Structure - Activity relationships of precursors and analogs of natural 3-enoyl-tetramic acids

Article abstract of DOI:10.1002/cbdv.201000179

Fragments and synthetic precursors prepared en route to the macrocyclic 3-acyltetramic acids (=3-acyl-1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) aburatubolactam and macrocidin A, as well as other analogs with variance in the ring heteroatom (N, O, S), and the residues at N(1), C(3), and C(5) were tested for cytotoxic and antimicrobial effects. Anticancer activity against various tumor cell lines in vitro did not necessarily require an intact pyrrolidin-2,4-dione ring. An acyclic β-hydroxy-octatrienoyl amide precursor to aburatubolactam also exhibited distinct activity with an IC ₅₀ (120 h) value of <2.5 IμM. The length of 3-oligoenoyl residues had little influence on the anticancer activity, but 3-alka-oligoenoyl tetramic acids were far more efficacious than their 3-(4-methoxycinnamoyl) congeners. N-H-3-acyltetramic acids were generally more active than their N-Me or N-Boc analogs, unless further polar groups necessitated an increased lipophilicity for sufficient uptake. Tetronic and thiotetronic acids were far less antiproliferative in cancer cells when compared with identically substituted tetramic acids. Copyright

Full text of DOI:10.1002/cbdv.201000179

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Structure–Activity Relationships of Precursors and Analogs of Natural
3-Enoyl-tetramic Acids
by Bertram Barnickela), Frances Bayliffeb), Randi Diestelc), Karl Kempfa), Sabine Laschatb),
Steffen Pachalib), Florenz Sassec), Andrea Schlenka), and Rainer Schobert*a)
a) Organisch-chemisches Laboratorium der Universitꢀt Bayreuth, Universitꢀtsstrasse 30, NW 1,
D-95447 Bayreuth (fax: þ49(0)921552671; e-mail: Rainer.Schobert@uni-bayreuth.de)
b) Institut fꢁr Organische Chemie der Universitꢀt Stuttgart, Pfaffenwaldring 55, D-70569 Stuttgart
c) Helmholtz Centre for Infection Research (HZI), Department of Chemical Biology, Inhoffenstrasse 7,
D-38124 Braunschweig
Fragments and synthetic precursors prepared en route to the macrocyclic 3-acyltetramic acids (¼ 3-
acyl-1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) aburatubolactam and macrocidin A, as well as other
analogs with variance in the ring heteroatom (N, O, S), and the residues at N(1), C(3), and C(5) were
tested for cytotoxic and antimicrobial effects. Anticancer activity against various tumor cell lines in vitro
did not necessarily require an intact pyrrolidin-2,4-dione ring. An acyclic b-hydroxy-octatrienoyl amide
precursor to aburatubolactam also exhibited distinct activity with an IC50 (120 h) value of <2.5 mm. The
length of 3-oligoenoyl residues had little influence on the anticancer activity, but 3-alka-oligoenoyl
tetramic acids were far more efficacious than their 3-(4-methoxycinnamoyl) congeners. N-H-3-
acyltetramic acids were generally more active than their N-Me or N-Boc analogs, unless further polar
groups necessitated an increased lipophilicity for sufficient uptake. Tetronic and thiotetronic acids were
far less antiproliferative in cancer cells when compared with identically substituted tetramic acids.
Introduction. – Tetramic acids (i.e., 1,5-dihydro-4-hydroxy-2H-pyrrol-2-ones) are
produced by a variety of marine and terrestrial organisms such as sponges,
cyanobacteria, bacteria, and fungi. They frequently show biological activity in man,
including antibiotic, anticancer and anti-inflammatory effects, which had been assessed
in reviews by Rosen [1], Royles [2], Ghisalberti [3], Gossauer [4], and Schobert and
Schlenk [5]. Pharmacologically most interesting are 3-acyltetramic acids 1 (Fig.). They
are thought to act by chelating essential metal ions and by mimicking phosphate groups
in the binding site of kinases and phosphatases. Their precise molecular targets are
mostly unknown. Lately, Waldmann and co-workers [6] showed that the sponge
metabolite melophlin A [7][8] targets dynamins in cells and thus modulates signal
transduction via the RAS network leading to a phenotype reversal in MDCK-F3 cells.
As a sideline to our synthetic studies of natural bioactive 3-acyl-tetramic acids
featuring macrocyclic rings, e.g., cylindramide [9][10], aburatubolactam (2) [11][12],
and macrocidin A (3) [13], we looked for potential anticancer and antimicrobial
activity in fragments and synthetic precursors prepared en route. This study was to
clarify whether an intact heterocyclic ring is a prerequisite for activity, and how the
latter is affected by the nature of the heteroatom, and the residues at N(1), C(3), and
ꢂ 2010 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2831
Figure. Structures of 3-acyl-tetramic acids in general, 1, and of natural macrocyclic derivatives
aburatubolactam (2) and macrocidin A (3)
C(5). The far aim was to simplify the structures of the natural leads while retaining
their biological efficacy.
Results and Discussion. – Chemistry. Precursors to Aburatubolactam (2) and Their
Analogs. The aburatubolactam fragment 8a was prepared in six steps from the known
a-azido ester 4 [9] (Scheme 1). Its reduction with SnCl2 afforded the amino ester 5,
which was then selectively monomethylated to compound 6 by a sequence comprised of
N-nosylation (¼4-nitrobenzenesulfonylation), N-methylation of the nosylated inter-
mediate, and finally cleavage of the Nos group with PhSH and K2CO3 in an overall yield
of 70%. Compound 6 was acylated with 2,2-dimethyl-6-[(1E,3E)-penta-1,3-dienyl]-4H-
[1,3]-dioxin-4-one to give amide 7 in 82% yield which, in turn, was cyclized under
Lacey–Dieckmann conditions to 3-(hexadienoyl)tetramic acid 8a in 93% yield. Its 5-
benzyl analog 8b was prepared on a different route. The tetramic acid 9 was first
reacted with the ylide Ph3PCCO to give the corresponding 3-[(triphenylphosphor-
anylidene)acetyl]tetramic acid, which was then deprotonated with tBuOK and treated
with crotonaldehyde to give 3-(hexadienoyl)tetramic acid 8b as the product of a Wittig
olefination. N-Me Analogs 8c and 8d and N-H congeners 11c and 11d were obtained
likewise from the respective tetramic acids 9 or 10, and the appropriate aldehyde [14].
3-Enoyl-tetramic Acids 8e and 8f, 3-Enoyl-tetronic Acids 16, and 3-Enoyl-
thiotetronic Acid 17. These compounds were also synthesized in two steps from the
respective known parent heterocycles 12–15, ylide Ph3PCCO, and the appropriate
aldehydes (Scheme 2).
Precursors to Macrocidin A (3). N-Boc-O-allyl diprotected 3-alkanoyl-tetramic
acids 19, differing in the length of the side chain at C(3), were prepared by Yoshii
acylation [15] of tetramic acid 18 with carboxylic acids in the presence of DMAP and
DCC (Scheme 3). For comparative biological tests, the products 19 were also
deprotected to give the corresponding 3-acyl-tetramic acids 11g–11j. Some of these
were de-allylated affording the respective tetramic acids 11k–11n.
Biological Evaluation. We tested antiproliferative effects of the 3-acyl-tetramic
acids 8 (R1 ¼Me), 11 (R1 ¼H), and 19 (R1 ¼Boc), of the 3-acyl-tetronic acids 16 and of
the 3-acyl-thiotetronic acids 17 in cells of human KB-3-1 cervix, human epithelial A-498
kidney carcinoma, and U-937 leukaemia as well as L929 mouse fibroblasts (Table 1).

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Scheme 1. Synthetic Precursors 4–8a en route to Aburatubolactam (2) and Some Analogs, 8b–8d, and
11c and 11d
i) SnCl2, MeOH, r.t., 5 h; 95%. ii) 1. NosCl (Nos¼4-nitrobenzenesulfonyl), iPr2NEt, CH2Cl2, r.t., 24 h;
83%; 2. MeI, K2CO3, DMF, r.t., 16 h; 84%; 3. PhSH, K2CO3, DMF, r.t., 3 h; 100%. iii) 2,2-Dimethyl-6-
[(1E,3E)-penta-[1,3]-dienyl]-4H-[1,3]dioxin-4-one (0.05m), toluene, 1208, 4 h; 82%. iv) MeONa
t
(0.025m), MeOH, r.t., 3 h; 93%. v) Ph3PCCO, THF, reflux, 6 h; 98%. vi) 1. BuOK, THF, 508, 20 min;
2. crotonaldehyde, reflux, 6 h; 64%. vii) 1. tBuOK, THF, 508, 20 min; 2. R3CHO, reflux, 6 h.
Scheme 2. Synthesis of 3-[(2E)-Enoyl]-5-methyltetramic, -tetronic, and -thiotetronic Acids
i) Ph3PCCO, THF, reflux, 6 h. ii) 1. tBuOK, THF, 508, 20 min; 2. R3CHO, reflux, 6 h.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2833
Scheme 3. Synthetic Precursors to Macrocidin A (3) and Analogs
i) R3CO2H, dicyclohexylcarbodiimide (DCC), 4-(dimethylamino)pyridine (DMAP), Et3N, CH2Cl2,
reflux, 24 h. ii) Trifluoroacetic acid (TFA), CH2Cl2, 208, 2 h. iii) Pd(PPh3)4, K2CO3, THF/MeOH, reflux,
24 h.
The starting a-amino esters 5 and 6 leading up to aburatubolactam fragment 8a were
only weakly efficacious against the test cells. A significant increase of cytotoxicity with
IC50 values below 2.5 mm in all cancer cell lines was found for the b-hydroxytrienyl
amide 7. Fragment 8a featuring an intact lactam ring was twice as active as its
immediate precursor 7 against kidney carcinoma and leukemia cells, and of a similar
activity against the cervix carcinoma cells. Apparently, the b-hydroxyoligoenyl amide
moiety is more of a prerequisite for anticancer activity against the tested cell lines than
the integrity of the heterocyclic core. A comparison of the tetramic acids 8a and 8b,
featuring identical residues at N(1) and C(3), underscores the influence of the residue
at C(5). The 5-Bn derivative 8b was more than 20-fold less active than the analog 8a.
The two series of 5-benzyl-N-methyl-3-enoyl-tetramic acids 8b–8d and N,5-dimethyl-3-
enoyl-tetramic acids 8e and 8f revealed that the length of a 3-alkenoyl substituent and
even the number of its C¼C bonds has little influence on the cytotoxicity while a 3-(4-
methoxycinnamoyl) residue is detrimental. A pairwise comparison of tetramic acids 8c/
11c, 8d/11d, and 8e/11e, which differed only in R1 being either Me or H, showed a
distinctly higher activity for the compounds featuring an N-H group. However, there
was virtually no activity difference between N-Boc-protected macrocidin fragment
analogues 19a–19d and their deprotected counterparts 11g–11j (R1 ¼H), which
corroborates the assumption of a rapid intracellular cleavage of the Boc group.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Table 1. Inhibitory Concentrationsa), IC50 (120 h; in mm), of the New Tetramic, Tetronic, and Thiotetronic
Acids When Applied to Cells of Human KB-3-1 Cervix Carcinoma, A-498 Kidney Carcinoma, U-937
Leukemia, and to L929 Mouse Fibroblasts
Compound
Cell lines
KB-3-1
A-498
U-937
L929
53
38
9.5
4.0
91
37
>100
42
5
6
58
44
98
56
2.1
0.93
44
58
37
7
1.7
1.6
0.75
13
8.6
83
8a
8b
2.0
50
8c
16
16
>100
16
8d
88
8e
36
14
8f
>100
7.6
57
6.1
>100
16
>100
11
57
7.5
>100
13
>100
>100
16
106
17
>100
41
11c
11d
11e
11f
11g
11h
11i
11j
11k
11l
11m
11n
16e
16f
17f
18
5.4
66
6.8
27
70
15
22
14
39
76
60
72
82
29
26
12
13
64
22
25
7.3
8.0
>100
>100
45
45
37
>100
>100
23
>100
>100
54
64
51
>100
>100
87
>100
>100
66
61
78
>100
>100
52
44
>100
58
19a
19b
19c
19d
19e
19f
19g
19h
Cisplatin
26
15
8.5
44
22
17
30
5
1.1
29
19
15
20
53
32
46
14
24
17
8.5
52
27
30
>75
64
50
46
15
2.5
13
27
22
24
7
3.1
–b)
a) Values are derived from concentration–response curves obtained by measuring the percentage of
grown cells relative to untreated controls after 5 d of incubation using an MTT assay. Values represent
means of two experiments in parallel. b) Not measured.
However, of the 5-(4-hydroxybenzyl)tetramic acids 11k–11n (R1 ¼H) and their
congeners 19e–19h (R1 ¼Boc), the latter were noticeably more cytotoxic than the
former, possibly due to a greater lipophilicity and thus an enhanced uptake. Tetronic
acid 16e which bears the same 5-methyl-3-dodec-2-enoyl residues as tetramic acids 8e
and 11e proved far less cytotoxic than these in all tested cells lines. Among the

