N, N′ -methyleno-didemnin A from the ascidian trididemnum solidum. Complete NMR assignments and confirmation of the imidazolidinone ring by strategic analysis of 1 J CH

Article abstract of DOI:10.1021/np100846s

The complete NMR assignments of N,N′-methyleno-didemnin A from Trididemnum solidum Van Name 1902 are reported along with a strategic analysis of ¹J_CH coupling constants that confirm the presence of the imidazolidinone ring.

Full text of DOI:10.1021/np100846s

NOTE
pubs.acs.org/jnp
N,N0-Methyleno-didemnin A from the Ascidian Trididemnum solidum.
Complete NMR Assignments and Confirmation of the Imidazolidinone
Ring by Strategic Analysis of 1JCH
Tadeusz F. Molinski,*,†,‡ Jaeyoung Ko,† Kirk A. Reynolds,† Sarah C. Lievens,§ and Katrina R. Skarda§
†Department of Chemistry and Biochemistry, and ‡Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California,
San Diego, 9500 Gilman Drive MC0358, La Jolla, California 92093-0358, United States
§Department of Chemistry, University of California, Davis, 1 Shields Avenue, Davis, California 95616, United States
S Supporting Information
b
ABSTRACT: The complete NMR assignments of N,N0-methyleno-
didemnin A from Trididemnum solidum Van Name 1902 are reported
along with a strategic analysis of 1JCH coupling constants that confirm
the presence of the imidazolidinone ring.
he Caribbean ascidian Trididemnum solidum Van Name
1902 is the source of the highly cytotoxic compounds
T
didemnins (Figure 1), which have been advanced to human
clinical trials as potential anticancer agents.1,2 The first described
members of the series, didemnins A (1) and B (2),1 are cyclic
depsipeptides containing the uncommon amino acids statine and
N,O-dimethyltyrosine.3,4 Advanced clinical trials of didemnin B
(2) for treatment of various solid tumors5 have been discon-
tinued; however, dehydro-didemnin B (3, aplidine) has been
launched into phase II clinical trials. Despite considerable
investigations, the mechanism of action of didemnin B and its
induction of apoptosis in cancer cells is not completely under-
stood. Our collections of T. solidum from the Bahamas gave
extracts—not unexpectedly—that displayed strong cytotoxicity
against cultured human colon tumor cells (HCT-116) due to the
presence of 1, 2, and their congeners. We describe here the
complete NMR characterization of N,N0-methyleno-didemnin A
(4), a derivative of didemnin A that co-occurs with 1 and 2 in T.
solidum in which the terminal N-methyl leucyl residue has
undergone cyclization to a substituted N-methyl 1-methylimida-
zolidin-4-one.6 Interpretation of the scalar heteronuclear cou-
pling constants of 4 and three synthetic 1-methylimidazolidin-4-
ones reveals useful signature values of 1JCH values that confirm
the identity of the heterocyclic ring.
Figure 1. Structures of didemnins A (1) and B (2), aplidine (dehydro-
didemnin B, 3), and N,N0-methyleno-didemnin A (4).
portion was fractionated by Sephadex LH-20 size exclusion
chromatography eluting with MeOH and finally purified by
reversed-phase HPLC to provide 4 along with didemnins A
T. solidum was exhaustively extracted with MeOH, and the
concentrated extract (IC50 <38 ng/mL against cultured human colon
cancer cells, HCT-116) was triturated with anhydrous MeOH
and CHCl3 to remove salts. The concentrated MeOH-soluble
Received: November 21, 2010
Published: February 22, 2011
r
Copyright
2011 American Chemical Society and
882
dx.doi.org/10.1021/np100846s J. Nat. Prod. 2011, 74, 882–887
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
(1) and B (2), which were identified by comparison of MS and
1H NMR data (C6D6) with literature data.7 Compound 4 was
isolated as a clear glass (HRMS [M þ H]þ = 955.5733) with a
formula C50H78N6O12, corresponding to the addition of one C
atom to 1, C49H78N6O12. Due to severe signal overlap of signals
of 4 in CD3OD, the 1H NMR spectrum was rerecorded in several
other solvents, including CDCl3, C6D6, C5D5N, and (CD3)2SO.
