Total synthesis of griseusins and elucidation of the griseusin mechanism of action

Article abstract of DOI:10.1039/c9sc02289a

A divergent modular strategy for the enantioselective total synthesis of 12 naturally-occurring griseusin type pyranonaphthoquinones and 8 structurally-similar analogues is described. Key synthetic highlights include Cu-catalyzed enantioselective boration-hydroxylation and hydroxyl-directed C-H olefination to afford the central pharmacophore followed by epoxidation-cyclization and maturation via diastereoselective reduction and regioselective acetylation. Structural revision of griseusin D and absolute structural assignment of 2a,8a-epoxy-epi-4′-deacetyl griseusin B are also reported. Subsequent mechanistic studies establish, for the first time, griseusins as potent inhibitors of peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3). Biological evaluation, including comparative cancer cell line cytotoxicity and axolotl embryo tail inhibition studies, highlights the potential of griseusins as potent molecular probes and/or early stage leads in cancer and regenerative biology.

Full text of DOI:10.1039/c9sc02289a

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DOI: 10.1039/C9SC02289A
ARTICLE
Total synthesis of griseusins and elucidation of the
griseusin mechanism of action
Received 00th January 20xx,
Accepted 00th January 20xx
Yinan Zhang,a,b,c,† Qing Ye,d,† Larissa V. Ponomareva,b,c Yanan Cao,d Yang Liu,b,c Zheng Cui,c Steven G.
Van Lanen,c S. Randal Voss,e Qing-Bai She,*,d Jon S. Thorson*,b,c
DOI: 10.1039/x0xx00000x
A
divergent modular strategy for the enantioselective total synthesis of 12 naturally-occurring griseusin type
pyranonaphthoquinones and 8 structurally-similar analogues is described. Key synthetic highlights include Cu-catalyzed
enantioselective boration-hydroxylation and hydroxyl-directed C-H olefination to afford the central pharmacophore
followed by epoxidation-cyclization and maturation via diastereoselective reduction and regioselective acetylation.
Structural revision of griseusin D and absolute structural assignment of 2a,8a-epoxy-epi-4’-deacetyl griseusin B are also
reported. Subsequent mechanistic studies establish, for the first time, griseusins as potent inhibitors of peroxiredoxin 1 (Prx1)
and glutaredoxin 3 (Grx3). Biological evaluation, including comparative cancer cell line cytotoxicity and axolotl embryo tail
inhibition studies, highlights the potential of griseusins as potent molecular probes and/or early stage leads in cancer and
regenerative biology.
unique to FB or more broadly attributed to the other diverse
PNQ family members remains to be determined.
Introduction
First reported in 1976, the griseusin-based PNQ metabolites
Pyranonaphthoquinone (PNQ) natural products represent an
array of structurally and functionally diverse bacterial
secondary metabolites.1 Despite extensive advances in PNQ
synthetic methodology,2-3 the commercial application of PNQs
as feedstock additives and the demonstrated anticoccidial and
antimalarial efficacy of PNQs,4 the mechanism of action for
PNQs remained unresolved. Enabled by our efficient divergent
strategy for the synthesis of frenolicin B (FB, a prototypical PNQ-
based natural product; Figure 1),2f systematic SAR and
mechanistic studies revealed FB as the most potent and
selective peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3)
inhibitors reported to date.5 These studies demonstrated
Prx1/Grx3 inhibition by FB led to increased intracellular ROS,
the consequence of which was inhibition of mTORC1-mediated
4E-BP1 phosphorylation, apoptosis induction and tumor
suppression. Whether this fundamental mechanism of action is
are structurally distinguished by their fused spiro-ring C/E
system (Figure 1) in lieu of the FB C1-alkyl side chain of FB.6
Griseusin members are further differentiated via ring D forms
(open or closed), relative stereochemistry, oxidation,
glycosylation, and/or O- or C-acetylation. Like their frenolicin
counterparts, many of the griseusins display potent cancer cell
line cytotoxicity. Representative griseusins have also been
reported as COMPARE negative (indicating a novel mechanism
of activity)6g and potentially synergistic with tumor necrosis
Me
O
R1
Me
O
OH
6'
4'
13
11
OH
E
OH
O
OH
3'
O
O
9
1
9
10a
OR2
9a
OH
10a
9a
1
C
O
O
3
O
B
A
3
4
5
5
14
4
O
D
11
O
6
6
N
15
O
O
O
O
O
O
12
O
SG
griseusin A6a,b
O
ent-4'-deacetyl-griseusin A (5)6h
(
1, R =-OAc, R =H)
frenolicin B (
FB
)
1
2
4'-deacetyl-griseusin A6d (2, R1=-OH, R2=H)
griseusin C6e,g (3, R1=O, R2=H)
epi-4'-deacetylgriseusin A6g (4, R1=-OH, R2=H)
3'-O--D-forosaminyl-griseusin A6c (R1=-OAc, R2=SG)
Me
R1
Me
O
OH
OH
Me
O
OH
OH
OH
O
OH
OH
O
O
O
OH
O
O
O
O
O
O
a. Jiangsu Key Laboratory for Functional Substances of Chinese Medicine, School of
Pharmacy, Nanjing University of Chinese Medicine, Nanjing, Jiangsu, 210023,
China.
