Synthesis and anticancer activity of the proposed structure of caldoramide, an N-peptidyltetramate from the cyanobacterium Caldora penicillata

Article abstract of DOI:10.1016/j.tet.2018.04.004

The structure proposed in the literature for caldoramide, a formal pentapeptide metabolite of the marine cyanobacterium Caldora penicillata, was synthesised in 12 steps and 16% yield (longest linear sequence from isoleucine) following the strategy of a stepwise consecutive extension of an N-oligopeptidyl chain on a tetramate anchor. The synthetic product differed from the natural isolate in optical rotation, some NMR data, and cytotoxicities. Its antiproliferative effect on three human cancer cell lines was distinctly lower than that of related dolastatin 10 and belamide A.

Full text of DOI:10.1016/j.tet.2018.04.004

Tetrahedron xxx (2018) 1e5
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis and anticancer activity of the proposed structure of
caldoramide, an N-peptidyltetramate from the cyanobacterium
Caldora penicillata
*
Anja Wunder, Matthias Rothemund, Rainer Schobert
Department of Chemistry, University Bayreuth, Universitaetsstr. 30, D-95440 Bayreuth, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 February 2018
Received in revised form
The structure proposed in the literature for caldoramide, a formal pentapeptide metabolite of the marine
cyanobacterium Caldora penicillata, was synthesised in 12 steps and 16% yield (longest linear sequence
from isoleucine) following the strategy of a stepwise consecutive extension of an N-oligopeptidyl chain
on a tetramate anchor. The synthetic product differed from the natural isolate in optical rotation, some
NMR data, and cytotoxicities. Its antiproliferative effect on three human cancer cell lines was distinctly
lower than that of related dolastatin 10 and belamide A.
29 March 2018
Accepted 3 April 2018
Available online xxx
©
2018 Elsevier Ltd. All rights reserved.
Keywords:
Caldoramide
Tetramic acids
Anticancer drugs
Oligopeptides
1
. Introduction
condensation steps on a preformed tetramate core. Thus we first
converted -phenylalanine 3 via its methylester 4 to the known
methyl 5-benzyltetramate by our established domino
additioneintramolecular Wittig olefination reaction employing the
L
Caldoramide was isolated, together with the known highly
potent anticancer compound dolastatin 10, from the tropical ma-
rine cyanobacterium Caldora penicillata collected at the Florida keys
6
5
3
cumulated phosphorus ylide Ph PCCO 5. The N-acylation of 6 with
(
Fig. 1).1 Interestingly, geographical dispersed collections of
an N-Boc-protected isoleucine imidazolylamide 10 was possible
ꢁ
C. penicillata all produced these metabolites which may thus be
used as chemotaxonomic markers for the identification of this
cyanobacterium.2 The isolating group assigned caldoramide the
structural formula 1 on grounds of 2D-NMR spectra and oxidative
breakdown reactions to give individual amino acids. Although only
moderately cytotoxic, natural caldoramide shares with dolastatin
under basic conditions at ꢀ40 C to give 12 in good yield after N-
deprotection of carbamate 11 with TFA (Scheme 1).
Isoleucine amide 10 was itself accessible in three steps and 67%
yield from -isoleucine 7 via N-Boc protection, and N-methylation
L
of the resulting carbamate 8 with NaH/MeI to give N-methyl-
0
isoleucine
9
which was finally treated with 1,1 -carbon-
1
HIF-
0 2 a selectivity for cancer cells positive in oncogenic KRAS and
yldiimidazole (Staab's reagent).
1
a
transcription factors. Herein, we report the first synthesis as
During the synthesis we realised that the coupling of N-meth-
ylated amino acids could not be efficiently achieved using standard
solid-phase protocols. Activation of the mostly carbamate protected
well as additional anticancer activities of the proposed structure 1
of caldoramide.
(
e.g. Fmoc, Boc) N-methylamino acids leads to racemisation or N-
6
2
. Results and discussion
carboxyanhydride formation and therefore low coupling yields.
This necessitated workarounds, e.g. for the attachment of an -N-
methylvaline residue to N-methylated -amino imide 12. First,
valine was converted with p-nitrobenzenesulfonylchloride to N-p-
nosyl- -valine 13 which should allow a selective downstream N-
monomethylation as previously demonstrated by us for threoni-
L
Unlike other syntheses of similar polypeptides, e.g. those of
a
L-
belamide A3 or dolastatin 10 which connect oligopeptide frag-
4
ments, ours builds up the tetrapeptidyl residue by four individual
L
5
a,7
8
ne.
Following a general protocol by Chaume et al. amino acid 13
*
Corresponding author.
E-mail address: Rainer.Schobert@uni-bayreuth.de (R. Schobert).
was converted to the corresponding acid chloride with 1-chloro-
https://doi.org/10.1016/j.tet.2018.04.004
040-4020/© 2018 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Wunder A, et al., Synthesis and anticancer activity of the proposed structure of caldoramide, an N-
peptidyltetramate from the cyanobacterium Caldora penicillata, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.04.004

2
A. Wunder et al. / Tetrahedron xxx (2018) 1e5
amine was immediately reacted with pNos-Val-Cl, freshly prepared
once more from N-nosylated valine 13 and 1-chloro-(N,N,2-
trimethyl)-1-propenylamine as for the conversion of 12 to 14. The
product N-trispeptidyltetramate 16 was de-nosylated, again with
2 3
2-methyl-5-tert-butylthiophenol and K CO in DMF, to afford the
corresponding primary amine. This was isolated and reacted
without further purification with dimethylvaline and HATU/DIPEA
to afford the proposed structure of caldoramide as a colourless
amorphous solid in 64% yield after column chromatography
(Scheme 3).
