Synthesis and cancer stem cell-based activity of substituted 5-morpholino-7H-thieno[3,2-b]pyran-7-ones designed as next generation PI3K inhibitors

Article abstract of DOI:10.1021/jm301522m

Dysregulation of the phosphatidylinositol-3-kinase (PI3K) pathway in a wide range of tumors has made PI3K a consensus target to inhibit as illustrated by more than 15 inhibitors now in clinical trials. Our previous work, built on the early pioneering multikinase inhibitor LY294002, resulted in the only PI3K vascular-targeted PI3K inhibitor prodrug, SF1126, which has now completed Phase I clinical trials. This inhibitor has properties that impart more in vivo activity than should be warranted by its enzymatic potency, which in general is much lower than other clinical stage PI3K inhibitors. We embarked on the exploration of scaffolds that retained such properties while simultaneously exhibiting an increased potency toward PI3K. This work resulted in the discovery of the 5-morpholino-7H-thieno[3,2-b]pyran-7-one system as the foundation of a new compound class of potential PI3K inhibitors having improved potency toward PI3K. The synthesis and cancer stem cell-based activity of these compounds are reported herein.

Full text of DOI:10.1021/jm301522m

Article
pubs.acs.org/jmc
Synthesis and Cancer Stem Cell-Based Activity of Substituted
5‑Morpholino‑7H‑thieno[3,2‑b]pyran-7-ones Designed as Next
Generation PI3K Inhibitors
Guillermo A. Morales,*,†,§,⊥ Joseph R. Garlich,‡,⊥,§ Jingdong Su,§ Xiaodong Peng,§ Jessica Newblom,§
Kevin Weber,§ and Donald L. Durden*,∥,‡
†BIO5 Institute (Oro Valley), The University of Arizona, 1580 East Hanley Boulevard, Oro Valley, Arizona 85737, United States
‡SignalRx Pharmaceuticals, 12545 El Camino Real, Suite C, San Diego, California 92130, United States
§Semafore Pharmaceuticals Inc., 8496 Georgetown Road, Indianapolis, Indiana 46268, United States
∥Department of Pediatrics, UCSD Rady Children’s Hospital, Division of Pediatric Hematology−Oncology, Moores Cancer Center,
UC San Diego Health System, La Jolla, California 92123, United States
S
* Supporting Information
ABSTRACT: Dysregulation of the phosphatidylinositol-3-
kinase (PI3K) pathway in a wide range of tumors has made
PI3K a consensus target to inhibit as illustrated by more than
15 inhibitors now in clinical trials. Our previous work, built on
the early pioneering multikinase inhibitor LY294002, resulted
in the only PI3K vascular-targeted PI3K inhibitor prodrug,
SF1126, which has now completed Phase I clinical trials. This
inhibitor has properties that impart more in vivo activity than
should be warranted by its enzymatic potency, which in general
is much lower than other clinical stage PI3K inhibitors. We
embarked on the exploration of scaffolds that retained such
properties while simultaneously exhibiting an increased potency
toward PI3K. This work resulted in the discovery of the 5-
morpholino-7H-thieno[3,2-b]pyran-7-one system as the foundation of a new compound class of potential PI3K inhibitors having
improved potency toward PI3K. The synthesis and cancer stem cell-based activity of these compounds are reported herein.