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2835
macrocidin A precursors, compound 11i that features a 3-octanoyl side chain like
macrocidin A itself was found most active.
3-Acyl-tetronic and -tetramic acids are also frequently distinguished by high
antimicrobial activities. In tests against a broad panel of bacteria, yeasts, and molds,
only the tetramic acids listed in Table 2 showed any activity. As structurally closely
related derivatives gave rise to quite different antimicrobial effects, a cogent structure–
activity relationship cannot be deduced here. None of the compounds had any effect on
the growth of the yeasts, molds, and Gram-negative bacteria indicated in the legend to
Table 2.
Table 2. Tetramic Acids Exhibiting Antimicrobial Activity in Selected Bacteriaa)b)
8a
11e
11h
11i
19h
E. coli tolC (Gram-neg.)
0
8
0
0
7
10
10
12
9
11
11
11
0
0
7
8
0
8
9
9
Staphylococcus aureus (Gram-pos.)
Micrococcus luteus (Gram-pos.)
Mycobacterium phlei (Gram-pos.)
a) Agar plates inoculated with the respective microorganisms were incubated with 6-mm cellulose discs
containing 20 ml of a MeOH solution (1 mg mlꢀ1) of the compounds tested. The diameters [mm] of the
resulting growth-inhibition zones were determined after 24 h of incubation at 308 and are cited here.
b) None of the compounds inhibited the growth of the yeasts Hansenula anomala and Saccharomyces
cerevisiae, or of the molds Aspergillus fumigatus, Botrytis cinerea, and Pythium debaryanum, or of the
Gram-negative bacteria Klebsiella pneumoniae and Pseudomonas aeruginosa. All other tetramic acids
and their precursors were virtually inactive against all tested bacteria.
Conclusions. – 3-Oligoenoyl-tetramic acids exhibit anticancer activities against
various tumor cell lines in vitro the magnitude of which depend mainly on structural
parameters such as the nature of the residues at N(1), C(3), and C(5). 3-Alka-
oligoenoyl tetramic acids were more efficacious than their 3-(4-methoxycinnamoyl)
congeners but did not vary significantly in activity with the length of the alkyl chain. N-
H 3-acyltetramic acids were generally more active than their N-Me or N-Boc analogs.
Tetramates with different residues of similar polarity at C(5) exhibited comparable
anticancer activities, e.g., the Bn/Me couples 8c/8e and 11c/11e. In contrast, the
activities of 8a and 8b that bear residues of markedly divergent polarities at C(5)
differed by a factor of 20 in favor of the more polar 8a. This might be due to a better
solubility or uptake of the latter. Tetronic and thiotetronic acids were far less
antiproliferative in cancer cells when compared with identically substituted tetramic
acids. Interestingly, the acyclic b-hydroxyoctatrienoyl amide 7 also exhibited distinct
activity with an IC50 (120 h) value of <2.5 mm against all tested cells despite its lack of
an intact pyrrolidin-2,4-dione ring. Further investigations to elucidate the uptake
pathways of 3-oligoenoyltetramic acids as a function of the polarity and size of their key
substituents, as well as their main biochemical targets, are under way.
This work was financially supported by the Deutsche Forschungsgemeinschaft (grants La 907/8-2, Sa
365/3-2, and Scho 402/9-2), the EU (ERASMUS fellowship for F. B.), and the Fonds der Chemischen
Industrie.

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Experimental Part
General. Abbreviations. MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide;
PBS, phosphate buffered saline. All reagent-grade chemicals were purchased from Aldrich, Merck,
Acros, Alfa Aesar, and ABCR. All solvents were dried and distilled before use. Column chromatography
(CC): silica gel 60 (230–400 mesh). M.p.: Gallenkamp or a Bꢀchi SMP 20 apparatus; uncorrected.
Optical rotations: at 589 nm with a Perkin-Elmer polarimeter 241. IR Spectra: Perkin-Elmer Spectrum
One FT-IR spectrophotometer equipped with an ATR sampling unit, ˜n in cmꢀ1. NMR Spectra: Bruker
Avance 300 and 500 spectrometer; chemical shifts (d) are given in ppm downfield from Me4Si as internal
standard for 1H and 13C; coupling constants (J) in Hz, ꢃ(taut.)ꢄ denotes signals of identical atoms in
different tautomers with the major tautomer marked by *. EI-MS: Varian MAT 311A. ESI-TOF-MS:
Bruker Daltonics micrOTOF_Q. GC/MS: HP 5890 series II, HP-5 column (Hewlett-Packard) with He as
carrier gas. A Knauer system with UV detector K-2000 and pump K-1800 was used for prep. HPLC. Anal.
HPLC was performed on a Beckman system with solvent module 126 and a diode array detector 168
equipped with a Nucleodex CD-b-PM column (Macherey–Nagel). LiChrosolv solvents (HPLC grade)
were purchased from Merck. Microanalyses: Perkin-Elmer 2400 CHN elemental analyser; correct
analyses (within ꢁ0.4% for C, H, N) were obtained for all new compounds. 2,2-Dimethyl-6-[(1E,3E)-
penta-[1,3]-dienyl]-4H-[1,3]-dioxin-4-one was prepared according to [16].
Chemistry1). Synthesis and Characterization of the Aburatubolactam Fragment 8a. Synthesis of
Methyl (3S)-N5-[(tert-Butoxy)carbonyl]-3-{[(tert-butyl)(dimethyl)silyl]oxy}-l-ornithinate (5). A soln.
of azido compound 4 (805 mg, 2.00 mmol) [9] in dry MeOH (50 ml) was treated with freshly dried SnCl2
(773 mg, 4.00 mmol) and stirred at r.t. for 5 h. The soln. was concentrated under reduced pressure, diluted
with 50 ml of AcOEt, washed successively with 50 ml each of sat. aq. NaHCO3 and brine, and dried
(MgSO4). Filtration over a pad of Celite gave 5 (700 mg, 93%). Faintly yellow oil. Rf (AcOEt/hexane
1:3) 0.10. [a]2D0 ¼ þ1.9 (c ¼ 1.00, CHCl3). IR: 3392, 2954, 2932, 2858, 2366, 1967, 1700, 1509, 1467, 1390,
1365, 1250, 1169, 1092, 1048, 1006, 912, 836, 776, 752, 646. 1H-NMR (500 MHz, CDCl3): 0.09 (s, 3 H); 0.11
(s, 3 H); 0.91 (s, 9 H); 1.41 (s, 9 H); 1.50–1.53 (m, 1 H); 1.73–1.88 (m, 3 H); 3.21 (dd, J¼12.3, 6.5, 2 H);
3.65 (d, J¼4.6, 1 H); 3.73 (s, 3 H); 4.02 (dt, J¼7.5, 4.6, 1 H); 4.78 (br., 1 H); 10.8–9.8 (br., 1 H).
13C-NMR (125 MHz, CDCl3): ꢀ4.7; ꢀ4.8; 17.9; 25.7; 28.4; 32.0; 37.2; 52.6; 59.1; 72.6; 78.9; 155.8; 173.3.
APCI-MS: 377 (62), 321 (92), 277 (100), 260 (29), 245 (33). HR-APCI-MS: 377.2463 ([MþH]þ
,
C17H37N2O5Siþ ; calc. 377.2466).
Synthesis of Methyl (3S)-N5-[(tert-Butoxy)carbonyl]-3-{[(tert-butyl)(dimethyl)silyl]oxy}-N2-meth-
yl-l-ornithinate (6). A soln. of 5 (113 mg, 0.30 mmol) in dry CH2Cl2 (10 ml) was treated with Hꢀnigꢄs base
(56.1 ml, 42.0 mg, 0.33 mmol) and 4-nitrobenzenesulfonyl chloride (NosCl; 81.0 mg, 0.30 mmol) and
stirred for 24 h at r.t. After hydrolysis with H2O (10 ml) and dilution with AcOEt (50 ml), the layers were
separated, and the aq. layer was extracted with AcOEt (3ꢂ20 ml), and the combined org. layers were
washed with brine (20 ml), dried (MgSO4), and evaporated, followed by CC (silica gel; hexane/AcOEt
6 :1; Rf 0.42) to yield 4-nitrobenzenesulfonamide (0.25 mmol, 83%) as a pale yellow oil. [a]2D0 ¼ þ39.8
(c ¼ 1.00, CHCl3). IR: 3352, 2932, 2858, 2361, 1745, 1685, 1607, 1531, 1436, 1348, 1311, 1252, 1164, 1091,
1010, 911, 836, 777, 734, 684, 613. 1H-NMR (300 MHz, CDCl3): 0.07 (s, 3 H); 0.08 (s, 3 H); 0.85 (s, 9 H);
1.45 (s, 9 H); 1.74–1.83 (m, 2 H); 3.17–3.31 (m, 2 H); 3.53 (s, 3 H); 4.03–4.18 (m, 2 H); 4.62 (br. s, 1 H);
5.90 (d, J ¼ 7.6, 1 H); 8.03–8.08 (m, 2 H); 8.32–8.38 (m, 2 H). 13C-NMR (75 MHz, CDCl3): ꢀ5.7; ꢀ3.9;
17.8; 25.5; 28.3; 34.2; 36.9; 52.4; 60.3; 72.1; 79.4; 124.2; 128.4; 145.8; 150.0; 155.9; 169.4. ESI-MS: 584
([MþNa]þ ), 528, 484, 463, 278. HR-ESI-MS: 584.2071 ([MþNa]þ , C23H39N3NaO9SSiþ ; calc.
584.2068).
To a soln. of 4-nitrobenzenesulfonamide (280 mg, 0.50 mmol) in dry DMF (12 ml) were added
K2CO3 (137 mg, 1.00 mmol) and MeI (34.0 ml, 77.7 mg, 0.55 mmol), and the mixture was stirred for 16 h
at r.t. After evaporation of the solvent, the crude product was purified by CC (silica gel; hexane/AcOEt
4 :1) to yield the N-methyl-4-nitrobenzenesulfonamide (250 mg, 0.42 mmol, 84%) as a yellow oil. Rf
(hexane/AcOEt 4 :1) 0.46. [a]2D0 ¼ þ55.0 (c ¼ 1.00, CHCl3). IR: 2933, 2859, 2559, 2364, 1742, 1709, 1675,
1
)
Synthetic details and characterizations of all new compounds are available upon request from the
authors.