For a thorough comparison of its physical data with those of the
natural isolate and for biological tests, ca. 10 mg of synthetic 1 were
purified by HPLC. Its NMR spectra were difficult to compare to those
1
13
published for the natural isolate. The C NMR shifts of the latter
show a general high-field offset relative to those of synthetic 1,
superimposed by conspicuous deviations from this trend for spe-
cific carbon atoms. Discrepancies were also observed for some
Fig. 1. Structures of caldoramide (proposed)1 and dolastatin 10.
1
coupling constants in the H NMR spectra, while HSQC and HMBC
spectra were in acceptable consistence (cf. Supplementary data).
2
0
The specific optical rotation of synthetic 1 was [
a
]
D
ꢀ13.2 (c 0.50,
1
25
MeOH) while the natural isolate was reported to have [
c 0.36, MeOH).
The isolating group also reported the cytotoxicities of natural
caldoramide in MTT assays against three isogenic human colon
carcinoma HCT-116 cell lines. For the wild type cell line HCT-116
a
]
D
þ11.1
(
9
wt
they reported an IC50 (48 h) value of 3.9
derived from dose-response curves not reaching zero cell viability.
For synthetic 1 we found an IC50 (48 h) value of ca. 44 M. We also
tested it against human HT-29 colon carcinoma and MCF-7 mamma
mM, quite unusually
m
3
a
carcinoma cells. The latter were reported to be sensitive to the
congeneric N-trispeptidyltetramate belamide A. The activities of 1
are listed in Table 1.
3. Conclusion
We reported the first synthesis of the structure proposed in the
literature for the cyanobacterial metabolite caldoramide in 12 steps
Scheme 1. Synthesis and N-acylation of anchor tetramate 6.
(N,N,2-trimethyl)-1-propenylamine, and reacted right away with
a-
amino imide 12 dissolved in a suspension of NaHCO in H O/CH Cl
3
2
2
2
to give N-dipeptidyltetramate 14 in 78% yield. The latter was then
quantitatively methylated at the terminal nosylated amino group
with MeI/K
For the attachment of the next
group of compound 15 was removed by treatment with 2-methyl-
-tert-butylthiophenol and K CO in DMF. The resulting crude
2 3
CO in DMF (Scheme 2).
L-valine residue the p-nosyl
5
2
3
Scheme 3. Completion of the synthesis of caldoramide 1. HATU ¼ 1-[bis(dimethyla-
mino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate;
DIPEA ¼ N,N-diisopropylethylamine.
2 6 4 2
Scheme 2. pNos ¼ p-NO (C H )SO .
Please cite this article in press as: Wunder A, et al., Synthesis and anticancer activity of the proposed structure of caldoramide, an N-
peptidyltetramate from the cyanobacterium Caldora penicillata, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.04.004

A. Wunder et al. / Tetrahedron xxx (2018) 1e5
3
Table 1
NCHCHN), 8.43 (1H, s, NCHN);
5.8 (CH CH), 23.9 (CH CH ), 28.4 ((CH
MeCH), 61.3 (CHCO), 81.4 ((CH C), 116.6 (NCHCHN), 131.2
(NCHCHN), 137.2 (NCHN), 155.9 (NCH CO), 167.7 (CHCON).
d
(125 MHz, CDCl
) 10.7 (CH
CH
),
C
3
3
2
Inhibitory concentrations IC50 (48 h)a in
mM of synthetic compound 1 when applied
1
(
3
3
2
3
)
3
C), 29.5 (NCH
3
), 32.3
topo
wt
to MCF-7
mamma, HT-29 colon and HCT-116 colon carcinoma cells.
3 3
)
cell line/compound
MCF-7topo
HCT-116wt
HT-29
3
1
33.9 ± 1.3
43.8 ± 3.7
77.5 ± 1.3
a
Values are derived from concentration-response curves obtained by measuring
the percentual absorbance of viable cells relative to untreated controls (100%) after
4.2.2. (5S)-1-[(N-Boc,N-methyl)-L-isoleucinoyl]-5-benzyl-4-
4
8 h exposure in MTT assays. Values represent means of four independent
methoxy-2,5-dihydropyrrol-2-one (11)
runs ± SD.
