Preparation and evaluation of deconstruction analogues of 7-deoxykalafungin as AKT kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2013.11.020

The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify structural features of the natural product scaffold that are essential for potency and selectivity. Using a deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the 3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT. We have also unexpectedly identified a new submicromolar inhibitor of PKA.

Full text of DOI:10.1016/j.bmcl.2013.11.020

Bioorganic & Medicinal Chemistry Letters 24 (2014) 271–274
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation and evaluation of deconstruction analogues of
7-deoxykalafungin as AKT kinase inhibitors
⇑
Sudha Korwar, Thuy Nguyen, Keith C. Ellis
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Biotech 1, 800 East Leigh Street, Richmond, VA 23298, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2013
Revised 6 November 2013
Accepted 11 November 2013
Available online 20 November 2013
The pyranonaphthoquinone (PNQ) lactone natural products, including 7-deoxykalafungin, have been
reported to be potent and selective covalent inhibitors of AKT kinase. In this work we seek to identify
structural features of the natural product scaffold that are essential for potency and selectivity. Using a
deconstruction approach, we designed and prepared simplified analogues of 7-deoxykalafungin. Testing
of the compounds for their ability to inhibit AKT and the closely related kinase PKA revealed that the
3,6-dihydro-2H-pyran ring of the PNQ lactones is required for potent and selective inhibition of AKT.
We have also unexpectedly identified a new submicromolar inhibitor of PKA.
Keywords:
Pyranonaphthoquinone lactone
Covalent inhibitor
AKT kinase
Ó 2013 Elsevier Ltd. All rights reserved.
Deconstruction analogues
7-Deoxykalafungin
Activity-based protein profiling (ABPP) is a chemical proteomics
strategy that can be used to selectively identify the activated form
of a protein in cells of various phenotypes both in vitro and
in vivo.1–3 ABPP is a particularly powerful technique because it
moves beyond simply quantifying the overall amount of a protein,
active and inactive, in the cellular environment. ABPP detects only
the fully activated, functional form of the protein of interest and
does not detect forms that are overexpressed but down regulated,
inactive because of posttranslational modifications, or not present
in the cellular location of interest. ABPP has been used to elucidate
the roles of multiple proteins and protein families in diseases
including cancer,4,5 inflammation,6,7 rheumatoid arthritis,8,9 and
depression.10
AKT is a member of the AGC family of kinases that regulates
multiple physiological processes including cell proliferation,
metabolism, and survival.20 Hyperactivation of AKT kinase has
been implicated in several disease states including cancer,21
type-II diabetes,22 and neurodegenerative diseases.23 To date, no
activity-based probes targeting AKT kinase have been reported.
The pyranonaphthoquinone (PNQ) lactone natural products lac-
toquinomycin and frenolicin B were identified as potent and selec-
tive inhibitors of AKT kinase in a high-throughput screen (Fig. 1).24
Further work demonstrated that the PNQ lactones inhibit AKT by
ABPP requires activity-based probes (ABPs) that contain three
essential elements: (1) the probes must covalently modify a resi-
due in the target protein that is reactive only in the activated form
of the protein; (2) the probes must contain a scaffold that binds
selectively to the targeted enzyme family or single enzyme; and
(3) the probes must contain a tag that can be used for isolation
or detection.2
One major source of ABPs has been protein-reactive natural
products.11–13 Activity-based probes have been developed from
the natural products wortmannin (targeting PI3K),14–16 E-64 (tar-
geting the cathepsins),17 b-lactams (targeting penicillin binding
proteins and b-lactamases),18,19 and several others (reviewed in
Ref. 6).
⇑
Corresponding author.
E-mail address: kcellis@vcu.edu (K.C. Ellis).
Figure 1. Structures of the pyranonaphthoquinone lactone natural products that
inhibit AKT kinase.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.11.020

