Total Synthesis of Reniochalistatin e

Article abstract of DOI:10.1021/acs.jnatprod.7b00656

Reniochalistatin E (1) is one of the five related cyclic peptides isolated from the marine sponge Reniochalina stalagmitis. The discovery of these compounds resulted from a screening program directed toward the identification of proline-rich bioactive compounds. Reniochalistatin E is the only member of the family to possess a tryptophan amino acid residue. Given the cytotoxicity observed for 1, efforts were directed toward developing a synthetic route to 1. The first total synthesis of 1 has been accomplished in a 15-step route in an overall 5.0% yield. The synthetic sample of reniochalistatin E was shown to have similar activity toward HeLa and RPMI-8226 cell lines compared to the natural sample, with IC₅₀ values of 16.9 vs 17.3 μM and 4.5 vs 4.9 μM, respectively. Interestingly, both of the fully deprotected octapeptides constructed toward the synthesis of reniochalistatin E were shown to have cytotoxicity. The route provides a means to probe the structure-activity relationship of 1 and further biological investigations.

Full text of DOI:10.1021/acs.jnatprod.7b00656

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Total Synthesis of Reniochalistatin E
Anthony Fatino, Giovanna Baca, Chamitha Weeramange, and Ryan J. Rafferty*
Department of Chemistry, Kansas State University, Manhattan, Kansas 66506, United States
S
* Supporting Information
ABSTRACT: Reniochalistatin E (1) is one of the five related
cyclic peptides isolated from the marine sponge Reniochalina
stalagmitis. The discovery of these compounds resulted from a
screening program directed toward the identification of proline-
rich bioactive compounds. Reniochalistatin E is the only
member of the family to possess a tryptophan amino acid
residue. Given the cytotoxicity observed for 1, efforts were
directed toward developing a synthetic route to 1. The first total
synthesis of 1 has been accomplished in a 15-step route in an
overall 5.0% yield. The synthetic sample of reniochalistatin E
was shown to have similar activity toward HeLa and RPMI-
8226 cell lines compared to the natural sample, with IC50 values
of 16.9 vs 17.3 μM and 4.5 vs 4.9 μM, respectively.
Interestingly, both of the fully deprotected octapeptides
constructed toward the synthesis of reniochalistatin E were shown to have cytotoxicity. The route provides a means to probe
the structure−activity relationship of 1 and further biological investigations.
he reniochalistatin family of cyclic peptides was isolated
According to the retrosynthetic plan outlined in Scheme 1,
Tfrom the marine sponge Reniochalina stalagmitis near efforts were directed toward the construction of tetrapeptide 2
(Scheme 2). Starting from the Boc-protected L-isoleucine (4) an
amidation was performed though a benzotriazole-L-yl-oxy-tris-
pyrrolidino-phosphonium hexafluorophosphate (PyBOP)-medi-
ated coupling with the methyl ester of L-proline (5), affording the
dipeptide 6 in 92% yield. Analogously, Boc-L-leucine (7) was
coupled to the methyl ester of L-tryptophan (8) under the same
conditions to access dipeptide 9 in 97% yield. Saponification of
dipeptide 6 furnished the free acid 10, and Boc deprotection of 9
accessed the amine hydrochloride salt 11. Both reactions
provided quantitative yields. The coupling of these two
dipeptides was accomplished with PyBOP under standard
conditions to give tetrapeptide 2 in 55% yield. Employing 1-
ethyl-3-(3'-dimethylaminopropyl)carbodiimide hydrochloride
(EDC·HCl) with HOBt as the coupling conditions, yields were
optimized at 86%.
With tetrapeptide 2 in hand, efforts were directed toward the
construction of tetrapeptide 3 (Scheme 3), which will allow for
access to 1. The coupling of Boc-L-proline (12) to the methyl
ester of L-leucine (13) was performed with PyBOP to furnish the
dipeptide 14 in 97% yield. Saponification of 14 gave access to the
free acid dipeptide 15 in quantitative yields. The second half of
the tetrapeptide was accessed from the Boc deprotection of
dipeptide 6 in quantitative yield. The coupling of 15 to 16 under
the previously employed PyBOP conditions afforded the desired
tetrapeptide 3, but unfortunately as an impure mixture based
upon the 1H NMR spectrum obtained. All attempts at
Yongxing Island in the South China Sea in 2014 by Lin and co-
workers.1 The family was discovered as part of a screening
program designed to find new bioactive cyclic peptides from
marine sponges in the South China Sea.2−5 Cyclic peptides that
are proline-rich have been shown to have a variety and wide
scope of biological activity, such as antiviral, antitumor,
antimicrobial, and general cytotoxic properties.6−8 The members
of the reniochalstatin family were found to be proline-rich and
composed of apolar and aromatic amino acid residues. Leucine
and isoleucine are present in all five members, along with
multiple proline units (two within reniochalistatins A, B, and C
and three within D and E). The aromatic amino acid residue
varies among the members of the family; B, C, and D contain
phenylalanine, whereas E is the only one with a tryptophan
residue.8 Reniochalistatins A through D are cyclic heptaptides,
whereas E is the only cyclic octapeptide that was isolated. Only
reniochalistatin E showed biological activity, with cytotoxicity
toward myeloma (RPMI-8226, IC50 of 4.9 μM) and gastric
(MGC-803, IC50 of 9.7 μM) cancer cell lines. It was the activity
toward myeloma that drew the attention of our laboratory and
resulted in a total synthesis campaign to be undertaken.
We envisioned that 1 could be synthesized via successive
amino acid couplings in tandem with selective deprotection
steps. The cyclic compound could be obtained from the coupling
and macrocyclization of the two desired tetrapeptides (2 and 3).
Each of these tetrapeptides could be constructed from their
corresponding commercially available L-amino acids, with
selective protection about either the free carboxylic acid or
amine (Scheme 1).
Received: July 29, 2017
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00656
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Scheme 1. Retrosynthetic Approach to Reniochalistatin E
Scheme 2. Assembly of Tetrapeptide 2 with Successive PyBOP Coupling and Deprotection Steps
Scheme 3. Construction of Tetrapeptide 3 through Successive Amide Forming Coupling Reactions along with Deprotection Steps
Scheme 4. Tetrapeptide Couplings and Cyclization Strategies Accessing Reniochalistatin E (1)
purification (workup procedures, chromatographic eluents, and
saponification or Boc deprotection, none of the desired material
was collected. Pure tetrapeptide 3 was successfully accessed
through the use of EDC·HCl and HOBt in 78% yield.
silica) of this material continued to provide the same
1
contaminant peaks within the acquired H NMR spectrum.