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2837
1532, 1466, 1353, 1311, 1252, 1172, 1095, 1008, 905, 834, 780, 735. 1H-NMR (500 MHz, CDCl3): ꢀ0.01 (s,
3 H); 0.02 (s, 3 H); 0.82 (s, 9 H); 1.44 (s, 9 H); 1.75–1.84 (m, 1 H); 1.90–2.00 (m, 1 H); 2.91 (s, 3 H);
3.26–3.36 (m, 2 H); 3.38 (s, 3 H); 4.20 (ddd, J ¼ 8.4, 5.3, 3.9, 1 H); 4.53 (d, J ¼ 8.4, 1 H); 4.78 (br., 1 H);
7.96–8.00 (m, 2 H); 8.32–8.35 (m, 2 H). 13C-NMR (75 MHz, CDCl3): ꢀ5.3; ꢀ3.9; 18.0; 25.8; 28.4; 31.5;
33.5; 35.9; 51.5; 61.7; 69.6; 76.5; 124.3; 128.9; 144.0; 150.4; 156.0; 168.9. ESI-MS: 598 ([MþNa]þ ), 542,
498, 476, 400, 363, 258, 128. HR-ESI-MS: 598.2231 ([MþNa]þ , C24H41N3NaO9SSiþ ; calc. 598.2225).
A soln. of N-methyl-4-nitrobenzenesulfonamide (125 mg, 0.21 mmol) in DMF (2 ml) was treated
with PhSH (24.6 ml, 26.6 mg, 0.24 mmol) and K2CO3 (83 mg, 0.60 mmol), and stirred for 3 h at r.t. After
quenching the reaction with sat. NaHCO3 soln. (10 ml), the mixture was extracted three times with
AcOEt (100 ml). The combined org. layers were washed with H2O (3ꢂ20 ml), the solvent was
evaporated, and the crude product was purified by CC (silica gel, hexane/AcOEt 2 :1) to yield 6 (98 mg,
0.25 mmol). Yellow oil. Rf (hexane/AcOEt 3 :1) 0.13. [a]2D0 ¼ þ15.6 (c ¼ 1.00, CHCl3). FT-IR (ATR):
3350, 2932, 2890, 2858, 2802, 2548, 2366, 2176, 2005, 1703, 1509, 1457, 1390, 1364, 1249, 1169, 1092, 1058,
1034, 1006, 936, 834, 775, 666. 1H-NMR (500 MHz, CDCl3): 0.06 (s, 3 H); 0.09 (s, 3 H); 0.88 (s, 9 H); 1.43
(s, 9 H); 1.62–1.72 (m, 1 H); 1.82 (br., 1 H); 1.84–1.93 (m, 1 H); 2.29 (s, 3 H); 3.12–3.28 (m, 3 H); 3.65
(s, 3 H); 3.96 (dd, J¼10.7, 5.7, 5.6, 1 H); 5.14 (br. s, 1 H). 13C-NMR (125 MHz, CDCl3): ꢀ5.0; ꢀ4.6; 17.8;
25.6; 28.3; 33.3; 35.0; 36.8; 51.6; 67.9; 72.2; 78.7; 155.8; 173.6. APCI-MS: 391 (100, [MþH]þ ), 335 (58),
291 (16), 274 (9), 259 (3). HR-APCI-MS: 391.2622 ([MþH]þ , C18H39N2O5Siþ ; calc. 391.2623).
Synthesis of Methyl (3S)-N5-[(tert-Butoxy)carbonyl]-3-{[(tert-butyl)(dimethyl)silyl]oxy}-N2-
[(2Z,4E,6E)-3-hydroxyocta-2,4,6-trienoyl]-N2-methyl-l-ornithinate (7). To a soln. of 2,2-dimethyl-6-
[(1E,3E)-penta-1,3-dienyl]-4H-[1,3]-dioxin-4-one (97.0 mg, 0.50 mmol) in dry toluene (10 ml) was added
6 (196 mg, 0.50 mmol), and the mixture was stirred for 4 h at 1108. Concentration under reduced pressure
and CC (silica gel; hexane/AcOEt 4 :1; Rf 0.25) afforded 7 (0.41 mmol, 82%). Pale yellow oil. Due to the
presence of keto–enol tautomers and two different rotamers of the enol form of 7, severe overlap of 1H-
and 13C-NMR signals of the isomers was observed, and thus no detailed NMR spectroscopic data are
given. However, the presence of a signal at 14.0 ppm (s, 1 H, ¼C(OH)ꢀ) in the 1H-NMR and a signal at
88.5 ppm (ꢀC(OH)¼CHꢀC¼O) in the 13C-NMR indicated the enol form, whereas the presence of a
signal a 3.70 ppm (m, COCH2C¼O) in the 1H-NMR and a signal at 193.2 ppm (COCH2C¼O) indicated
the keto form. IR: 2933, 2858, 2365, 2182, 1971, 1741, 1708, 1635, 1530, 1472, 1411, 1367, 1254, 1168, 1109,
1041, 998, 838, 779, 677. ESI-MS: 549 ([MþNa]þ ), 493, 449, 413, 361, 313, 181. HR-ESI-MS: 549.2966
([MþNa]þ , C26H46N2NaO7Siþ ; calc. 549.2966).
Synthesis of tert-Butyl ((3S)-3-{[(tert-Butyl)(dimethyl)silyl]oxy}-3-{(2S,4Z)-4-[(2E,4E)-1-hydroxy-
hexa-2,4-dien-1-ylidene]-1-methyl-3,5-dioxopyrrolidin-2-yl}propyl)carbamate (8a). A soln. of 7 (109 mg,
205 mmol) in abs. MeOH (1 ml) was treated with a freshly prepared MeONa soln. (0.025m, 4.12 ml,
103 mmol) and stirred vigorously at r.t. for 4 h. The soln. was poured into a mixture of AcOEt/H2O 1:1
(50 ml), acidified with HCl (pH 1) beforehand. The soln. was extracted with AcOEt (3ꢂ150 ml), dried
(MgSO4), and concentrated under reduced pressure. CC (silica gel; hexane/AcOEt 4 :1; Rf 0.05) gave 8a
(94.0 mg, 190 mmol, 93%). Brown oil. [a]2D0 ¼ ꢀ43.0 (c¼1.00, MeOH). IR: 2934, 2859, 2362, 1702, 1524,
1570, 1449, 1410, 1368, 1254, 1163, 1099, 1001, 837, 778. 1H-NMR (300 MHz, CDCl3): 0.11 (s, 6 H); 0.88 (s,
9 H); 1.41 (s, 9 H); 1.52–1.74 (m, 2 H); 1.90 (d, J¼5.7, 3 H); 3.07 (s, 3 H); 3.10–3.23 (m, 2 H); 3.79 (d, J¼
1.9, 1 H); 3.17–4.25 (m, 1 H); 6.19–6.45 (m, 2 H); 7.01 (d, J¼15.5, 1 H); 7.42 (dd, J¼15.5, 10.0, 1 H).
13C-NMR (62.5 MHz, CDCl3): ꢀ4.5; 4.9; 17.8; 19.0; 25.7; 26.9; 28.4; 32.0; 37.6; 71.0; 71.4; 79.2; 100.9;
119.4; 131.0; 142.3; 145.0; 155.8; 173.5; 174.1; 191.7. ESI-MS: 495 ([MþH]þ ), 427, 395, 377, 335, 263, 245,
195, 177, 102. HR-ESI-MS: 495.2895 ([MþH]þ , C25H43N2O6Siþ ; calc. 495.2885).
General Procedure for the Synthesis of Compounds 8b–8f, 11c–11f, 16e, 16f, and 17 [14]. Under an
inert atmosphere, a soln. of Ph3PCCO (302 mg, 1.0 mmol) in dry THF (20 ml) was added dropwise over a
period of 20 min to a refluxing soln. of the respective tetramic acid (i.e., 9, 10, 12, 13), tetronic acid (i.e.,
14), or thiotetronic acid (i.e., 15) (1.0 mmol) in dry THF (60 ml). Heating was continued for 16 h, then
half of the solvent was evaporated, pentane was added to the residue, and the product 3-
(phosphoranylideneacetyl)-pyrrolidinone or -dihydrofurandione or -dihydrothiophendione, resp., was
allowed to precipitate. It was collected by filtration, washed, and dried or recrystallized. Under an inert
atmosphere, a soln. of the so obtained heterocyclic ylide (1.0 mmol) in dry THF (40 ml) was treated with
tBuOK (112 mg, 1.0 mmol), and the resulting mixture was heated at reflux for 20 min. A soln. of the