The best dispersion of NMR signals for 4 was achieved in C6D6,
which also permitted the observation and correlation of the
the C-64 CH2 group of 4 revealing two different couplings
(1JCH = 159.6 and 146.4 Hz) for the diastereotopic H-64R and
H-64β protons (Table 2), respectively. In comparison, N-CH3
groups showed lower scalar coupling values (e.g., 1JCH (C-63) =
135.0 Hz; (C-36) = 138.6 Hz), while R-CH signals show
characteristic couplings (e.g., 1JCH (C-11) = 148.2 Hz). Clearly,
C-64 is substituted with two N atoms. It is of interest to note that
1JCH of the diastereotopic C-64 geminal C-H couplets are not
equal and that subtle stereoelectronic effects also influence the
magnitude of the heteronuclear coupling, albeit with an attenu-
ated effect (ΔJ ≈ 5% of the value of 1JCH).
1
exchangeable NH and OH protons. The H NMR data of 4
(C6D6, Table 1) were highly suggestive of a peptide of the
didemnin class with spin systems corresponding to an approxi-
mated A2B2 pattern for a para-substituted phenolic ring (δH
6.78, d, J = 8.4 Hz. δC 130.4, d; 6.70, d, J = 8.4 Hz. δC113.6), two
O-CH, eight CH attached to electronegative substituents, two
secondary amide protons, (CdO)NH, a hydroxy proton, nine
methyl doublets, one methyl triplet, two N-methyl singlets, and
an O-methyl singlet. These structural features were readily
correlated with spin systems found in 1, except for two changes:
amine N-H and one amide-N-H were replaced by a downfield
shifted CH2 group (δH 5.05, d, J = -4.2 Hz; 3.35, dd, J = -4.2,
1.2 Hz; δC 68.9, d).8 The H-64R and H-64β diastereotopic
signals were correlated by COSY cross-peaks to H-589 and
HMBC cross-peaks to C-63 and C-58. Taken together, the
signals of 4 could be accommodated by placement of the protons
within an imidazolidinone ring. Finally, compound 4, prepared
from 1 (paraformaldehyde, benzene, 80 °C), was shown to be
identical with the sample isolated from T. solidum (1H NMR and
MS). The structure of 4 was first proposed by Gloer;10 however,
incomplete MS and NMR data allowed only a tentative
assignment.
Finally, we prepared the model compounds 5-8 (Scheme 1)
1
and compared the measured JCH values with those of 4.
Coupling (EDCI) of (S)-N-methyl-N-Boc-leucine, obtained by
hydrolysis of the corresponding methyl ester 9,13 with phenethy-
lamine or isopropylamine followed by deprotection (TFA) and
treatment of the amide products with paraformaldehyde in
benzene gave the N-methylimidazolidin-4-ones 5 and 6, respec-
tively. Ammoniolysis of a mixed anhydride derived from 9 gave
the corresponding leucinamide, which was converted through a
similar sequence to 7; an N-hydroxymethyl imidazolidinone
formed by addition of a second formaldehyde equivalent to the
amide nitrogen. Finally, pseudoephedrine was converted to the
known oxazolidine 814 in a similar manner. The coupled HSQC
spectra of 5-7 showed scalar heteronuclear couplings for the AB
signals15 corresponding to the diastereotopic N-CH2-N protons
(1JCH = 154.3, 144.4 Hz; 154.4, 143.8 Hz, 157.1, and 145.5 Hz,
respectively). Notably, the second 1H NMR AB spin system in 7,
corresponding to the N-CH2-OH group, showed slightly larger
and almost equivalent heteronuclear scalar couplings (1JCH
=
157.1, 156.7 Hz), as would be expected for an aminal group that
replaces an N atom for a more electronegative O. Larger coupling
constants were found for the diastereotopic N-CH2-O group in
oxazolidine 8 (1JCH = 159.6, 151.7 Hz), which lacks an electron-
withdrawing N-CdO group. In contrast, carbons substituted
with a single N atom have a much lower heteronuclear coupling
constant; for 5-8 the values for N-CH3 groups fell into the
narrow range 1JCH = 133.5-135.5 Hz. The differential values of
1JCH in the diastereotopic N-CH2-N groups seen in 5-7 were
entirely comparable to those of 4 and consistent with the
depicted structure. These observations may be put to further
practical use, not only in elimination of carbinolamines as
possible structures for 416 but for differentiation of 2-unsubsti-
tuted oxazolidines from imidazolidinones. Given the sensitivity
of modern microcryoprobe NMR spectrometers, measurement
of 1JCH values by 13C-coupled 1H-13C HSQC is practical, even
with only microgram amounts of natural products.17
While the above deductive reasoning supported the presence
of an imidazolidinone ring system in 4, we sought independent
1
spectroscopic evidence in the form of JCH coupling constants.
