chemistry of Pseudomonic Acid. 17. Dual-action C-1
Oxazole Derivatives of Pseudomonic Acid having an extended
Spectrum of Antibacterial Activity. J . Med. Chem. 1996, 39,
3596.
(2) Basker, M. J .; Comber, K. R.; Clayton, J . P.; et al. Ethyl Monate
A: a Semisynthetic Antibiotic Derived from Pseudomonic Acid
A. In Current Chemotherapy and Infectious Diseases; Nelson, J .
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ington, 1980; Vol. 1, p 471.
(3) (a) Hughes, J .; Mellows, G. Inhibition of Isoleucyl Transfer-
Ribonucleic Acid Synthetase in Escherichia Coli by Pseudomonic
Acid. Biochem. J . 1978, 176, 305-318. (b) Hughes, J .; Mellows,
G. Interaction of Pseudomonic Acid with Escherichia Coli B
Isoleucyl tRNA Synthetase. Biochem. J . 1980, 191, 209-219.
(4) Cusack, S. Eleven down and Nine to go. Nature Struct. Biol.
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(5) Klein, L. L.; Yeung, C. M.; Kurath, P.; Mao, J . C.; Fernandes,
P. B.; Lartey, P.A.; Pernet, A. G. Synthesis and Activity of
Nonhydrolysable Pseudomonic Acid Analogues. J . Med. Chem.
1989, 32, 151.
(6) (a) Walker, G.; Brown, P.; Forrest, A. K.; O’Hanlon, P. J .; Pons,
J . E. New Antibacterial Agents: Synthesis and Antibacterial
Activity of Heterocyclic Derivatives of Pseudomonic Acid. In
Recent Advances in the Chemistry of Anti-infective Agents; Royal
Society of Chemistry: London, 1993, p 106. (b) Crimmin, M. J .;
O’Hanlon, P. J .; Rogers, N. H.; Sime, F. M.; Walker, G. The
Chemistry of Pseudomonic Acid. Part 11. Dehydrative Cycliza-
tion of R-Acylamino Ketones to Oxazoles. J . Chem. Soc., Perkin
Trans. 1 1989, 2059-2063.
(7) Crimmin, M. J .; O’Hanlon, P. J .; Rogers, N. H.; Walker, G. The
Chemistry of Pseudomonic Acid. Part 10. Preparation of Het-
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2057.
(8) Mukaiyama, T.; Hoshino, T. The reaction of Primary Nitropar-
affins with Isocyanates. J . Am. Chem. Soc. 1960, 82, 5339.
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WO93/06118, 1993.
(10) Quilico, A. The Chemistry of Heterocyclic Compounds; wiley:
New York, 1962; Volume 17, 5-and 6-membered Compounds
with Nitrogen and Oxygen; p 6.
(11) Clayton, J . P.; Oliver, R. S.; Rogers, N. H.; King, T. J . The
Chemistry of Pseudomonic Acid. Part 3. The rearrangement of
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Isoleu cyl t R NA Syn t h et a se (IR S) In h ib it ion . Staph.
aureus strains were grown to late stationary phase in shake
flasks (240 rpm) containing Nutrient Broth No. 2. For
extraction of IRS, cells were harvested by centrifugation at
5000g and washed several times in cold phosphate-buffered
saline (PBS). Bacterial synthetases were extracted by soni-
cation in the presence of lysostaphin (150 µg/mL) in the same
buffer, followed by treatment with DNase (10 µg/mL) and
overnight dialysis against 20 mM Tris‚HCl pH 8, containing
5 mM MgCl2 and 2 mM dithiothreitol (DTT), and ultracen-
trifugation at 200 000g for 1h. All enzymes were stored at
-20 °C in the presence of 30% glycerol.
(12) Brown, P.; O’Hanlon, P. J . Novel Synthesis of a Mixed Phos-
phonic Anhydride. A route to Carboxylic Acid Derivatives from
a Methyl Ketone. Tetrahedron Lett. 1994, 35, 9619.
(13) Clapp, L. B. 1,2,4-Oxadiazoles. Adv. in Heterocyc. Chem. 1976,
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(14) Turchi, I. J . The Chemistry of Heterocyclic Compounds; Wiley:
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(16) Liu, K. C.; Shelton, B. R.; Howe, R. K. A particularly Convenient
Preparation of Benzohydroximinoyl Chlorides (Nitrile Oxide
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(17) Chemical Design Ltd., Oxon, England.
IRS activity was assayed as the charging of tRNAIle with
14C-Ile using the method of Durekovic21 under conditions where
counts were approximately proportional to time and enzyme
concentration. Assay mixtures contained 30 mM Tris, 2 mM
DTT, 10 mM MgCl2, 70 mM KCl, 1.56 mg/mL E. coli tRNA, 5
mg/ mL equine ATP, and 4.8 µM [U-14C]-isoleucine. The
concentration of each compound resulting in 50% inhibition
of 14C-Ile incorporation (IC50) was determined after preincu-
bation of increasing concentrations of the compound with IRS
for 5 min, followed by addition of substrates and cofactors and
reaction for 10 min at 37°C.
(18) Dewar, M. J . S.; Zoebisch, E. G.; Healey, E. P.; Stewart, J . J . P.
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(19) Ferenczy, G. G.; Reynolds, C. A.; Richards, W. G. Semiempirical
AM1 Potential Derived Charges: A Comparison with ab initio
Values. J . Comput. Chem. 1990, 11, 159-169.
(20) Spartan version 4.1.1, Wavefunction Inc, 18401 von Karman Ave
no. 370, Irvine, CA 92715. Copyright) 1995 Wavefunction Inc.
(21) Durekovic, A.; Flossdorf, J .; Kula, M.-R. Isolation and Properties
of Isoleucyl tRNA Synthetase from Escherichia coli MRE 600.
Eur. J . Biochem. 1973, 36, 528-533.
J M960738K