Synthesis, stereochemical assignment, and bioactivity of the Penicillium metabolites penicillenols B1 and B2

Article abstract of DOI:10.1016/j.tet.2015.05.116

The Penicillium metabolites penicillenols B₁ and B₂ were synthesised for the first time by elimination of a mesylated penicillenol A precursor as obtained from an l-threonine derived tetramic acid. The (Z,S)- and (E,S)-configured diastereomers were identical to the natural compounds as to NMR spectra and optical rotations. Both isomers showed antiproliferative effects against cancer and endothelial cell lines and penicillenol B₁ was also notably antibiotic against Staphylococcus aureus.

Full text of DOI:10.1016/j.tet.2015.05.116

Tetrahedron 71 (2015) 5064e5068
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis, stereochemical assignment, and bioactivity of the
Penicillium metabolites penicillenols B1 and B2
,
Karl Kempf a, Florian Schmitt a, Ursula Bilitewski b, Rainer Schobert a
*
a Organic Chemistry Laboratory, University Bayreuth, Universitaetsstr. 30, D-95440 Bayreuth, Germany
b Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The Penicillium metabolites penicillenols B1 and B2 were synthesised for the first time by elimination of
a mesylated penicillenol A precursor as obtained from an L-threonine derived tetramic acid. The (Z,S)-
and (E,S)-configured diastereomers were identical to the natural compounds as to NMR spectra and
optical rotations. Both isomers showed antiproliferative effects against cancer and endothelial cell lines
and penicillenol B1 was also notably antibiotic against Staphylococcus aureus.
Ó 2015 Elsevier Ltd. All rights reserved.
Received 20 February 2015
Received in revised form 28 May 2015
Accepted 29 May 2015
Available online 4 June 2015
Keywords:
Penicillenol
Tetramic acids
Antibiotics
Anticancer drugs
1. Introduction
penicillenols A1 1a and A2 1b were synthesised by Yoda et al. via
cyclisation of N,O-protected L-threonine and D-allothreonine, re-
The penicillenols 1 were isolated from Penicillium sp. GQ-7, an
endophytic fungus associated with Aegiceras corniculatum.1 They
are N-methylated pyrrolidine-2,4-diones (tetramic acids)2 bearing
spectively, with Meldrum’s acid and subsequent 3-acylation of the
so-formed tetramic acids.3 Our group reported the synthesis of
penicillenol C1 1e via Wittig cyclisation of a protected benzyl L-N-
an
a
-methyl branched C8-fatty acyl residue at C-3 (Fig. 1). The
methylthreoninate with the phosphorus ylide Ph3PCCO 2.4 In either
case the comparison of the NMR and optical rotation data of the
synthetic products allowed the configurational assignment of the
natural products. In contrast, the penicillenols B1 1c and B2 1d,
which are formal dehydration products of penicillenol A have
neither been synthesised nor had their configuration at C-9 been
assigned.
Herein we report the first synthesis of 1c and 1d employing an
alternative third route to the threonine derived tetramic acid core,
and the assignment of the configuration of C-9 of the natural
penicillenols B1 and B2. Since they had shown antiproliferative
activity against HL-60 leukaemia with single-digit micromolar IC50
values we also assessed their efficacy against further cancer and
endothelial cell lines. As a first test for antibiotic activity we mea-
sured their dose-dependent effects on Staphylococcus aureus.
2. Results and discussion
In our previous synthesis of 1e we prepared the N-methylated
and O-TIPS protected benzyl threoninate 6, required for the cycli-
Fig. 1. Structures of penicillenols 1aef.
sation with ylide Ph3PCCO 2 to give tetramic acid 8, from L-threo-
nine in seven steps, which included N-nosylation to enable N-
* Corresponding author. Tel.: þ49 921 55 2679; fax: þ49 921 55 2671; e-mail
address: Rainer.Schobert@uni-bayreuth.de (R. Schobert).
mono-methylation.4 Although we still deem this a viable approach,
http://dx.doi.org/10.1016/j.tet.2015.05.116
0040-4020/Ó 2015 Elsevier Ltd. All rights reserved.