When this material was carried forward, either through
B
DOI: 10.1021/acs.jnatprod.7b00656
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Journal of Natural Products
Article
Having accessed both of the desired tetrapeptides 2 and 3, the
coupling and macrocyclization were performed. The initial
approach to the linear octapeptide of 1 was through the
saponification of 2 and the Boc deprotection of 3. Coupling of
the free acid of 2 to the free base of 3 with EDC·HCl furnished
octapeptide 17 in 34% yield over three steps (Scheme 4).
Compound 17 was subjected to saponification followed by Boc
deprotection to afford its fully deprotected form 17a in 97%
yield. The attempted macrocyclization of this material under
PyBOP conditions failed to provide any of the desired product,
but rather resulted in the return of starting material (55%) and
decomposition. It is hypothesized that the bulk of both the
secondary amine within the proline residue and the BOP-
activated ester of the tryptophan residue could provide sufficient
steric congestion, thereby preventing cyclization from occurring.
To reduce the steric hindrance about these centers, aminolysis
was then attempted for ring closure. For this, the Boc group on
17 was removed to afford 17b, with the methyl ester intact.
Unfortunately, all attempts at cyclization in this fashion failed to
provide 1 and resulted in the return of starting material (67%)
and decomposition. Recognizing the difficulty in macrocycliza-
tion of these two sterically encumbered proline and tryptophan
residues of 17a, it was envisioned that coupling of these two
centers to access the linear octapeptide would be more attainable
followed then by macrocyclization. As such, 2 was saponified to
its free acid and Boc deprotection of 3 afforded the free amine,
both accomplished in quantitative yields. The EDC·HCl
coupling of these two units furnished the linear octapeptide 18
in 61% yield over three steps. Compound 18 was transformed
into its free acid and free amine counterpart in 95% yield and
then subjected to EDC·HCl coupling conditions to afford the
Table 1. Screening of Coupling Conditions of Fully
Deprotected 17a and 18a
entry
cmpd
coupling agent
additive
result
1
17a
EDC·HCl
EDC·HCl
DEBPT
DEBPT
HBTU
N/A
6%
8%
2
HOBt
N/A
3
4
CsCl
N/A
5
6
HBTU
DMAP
DMAP
N/A
4%
7
TBTU
trace
trace
9%
8
18a
PyBOP
EDC·HCl
EDC·HCl
DEBPT
DEBPT
HBTU
9
N/A
10
11
12
13
14
HOBt
N/A
15%
CsCl
N/A
5%
HBTU
DMAP
13%
yl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
(HBTU)10−13 and N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-
1-yl)uronium tetrafluoroborate (TBTU)13−16 were both at-
tempted with 17a and 18a in the absence and presence of 4-
(dimethylamino)pyridine (DMAP).17,18 In all attempted cycliza-
tions, these uronium-based reagents provided either none or low
yields of 1. Employing HBTU with DMAP to cyclize 18a
provided a 13% yield of 1 (entry 14), but in our hands, EDC·HCl
with HOBt gave the best, albeit low, 15% yield of the natural
product.
With reniochalistatin E and numerous di-, tetra-, and
octapeptides in both fully protected and deprotected states in
hand, all compounds were screened for general cytotoxicity
against six human cancer cell lines (A549, lung; HeLa, cervical;
MiaPaca, pancreatic; U937, lymphoma, RPMI-8226, myeloma;
and MM.1R, multiple myeloma). None of the di- or tetrapeptides
in protected and partial deprotected states were found to have
IC50 values lower than 30 μM against these cell lines. Our
synthetic sample of reniochalistatin E possessed approximately
the same IC50 value toward the HeLa and RPMI-8226 cell lines:
IC50 of 16.9 μM ( 1.9) relative to the reported 17.3 μM in HeLa
and an IC50 of 4.5 ( 2.2) relative to the reported 4.9 μM in
RPMI-8226 (Table 1). Reniochalistatin E lacked cytotoxicity
against all of the tested cell lines (cytotoxicity = IC50 < 10 μM).
Both 17 and 18, diprotected octapeptides, were shown to have
no cytotoxicity. Interestingly, the free acid/amine 17a was shown
to have borderline cytotoxicity toward the U937 cell line with an
IC50 of 9.5 2.1 μM. In comparison to the free acid/amine 18a,
no cytotoxicity was observed. No further investigational studies
at this time have been performed that could draw conclusions
regarding structure−activity relationships based upon the results
obtained.
1
desired natural product 1 in 9% yield. The H and 13C NMR
spectra as well as the MS data of the synthesized 1 match with the
literature data (see the Supporting Information). Confirmation
of the trans prolines within 1 was confirmed based on the
comparison of the reported ΔδCβ‑Cγ values of proline residues
(4.9, 4.2, and 4.1 ppm for Pro,1 Pro,2 and Pro,3 respectively). The
successful macrocyclization of the free acid/amine of 18 with
EDC·HCl on the first attempt led us to attempt the same
conditions for cyclization upon the unsuccessful closure of the
free acid/amine of 17. To our surprise, treating 17a with EDC·
HCl afforded reniochalistatin E in 6% yield. All spectroscopic
(NMR and IR) and MS data matched both the literature and the
previously synthesized material.
Screening various coupling reagents and conditions was then
performed to increase the yield of 1 (Table 1). Entries 1 and 9
highlight the coupling affording 1 outlined in Scheme 4 from 17a
and 18a, respectively. The attempted PyBOP-mediated coupling
of 18a (entry 8) provided only recovered starting material (78%)
and decomposition. Employing HOBt, a commonly used
additive in peptide couplings, with EDC·HCl to cyclize 17a
and 18a (entries 2 and 10) resulted in low yields of 1 of 2% and
6%, respectively. Entry 10 reflects the highest yield of 1 in our
synthetic approach via 18a. Next, macrocyclization was
attempted employing 3-(diethoxyphosphoroyloxyl)-1,2,3-ben-
zotriazine-4(3H)-one (DEPBT), a reagent that has been
successful in the cyclization of similar amino acid based
macrocycles.9 Unfortunately, all attempts at cyclization with
this reagent of 17a and 18a (entries 3 and 11) as well as using
cesium chloride as an additive to promote carbonyl coordination
(entries 4 and 12) failed to provide 1 in any increased yield.