O
OH
OR1
OH
O
O
O
griseusin B6a,b (6, R1=-OAc)
ent-4'-deacetyl-griseusin B6h (9, R1=H)
ent-4'-deacetyl-griseusin B methyl
4a,10a-epoxy-epi-
deacetylgriseusin B (11)6g
b. Center for Pharmaceutical Research and Innovation
c. College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA. E-mail:
jsthorson@uky.edu
4'-deacetyl-griseusin B6d (7, R1=-OH)
epi-4'-deacetyl-griseusin B6g (8, R1=-OH) ester6h (10, R1=Me)
Me
O
R1
Me
O
R1
Me
O
O
d. Markey Cancer Center and Department of Pharmacology and Nutritional
Sciences, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.
E-mail: qing-bshe@uky.edu
OH
OH
OH
O
O
O
OH
OH
OH
O
O
O
O
O
e. Department of Neuroscience, Spinal Cord and Brain Injury Research Center,
Ambystoma Genetic Stock Center, University of Kentucky, Lexington, KY 40536,
USA
OMe
OMe
O
O
O
O
OH
O
OH
O
griseusin F6i (R1=OH)
griseusin G6i (R1= O)
griseusin D (isolation report6f
)
ent-griseusin D (this report) (12, R1=O)
† These authors contributed equally.
griseusin E6h (R1=OAc)
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
Figure 1. Naturally-occurring frenolicin prototype (FB) and griseusins.
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Journal Name
O
4'
O
OH
6'
O
OTBS
Me
O
R1
O
would derive from simple convergent C-H olefinaVtiieownArutisclienOgnl2in0e
DOI: 10.1039/C9SC02289A
and 22R, the latter of which would result from Sharpless
3'
1
O
O
OH
O
O
O
O
O
O
9
OR2
22R
9a
OH
OH
O
O
asymmetric dihydroxylation9 of the Heck-coupled product of
methyl 3-butenoate and bromine 24. Following the same
strategy, ent-griseusin C (and the griseusin D/E subclass) may
derive from enantiomer 25 and AD-mix-. For the griseusin B
series, alcohol 28 would stem from copper-catalyzed
enantioselective boration-hydroxylation10 of 27, accessible via a
palladium coupling between bromine 26 and a 24-derived
boronic acid. Final maturation/tailoring reactions would also
leverage the C4′ ketone of precursors 23, 24 and 30 where
applicable.
3
4
14
6
5
O
OMe
O
O
O
OMe
OMe
O
15
O
O
20
O
23
O
21
[griseusin A/C (1-4)]
O
Me
O
R1
OH
O
O
O
O
OH
O
O
OH
OH
O
O
O
Br
OMe
OH
O
OMe
24
OMe
25
O
OH
1,5-dihydroxy
naphthalene
O
[ent-griseusin A/C (5)]
[griseusin D/E (12)]
COOMe
26
Br
O
6'
O
4'
Me
O
R1
R2
O
O
O
O
O
O
3'
1
4
OH
9
OH
9a
O
OH
O
O
3
COOMe
COOMe
COOMe
15
5
6
14
COOH
2.2 Synthesis of key griseusin A/C precursors.
MeO
MeO
OMe
O
28
27
30
A range of conditions were explored for reduction of the 323m
C4′ ketone, the primary goal of which was to establish a general
reductive method with bilateral C4′ diastereoselectivity (Table
S1 and Scheme 2). Among the common borohydride reagents
(Table S1, entries 1-2), K selectride provided the desired -
alcohol 33 exclusively in a 94% isolated yield compared to the
observed 4:1 diastereoselectivity with NaBH4 (Table S1, entry
1). Non-ionic boranes favored formation of the desired -alcohol
34 with the borane 2-picoline complex in the presence of HCl
providing the best diastereoselectivity (Table S1, entries 3-11).11
Solvent optimization (Table S1, entries 12-15) revealed that
reactions in ether provided the desired -alcohol in 75% isolated
yield with 1:10 diastereoselectivity (Table S1, entry 15). Putative
mechanistic rationale for diastereoselectivity in this reaction is
based on sterics (favoring hydride attack from the si-face to give
(R)-alcohol 33) or partial spiroketal oxygen-borane coordination
(favoring hydride attack from the re-face to give (S)-alcohol 34).