treatments, so combinations of PI3K inhibitors with other
drugs are being investigated in early clinical trials.5 PI3K
inhibitors are also being investigated for the treatment of
inflammatory diseases.6
INTRODUCTION
■
Phosphatidylinositol 3-kinase (PI3K) is a family of intracellular
enzymes that transduce signals arising from cell surface
receptors such as G-protein-coupled receptors and tyrosine
kinase receptors to inside of the cell. There are several classes of
PI3K of which the class I PI3Ks has been the most studied and
consists of the α, β, γ, and δ isoforms.1 All of these enzymes
phosphorylate the 3-position hydroxyl group of the inositol ring
of phosphatidylinositol and in particular convert PIP2
(phosphatidylinositol(4,5)-bisphosphate) into PIP3
(phosphatidylinositol(3,4,5)-trisphosphate). This process is
opposed by the well-studied tumor suppressor PTEN, which
is a phosphatase that converts PIP3 back into PIP2. Together,
PI3K and PTEN are considered the “Yin and Yang” of
oncogenic signaling.2 The PIP3 that is produced by PI3K in
turn is a crucial activator of downstream signaling elements, of
which the most important and most studied is AKT. Because of
PI3K’s critical role in cell functions, it is a highly desirable target
to inhibit for treating cancer.3 This is best illustrated by more
than 15 different PI3K inhibitors reaching phase I clinical
trials.4 Additionally, the activation of PI3K by mutations or by
aberrant signaling decreases the effects of many anticancer
The natural product wortmannin was discovered as the first
PI3K inhibitor and was found to covalently bind to the catalytic
ATP pocket of PI3K and termed an irreversible inhibitor.7
There was considerable toxicity associated with wortmannin,
perhaps due to its irreversible binding, which may have tainted
this class of compounds and set back interest for several years.7
The natural product quercetin was also found to be a PI3K
inhibitor, but differed mechanistically from wortmannin in that
it was a competitive inhibitor by competing with ATP for the
kinase catalytic site of PI3K. Analogues of quercetin were
synthesized and led to the first generation of designed small
molecule PI3K inhibitors, of which LY294002 (1) (Figure 1)
was found to be the most potent. An SAR study revealed that
the phenyl group (8-position) and the morpholino group (2-
position) on the chromenone system were crucial for PI3K
Received: October 19, 2012
Published: February 14, 2013
© 2013 American Chemical Society
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The pan-PI3K inhibitor 1 has served as a research tool for
the past 22 years, generating an avalanche of studies cited in
over 6000 publications listed on PubMed. Compound 1 suffers
from poor oral availability and very fast metabolism.18
Compound 1 is a pan-class I isoform inhibitor of PI3K (α, β,
γ, δ) with moderate potency.19 It has also been shown to inhibit
related kinases important to cancer survival and progression
such as PIM-1, PLK-1, DNA-PK, and mTOR kinase.20−24
Compound 1 has shown properties independent of its PI3K
inhibition that may be unique among synthetic PI3K inhibitors
including inducing oxidative stress25 and inhibition of drug
efflux mechanisms.26 Despite the poor drug-like properties and
modest potency, 1 has demonstrated significant anticancer
activity in animal models at competitive dose levels.27,28 One
approach to building on these unique properties has been to
create prodrugs of 1. One such compound is SF1126, a vascular
targeted prodrug of 1, shown to have improved preclinical12
activity as well as demonstrated safety and signs of clinical
efficacy in phase I trials in patients with solid tumors29 and B-
cell malignancies.30
At the time we began the research described herein (early
2007), there were very limited examples of small molecule
PI3K inhibitors beyond 1. On the basis of the compelling
medical interest in PI3K inhibitors coupled with our extensive
experience with 1 and its prodrugs, we sought to create novel
small molecule analogues with greater potency and improved
drug-like properties while trying to maintain the desirable
spectrum of kinase inhibition and unique properties such as
induction of oxidative stress.
After a thorough analysis of the reported chemotypes
exhibiting PI3K affinity, it was noted that a direct thiophene
analogue of 1’s chromenone core had not been explored for
PI3K inhibition. The use of thiophene as a bioisostere of the
phenyl ring moiety is well documented, but because the phenyl
ring is a central part of the scaffold that fits within the catalytic
site, it was not known how the thiophene substitution might
affect the overall fit within the enzyme. Therefore, we pursued
the construction and exploration of the 5-morpholino-7H-
thieno[3,2-b]pyran-7-one (2) system as the foundation of a
new compound class of potential PI3K inhibitors as anticancer
agents focusing primarily on PI3K-α inhibition.
Figure 1. 2-Morpholino-4H-chromen-4-one (1) and 5-morpholino-
7H-thieno[3,2-b]pyran-7-one (2) scaffolds.