Uronium-based coupling reagents are well reported for their
success in macrocyclizations, and as such, N,N,N′,N′-tetrameth-
In conclusion, we have accomplished the first total synthesis of
reniochalistatin E in 15 steps with an overall 5.0% yield from
commercially available materials. Cytotoxicity studies with
reniochalistatin E and all intermediates accessed within the
route revealed a general lack of cytotoxicity. However, the
synthetic and isolated reniochalistatin E are shown to possess
nearly the same activity toward the HeLa cervical cancer cell line,
further supporting the total synthesis outlined herein. In
addition, an interesting and unexpected cytotoxicity was
observed for the free acid/amine of 17a (linear octapeptide
precursors to the natural product) toward the U937 cell line.
With a route established to this cyclic octapeptide, efforts are
C
DOI: 10.1021/acs.jnatprod.7b00656
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Table 2. Evaluation of Reniochalistatin E and Linear Octapeptides for Cytotoxicity
a
observed IC50 (μM)
cmpd
A549
HeLa
MiaPaca
U937
RPMI-8226
MM.1R
1
>20
>20
>20
>20
>20
16.9 1.9
>20
>20 μM
19.1 1.3
>20
12.4 2.4
>20
4.5 1.8
>20
>11.2 1.0
>20
17
17a
18
18a
>20
9.5 2.1
>20
>20
>20
>20
>20
>20
>20
18.4 1.7
>20
>20
16.4 3.5
>20
a
Cytotoxicity evaluated in 384-well plates, 1500 (2000 U937) cells/well, 72 h incubation period, and evaluated via Alamar Blue.
[α]2D5 +27 (c 0.15, CHCl3); IR (film) νmax 3418, 3018, 2872, 1739, 1513,
1215 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 8.06 (d, J =
7.4 Hz, 1H), 7.45 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.1 Hz, 1H), 7.13 (d, J
= 2.4 Hz, 1H), 7.07−7.02 (m, 1H), 6.96 (td, J = 7.5, 6.9, 1.1 Hz, 1H),
6.82 (d, J = 8.5 Hz, 1H), 4.51 (q, J = 7.1 Hz, 1H), 3.98 (td, J = 8.7, 6.0 Hz,
1H), 3.55 (s, 3H), 3.13−3.05 (m, 2H), 1.53 (dd, J = 13.6, 6.9 Hz, 2H),
1.35 (s, 9H), 1.26 (s, 1H), 0.84 (d, J = 6.6 Hz, 3H), 0.81 (d, J = 6.5 Hz,
3H); 13C NMR (101 MHz, CDCl3) δ 172.99, 172.44, 155.95, 136.45,
127.76, 123.62, 122.15, 119.58, 118.63, 111.71, 109.49, 80.22, 53.46,
53.29, 52.54, 41.55, 28.52, 27.81, 24.92, 23.18, 22.04; HRESIMS m/z
454.2308 [M + Na]+ (calcd for C23H33N3O5Na+, 454.2312).
N-Boc-L-Ile-L-Pro-OH (10). To a stirring solution of 6 (556 mg, 1.62
mmol, 1.0 equiv) in 1:1 THF/H2O (16 mL) was added LiOH (194 mg,
8.12 mmol, 5.0 equiv). The mixture was left to stir at rt for 5 h. The
reaction was quenched with 1 M HCl (1 vol equiv), and the product was
extracted with CH2Cl2, washed with NaHCO3(satd), dried over sodium
sulfate, and concentrated under reduced pressure. The product 10 was
obtained in quantitative yield as an oily residue, which was used without
further purification. IR (film) νmax 3456, 3304, 2977, 1704, 1314 cm−1;
1H NMR (400 MHz, CDCl3) δ 8.31 (s, 1H), 5.35 (d, J = 9.5 Hz, 1H),
4.57 (t, J = 6.4 Hz, 1H), 4.32−4.25 (m, 1H), 3.82 (dt, J = 9.6, 7.2 Hz,
1H), 3.64 (ddd, J = 9.7, 7.5, 5.4 Hz, 1H), 2.18 (dd, J = 10.0, 3.9 Hz, 2H),
2.07−1.99 (m, 2H), 1.76 (td, J = 7.0, 3.4 Hz, 1H), 1.64−1.48 (m, 2H),
1.41 (d, J = 7.7 Hz, 9H), 0.95 (d, J = 6.7 Hz, 3H), 0.87 (t, J = 7.2 Hz, 3H).
N-Boc-L-Leu-L-Trp-OH (11). To a RBF was added 1 M HCl in
dioxane (4.6 mL), which cooled to 0 °C, at which point 9 (500 mg, 1.16
mmol) was added. The reaction was allowed to stir for 1.5 h. The solvent
was removed under reduced pressure to afford a crude white solid (11),
which was used without further purification. IR (film) νmax 3416, 2879,
1651, 1176 cm−1; 1H NMR (400 MHz, DMSO-d6) δ 9.04 (d, J = 7.2 Hz,
1H), 8.25 (s, 3H), 7.49 (d, J = 7.8 Hz, 1H), 7.35 (dt, J = 8.1, 1.0 Hz, 1H),
7.24 (d, J = 2.3 Hz, 1H), 7.07 (ddd, J = 8.1, 7.0, 1.3 Hz, 1H), 7.00 (ddd, J
= 8.0, 7.0, 1.1 Hz, 1H), 4.63−4.54 (m, 1H), 3.81 (t, J = 7.2 Hz, 1H), 3.58
(s, 3H), 3.21−3.13 (m, 2H), 1.68 (dt, J = 13.3, 6.6 Hz, 2H), 1.57 (s, 2H),
0.90 (dd, J = 7.9, 6.4 Hz, 6H).