A similar optimization strategy was pursued for reduction of
the 23 C4′-carbonyl to set the stage for the synthesis of C3′/4′-
diols (Table S2). Electrophilic reducing agents, such as sodium
borohydride and lithium borohydride, provided the desired C4’
cis-diol 35 in high yield and si-facial selectivity (Scheme 2). In
contrast, this study failed to identify suitable reductive
conditions to furnish the corresponding desired trans-diol 36,
potentially due to C3′-OH infringement on the putative
[griseusin B (6-8, 9-11)]
Scheme 1. Retrosynthetic analysis for griseusin subclasses A-E
factor-related apoptosis-inducing ligand (TRAIL) in TRAIL-
resistant gastric adenocarcinoma cell lines.6h Yet, the molecular
target and mechanism of action for griseusin remains unknown.
Enabled by our recently reported synthetic approach to
griseusin A (1), 4’-deacetyl-griseusin A (2), and griseusin C (3),3m
herein we report the further development and implementation
of the first concise divergent synthetic approach to a broad set
of griseusin A-E analogs (twelve naturally-occurring griseusins
and eight additional synthetic analogues). Methodological
highlights include mechanistic investigation of C-H olefination
en route to the griseusin C-ring, enantioselective Cu-catalyzed
boration-hydroxylation to afford open D-ring griseusin B
members and a series of stereoselective and regioselective
transformations for E-ring diversification. This enabling
chemistry also facilitated revision of the previous reported
griseusin D structure, assignment of stereocenters in 4a,10a-
epoxy-epi-4’-deacetyl griseusin B and the broadest griseusin
comparative cancer cell line cytotoxicity SAR analysis to date.
Subsequent biochemical and cell-based studies revealed
representative griseusins to inhibit Prx1/Grx3 and 4E-BP1
phosphorylation in manner similar to FB and to inhibit tail
regeneration in our recently developed axolotl tail regeneration
assay.7 Given the notable demonstrated in vivo efficacy of
optimized FB analogs,5 the current study considerably expands
the range PNQ pharmacophores available for further
optimization of Prx1/Grx3 selectivity and/or ADMET.
spiroketal
oxygen-borane
coordination
required
for
stereoselective hydride delivery. As an alternative, benzyl
protective intermediate 37 led to the re-face C4′-carbonyl
stereospecific reduction reminiscent to that for (S)-alcohol 34
(Table
S3).
The
corresponding
23
protection/reduction/deprotection protocol afforded the desired
trans-diol 36 in a 54% cumulative yield for three steps (Scheme
2).
Results and discussion
2.1 Retrosynthetic analysis and rationale.
With diol 35 in hand, we next evaluated regioselective O-
acetylation (Table S4). Initial attempts to obtain the desired
products 39-41 via mild transesterification12 were hampered by
poor diol reactivity (Table S4, entries 1-2). Use of acetic
anhydride and base optimization led to the desired products in
variable yields (Table S4, entries 3-8). Specifically, use of a
sterically-hindered base favored the desired C4′-acetylation
(Table S4, entry 6) while relatively weak base gave exclusive
C3′-acetylation (Table S4, entry 7). Reduction of the amount of
Scheme 1 highlights a conceptual overview of the envisioned
synthetic route to griseusin-type PNQs. C1 epimerization within
the context of spiropyran construction and/or subsequent
scaffold maturation presents a primary hurdle to griseusin total
synthesis. We postulated that diastereoselective C1-C3′
epoxidation and subsequent C6′-OH intramolecular cyclization
could provide the key spiro-pyrano core 23, the C1 stereocenter
of which should be stable to late-stage deprotection of the
acetonide.8 For this approach, the critical 21 C1-C3′-enone
2 | J. Name., 2012, 00, 1-3
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ARTICLE
base and acetic anhydride further improved yield (Table S4, subsequent modification of reaction solvent (TableVSie6w,AertnictlreiOesnli2ne-
DOI: 10.1039/C9SC02289A
entry 8) while the use of a nucleophilic strong base favored 4) and catalyst/oxidant loading (Table S6, entries 4-8) gave the
peracetylated product 41 (Table S4, entry 9).