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
respective aldehyde (1.0 mmol) in dry THF (10 m) was added dropwise, and the mixture thus obtained
was heated at reflux for another 12 h [14]. The resulting mixture was treated with 2 equiv. AcOH and
evaporated. The crude product was purified by HPLC (Prontosil column RP-18 250ꢂ20 mm, 5 mm; flow
rate: 10 ml/min).
(5RS)-5-Benzyl-3-[(2E,4E)-1-hydroxyhexa-2,4-dien-1-ylidene]-1-methylpyrrolidine-2,4-dione (8b).
According to the general procedure, 8b (45 mg, 30%) was obtained as a yellow solid from crotonaldehyde
(62 ml, 0.75 mmol) and 1-methyl-5-benzyl-3-[(triphenylphosphoranylidene)acetyl]pyrrolidine-2,4-dione
(505 mg, 1 mmol) [14]. CH2Cl2 was used as solvent instead of abs. THF, and the mixture was stirred at r.t.
The crude product was purified by HPLC (MeOH/H2O 65 :35). M.p. 568 (dec.). IR (ATR): 3028, 2923,
2853, 1698, 1614, 1563, 1441, 1402, 1376, 1354, 1290, 1256, 1178, 1079, 1000, 937, 909, 855, 799, 730, 699,
647, 628, 609, 593, 551, 528. 1H-NMR (300 MHz, CDCl3): 1.88 (d, J ¼ 6.3, 3 H); 2.87*, 2.88, 2.89 (3ꢂtaut.,
s, 3 H); 3.07, 3.17 (dd, J ¼ 14.6, 5.8, 4.6, 1 H); 3.92*, 4.04 (taut., dd, J ¼ 5.8, 4.6, 1 H); 6.23 (dd, J ¼ 15.1,
6.3, 1 H); 6.34 (dd, J ¼ 15.1, 10.2, 1 H); 7.02 (d, 15.3, 1 H); 7.08–7.25 (m, 5 H); 7.36 (dd, J ¼ 15.3, 10.2,
1 H); 13C-NMR (75.5 MHz, CDCl3): 19.0; 27.7; 35.9; 100.2; 119.4; 127.0; 129.0; 129.3; 131.1; 135.7; 142.1;
144.9; 173.4; 173.7; 193.5. EI-MS (70 eV): 297 (60; Mþ ), 206 (100), 138 (69), 95 (33), 91 (15), 67 (14), 42
(16).
(ꢁ)-5-Benzyl-3-[(E)-1-hydroxydodec-2-en-1-ylidene]-1-methylpyrrolidine-2,4-dione (8c). Analo-
gously to 8b, 8c (46 mg, 0.12 mmol, 12%) was obtained as a red oil from decanal (156 mg, 1 mmol)
and
5-benzyl-1-methyl-3-[(triphenylphosphoranylidene)acetyl]pyrrolidine-2,4-dione
(505 mg,
1.0 mmol) as prepared from Ph3PCCO (302 mg, 1 mol) and 5-benzyl-1-methyltetramic acid (9;
203 mg, 1 mmol) [14]. It was purified by HPLC (elution with neat MeOH after washing the polar by-
products off the column with MeOH/H2O 85 :15). IR (ATR): 2924, 1705, 1643, 1580, 1496, 1455, 1398,
1
1255, 1203, 1079, 1007, 913, 853, 699. H-NMR (300 MHz, CDCl3): 0.86 (t, J ¼ 6.9, 3 H); 1.22–1.33 (m,
10 H); 1.38–1.52 (m, 2 H); 2.30 (dt, J ¼ 7.6, 5.9, 2 H); 2.86*, 2.89 (taut., s, 3 H); 3.07 (dd, J ¼ 14.6, 5.6,
1 H); 3.18 (dd, J ¼ 14.6, 4.7, 1 H); 3.92*, 4.05 (taut., dd, J ¼ 5.6, 4.7, 1 H); 7.04 (d, J ¼ 15.7, 1 H); 7.1–7.6
(m, 6 H). 13C-NMR (75.5 MHz, CDCl3): 14.1; 22.6; 27.7; 28.2; 29.3; 29.4; 29.5; 31.8; 33.3; 35.9; 67.8; 99.7*,
102.4 (taut.); 120.6, 121.3* (taut.); 127.0; 128.5; 129.3; 135.2, 135.7* (taut.); 150.6*, 151.6 (taut.); 173.3;
173.8; 193.6. EI-MS (70 eV): 383 (37; Mþ ), 292 (100), 230 (8), 152 (6), 140 (11), 91 (18), 43 (22).
(ꢁ)-5-Benzyl-3-[(E)-1-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]-1-methylpyrrolidine-2,4-
dione (8d; 119.9 mg, 0.33 mmol, 33%) was obtained from Ph3PCCO (302 mg, 1.0 mmol), 9 (203 mg,
1.0 mmol), and 4-methoxybenzaldehyde (136 mg, 1.0 mmol). Purification by HPLC (elution with a
gradient MeOH/H2O 55 :45 to 100 :0 within 15 min) left a yellow solid. M.p. 145–1488. IR (ATR): 2928,
2172, 1694, 1628, 1580, 1567, 1510, 1456, 1405, 1353, 1288, 1255, 1171, 1139, 1116, 1024, 988, 938, 921, 868,
849, 834, 812, 756, 725, 705. 1H-NMR (300 MHz, CDCl3): 2.89, 2.92 (taut., s, 3 H); 3.10 (dd, J ¼ 14.4, 5.5,
1 H); 3.20 (dd, J ¼ 14.4, 4.6, 1 H); 3.84 (s, 3 H); 3.96 (t, J ¼ 4.6, 1 H); 6.91 (d, J ¼ 10.2, 2 H); 7.11–7.25 (m,
5 H); 7.59 (d, J ¼ 14.4, 1 H); 7.61 (d, J ¼ 10.2, 2 H); 7.76 (d, J ¼ 14.4, 1 H). 13C-NMR (75.5 MHz, CDCl3):
27.6; 35.8; 55.4; 67.8; 100.1; 114.4; 115.5; 126.9; 127.4; 128.56; 129.25; 130.8; 135.7; 144.0; 162.09; 173.25;
173.73; 193.58. EI-MS (70 eV): 363 (22; Mþ ), 277 (70), 272 (44), 230 (14), 201 (16), 161 (28), 138 (45),
83 (92), 48 (100). HR-EI-MS: 362.1392 ([Mꢀ1]þ ; calc. 362.1392).
(ꢁ)-3-[(E)-1-Hydroxydodec-2-en-1-ylidene]-1,5-dimethylpyrrolidine-2,4-dione (8e; 61.5 mg,
0.2 mmol, 20%) was obtained from Ph3PCCO (302 mg, 1.0 mmol), 1,5-dimethyltetramic acid ((ꢁ)-12;
127 mg, 1.0 mmol), and decanal (156 mg, 1.0 mmol). It was purified by HPLC (elution with neat MeOH
after washing the polar by-products off with MeOH/H2O 85 :15) to afford a red oil. IR (ATR): 2924,
2853, 1706, 1644, 1580, 1484, 1445, 1401, 1367, 1306, 1236, 1152, 1119, 1068, 985, 927, 790, 748, 721, 695.
1H-NMR (300 MHz, CDCl3): 0.85 (t, J ¼ 6.6, 3 H); 1.20–1.30 (m, 12 H); 1.33 (d, J ¼ 7.0, 3 H); 1.43–1.49
(m, 2 H); 2.29 (q, J ¼ 6.8, 2 H); 2.94, 2.96* (taut., s, 3 H); 3.67*, 3.80 (taut., q, J ¼ 7.0, 1 H); 7.07 (d, J ¼
16.0, 1 H); 7.10–7.30 (m, 1 H). 13C-NMR (75.5 MHz, CDCl3): 14.1; 14.9; 22.6; 26.3; 27.9; 28.2; 29.25;
29.3; 29.4; 31.8; 33.2; 62.3; 98.9; 121.3; 150.4; 173.1; 173.6; 194.8. EI-MS (70 eV): 307 (44, Mþ ), 277 (28),
222 (3), 208 (11), 180 (72), 169 (46), 154 (100), 128 (13), 97 (19), 83 (41), 58 (39).
(ꢁ)-3-[(E)-1-Hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]-1,5-dimethylpyrrolidine-2,4-dione
(8f; 72 mg, 0.25 mmol, 25%) was obtained from Ph3PCCO (302 mg, 1.0 mmol), (ꢁ)-12 (127 mg,
1.0 mmol), and 4-methoxybenzaldehyde (136 mg, 1.0 mmol). HPLC Purification (MeOH/H2O gradient
from 55 :45 to 100 :0 within 15 min) gave a yellow solid. M.p. 1328. IR (ATR): 3398, 2973, 2901, 2837,

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2839
2347, 1694, 1597, 1579, 1511, 1441, 1423, 1398, 1370, 1307, 1289, 1248, 1171, 1112, 1029, 981, 932, 828, 787,
1
735, 702. H-NMR (300 MHz, CDCl3): 1.36 (d, J ¼ 7.5, 3 H); 2.95, 2.98 (taut., s, 3 H); 3.71 (q, J ¼ 7.5,
1 H); 3.73, 3.83 (taut., s, 3 H); 6.90 (d, J ¼ 8.8, 2 H); 7.59 (d, J ¼ 8.8, 2 H); 7.63 (d, J ¼ 15.9, 1 H); 7.79 (d,
J ¼ 15.9, 1 H). 13C-NMR (75.5 MHz, CDCl3): 15.0; 16.5; 26.4; 53.4; 55.4; 60.3; 62.6; 99.4; 113.6; 115.5;
114.5; 127.4; 128.4; 131.0; 143.9; 159.5; 162.1; 173.1; 173.7; 194.9*, 196.6 (taut.). EI-MS (70 eV): 287 (100,
Mþ ), 258 (24), 244 (49), 201 (25), 174 (28), 161 (52), 134 (36), 103 (11), 77 (14), 58 (65).
(5S)-5-Benzyl-3-[(E)-1-hydroxydodec-2-en-1-ylidene]pyrrolidine-2,4-dione (11c). Analogously to
8c, 11c (120 mg, 38%) was obtained as a white solid from Ph3PCCO (302 mg, 1.0 mmol), (S)-5-
benzyltetramic acid (10; 189 mg, 1.0 mmol), and decanal (156 mg, 1.0 mmol). It was purified by HPLC
(MeOH/H2O 85 :15). M.p. 120–1258 (MeOH/pentane 1:2). [a]2D0 ¼ ꢀ31 (c¼0.025, CHCl3). IR (ATR):
3200, 2920, 2848, 1707, 1647, 1594, 1428, 1280, 984, 879, 722, 693. 1H-NMR (300 MHz, (D6)DMSO): 0.85
(t, J ¼ 6.6, 3 H); 1.18–1.40 (m, 12 H); 1.45–1.64 (m, 2 H); 2.30 (q, J ¼ 6.6, 2 H); 2.92–2.96 (m, 2 H); 4.21
(t, J ¼ 6.6, 1 H); 7.05 (d, J ¼ 15.4, 1 H); 7.11–7.28 (m, 5 H); 7.21–7.30 (m, 1 H). 13C-NMR (75.5 MHz,
(D6)DMSO): 13.9; 22.1; 27.6; 29.0; 29.1; 29.2; 29.3; 29.4; 31.6; 36.7; 64.5; 100.2; 121.2; 126.8; 128.5; 130.1;
136.3; 150.3; 173.3; 175.5; 195.4. EI-MS (70 eV): 313 (100, Mþ ), 270 (10), 242 (100), 189 (10), 120 (70),
91 (100), 55 (45).
(5S)-5-Benzyl-3-[(E)-1-hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]pyrrolidine-2,4-dione
(11d; 109 mg, 31%) was obtained as a yellow solid from Ph3PCCO (302 mg, 1.0 mmol), 10 (189 mg,
1 mmol), and 4-methoxybenzaldehyde (136 mg, 1 mmol). M.p. 158–1608 (MeOH/pentane 1:2). [a]D25
¼
ꢀ44 (c¼0.05, CHCl3). IR (ATR): 3185, 1661, 1630, 1553, 1421, 1252, 1171, 977, 824, 698. 1H-NMR
(300 MHz, (D6)DMSO): 2.95–2.98 (m, 2 H); 3.83 (s, 3 H); 4.21 (t, J ¼ 6.0, 1 H); 7.04 (d, J ¼ 8.4, 2 H);
7.11–7.28 (m, 5 H); 7.47 (d, J ¼ 15.6, 1 H); 7.66 (d, J ¼ 8.4, 2 H); 7.75 (d, J ¼ 15.6, 1 H); 8.92 (s, 1 H);
13C-NMR (75.5 MHz, (D6)DMSO): 36.7; 55.5; 103.5; 114.8; 114.9; 126.6; 126.8; 128.1; 129.7; 130.7; 136.0;
143.4; 162.0; 173.0; 175.1; 194.9. EI-MS (70 eV): 350 (100, Mþ ), 258 (100), 201 (12), 161 (80), 133 (19),
91 (48). HR-EI-MS: 348.1236 (C21H18NO4ꢀ ; calc. 348.1236), 350.1502 (C21H20NOþ4 ; calc. 350.1507).
(5S)-3-[(E)-1-Hydroxydodec-2-en-1-ylidene]-5-methylpyrrolidine-2,4-dione (11e; 103 mg, 37%) was
obtained as a white solid from Ph3PCCO (302 mg, 1.0 mmol), (S)-5-methyltetramic acid (13; 113 mg,
1.0 mmol), and decanal (156 mg, 1.0 mmol). M.p. 100–1038 (MeOH/pentane 1:2). [a]2D0 ¼ ꢀ38 (c¼0.1,
1
CHCl3). IR (ATR): 3232, 2924, 2854, 1707, 1641, 1575, 1429, 1230, 907, 729, 665. H-NMR (300 MHz,
CDCl3): 0.85 (t, J ¼ 7.4, 3 H); 1.15–1.31 (m, 12 H); 1.34 (d, J ¼ 7.1, 3 H); 1.42–1.54 (m, 2 H); 2.30 (q, J ¼
7.1, 2 H); 3.86, 4.04 (taut., q, J ¼ 7.1, 1 H); 6.15*, 6.33 (taut., s, 1 H); 7.09 (d, J ¼ 15.8, 1 H); 7.14–7.23 (m,
1 H). 13C-NMR (75.5 MHz, CDCl3): 14.1; 17.5; 22.6; 28.1; 28.2; 29.2; 29.3; 29.4; 29.5; 31.8; 33.3; 33.5;
55.5*, 57.7 (taut.); 98.7*, 101.3 (taut.); 120.9*, 121.3 (taut.); 151.3*, 152.3 (taut.); 168.8*, 174.9 (taut.);
175.5*, 175.9 (taut.); 195.8*, 204.9 (taut.). EI-MS (70 eV): 273 (60, Mþ ), 230 (35), 201 (55), 174 (20), 161
(10), 135 (25), 103 (36), 77 (70), 56 (39), 44 (30).
(5S)-3-[(E)-1-Hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]-5-methylpyrrolidine-2,4-dione
(11f; 107 mg, 36%) was obtained as a yellow solid from Ph3PCCO (302 mg, 1.0 mmol), 13 (113 mg,
1.0 mmol), and 4-methoxybenzaldehyde (136 mg, 1.0 mmol). It was purified by HPLC (MeOH/H2O
95 :5). M.p. 120–1258. [a]2D0 ¼ ꢀ64 (c¼0.1, CHCl3). IR (ATR): 3207, 3066, 2837, 1654, 1623, 1559, 1509,
1
1417, 1249, 1165, 1029, 911, 825, 725, 693. H-NMR (300 MHz, CDCl3): 1.37 (d, J ¼ 6.9, 3 H); 3.82 (s,
3 H); 4.62 (q, J ¼ 6.9, 1 H); 6.73 (s, 1 H); 6.90 (d, J ¼ 8.9, 2 H); 7.57 (d, J ¼ 8.9, 2 H); 7.62 (d, J ¼ 15.9,
1 H); 7.80 (d, J ¼ 15.9, 1 H). 13C-NMR (75.5 MHz, CDCl3): 17.5; 55.4; 57.7; 98.9; 114.5; 115.4; 127.3;
130.9; 144.6; 162.2; 174.9; 175.5; 196.0. EI-MS (70 eV): 293 (85, Mþ ), 275 (65), 194 (25), 166 (52), 155
(38), 110 (18), 81 (25), 44 (45).
(5S)-3-[(E)-1-Hydroxydodec-2-en-1-ylidene]-5-methylfuran-2,4(3H,5H)-dione (16e). Analogously
to the tetramic acids 8, 16e was obtained as a red oil (110 mg, 37%) from Ph3PCCO (302 mg, 1.0 mmol),
(S)-5-methyltetronic acid (14; 114 mg, 1.0 mmol), and decanal (156 mg, 1.0 mmol) after purification by
HPLC (Prontosil RP-18, 250ꢂ20 mm, 5 mm; flow rate: 10 ml/min, MeOH/H2O 95 :15). [a]2D0 ¼ ꢀ25 (c¼
0.05, CHCl3). IR (ATR): 2956, 2923, 2851, 1750, 1692, 1638, 1575, 1401, 1378, 1314, 1187, 1084, 1056, 1006,
1
978, 954, 852, 723. H-NMR (300 MHz, CDCl3): 0.85 (t, J ¼ 7.0, 3 H); 1.17–1.34 (m, 12 H); 1.48*, 1.51
(taut., d, J ¼ 7.0, 3 H); 2.30–2.40 (m, 2 H); 4.67*, 4.75 (taut., q, J ¼ 7.0, 1 H); 7.11*, 7.14 (taut., d, J ¼ 15.8,
1 H); 7.33–7.48 (m, 2 H, both tautomers). 13C-NMR (75.5 MHz, CDCl3): 14.3; 16.9; 22.6; 27.9; 29.0; 29.2;
29.3; 29.4; 29.5; 29.6; 29.7; 31.8; 33.6; 56.9; 96.7*, 102.3 (taut.); 129.0, 129.2 (taut.); 133.5, 133.7 (taut.);