N-Boc-L-Ile-L-Pro-L-Leu-L-Trp-OMe (2). Compound 10 (1.32 g,
4.02 mmol, 1 equiv), compound 11 (1.08 g, 2.94 mmol, 1 equiv), and
HOBt (473 mg, 3.09 mmol, 1.05 equiv) were dissolved in dry THF (40
mL) at rt under an argon atmosphere. The solution was cooled to 0 °C
and stirred for 20 min, after which Et3N (1.43 mL, 10.29 mmol, 3.5
equiv) was then added and stirred. Lastly, 20 min later EDC·HCl (592
mg, 3.09 mmol, 1.05 equiv) was added, and the reaction mixture was left
to stir overnight. The reaction was quenched with H2O (1 vol equiv),
and the product was extracted with CH2Cl2, dried over sodium sulfate,
and concentrated under reduced pressure. The crude material was
purified via flash silica gel chromatography (3:1 EtOAc/hexane) to give
afford 2 (2.22 g, 86% yield) as a clear liquid: [α]2D5 +48.1 (c 1.75, CHCl3);
IR (film) νmax 3305, 3017, 2876, 1741, 1631, 1506, 1392, 1215 cm−1; 1H
NMR (CDCl3, 400 MHz) δ 8.29 (s, 1H), 7.51 (d, J = 8.6 Hz, 1H), 7.33
(d, J = 8.0 Hz, 1H), 7.16 (td, J = 8.2, 7.6, 1.3 Hz, 1H), 7.13−7.05 (m,
1H), 7.03 (d, J = 2.4 Hz, 1H), 6.97 (d, J = 7.8 Hz, 1H), 6.70 (d, J = 7.9
Hz, 1H), 5.20 (d, J = 9.4 Hz, 1H), 4.90 (dt, J = 7.9, 5.5 Hz, 1H), 4.37 (d, J
= 6.0 Hz, 1H), 4.27 (dd, J = 9.3, 7.0 Hz, 1H), 3.71 (d, J = 8.9 Hz, 1H),
3.66 (s, 3H), 3.60−3.51 (m, 1H), 3.30 (d, J = 5.5 Hz, 2H), 2.12−1.87
(m, 3H), 1.73−1.56 (m, 5H), 1.43 (s, 9H), 1.13−1.05 (m, 1H), 0.92 (d,
J = 6.8 Hz, 3H), 0.90−0.80 (m, 11H); 13C NMR (CDCl3, 101 MHz) δ
172.86, 172.49, 172.08, 171.74, 156.08, 136.32, 127.75, 123.57, 122.11,
currently being pursued toward probing the effects of single
amino acid variations as well as the transposition of phenyl-
alanine for tryptophan. The resulting analogues of 1 will be
evaluated for cytotoxicity.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
measured on an Atago Polax-2L polarimeter with a sodium lamp. IR
spectra were recorded on a Cary 630 FT-IR spectrometer as thin films.
Only the strongest and/or structurally important absorptions of IR
spectra were reported in wavenumbers (cm−1). 1H (400, 500 MHz) and
13C (100, 151 MHz) spectra were obtained on Varian and Bruker-
Ascend spectrometers. The chemical shifts are given in parts per million
(ppm) relative to residual CHCl3 at δ 7.26 ppm for proton spectra and
relative to CDCl3 at δ 77.23 ppm for carbon spectra, unless otherwise
noted. High-resolution mass spectra were obtained using an LCT
Premier time-of-flight mass spectrometer. Flash column chromatog-
raphy was performed with silica gel grade 60 (230−400 mesh).
Dichloromethane (CH2Cl2), tetrahydrofuran (THF), toluene (PhMe),
N,N-dimethylformamide (DMF), CH3CN, triethylamine (Et3N), and
MeOH were all degassed with argon and passed through a solvent
purification system containing alumina or molecular sieves. All
commercially available reagents were used as received.
N-Boc-L-Ile-L-Pro-OMe (6). To a stirring solution of N-Boc-L-
isoleucine (4; 1.0 g, 4.16 mmol, 1.0 equiv) in CH2Cl2 (42 mL) were
added PyBOP (2.27 g, 4.37 mmol, 1.05 equiv) and iPr2EtN (1.09 mL,
6.24 mmol, 1.5 equiv). To a separate round-bottom flask (RBF) were
added L-proline methyl ester (5; 1.03 g, 6.24 mmol, 1.5 equiv), iPr2EtN
(2.17 mL, 12.48 mmol, 3.0 equiv), and CH2Cl2 (6.3 mL). Both reaction
mixtures were left to stir for 1 h, at which time they were combined and
left to stir overnight at room temperature (rt). The reaction was
quenched with 1 M HCl (1 vol equiv), and the product was extracted
with CH2Cl2 (×2), washed with NaHCO3(satd), dried over sodium
sulfate, and concentrated under reduced pressure. The crude material
was purified via flash silica gel chromatography (1:2 EtOAc/hexane) to
afford 6 (1.30 g, 92% yield): [α]2D5 +40.3 (c 1.23, CHCl3); IR (film) νmax
3436, 2973, 1746, 1642, 1436, 1217 cm−1; 1H NMR (CDCl3, 400 MHz)
δ 5.11 (d, J = 9.5 Hz, 1H), 4.52−4.44 (m, 1H), 4.24 (dd, J = 9.5, 7.2 Hz,
1H), 3.80−3.73 (m, 1H), 3.66 (s, 3H), 3.65−3.58 (m, 1H), 2.22−2.12
(m, 1H), 2.01−1.90 (m, 3H), 1.71 (ddq, J = 10.3, 6.5, 3.5 Hz, 1H), 1.53
(td, J = 7.2, 6.6, 3.2 Hz, 1H), 1.37 (s, 9H), 1.07 (ddd, J = 13.4, 6.7, 2.3 Hz,
1H), 0.96 (d, J = 6.8 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H); 13C NMR
(CDCl3, 101 MHz) δ 172.64, 171.65, 156.01, 79.70, 59.04, 56.46, 52.35,
47.42, 38.13, 29.29, 28.56, 25.17, 24.38, 15.47, 11.46; HRESIMS m/z
343.2193 [M + H]+ (calcd for C17H30N2O5, 343.2188).