The unique feature of the heterocyclic ring is two N atoms
bonded to a sp3-hybridized CH2 group. The magnitude of 1JCH
in sp3 C-C-H couplets changes with the replacement of C with
electronegative substituents and can be used to identify the
attached atoms. For example, the 1JCH was used to discriminate
two possible constitutional isomers of varamines A and B and
supports the presence of an S-Me over an alternative N-Me
group.11 The one-bond heteronuclear coupling constant 1JCH is
known to be primarily dependent upon C hybridization (% s
character of the associated molecular orbitals), electronegativity
of attached atoms, the presence of electron-withdrawing groups,
stereoelectronic effects, and, in the case of cyclic structures, ring
1
strain. Values for JCH in unstrained alkanes (R3C-H) are
essentially invariant within a narrow range of 125-130 Hz.
In order to gain insight into the assignment of the two well-
dispersed N-CH2-N0 proton signals in 4-7, molecular me-
chanics calculations (MMFF) were carried out on 6 to ascertain
the preferred conformations. Minimized structures were found
(N = 25), and Monte Carlo searching revealed that the lowest
four conformers accounted for 73% of the conformer distribu-
tion. Figure 2 depicts models of the two lowest conformers with
energies and Boltzmann weightings of -1.8 kcal mol-1 (36.3%)
and -0.56 kcal mol-1 (21.3%). The imidazolidinone is relatively
flat except for the N-Me that lies below the plane of the ring syn to
HR in order to reduce nonbonded interactions. It is likely that, of
the two diastereotopic protons on the N-CH2-N0 group, the
proton syn-facial to HR 4 in the imidazolidinone participates in
Substitution of the C-H couplet with an N-substituent increases
1
the magnitude of JCH to 135-140 Hz, while the greater
1
electronegativity of O-substituents further increases JCH to
145-150 Hz. The bonding of two electronegative atoms to
C-H (e.g., O-CH-O, N-CH-O, and N-CH-N groups) raises the
1
value of JCH over their monosubstituted analogues. The best
1
studied cases of JCH include the anomeric O-CH-O groups in
aldoses and glycosides, which show values of O-CH-O in the
ranges 1JCH = 169-171 and 158-162 Hz for axial and equatorial
substituents, respectively.12
The values of 1JCH in 4 were conveniently obtained from 13C-
coupled HSQC spectra (measured with the 13C decoupler
turned off during FID acquisition; see Table 2). 13C-coupled
cross-peaks were observed for the AB system corresponding to
4
1
the consistently observed long-range JHH in the H NMR
spectra of 4-7 as the two H's and intervening bonds conform
883
dx.doi.org/10.1021/np100846s |J. Nat. Prod. 2011, 74, 882–887

Journal of Natural Products
NOTE
Table 1. NMR Assignments of N,N’-Methyleno-didemnin A (4) (600 MHz, C6D6)
a,b
c
no.