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
167.8*, 177.0 (taut.); 178.3*, 179.6 (taut.); 195.0*, 204.2 (taut.). EI-MS (70 eV): 294 (100, Mþ ), 276 (50),
167 (30), 156 (20), 110 (30), 81 (70), 44 (50).
(5S)-3-[(E)-1-Hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]-5-methylfuran-2,4(3H,5H)-dione
(16f) was obtained as a yellow solid (90 mg, 33%) from Ph3PCCO (302 mg, 1.0 mmol), (S)-5-
methyltetronic acid (14; 113 mg, 1.0 mmol), and 4-methoxybenzaldehyde (136 mg, 1 mmol) after
purification by HPLC (MeOH/H2O 85 :15). M.p. 97–988. [a]2D5 ¼ ꢀ56 (c¼0.05, CHCl3). IR (ATR):
2933, 1753, 1683, 1624, 1593, 1576, 1512, 1375, 1259, 1171, 1020. 1H-NMR (300 MHz, CDCl3): 1.45 (d, J ¼
6.9, 3 H); 3.80 (s, 3 H); 4.57–4.75 (m, 1 H); 6.86 (d, J ¼ 8.9, 2 H); 7.51 (d, J ¼ 15.2, 1 H); 7.58 (d, J ¼ 8.9,
2 H); 7.92 (d, J ¼ 15.2, 1 H); 10.45 (br. s, 1 H). 13C-NMR (75.5 MHz, CDCl3): 16.6; 55.3; 81.2; 96.3; 113.6;
114.5; 126.6; 131.6; 147.7; 163.0; 176.6; 181.2; 203.9. EI-MS (70 eV): 275 (100, Mþ ), 245 (12), 201 (50),
174 (73), 131 (40), 77 (30). HR-EI-MS: 273.0763 (C15H13Oꢀ5 ; calc. 273.0768), 275.0919 (C15H15O5þ ; calc.
275.0914).
(ꢁ)-3-[(E)-1-Hydroxy-3-(4-methoxyphenyl)prop-2-en-1-ylidene]-5-methylthiophene-2,4(3H,5H)-
dione (17f). Analogously to the tetramic acids 8, 17f was obtained as a yellow solid (73 mg, 25%) from
Ph3PCCO (302 mg, 1.0 mmol), (ꢁ)-5-methylthiotetronic acid (15; 130 mg, 1.0 mmol), and 4-methoxy-
benzaldehyde (136 mg, 1.0 mmol) after purification by HPLC (MeOH/H2O gradient from 55 :45 to
100 :0). M.p. 97–998. IR (ATR): 2918, 2850, 1677, 1589, 1539, 1509, 1450, 1400, 1291, 1247, 1169, 1113,
1027, 986, 934, 889, 828, 780. 1H-NMR (300 MHz, CDCl3): 1.64 (d, J ¼ 6.9, 3 H); 3.85 (s, 3 H); 4.11 (q, J ¼
6.9, 1 H); 6.92 (d, J ¼ 8.8, 2 H); 7.64 (d, J ¼ 8.8, 2 H); 7.92 (d, J ¼ 15.7, 1 H); 7.99 (d, J ¼ 15.7, 1 H).
13C-NMR (75.5 MHz, CDCl3): 18.8; 46.4*, 48.8 (taut.); 55.5; 104.8, 106.1* (taut.); 114.7, 115.0* (taut.);
127.11; 128.61; 131.68; 146.4, 148.5* (taut.); 161.6, 163.00* (taut.); 177.1, 179.8* (taut.); 191.6*, 197.2
(taut.); 202.5, 205.9* (taut.). EI-MS (70 eV): 289 (100, Mþ ), 255 (14), 201 (87), 174 (60), 131 (40), 77
(23), 63 (8).
tert-Butyl (2S)-3,5-Dioxo-2-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-1-carboxylate (18). Boc-All-
yl-l-tyrosine (3.21 g, 10.0 mmol) was dissolved in anh. CH2Cl2 (40 ml), and Meldrumꢄs acid (1.59 g,
11.0 mmol), DCC (2.46 g, 12.0 mmol), and DMAP (2.44 g, 20.0 mmol) were added successively. After
stirring at r.t. for 2.5 h, the formed precipitate was removed by filtration, washed with CH2Cl2 (2ꢂ20 ml),
and the solvent was removed under reduced pressure. The residue was dissolved in AcOEt (120 ml), and
washed with 2m HCl (3ꢂ40 ml) and brine (40 ml). Subsequently, the org. layer was heated under reflux
for 1 h, and the solvent was removed under reduced pressure, to yield 18 (3.45 g, 10.0 mmol, quant.).
White amorphous solid. Rf (cyclohexane/EtOH 2 :1) 0.38. [a]2D6 ¼ þ75.3 (c¼1.0, CHCl3). IR (ATR):
3166, 2978, 2933, 1754, 1711, 1610, 1511, 1362, 1298, 1241, 1150, 1077, 1021, 998, 927, 830. 1H-NMR
(300 MHz, CD3OD): 1.60 (s, 9 H); 3.09 (dd, J¼14.0, 2.6, 1 H); 3.41 (dd, J¼14.0, 5.2, 1 H); 4.47 (ddd, J¼
5.2, 1.6, 1.5, 2 H); 4.64 (dd, J¼5.2, 2.6, 1 H); 5.22 (ddt, J¼10.6, 1.6, 1.5, 1 H); 5.36 (ddt, J¼17.3, 1.6, 1.5,
1 H); 6.03 (ddt, J¼17.3, 10.6, 5.2, 1 H); 6.78 (d, J¼8.7, 2 H); 6.97 (d, J¼8.7, 2 H); HꢀC(3) was not
observed due to D exchange. 13C-NMR (75 MHz, CD3OD): 28.5; 35.0; 62.5; 69.7; 83.6; 96.4; 115.3; 117.4;
127.5; 131.8; 135.0; 150.9; 159.2; 173.3; 178.1. EI-MS (70 eV): 345 (4, Mþ ), 272 (4), 245 (7), 220 (3), 176
(7), 147 (100), 119 (6), 107 (11).
General Procedure for the Synthesis of (5S)-1-Boc-3-(1-hydroxyalkylidene)-5-[4-(prop-2-en-1-
yloxy)benzyl]pyrrolidine-2,4-diones 19a–19d [15]. To a soln. of 18 (345 mg, 1.0 mmol) in dry CH2Cl2
(10 ml) at 08 were successively added DMAP (41 mg, 0.33 mmol), the appropriate alkanoic acid
(1.1 mmol), and DCC (250 mg, 1.2 mmol). After stirring at this temp. for 15 min, the mixture was
allowed to reach r.t. and stirred for a further 1.5 h. After cooling to 08, dry Et3N (0.15 ml, 1.1 mmol) was
added. The mixture was stirred at 08 for 15 min and subsequently refluxed for 24 h. After cooling to r.t.,
the precipitate was removed by filtration, and the org. layer was washed with 2m HCl (3ꢂ30 ml). The aq.
layer was extracted with CH2Cl2 (30 ml), dried (Na2SO4), and the solvent was removed under reduced
pressure. CC (silica gel; solvent as indicated) gave tetramic acids 19 mainly as orange to red oils. Prior to
running NMR spectra, the products were dissolved in AcOEt and washed with 0.05m Na2EDTA soln.
tert-Butyl (5S)-3-(1-Hydroxybutylidene)-2,4-dioxo-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-1-
carboxylate (19a). According to the general procedure (CC; cyclohexane/EtOH gradient progressing
from 2 :1 to 2 :1), 19a was obtained from butanoic acid (97 mg) as an orange crystalline solid (208 mg,
0.52 mmol, 52 %). Rf (cyclohexane/EtOH 3 :1) 0.40. M.p. 868. [a]2D5 ¼ ꢀ10.3 (c¼1.0, CHCl3). IR (ATR):
1
3349, 2932, 1713, 1662, 1609, 1510, 1348, 1301, 1246, 1223, 1148, 1022, 925, 800. H-NMR (300 MHz,