N-Boc-L-Leu-L-Trp-OMe (9). To a stirring solution of compound N-
Boc-L-leucine (7; 1.0 g, 4.32 mmol, 1.0 equiv) in CH2Cl2 (43 mL) were
added PyBOP (2.36 g, 4.54 mmol, 1.05 equiv) and iPr2EtN (1.13 mL,
6.48 mmol, 1.5 equiv). To a separate RBF were added L-tryptophan
methyl ester (8; 1.65 g, 6.48 mmol, 1.5 equiv), iPr2EtN (2.26 mL, 12.96
mmol, 3.0 equiv), and CH2Cl2 (6.5 mL). Both reaction mixtures were
left to stir for 1 h, at which time they were combined and left to stir
overnight at rt. The reaction was quenched with 1 M HCl (1 vol equiv),
and the product was extracted with CH2Cl2 (×2), washed with
NaHCO3(satd), dried over sodium sulfate, and concentrated under
reduced pressure. The crude material was purified via flash silica gel
chromatography (1:1 EtOAc/hexane) to afford 9 (1.81 g, 97% yield):
D
DOI: 10.1021/acs.jnatprod.7b00656
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Journal of Natural Products
Article
119.61, 118.60, 111.55, 109.58, 79.89, 60.26, 56.61, 52.95, 52.56, 52.15,
47.99, 41.15, 37.95, 28.60, 25.58, 27.76, 25.31, 24.79, 24.42, 23.10, 23.05,
22.06, 15.65, 11.31; HRESIMS m/z 642.3817 [M + H]+ (calcd for
C34H52N5O7, 642.3822).
dioxane (6.4 mL), and the solution was cooled to 0 °C. The reaction
mixture was allowed to stir at the same temperature for 3 h. The solvent
was removed under reduced pressure to afford a crude white solid (free
base of 2), which was used without further purification. IR (film) νmax
3323, 3018, 2879, 1633, 1449, 1216 cm−1; 1H NMR (CDCl3, 400 MHz)
δ 8.17 (d, J = 44.9 Hz, 2H), 7.51 (d, J = 7.7 Hz, 1H), 7.32 (d, J = 8.0 Hz,
1H), 7.14 (t, J = 7.5 Hz, 1H), 7.10−7.05 (m, 2H), 6.83 (d, J = 8.3 Hz,
1H), 4.96 (d, J = 7.8 Hz, 1H), 4.83−4.63 (m, 4H), 4.42 (q, J = 7.8 Hz,
2H), 3.91 (d, J = 8.3 Hz, 1H), 3.79−3.70 (m, 2H), 3.65 (s, 5H), 3.30 (d, J
= 6.0 Hz, 2H), 2.96 (ddt, J = 22.3, 10.3, 6.4 Hz, 2H), 1.95−1.81 (m, 7H),
1.29−1.22 (m, 3H), 0.89−0.76 (m, 22H).
N-Boc-L-Pro-L-Val-OMe (14). Compounds 12 (3.0 g, 13.94 mmol, 1
equiv) and 13 (2.34 g, 13.94 mmol, 1 equiv) were added to a stirring
solution of HOBt (2.24 g, 14.63 mmol, 1.05 equiv) in dry THF (139
mL) at rt under an argon atmosphere. The solution was then cooled to 0
°C and stirred for 20 min, after which Et3N (6.8 mL, 48.79 mmol, 3.5
equiv) was added. After an additional 20 min of stirring EDC·HCl (2.8 g,
14.63 mmol, 1.05 equiv) was added, and the reaction mixture was left to
stir overnight. The reaction was quenched with H2O (1 vol equiv), and
the product was extracted with CH2Cl2, dried over sodium sulfate, and
concentrated under reduced pressure. The crude material was purified
via flash silica gel chromatography (1:1 EtOAc/hexane) to give 14 (4.45
g, 97% yield) as a clear liquid: [α]2D5 = +80.8 (c 2.05, CHCl3); IR (film)
N-Boc-L-Pro-L-Val-L-Ile-L-Pro-OH (Free Acid of Tetrapeptide
3). To a stirring solution of 3 (200 mg, 0.37 mmol, 1 equiv) in 1:1 THF/
H2O (1.5 mL) was added LiOH (45 mg, 1.86 mmol, 5 equiv), and the
solution was left to stir at rt for 3 h. The reaction was quenched with 1 M
HCl (1 vol equiv), and the product was extracted with CH2Cl2, washed
with NaHCO3(satd), dried over sodium sulfate, and concentrated under
reduced pressure. The crude material (free acid of 3), obtained in
quantitative yield as an oily residue, was used without further
1
νmax 3680, 3323, 3415, 2973, 2879, 1740, 1681, 1216 cm−1; H NMR
(CDCl3, 400 MHz) δ 4.50 (dd, J = 8.6, 5.1 Hz, 1H), 4.32 (s, 1H), 3.72 (s,
3H), 3.42 (s, 2H), 2.29 (s, 1H), 2.15 (pd, J = 6.9, 5.1 Hz, 2H), 1.95−1.84
(m, 3H), 1.48 (s, 9H), 0.92 (dd, J = 8.3, 6.8 Hz, 6H); 13C NMR (DMSO-
d6, 101 MHz) δ 172.71, 78.98, 59.71, 58.16, 52.30, 47.18, 31.67, 30.41,
28.80, 28.68, 23.61, 19.77, 18.99, 14.76; HRESIMS m/z 329.2025 [M +
H]+ (calcd for C16H29N2O5, 329.2032).
1
purification. IR (film) νmax 3311, 3016, 2965, 1632, 1215 cm−1; H
NMR (CDCl3, 400 MHz) δ 7.50 (s, 0H), 4.63 (t, J = 8.9 Hz, 0H), 4.41
(d, J = 65.5 Hz, 4H), 3.88 (d, J = 10.7 Hz, 1H), 3.67 (s, 2H), 3.43 (s, 2H),
2.16 (s, 7H), 1.96−1.74 (m, 5H), 1.46 (s, 9H), 1.21−0.74 (m, 12H).
Octapeptide N-Boc-L-Pro-L-Val-L-Ile-L-Pro-L-Ile-L-Pro-L-Leu-L-
Trp-OMe (17). Free base of 2 (915 mg, 1.69 mmol, 1 equiv), free
acid of 3 (784.5 mg, 1.5 mmol, 1 equiv), and HOBt (240 mg, 1.56 mmol,
1.05 equiv) were dissolved in dry THF (17 mL) at rt, under an argon
atmosphere. The solution was then cooled to 0 °C and stirred for 20
min, after which Et3N (0.731 mL, 5.25 mmol, 3.5 equiv) was added.