δC
δHb, mult., J (Hz)
δN
COSYb
HMBC (#C)b
1
2
4
5
168.4
65.9
169.7d
3.13, dd (11.3, 4.5)
28R, 28β
36
36
57.3
4.33, dd (8.7, 5.1)
1.07, m
6β
6R
6β
22.8
7R, 8β
7
5
1.28, m
5, 7β
7R
7β
24.1
46.5
1.00, m
6β, 7β, 8R, 8β
7R, 8R, 8β
6R, 7R, 7β
6R, 7R, 7β
1.40, m
8R
8β
2.95, ddd (-13.2, 9.6, 2.4)
3.27, ddd (-13.2, 9.0, 2.4)
5
7
10
170.7d
50.0
39
11
5.16, td (9.0, 2.7)
8.61, d (9.0)
12, 39, 40
11
12N
13
119.2
13
170.1
50.1
14
4.51, q (6.2)
5.79, d (3.0)
44
46
13, 15, 44
15, 18, 46
15
204.3
80.9
16
18
172.7
39.3
19R
19β
20
2.60, dd (-17.6, 10.2)
3.65, d (-17.6)
19β, 20
18, 20
18
19R, 21
67.3
54.4
4.37, td (10.2, 2.0)
4.54, td (10.2, 3.0)
7.65, d (10.0)
19R, 21, 50
19β, 20, 22, 51
21
20, 54
22N
23
117.4
169.0
57.5
69.9
33.8
24
5.32, d (2.3)
1, 23, 25, 65
23, 24
2
25
5.42, qd (6.4, 2.3)
3.46, dd (-14.1, 4.5)
3.56, dd (-14.1, 11.3)
65
28R
28β
29
2, 28β
2, 28R
2, 29
130.0
130.4
113.6
158.8
54.4
30,34
31,33
32
6.78, d (8.4)
6.70, d (8.4)
31/33
30/34
29,32
34,
35
3.33, s
32
2
36
38.1
2.04, s
39R
39β
40
42.2
1.85, m
11, 40
1.85, m
11, 40
24.9
23.6
21.0
15.0
30.6
16.4
18.8
1.92, m
11, 39, 41, 42
41
0.88, d (6.6)
1.08, d (6.0)
1.77, d (6.2)
2.49, hep.d (7.2, 3.0)
0.84, d (7.2)
0.75, d (7.2)
2.95, d (2.0)
2.35, hex.d (6.6, 3.0)
1.40, m
40
39, 40, 42
39, 40, 41
13, 14, 15
42
40
44
14
46
16, 47, 48
47
46
16, 46, 48
16, 46, 47
48
46
50 OH
51
20
34.0
27.7
21, 52R, 52β, 54
51, 52β
52R
52β
53
21, 51
1.69, m
51, 52R, 53
52β
51, 53, 54
51, 52
13.0
14.6
174.4
64.3
37.8
1.18, t (7.2)
1.03, d (7.2)
54
51
21, 51, 52
57
58
2.77, t (5.7)
1.57, ddd (13.2, 8.4, 4.8)
1.69, m
59R, 59β, 64R, 64β
58, 59β, 60
57
59R
59β
60
57, 58
57, 58
59
58, 59R, 60
24.4
2.07, m
59R, 59β, 61, 62
884
dx.doi.org/10.1021/np100846s |J. Nat. Prod. 2011, 74, 882–887

Journal of Natural Products
NOTE
Table 1. Continued
COSYb
HMBC (#C)b
a,b
c
no.
δC
δHb, mult., J (Hz)
0.93, d (6.6)
δN
61
23.2
22.4
40.0
68.9
60
60
59, 60, 62
59, 60, 61
59, 64
62
0.97, d (6.6)
1.96, s
63
64R
64β
65
3.35, dd (-4.2, 1.2)
5.05, d (-4.2)
1.45, d (6.4)
58, 64β
58, 64R
25
63
17.9
25
a Chemical shifts from HSQC, HMBC cross-peaks (600 MHz), room-temperature probe. b Spectra referenced to C6D5H (δH 7.16) and C6D6 (δC
128.06). c Measured from 1H-15N HSQC cross-peaks. d gHMBC (2,3J = 8.4 Hz), cryomicroprobe.
Scheme 1. Syntheses of Model Compounds 5-8a
Figure 2. Molecular mechanics minimized structures for 6 (MMFF,
Spartan 08) and Boltzman-weighted energies: (a) E = -1.8 kcal mol-1
(36.3%); (b) E = -0.56 kcal mol-1 (21.3%).
MHz instrument, equipped with a 1.7 mm {13C,15N}1H CPTXI
microcryoprobe, a Jeol ECA 500 MHz spectrometer equipped with a
5 mm {13C}1H probe, or a Varian Mercury 400 MHz instrument
equipped with a 5 mm {1H}13C probe and measured at 23 °C in CDCl3
a See Table 2 for 1JCH values.
or C6D6 with reference to residual solvent signals (CDCl3 1H, δ 7.26
ppm; 13C, δ 77.16 ppm; C6D6 1H, δ 7.15 ppm; 13C, δ 128.1 ppm).
to an approximate “W” conformation, although this must be
considered only a tentative assignment. No useful NOEs were
observed in the NMR spectra of 4 that would otherwise clarify
this assignment.
