Total Synthesis of Laucysteinamide A, a Monomeric Congener of Somocystinamide A

Article abstract of DOI:10.1021/acs.jnatprod.0c01317

Laucysteinamide A (4) is a marine natural product isolated from the cyanobacterium Caldora penicillata and contains structural motifs found in promising cancer drug leads. The first total synthesis of 4 and its analogues was achieved, which also enabled a concise formal synthesis of somocystinamide A (3), a dimeric congener of 4 that previously showed extremely potent antiproliferative activities. This work provides further insights on structure-activity relationships in this class of natural products.

Full text of DOI:10.1021/acs.jnatprod.0c01317

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Total Synthesis of Laucysteinamide A, a Monomeric Congener of
Somocystinamide A
Kimberly S. Taylor, Chen Zhang, Evgenia Glukhov, William H. Gerwick, and Takashi L. Suyama*
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ABSTRACT: Laucysteinamide A (4) is a marine natural product isolated from
the cyanobacterium Caldora penicillata and contains structural motifs found in
promising cancer drug leads. The first total synthesis of 4 and its analogues was
achieved, which also enabled a concise formal synthesis of somocystinamide A
(3), a dimeric congener of 4 that previously showed extremely potent
antiproliferative activities. This work provides further insights on structure−
activity relationships in this class of natural products.
arine cyanobacteria have been an incredibly prolific
source of bioactive natural products and have yielded
for this apoptosis pathway. This observation may imply that
the dimeric character of 3 is important for its activity. Indeed,
the sulfhydryl derivative of 3, obtained by chemically reducing
the disulfide, showed no cytotoxicity.12
M
medicinally important compounds including those that feature
enamide1,2 and thiazoline moieties.3 These structural features
represent a synthetic challenge due to their labile nature.
Curacin A (1), a potent tubulin inhibitor containing a
thiazoline moiety and a highly lipophilic chain with four
olefins, was isolated from the marine cyanobacterium Moorena
producens (a subset of its former characterization, Lyngbya
majuscula).4−6 Tubulin dimerization and subsequent polymer-
ization requiring GTP binding is a critical step in cell division.7
Curacin A (1) inhibits this assembly process, thus inhibiting
cell division and growth. This is of the utmost importance in
combating cancers, as rapid growth is a key characteristic of
tumor cells. The thiazoline and exocyclic olefin present in 1
were shown to be essential for its cytotoxicity.8 This
heterocycle moiety proved to be a source of significant
instability and required great care during handling and storage
of the molecule.7,8 Kalkitoxin A (2), another thiazoline-
containing metabolite with similar structural motifs and an
isosteric profile to 1, was shown to be a potent neurotoxic9 and
antiangiogenic agent.10
Somocystinamide A (3) is a dimeric natural product with
two tertiary enamides. It was isolated from a mixed assemblage
of Lyngbya majuscula and Schitzothrix.11 Remarkably, metab-
olite 3 showed antiproliferative activity against primary human
endothelial cells (HUVECs) at 500 fM.12 Like 1, this activity
also inhibits cancer growth because tumor tissues need an
increased supply of nutrients, resulting in rapid blood vessel
proliferation. It was shown that compound 3 promotes
apoptosis through activating caspase 8.12 Recruitment of two
procaspase-8 units and subsequent self-activation is necessary
More recently, laucysteinamide A (4) was also isolated from
a related marine cyanobacterium, Caldora penicillata.13,14
Laucysteinamide A (4) had an IC50 value of 11 μM against
the H460 lung cancer cell line, but its full biological activity
could not be evaluated due to sample decomposition.13 Due to
the similarities in structure to compounds 1−3, which have
shown promising cancer therapy potential, a more thorough
evaluation of the biological properties of 4 was deemed
desirable. Moreover, biological activity data obtained for 4 will
be insightful toward understanding the structure−activity
relationships of 3 because 4 is essentially a monomeric
analogue of 3. While showing a promising bioactivity profile, 3
has suffered from its poor solubility in water.12,15 With the rise
of therapeutics consisting of biologic macromolecules con-
jugated to small synthetic molecules, such as antibody−drug
conjugates (ADCs), natural products with high lipophilicity
such as 1−4 and their derivatives can redeem their in vivo
efficacy through being employed as an ADC payload.16 Herein
reported are the first total syntheses of laucysteinamide A (4)
Special Issue: Special Issue in Honor of A. Douglas
Kinghorn
Received: December 7, 2020
Published: February 26, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
https://doi.org/10.1021/acs.jnatprod.0c01317
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Chart 1
Scheme 1. Total Synthesis of Laucysteinamide A (4) and Its Partially Saturated Analogue 11
and its analogue as well as a more efficient and shorter formal
synthesis of somocystinamide A (3).15
Information) and coupled with methylamine to afford the
amide 6.20,21 Coupling of 6 and 7 by Hoveyda−Grubbs II
catalyst was achieved with an excellent yield. The resulting
long-chain amide 8 was deprotected via Birch reduction to
afford a thiol, which was then acetylated, forming thioester 9.
