The rational design of a novel potent analogue of the 5′-AMP- activated protein kinase inhibitor compound C with improved selectivity and cellular activity

Article abstract of DOI:10.1016/j.bmcl.2010.09.088

We have designed and synthesized analogues of compound C, a non-specific inhibitor of 5′-AMP-activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach. Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in the inhibition of the human AMPK (hAMPK) α2 subunit in the heterotrimeric complex in vitro, exhibited significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced cellular inhibition of AMPK.

Full text of DOI:10.1016/j.bmcl.2010.09.088

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6394–6399
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The rational design of a novel potent analogue of the 50-AMP-activated
protein kinase inhibitor compound C with improved selectivity and cellular
activity
Fouzia Machrouhi a, Nouara Ouhamou a, Keith Laderoute b, Joy Calaoagan b, Marina Bukhtiyarova a,
Paula J. Ehrlich a, Anthony E. Klon a,
⇑
a Ansaris, Four Valley Square, 512 East Township Line Rd, Blue Bell, PA 19422, United States
b SRI International, Menlo Park, CA 94025, United States
a r t i c l e i n f o
a b s t r a c t
We have designed and synthesized analogues of compound C, a non-specific inhibitor of 50-AMP-
activated protein kinase (AMPK), using a computational fragment-based drug design (FBDD) approach.
Synthesizing only twenty-seven analogues yielded a compound that was equipotent to compound C in
the inhibition of the human AMPK (hAMPK) a2 subunit in the heterotrimeric complex in vitro, exhibited
significantly improved selectivity against a subset of relevant kinases, and demonstrated enhanced
Article history:
Received 10 August 2010
Revised 14 September 2010
Accepted 15 September 2010
Available online 19 September 2010
cellular inhibition of AMPK.
Keywords:
Ó 2010 Elsevier Ltd. All rights reserved.
Fragment-based drug design
FBDD
50-AMP-activated protein kinase
AMPK
Compound C
50-AMP-activated protein kinase (AMPK) is a major regulator of
normal cellular energy metabolism.1 AMPK activity is sensitive to
the cellular AMP:ATP ratio, which is increased by physiological
stresses such decreased oxygen and glucose concentrations
(hypoxia and hypoglycemia). AMPK activity is also associated with
the growth of solid tumors, which commonly contain regions of
pathologic hypoxia and hypoglycemia.2 AMPK cooperates with
hypoxia-inducible factor-1 (HIF-1), the primary transcriptional reg-
ulator of the mammalian response to hypoxia and other metabolic
stresses that lower cellular ATP levels, in pathophysiological tumor
microenvironments.3,4 Thus, in the context of microenvironmental
stress, AMPK may represent a novel target for the treatment of solid
tumors. The successful design of potent and selective inhibitors of
AMPK that exhibit activity is an important first step toward estab-
lishing the proof-of-concept for the therapeutic value of AMPK
inhibition in solid tumor microenvironments.
C ([4-(2-piperidin-1-yl-ethoxy)-phenyl])-3-pyridin-4-yl-pyrrazol-
o[1,5-a]-pyrimidine) is considered to act as a competitive inhibitor
of ATP binding to the catalytic
a
subunit of AMPK.5,11 However,
compound C has significantly limited specificity as an AMPK
inhibitor.5
We discuss the SAR of a series of pyrazolopyrimidine and
aminooxazole analogues of compound C and present their general
binding mode obtained through the use of homology modeling and
Grand Canonical Monte Carlo (GCMC) fragment simulations.9 A fo-
cused kinase selectivity profile for the most active subset of these
compounds is presented, as well as the cellular AMPK inhibition
data for the most promising pyrazolopyrimidine.
This Letter describes our efforts to design a Type I inhibitor of
AMPK with a superior biological profile than that of compound C
(Fig. 1), which is a commonly used experimental direct inhibitor
of the enzyme,5 through the use of our proprietary fragment-based
drug design (FBDD) software6–9 and Imagiro™,10 our proprietary
torsion-space molecular mechanics software package. Compound
⇑
Corresponding author. Tel.: +1 215 358 2029; fax: +1 215 358 2020.
E-mail address: aklon@ansarisbio.com (A.E. Klon).