The cytotoxicity of 1 and 4 toward cultured human colon
tumor cell lines (HCT-116) was briefly evaluated. N,N0-Methy-
leno-didemnin A (4) was found to be slightly more active (IC50
24 nM) than didemnin A (1; IC50 32 nM), but less so than the
very potent didemnin B (2; IC50 9.0 nM).
The higher didemnins have only been partially characterized.
Although the closely related formamide didemnin G (NR-formyl-
didemnin A) and didemnin X,1b a higher molecular mass
imidazolidinone, were isolated from T. solidum, compound 4
was known previously as a condensation product of formalde-
hyde with didemnin A (1).10 Here, we provide the first complete
structural characterization of 4 and confirm its occurrence as a
natural product in T. solidum from the Bahamas.10,18
In summary, we have isolated and completely assigned the
NMR spectra of 4, an N,N0-methyleno derivative of didemnin A
from T. solidum that shows similar cytotoxicity to didemnin A (1)
against HCT-116 cells. A useful method, based on 1JCH values, to
differentiate oxazolidinones, carbinolamines, and imidazolidi-
none-modified peptides was validated by critical comparison
with synthetic model compounds.
HRMS measurements were measured by MALDI (UC Riverside) or ESI
conditions with an Agilent 6000 series TOF mass spectrometer (Scripps
Research Institute MS facility) or a ThermoFinnigan Orbitrap mass
spectrometer (UC San Diego, Small Molecule MS facility). Semipre-
parative HPLC was carried out with a Varian high-capacity dual-pump
system coupled to a Rainin UV-1 high-dynamic UV detector under
specified conditions. All solvents for HPLC purification were of
commercial HPLC grade.
Animal Material. Trididemnum solidum (99-09-048) was collected
by hand using scuba from Little San Salvador, Bahamas, in 1999 at a
depth of 20 m and stored frozen until extracted. A second collection
(07-35-213) was made in the same area in 2008. The specimen consisted
of thick, tough-to-tear sheets that were pale green on the exposed surface
and pale pinkish on the attached underside due to the presence of
cyanobacterial phycoerythrin. The surface of this colonial ascidian was
very finely textured with darker colored zooids embedded within a pale
green tunic. Additionally, the MeOH extract of the animal was deeply
pigmented with chlorophyll, a signature property for the presence of
cyanobacteria. Together with other morphological features, these traits
are uniquely associated with the common T. solidum of the Caribbean
Sea. A preserved type sample is archived at the University of California,
San Diego.
Extraction and Isolation. The tunicate (402.3 g, frozen) was
mascerated and extracted four times with MeOH (total of 6 L). The
combined extracts were concentrated under reduced pressure and the
green-white solids triturated with anhydrous MeOH, followed by
CHCl3. The deep-green MeOH-soluble fraction was concentrated
under reduced pressure before separation by size exclusion chromatog-
raphy over a column of Sephadex LH-20 eluting with MeOH. The
didemnin-containing fraction was further purified by reversed-phase
’ EXPERIMENTAL SECTION
General Experimental Procedures. 1H and 13C NMR spectra
were recorded in CDCl3 on several instruments: a Bruker DRX 600
885
dx.doi.org/10.1021/np100846s |J. Nat. Prod. 2011, 74, 882–887

Journal of Natural Products
NOTE
Table 2. Selected 1JCH for 4, Imidazolidinones 5-7, and
taken up in CH2Cl2 (0.5 mL) and stirred in the presence of TFA
(0.5 mL) until TLC indicated the complete deprotection to the
corresponding secondary amine TFA salt (ninhydrin). The crude
product was suspended in benzene containing excess paraformaldehyde
(52 mg) and heated at 80 °C for 4 h. After cooling, the solution was
washed sequentially with aqueous NaHCO3 and brine, then dried over
Na2SO4. The mixture was concentrated under reduced pressure and
purified by flash chromatography (SiO2, 1:2 EtOAc/hexane) to afford 5
Oxazolidine 8
cmpd
#Ha
#C
1JCHb/Hz
group
4
4
4
4
4
4
4
4
4
4
5
6
7
7
8
5
6
7
8
11
11
20
21
31
36
41
51
63
59
64
2
148.2
NH-CHR-C(O)
R2-CH-O
20
147.9
21
144.6
R2-CH-NHC(O)
Ph
1
31
156.6
(24 mg, 53%): [R]D þ24.5 (c 0.01, CHCl3); H NMR (600 MHz,
36
138.6
N-CH3
CDCl3) δ 7.24 (m, 2H), 7.17 (m, 3H), 4.17 (d, 1H, J = 5.2 Hz), 3.55 (dd,
1H, J = 5.2, 1.5 Hz), 3.49 (m, 2H), 2.80 (m, 3H), 2.32 (s, 3H), 1.85 (m,
1H), 1.53 (m, 1H), 1.36 (m, 1H), 0.90 (d, 3H, J = 6.8 Hz), 0.88 (d, 3H,
J = 6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 173.8, 138.6, 128.8, 128.7,
126.7, 70.6, 65.2, 42.7, 41.1, 39.5, 34.2, 24.9, 23.1, 22.7; 13C-coupled
HSQC, see Table 2; HRESIMS m/z 261.1961 [M þ H]þ (calcd for
C16H25N2O, 261.1967).