A solution of (5S)-benzyl-4-methoxy-2,5-dihydropyrrol-2-one 6
ꢁ
(
203 mg, 1.0 mmol, 1.00 equiv) in 10 mL THF was cooled to ꢀ40 C
and slowly treated with LiHMDS (1.05 mL, 1 M solution in THF,
.05 mmol, 1.05 equiv). The yellow solution was stirred for 10 min
(
longest linear sequence from isoleucine) and 16% yield. Our
1
strategy of a stepwise consecutive extension of an N-oligopeptidyl
chain on a tetramate anchor proved feasible. The cytotoxic activities
of synthetic 1 against selected human cancer cell lines were rela-
tively low when compared to those reported for the closely related
and then treated dropwise (0.1 mL/min) with a solution of freshly
prepared isoleucine amide 10 (295 mg, 1.0 mmol, 1.00 equiv) in THF
(
H
4 mL). The resulting mixture was stirred for 1 h, quenched with
O (0.5 mL), warmed to room temperature, and diluted with ethyl
acetate. The layers were separated and the organic phase was
washed with 1 M NaOH, 1 M HCl and brine, dried over Na SO and
2
methyl
belamide A) and for the natural isolate of caldoramide. After all,
we cannot confirm with certainty the identity of synthetic product
and the natural caldoramide isolate due to some discrepancies in
their NMR data, specific optical rotations, and cytotoxicities.
2
N-(Me N-ValeMeN-ValeMeN-Phe)-5-benzyltetramate
(
2
4
concentrated in vacuo. The remainder was purified by column
chromatography (cyclohexane/ethyl acetate, 4:1) to yield 11
1
(
237 mg, 55%) as a waxy white solid which solidified upon pro-
ꢁ
20
longed storage (m.p. 77e84 C); [
031, 2969, 2936, 2882, 1730, 1679, 1629, 1455, 1429, 1407, 1380,
365, 1301, 1247, 1228, 1190, 1148, 1120, 1079, 1048, 1023, 965, 872,
a]
D
þ 150.7 (c 0.90, CHCl
3 max
); n
4
. Experimental
3
1
4
.1. General
ꢀ1
; d
8
0
03, 699 cm
.82e0.95 (6H, m, CH
H
(500 MHz, CDCl
CH, CH CH ), 1.11e1.21 (1H, m, CH
C), 1.42e1.49 (1H, m, partly hidden, CH
.08e2.19 (1H, m, CHCH ), 3.02 and 3.03 (3H, s, NCH ), 3.10 (1H, dd,
HH 14.1, 2.9 Hz, Ph-CHH), 3.44 (1H, dd, JHH 14.1, 4.9 Hz, Ph-CHH),
.76 and 3.83 (3H, s, OCH ), 4.80 and 4.84 (1H, s, C]CHCO), 4.91
1H, dd, JHH 4.9, 2.9 Hz, Bn-CH), 6.18 (1H, d, JHH 11.1 Hz, NCHCON),
3
) 1:3.3 mixture of two rotamers:
CHH), 1.42
CHH),
3
3
2
3
Melting points (uncorrected): Büchi melting point apparatus M-
65. IR: Perkin-Elmer Spectrum 100 FT-IR spectrophotometer with
and 1.51 (9H, s, (CH
2
J
3
3
)
3
3
5
3
3
1
13
ATR sampling unit. NMR: H and C NMR spectra were recorded on
Bruker DRX 500 and Avance 300 spectrometers; chemical shifts are
given in parts per million (d) using the residual solvent peak as an
internal standard. Mass spectra: Finnigan MAT 8500 (EI, 70 eV).
HRMS: UPLC/Orbitrap MS system in ESI mode. Optical rotations:
3
(
ar
6
.89e7.25 (5H, m, H );
CH CH), 24.7 (CH CH
NCH ), 34.7/35.0 (CH
NCHCON), 80.2 ((CH C), 95.1 (C]CHCO), 127.1 (C ), 128.3 (C ),
d
C
(125 MHz, CDCl
), 28.5/28.6 ((CH
Ph), 58.5 (CH O), 59.4 (CHBn), 59.8
3
) 10.4 (CH
3 2
CH ), 14.7
) C), 31.1 (CHEt), 33.8
(
(
(
3
3
2
3 3
Perkin-Elmer Polarimeter 241 (
l
¼ 589 nm); [
in 10 deg cm g . For column chromatography Merck silica gel
0 (230e400 mesh) was used. Analytical TLC was carried out using
D
a] values are given
3
2
3
ꢀ1
2
ꢀ1
ar
ar
3 3
)
6
ar
ar
ar
ar
1
28.5 (C ), 129.6 (C ), 129.74 (C ), 134.3 (C ), 156.4 (OCON), 169.2
Merck silica gel 60 F254s foil plates. Analytical HPLC was performed
on a Beckman System Gold Programmable Solvent Module 126
using Phenomenex Kinetex
2
(
[
¼CHCON), 171.6 (NCHCON), 177.7 (COMe). HRMS: m/z calcd for
þ
MþNa, C24
34 2 5
H N O Na ]: 453.23599; found: 453.23503.
®
C-18-HPLC column, length
m; detection by a
50 ꢂ 4.6 mm, pore size 100 Å, particle size 5
m
Beckman Instruments Diode Array Detection Module 168. Prepar-
ative HPLC was performed on an Amersham Biosciences System
Programmable Solvent Module P-900 using Phenomenex Gemini-
NX C-18-HPLC column, length 250 ꢂ 10.00 mm, pore size 110 Å,
4.2.3. (5S)-1-(N-methyl-
L-isoleucinoyl)-5-benzyl-4-methoxy-2,5-
dihydropyrrol-2-one (12)
A solution of carbamate 11 (0.25 g, 0.58 mmol, 1.0 equiv) in
ꢁ
CH
2
Cl
2
(8 mL) was cooled to 0 C and slowly treated with 0.67 mL
particle size 5 mm, detection by an Amersham Biosciences UV-900
trifluoroacetic acid (0.67 mL, 8.71 mmol,15 equiv). The solution was
warmed to room temperature, stirred for 4 h, and then diluted with
Module. Starting compounds were bought from the usual sources
and used without further purification.