K. Kempf et al. / Tetrahedron 71 (2015) 5064e5068
5065
we now explored a new strategy for the synthesis of ester 6 that
achieves N-mono-methylation without preceding nosylation. The
yield of this route to 6 is higher (46%) than that of the previous one
(34%), it is upscalable since its intermediates are easier to purify
without columning, and it is more atom-economical. We assumed
that the unknown configuration at C-9 of 1c and 1d would be S as in
the case of penicillenols A and C.
gave 10, which was treated overnight with 0.1 N aqueous NaOH/
THF to give a 3:1 mixture of both penicillenols B. It was base-line
separated on
a prep HPLC column (Kinetex 5 mm, C18,
250ꢀ21.2 mm) to afford the pure isomers 1c (35%) and 1d (11%),
each as a 5:6 mixture of two exo-enol tautomers.
Originally, we intended to introduce the C5]C6 bond early in
the synthesis of penicillenols B to avoid the necessity of protecting
According to literature5
L-threonine was converted in five steps
the secondary alcohol of threonine. N-Boc-L-threonine 11 gave the
and 64% yield to its N-methyl-N-Fmoc derivative 3 (Scheme 1). This
sequence was comprised of O-acetylation and N-Fmoc protection of
N-methylated aminobutenoate 12 in one step and as a 3:1-
mixture of Z- and E-isomers when treated with NaH and Me2SO4
(Scheme 2). However, all attempts at cleaving the Boc group to
liberate unsaturated amino esters such as 15, required for the
cyclisation with ylide 2, failed for unknown reasons. So this
approach had to be abandoned.
threonine,
a subsequent oxazolidinone formation with para-
formaldehyde, and the reduction of the latter to give the N-methyl
derivative. Hydrolysis left acid 3, which was esterified with benzyl
bromide and Cs2CO3 yielding the hitherto unknown ester 4 as
colourless crystals in over 80%. O-TIPS protection afforded 5, the
Fmoc group of which was removed with piperidine to leave the
amino ester 6 in 86% over the last two steps, which can be carried
out in one pot. Refluxing amino ester 6 with ylide 2 in toluene gave
benzyl tetramate 7 in 55% yield via a domino sequence comprised
of amine addition to the C]C bond of 2 followed by an intra-
molecular Wittig olefination of the so-formed amide ylide.
Hydrogenolysis of 7 led to tetramic acid 8, which was 3-acylated
according to the Yoda protocol3 with S-2-methyloctanoic acid6
prepared by a-methylation of octanoic acid using an Evans auxil-
Scheme 2. TMSE¼Me3SiCH2CH2.
iary. Pure tetramic acid 9 was obtained in 68% yield upon chro-
matography of the crude on a reversed stationary phase (Merck
LiChrorep RP-18). Its desilylation with 70% HF/pyridine and sub-
sequent purification by another RP column chromatography fur-
nished penicillenol A1 1a in 88% yield. Mesylation of the 6-OH of 1a
The configurations of the C5]C6 bond of our synthetic
penicillenols
B were assigned by NOESY experiments (cf.
Supplementary data). For the assignment of the configuration at
C-9 of the natural penicillenols B we compared the known chemical
shifts in their 13C NMR spectra1 with those of our synthetic (9S)-
configured Z- and E-diastereomers. Table 1 shows a perfect match
of indicative carbon signals. The specific rotations of our samples of
24
24
penicillenols B were [
a
]
ꢁ15 (c 0.23, MeOH) for 1c and [
a
]
D
ꢁ6
D
(c 0.13, MeOH) for 1d, which is the other way round as had been
24
24
reported in literature1 (1c: [
a
]
ꢁ7.8; 1d: [
a
]
ꢁ15.9). Given the
D
D
matching NMR spectra, the congruence of the HPLC retention times
(Z-products eluted first in our hands and in literature), and the fact
that 1a did not racemise under the basic conditions of the final
elimination step, we assume a mixing up of samples or data by the
isolating group.
This group also reported the cytotoxicities of penicillenols AeC
in MTT assays7 against four cancer cell lines of which only leu-
kaemia HL-60 responded to penicillenols B. Since these activities
lay within the range of clinical applicability we tested 1c and 1d for
growth inhibition of cells of highly invasive 518A2 melanoma and
cisplatin-resistant HT-29 colon carcinoma and of the hybrid en-
dothelial cell line Ea.hy926. Both compounds are equally active and
even exceed the effect of cisplatin against HT-29. However, they are
still far from clinical usability (Table 2).