2.3 Synthesis of key griseusin B precursors.
desired 1-methylene isochroman 43 in 66% on gram scale (Table
S6, entry 9). The lower observed abundance of side product 52
in these C1 olefination studies implicated the flexible secondary
alcohol of 28 (compared to the corresponding rigid secondary
alcohol of 20) as advantageous. Access to 53 also facilitated
further mechanistic investigation of this C-H olefination event
(Scheme 4). Specifically, intermediate 53 results from initial
hydroxyl-assisted C-H insertion of Pd (II) followed by -
elimination and subsequently enters the second round of Pd-
catalyzed intra-molecular oxidative cyclization (eq 1). In
contrast, side product 52 cannot undergo -elimination (eq 3)
and therefore irreversibly consumes 53 as a major competing
reaction (eq 2). The bifurcation between the desired product
(e.g., 1-methylene isochroman 43) and undesired shunt products
(e.g., 52) is presumably dictated by a propensity for palladium
The route to griseusin B analogues began with the synthesis of
42 following standard methodology for lithium-bromine
exchange-mediated boronic acid generation (Scheme 3).
Palladium-catalyzed Suzuki coupling13 of 42 with
bromobutenoate (26) gave 27. With -unsaturated ester 27 in
hand, we investigated a range of -borylation conditions for
enantioselective -conjugate addition (Table S5). This small
study revealed chiral diphosphine ligand10 as advantageous over
bisoxazoline and N-heterocyclic carbene catalysts,14 enabling
quantitative yield with 90% enantioselectivity on gram scale.
Subsequent installation of 29R (to access the desired 1-
methylene isochroman 43, Scheme 4) began with our C-H
activation C1 olefination conditions for 20.3m While these initial
conditions (Table S6, entry 1) were sub-optimal in this case,
O
O
O
O
O
O
Br
COOMe
O
OTBS
Me
O
OH
Me
a)
26
O
OTBS
COOMe
O
O
O
O
O
O
b)
B(OH)2
Br
22R
a)
b)
OMe
OH
O
OMe
OMe
O
42
27
24
O
O
OTES
O
OMe
O
OMe
O
OMe
O
OTES
O
O
Me
O
O
O
O
O
21-TBS
21
20
c)
Table S5
29R
Table S6
OH
OH
(R)
c)
f)
COOMe
COOMe
d),
O
O
O
d)
OMe
28
OMe
43
O
O
O
4'
O
O
O
O
O
O
O
OMe
OH
e)
O
f), g)
O
Table S1
O
33
H
H
4'
OH
(S)
O
O
OMe
O
OMe
O
O
O
O
O
O
O
O
O
O
O
O
O
O
32
23
3'
O
e)
O
O
O
OH
OBn
j)
COOMe
O
O
COOMe
COOMe
O
OMe
OMe
OMe
OMe
47
O
34
h)
Table S2
g),
44
30
h)
H
H
H
H
OH
OH
OH
OH
OR
O
O
O
O
O
O
O
O
O
O
O
O
O
OH
O
OBn
Table S3
i),
O
O
O
COOR
O
OMe
OMe
O
O
OMe
OMe
45
46
(R = Me, >10:1 dr)
(R = H)
O
k)
h)
35
(>10:1 dr)
O
O
i)
37
38
36
(9:1 dr), R = Bn
, R = H
j)
Table S4
H
H
H
O
H
O
OH
OR
OH
OAc
OBn
k)
l)
m)
O
O
O
O
O
O
O
O
OBn
OAc
OAc
OH
OAc
m)
O
O
O
O
O
O
O
O
O
COOMe
COOMe
COOH
O
O
OAc
OH
O
O
O
OMe
OMe
50
OMe
(>10:1 dr)
51
48
49
(R = Bn, 10:1 dr)
(R = H)
l)
O
OMe
O
O
OMe
OMe
O
O
O
41
39
40
Scheme 3. Synthesis of griseusin B-type analogues. Key NOE crosspeaks are highlighted
(red arrows). Reagents and conditions: (a) nBuLi, B(OiPr)3, THF, -78 oC, 2 h, 80% (b) 26,
Pd(OAc)2, KF, dioxane, rt, 2 h, 74%; (c) 1.5 mol% CuCl, 3 mol% S-Rp-josiphos, 2.5 mol%
NaOtBu, Bpin2, THF, 0 oC-rt, 16 h; then NaBO3, H2O, rt, 2h, 91%,  ee; (d) 29, 40 mol%
Pd(OAc)2, Ag2CO3 (2eq), Li2CO3, CHCl3, 80 oC, 16 h, 63%; (e) HF·pyridine, CH3CN, rt, 12h,
81%; f) HF·pyridine, TEA, dioxane, rt, 1h, 88%; g) DMDO, 50 mol% TfOH, DCM, -78 oC-(-
20)oC, 2h, 93%; h) LiBH4 (2 eq), THF, -78 oC, 0.5 h, 86%, :  i) 1N LiOH, MeOH, rt,
1h, 95%; j) BnBr, Ag2O, NMP, rt, 36 h, 76%; (k) BH3·Me3N (2 eq), TFA (3.0 eq), ether, rt,
36 h, 77%,  = 10:1; (l) Pd/C, H2, MeOH, rt, 4 h, 93%; m) 1N LiOH, MeOH, rt, 1h; Ac2O,
DABCO, DCM, rt, 40 h; 65% for two steps.