CH
NaHCO
with CH
Na SO and concentrated in vacuo to yield 11 (0.18 g, 94%) as a
colourless oil; [
2
Cl
2
, cooled in an ice-bath and quenched with sat. aqueous
. The layers were separated, the aqueous one was extracted
Cl , and the combined organic phases were dried over
3
4
4
0
.2. Syntheses and characterisation
2
2
2
4
2
0
.2.1.
L
-N-Boc-isoleucine imidazole-1-ylamide (10)
a]
D
þ 204.2 (c 1.0, CHCl
3
); nmax 3336, 3031, 2964,
An ice-cold solution of N-Boc-N-methyl-
.33 mmol, 1.0 equiv) in THF was treated with 1,1 -carbon-
L
-isoleucine 9 (80 mg,
1724, 1674, 1627, 1455, 1379, 1351, 1304, 1262, 1245, 1188, 1115, 963,
0
ꢀ1
799, 732, 700 cm
CH CH
10.2, 7.3 Hz, CH
1.60 (1H, dqdd, JHH 10.2, 6.7, 4.6, 3.1 Hz, CHEt), 2.44 (3H, s, NCH
3.18 (1H, dd, JHH 14.0, 2.9 Hz, PhCHH), 3.65 (1H, dd, JHH 14.0, 5.2 Hz,
PhCHH), 3.85 (3H, s, CH O), 4.30 (1H, d, JHH 4.6 Hz, NCHCON), 4.85
(1H, s, C]CHCO), 4.86 (1H, dd, JHH 5.2, 2.9 Hz, BnCH), 6.98e7.24
;
d
H
(500 MHz, CDCl
CH), 1.12 (1H, ddq, JHH 13.3,
CHH), 1.49 (1H, dqd, JHH 13.3, 7.3, 3.1 Hz, CH CHH),
),
3
) 0.85 (3H, t, JHH 7.3 Hz,
yldiimidazole (63 mg, 0.39 mmol,1.2 equiv) in several portions over
min and stirred for another 2.5 h. The mixture was diluted with
diethyl ether, washed with water and brine, and dried over Na SO
3
2 3
), 0.99 (3H, d, JHH 6.7 Hz, CH
5
3
3
2
4
.
3
Removal of the volatiles left 75 mg (78%) of crude 10 as a colourless
oil which was submitted to the next step without further purifi-
cation.
1
7
rotamer: 0.89e1.00 (6H, m, CH
1
2
3
n
max 3123, 2970, 2933, 2881, 1734, 1684, 1531, 1473, 1390,
ar
368, 1308, 1275, 1233, 1214, 1153, 1118, 1094, 1062, 933, 871, 808,
(5H, m, H );
d
C
(125 MHz, CDCl
3
) 11.9 (CH
3
CH
2
), 16.6 (CH
3
CH), 23.4
O),
ꢀ
1
48 cm
;
d
H
(500 MHz, CDCl
3
)
mixture of rotamers; major
CH ), 1.07 (1H, m, CH CHH),
CHH), 1.46 (9H, s, (CH C),
.18e2.30 (1H, m, MeCH), 2.72 (3H, s, NCH ), 5.11 (1H, d, JHH 10.9 Hz,
(CH CH ), 35.2 (PhCH
3
2
2
), 35.7 (NCH
3
), 38.0 (CH
3
CH), 58.5 (CH
3
ar
ar
3
CH, CH
3
2
3
60.3 (BnCH), 67.7 (NCHCON), 95.3 (¼CHCO), 127.3 (C ), 128.2 (C ),
ar ar
.36e1.46 (1H, m, partly hidden, CH
3
3
)
3
129.7 (C ), 134.3 (ipso-C ), 170.1 (¼CHCON), 176.4 (NCHCON), 178.1
þ
3
(COMe). HRMS: m/z calcd for [MþH, C19
H
27
N
2
O
3
]: 331.20162;
CHCO), 7.08 (1H, d, JHH 0.9 Hz, NCHCHN), 7.69 (1H, d, JHH 0.9 Hz,
found: 331.20115.
Please cite this article in press as: Wunder A, et al., Synthesis and anticancer activity of the proposed structure of caldoramide, an N-
peptidyltetramate from the cyanobacterium Caldora penicillata, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.04.004

4
A. Wunder et al. / Tetrahedron xxx (2018) 1e5
4
.2.4. pNos-
L
-Val-(NMe-
L
-Ile)-(5-benzyl-4-methoxy-2,5-
3 2 3
(CH N), 34.5 (CHEt), 35.7 (PhCH ), 58.5 (CH O), 58.8 (NCHCON),
ar
ar
dihydropyrrol-2-one) (14)
In a Schlenk tube N-p-nosyl-
equiv) was suspended in 2.5 mL of dry CH
59.9 (BnCH), 60.4 (SNCH), 94.9 (¼CHCO), 124.4 (C ), 127.2 (C ),
ar ar ar ar
L-valine 13 (98 mg, 0.32 mmol, 1.3
128.3 (C ), 128.4 (C ), 129.7 (C ), 134.9 (ipso-C ), 145.8/150.0
ꢁ
ar
2
Cl
2
, cooled to 0 C and
(ipso-C ), 169.3 (¼CHCON), 171.8 (MeNCO), 172.7 (NCHCON), 178.3
þ
then treated with 1-chloro-N,N,2-trimethyl-1-propenylamine
(COMe). HRMS: m/z calcd for [MþH, C31
H
40
N
4
O
8
NaS ]: 651.24591;
ꢁ
(
43
mL, 0.32 mmol, 1.3 equiv). The reaction was stirred at 0 C for
found: 651.24377.