Deprotection of 9 in TFA and subsequent reflux in benzene
promoted dehydrative cyclization to afford the labile thiazoline
10 in a manner similar to the curacin A synthesis by White et
al.8 Without purification, the enamide moiety was immediately
installed by treatment with 4-pentenal and toluenesulfonic acid
in dichloroethane under reflux to afford the natural product 4.
In order to achieve removal of H2O from this reversible
reaction in a halogenated solvent, a Hickman still equipped
with molecular sieves was employed in place of a Dean−Stark
apparatus. While the yield for the last steps was not ideal, it is
within the expected range based on the 30% yield for
cyclization to form the thiazoline ring in curacin A.8 This
material was extensively studied by 1D and 2D NMR (see
Supporting Information for 1D spectra) and was shown to
have a structure identical to that of the natural product. A
critique of the application of the exciton chirality method
employed for 4 has been raised in the literature;22 however, the
original configurational assignment has been confirmed by this
synthesis as evidenced by the specific rotation value of the
synthetic sample (+41.3 vs natural +17.1).13 The overall yield
of the synthesis was 9.4% from the known olefin 7.
Initially, a synthesis was envisioned in which the thiazoline
moiety would be constructed early to avoid the use of
protecting groups. However, this approach was not successful,
presumably due to the labile nature of the thiazoline moiety.
This agrees with previous reports of thiazoline-containing
natural products that have been found to decompose even
under cold storage conditions.4,7,8 Thus, a synthetic route was
developed in which the thiazoline ring was installed at a late
stage. Due to the labile nature of the enamide, we planned its
installation last using the condensation method employed in
the total synthesis of somocystinamide A (3).15 The putative
biosynthetic origin and assigned absolute configuration
indicate laucysteinamide A (4) is likely derived from L-cysteine
via an NRPS mechanism13 and provided an obvious synthon.
Stereochemical requirements prevented us from employing
traditional olefination techniques such as the Wittig reaction.
As with somocystinamide A (3), olefin cross metathesis was
the chosen coupling method.15
Accordingly, olefin cross metathesis substrates 6 and 7 were
prepared. The olefin 7 was synthesized from L-cysteine in five
steps as reported with orthogonal protection by the phenyl
thiazolidine and N-Boc.15,17,18 Commercially available 11-
bromo-1-undecene was homologated19 by a malonate ester
and subsequently decarboxylated to 5 (see Supporting
866
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Scheme 2. Concise Formal Synthesis of Somocystinamide A
While handling 4, we found that removing solvents in vacuo
and storing it as a neat oil encouraged decomposition.
According to the literature, 1 was stored frozen in benzene
to avoid decomposition.4,7,8 Likewise, we stored 4 in benzene
that contained ∼0.1% triethylamine at −20 °C. The partially
saturated enamide analogue 11 was synthesized from the
secondary amide intermediate 10 by employing valeraldehyde
rather than 4-pentenal. To investigate the role of the enamide
functional group, the saturated amide (N,N-methyl-(4-
pentenyl)amide) analogue of 8 was prepared and subjected
to the Birch reduction conditions; however, this led to
decomposition, presumably due to reduction of the amide.
Furthermore, preparation of the carboxylic acid analogue of 9
in anticipation of amidation with N,N-methyl-(4-pentenyl)-
amine was not fruitful.