Scheme 2. Synthesis of griseusin A/C-type analogues. Key NOE crosspeaks are
highlighted (red arrows). Reagents and conditions: (a) Pd(OAc)2 (20 mol%), Li2CO3,
Ag2CO3 (4 eq), DCE, 80 oC, 16 h, 40%; (b) HF·pyridine (10 eq), TEA (2 eq), dioxane, rt, 16
h, 85%; (c) HF·pyridine (10 eq), CH3CN, rt, 16 h, 91%; (d) K-selectride, THF, -78 oC, 2 h,
94%; (e) BH3·2-picoline, HCl, Et2O, 16 h, 75%; (f) DMDO, TfOH, DCM, -78 oC-0 oC, >10:1
dr, 80%; (g) LiBH4 (2 eq), THF, -78 oC, 0.5 h, 85%, :  (h) KOtBu (2 eq), BnBr (2 eq),
THF, 0 oC-rt, 16 h, 77%; (i) BH3·Me3N (2 eq), TFA (3.0 eq), ether, rt, 36 h, 74%,  = 9:1;
(j) Pd/C, H2, MeOH, rt, 4 h, 95%; (k) Ac2O (10 eq), dichylohexymethyl amine (10 eq), DCM,
rt, 40 h, 70%; (l) Ac2O (5 eq), pyridine (5 eq), DCM, rt, 16 h, 88%; (m) Ac2O (10 eq), DABCO
(10 eq), DCM, rt, 16 h, 93%.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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2.4 Completion of total syntheses.
View Article Online
O
O
OTES
The synthesis of ent-precursors mirroredDthOaI:t1c0o.1n0d39u/cCt9eSdCf0o2r28th9Ae
preparation of griseusin A/C and B subclasses. For example, ent-
45 employed R-Sp-josiphos as a chiral catalyst in the
enantioselective -borylation of 27 and -unsaturated ketone
handle 29S in the C-H olefination step, while AD-mix- and
ketone 22S were applied to construct ent-griseusin C and D
scaffolds. With all key griseusin and ent-griseusin precursors in
hand, final deprotection was accomplished with silver oxide
under acidic conditions as summarized in Table 1. Following
previously reported oxidative strategies,16 hydrogen peroxide
and DABCO furnished the desired 11 C-4a/C-10a epoxide with
8:1 diastereoselectivity (Scheme 5 and Table S7). In summary,
starting from 1,5-dihydroxynaphthalene the divergent strategy
put forth enabled access to eleven naturally-occurring griseusins
and seven new griseusin analogues in 10-18 steps with total
yields ranging from 3-8%.
O
O
condition as
entry 3, Table S6
(1)
O
OTES
90% conversion,
COOMe
OTES
53
0%
left
O
O
OMe
43
OH
COOMe
O
O
0% Pd(OAc)2,
other condition as
entry 3, Table S6
OMe
O
O
53
(2)
COOMe
OTES
45% conversion
OMe
52
O
O
OTES
O
O
O
O
O
condition as
entry 3, Table S6
O
(3)
COOMe
43
100%
COOMe
0%
produced,
52
left
OMe
OMe
52
43
O
2.5 Configuration assignment of griseusin B epoxide (11).
O
O
OEt
O
O
condition as
While the spectroscopic data for synthetic 11 and previously-
isolated 11 were consistent (Table S8), the reported epoxide 11
isolated from Nocardiopsis sp. YIM80133 lacked assignment of
absolute stereochemistry.4g To address this, NMR studies of
tetrol 55, produced via hydrogenation of 11, enabled assignment
of the epoxide absolute stereochemistry as C-4aR, 10aS (Scheme
5). As biosynthetic transformations are commonly conserved
within a given natural product classes, this definitive
stereochemical assignment may also be relevant to other
naturally-occurring C-4a/C-10a-epoxy PNQs.1a, 1c, 1m
entry 1, Table S6
O
OH
(4)
81%
O
OMe
O
OMe
54
O
20
O
Scheme 4. Reactions relevant to C-H olefination mechanistic considerations.