The penicillenols B also showed dose-dependent antimicrobial
activity against Gram-positive bacteria such as S. aureus (Fig. 2) and
Gram-negative bacteria such as the export-limited Escherichia coli
tolC mutant (not shown). Interestingly, penicillenol B1 was dis-
tinctly more active than penicillenol B2.
3. Conclusion
We reported a new synthetic route to penicillenol A1 and, by
mesylationeelimination of this, also the first access to both pen-
icillenols B1 and B2. We assigned the stereochemistry of C-9 in the
3-acyl sidechain as S. The penicillenols B were efficacious against
two cancer cell lines and they also showed antimicrobial activity
against Gram-positive and -negative bacteria. It is worthy of note
that penicillenol B1 was conspicuously more active than its B2
Scheme 1. Reagents and conditions: (i) 28 equiv AcCl/AcOH, 6 N HCl 1:1, 0 ꢂC, 45 min;
(ii) FmocONSu, NEt3, dioxane/THF 1:1, 12 h, rt; (iii) HO(CH2O)nH, pTsOH, tol., 1 h, refl.;
(iv) AlCl3, Et3SiH, CH2Cl2, 4 h, rt; (v) dioxane/2 M aq HCl 1:1, 60 ꢂC, 12 h; (vi) Cs2CO3,
BnBr, rt; (vii) TIPSOTf, NEt3, 12 h, 0 ꢂC; (viii) 20% piperidine, CH2Cl2; (ix) 1.2 equiv 2, tol.,
refl., 16 h; (x) 1 bar H2, Pd/C, MeOH; (xi) S-2-methyloctanoic acid, 1.2 equiv EDC,
0.2 equiv DMAP, CH2Cl2, 12 h, rt, then 1.5 equiv CaCl2, 1.5 equiv NEt3, rt, 12 h, then 3.5 h
refl.; (xii) 8 equiv HF/pyridine (88%), THF; (xiii) 2 equiv MesCl, 4 equiv NEt3, 0.4 equiv
DMAP; (xiv) THF, 0.1 N NaOH 2:1, rt.

5066
K. Kempf et al. / Tetrahedron 71 (2015) 5064e5068
Table 1
Chemical shifts da [ppm] of indicative hydrogen and carbon atoms of natural and synthetic penicillenols B1 and B2
Position
Nat 1c1 exoA/exoB
Synt 1c exoA/exoB
Nat 1d1 exoA/exoB
Synt 1d exoA/exoB
6 (1H)
5.89, q, J 7.7
2.03, d, J 8.2
3.39, s
172.5/165.7
99.6/101.8
191.2/197.4
136.7
107.5
181.0/185.0
27.9
5.87, q, J 8.0
2.01, d, J 8.0
3.37, s
172.5/165.6
99.5/101.8
191.1/197.4
136.7
107.3
180.9/184.8
27.8
5.38, q, J 7.8
2.23, d, J 7.8
2.99, s
170.8/164.0
101.3/103.7
190.8/196.6
134.2
111.8
182.8/187.3
24.7
5.38, q, J 7.7
2.23, d, J 7.7
3.04, s
170.9/164.1
101.3/103.7
190.8/196.6
134.3
111.8
183.1/187.3
24.7/24.9
7 (1H)
17 (1H)
2 (13C)
3 (13C)
4 (13C)
5 (13C)
6 (13C)
8 (13C)
17 (13C)
a
13C NMR spectra were recorded in CDCl3 at 150 MHz (natural 1c,d)1 and 75 MHz (synthetic 1c,d). 1H NMR spectra were recorded in CDCl3 at 600 MHz (natural 1c,d)1 and
300 MHz (synthetic 1c,d).
Table 2
(230e400 mesh) or Merck LiChrorep RP-18 (40e63
m
m) was used.
Inhibitory concentrations IC50 (72 h)a in
mM of penicillenols B1 1c and B2 1d when
HPLC columns: Phenomenex Kinetex 5
m
C18 250ꢀ4.6 mm, flow
applied to 518A2 melanoma, HT-29 colon carcinoma, and Ea.hy926 hybrid endo-
thelial cells
rate 0.7 mL/min (analyt.) and 250ꢀ21.2, 14.9 mL/min (prep.). Sol-
vents were dried and distilled (diethyl ether, toluene and THF over
Na, CH2Cl2 over CaH2) and stored under an atmosphere of dry ar-
gon. Starting compounds were bought from the usual sources and
used without further purification. For the synthesis of compounds
12e14 see Supplementary data.