272
S. Korwar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 271–274
covalently alkylating Cys310 on the kinase activation loop.25 De-
spite the fact that this cysteine is conserved in over half of the en-
zymes in the AGC kinase family, the PNQ lactones are selective for
AKT over these other family members.
The preliminary structure-activity relationship (SAR) for the C5
position of the PNQ lactone 7-deoxykalafungin shows that mono-
substitution at this position with both small (R = H, Me) and large
(R = CH2OBn, 2-thienyl) groups are tolerated but that di-substitu-
tion leads to a 10-fold decrease in inhibition.25 This study did not
investigate selectivity.25
As the first step in a program to explore the development of the
PNQ lactones as an AKT-selective activity-based probe, we sought
to understand the structural features of the natural product that
impart potency and selectivity to this scaffold. In this communica-
tion, we have designed and prepared deconstruction analogues of
7-deoxykalafungin and evaluated their potency against recombi-
nant AKT1 and selectivity for AKT1 over the closely related AGC ki-
nase PKAa.
The PNQ lactones must be activated in order to function as
covalent inhibitors. Under in vitro and in vivo conditions the qui-
none is reduced and rearranges to the activated quinone methide
(1, Scheme 1), which acts as a Michael acceptor (shown in
magenta).26 The nucleophilic Cys310 of AKT then attacks the acti-
vated quinone methide 1, resulting in PNQ lactone—AKT adduct 2.
The formation of the activated quinone methide 1 by reduction is
essential for activity. Analogues missing the lactone ring are inac-
tive as AKT inhibitors up to concentrations of 20 l
M.25
The deconstruction analogues that we have designed and pre-
pared are shown in Scheme 2. We designed these analogues to
either preserve the latent electrophile by retaining the quinone
(analogues 3 and 8) or replace this motif with an already-formed
a,b-unsaturated ketone Michael acceptor (shown in magenta, ana-
logues 5–7). We also designed these analogues to systematically
remove structural elements from the natural product scaffold that
provided steric bulk and conformational rigidity to determine how
these elements contributed to both potency and selectivity.
Several synthetic approaches to the PNQ lactones have been
previously described.27–31 We chose to synthesize 7-deoxykalafun-
gin by a variation of the route of Eid and co-workers32 starting
Scheme 2. Deconstruction analysis of 7-deoxykalafungin and the structure of
deconstruction analogues 3–8.
from commercially available 1,4-dimethoxynaphthalene
9 as
shown in Scheme 3. Bromination, Heck coupling, Sharpless asym-
metric dihydroxylation with concomitant lactone formation, oxa-
Pictet–Spengler reaction, and finally oxidation with CAN afforded
the desired 7-deoxykalafungin. Deconstruction analogue 3 was
prepared by treatment of intermediate 12 from the 7-deoxykala-
fungin synthesis with CAN (Scheme 4).
Deconstruction analogues 5–7 were prepared by a common
route starting from benzoic acid 19 (Scheme 5). EDC coupling affor-
ded the Weinreb amide 20, which was treated with vinylmagne-
sium bromide in ethyl ether to afford the vinyl ketone 7.
Subsequent treatment of 7 with a 2nd generation cross metathesis
catalyst (CAS# 253688-91-4) and either 3-methoxyprop-1-ene or
methyl pent-4-enoate in refluxing DCM afforded 5 and 6, respec-
tively, both in >20:1 E/Z selectivity.
Finally, naphthaquinone 8 was prepared by CAN oxidation of
1,4-dimethoxynaphthalene 9 (not shown).
7-Deoxykalafungin and compounds 3 and 5–8 were tested in
the Z’-LYTE assay (Invitrogen) for their ability to inhibit kinase
function of AKT1 and PKAa (Table 1). This FRET-based assay mea-
sures the ability of a kinase enzyme to phosphorylate a peptide
substrate and detects functional inhibitors of kinase activity inde-
pendent of mechanism of action. Our data confirmed the previous
report that 7-deoxykalafungin is a potent inhibitor of AKT1 but
Scheme 1. Activation of 7-deoxykalafungin to form activated quinone methide 1 and AKT adduct 2.