The synthetic route employed for 4 was applied to
somocystinamide A (3) as seen in Scheme 2.23 This formal
synthesis shortened the reported total synthesis of 3 by three
steps and improved the overall yield from 11.6% to 16.1%
(39% increase).15 The use of a potentially dangerous and
highly toxic reagent, diazomethane, was eliminated in this
improved synthesis, and it makes a larger scale synthesis more
practical for further biological testing.
We had postulated that the activity in the cytotoxicity assay
observed for the natural sample of 4 (IC50 = 11 μM)13
compared to that of 3 (IC50 = 46 nM against A549 lung
carcinoma)12 might have been due to partial decomposition of
the sample and/or solubility issues. Indeed, a DMSO solution
of synthetic 4 showed visual signs of insolubility at high
concentrations in aqueous media. However, even with the aid
of an emulsifying agent, PEG400, the synthetic sample of 4 and
its analogue 11 only had IC50 values of 20 and 33 μM,
respectively, against the H460 cell line (Table 1). In order to
further investigate the importance of the terminal olefin-
enamide moiety in 3 and 4, small enamide analogues (16 and
17) were prepared in the same manner as 4 (Supporting
Information). Both analogues showed no significant lethality in
a brine shrimp assay. Interestingly, the highly antiproliferative
agent 3 was also not lethal to brine shrimp even at 130 μM.15
Because H460 cells are known to express procaspase-8,24
compounds 4, 16, and 17 should show cytotoxicity against
them while showing no toxicity to brine shrimp if they induce
apoptosis in the same manner as 3. However, 16 and 17
showed no activity in the H460 assay. Hence, the terminal
PKS-NRPS enamide motif alone is likely not responsible for
the observed activities of 3 or 4. These results along with the
apparent inactivity of the thiol monomeric derivative 1812
imply that the dimeric nature of 3 is important to impart high
potency.
This first total synthesis of the marine cyanobacterial
metabolite laucysteinamide A (4) and its analogue has led to
confirmation of its bioactivity profile as well as its absolute
configuration. This work also led to a shorter and more robust
formal synthesis of a potent congener of 4, somocystinamide A
(3). Finally, we gained further insight into structure−activity
relationships with regard to 3, an important compound with
development potential as a novel cancer therapeutic.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
acquired on a JASCO J-810 spectrophotometer. IR spectra were
acquired on a PerkinElmer Spectrum Two FT-IR spectrometer. NMR
spectra were obtained using a Varian 600 MHz INOVAUnity NMR
instrument, and 1H NMR reference values were 7.26 ppm for CDCl3
and 7.16 ppm for C6D6. 13C NMR reference values were 77.16 ppm
for CDCl3 and 128.06 ppm for C6D6. HRMS data were acquired on
an Agilent 6230 TOF-MS under positive ion ESI-TOF-MS conditions
and on a Thermo Scientific QExactive MS under positive ion HESI
conditions. Optical density in the cytotoxicity assay was recorded on a
SpectraMax M2 microplate reader with SoftMax Pro microplate data
acquisition (Molecular Devices, LLC). NCI-H460 hypotriploid
human lung carcinoma cells were acquired from the American Type
Culture Collection. The second-generation Grubbs−Hoveyda ruthe-
nium catalyst was purchased from Sigma-Aldrich. All other reagents
and solvents were purchased from Fisher Scientific. CH2Cl2,
dichloroethane, triethylamine, and benzene were distilled from
CaH2. NaH was purchased as a ∼60% w/w dispersion in oil and
was used as-is. TFA and acetic anhydride were distilled immediately
before use. Anhydrous THF was purchased and used as-is. All other
reagents and solvents were used as purchased. All reactions were
carried out under a N2 atmosphere unless otherwise specified.