After an additional 20 min of stirring, EDC·HCl (299 mg, 1.56 mmol,
1.05 equiv) was added, and the mixture was left to stir overnight. The
reaction was quenched with H2O (1 vol equiv), and the product was
extracted with CH2Cl2, dried over sodium sulfate, and concentrated
under reduced pressure. The crude material was purified via flash silica
gel chromatography (1:9 EtOAc/MeOH) to afford 17 (605 mg, 34%
yield) as an amber liquid: [α]2D5 −26 (c 0.21, MeOH); 1H NMR (CDCl3,
400 MHz) δ 7.50 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.13 (t, J =
7.2 Hz, 1H), 7.10−7.03 (m, 2H), 6.82 (s, 1H), 4.97 (s, 1H), 4.65 (s, 2H),
4.42 (s, 2H), 4.25 (s, 1H), 3.88 (d, J = 9.4 Hz, 1H), 3.69 (s, 2H), 3.66 (s,
3H), 3.30 (d, J = 4.9 Hz, 2H), 2.07 (d, J = 145.9 Hz, 24H), 1.44 (s, 4H),
1.31−1.21 (m, 1H), 0.90−0.75 (m, 25H); 13C NMR (CDCl3, 101
MHz) δ 17.38, 172.39, 151.82, 142.80, 140.68, 136.40, 127.78, 122.15,
119.63, 118.69, 111.44, 52.59, 48.15, 28.55, 24.87, 24.87, 23.06, 23.06,
19.62, 19.62, 15.33, 11.24; HRESIMS m/z 1070.6253 [M + H]+ (calcd
for C55H85N9O11Na, 1070.6267).
H2N-L-Pro-L-Val-L-Ile-L-Pro-OMe (Free Amine of Tetrapeptide
3). To an RBF containing 3 (151 mg, 0.28 mmol) was added 1.0 M HCl
in dioxane (1.1 mL), and the solution was cooled to 0 °C. The reaction
was allowed to stir at the same temperature for 3 h. The solvent was
removed under reduced pressure, forming a crude white solid (free base
3), which was used without further purification. IR (film) νmax 3323,
2878, 1742, 1633, 1216 cm−1; 1H NMR (CDCl3, 400 MHz) δ 10.92 (s,
1H), 8.76 (s, 1H), 8.16 (s, 1H), 7.83 (d, J = 9.0 Hz, 1H), 4.92 (s, 1H),
4.76−4.68 (m, 1H), 4.52−4.35 (m, 2H), 3.87 (s, 2H), 3.69 (d, J = 2.2
Hz, 3H), 3.64 (dd, J = 8.5, 5.1 Hz, 1H), 3.44 (s, 3H), 2.63 (s, 1H), 2.46−
2.17 (m, 5H), 2.00 (dd, J = 18.0, 6.0 Hz, 10H), 1.52−1.39 (m, 2H),
1.01−0.79 (m, 13H).
N-Boc-L-Pro-L-Val-OH (15). To a stirring solution of 14 (360 mg,
1.1 mmol, 1 equiv) in 1:1 THF/H2O (4.4 mL) was added LiOH (131
mg, 5.5 mmol, 5 equiv), and the mixture was left to stir at rt for 3 h. The
reaction was quenched with 1 M HCl (1 vol equiv), and the product was
extracted with CH2Cl2, washed with NaHCO3(satd), dried over sodium
sulfate, and concentrated under reduced pressure. Crude 15 was
obtained in quantitative yield as an oily residue and was used without
1
further purification: IR (film) νmax 3410 (b), 1676 cm−1; H NMR
(CDCl3, 400 MHz) δ 10.37 (s, 1H), 7.51 (s, 1H), 4.50 (s, 1H), 4.40−
4.19 (m, 1H), 3.50−3.28 (m, 2H), 2.31−2.09 (m, 3H), 1.94−1.79 (m,
2H), 1.43 (s, 9H), 0.91 (dd, J = 9.9, 6.9 Hz, 6H).
H2N-L-Leu-L-Trp-OMe (16). To a RBF containing 6 (857 mg, 2.5
mmol) was added 1.0 M HCl in dioxane (10 mL), and the mixture was
cooled to 0 °C. The reaction mixture was allowed to stir at the same
temperature for 3 h. The solvent was removed under reduced pressure
to afford crude 16 in quantitative yield as a white solid, which was used
without further purification. IR (film) νmax 3416, 2879, 1651, 1454 cm−1;
1H NMR (CDCl3, 400 MHz) δ 8.34−8.27 (m, 3H), 4.61 (dd, J = 8.5, 5.3
Hz, 1H), 4.25 (s, 1H), 3.97 (s, 1H), 3.68 (s, 3H), 3.52 (d, J = 9.0 Hz,
1H), 2.28 (s, 1H), 2.05−1.91 (m, 4H), 1.68 (ddd, J = 13.5, 7.5, 3.3 Hz,
1H), 1.35 (ddd, J = 13.3, 10.0, 7.1 Hz, 1H), 1.12 (d, J = 6.8 Hz, 3H), 0.94
(t, J = 7.3 Hz, 3H); HSESIMS m/z 243.1658 [M + H]+ (calcd for
C12H23N2O3, 243.1664).
N-Boc-L-Pro-L-Val-L-Ile-L-Pro-OMe (3). Compounds 16 (1.71 g,
7.06 mmol, 1 equiv) and 15 (2.24 g, 7.13 mmol, 1 equiv) were added to a
stirring solution of HOBt (1.13 g, 7.41 mmol, 1.05 equiv) and dry THF
(71 mL) at rt under an argon atmosphere. The solution was cooled to 0
°C and stirred for 20 min, after which Et3N (3.46 mL, 24.71 mmol, 3.5
equiv) was added. After an additional 20 min of stirring, EDC·HCl (1.42
g, 7.41 mmol, 1.05 equiv) was added, and the mixture was stirred
overnight. The reaction was quenched with H2O (1 vol equiv), and the
product was extracted with CH2Cl2, dried over sodium sulfate, and
concentrated under reduced pressure. The crude material was purified
via flash silica gel chromatography (7:1 EtOAc/hexane) to give 3 (1.52
g, 78% yield) as a clear liquid: [α]2D5 +72.3 (c 3.21, CHCl3); IR (film) νmax
3673, 3412, 3306, 2879, 1743, 1632, 1368 cm−1; 1H NMR (CDCl3, 400
MHz) δ 7.48 (s, 1H), 6.65 (s, 1H), 6.42 (s, 1H), 4.58 (t, J = 8.2 Hz, 1H),
4.47 (dd, J = 8.6, 4.9 Hz, 1H), 4.31 (s, 1H), 4.26 (dd, J = 8.6, 5.6 Hz, 1H),
3.81 (dt, J = 9.8, 6.3 Hz, 1H), 3.68 (s, 3H), 3.64−3.59 (m, 1H), 3.46−
3.28 (m, 2H), 2.33 (s, 1H), 2.19 (ddq, J = 12.9, 6.8, 3.7 Hz, 2H), 2.02−
1.79 (m, 8H), 1.43 (s, 9H), 0.97 (d, J = 6.8 Hz, 3H), 0.89−0.80 (m, 9H);
13C NMR (CDCl3, 101 MHz) δ 172.63, 172.40, 171.29, 171.25, 170.63,
N-Boc-L-Ile-L-Pro-L-Leu-L-Trp-OH (Free Acid of Tetrapeptide
2). To a stirring solution of 14 (100 mg, 0.16 mmol, 1 equiv) in 1:1
THF/H2O (640 mL) was added LiOH (19 mg, 0.78 mmol, 5 equiv),
and the solution was left to stir at rt for 3 h. The reaction was quenched
with 1 M HCl (1 vol equiv), and the product was extracted with CH2Cl2,
washed with NaHCO3(satd), dried over sodium sulfate, and concentrated
under reduced pressure. Crude 15 was obtained in quantitative yield as
an oily residue, which was used without further purification. IR (film)
νmax 3311 (bs), 2965, 1632, 1215 cm−1; 1H NMR (CDCl3, 400 MHz) δ
8.42 (s, 1H), 7.57 (d, J = 7.9 Hz, 1H), 7.31 (s, 1H), 7.12 (dq, J = 24.7, 9.6,
7.3 Hz, 3H), 5.22 (s, 1H), 4.83 (d, J = 7.5 Hz, 1H), 4.31 (d, J = 41.9 Hz,
2H), 3.73 (s, 2H), 3.54 (s, 1H), 3.30 (s, 2H), 2.08−1.72 (m, 0H), 1.43
(s, 9H), 1.10−0.57 (m, 12H).