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2841
CD3OD): 0.83 (t, J¼7.4, 3 H); 1.50 (tq, J¼7.5, 7.4, 2 H); 1.59 (s, 9 H); 2.68 (t, J¼7.5, 2 H); 3.14 (dd, J¼
14.1, 2.6, 1 H); 3.33 (dd, J¼14.1, 5.4, 1 H); 4.42 (ddd, J¼5.2, 1.7, 1.5, 2 H); 4.51 (dd, J¼5.4, 2.6, 1 H); 5.17
(ddt, J¼10.5, 1.5, 1.5, 1 H); 5.31 (ddt, J¼17.3, 1.7, 1.5, 1 H); 5.97 (ddt, J¼17.3, 10.5, 5.2, 1 H); 6.72 (d, J¼
8.6, 2 H); 6.86 (d, J¼8.6, 2 H). 13C-NMR (75 MHz, CD3OD): 14.3; 20.4; 28.6; 34.9; 36.0; 65.0; 69.8; 84.9;
105.5; 115.7; 117.6; 127.6; 132.0; 135.0; 150.9; 159.4; 174.9; 195.2; 195.6. EI-MS (70 eV): 415 (9, Mþ ), 385
(4), 342 (5), 315 (26), 176 (8), 147 (100), 107 (29), 99 (42), 56 (100).
tert-Butyl (5S)-3-(1-Hydroxyhexylidene)-2,4-dioxo-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-1-
carboxylate (19b). Analogously to 19a, 19b (328 mg, 0.74 mmol, 74%) was obtained from hexanoic
acid (128 mg) as an orange oil after CC (gradient elution with cyclohexane/AcOEt 3 :1 to cyclohexane/
EtOH 3 :1). Rf (cyclohexane/EtOH 4 :1) 0.30. [a]2D5 ¼ ꢀ10.7 (c¼1.0, CHCl3). IR (ATR): 3349, 2931,
2857, 1714, 1663, 1608, 1510, 1348, 1301, 1246, 1158, 1025, 925. 1H-NMR (300 MHz, CD3OD): 0.88 (t, J¼
6.8, 3 H); 1.05–1.54 (m, 6 H); 1.61 (s, 9 H); 2.71 (t, J¼7.3, 2 H); 3.16 (dd, J¼14.1, 2.5, 1 H); 3.35 (dd, J¼
14.1, 5.3, 1 H); 4.44 (ddd, J¼5.2, 1.7, 1.5, 2 H); 4.52–4.66 (m, 1 H); 5.20 (ddt, J¼10.5, 1.5, 1.5, 1 H); 5.35
(ddt, J¼17.3, 1.7, 1.5, 1 H); 5.99 (ddt, J¼17.3, 10.6, 5.2, 1 H); 6.74 (d, J¼8.6, 2 H); 6.88 (d, J¼8.6, 2 H).
13C-NMR (75 MHz, CD3OD): 14.4; 23.4; 26.1; 28.6; 32.4; 34.8; 35.9; 64.9; 69.7; 84.8; 105.2; 115.6; 117.6;
127.4; 131.9; 134.8; 150.7; 159.2; 174.9; 195.2; 195.5. EI-MS (70 eV): 443 (12, Mþ ), 370 (7), 343 (36), 187
(8), 147 (100), 107 (37), 99 (45), 56 (100).
tert-Butyl (5S)-3-(1-Hydroxyoctylidene)-2,4-dioxo-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-1-
carboxylate (19c). Analogously to 19a, 19c (247 mg, 0.53 mmol, 53%) was obtained from octanoic acid
(159 mg) as an orange oil after CC (gradient elution with cyclohexane/AcOEt 3 :1 to cyclohexane/EtOH
3 :1). Rf (cyclohexane/EtOH 2 :1) 0.64. [a]2D5 ¼ ꢀ15.2 (c¼1.0, CHCl3). IR (ATR): 2928, 2857, 1713, 1661,
1
1603, 1509, 1347, 1300, 1244, 1222, 1150, 1022, 997, 922. H-NMR (300 MHz, CD3OD): 0.91 (t, J¼7.0,
3 H); 1.19–1.55 (m, 10 H); 1.62 (s, 9 H); 2.73 (t, J¼7.3, 2 H); 3.18 (dd, J¼14.1, 2.6, 1 H); 3.37 (dd, J¼
14.1, 5.4, 1 H); 4.46 (ddd, J¼5.2, 1.7, 1.5, 2 H); 4.56 (dd, J¼5.4, 2.6, 1 H); 5.21 (ddt, J¼10.5, 1.5, 1.5, 1 H);
5.35 (ddt, J¼17.3, 1.7, 1.5, 1 H); 6.01 (ddt, J¼17.3, 10.6, 5.2, 1 H); 6.76 (d, J¼8.7, 2 H); 6.90 (d, J¼8.7,
2 H). 13C-NMR (75 MHz, CD3OD): 14.6; 23.8; 27.0; 28.6; 30.2; 30.4; 32.9; 36.0; 38.9; 65.0; 69.8; 84.9;
105.4; 115.7; 117.5; 127.6; 132.0; 135.0; 151.0; 159.4; 175.2; 195.3; 195.7. EI-MS (70 eV): 471 (18, Mþ ), 398
(11), 371 (65), 225 (5), 187 (8), 176 (5), 147 (98), 146 (100), 107 (57), 91 (24), 56 (89), 42 (98).
tert-Butyl (5S)-3-(1-Hydroxydecylidene)-2,4-dioxo-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-1-
carboxylate (19d). Analogously to 19c, 19d (263 mg, 0.53 mmol, 53%) was obtained from decanoic
acid (189 mg) as an orange oil after CC (gradient elution with cyclohexane/AcOEt 3 :1 to cyclohexane/
EtOH 3 :1). Rf (cyclohexane/EtOH 3 :1) 0.42. [a]2D4 ¼ ꢀ9.8 (c¼0.5, CHCl3). IR (ATR): 3375, 2925, 2855,
1714, 1669, 1601, 1510, 1456, 1348, 1300, 1245, 1223, 1148, 1023, 922, 803. 1H-NMR (300 MHz, CD3OD):
0.90 (t, J¼6.8, 3 H); 1.20–1.57 (m, 14 H); 1.62 (s, 9 H); 2.73 (t, J¼7.4, 2 H); 3.18 (dd, J¼14.0, 2.5, 1 H);
3.38 (dd, J¼14.0, 5.3, 1 H); 4.46 (ddd, J¼5.1, 1.7, 1.5, 2 H); 4.57 (dd, J¼5.3, 2.5, 1 H); 5.21 (ddt, J¼10.6,
1.5, 1.5, 1 H); 5.35 (ddt, J¼17.3, 1.7, 1.5, 1 H); 6.01 (ddt, J¼17.3, 10.6, 5.1, 1 H); 6.76 (d, J¼8.7, 2 H); 6.90
(d, J¼8.7, 2 H). 13C-NMR (75 MHz, CD3OD): 13.1; 22.3; 25.4; 27.0; 28.8; 28.9; 29.0; 29.1; 31.6; 34.4; 35.9;
63.6; 68.3; 82.8; 103.3; 114.1; 116.0; 126.1; 130.5; 133.5; 150.0; 157.9; 171.2; 193.2; 195.4. EI-MS (70 eV):
499 (2, Mþ ), 399 (15), 253 (2), 227 (2), 187 (4), 176 (2), 147 (100), 107 (23), 91 (23), 57 (50).
General Procedure for the Synthesis of (5S)-N-Boc-3-(1-Hydroxyalkylidene)-5-(4-hydroxybenzyl)-
pyrrolidine-2,4-diones 19e–19h [17]. A soln. of the respective allyloxy ester 19a–19d in dry THF/MeOH
(5 :1, 24 ml) was successively treated with K2CO3 (3 equiv.) and Pd(PPh3)4 (2 mol-%). The resulting
mixture was stirred under reflux for 24 h, the solvent was removed under reduced pressure, the residue
was dissolved in CH2Cl2 (30 ml), and the org. layer was extracted with 1m NaOH (2ꢂ30 ml). The aq.
layer was acidified to pH 2 with conc. HCl, and extracted with AcOEt (3ꢂ30 ml). Drying (Na2SO4) and
removal of the volatiles under reduced pressure gave the deprotected tetramic acids 19e–19h. To obtain
well-resolved NMR spectra, these compounds were dissolved in AcOEt and washed with 0.05m
Na2EDTA soln.
tert-Butyl (5S)-3-(1-Hydroxybutylidene)-5-(4-hydroxybenzyl)-2,4-dioxopyrrolidine-1-carboxylate
(19e; 95 mg, 0.25 mmol, 64%) was obtained from 19a (162 mg, 0.39 mmol). Reddish crystalline solid.
M.p. 818. Rf (cyclohexane/EtOH 1:1) 0.56. [a]2D6 ¼ ꢀ31.8 (c¼0.5, MeOH). IR (ATR): 3283, 2967, 1594,
1514, 1444, 1351, 1223, 1148, 814. 1H-NMR (300 MHz, CD3OD): 0.87 (t, J¼7.6, 3 H); 0.90* (taut., t, J¼
7.3, 3 H); 1.48–1.65 (m, 2 H); 1.61 (s, 9 H); 2.59–3.04 (m, 2 H); 3.14 (br. d, J¼13.6, 1 H); 3.33 (dd, J¼