Table 1. IC50 Values of Enamide Natural Products and
Analogues
compound
H460
brine shrimp
other
3
na
>130 μM15
A549: 46 nM12
HUVEC: 500 fM12
na
4
11 μM (natural)13
20 3 μM (synthetic)
33 12 μM
>248 μM
>300 μM
>100 μM
11
16
17
na
na
na
na
>100 μM
>100 μM
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Chart 2
Synthesis. N-Methyl 12-Tridecenamide (6). To a suspension of 5
(1.24 g, 5.85 mmol) in CH2Cl2 (40 mL) were added CH3NH2·HCl
(594 mg, 8.80 mmol, 1.50 equiv), Et3N (1.63 mL, 11.7 mmol, 2.00
equiv), EDC·HCl (1.23 g, 6.44 mmol, 1.10 equiv), and DMAP (72
mg, 0.59 mmol, 0.10 equiv) successively at 0 °C. The solution was
stirred overnight as it warmed to rt. Consumption of 5 was monitored
and confirmed by TLC analysis. The solvents were removed in vacuo,
and the resulting concentration was triturated with EtOAc to separate
the urea byproduct. The resulting solution was washed with 0.5 M
HCl (×3) followed by H2O, NaHCO3, H2O, and brine successively.
The organic layer was dried over Na2SO4 and concentrated to a solid,
which was eluted through a plug of silica as an EtOAc solution to
afford N-methyl 12-tridecenamide (6) as a white solid (956 mg, 4.24
mmol, 73% yield) upon removal of the solvents under vacuum. TLC
Rf 0.22 EtOAc/hexanes (1:1) KMnO4 stain; IR νmax 3296, 3085,
2979, 2916, 2849, 1636, 1560, 912 cm−1; 1H NMR (600 MHz,
CDCl3) δ 6.43 (1H, s), 5.72 (1H, ddt, J = 17.0, 10.2, 6.7 Hz), 4.90
(1H, d, J = 17.0 Hz), 4.84 (1H, d, J = 10.2 Hz), 2.70 (3H, d, J = 4.8
Hz), 2.10 (2H, t, J = 7.7 Hz), 1.95 (2H, q, J = 7.2 Hz), 1.54 (2H, t, J =
7.3 Hz), 1.29 (2H, p, J = 6.9 Hz), 1.24−1.13 (12H, m); 13C NMR
(150 MHz, CDCl3) δ 174.2, 139.1, 114.0, 36.5, 33.7, 29.5, 29.5, 29.4,
29.4, 29.3, 29.1, 28.9, 26.1, 25.8; HR-ESI-TOFMS m/z [M + H]+
226.2166 (calcd for C14H28NO, 226.2165).
(2R,4R)-3-tert-Butoxycarbonyl-4-((E)-13-amine-13-oxotridec-1-
enyl)-2-phenylthiazolidine (8). N-Methyl 12-tridecenamide (6) (730
mg, 3.24 mmol) was added to a solution of (2R,4R)-2-phenyl-4-
vinylthiazolidine-3-tert-butyl carboxylate (7) (535 mg, 1.84 mmol) in
CH2Cl2 (25 mL). The solution was degassed under vacuum with
sonication three times. Hoveyda−Grubbs generation II catalyst (57
mg, 0.091 mmol) was added, turning the solution bright green. The
solution was heated to reflux then refluxed overnight. The reaction
progress was monitored by TLC analysis. The solution was
concentrated under vacuum and was subjected to flash column
chromatography (hexanes to 1:1 hexanes/EtOAc) to yield a pale
yellow oil (759 mg, 1.55 mmol, 87%). TLC Rf 0.24 EtOAc/hexanes
(2:1) KMnO4 stain; [α]D25 +198 (c 0.17, CH2Cl2); IR νmax 3300 (br),
3031, 3064, 2976, 2924, 2852, 1733, 1696, 1648, 1365, 1157 cm−1;
1H NMR (600 MHz, CDCl3) δ 7.36 (2H, d, J = 7.5 Hz), 7.30 (2H, t,
J = 7.5 Hz), 7.23 (1H, t, J = 7.3 Hz), 6.07 (1H, s), 5.77 (1H, dt, J =
14.1, 6.2 Hz), 5.63 (1H, dd, J = 14.1, 7.5 Hz), 5.53 (1H, m,), 4.81
(1H, bs), 3.24 (1H, dd, J = 11.7, 6.4 Hz). 2.80 (1H, dd, J = 11.7, 4.6
Hz), 2.79/2.78 (3H, s, rotamers), 2.14 (2H, m), 2.06 (2H, q, J = 7.0
Hz), 1.60 (2H, m), 1.38 (2H, quin, J = 7.5 Hz), 1.34−1.26 (21H, m);
13C NMR (150 MHz, CDCl3) δ 173.9, 153.8, 142.0, 134.2, 129.5,
128.3, 127.6, 126.4, 80.6, 66.2, 64.0, 36.9, 36.8, 32.3, 29.7, 29.6, 29.6,
29.5, 29.4, 29.2, 28.4, 28.4, 26.4, 25.9; HR-ESI-TOFMS m/z [M +
Na]+ 511.2959 (calcd for C28H44N2O3SNa, 511.2965).