Cell line/compound
518A2
HT-29
Ea.hy926
1c
1d
Cisplatin
24.7ꢃ3.1
21.1ꢃ1.3
9.0ꢃ2.0
28.0ꢃ1.1
26.0ꢃ1.8
>100
32.7ꢃ2.2
32.6ꢃ4.0
d
a
Values are derived from concentrationeresponse curves obtained by measuring
the percentage absorbance of viable cells relative to untreated controls (100%) after
72 h exposure in MTT assays. Values represent means of four independent runsꢃSD.
4.2. Syntheses and characterisation
4.2.1. Benzyl
methyl-threoninate (4). Analogously to a general protocol8 a solu-
tion of N-Fmoc-N-methyl-
-threonine (3)5 (6.90 g, 19.5 mmol) in
(2S,3R)-N-[(9H-fluoren-9-yl)methoxycarbonyl]-N-
L
methanol (100 mL) was treated with Cs2CO3 (3.25 g, 10.0 mmol)
and stirred at room temperature until it had completely dissolved.
The solvent was evaporated and the remainder was suspended
twice in CHCl3 and evaporated each time under reduced pressure to
remove residual water. The resulting solid was treated with DMF
(300 mL) and benzylbromide (2.6 mL, 22.0 mmol) and the mixture
thus obtained was vigorously stirred for 12 h and then evaporated
under reduced pressure. The residue was partitioned between
diethyl ether and water, the aqueous phase was extracted twice
with diethyl ether, and the combined organic layers were finally
washed with brine, dried over Na2SO4, and evaporated in vacuum.
The crude product was purified by column chromatography (silica
gel; hexane/ethyl acetate, 10:1 to 3:1) to afford ester 4 (7.25 g,
16.3 mmol, 84%) as white needles of mp 91 ꢂC; Rf 0.20 (hexane/ethyl
20
Fig. 2. Dose-dependent antimicrobial effects of penicillenols B1 1c and B2 1d on
Staphylococcus aureus. Bacterial growth was measured as ‘turbidity’ (OD at 600 nm).
Minimal inhibitory concentrations (MIC) were ca. 8 mM for 1c and 70 mM for 1d.
acetate, 3:1); [
a
]
ꢁ2.0 (c 1.00, CH2Cl2); nmax 3481, 3035, 2961,
D
1742, 1699, 1479, 1451, 1401, 1373, 1308, 1236, 1195, 1146, 1104, 1035,
988, 879, 757, 738, 697 cmꢁ1
;
dH (CDCl3) 1.22 (3H, d, JHH 6.5 Hz,
CCH3), 3.02 (3H, s, NCH3), 4.27 (1H, t, JHH 7.0 Hz, FmocCH), 4.45 (2H,
d, JHH 7.0 Hz, FmocCH2), 4.50 (1H, dq, JHH 4.9, 6.5 Hz, 3-H), 4.66 (1H,
d, JHH 4.9 Hz, 2-H), 5.19 (1H, d, JHH 12.4 Hz, CHHaPh), 5.26 (1H, d, JHH
12.4 Hz, CHHbPh), 7.29e7.37 (7H, m, ArH), 7.41 (2H, t, JHH 7.4 Hz,
FmocArH), 7.60 (2H, d, JHH 7.4 Hz, FmocArH), 7.78 (2H, d, JHH 7.7 Hz,
FmocArH); dC (CDCl3) 19.5 (C-4), 33.9 (NMe), 47.1 (FmocCH), 64.7
(C-2), 67.0 (CH2Ph), 67.1 (C-3), 67.9 (FmocCH2), 119.8, 124.9, 126.9,
127.6, 128.1, 128.3, 128.5, 135.2, 141.2, 143.7, 157.5, 169.8. HRMS: m/z
calcd for [MþH, C27H28NO5þ]: 446.19620; found: 446.19553.
congener against the bacteria selected by us. A more comprehen-
sive screening for antimicrobial activity is currently underway.