41
124.8
R-CH3
51
129.6
R-CH3
63
135.0
N-CH3
R-CH2-R0
59R,β
64R,β
2R,β
2R,β
2R,β
123.0, 128.4
159.6, 146.4
154.3, 144.5
154.4, 143.8
157.1, 145.2
157.1, 156.7
159.6, 151.7
135.5
NR-CH2-NHC(O)
NR-CH2-NHC(O)
NR-CH2-NHC(O)
NR-CH2-NHC(O)
NH-CH2OH
N-CH2-O
(S)-5-Isobutyl-1-methyl-3-isopropylimidazolidin-4-one
(6). Compound (S)-6 was prepared using the above procedure. (S)-6:
[R]D þ26 (c 0.008, CHCl3); 1H NMR (600 MHz, CDCl3) δ 4.34 (dd,
1H, J = 5.3, 0.8 Hz), 4.29 (m, 1H), 3.68 (dd, 1H, J = 5.3, 1.7 Hz), 2.86
(m, 1H), 2.43 (s, 3H), 1.92 (m, 1H), 1.61 (m, 1H), 1.41 (m, 1H), 1.53
(d, 3H, J = 6.8 Hz), 1.14 (d, 3H, J = 6.8 Hz), 0.94 (d, 3H, J = 6.8 Hz), 0.92
(d, 3H, J = 6.8 Hz); 13C NMR (100 MHz, CDCl3) δ 172.8, 65.8, 65.7,
42.3, 40.9, 39.4, 25.0, 23.0, 22.8, 20.2, 20.1; 13C-coupled HSQC, see
Table 2; HRESIMS m/z 199.1804 [M þ H]þ(calcd for C11H23-
N2O,199.1805).
(S)-5-Isobutyl-1-methyl-3-hydroxymethylimidazolidin-4-
one (7). To a stirred solution of (S)-N-Boc-N-methylleucine (81 mg,
0.33 mmol) and N-methylmorpholine (7 μL, 0.33 mmol) in dry THF
was added isobutyl chloroformate (43 μL, 0.33 mmol) at -15 °C. After
the reaction mixture was stirred for 20 min at -15 °C, NH3 gas was
bubbled into the reaction mixture for 5 min at -15 °C and then for an
additional 1 h before diluting with H2O and extraction with EtOAc
(ꢀ3). The combined organic layers were dried (Na2SO4) and concen-
trated under reduced pressure, and the residue was purified by flash
chromatography (SiO2, 1:2 EtOAc/hexane) to give the corresponding
leucinamide (62 mg, 77%), which was used to prepare (S)-7 as described
above. (S)-7: [R]D þ29 (c 0.01, CHCl3); 1H NMR (600 MHz, CDCl3)
δ 4.89 (d, 1H, J = 10.9 Hz), 4.73(d, 1H, J = 10.9 Hz), 4.49 (d, 1H, J = 5.3
Hz), 3.94 (dd, 1H, J = 5.3, 1.5 Hz), 2.90 (m, 1H), 2.45 (s, 3H), 1.90 (m,
1H), 1.61 (m, 1H), 1.44 (m, 1H), 0.96 (d, 3H, J = 6.4 Hz), 0.93 (d, 3H,
J = 6.4 Hz); 13C NMR (100 MHz, CDCl3) δ 174.7, 69.0, 65.4, 65.3, 40.6,
39.1, 24.9, 22.9, 22.8; 13C-coupled HSQC, see Table 2; HRESIMS m/z
187.1441 [M þ H]þ (calcd for C9H19N2O2 187.1447).