(R,E)-N-Methyl 14-((tert-Butoxycarbonyl)amino)-15-(acetylthio)-
pentadec-12-enamide (9). Liquid ammonia was prepared from
combining solid NH4Cl and powdered NaOH at room temperature.
The resulting ammonia gas was passed through Drierite and was
condensed at −78 °C. This liquid ammonia was distilled from sodium
into the reaction flask at −78 °C. A solution of (2R,4R)-3-tert-
butoxycarbonyl-4-((E)-13-amine-13-oxotridec-1-enyl)-2-phenylthia-
zolidine (8) (819 mg, 1.68 mmol) in THF (3 mL) was added to the
liquid ammonia (30 mL) at −78 °C. Sodium metal (∼150 mg) was
added to keep the solution a midnight blue color. The solution was
allowed to warm to rt and refluxed for approximately 2 h, during
which the blue color persisted. Solid NH4Cl was added to quench the
reaction. The solution was dried under a stream of nitrogen gas. Then,
the residue was extracted with CH2Cl2 and filtered through a fritted
funnel. The filtrate was then concentrated in vacuo to yield a pale
brown oil. CH2Cl2 (40 mL) was added to the concentrated oil, and
the solution was cooled. Subsequently, Ac2O (750 μL, 7.93 mmol)
and Et3N (2.2 mL 16 mmol) were added successively. The solution
was left to warm to rt under stirring overnight. The solvents were
removed in vacuo, yielding a white solid, which was dissolved in Et2O.
To the organic phase were added successively aqueous Na2CO3,
aqueous HCl, and brine to wash. The organic layer was then dried
over MgSO4. The resulting solution was concentrated in vacuo to
yield a pale yellow solid, which was then subjected to flash column
chromatography (hexanes to 1:9 MeOH/EtOAc), yielding a white
amorphous solid (332 mg, 0.751 mmol, 45%). TLC Rf 0.49 EtOAc;
[α]2D5 −2 (c 0.03, CH2Cl2); IR νmax 3299 (br), 3094, 2924, 2853,
1
1695, 1650, 1551, 1241, 1171 cm−1; H NMR (600 MHz, CDCl3) δ
5.62 (1H, td, J = 15.3, 6.9 Hz), 5.43 (1H, bs), 5.33 (1H, dd, J = 15.3,
6.1 Hz), 4.63 (1H, m), 4.25 (1H, bs), 3.09 (1H, dd, J = 14.0, 4.1 Hz),
3.04 (1H, dd, J = 14.0, 7.2 Hz), 2.81/2.80 (3H, s, rotamers), 2.34
(3H, s), 2.16 (2H, t, J = 7.6 Hz), 2.00 (2H, q, J = 7.1 Hz), 1.62 (4H,
m), 1.44 (9H, s), 1.35−1.25 (12H, m); 13C NMR (150 MHz, CDCl3)
δ 195.7, 173.9, 153.3, 133.1, 128.5, 56.1, 36.9, 34.2, 32.3, 30.7, 29.6,
29.6, 29.5, 29.5, 29.2, 28.5, 26.4, 25.9, 14.3; HR-ESI-TOFMS m/z [M
+ Na]+ 465.2754 (calcd for C23H42N2O4SNa, 465.2757).