Figure 1. Chemical structure of compound C.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.088

F. Machrouhi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6394–6399
6395
We generated a model of the catalytic
the DFG-loop in the ‘in’ conformation using MOE.12 The crystal
structure of the AMPK
2 subunit (RCSB ID# 2H6D, 1.85 Å)13 was
used as the overall template for the protein model. The activation
loop (A-loop) was not crystallographically resolved in this
structure and the DFG loop is in the ‘out’ conformation. Residues
157-179 of the microtubule affinity regulating kinase-1 (MARK-1)
crystal structure (RCSB ID# 2HAK, 2.60 Å)14 were used as a tem-
plate for the ‘in’ conformation of the DFG-loop and the missing res-
idues for the A-loop. MARK-1 was selected as the template for the
a
2 subunit of AMPK with
acceptor with the backbone nitrogen of residue Val-96 in the hinge
region, while the 4-pyridine fragment forms a hydrogen bond with
the side chain of Lys-45. The piperidine fragment is oriented to-
wards the solvent and does not appear to form specific interactions
with AMPK. We hypothesize that this fragment performs the func-
tion of a solubilizing group. The P-loop (residues 23–30) is moved
slightly towards the N-terminal lobe of the kinase in the final mod-
el relative to the 2H6D crystal structure of AMPK to allow for better
steric contact with the pyridinyl moiety of compound C. The over-
a
all RMSD of the Ca atoms between the two structures is 2.4 Å.
A-loop conformation of the AMPK
a
2 subunit due to the 50% se-
In order to design inhibitors of AMPK, the binding of 1785 small
molecular fragments16 was simulated against the homology model
using the GCMC approach. Hypothetical compounds were designed
using the results of the fragment simulations to find replacements
for the pyridine fragment that maximized interactions with Lys-45
as well as Asp-157 in the DFG-loop as shown in Figure 2B. Replace-
ments for the pyrazolo[1,5-a]pyrimidine fragment were explored,
with the most successful being aminooxazole. All designed com-
pounds were evaluated using computational ADME filters17 and
prioritized for synthesis on the basis of their calculated binding
affinities and ADME properties.
The synthesis of designed substituted pyrazolo[1,5-a]pyrimi-
dine derivatives was accomplished according to Scheme 1. The
bromide intermediate 2 was readily obtained from the commer-
cially available 118 via an SN2 type reaction with 4-(2-chloroethyl)
morpholine hydrochloride salt under basic conditions. The bro-
mide 2 was then reacted with selected commercial available or
readily synthesized boronates or boronic acids in microwave palla-
dium catalyzed Suzuki cross-coupling reactions with good to mod-
erate yields to give compounds 3–1019 Compound 11 was obtained
after a subsequent Suzuki coupling reaction between intermediate
15 and bromide 2 followed by deprotection of the PMB groups. The
boronate 15 was prepared after a PMB protection of commercially
available 4-bromobenzene-1,2-diol 14 followed by typical boro-
nate formation in good yield.
quence identity between the AMPK and MARK-1 kinase domains.
Compound C, which inhibits AMPK with reported IC50 values rang-
ing from 0.1 to 0.2 l
M15 was used to model the inhibitor-bound
protein complex. The three fragments comprising the core struc-
ture of compound C (benzene, pyridine, and pyrazolo[1,5-a]pyrim-
idine, Fig. 1) were simulated for binding to the AMPK homology
model using our Grand Canonical Monte Carlo (GCMC) simulation
approach.6–8 Compound C was assembled from the resulting frag-
ment simulation data and found to bind in the ATP site. The result-
ing complex was subjected to energy minimization, which was
terminated when the energy gradient reached 0.001, followed by
259 ps of molecular dynamics simulations in torsion space using
Imagiro10 to ensure that the kinetic and potential energies of the
system were equilibrated, arriving at the optimized homology
model (Fig. 2A).