S. Korwar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 271–274
273
Table 1
Inhibition of recombinant AKT1 and PKA
a
by 7-deoxykalafungin and analogues 3-8
a
Compound
IC50
(
lM)
AKT1
PKAa
7-Deoxykalafungin
0.28
2.94
50.0
13.7
12.3
11.0
>100
0.43
3
5
6
7
8
>100
>100
>100
b
n.d.
a
IC50’s were determined using Z’-LYTE assay kits (Invitrogen). 95% confidence
intervals can be found in the Supporting information.
b
Not determined.
Scheme 3. Reagents and conditions: (a) NBS, DCM, 96%. (b) isobutyl but-3-enoate,
Pd(tBu3P)2, Cy2NMe, toluene, 81%. (c) K3Fe(CN)6, K2CO3, NaHCO3, (DHQD)2PHAL,
CH3SO2NH2, K2OsO4Á2H2O, tBuOH, H2O, 53%, dr >95:5. (d) CH3CHO, BF3ÁOEt2, DCM,
93%, dr >95:5. (e) CAN, ACN, H2O, 72%.
Figure 2. PNQ lactone analogue 21.25
and selective inhibition of AKT. Of note is previously reported
PNQ lactone analogue 21 (Fig. 2),25 which contains the 3,6-dihy-
dro-2H-pyran ring but lacks a substituent at C5. These data, com-
bined with that reported in this work, demonstrate that the
rigidity of 3,6-dihydro-2H-pyran ring is essential for potent and
selective inhibition of AKT1. Our data also demonstrates that the
activated quinone methide (as seen in 1 and 3a, Schemes 2 and
3) is necessary for submicromolar inhibiton of AKT and that it can-
Scheme 4. Reagents and conditions: (a) CAN, ACN, H2O, 65%.
not be replaced with an a,b-unsaturated ketone with similar struc-
tural elements (such as 5–7). We have summarized the known data
into a pharmacophore model (Fig. 3).
The most unexpected result of this study is that deconstruction
analogue 3 inhibits PKA at submicromolar concentrations and that
it is a better inhibitor of PKA than AKT. PKA also contains a cysteine
in the activation loop in a similar position to that of AKT (Cys199 of
PKAa vs Cys310 of AKT1). The increased flexibility of deconstruc-
tion analogue 3 could be allowing this compound to alkylate PKA
a
Cys199. Deconstruction analogue 3 represents a new electrophile
to explore for the development of an activity-based probe for
PKAa.
In summary, we have demonstrated that 3,6-dihydro-2H-pyran
ring of the PNQ lactones is an essential structural feature for po-
tency and selectivity. With a pharmacophore in hand for inhibition
of AKT by the PNQ lactones, we can now begin to develop this
scaffold into an AKT-selective activity-based probe. We have also
identified a new electrophile and scaffold to explore for the devel-
Scheme 5. Reagents and conditions: (a) EDCÁHCl, NMM, MeON(H)MeÁHCl, DCM,
84%. (b) vinylmagnesium bromide, Et2O, 55%. (c) 3-methoxyprop-1-ene, cross
metathesis catalyst, DCM, 40 °C, 30%, >20:1 E/Z. (d) methyl pent-4-enoate, cross
metathesis catalyst, DCM, 40 °C, 41%, >20:1 E/Z.
opment of an activity-based probe for PKAa.
does not inhibit PKAa. Surprisingly, however, deconstruction ana-
logue 3 showed a reversal of this inhibition pattern. Analogue 3,
which presumably forms activated intermediate 3a (Scheme 2),
inhibited PKA
a at submicromolar concentration but was 10-fold
less active toward AKT1 than parent 7-deoxykalafungin. The
remaining deconstruction analogues 5–8 were double-digit mi-
cro-molar inhibitors of AKT1 and did not inhibit PKAa.
The data above, when combined with that previously pub-
lished,25 gives us insight into the minimum structural elements
of the PNQ lactone natural products that are required for potent
Figure 3. Pharmacophore model for AKT inhibition by the PNQ lactones.

274
S. Korwar et al. / Bioorg. Med. Chem. Lett. 24 (2014) 271–274
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Supplementary data (experimental data for the synthesis and
characterization of 7-deoxykalafungin, analogues 3, 5–8, and inter-
mediates 10–20, and assay protocols for inhibition of AKT and PKA)
associated with this article can be found, in the online version, at
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