(R)-2-Methyl-4-((E)-11-(methylamino)-11-oxotridec-1-enyl)-2-
thiazoline (10). To a solution of 9 (153.3 mg, 0.3466 mmol) in
CH2Cl2 (16 mL) at 0 °C was added TFA (5.4 mL). The solution was
allowed to warm to rt while stirring for 1 h, forming a pale pink film
upon concentration in vacuo, which was immediately solubilized in
benzene (5 mL). The benzene solution was then refluxed under N2
for 2 h with a Hickman still filled with 3 Å molecular sieves on top of
the reaction vessel. The reaction progress was monitored by TLC and
LCMS analysis. Once the reaction appeared to reach completion, the
solvent was removed in vacuo and the crude oil that resulted was
immediately used in the next step without purification.
General Procedures for Laucysteinamide A (4) and the Partially
Saturated Analogue (11). To a solution of crude thiazoline (10) in
CH2Cl (35 mL) were added 4-pentenal (150 μL, 1.43 mmol) and
TsOH·H2O (16.9 mg, 0.0981 mmol) at rt. The solution was degassed
under vacuum with sonication three times. The reaction vessel was
equipped with a Hickman still filled with 3 Å molecular sieves and a
condenser. The reaction mixture was vigorously refluxed overnight,
during which two additional amounts of 4-pentenal were added (a
total of 225 μL, 2.13 mmol). A color change from pale yellow to dark
brown was noted. The reaction was monitored using TLC and LCMS
analysis, which showed the desired mass and diminished reactant
concentration. Then the solution was cooled to 0 °C, and Et3N (75
μL, 0.54 mmol) was added. The solution was concentrated in vacuo
and was directly subjected to flash column chromatography (hexanes
to EtOAc) in a triethylamine buffer, yielding a pale yellow oil 4 (32.8
mg, 0.0841 mmol, 24% across three steps). TLC Rf 0.60 EtOAc/
hexanes (1:1); [α]2D5 +41 (c 0.02, CHCl3); IR νmax 3031, 3085, 3006,
2923, 2851, 1708, 1679, 1646, 1390, 1289, 1090 cm−1; 1H NMR (600
MHz, C6D6 including 0.1% Et3N) δ 7.79 (0.3H, rotamer, d, J = 14.4
Hz), 6.43 (0.7H, rotamer, d, J = 13.8 Hz), 5.77 (1H, ddt, J = 16.6,
10.1, 6.2 Hz), 5.69 (1H, dt, J = 14.9, 6.8 Hz), 5.55 (1H, dd, J = 14.9,
6.7 Hz), 5.08−4.96 (2H, m), 4.80 (1H, q, J = 8.2 Hz), 4.67 (0.3H,
rotamer, dt, J = 14.4, 7.0 Hz), 4.61 (0.7H, rotamer, dt, J = 13.8, 6.9
Hz), 3.02 (1H, dd, J = 10.8, 8.2 Hz), 2.90 (2.1H, rotamer, s), 2.79
(1H, dd, J = 10.8, 8.2 Hz), 2.68 (0.6H, rotamer, t, J = 6.6 Hz), 2.61
(1.4H, rotamer, dq, J = 6.6, 1.5 Hz), 2.36 (0.9H, rotamer, s), 2.09
(1.4H, rotamer, dd, J = 8.6, 6.1 Hz), 1.98 (2H, d, J = 1.6 Hz), 1.93
(0.6H, rotamer, t, J = 7.4 Hz), 1.73−1.65 (2H, m), 1.37−1.20 (12H,
m); 13C NMR (150 MHz, C6D6 including 0.1% Et3N) δ 170.5, 170.3,
164.7, 138.1, 137.6, 132.1, 130.4, 130.1, 129.1, 115.2, 114.9, 107.2,
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107.0, 79.2, 40.4, 34.9, 34.8, 34.2, 33.7, 32.8, 31.5, 30.0, 29.9, 29.8,
29.6, 29.4, 25.3, 25.3, 20.2; HR-ESI-TOFMS m/z [M + H]+ calcd for
C23H39N2OS 391.2778, found 391.2774.