In the final model, the pyrazolo[1,5-a]pyrimidine core was
observed to present one ring nitrogen to act as a hydrogen bond
Two synthetic routes were developed for analog 5. The overall
reaction in Scheme 1 was followed. The boronate 13 was prepared
after PMB protection of compound 12, followed by standard boro-
nate formation in good yield. The product was then reacted with
bromide 2 under Suzuki cross coupling conditions to give the de-
sired analog 5 after deprotection. Inhibitor 9 was also converted
to analog 5 via a one pot two step synthesis consisting of diazoti-
zation and hydrolysis in moderate yield as outlined in Scheme 2.20
The inhibitory activities of all analogs were tested against the
hAMPK
2, b1,
a
2 subunit in complex with the heterotrimer (hAMPK
a
c1). The structure–activity relationship of the pyrazol-
o[1,5-a]pyrimidines is summarized in Table 1. Synthetic interme-
diates 1 and 2 were included to test the contribution of each
fragment on the binding of the pyrazolo[1,5-a]pyrimidines to
AMPK. The addition of the morpholinoethyl resulted in a twofold
improvement in potency observed in 2 relative to 1. Replacement
of the piperidine tail in compound C (Fig. 1) with the morpholino-
ethyl group in 4 resulted in a sevenfold loss in potency. Addition-
ally, the ADME filters used to prioritize the designed compounds
for synthesis predicted that compounds with the morpholino frag-
ment would have better human intestinal absorption.17 The en-
hanced potency of 4 relative to 3 was attributed to the ability of
the nitrogen on the pyridine ring to form a hydrogen bond with
the side chain of Lys-45. We analyzed the results of the fragment
simulation data to identify fragments that could form an additional
hydrogen bond with the side chain of Asp-157 on the DFG-loop
while retaining contact with Lys-45. Compounds 5, 6, 7, 8, 9, and
11 were designed using this approach.
Figure 2. Minimized predicted lowest-energy binding pose of compound C (A) and
compound 5 (B) (carbons in magenta) in complex with the hAMPK
a2 subunit
without their respective ethylpiperidinyl or ethylmorpholino tails. The hydrogen-
bonds between the pyridinone oxygen and the zeta nitrogen of Lys-45; the
pyridinone nitrogen and the delta oxygen of Asp-157 (B) and between the
pyrazolopyrimidine core and the backbone nitrogen of Val-96 in the hinge region
(A and B) are shown in green. Note that the DFG loop (orange) is in the ‘in’
conformation.
The pyridinone fragment of compounds 5 (Fig. 2) and 6 showed
favorable interactions with the Lys-45/Asp-157 pair and two plau-
sible attachment points to the pyrazolo[1,2-a]pyrimidine core.

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F. Machrouhi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6394–6399
Scheme 1. Synthesis of pyrazolo[1,5-a]pyrimidine compounds. Reagents and conditions: (a) K2CO3, DMF, 80 °C, 90%, 4-(2-chloroethyl)morpholine HCl; (b) 10 mol %
PdCl2(PPh3)2, Na2CO3 2 M, ArB(OH)2; (c) PMBCl, cat. NaI, DCM, rt, 80%; (d) 10% PdCl2 dppf.dcm, Bis (pinacolato) diborane, NaOAc, DMF, 80 °C, 75%.
Scheme 2. Alternative synthetic route to 5.
Both variants were made to test the preference for the geometry of
the Lys-45/Asp-157 interaction. Compound 6 showed no improve-
ment over 4, while 5 displayed equipotent activity compared to
compound C. Compound 7 displayed twofold improvement in
activity relative to 4. As with the pyridinone fragment, the GCMC
simulations in indicated that the 2-aminopyridine fragment would
form profitable interactions with the Lys-45/Asp-157 pair, and two
compounds could be designed with different attachment points to
the pyridine ring that preserve the hydrogen bonds. Compound 8
was found to be equipotent with 4, while 9 was 10-fold weaker.
The catechol was also identified as a potentially favorable fragment
that might preserved the key interactions, so 11 was synthesized
but found to be fourfold weaker than 4. The advanced intermediate
10, lacking a hydrogen bond donor, was twofold less potent than
the target compound 11. Another reason for the reduced potency
of 10 relative to 11 is likely due to steric clashes between the meth-
oxy groups and the side chain atoms of either Lys-45 or Asp-157.