1
h while being monitored by tlc (cyclohexane/ethyl acetate, 3:1).
To this end an aliquot was quenched with CH OH and the resulting
3
4.2.6. pNos-L-Val-(NMe-L-Val)-(NMe-L-Ile)-(5-benzyl-4-methoxy-
methyl ester taken as a measure of consumption of the starting
material. Eventually, a suspension of amine 12 (82 mg, 0.25 mmol,
2,5-dihydropyrrol-2-one) (16)
A solution of 15 (45 mg, 72
m
mol, 1.0 equiv) in DMF (0.7 mL) was
mol, 6.0 equiv) and 2-methyl-5-
mol, 2.5 equiv), stirred for an
1
.0 equiv) in CH
2
Cl
2
(2.5 mL) and 10% aqueous NaHCO
3
(2.5 mL) was
treated with K
2 3
CO (59 mg, 429 m
tert-butylthiophenol (32 mg, 179
added to the so prepared solution of acid chloride and the resulting
mixture was stirred for 1 h. The layers were separated, the aqueous
one was extracted three times with ethyl acetate, and the combined
m
hour and quenched with 1 M HCl and brine. The aqueous layer was
repeatedly extracted with diethyl ether until no thiophenol could
be detected in the extract by tlc (cyclohexane/ethyl acetate, 1:1).
2 4
organic phases were washed with brine, dried over Na SO and
concentrated in vacuo. After column chromatography (cyclo-
The pH value of the aqueous layer was adjusted to 9 with Na
and 2 mL of CH Cl were added. This solution was directly used for
the subsequent coupling. In a separate Schlenk tube N-p-nosyl-L-
2 3
CO
hexane/diethyl ether, 3:1 to 4:1) 14 (118 mg, 78%) was obtained as a
2
2
ꢁ
20
colourless solid of m. p. 90e95 C; [
a
]
D
þ118.0 (c 1.0, CHCl
3
);
n
max
2
964, 2931, 1733, 1677, 1628, 1530, 1456, 1378, 1369, 1350, 1306,
valine 13 (69 mg, 215
mmol, 3.0 equiv) was suspended in 1 mL of dry
ꢀ
1
ꢁ
1171, 737, 616 cm
;
d
H
(500 MHz, CDCl
3
) 78:22 mixture of rotam-
CH
2
Cl
2
, cooled to 0 C, and treated with 1-chloro-N,N,2-trimethyl-
ers; major rotamer: 0.56e0.63 (2H, m, CH CH ), 0.66 (3H, t, JHH
.6 Hz, CH CH ), 0.77 (3H, d, JHH 6.4 Hz, CH CH ), 0.80 and 1.11 (3H
each, d, JHH 6.7 Hz, (CH CH), 1.86e1.95 (2H, m, CHEt, (CH CH),
.97 (3H, s, NCH ), 3.11 (1H, dd, JHH 14.0, 3.2 Hz, PhCHH), 3.29 (1H,
dd, JHH 14.0, 5.5 Hz, PhCHH), 3.77 (3H, s, CH O), 4.01 (1H, m, SNCH),
.78 (1H, dd, JHH 5.5, 3.2 Hz, BnCH), 4.84 (1H, s, C]CHCO), 5.96 (1H,
br d, NH), 6.17 (1H, d, JHH 10.7 Hz, NCHCON), 6.92e6.99 (2H, m,
3
2
1-propenylamine (34
m
L, 215
m
mol, 3.0 equiv). The mixture was
ile
ꢁ
7
3
2
3
stirred at 0 C for 1 h while being monitored by tlc as described for
the synthesis of 14. After consumption of all starting material the
previously prepared solution of the de-nosylated amine derived
from 15 was added via syringe, the mixture was stirred for 30 min,
the layers were separated, and the aqueous one was extracted with
3
)
2
3 2
)
2
3
3
4
CH
2
Cl
2
(3ꢂ). The combined organic phases were washed with 1 M
ar
ar
meta-H ), 7.16e7.23 (3H, m, H ), 8.01e8.06 (2H, m, CHCNO
.31e8.35 (2H, m, 2H, CHCS); (125 MHz, CDCl ) 10.8 (CH CH
4.5 (CH CH), 16.0 and 20.2 ((CH CH), 24.9 (CH CH
Me CH), 31.3 (CH N), 33.9 (CHEt), 35.6 (PhCH ), 58.6 (CH O), 58.8
SNCH), 59.4 (NCHCON), 59.9 (BnCH), 94.8 (¼CHCO), 124.3 (C ),
2
),
NaOH, dried over Na
2
SO and concentrated in vacuo. The remainder
4
8
d
C
3
3
2
),
was purified by column chromatography (cyclohexane/ethyl ace-
tate,1:1) to yield 16 (44 mg, 85%) as an amorphous white solid of m.