80.70, 61.37, 59.96, 59.00, 58.70, 55.04, 52.35, 47.51, 47.25, 37.65, 31.41,
30.58, 29.29, 28.55, 25.17, 24.52, 19.50, 17.45, 15.41, 11.30; HSESIMS
m/z 561.3198 [M + H]+ (calcd for C27H46N4O7Na, 561.3264).
H2N-L-Ile-L-Pro-L-Leu-L-Trp-OMe (Free Amine of Tetrapeptide
2). To a RBF containing 2 (1.03 g, 1.6 mmol) was added 1.0 M HCl in
E
DOI: 10.1021/acs.jnatprod.7b00656
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Octapeptide N-Boc-L-Ile-L-Pro-L-Leu-L-Trp-L-Pro-L-Val-L-Ile-L-
Pro-OMe (18). Free acid of 2 (137 mg, 0.31 mmol, 2.1 equiv) and
free base of 3 (92 mg, 0.15 mmol, 1 equiv) were added to a stirring
solution of HOBt (25 mg, 0.16 mmol, 1.05 equiv) in dry THF (3.1 mL)
at rt under an argon atmosphere. The solution was cooled to 0 °C and
stirred for 20 min, after which Et3N (0.07 mL, 0.53 mmol, 3.5 equiv) was
added and stirred. After an additional 20 min of stirring, EDC·HCl (31
mg, 0.16 mmol, 1.05 equiv) was added and the reaction mixture was left
to stir overnight. The reaction was quenched with H2O (1 vol equiv),
and the product was extracted with CH2Cl2, dried over sodium sulfate,
and concentrated under reduced pressure. The crude material was
purified via flash silica gel chromatography (9:1 EtOAc/MeOH) to
afford 18 (198 mg, 61% yield) as an amber liquid: [α]2D5 −29 (c 0.78,
MeOH); IR (film) νmax 3423, 2925, 1741, 1687, 1612, 1452 cm−1; 1H
NMR (CDCl3, 400 MHz) δ 8.67 (s, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.62−
7.27 (m, 4H), 7.20−6.85 (m, 6H), 5.21 (d, J = 9.3 Hz, 1H), 5.01 (q, J =
7.2 Hz, 1H), 4.75−4.00 (m, 10H), 3.80 (dt, J = 35.8, 5.2 Hz, 3H), 3.72−
3.66 (m, 3H), 3.58 (s, 3H), 3.18 (d, J = 7.3 Hz, 1H), 2.36−1.85 (m,
16H), 1.42 (d, J = 4.6 Hz, 9H), 1.04−0.83 (m, 24H); 13C NMR (CDCl3,
101 MHz) δ 172.76, 171.84, 171.74, 171.58, 170.90, 156.03, 136.30,
124.14, 123.58, 121.96, 119.58, 118.68, 111.55, 109.77, 79.70, 77.52,
77.04, 60.50, 60.27, 59.34, 59.11, 56.57, 55.13, 55.03, 52.31, 52.26, 52.10,
51.68, 48.01, 47.63, 47.55, 41.19, 37.93, 37.59, 37.53, 30.91, 30.26, 29.26,
28.58, 28.56, 28.11, 27.78, 25.36, 25.18, 24.80, 24.59, 24.53, 23.32, 23.01,
21.92, 19.59, 19.04, 18.36, 15.70, 15.60, 15.39, 11.33, 11.27; HSESIMS
m/z 1070.6253 [M + H]+ (calcd for C55H85N9O11Na, 1070.6267).
Reniochalistatin E (1) via 18. To a stirring solution of 18 (470 mg,
0.45 mmol, 1 equiv) in a 1:1 THF/H2O (1.8 mL) was added LiOH (54
mg, 2.24 mmol, 5 equiv), and the solution was left to stir at rt for 3 h. The
reaction was quenched with 1 M HCl (1 vol equiv), and the product was
extracted with CH2Cl2, dried with brine, dried over sodium sulfate, and
concentrated under reduced pressure to afford the free acid of 18, which
was used without further purification. The free acid of 18 (365 mg, 0.72
mmol) was added to an RBF, and the flask was cooled to 0 °C, to which
was added 1.0 M HCl in dioxane (2.9 mL). The reaction mixture was
stirred for 3 h at rt. The solvent was removed under reduced pressure to
afford the crude material 18a, a white/orange solid that was used
without further purification. Compound 18a (115 mg, 0.12 mmol, 1
equiv) was added to a stirring solution of HOBt (18 mg, 0.12 mmol, 1
equiv) in dry THF (12 mL) at rt under an argon atmosphere. The
solution was cooled to 0 °C and stirred for 20 min, after which Et3N
(0.06 mL, 0.42 mmol, 3.5 equiv) was added. After an additional 20 min
of stirring, EDC·HCl (25 mg, 0.13 mmol, 1.05 equiv) was added and the
reaction mixture was left to stir overnight. The reaction was quenched
with H2O (2 vol equiv), and the product was extracted with CH2Cl2,
dried over sodium sulfate, and concentrated under reduced pressure.