Taken together, the resulting SAR for the pairs of 5 and 6 as well
as 8 and 9 demonstrate a clear preference for the trajectory from
the core for the hydrogen bond acceptor and donor atoms, while
compounds 5 and 7 show that the addition of a hydrogen bond do-
nor to exploit the interaction with Asp-157 can result in improved
potency against hAMPK.
fragment simulations were used to design new compounds with
reduced molecular weight that preserved the hydrogen bonding
interaction with the backbone nitrogen of Val-96. These included
2-aminooxazoles (Table 2), pyridines, pyrimidines, 2-aminothiaz-
oles, 2-aminooxazoles, pyrazolo[1,5-a]pyrimidine-7-amines, and
pyrazolo[1,5-a]pyrimidin-2-amines. Due to synthetic feasibility
concerns, only the designs containing pyridine, 2-aminooxazole,
and pyrazolo[1,5-a]pyrimidine-7-amine cores were synthesized
and tested. The pyridine and pyrazolopyrimidine-7-amine cores
showed weak or no activity (>10 lM) in the APMK assay (data
not shown). The most active of these core replacements was the
2-aminooxazole series. The 2-aminooxazoles are predicted to bind
to AMPK with the oxazole ring nitrogen forming the hydrogen
bond with the backbone of Val-96. The 2-amino group is predicted
to form an additional hydrogen bond contact with the backbone
carbonyl of Ser-97. Replacements for the 2-(piperidin-1-yl)ethoxy
fragment were also investigated and the most promising one iden-
tified from the GCMC simulations was benzene sulfonamide. The
oxygen of the sulfonamide was predicted to form a hydrogen bond
with the backbone nitrogen of Glu-100, while the nitrogen acts as a
hydrogen bond donor to a side chain oxygen of the same residue.
The synthesis of the 2-aminooxazole compounds using an
iminophosphorane-mediated cyclization is outlined in Scheme
3.21 The reaction of an isothiocyanate with an acyl azide and tri-
phenylphosphine in dioxane at 95 °C or in dichloromethane at
room temperature provided the 2-aminooxazole.22 The acyl azide
20 was obtained from the available 2-chloro-1-(3,4-dihydrophe-
nyl)ethanone via a Finkelstein reaction with sodium azide.23,24
We investigated the replacement of the pyrazolo[1,5-a]pyrimi-
dine core by searching for fragments that would form favorable
interactions with the hinge region while preserving the orientation
of fragments that were believed to form favorable contacts with
Lys-45 and Asp-157. Several core replacements suggested by the

F. Machrouhi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6394–6399
6397
Table 1
Table 2
Inhibition of hAMPK (
a
2, b1,
c1) by pyrazolopyrimidines
Inhibition of hAMPK a2 by aminooxazoles
a
No.
21
R
IC50
2.9
(lM)
R1
R2
IC50
7.7
(lM)
a
No.
1
2
3
4
3.5
1.8
22
5.0
a
As in Table 1.
0.28
0.06
5
6
7
8
0.27
0.14
0.22
Scheme 3. General method for the synthesis of 2-aminooxazoles.
9
2.1
2.2
10
11
1.1
a
Values were measured using the Hot Spot™ filtration binding 33P Kinase assay,
Reaction Biology, Malvern, PA. Compounds were tested in ten-dose IC50 mode with
threefold dilutions starting at 10 M. All reactions were carried out in the presence
of 10 M ATP.
l
l
The acyl azide intermediate 19 was synthesized from the corre-
sponding acyl bromide 18 as shown in Scheme 4. Compound 17
was generated via Stille cross-coupling from 16 (see also Scheme
1).25,26 Compounds 19 and 20 were converted to the intermediate
iminophosphorane upon treatment with PPh3, which was followed
by rearrangement and deprotection to yield 21 and 22.
Compound C has previously been reported to be a potent inhib-
itor of a number of kinases in addition to AMPK, including Eph-A2,
c-Src, Lck, KDR, and MNK-1.15 A kinase selectivity screen was car-
ried out using these six kinases plus the relevant cancer kinases
Flt1, Flt3, and Rsk1. We tested the most potent pyrazolo[1,5-
a]pyrimidines in addition to two 2-aminooxazoles along with com-
pound C. As seen in Table 3, compound C is non-selective against
the kinases in screen, inhibiting seven of the eight kinases more
potently than AMPK. Compound 5, which is equipotent with
compound C for AMPK, is substantially more selective. With the
exception of Flt3, 5 showed improvements in selectivity between
4.5-fold for Mnk1 and 60-fold for Flt1 as compared to compound C.