School of Pharmacy and Pharmaceutical Sciences, University
of California San Diego, La Jolla, California 92093, United
States
Partially Saturated Laucysteinamide A Analogue (11). Likewise,
valeraldehyde was used in place of 4-pentenal with thiazoline 10 (44.0
mg, 0.136 mmol) to afford a colorless film (6.9 mg, 0.018 mmol, 13%
from 9). TLC Rf 0.69 EtOAc/hexanes (1:1); [α]2D5 +33 (c 0.03,
CHCl3); IR νmax 3090, 3035, 2923, 2855, 1738, 1670, 1646, 1373,
1218, 1090 cm−1; 1H NMR (600 MHz, C6D6 including 0.1% Et3N) δ
7.82 (0.3H, rotamer, d, J = 14.5 Hz), 6.47 (0.7H, rotamer, d, J = 13.8
Hz), 5.69 (1H, dt, J = 14.8, 6.7 Hz), 5.55 (1H, dd, J = 14.8, 6.7 Hz),
4.80 (1H, q, J = 8.1 Hz), 4.70 (0.3H, rotamer, dt, J = 14.5, 7.1 Hz),
4.64 (0.7H, rotamer, dt, J = 13.8, 7.2 Hz), 3.02 (1H, dd, J = 10.8, 8.1
Hz), 2.94 (2.1H, rotamer, s), 2.79 (1H, dt, J = 10.8, 8.1 Hz), 2.41
(0.9H, rotamer, s), 2.15 (2H, t, J = 7.4 Hz), 1.88 (2H, q, J = 7.2 Hz),
1.77−1.67 (2H, m), 1.41−1.16 (21H, m), 0.88 (3H, t, J = 7.3 Hz);
13C NMR (150 MHz, C6D6 including 0.1% Et3N) δ 170.5, 170.2,
164.6, 133.5, 132.1, 130.4, 140.1, 129.1, 110.0, 109.3, 79.2, 40.4, 34.3,
33.8, 32.9, 32.8, 30.0, 29.9, 29.8, 29.8, 29.6, 29.6, 29.5, 25.4, 25.3,
24.1, 23.9, 20.2, 13.8; HR-ESI-TOFMS m/z [M + H]+ 393.2929
(calcd for C23H41N2OS, 393.2934).
Cytotoxicity Assay Protocols. NCI-H460 cells were maintained
in the lab. On the first day cells were seeded into 96-well plates at 6.66
× 103 cells/mL of RPMI 1640 medium with standard fetal bovine
serum 180 μL/well and incubated for 24 h (37 °C, 5% CO2). Then
each one of the four tested compounds (4, 11, 16, or 17) was
dissolved in RPMI 1640 followed by nine half-log serial dilutions in
the presence of 10% v/v DMSO and PEG400 in triplicates, and 20 μL
of each of these formulations was added to the attached cells in
duplicates. This way each concentration of each compound was tested
six times in the presence of 1% v/v of DMSO and PEG400. The
highest tested concentrations for compounds 4, 11, 16, and 17 were
128, 127, 248, and 300 μM, respectively. Plates were incubated for an
additional 48 h and then stained with 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) for 25 min, and after being
dissolved in DMSO the optical densities were recorded at 630 and
570 nm for each well. Dose−response curves and IC50 values were
generated with GraphPad Software.
William H. Gerwick − Center for Marine Biotechnology and
Biomedicine, Scripps Institution of Oceanography and Skaggs
School of Pharmacy and Pharmaceutical Sciences, University
of California San Diego, La Jolla, California 92093, United
States; orcid.org/0000-0003-1403-4458
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01317
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Y. Su at UCSD Molecular Mass Spectrometry
Facility and S. N. Majuta at WVU Shared Research Facility for
assistance with acquisition of HRMS data. The H and 13C
1
NMR spectra were acquired on a Varian 600 MHz INOVAUnity
NMR instrument at West Virginia University. The Department
of Chemistry and Forensics at Waynesburg University is
acknowledged for reagents and supplies used in this project.
K.S.T. gratefully acknowledges Sigma Xi research grant
G20200315110993198, which was used to purchase equip-
ment and chromatography supplies.
DEDICATION
■
Dedicated to Dr. A. Douglas Kinghorn, The Ohio State
University, for his pioneering work on bioactive natural
products.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01317.
■
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AUTHOR INFORMATION
Corresponding Author
Takashi L. Suyama − Department of Chemistry and Forensic
Science, Waynesburg University, Waynesburg, Pennsylvania
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Email: tsuyama@waynesburg.edu
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