4. Experimental
4.1. General
€
Melting points (uncorrected): Buchi melting point apparatus M-
565. IR: PerkineElmer Spectrum 100 FT-IR spectro-photometer
with ATR sampling unit. NMR: if not indicated otherwise, measured
in CDCl3 at 300 MHz (1H) or 75.5 MHz (13C) on a Bruker Avance 300
4.2.2. Benzyl (2S,3R)-N-methylthreoninate (6). A solution of benzyl
N-Fmoc-N-methyl-threoninate 4 (2.98 g, 6.7 mmol) in 60 CH2Cl2
(60 mL) was kept under argon atmosphere at 0 ꢂC and treated with
NEt3 (1.1 mL, 810 mg, 8.0 mmol) and triisopropylsilyltriflate
(1.98 mL, 2.25 g, 7.4 mmol). The mixture was allowed to warm to
room temperature and stirred until the reaction was complete by
TLC (hexane/ethyl acetate, 3:1). The solution was cooled to 0 ꢂC,
treated with piperidine (18 mL), stirred for 10 min at 0 ꢂC, and
spectrometer. Chemical shifts are given in parts per million (d)
downfield from Me4Si as internal standard for 1H and 13C. Mass
spectra: Finnigan MAT 8500 (EI, 70 eV). HRMS: UPLC/Orbitrap MS
system in ESI mode. Optical rotations: PerkineElmer Polarimeter
343 (l¼589 nm). For column chromatography Merck silica gel 60

K. Kempf et al. / Tetrahedron 71 (2015) 5064e5068
5067
eventually concentrated in vacuo at 25 ꢂC. The remaining piperi-
dine solution was immediately loaded onto a column and separated
(silica gel; hexane/ethyl acetate, 20:0 to 20:1) to afford ester 6
(2.16 g, 5.7 mmol, 86% after two steps) as a colourless oil. Rf¼0.63
(0.35 mmol, 35% over two steps) of penicillenol B1 as a red oil in the
first fraction and 29 mg (0.17 mmol, 11% over two steps) of pen-
icillenol B2 in the second fraction. Analytic HPLC: elution with 85%
MeOH/0.1% HCOOH for 10 min, then gradient rise to 95% MeOH
within 3 min; tR of 1c: 16.3 min, lmax¼270 nm, tR of 1d: 17.8 min,
lmax¼270 nm.
(cyclohexane/EtOAc, 2:1, þ1% NEt3). [
a]
20 2.2 (c 1.00, CH2Cl2); nmax
D
2942, 2895, 2866, 1742, 1732, 1463, 1456, 1373, 1212, 1152, 1124,
1099, 1060, 1014, 998, 967, 949, 918, 882, 746, 733, 696, 676 cmꢁ1
;
dH (CDCl3) 0.95e1.07 (21H, m, TIPS), 1.26 (3H, d, JHH 6.3 Hz, CCH3),
1.71 (1H, br s, NH), 2.40 (3H, s, NCH3), 3.14 (1H, JHH 3.6 Hz, CHN),
4.32 (1H, qd, JHH 6.3, 3.6 Hz, CHCH3), 5.10 (1H, d, JHH 12.2 Hz,
CHa2Ph), 5.22 (1H, d, JHH 12.2 Hz, CHb2Ph), 7.27e7.40 (5H, m, ArH); dC
(CDCl3) 12.6 (SiCH),18.0 (SiCCHa3),18.1 (SiCCH3b), 20.5 (CHCH3), 35.3
(NMe), 66.4 (CH2), 69.4 (CHCH3), 69.9 (CHN), 128.2 (ArH), 128.4
(ArH), 128.5 (ArH), 135.8 (ArCq), 173.3 (CO).