2
2
NR-Me
133.5
NR-Me
134.8
NR-Me
134.0
N-Me
a Note different locant numberings; see Figure 1 and Scheme 1. b 1JCH
measured from 13C-coupled HSQC spectra (600 MHz, C6D6).
HPLC eluting with 85:15 MeOH/H2O to give didemnin B (2) (86.7
mg, 0.022% wet weight) and N,N0-methyleno-didemnin A (4) (6.83 mg,
0.0017% wet weight). The second collection (07-35-213) was processed
in a different manner. The frozen tunicate (56.3 wet wt) was homo-
genized with MeOH (2 ꢀ 400 mL) using a hand blender, and the
combined deep-green aqueous MeOH extracts were partitioned se-
quentially against hexanes, CHCl3, and n-BuOH. The CHCl3-soluble
fraction was separated by silica gel chromatography (MeOH/CHCl3,
stepped gradient), and the didemnin-containing fraction subjected to
HPLC (Phenomenex Luna C18, 10 ꢀ 250 mm, 85:15 MeOH/H2O) to
give didemnin A (1, 8.63 mg, 0.015% w/w wet wt).1
N,N0-Methyleno-didemnin A (4):. colorless glass; [R]D -153
(c 0.38, MeOH); 1H NMR, 13C NMR, see Table 1; 13C-coupled HSQC,
see Table 2; MALDI-HRMS m/z 955.5733 [M þ H]þ (calcd for
C50H79N6O12, 955.5755).
Synthesis of N,N0-Methyleno-didemnin A (4) from Didem-
nin A (1). Compound 4 was prepared from didemnin A (1) using a
variant of Gloer’s method.10 A solution of 1 (2.18 mg, 2.31 μmol) in
benzene (2 mL) was treated with excess paraformaldehyde (0.71 mg).
The mixture was heated at 80 °C (1.5 h) until LC-MS indicated
consumption of starting material. The benzene layer was washed with
aqueous K2CO3 (10% w/v), dried over solid K2CO3, and concentrated
under a stream of N2. Separation of the crude product by HPLC (C18
reversed phase, 85:15 MeOH/H2O) gave pure 4 (0.45 mg), identical
with the natural product (1H NMR, MS). Without alkali workup,
product 4 was unstable to the conditions of purification.
Molecular Modeling. MMFF calculations of 6 were conducted
with Spartan 08 (Wavefunction) using the MM-minimized conforma-
tion as a starting point. The 25 lowest energy conformations were found
(Monte Carlo), and the relative energies and Boltzmann weightings (%)
of the lowest four were calculated to be -1.88 kcal mol-1 (36.4%), -
0.56 (21.3), 1.47 (9.4), and 2.42 (6.4). See Figure 2.