OH
OH
OH
OH
OH
OH
OH
O
OH
O
OH
O
O
O
O
Table S7
a)
b)
COOH
O
O
O
10a
4a
OH
O
COOH
COOH
2.6 Structural revision of griseusin D (12).
H
H
H
H
HO
O
O
8
Methanolysis of 18 afforded E-ring lactone 12 in good yield
(Scheme 6). While the spectroscopic data for compound 12 was
in strong agreement with the reported griseusin D (Table S9),6f
the determined optical rotation of 12 was diametrically opposed
to that reported for griseusin D. Thus, we postulate 12 to be ent-
griseusin D and the structure of reported griseusin D to be that
illustrated in Scheme 6. Consistent with this, most griseusins
previously reported from the griseusin D-producing strain
(Nocardiopsis sp. YIM80133) also displayed negative optical
rotations.6g
NOE
11
55
Scheme 5. Synthesis and structural determination of 4a,10aepoxy-epi-
deacetylgriseusin B (11). Key NOE crosspeaks are highlighted (red arrows). Reagents and
conditions: (a) H2O2, DABCO, DCM, 0 oC-rt, 2 h, 88%; (b) H2, Pd/C, MeOH, 2 h, 42%
re-coordination versus nucleophilic attack of the participant
hydroxyl (which can be influenced via nucleophile orientation
and/or nucleophilicity as noted in Table S6). For example, the
higher yield of methylene isochroman 54 is attributed to lower
nucleophilicity of the 20-derived -unsaturated ester (Scheme
4). Cumulatively, these studies provide further support of Yu’s15
and our3m previously proposed step-wise mechanism for this
oxidative cyclization event.
Guided by our prior success with griseusin A-type spiropyran
ring formation, unsaturated ketone 43 was converted to C3’-
deshydroxy substrate 44 and C3′-(S)-hydroxy substrate 30,
respectively (Scheme 3). The C4′-carbonyl of intermediate 30
was subsequently stereoselectively reduced to obtain 45
followed by hydrolysis with LiOH to give intermediate 46. In
parallel, 30 C3′-O-benzyl protection under a mild conditions
gave intermediate 47. Subsequent diastereoselective 47 C4′-
ketone reduction with either borane or borohydride gave 4’-(S)-
alcohol 48 or 4′-R-alcohol 50, respectively. Final standard
2.7 Griseusin comparative cancer cell line cytotoxicity.
All compounds were tested against four cancer cell lines (non-
small cell lung A549, prostate PC3 and colorectal HCT116 and
DLD-1) (Table 2). While most griseusins were found to be
cytotoxic across this panel, A549 was generally (2- to 10-fold)
less susceptible to PNQs. An intact D-lactone ring (e.g., as found
in griseusins A and C, compounds 1-4) was generally
advantageous as compared to D-ring open comparators (e.g.,
griseusins B and D, compounds 6-8, 12). Surprisingly, active
enantiomeric comparators (e.g., 1 vs 5 or 7 vs 9) displayed
similar potencies. E-ring C3’-substitution generally had a
negative impact potency (e.g., 2 vs 16, 3 vs 13, or 4 vs 17), while
bioactivity was less impacted by C4’-alterations (ketone, S-
hydroxyl, R-hydroxyl). Destruction of the naphthoquinone core
also abolished activity (e.g., compounds 11 and 55).
protecting group manipulations gave key subclass
intermediates 49 and 51.
B
4 | J. Name., 2012, 00, 1-3
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Table 2. Cancer cell cytotoxicity and inhibition of axolotl tail regeneration.a
O
Me
O
O
View Article Online
Me
O
O
OH
O
DOI: 10.1039/C9SC02289A
OH
OH
O
O
O
HCl, MeOH
66%
OH
OH
OH
axolotl
tailc
+
+
+
-
N.D.