4.2.4.1. Penicillenol B1 (1c). [
a
]
24 ꢁ15 (c 0.23, MeOH); nmax 2927,
D
2857, 1705, 1676, 1610, 1450, 1407, 1370, 1341, 1322, 1239, 1217, 1170,
1111, 1093, 1031, 997, 903, 826, 789, 726, 618, 584, 574, 562 cmꢁ1
; dH
(CDCl3, 6:5 mixture of tautomera and tautomerb) 0.79e0.89 (3H, m,
15-H), 1.15 (3H, d, JHH 6.9 Hz, 16-H), 1.19e1.33 (8H, m, CHa2þb),
1.38e1.52 (1H, m, 10-H), 1.60e1.75 (1H, m, 10-H), 2.01 (3H, d, JHH
8.0 Hz, 7-Ha), 2.07 (3H, d, JHH 8.0 Hz, 7-Hb), 3.34 (3H, s, NMeb), 3.37
(3H, s, NMea), 3.62e3.75 (1H, m, 9-H), 5.85 (1H, q, JHH 8.0 Hz, 6-Ha),
5.87 (1H, q, JHH 8.0 Hz, 6-Hb); dC (CDCl3) 11.4 (C-7a), 11.8 (C-7b), 14.0
(C-15a/b), 17.0 (C-16b), 17.2 (C-16a), 22.5 (C-13a/b), 27.1 (C-11), 27.8
(C-17), 29.2 (C-12a), 29.25 (C-12b), 31.6 (C-14), 33.3 (C-10b), 33.7 (C-
10a), 35.8 (C-9a), 37.4 (C-9b), 99.5 (C-3a), 101.8 (C-3b), 107.3 (C-6a),
108.1 (C-6b), 134.4 (C-5b), 136.7 (C-5a), 165.6 (C-2b), 172.5 (C-2a),
180.9 (C-8a), 184.8 (C-8b), 191.1 (C-4a), 197.4 (C-4b); MS (EI), m/z (%)
279 (95, Mþ), 251 (7), 232 (8), 222 (12), 208 (76), 195 (99), 177 (16),
166 (100), 139 (42), 127 (4), 113 (6), 86 (15), 70 (30), 57 (25), 44 (21);
HRMS: m/z calcd for [MþH, C16H26NO3þ]: 280.19072, found:
280.19022.
4.2.3. 3-[10-Hydroxy-(20S)-methyl-oct-10-ylidene]-(5S)-[(10R)-triiso-
propylsilyloxy-ethyl]-1-methylpyrrolidine-2,4-dione (9). To a solu-
tion of (S)-2-methyloctanoic acid6 (0.33 g, 2.1 mmol) in CH2Cl2
(20 mL) at room temperature were added EDC$HCl (0.48 g,
2.5 mmol), tetramic acid 84 (0.55 g, 1.75 mmol), and DMAP (0.05 g,
0.4 mmol), and the resulting mixture was stirred for 12 h. NEt3
(0.62 mL, 4.5 mmol) and dry CaCl2 (0.29 g, 2.6 mmol) were added
and stirring was continued for 24 h. After dilution with 20 mL of
CH2Cl2 the organic phase was washed with 1 M NaHSO4 and 0.05 M
Na2-EDTA solutions. The aqueous phases were re-extracted with
CH2Cl2 and the combined organic layers were dried and concen-
trated in vacuum. The crude product was purified by a reversed
phase silica gel column (22ꢀ1.2 cm, rinsing with 85% MeOH, then
elution with 100% MeOH) to afford 540 mg (1.19 mmol, 68%) of 9 as
a red oil (analytic HPLC: 85% MeOH, after 15 min in 10 min to 100%
with 0.1% HCOOH in the water fraction; tR of product (3-acyl tet-
4.2.4.2. Penicillenol B2 (1d). [
a
]
24 ꢁ6 (c 0.13, MeOH); nmax 2928,
D
2858, 2043, 2003, 1703, 1671, 1615, 1445, 1398, 1374, 1341, 1315,
1278, 1206, 1164, 1091, 1050, 883, 826, 802, 726, 681, 606, 584,
573 cmꢁ1 dH (CDCl3, 6:5 mixture of tautomera and tautomerb)
;
0.81e0.90 (3H, m, 15-H), 1.19 (3H, d, JHH 7.0 Hz, 16-H), 1.22e1.29
(8H, m, CHa2=b), 1.42e1.51 (1H, m, 10-H), 1.65e1.75 (1H, m, 10-H),
2.23 (3H, d, JHH 7.7 Hz, 7-Haþb), 3.04 (3H, s, NMeb), 3.05 (3H, s,
NMea), 3.70 (1H, pseudo-sxt, JHH 7.0 Hz, 9-H), 5.38 (1H, q, JHH 7.7 Hz,
6-Ha), 5.46 (1H, q, JHH 7.7 Hz, 6-Hb); dC (CDCl3) 11.8 (C-7a), 12.4 (C-
7b), 14.0 (C-15a/b), 17.0 (C-16b), 17.2 (C-16a), 22.6 (C-13a/b), 24.7 (C-
17a), 24.9 (C-17b), 27.2 (C-11a/b), 29.3 (C-12a/b), 31.6 (C-14a/b), 33.6
(C-10a/b), 35.7 (C-9a), 37.1 (C-9b), 101.3 (C-3a), 103.7 (C-3b), 111.8 (C-
6a), 112.6 (C-6b), 134.3 (C-5a), 136.0 (C-5b), 164.1 (C-2b), 170.9 (C-2a),
183.1 (C-8a), 187.3 (C-8b), 190.8 (C-4a), 196.6 (C-4b); m/z (%) 279 (99,
Mþ), 251 (7), 232 (8), 222 (12), 208 (66), 195 (99), 177 (15), 166
(100), 139 (35), 113 (4), 85 (7), 70 (24), 57 (14), 44 (13); HRMS: m/z
calcd for [MþH, C16H26NO3þ]: 280.19072, found: 280.19040.