1
3
3
)
2
3
2
), 31.0
ꢁ
20
(
(
1
2
3
2
3
p. 120e128 C; [
a
]
D
þ61.7 (c 0.94, CHCl
3
); nmax 3200, 3106, 3034,
ar
2965, 2931, 2876, 1728, 1626, 1531, 1456, 1379, 1349, 1305, 1246,
ar
ar
ar
ar
ar
27.2 (C ), 128.4 (C ), 128.6 (C ), 129.5 (C ), 134.7 (ipso-C ), 145.9
1193, 1170, 1089, 965, 854, 738, 719, 700, 686, 671, 615, 565,
ar
ꢀ1
and 150.0 (ipso-C ), 169.1 (¼CHCON), 171.2 (MeNCO), 171.3
554 cm
(CH
6.8 Hz, (CH
;
d
H
(500 MHz, CDCl
3
)
0.17 (3H, d,
J
HH 6.7 Hz,
CH ), 0.82 (3H, d, JHH
CH ), 0.83 (3H, t, JHH 7.3 Hz, overlapped, CH CH
.88 (3H, d, JHH 6.7 Hz, CH CH), 1.06 (1H, ddq, JHH 13.5, 9.0, 7.3 Hz,
val1
val1
(
C
NCHCON), 178.4 (COMe). HRMS: m/z calcd for [MþNa,
3 2 3 2
) CH ), 0.80 (3H, d, JHH 6.5 Hz, (CH )
þ
val2
30
H
38
N
4
O
8
NaS ]: 637.23026; found: 637.23010.
3
)
2
3
2
)
0
3
val2
4
.2.5. pNos-NMe-
dihydropyrrol-2-one) (15)
A solution of sulfonamide 14 (166 mg, 0.27 mmol, 1.0 equiv) in
DMF (2.7 mL) was treated with K CO (75 mg, 0.54 mmol, 2.0 equiv)
and CH I (34 L, 0.54 mmol, 2.0 equiv). The resulting mixture was
stirred at room temperature for 1 h, quenched with H O and diluted
L
-Val-(NMe-
L
-Ile)-(5-benzyl-4-methoxy-2,5-
CH
3
CHH), 1.12 (3H, d, J ¼ 6.8 Hz, (CH
3
)
2
CH ) 1.20 (1H, dqd, JHH
13.5, 7.3, 3.2 Hz, CH
3
CHH), 1.88 (1H, sptd, JHH 6.8, 3.1, Me
2
CHval2),
2.08 (1H, ddqd, JHH 10.8, 9.0, 7.0, 3.2 Hz, CHEt), 2.22 (1H, dqq, JHH
val1
val1
2
3
10.8, 6.7, 6.5 Hz, Me
2
CH ), 2.88 (3H, s, CH
3
N
), 3.09 (1H, dd, JHH
ile
3
m
14.0, 3.4 Hz, PhCHH), 3.15 (3H, s, CH
4.9 Hz, PhCHH), 3.75 (3H, s, CH
3
N
), 3.31 (1H, dd, JHH 14.0,
2
3
O), 4.01 (1H, dd, JHH 9.2, 3.1 Hz,
with diethyl ether. The layers are separated and the aqueous phase
was extracted with diethyl ether. The combined organic phases
SNCH), 4.81 (1H, s, C]CHCO), 4.83 (1H, dd, JHH 4.9, 3.4 Hz, BnH),
val1
5.02 (1H, d, JHH 10.8 Hz, NCHCO ), 5.86 (1H, d, JHH 9.2 Hz, NH),
ile
were washed with brine, dried over Na
2
SO
4
and concentrated in
6.44 (1H, d, JHH 10.8 Hz, NCHCO ), 6.96e7.00 (2H, m, ortho-Ph),
vacuo. Purification by column chromatography (cyclohexane/ethyl
acetate, 2:1) afforded 15 (168 mg, 99%) as a colourless solid of m. p.