+
O
O
A549b
PC3b
HCT116b
DLD-1b
O
O
Cmpd
O
OMe
OMe
O
O
18
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
55
1.0±0.2
3.4±1.2
1.8±0.03
10.3±7.9
6.9±0.1
4.1±1.0
11.6±9.0
>20
0.1±0.08
0.9±0.3
0.1±0.1
1.9±0.3
0.6±0.02
2.1±0.9
0.9±0.5
2.9±0.6
0.9±0.08
0.7±0.01
>20
1.0±0.3
0.1±0.02
1.1±0.3
0.2±0.06
0.2±0.1
0.2±0.04
0.6±0.3
0.3±0.05
>20
0.2±0.08
0.5±0.1
0.1±0.02
>2
0.2±0.01
1.2±0.9
>2
1.2±0.6
0.4±0.05
0.8±0.5
>2
0.1±0.02
0.1±0.08
0.1±0.004
>2
0.2±0.06
0.6±0.3
0.3±0.1
>2
O
OH
D = +75o
synthesized ent-griseusin D
O
OH
isolated, []D = -67o
proposed revised griseusin D
O
12,

]
[
Scheme 6. Synthesis of ent-griseusin D (12) and proposed griseusin D structural revision.
Table 1. Total synthesis of natural-occurring griseusins (1-10) and their analogs (13-19).a
-
-
R1
R2
R3
R1
R2
R3
O
O
OH
O
O
O
15.1±0.2
5.0±0.1
>20
0.3±0.01
0.5±0.4
>2
-
toxic
-
+
toxic
-
+
toxic
+
method A or B
O
O
OMe R4
O
R4
3.2±0.6
0.7±0.3
2.1±0.1
1.2±0.3
1.0±0.2
1.2±0.2
2.7±0.5
1.2±0.2
>20
>2
0.1+0.02
0.1±0.03
0.9±0.4
0.1±0.01
0.1±0.01
0.04±0.01
0.1±0.03
0.1±0.06
>2
0.1±0.06
0.7±0.01
0.2±0.02
0.1±0.04
0.1±0.01
0.2±0.03
0.1±0.05
>2
reactant
(method b)
39 (A)
product
(yieldc)
1 (81%)
2 (85%)
reactant
product
(yieldc)
10 (79%)
13 (73%)
entry
entry
(method b)
ent-46 (A)
32 (A)
1
2
10
11
35 (A)
1,3’-epi-23f,
(A)
40 (A)
33 (A)
34 (A)
+
3
23 (A)
3 (63%)
12
14 (63%)
toxic
N.D.
4
5
6
7
8
9
36 (A)
ent-35 (A)
51 (B)
46 (B)
49 (B)
4 (62%)
5 (77%)
6 (54%d)
7 (68%)
8 (56% e)
9 (55% e)
13
14
15
16
17
15 (75%)
16 (78%)
17 (75%)
18 (63%)
19 (52% d)
aSee SI for experimental details. bIC50±SD values (M) from experiments performed
in triplicate. The highest concentrations tested were 20 M (A549 and PC3) and 2
M (HCT116 and DLD-1), respectively. cResults from single dose (10 M)
experiments performed in triplicate where ‘+’ indicates complete inhibition, ‘-‘
reflects no effect and ‘toxic’ denotes lethality.
25 (A)
44 (B)
ent-45 (B)
aSee SI for experimental details. bMethod A: 5 eq. AgO, 10 eq. 6N HNO3 at 0 oC for
10 min; method B: 2.2 eq. AgO, 6 eq. 3N HNO3 at -10 oC for 30 min. cIsolated yields.
dDeprotection of 51 benzyl ether (hydrogenolysis) was conducted before the
deprotection step and reported yield included this conversion. eEster hydrolysis of
49 and ent-45 were conducted before the deprotection step and the reported yield
included this conversion. fSee ref 3m for structural detail.
2.8 Griseusins inhibit peroxiredoxin 1 (Prx1) and glutaredoxin 3
(Grx3) and thereby influence 4E-BP1 phosphorylation.
We recently identified the PNQ FB as a potent inhibitor of
peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3) and
determined FB’s antitumor effect to be mediated by inhibition of
phosphorylation of 4E-BP1.5 While 4E-BP1 is a cap-dependent
translation repressor of oncogenic mRNA translation and
tumorigenesis, phosphorylation of 4E-BP1 by mTORC1 relieves
its inhibitory control leading to tumor progression.17 To assess
whether griseusin-type PNQs can also bind Prx1 and/or Grx3,
we conducted a simple competition assay with model griseusin
13 using the FB-based biotinylated probe set from our
previously-reported target identification study (Figure 2a).5 In
this study (Figure 2b), 13 could completely block the binding of
the active FB-based Probe 1 to Prx1 or Grx3 in HCT116 crude
extracts. Consistent with this, 13 also directly inhibited Prx1 and
Prx2 catalytic activity with apparent IC50s of 2.3 M and 7.3 M,
respectively (Figure S1). Similar to the inhibitory effect of FB
on 4E-BP1 phosphorylation, the representative cytotoxic
griseusins tested (e.g., 3, 13, 17, and 19) inhibited 4E-BP1
phosphorylation while non-cytotoxic analogues (e.g., 11) had no
effect on 4E-BP1p (Figure 2c). Collectively, these findings
suggest that griseusin-type and FB-type PNQs exhibit a similar
mechanism of action.