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
13.6, 4.9, 1 H); 3.63 (br. s, 1 H); 4.05–4.10 (m, 1 H); 4.47–4.57 (m, 1 H); 6.50–7.26 (m, 4 H). 13C-NMR
(75 MHz, CD3OD): 13.8; 20.0; 28.4; 35.5; 35.6, 37.1* (taut.); 63.4, 64.5* (taut.); 71.3; 84.7; 115.9; 127.2;
131.6; 150.9; 157.2; 172.9; 190.6; 195.3. EI-MS (70 eV): 351 (26), 274 (62), 259 (9), 230 (8), 199 (13), 198
(17), 169 (20), 152 (9), 107 (100), 77 (16), 56 (46), 41 (97).
tert-Butyl (5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxyhexylidene)-2,4-dioxopyrrolidine-1-carboxylate
(19f; 29 mg, 50%) was obatined from 19b (62 mg, 0.14 mmol). Reddish oil. Rf (cyclohexane/EtOH
1:1) 0.55. [a]2D6 ¼ ꢀ28.1 (c¼0.5, MeOH). IR (ATR): 3285, 2929, 1594, 1514, 1445, 1351, 1257, 1151, 1102,
1
1016, 811. H-NMR (300 MHz, CD3OD): 0.91 (t, J¼6.7, 3 H); 1.17–1.64 (m, 6 H); 1.61 (s, 9 H); 2.59–
2.84 (taut., m, 2 H); 2.89* (dd, J¼14.0, 5.4, 1 H); 2.98* (dd, J¼14.0, 4.2, 1 H); 3.05–3.21 (m, 2 H); 3.53–
3.69 (m, 1 H); 4.39–4.49, 4.59–4.69 (taut., m, 1 H); 6.56–7.10 (m, 4 H). 13C-NMR (75 MHz, CD3OD):
14.0; 23.5; 26.8; 28.6; 29.3; 32.5; 37.5; 64.1; 71.6; 84.2; 103.4; 116.2; 125.1, 127.5 (taut.); 132.0; 151.3; 157.1;
173.2; 198.0. EI-MS (70 eV): 331 (2, [MꢀOtBu]þ ), 303 (15, [MꢀBoc]þ ), 277 (6), 226 (6), 197 (46), 164
(5), 147 (7), 141 (8), 126 (10), 107 (100), 91 (7), 77 (16), 57 (14).
tert-Butyl (2S)-2-(4-Hydroxybenzyl)-4-(1-hydroxyoctylidene)-3,5-dioxopyrrolidine-1-carboxylate
(19g; 45 mg, 0.10 mmol, 14%) was obtained from 19c (236 mg, 0.72 mmol). Reddish oil. Rf (cyclo-
hexane/EtOH 2 :1) 0.62. [a]2D6 ¼ ꢀ41 (c¼0.5, MeOH). IR (ATR): 3293, 2929, 1647, 1595, 1514, 1446,
1350, 1225, 1149, 816. 1H-NMR (300 MHz, CD3OD): 0.83–0.96 (m, 3 H); 1.14–1.41 (m, 10 H); 1.61 (s,
9 H); 2.60–3.03 (m, 2 H); 3.16 (br. d, J¼14.0, 1 H); 3.33 (br. d, J¼14.0, 1 H); 3.64 (br. s, 1 H); 4.03–4.14,
4.47–4.56 (taut., m, 1 H); 6.52–7.15 (m, 4 H). 13C-NMR (75 MHz, CD3OD): 14.6; 23.8; 26.2, 26.8 (taut.);
28.6; 30.1; 30.3; 32.9; 34.1, 34.9*, 35.1 (3ꢂtaut.); 36.0; 37.5; 64.5, 65.1 (taut.); 71.5; 84.8; 105.3; 115.6,
116.2*, 117.5 (3ꢂtaut.); 126.0, 127.5 (taut.); 132.0*, 133.1 (taut.); 150.9; 157.5; 157.8; 177.8; 195.5. EI-MS
(70 eV): 431 (1, Mþ ), 371 (4), 331 (9), 225 (33), 107 (100), 56 (44).
tert-Butyl (5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxydecylidene)-2,4-dioxopyrrolidine-1-carboxylate
(19h; 63 mg, 0.14 mmol, 41%) was obtained from 19d (170 mg, 0.34 mmol) as a reddish oil. Rf
(cyclohexane/EtOH 2 :1) 0.55. [a]2D6 ¼ ꢀ27 (c¼1.0, MeOH). IR (ATR): 3302, 2925, 1648, 1595, 1514,
1442, 1348, 1258, 1220, 1151, 1103, 1018, 812. 1H-NMR (300 MHz, CD3OD): 0.90 (br. t, 3J¼6.8, 3 H); 1.29
(br. s, 14 H); 1.61 (s, 9 H); 2.27 (t, J¼7.4, 2 H); 2.59–3.07 (m, 2 H); 3.08–3.20 (m, 1 H); 3.11–3.44 (m,
1 H); 3.62 (d, J¼2.0, 1 H); 4.03–4.10, 4.46–4.54, 4.57–4.67 (3ꢂtaut., m, 1 H); 6.54–7.23 (m, 4 H).
13C-NMR (75 MHz, CD3OD): 14.6; 23.8*, 25.8 (taut.); 26.2*, 26.8, 26.9, 27.1 (4ꢂtaut.); 28.6*, 28.8, 29.3
(3ꢂtaut.); 30.3, 30.4, 30.5*, 30.6, 30.7 (5ꢂtaut.); 33.1*, 33.2 (taut.); 34.1, 34.9 (taut.); 35.1, 36.0* (taut.);
37.5*, 38.9 (taut.); 63.9*, 65.1 (taut.); 69.8, 71.5 (taut.); 80.6, 84.8* (taut.); 105.4, 105.7 (taut.); 116.1,
116.2*, 116.3, 116.4 (4ꢂtaut.); 126.0, 126.3, 127.5*, 128.6 (4ꢂtaut.); 1313, 131.4, 131.9, 132.0* (4ꢂtaut.);
150.9; 157.4*, 157.5, 157.7, 157.8 (4ꢂtaut.); 173.8, 175.1, 175.6, 178.1* (4ꢂtaut.); 191.0; 195.4, 195.9
(taut.). EI-MS (70 eV): 406 (6), 358 (10, [MꢀBoc]þ ), 252 (53), 172 (10), 147 (36), 126 (11), 107 (100),
77 (14).
General Procedure for the Synthesis of (5S)-3-(1-Hydroxyalkylidene)-5-[4-(prop-2-en-1-yloxy)ben-
zyl]pyrrolidine-2,4-diones 11g–11j. A soln. of tetramic acid 19 in dry CH2Cl2 (5 ml) was treated with
TFA (0.5 ml), and the mixture was stirred at r.t. for 2 h. The solvent was evaporated, and residual TFA
was removed by azeotropic distillation with toluene. To obtain well-resolved NMR spectra, the products
11g–11j were dissolved in AcOEt and washed with 0.05m Na2EDTA soln.
(5S)-3-(1-Hydroxybutylidene)-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-2,4-dione (11g; 73 mg,
0.21 mmol, 88%) was obtained from 19a (100 mg, 0.24 mmol). Orange oil. Rf (cyclohexane/EtOH
2 :1) 0.22. [a]2D5 ¼ ꢀ28.2 (c¼1.0, CHCl3). IR (ATR): 3321, 2929, 2853, 1659, 1609, 1509, 1346, 1241, 1219,
1021, 924, 801. 1H-NMR (300 MHz, CD3OD): 0.89*, 1.01 (taut. t, J¼7.4, 3 H); 1.51–1.67 (m, 2 H); 2.58–
2.81 (taut., m, 4 H); 2.87* (t, J¼7.3, 2 H); 2.90* (dd, J¼14.1, 5.6, 1 H); 3.00* (dd, J¼14.1, 4.5, 1 H); 4.08*
(dd, J¼5.6, 4.5, 1 H); 4.43, 4.46* (taut., d, J¼5.2, 2 H); 4.64 (taut., dd, J¼5.4, 2.2, 1 H); 5.20 (ddt, J¼
10.6, 1.6, 1.4, 1 H); 5.33, 5.35* (taut., ddt, J¼17.2, 1.6, 1.6, 1 H); 6.00 (ddt, J¼17.2, 10.6, 5.2, 1 H); 6.72,
6.78* (taut., d, J¼8.6, 2 H); 6.82, 7.05* (taut., d, J¼8.6, 2 H). 13C-NMR (75 MHz, CD3OD): 14.1*, 14.2,
14.4 (3ꢂtaut.); 19.1, 20.2, 20.6* (3ꢂtaut.); 35.9, 37.5* (taut.); 37.7, 40.8* (taut.); 63.8, 63.9* (taut.); 69.8;
103.3, 105.8* (taut.); 115.6, 115.7* (taut.); 117.5*, 117.6 (taut.); 127.8, 129.0*, 129.3, 130.0 (4ꢂtaut.);
131.9, 132.0* (taut.); 134.9, 135.0* (taut.); 159.1*, 159.3, 159.9 (3ꢂtaut.); 174.9*, 175.8 (taut.); 190.0*,
195.5 (taut.); 198.0. EI-MS (70 eV): 315 (3, Mþ ), 224 (7), 187 (4), 146 (100), 107 (24), 61 (15), 56 (59).

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
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(5S)-3-(1-Hydroxyhexylidene)-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-2,4-dione (11h; 68 mg,
0.20 mmol, quant.) was obtained from 19b (88 mg, 0.20 mmol). Yellow oil. Rf (cyclohexane/EtOH
2 :1) 0.34. [a]2D5 ¼ ꢀ25 (c¼1.0, CHCl3). IR (ATR): 3261, 2929, 2857, 1718, 1659, 1609, 1510, 1455, 1346,
1
1244, 1235, 1074, 1022, 800. H-NMR (300 MHz, CD3OD): 0.75–1.00 (m, 3 H); 1.04–1.77 (m, 6 H);
1.79–1.90* (m, 2 H); 2.59–2.81 (m, 2 H); 2.81–2.90 (taut., m, 2 H); 2.91 (dd, J¼14.1, 5.4, 1 H); 2.96 (dd,
J¼14.1, 4.3, 1 H); 3.05–3.24 (m, 2 H); 4.09* (dd, J¼5.4, 4.3, 1 H); 4.22–4.36 (taut., m, 1 H); 4.42, 4.46*
(taut., d, J¼5.2, 2 H); 4.47–4.54 (m, 1 H); 5.20 (br. d, J¼10.6, 1 H); 5.33 (br. d, J¼17.3, 1 H); 5.35*
(taut., ddt, J¼17.2, 1.6, 1.6, 1 H); 6.00 (ddt, J¼17.3, 10.6, 5.2, 1 H); 6.69 (d, J¼8.2, 2 H); 6.78* (taut., d,
J¼8.5, 2 H); 6.84 (d, J¼8.2, 2 H); 7.05* (taut., d, J¼8.5, 2 H); 7.56–7.63, 7.67–7.74 (taut., m, 2 H).
13C-NMR (75 MHz, CD3OD): 14.4*, 14.5 (taut.); 23.5*, 23.6, 23.7 (3ꢂtaut.); 26.8, 26.9* (taut.); 32.5*,
32.7, 32.8 (3ꢂtaut.); 34.2, 35.0* (taut.); 37.5; 63.8; 69.8; 103.2; 115.4, 115.6* (taut.); 117.5; 128.2, 129.0,
130.0* (3ꢂtaut.); 131.9, 132.0*, 132.5 (3ꢂtaut.); 133.7, 135.0 (taut.); 159.1; 175.8; 191.1; 198.0. EI-MS
(70 eV): 343 (6, Mþ ), 293 (8), 187 (7), 147 (100), 107 (35), 91 (12), 71 (14).
(5S)-3-(1-Hydroxyoctylidene)-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-2,4-dione (11i; 74 mg,
0.20 mmol, quant.) was obtained from 19c (94 mg, 0.20 mmol). Yellow oil. Rf (cyclohexane/EtOH
2 :1) 0.18. [a]2D5 ¼ ꢀ65 (c¼1.0, CHCl3). IR (ATR): 3206, 2927, 2855, 1716, 1651, 1609, 1510, 1454, 1346,
1243, 1235, 1097, 1021, 921, 801. 1H-NMR (300 MHz, CD3OD): 0.86–0.96 (m, 3 H); 1.09–1.75 (m, 10 H);
1.80–1.90 (m, 2 H); 2.59–2.86 (m, 2 H); 2.91* (dd, J¼14.1, 5.4, 1 H); 3.00* (dd, J¼14.1, 4.4, 1 H); 3.14
(taut., dd, J¼13.8, 2.2, 1 H); 3.40 (taut., dd, J¼13.8, 5.5, 1 H); 4.09* (dd, J¼5.4, 4.4, 1 H); 4.44, 4.47
(taut., ddd, J¼5.2, 1.7, 1.5, 2 H); 4.63 (taut., dd, J¼5.5, 2.2, 1 H); 5.21 (br. d, J¼10.6, 1 H); 5.34, 5.35*,
(taut., ddt, J¼17.3, 1.7, 1.7, 1 H); 6.01 (ddt, J¼17.3, 10.6, 5.4, 1 H); 6.75, 6.79* (taut., d, J¼8.7, 2 H); 6.83,
7.05* (taut., d, J¼8.7, 2 H). 13C-NMR (75 MHz, CD3OD): 14.5, 14.6*, 14.7 (3ꢂtaut.); 23.8*, 23.9 (taut.);
25.8, 26.2 (taut.); 26.9, 27.1* (taut.); 30.2*, 30.3, 30.4, 30.6 (4ꢂtaut.); 32.9*, 33.0, 33.1 (3ꢂtaut.); 34.1,
34.9*, 35.1, 36.0 (4ꢂtaut.); 37.5*, 38.9 (taut.); 63.9; 69.8; 103.2, 105.7* (taut.); 115.5, 115.6* (taut.);
117.5*, 117.6 (taut.); 127.9, 129.0* (taut.); 131.9, 132.0* (taut.); 134.9, 135.1* (taut.); 159.1*, 159.3 (taut.);
170.7, 175.1*, 177.8 (3ꢂtaut.); 191.0*, 195.6 (taut.); 196.0, 198.0 (taut.). EI-MS (70 eV): 371 (16, Mþ ),
147 (100), 107 (18), 91 (8), 57 (12), 41 (65).
(5S)-3-(1-Hydroxydecylidene)-5-[4-(prop-2-en-1-yloxy)benzyl]pyrrolidine-2,4-dione (11j; 79 mg,
0.20 mmol, quant.) was obtained from 19d (100 mg, 0.20 mmol). Yellow-to-orange oil. Rf (cyclo-
hexane/EtOH 2 :1) 0.29. [a]2D5 ¼ ꢀ40 (c¼0.5, CHCl3). IR (ATR): 3325, 2925, 2853, 1650, 1609, 1510,
1346, 1244, 1221, 1177, 1021, 922, 801. 1H-NMR (300 MHz, CD3OD): 0.90 (t, J¼6.9, 3 H); 1.05–1.76 (m,
14 H); 1.79–1.91, 2.58–2.82* (taut., m, 2 H); 2.82–3.20 (m, 2 H); 4.05–4.12*, 4.54–4.60 (taut., m, 1 H);
4.43, 4.47* (taut., d, J¼5.3, 2 H); 5.21 (d, J¼10.6, 1 H); 5.35 (d, J¼17.4, 1 H); 6.01 (ddt, J¼17.4, 10.6, 5.3,
1 H); 6.70, 6.78* (taut., d, J¼8.3, 2 H); 6.83, 7.05* (taut., d, J¼8.3, 2 H). 13C-NMR (75 MHz, CD3OD):
14.6, 14.7* (taut.); 23.9; 26.2; 26.9, 27.1* (taut.); 30.3*, 30.5*, 30.6*, 30.7, 30.8 (5ꢂtaut.); 33.2; 34.9; 37.5;
63.9; 69.7, 69.8* (taut.); 107.7; 115.5, 115.6* (taut.); 117.5; 128.9; 131.9, 132.0* (taut.); 134.9, 135.0*
(taut.); 159.1*, 159.2 (taut.); 175.2; 189.6; 196.5. EI-MS (70 eV): 399 (6, Mþ ), 147 (30), 146 (100), 119
(8), 107 (27), 91 (10), 57 (15).
General Procedure for the Synthesis of (5S)-3-(1-Hydroxyalkylidene)-5-(4-hydroxybenzyl)pyrroli-
dine-2,4-diones 11k–11n [17]. A soln. of tetramic acid 19 in dry CH2Cl2 (5 ml) was treated with TFA
(0.5 ml), and the mixture was stirred at r.t. for 2 h. The solvent was evaporated, and residual TFA was
removed by azeotropic distillation with toluene. The Boc-deprotected intermediates were dissolved in
dry THF/MeOH 5 :1 (24 ml) and treated with K2CO3 (3 equiv.) and Pd(PPh3)4 (2 mol-%). The mixture
was stirred under reflux for 24 h, the solvent was removed, the residue was dissolved in butan-2-one
(30 ml), and the resulting org. layer was extracted with 1m NaOH (2ꢂ30 ml). The aq. layer was acidified
to pH 2 with conc. HCl, and extracted with AcOEt (3ꢂ30 ml). Drying (Na2SO4), removal of the
volatiles under reduced pressure, and CC as indicated afforded the tetramic acids 11k–11n.
(5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxybutylidene)pyrrolidine-2,4-dione (11k; 11 mg, 0.04 mmol,
50%) was obtained from 19a (33 mg, 0.08 mmol) after CC (silica gel; gradient elution cyclohexane/
AcOEt 2 :1 to cyclohexane/EtOH 1:1). Orange oil. Rf (cyclohexane/EtOH 1:1) 0.29. [a]2D6 ¼ ꢀ9.5 (c¼
0.5, MeOH). IR (ATR): 3299, 2963, 1595, 1514, 1446, 1347, 1258, 1216, 1084, 1014, 794. 1H-NMR
(300 MHz, CD3OD): 0.91*, 1.00 (taut., t, J¼7.3, 3 H); 1.47–1.74 (m, 2 H); 2.60–3.02 (m, 2 H); 3.07–3.18
(m, 1 H); 3.30–3.42 (m, 1 H); 3.64 (s, 1 H); 3.88 (dd, J¼6.8, 5.0, 1 H); 4.03–4.10, 4.48–4.57 (taut., m,