7.18e7.23 (3H, m, Ph), 8.00e8.05 (2H, m, nosyl-H), 8.29e8.34 (2H,
m, nosyl-H);
d
C
(125 MHz, CDCl
3
) 10.6 (CH
3
CH
2
), 14.5 (CH
3
CH), 15.8
ꢁ
20
val2
val1
val2
1
83e187 C; [
a
]
D
þ79.0 (c 0.52, CHCl
3
);
n
max 2972, 2932, 2882,
((CH
25.2 (CH
31.8 (CH
3
)
2
CH ), 17.8 and 19.2 ((CH
3
)
2
CH ), 20.2 ((CH
3
)
2
CH ),
CH ),
val1
val1
val2
1729, 1677, 1643, 1625, 1529, 1453, 1384, 1340, 1304, 1272, 1240,
3
CH
N
2
), 27.0 (Me
2
CH ), 30.3 (CH
), 34.4 (CHEt), 35.6 (PhCH
(NCHCO ), 59.8 (BnCH), 94.9 (¼CHCO),124.5 (C ), 127.2 (C ), 128.4
3
N
), 30.8 (Me
2
ile
1
192, 1159, 1134, 1120, 1106, 1084, 968, 956, 936, 864, 852, 819, 797,
3
2
), 58.48*, 58.49*, 58.54*, 58.6
ꢀ1
ile
ar
ar
737, 720, 699, 602, 567 cm
;
d
H
(500 MHz, CDCl
3
) 95:5 mixture of
CH), 0.86
), 0.91 (3H, d, JHH 6.4 Hz, overlapped,
CH), 0.92 (3H, d, JHH 6.7, overlapped, CH CH), 1.12 (1H, ddq,
CHH), 1.31 (1H, dqd, JHH 13.7, 7.4, 2.9 Hz,
CHH), 2.13 (1H, ddqd, JHH 11, 8.0, 6.7, 2.9 Hz, CHEt), 2.32 (1H,
dqq, JHH 10.7, 6.7, 6.4 Hz, (CH CH), 3.04 (3H, s, SNCH ), 3.12 (1H,
dd, JHH 14.0, 3.4 Hz, PhCHH), 3.30 (1H, dd, JHH 14.0, 5.2 Hz, PhCHH),
ar
ar
ar
ar
ar
rotamers; major rotamer: 0.81 (3H, d, JHH 6.6 Hz, (CH
3
)
2
(C ), 128.6 (C ), 129.6 (C ), 134.7 (ipso-C ), 145.9/150.2 (ipso-C ),
val2
val1
(
(
3H, t, JHH 7.4 Hz, CH
CH
3
CH
2
169.2 (¼CHCON), 170.8 (CO ), 170.9 (CO ), 172.1 (NCHCON),
val1
3
)
2
3
178.3 (COMe); *assignment interchangeable: CH
3
O/NCHCO
/
þ
49 5 9
H N O NaS ]: 750.31432;
JHH 13.7, 8.0, 7.4 Hz, CH
3
SNCH. HRMS: m/z calcd for [MþNa, C36
CH
3
found: 750.31347.
3
)
2
3
4.2.7. Caldoramide (proposed structure 1)
Analogously to 15, compound 16 (57 mg, 79
3
.36 (3H, s, NCH
3
), 3.74 (3H, s, OCH
3
), 4.68 (1H, d, JHH 11 Hz, SNCH),
m
mol, 1.0 equiv) was
mol, 6.0 equiv) and 2-
mol, 2.5 equiv) in DMF
4
.82 (1H, s, C]CHCO), 4.84 (1H, dd, JHH 5.2, 3.4 Hz, BnCH), 6.44 (1H,
de-nosylated with K
2
CO
3
(65 mg, 473
m
phenyl
d, JHH 10.7 Hz, NCHCON), 7.02e7.08 (2H, m, H
m, H
), 7.17e7.26 (3H,
methyl-5-tert-butylthiophenol (35
mL, 189 m
phenyl
ar
ar
), 7.97e8.02 (2H, m, H ), 8.33e8.38 (2H, m, H );
d
C
(0.78 mL) to give the corresponding primary amine.
A solution of it in DMF (0.4 mL) and DIPEA (66 L, 379
equiv) was added to a Schlenk tube containing dimethyl-L
(
125 MHz, CDCl
3
3
) 10.6 (CH CH
2
), 14.5 (CH
3
CH), 18.9 and 19.8
m
m
mol, 4.8
-valine
(
(CH CH), 25.5 (CH
3
)
2
3
CH ), 28.8 (Me
2
2
CH), 30.4 (SNCH
3
), 32.3
Please cite this article in press as: Wunder A, et al., Synthesis and anticancer activity of the proposed structure of caldoramide, an N-
peptidyltetramate from the cyanobacterium Caldora penicillata, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.04.004

A. Wunder et al. / Tetrahedron xxx (2018) 1e5
5
(
14 mg, 95
m
mol, 1.2 equiv) and HATU (36 mg, 95
m
mol, 1.2 equiv),
course of 90 days via addition of increasing amounts of topotecan
(up to 500 nM) into the culture medium of MCF-7 breast cancer
cells.