Figure 2. (a) Structures of compound 13 and probes 1 and 2. Probe 1 is active (both
cytotoxic and as an inhibitor of 4E-BP1 phosphorylation) while the control Probe 2 is an
inactive comparator. (b) Compound 13 competes with Probe 1 binding to Prx1 and Grx3.
(c) Cytotoxic griseusin-type PNQs effectively inhibit phosphorylation of 4E-BP1 but not
AKT and ERK kinases.
2.9 Impact of griseusins on axolotl tail regeneration.
Recent studies in a highly regenerative salamander model (the
Mexican axolotl, Ambystoma mexicanum) revealed that ROS
rapidly increased in response to axolotl tail amputation and was
required for tail regeneration.18 These studies also demonstrated
pharmacological inhibition of ROS producing enzymes with
diphenyleneiodonium chloride (DPI) and VAS2870 reduced
ROS and led to inhibition of cellular proliferation and tail
This journal is © The Royal Society of Chemistry 20xx
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outgrowth inhibition. To investigate the impact of the DA016176 (YZ), the University of Kentucky CollegeVoiefwPAhrtaicrlemOanclinye,
DOI: 10.1039/C9SC02289A
context, the National Center for Advancing Translational Sciences
Prx1/Grx3-inhibiting
griseusins
within
this
representative griseusin analogues were evaluated in the same (UL1TR000117 and UL1TR001998), and the Start-up funding of
axolotl embryo tail regeneration (ETR) assay.7 Tail-amputated Jiangsu Specially-Appointed Professor and National Natural
axolotl embryos were incubated in microtiter plates in the Science Foundation of China (No. 21877062, YZ). We also thank
absence (vehicle control, DMSO) or presence of 10 M agent (1- Prof. Tyler D. McQuade (Florida State University) generously
4, 6-19) and imaged on day 1 (pre-treatment) and day 7. The providing McQuade I NHC copper catalyst.
initial single dose screen revealed a moderate correlation
between cancer cell line cytotoxicity and inhibition of tail Human colon (HCT116, DLD-1), prostate (PC3) and lung (A549)
regeneration (Table 2, Figure S2) where divergence may result, cancer cell lines were obtained from the American Type
in part, from yet to be determined factors that contribute to CultureCollection (ATCC, Manassas, VA) and cultured as per
differences in uptake and/or in vivo exposure. Importantly, these ATCC recommendations.
single dose studies, in conjunction with the subsequent
established dose response of both inhibition of tail regeneration
and lethality with representative 13 (Figure S3), are consistent
with a mechanistic relationship between Prx1/Grx3, ROS and
Notes and references
tail regeneration and provide preliminary evidence for in vivo
application of griseusin-based probes.
1
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Conclusions
This comprehensive study highlights the first concise and
divergent synthetic approach to prepare griseusin A-E analogs.
The enabling chemistry put forth facilitated
a broad
comparative cancer cell line cytotoxicity SAR analysis and in-
depth mechanistic studies that established griseusins as
inhibitors of Prx1/Grx3 and 4E-BP1 phosphorylation.
Importantly, Prx1 and Grx3 overexpression is a hallmark of a
variety of cancers and is associated with redox adaptation that
promotes tumor progression and resistance to many anticancer
agents and radiation.19 Consistent with this, knockdown of Prx1
and Grx3 expression in cancer cells leads to an increase in ROS
levels, resulting in inhibition of proliferation, survival, invasion,
metastasis, and sensitivity to chemotherapy and radiation.19d,20
These studies also suggest that similar subtle shifts in the
balance of [ROS] within the axolotl tail regeneration model may
also contribute to the observed anti-proliferative effects of
griseusins in vivo. Cumulatively, the facile access to the
griseusins described herein is expected to enable new
molecular probe development to advance the study the role of
Prx1 and Grx3 in biology and may also serve as a starting point
for early anticancer lead development. Given the established
mechanistic relationship between griseusins and FB (an
effective anticoccidial and antimalarial agent),1a,4 the current
study may also prompt similar functional evaluation of
griseusins.
2
3
Conflicts of interest
The authors declare the following competing financial interest:
J.S.T. is a co-founder of Centrose (Madison, WI, USA).
Acknowledgements
This work was supported by National Institutes of Health grants
R01 CA203257 (QBS, JST), R24 OD21479 (SRV, JST), T32
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