ramic acid): 31.2 min, lmax¼285 nm; tR of 4-O-acyl tetramate:
20
29.5 min, lmax¼226 nm); [
a
]
D
ꢁ39.1 (c 1.00, MeOH); nmax 2930,
2867, 1710, 1652, 1615, 1463, 1376, 1328, 1262, 1214, 1139, 1096,
1069, 999, 971, 922, 882, 797, 755, 703, 677 cmꢁ1
;
dH (CDCl3) 0.84
(3H, t, JHH 6.6 Hz, CH2CH3), 0.96e1.01 (21H, m, TIPS), 1.11 (3H, d, JHH
6.7 Hz, C-16), 1.19e1.31 (8H, m), 1.40e1.50 (1H, m, 10-Ha), 1.43 (3H,
d, JHH 6.9 Hz, C-7), 1.59e1.70 (1H, m, 10-Hb), 3.10 (3H, s, NMe), 3.47
(1H, d, JHH 1.6 Hz, 5-H), 3.57 (1H, ddqui, JHH 6.3, 1.9, 6.7 Hz, 9-H),
4.56 (IH, dq, JHH 1.6, 6.9 Hz, 6-H); dC (CDCl3) 12.6 (SiCH), 14.0 (C-15),
16.7 (C-16), 17.9 (SiCCH3), 22.5 (CH2), 22.9 (CHCH3), 27.1 (CH2), 29.0
(NMe), 29.2 and 31.6 (CH2), 34.0 (C-10), 35.8 (C-9), 68.0 (CHCH3),
72.4 (C-5),101.1 (C-3),174.4 (CON),190.6,193.2. MS (EI), m/z (%): 410
(100) C22H40NO4Siþ, 366 (27), 253 (29) C15H22NO3þ, 201 (28)
C11H25OSiþ, 157 (24). HRMS: m/z calcd for [MþH, C25H48NO4Siþ]:
453.3347, found: 453.3341.
4.3. Cell cultures and growth inhibition (MTT) assay
4.3.1. Cell cultures. The human 518A2 melanoma cells (Department
of Radiotherapy and Radiobiology, University Hospital Vienna)9 and
the human colon adenocarcinoma cell line HT-29 (DSMZ no.: ACC
299) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM)
or Roswell Park Memorial Institute (RPMI) Medium (HT-29), sup-
plemented with 10% foetal bovine serum (FBS), 1% antibiotic-anti-
4.2.4. Penicillenol B1, (5Z,20S)-5-ethylidene-3-(1-hydroxy-2-
methyloct-1-ylidene)-1-methylpyrrolidine-2,4-dione (1c) and pen-
icillenol B2, (5E,20S)-5-ethylidene-3-(1-hydroxy-2-methyloct-1-
ylidene)-1-methylpyrrolidine-2,4-dione (1d). Penicillenol A1 (310
mg, 1.04 mmol; for its synthesis, data, and spectra cf. Supplemen-
tary data) was dissolved in THF, treated with NEt3 (580
mL,
mycotic solution (all from Gibco) and 250
mg/mL gentamycin
4.2 mmol, 4 equiv), methanesulfonylchloride (170 L, 4.2 mmol,
m
(SERVA). The endothelial hybrid cell line Ea.hy926 (ATCC no.: CRL-
2922)10 was grown in conditioned DMEM with an endothelial
medium supplement (PAA/GE Healthcare) added. The cells were
maintained in a moisture-saturated atmosphere (5% CO2, 95% hu-
midity) at 37 ꢂC.