MTT assay : MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
2H-tetrazolium hydrobromide; ABCR] was used to identify the
ꢁ
dissolved in 0.3 mL DMF, cooled to 0 C, and having pre-reacted for
2
0 min. The resulting mixture was stirred for 2 h at room temper-
ature, diluted with ethyl acetate, washed with brine, dried over
Na SO and concentrated in vacuo. After purification by column
9
2
4
chromatography (ethyl acetate/EtOH, 96:4) caldoramide 1 (pro-
metabolic activity of vital cells which are capable of reducing it to a
topo
wt
4
posed structure) was obtained as a colourless, amorphous solid
violet formazan. MCF-7
were seeded (100 L/well) and cultured for 24 h in 96-well
microplates. Incubation of cells following treatment with the test
compound (final concentrations ranging from 50 nM to 200 M)
, HT-29, and HCT-116 cells (5ꢂ10 /mL)
ꢁ
20
1
(
[
1
8
34 mg, 64%) of m. p. 177e179 C; [
a
]
D
ꢀ13.2 (c 0.50, CH
max 3318, 2964, 2932,1733,1627,1497,
455, 1380, 1304, 1259, 1247, 1193, 1093, 1112, 1033, 1026, 965, 909,
3
OH) [lit :
m
2
0
a]
D
þ11.1 (c 0.36, CH
3
OH)]; n
m
ꢀ
1
07, 799, 729, 699, 670, 645 cm
;
d
H
(500 MHz, CDCl
3
) 0.78 (3H, d,
was continued for 48 h. Solvent controls with DMF were treated
ꢁ
J
HH 6.7 Hz, 25/26-H), 0.86 (3H, t, JHH 7.3 Hz, 18-H), 0.91 (3H, d, JHH
identically. Then, the plates were centrifuged (300 g, 4 C) for 5 min
6
6
6
.7 Hz, 19-H), 0.91 (3H, d, JHH 6.7 Hz, 25/26-H), 0.94 (3H, d, JHH
.7 Hz, 38/39-H), 0.97 (3H, d, JHH 6.7 Hz, 32/33-H), 1.01 (3H, d, JHH
.7 Hz, 32/33-H), 1.02 (3H, d, JHH 6.7 Hz, 38/39-H), 1.13 (1H, ddq, JHH
and the medium was discarded by a swift turn of the microplates.
MTT in phosphate-buffered saline (0.05%) was added. After 2 h the
precipitate of formazan was dissolved in DMSO containing 10%
sodium dodecylsulfate and 0.6% AcOH. The microplates were
centrifuged as before and swiftly turned to discard the MTT solu-
tion before adding the solvent mixture. The microplates were
a
b
1
3.9, 8.7, 7.3 Hz, 17-H ), 1.26e1.30 (1H, m, 17-H , overlapped), 1.99
1H, sptd, JHH 6.7, 5.9 Hz, 31-H), 2.09 (1H, sptd, JHH 6.7, 6.1 Hz, 37-H,
overlapped), 2.11 (1H, m, 16-H, overlapped), 2.27 (6H, s, 41/42-H),
(
ꢁ
2
.41 (1H, dspt, JHH 11.0, 6.7 Hz, 24-H), 2.49 (1H, d, JHH 6.1 Hz, 36-H),
incubated for 1 h at 37 C and absorbances at 570 nm and 630 nm
a
3
.11, (1H, dd, JHH 14.0, 3.4 Hz, 6-H ), 3.13 (3H, s, 28-H), 3.24 (3H, s,
(background) were measured with a Tecan F-200 plate reader. All
experiments were carried out in quadruplicate, and the percentage
of the viable cells was calculated with Graphpad Prism 7 as the
mean ± standard deviation relative to the control (100%).
b
21-H), 3.33 (1H, dd, JHH 14.0, 5.2 Hz, 6-H ), 3.75 (3H, s, 13-H), 4.83
(
3
1H, s, 2-H), 4.84 (1H, dd, JHH 8.9, 5.9 Hz, 30-H), 4.86 (1H, dd, JHH 5.2,
.4 Hz, H-4), 5.30 (1H, d, JHH 11.0 Hz, 23-H), 6.48 (1H, d, JHH 10.7 Hz,
ar
15-H), 6.99 (1H, d, JHH 8.9 Hz, 30-NH), 7.00e7.04 (2H, m, H ), 7.22
ar
(
C 3
3H, m, H ); d (125 MHz, CDCl ) 10.7 (C-18),14.6 (C-19), 17.8 (C-38/
3
2
9), 17.9 (C-32/33), 18.4 (C-25/26), 19.7 (C-25/26), 20.0 (C-32/33),
0.3 (C-38/39), 25.2 (C-17), 27.3 (C-24), 27.8 (C-37), 30.7 (C-28), 31.0
Appendix A. Supplementary data
(
3
9
1
1
C-31), 31.9 (C-21), 34.4 (C-16), 35.7 (C-6), 43.1 (C-41/42), 53.8 (C-
Supplementary data related to this article can be found at
https://doi.org/10.1016/j.tet.2018.04.004.
0), 58.4 (C-23), 58.5 (C-13), 58.7 (C-15), 59.8 (C-4), 76.6 (C-36),
ar
ar
ar
4.9 (C-2), 127.2 (para-C ), 128.4 (meta-C ), 129.7 (ortho-C ),
ar
34.8 (ipso-C ), 169.2 (C-1), 171.3 (C-22), 171.9 (C-35), 172.3 (C-14),
þ
60 5 6
H N O ]:
References
72.9 (C-29), 178.2 (C-3). HRMS: m/z calcd for [MþH, C37
6
70.45186; found: 670.45381.
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becco's Modified Eagle's Medium (DMEM), supplemented with 10%
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(
b) L o€ ffler J, Schobert R. J Chem Soc Perkin Trans. 1996;1:2799e2802.
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(b) Teixid oꢀ M, Albericio F, Giralt E. J Pept Res. 2005;65:153e166.
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ꢁ
atmosphere (5% CO2, 95% humidity) at 37 C. The BCRP over-
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topo
clone was generated over a
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Please cite this article in press as: Wunder A, et al., Synthesis and anticancer activity of the proposed structure of caldoramide, an N-
peptidyltetramate from the cyanobacterium Caldora penicillata, Tetrahedron (2018), https://doi.org/10.1016/j.tet.2018.04.004