Cytotoxicity Assays. Cytotoxicity was measured with cultured
HCT-116 cells (ATCC CCL-247) by measuring growth inhibition in the
presence of compound in 96-well microtiter plates. Cells (density = 1.2 ꢀ
104 cells/mL) in McCoy’s 5A Media containing 10% FBS (175 mL of cell
suspension per well) were incubated at 37 °C under 5% CO2 for 24 h prior
to addition of extract or compound (in triplicate). IC50 was measured from
treatments using 8 points in a dilution series from 125 to 0.00763 mg/mL or
125 to 0.00763 ng/mL. Cells were incubated with solutions of extract or pure
compound in DMSO (0.89% v/v) for 72 h (3 days) prior to addition of
MTS reagent (25 mL of 1.9 mg/mL MTS (3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) with 0.044
mg/mL PMS (phenazine methosulfate) in PBS) and further incubated for
3 h before measuring the absorbance at λ= 490 nm using a microplate reader
(Molecular Probes Datamax Pro) and analyzed with native instrument
(S)-5-Isobutyl-1-methyl-3-phenethylimidazolidin-4-one
(5). A mixture of (S)-N-Boc-N-methylleucine (200 mg, 0.82 mmol,
prepared by saponification of the corresponding methyl ester 913) and
EDCI (236 mg, 1.23 mmol) in THF (5 mL) was stirred for 15 min, then
treated with phenethylamine (149 mg, 1.23 mmol), then stirred for an
additional 8 h before diluting with H2O and extraction with EtOAc
(ꢀ3). The combined organic layers were dried (Na2SO4) and concen-
trated under reduced pressure, and the residue was purified by flash
chromatography (SiO2, 1:20 EtOAc/hexane) to give the corresponding
(S)-phenethylamide (121 mg, 59%). The crude product (60 mg) was
886
dx.doi.org/10.1021/np100846s |J. Nat. Prod. 2011, 74, 882–887

Journal of Natural Products
NOTE
software (SoftmaxPro). The corresponding IC50 values for the compounds
(13) Lee, G. M.; Molinski, T. F. Tetrahedron Lett. 1992, 33, 7671–
7674.
were measured and reported here as follows: 1, 32 nM; 2, 9.0 nM; 4, 24 nM.
(14) (a) von Pfanz, H.; Kirchner, G. Liebigs Ann. Chem. 1958, 614,
149–158. (b) Gesson, J. P.; Jacquesy, J. C.; Mondon, M. Synlett 1990,
669–670.
’ ASSOCIATED CONTENT
1
(15) Strictly, the H NMR spectrum of 4 is ABX, where the X
Supporting Information. 1H,13C NMR and 2D NMR
S
proton, the R-proton of the N-methyleucyl residue (H-58), shows weak
long-range homonuclear coupling (4J = 1.2 Hz) to only one of the N-
CH2-N signals: H64R but not H64β (the stereoassignments of these
protons have not been made). See Table 1. Collectively, synthetic
imidazolidinones 5-8 show variable 1H spin patterns from AB in 8, with
zero long-range homonuclear couplings, to ABX in 5-7 with 2JAB ≈ -
5.3 Hz, 4JAX ≈ 1.5-1.7 Hz, and 4JBX ≈ 0 Hz.
b
spectra of 4, 1H, 13C spectra of 5-8, and the 13C-coupled HSQC
spectrum of 5. These materials are available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(16) Carbinolamines that are formally derived as adducts of peptides
with formaldehyde would be expected to show pseudomolecular ions in
their MS spectra that are higher by 30 amu mass units, but—as we
observed for 7—their lability under ESIMS ionization conditions is
misleading. The reactive formaldehyde equivalent is easily lost, probably
in the heated capillary of the ionization stage, and the expected
pseudomolecular ions are absent or replaced by different product ions.
(17) (a) Molinski, T. F. Nat. Prod. Rep. 2010, 27, 321–329. (b)
Dalisay, D. S.; Molinski, T. F. J. Nat. Prod. 2009, 72, 739–744. (c)
Molinski, T. F. Curr. Opin. Biotechnol. 2010, 21, 819–826.
Corresponding Author
*Tel: þ1 (858) 534-7115. Fax: þ1 (858) 822-0386. E-mail:
tmolinski@ucsd.edu.
’ ACKNOWLEDGMENT
We thank J. Gloer (University of Iowa) for helpful discussions,
the University of California, Riverside, for providing HRMS
measurements, and B. Morinaka for assistance with NMR
measurements and cytotoxicity assays. The 500 MHz NMR
spectrometers were purchased with a grant from the NSF
(CRIF, CHE0741968). The author is grateful for generous
support of this work from the NIH (AI 039987, CA122256).
(18) We cannot rule out the possibility that 4 is the product of
adventitious condensation of didemnin A (1) with formaldehyde
present as a contaminant in the MeOH solvent used during isolation-
purification, although this seems unlikely, as the technical specifications
of the HPLC grade MeOH state “<0.001% formaldehyde”. LC-MS
analysis (C18 reversed phase) of the whole MeOH extract of T. solidum
showed the presence of a peak that could be assigned to 4 (m/z 955
[M þ H]þ, 977 [M þ Na]þ).
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dx.doi.org/10.1021/np100846s |J. Nat. Prod. 2011, 74, 882–887