Figure 3 compares the effect of 5 and compound C on basal
AMPK activity (detected by immunoblotting for specific phosphor-
ylation of the AMPK substrate acetyl CoA carboxylase 1 (ACC) on
Scheme 4. Synthesis of 2-aminooxazoles. Reagents and conditions: (a) 10 mol %
PdCl2(PPh3)2, LiCl, vinyltin reagent; (b) NBS THF/water; (c) NaN3 acetone/water; (d)
PPh3, ArNSC; (e) TFA, 100 °C, microwave.
Ser79; Phospho-ACC3,27) in a mouse embryonic fibroblast (MEF)
cell line. Transformed derivatives of these cells were previously
used to investigate the contribution of AMPK to experimental tu-
mor growth.3 Both 5 and compound C inhibited cellular AMPK
activity (P-ACC levels), but 5 was a more potent AMPK inhibitor
than compound C (e.g., 65
that both 5 and compound C appeared to decrease specific phos-
phorylation of the AMPK catalytic subunit (P-AMPK levels; results
lM, 6 h) in MEF cells. It is noteworthy
a

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F. Machrouhi et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6394–6399
Table 3
Near kinase selectivity panel of pyrazolopyrimidines and aminooxazoles.
a
No.
IC50 (lM)
hAMPK
a
2
c-Src
EphA2
Flt1
Flt3
KDR
Lck
Mnk1
Rsk1
Compound C
4
5
7
0.041
0.27
0.061
0.14
0.22
1.1
0.002
0.050
0.061
3.3
0.021
0.096
3.2
0.011
0.25
0.26
0.30
0.84
0.74
>10
0.011
0.21
0.47
0.004
0.040
0.002
0.074
>10
<0.001
0.001
<0.001
<0.001
0.001
0.001
0.13
0.004
0.056
0.10
0.010
0.058
0.001
0.100
>10
0.016
0.30
0.43
0.094
0.44
0.02
0.97
>10
0.011
0.13
0.045
0.027
0.164
0.95
0.21
2.1
2.1
0.22
1.1
0.56
3.4
8
11
21
22
2.8
5.0
2.2
1.10
8.2
5.6
0.12
>10
a
As in Table 1.
Figure 3. Effects of compound 5 and compound C on AMPK-dependent ACC phosphorylation (P-ACC) and phosphorylation of AMPK
in complete medium (DMEM + 25 mM HEPES [pH 7.4] + 10% fetal bovine serum [FBS]). A Representative immunoblots of total protein from MEFs harvested following
exposure to compound 5 or compound C (1, 5, or 10 M; 6 h). Replicate blots were probed for the relative levels of P-ACC or total ACC. B Corresponding histogram of
a on Thr172 (P-AMPK) in MEFs cultured
l
densitometry measurements from multiple immunoblots of total protein from untreated (control) MEFs and MEFs treated with compound 5 or compound C; data were
normalized to the control data points. Error bars, SD from independent cultures/data point (n = 3). Statistical comparisons were made between control and treated cells
(2-tailed unpaired t-test; p 6 0.05 was considered a significant difference): only significant differences between control and treated cells are shown (*p = 0.03, 5
lM
compound 5; **p = 0.03, 10 M compound 5; ***p = 0.04, 10
l
l
M compound C). Immunoblotting protocols have been described in detail.28
2. Pang, T.; Zhang, Z.-S.; Gu, M.; Qiu, B.-Y.; Yu, L.-F.; Cao, P.-R.; Shao, W.; Su, M.-B.;
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fect is direct or indirect.28 In summary, the results shown in Figure
3 demonstrate that 5 inhibited endogenous AMPK activity in cul-
tured mammalian cells.
In conclusion, pyrazolo[1,5-a]pyrimidine and 2-aminooxazole
inhibitors of the human AMPKa2 subunit were identified through
the use of FBDD and medicinal chemistry efforts. A binding model
was obtained from the results of GCMC fragment simulations in to
a homology model of the protein and is consistent with the ob-
served SAR. The most potent pyrazolo[1,5-a]pyrimidine is equipo-
tent with compound C in the in vitro AMPK assay and shows
improved kinase selectivity. Compound 5 also shows greater inhi-
bition against AMPK activity in a cellular assay relative to com-
pound C.
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Acknowledgements
This work was supported by the Grants CA132529 and CA73807
from the NIH, National Cancer Institute. We would like to thank Dr.
Kevin Moriarty and Dr. Martha Kelly for helpful discussions and
critical review of this Letter.
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