4 equiv), and DMAP (51 mg, 0.4 mmol, 0.4 equiv) and stirred for
12 h. The mixture was poured into water, acidified with
NaHSO4 and extracted three times with ethyl acetate. The com-
bined organic layers were washed with brine, dried over Na2SO4
and concentrated in vacuum to leave crude product 10. It was
dissolved in THF (60 mL), 0.1 N aqueous NaOH (30 mL) was added,
and the resulting solution was stirred at room temperature for 3 d.
NaHSO4 was added, the resulting mixture was extracted three
times with ethyl acetate, and the organic layers were washed with
brine, dried over Na2SO4 and evaporated in vacuum. The remainder
was purified by HPLC (70% MeOH/0.1% HCOOH) to afford 100 mg
4.3.2. MTT
assay.7 MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-2H-tetrazolium hydrobromide; ABCR] was used to iden-
tify the metabolic activity of vital cells, which are capable of re-
ducing it to a violet formazan. 518A2, HT-29, and Ea.hy926 cells
(5ꢀ104/mL) were seeded and cultured for 24 h in 96-well micro-
plates. Incubation of cells following treatment with the test

5068
K. Kempf et al. / Tetrahedron 71 (2015) 5064e5068
compounds was continued for 72 h. Blank and solvent controls
were treated identically. Then, MTT in phosphate-buffered saline
was added to a final concentration of 0.05% (518A2, Ea.hy926) or
0.1% (HT-29). After 2 h the precipitate of formazan was dissolved in
DMSO containing 10% sodium dodecylsulfate. The microplates were
swiftly turned to discard the medium before adding the solvent
mixture. The microplates were gently shaken in the dark for 30 min
and absorbances at 570 nm and 630 nm (background) were mea-
sured with a Tecan F-200 plate reader. All experiments were carried
out in quadruplicate, and the percentage of the viable cells was
calculated with Origin 8.1G as the meanꢃstandard deviation rela-
tive to the control (100%).
Supplementary data
Syntheses and data of compounds 1a,12, and 14; NMR spectra of
1a, 1c, 1d, 4, 6, 9, 12, and 14. Supplementary data associated with
this article can be found in the online version, at http://dx.doi.org/
10.1016/j.tet.2015.05.116.
References and notes
1. Lin, Z.-J.; Lu, Z.-Y.; Zhu, T.-J.; Fang, Y.-C.; Gu, Q.-Q.; Zhu, W.-M. Chem. Pharm. Bull.
2008, 56, 217e221.
2. Reviews on tetramic acids: (a) Henning, H.-G.; Gelbin, A. Adv. Heterocycl. Chem.
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Elsevier: 2003; Vol. 28/1, pp 109e163; (d) Gossauer, A. Monopyrrolic natural
compoundsincludingtetramicacid derivativesInHerz, W.,Falk, H., Kirby, G. W.,Eds..
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4.4. Antibacterial activity test
S. aureus (NCTC 8325) was cultivated in T-medium, which was
composed of tryptone (LP 0042, 17 g/L), neutralised soya peptone
(LP 0044, 3 g/L), NaCl (0.1 M), KCl (2 mM), MOPS (50 mM),
CaCl2$2H2O (2 mM) and glucose (1%). Medium (200 mL) was in-
oculated with an S. aureus colony and incubated at 37 ꢂC with
shaking overnight. An aliquot of the overnight culture was diluted
with fresh T-medium to obtain 50 mL with a start OD600 of 0.01.
This culture was allowed to grow for another 3 h at 37 ꢂC. The
resulting culture was added to the wells of a transparent 96-well
microtiter plate containing diluted DMSO solutions of the test
compounds. The total volume in each plate was 180 mL. The bacteria
7. Mosmann, T. J. Immunol. Methods 1983, 65, 55e63.
were incubated with the compounds at 37 ꢂC for 21 h. Bacterial
growth was followed via determination of the optical density (OD)
at 600 nm (turbidity). Blank and solvent controls were included on
the microtiter plate.
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