The crude material was purified via flash silica gel chromatography
(85:15 EtOAc/MeOH) to afford 1 (39 mg, 9% yield): [α]2D5 −100 (c
0.16, MeOH); IR (film) νmax 3290, 2960, 2927, 1670, 1615, 1501, 1445
cm−1; 1H NMR (CDCl3, 500 MHz) δ 10.96−10.84 (m, 1H), 8.11−7.78
(m, 1H), 7.52 (dd, J = 24.5, 7.9 Hz, 2H), 7.40 (d, J = 8.2 Hz, 1H), 7.32 (t,
J = 8.7 Hz, 1H), 7.02−6.92 (m, 2H), 4.38−4.33 (m, 1H), 3.36 (s, 23H),
3.33−3.22 (m, 1H), 2.48 (s, 1H), 1.99−1.70 (m, 2H), 1.24 (s, 2H),
0.96−0.75 (m, 8H), 0.83 (s, 27H); 13C NMR (DMSO-d6, 126 MHz) δ
172.37, 171.88, 171.81, 171.48, 171.31, 170.58, 170.22, 169.58, 136.59,
127.36, 124.18, 121.21, 118.60, 118.38, 111.71, 111.57, 61.20, 60.67,
59.59, 56.74, 55.85, 54.61, 54.39, 54.22, 47.81, 47.55, 47.41, 38.21, 37.49,
35.09, 33.21, 30.01, 23.92, 29.31, 29.28, 25.22, 25.12, 24.95, 24.83, 24.10,
23.77, 22.71, 20.87, 19.21, 18.88, 15.69, 15.10, 11.50, 9.81; HRESIMS
m/z 938.5475 [M + H]+ (calcd for C49H73N9O8Na, 938.5473).
reaction mixture was stirred for 3 h at the same temperature, then
allowed to warm to rt overnight. The solvent was removed under
reduced pressure to afford the crude material 17a, a white/orange solid
that was used without further purification. Compound 17a (152 mg,
0.16 mmol, 1 equiv) was added to the stirring solution of HOBt (25 mg,
0.16 mmol, 1 equiv) in dry THF (16 mL) at rt under an argon
atmosphere. The solution was cooled to 0 °C and stirred for 20 min,
after which Et3N (0.08 mL, 0.56 mmol, 3.5 equiv) was added. After an
additional 20 min of stirring, EDC·HCl (32 mg, 0.17 mmol, 1.05 equiv)
was added, and the reaction mixture was left to stir overnight. The
reaction was quenched with H2O (1.5 vol equiv), and the product was
extracted with CH2Cl2, washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure. The crude material was
purified via flash silica gel chromatography with a gradient solvent
system (9:1 EtOAc/MeOH; 7:3 EtOAc/MeOH) to afford 1 (8.6 mg,
6% yield). All NMR and MS data matched those of 1 accessed via 18.
Biological Assays. Cell Culture Information. Cells were grown in
media supplemented with fetal bovine serum (FBS) and antibiotics (100
μg/mL penicillin and 100 U/mL streptomycin). Specifically, experi-
ments were performed using the following cell lines and media
compositions: HeLa, A549, RPMI-8226, MM.1R, and U-937 (RPMI-
1640 + 10% FBS) and Mia PaCa-2 (DMEM + 10% FBS). Cells were
incubated at 37 °C in a 5% CO2, 95% humidity atmosphere for all
experiments.
IC50 Value Determination for Adherent Cells Using Alamar Blue.
Adherent cells were added to a 384-well plate (1500 cells/well) in 10 μL
of media and were allowed to adhere for 2−3 h. Compounds were
solubilized in DMSO (10 μM stock solutions) and added to a 96-well
plate over a range of concentrations (31.6 nM to 200 μM) with media,
and 40 μL was added to the 384-well plate in triplicate for each
concentration of compound. After 69 h of continuous exposure, 5 μL of
Alamar Blue was added to each well, and the cells were allowed to
incubate for an additional 3 h. The plates were then read for fluorescence
intensity with an excitation of 560 nm and emission of 590 nm on a
BioTek Synergy H1 plate reader. Doxorubin and etoposide were both
used as positive death controls, and wells with no compounds added as
negative death controls. IC50 values were determined from three or more
independent experiments using GraphPad Prism 7.0. (LaJolla, CA,
USA)
IC50 Value Determination for Nonadherent Cells Using Alamar
Blue. The same procedure for adherent cells was used, with the following
modifications. Cells (2000 cell/well) in media (10 μL) were added after
40 μL of compound in media was added to the 384-well plate. No time
was given to allow cells to adhere.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00656.
1H NMR and 13C NMR spectra for all new compounds
(PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail (R. J. Rafferty): rjraff@ksu.edu. Phone: 785-532-6624.
■
ORCID
Ryan J. Rafferty: 0000-0002-4835-6343
Notes
The authors declare no competing financial interest.
Reniochalistatin E via 17. To a stirring solution of 17 (249 mg,
0.24 mmol, 1 equiv) in 1:1 THF/H2O (1 mL) was added LiOH (29 mg,
1.2 mmol, 5 equiv), and the mixture was left to stir at rt for 3 h. The
reaction was quenched with 1 M HCl (1 vol equiv), and the product was
extracted with CH2Cl2, washed with brine, dried over sodium sulfate,
and concentrated under reduced pressure. The crude material (free acid
of 17) was obtained in 68% yield as a white powder, which was used
without further purification. The free acid of 17 (167 mg, 0.16 mmol)
was added to an RBF, and the flask was cooled to 0 °C, to which 1.0 M
HCl in dioxane (0.64 mL) and 1 mL of dioxane were added. The
ACKNOWLEDGMENTS
■
This work could not have been undertaken without the gracious
financial support from the Johnson Cancer Center of Kansas
State University and Startup Capital from Kansas State
University. This paper is warmly dedicated to Professor Richard
F
DOI: 10.1021/acs.jnatprod.7b00656
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
M. Hyslop in recognition of his mentorship, guidance, and
friendship.
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G
DOI: 10.1021/acs.jnatprod.7b00656
J. Nat. Prod. XXXX, XXX, XXX−XXX