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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
1 H); 6.62–7.06 (m, 4 H). 13C-NMR (75 MHz, CD3OD): 14.1, 14.2, 14.3, 14.4* (4ꢂtaut.); 19.3, 20.1, 20.6
(3ꢂtaut.); 35.1; 37.6, 40.8, 44.8 (3ꢂtaut.); 62.3, 65.0* (taut.); 71.5; 116.0, 116.2* (taut.); 127.9; 131.9,
132.0* (taut.); 157.5, 157.6 (taut.); 175.1; 198.4; 201.9. EI-MS (70 eV): 276 (11, [Mþ1]þ ), 181 (27), 149
(16), 107 (100), 89 (31), 57 (45), 45 (86).
(5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxyhexylidene)pyrrolidine-2,4-dione (11l; 69 mg, 0.23 mmol,
42%) was obtained from 19b (238 mg, 0.55 mmol) after CC (silica gel; gradient elution cyclohexane/
AcOEt 2 :1 to cyclohexane/EtOH 1:1). Orange oil. Rf (cyclohexane/EtOH 1:1) 0.54. [a]2D6 ¼ ꢀ127 (c¼
1.0, MeOH). IR (ATR): 3283, 2957, 2930, 2870, 1647, 1593, 1514, 1444, 1348, 1221, 1172, 1105, 860.
1H-NMR (300 MHz, CD3OD): 0.90 (t, J¼6.7, 3 H); 1.18–1.40 (m, 4 H); 1.50–1.61 (m, 2 H); 2.59–2.72
(m, 1 H); 2.72–2.84 (m, 1 H); 2.89 (br. d, J¼14.2, 1 H); 2.98 (br. d, J¼14.2, 1 H); 4.01–4.14 (m, 1 H);
6.65 (d, J¼7.0, 2 H); 6.97 (d, J¼7.0, 2 H). 13C-NMR (75 MHz, CD3OD): 14.4; 23.4; 26.8; 32.5; 34.1; 37.5;
64.0; 103.2; 116.2; 127.5; 132.0; 157.5; 175.8; 191.2; 198.2. EI-MS (70 eV): 303 (22, Mþ ), 197 (97), 107
(100), 73 (30).
(5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxyoctylidene)pyrrolidine-2,4-dione (11m; 108 mg, 0.33 mmol,
38%) was obtained from 19c (415 mg, 0.88 mmol) after CC (silica gel; gradient elution cyclohexane/
AcOEt 3 :1 to cyclohexane/EtOH 1:1). Orange oil. Rf (cyclohexane/EtOH 2 :1) 0.18. [a]2D6 ¼ ꢀ29 (c¼
0.5, MeOH). IR (ATR): 3297, 2925, 2856, 1648, 1594, 1514, 1440, 1346, 1229, 1171, 1105, 814. 1H-NMR
(300 MHz, CD3OD): 0.84–0.95 (m, 3 H); 1.21–1.40 (m, 8 H); 1.47–1.62 (m, 2 H); 2.27 (t, J¼7.3, 2 H);
2.59–2.89* (taut., m, 2 H); 2.82–3.16 (m, 2 H); 3.62 (br. s, 1 H); 4.02–4.11* (m, 1 H); 4.58–4.64 (taut.,
m, 1 H); 6.59 (taut., d, J¼8.2, 2 H); 6.66* (d, J¼7.6, 2 H); 6.73 (taut., d, J¼8.2, 2 H); 6.97* (d, J¼7.6,
2 H). 13C-NMR (75 MHz, CD3OD): 14.4*, 14.5 (taut.); 23.6; 26.9; 29.9; 30.1; 32.7; 34.0; 37.4; 63.9; 102.9,
105.6* (taut.); 115.9, 116.0* (taut.); 126.1, 127.4* (taut.); 131.7, 131.8* (taut.); 157.3*, 157.6 (taut.); 175.0*,
177.6 (taut.); 190.9; 195.6*, 197.8 (taut.). EI-MS (70 eV): 331 (18, Mþ ), 283 (10), 277 (15), 224 (83), 107
(100), 91 (9), 77 (23).
(5S)-5-(4-Hydroxybenzyl)-3-(1-hydroxydecylidene)pyrrolidine-2,4-dione (11n; 62 mg, 0.17 mmol,
61%) was obtained from 19d (137 mg, 0.28 mmol) after CC (silica gel; gradient elution cyclohexane/
AcOEt 3 :1 to cyclohexane/EtOH 1:1). Orange oil. Rf (cyclohexane/EtOH 2 :1) 0.43. [a]2D6 ¼ ꢀ6.9 (c¼
1.0, MeOH). IR (ATR): 3277, 2924, 2854, 1651, 1594, 1514, 1444, 1348, 1260, 1103, 1016, 806. 1H-NMR
(300 MHz, CD3OD): 0.90 (t, J¼6.7, 3 H); 1.22–1.34 (m, 12 H); 1.49–1.61 (m, 2 H); 2.59–2.84 (m, 2 H);
2.88 (dd, J¼13.8, 5.2, 1 H); 2.97 (dd, J¼13.8, 3.5, 1 H); 3.63 (br. s, 1 H); 4.03–4.11 (m, 1 H); 6.66 (d, J¼
8.1, 2 H); 6.97 (d, J¼8.1, 2 H). 13C-NMR (75 MHz, CD3OD): 14.5; 23.7; 26.9; 30.2; 30.3; 30 :4; 30.5; 33.0;
34.0; 37.4; 63.8; 71.1; 103.1; 116.0; 127.4; 131.8; 157.3; 175.5; 191.0; 198.0. EI-MS (70 eV): 359 (6, Mþ ), 353
(37), 107 (100), 77 (5), 55 (7), 41 (11).
Cell Lines and Culture Conditions. Human A-498 kidney cancer, KB-3-1 cervix carcinoma, and U-
937 leukemia cells, as well as murine L929 fibroblasts were obtained from the German Collection of
Microorganisms and Cell Cultures (DSMZ) and cultivated in the media recommended by the supplier at
378 and 10% CO2.
Inhibition of Cell Growth and Metabolic Activity (MTT assay). MTT (Sigma) was used to identify
the metabolic activity of cells which are capable of reducing it to a violet formazan product. Sixty ml of
serial dilutions of the test compounds were added to 120-ml aliquots of a cell suspension (50,000/ml) in 96-
well microplates. Blank and solvent controls were incubated under identical conditions. After 5 d, 20 ml of
MTT in PBS were added to a final concentration of 0.5 mg/ml. After 2 h, the precipitate of formazan
crystals was centrifuged, and the supernatant was discarded. The precipitate was washed with 100 ml of
PBS and dissolved in 100 ml of i-PrOH containing 0.4% HCl. The microplates were gently shaken for
20 min to ensure a complete dissolution of the formazan and finally measured at 595 nm with an ELISA
plate reader. All experiments were carried out in two parallel experiments, and the percentage of viable
cells was calculated as the mean with respect to the controls set to 100%.
Agar Diffusion Assay. Agar plates containing 15 ml of medium were inoculated with bacterial or
yeast suspensions in liquid broth to give a final OD of 0.01 (bacteria) or 0.1 (yeasts). The microorganisms
were from the Helmholtz Centre for Infection Research (HZI) collection and grown on standard medium.
In the case of molds, spores were collected from well-grown Petri dishes which were rinsed with 10 ml of
sterile aqua dest. One ml of the spore suspension was added to 100 ml of molten agar medium. Twenty ml
of test samples in MeOH (1 mg/ml) were applied onto 6-mm cellulose discs. MeOH was allowed to

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2845
evaporate, and the discs were placed upon the inoculated agar plate. The diameters in mm of the resulting
growth zones were determined after 24 h of incubation at 308.
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Received June 28, 2010