affinity.8 Six years later, PI3K-γ was cocrystallized with 1,
further revealing that (1) the oxygen of the morpholine group
forms a key hydrogen bond interaction with Val882 (hinge
region), (2) the phenyl group (8-position) resides at a location
where the ribose unit of ATP is accommodated, and (3) the
carbonyl group engages into a putative hydrogen-bond
interaction with Lys833.9 These findings led to the birth of
new PI3K-targeted scaffolds where the morpholine group was
used as a “warhead” to gain affinity toward PI3K’s ATP catalytic
pocket.10,11
The development of PI3K-targeted therapeutics faced two
paradigms, namely simultaneously inhibit all PI3K isoforms
(pan inhibition) or only one (isozyme selective). PI3K pan
inhibition is desirable based on the theory that if all PI3K
isoforms are blocked in cells wherein this pathway is
disregulated, such an approach will effectively block the ability
of tumor cells to survive, grow, and proliferate.12−14 A potential
concern this approach faces, however, is that this could lead to
unwanted toxicities. To address this potential concern, an
isozyme-selective PI3K inhibition approach has been proposed
as an alternate way to pharmacologically modulate the PI3K cell
signaling pathway.15 Genetic alterations of PIK3CA, the gene
for PI3K-α, in cells leads to PIK3CA mutations and
amplifications, where PI3K-α isoform selective inhibitors can
be of therapeutic use.16 For tumors that are PTEN-deficient, it
has been established that it is the PI3K-β isozyme that drives
growth and survival, and for such tumors, PI3K-β selective
inhibitors are expected to exhibit therapeutic benefits.17 While
PI3K-α and PI3K-β are more ubiquitously expressed, PI3K-δ is
primarily found in leukocytes, where its overexpression is
generally exhibited in lymphoma cancers and B-cell malig-
nancies affecting the immune system. Just like PI3K-δ, the
PI3K-γ isoform is also found mainly in leukocytes with a limited
tumorigenic role and a more prominent role in inflammatory
diseases.15
a
Scheme 1. Synthesis of the 5-Morpholino-7H-thieno[3,2-b]pyran-7-one Series 8
a
Reagents and conditions: (a) LDA, THF, 0 °C → RT; (b) Tf2O, DCM, RT; (c) Pd(PPh3)4 or PdCl2(dppf), Cs2CO3, DME, MW, 130 or 180 °C,
15 min.
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SYNTHESIS
Scheme 3. Selective Thiophene Carbon−Carbon Couplings
■
a
between 6 and Iodoaromatics
To validate scaffold 2, we pursued the synthesis of the phenyl-
containing thienopyranone 26 to compare it with its
chromenone counterpart 1.
The synthesis of the thienopyranone scaffold began with the
alkylation of methyl 4-bromo-3-hydroxythiophene-2-carboxy-
late 3 with N-acetylmorpholine 4 using LDA in THF to afford
1-(4-bromo-3-hydroxythiophen-2-yl)-3-morpholinopropane-
1,3-dione 5 which, upon treatment with triflic anhydride,
underwent intramolecular cyclization−dehydration to afford
the key intermediate 3-bromo-5-morpholino-7H-thieno[3,2-
b]pyran-7-one 6. With intermediate 6 in hand, we now have
the new thienopyranone core with a bromo atom strategically
placed to allow the introduction of a wide range of
functionalities and substituents via halogen replacement
reactions.
a
Reagents and conditions: (a) PdCl2(dppf), Cs2CO3, DME, MW, 140
°C, 15 min; (b) PdCl2(PPh3)2, AgNO3, KF, DMSO, MW, 120 °C, 15
The first thienopyranone derivatization round began with the
incorporation of diverse aromatic and heteroaromatic function-
alities via microwave-assisted Suzuki carbon−carbon coupling
conditions using commercially available aromatic and hetero-
aromatic boronic acids/esters 7 in the presence of either
Pd(PPh3)4 or PdCl2(dppf) in Cs2CO3/DME at 130 or 180 °C
to produce the desired thienopyranone analogues 8 (Scheme
1).
min.
in diisopropylamine at 100 °C under microwave irradiation
conditions (Scheme 4).
Scheme 4. Sonogashira Carbon−Carbon Couplings between
6 and Alkynes
a
To expand the scope of the Suzuki coupling reaction and
avoid being limited by the commercial availability of boronic
acids/esters, we reversed the role of intermediate 6 (electro-
phile) by converting it to its corresponding boronic acid
analogue 9 (nucleophile). In this case, 6 was borylated by
reacting it with pinacol borane in the presence of NiCl2(dppf)/
dppp and 1,3-bis(diphenylphosphino)propane at 160 °C in
dioxane under microwave irradiation conditions. Further
treatment with 1 M HCl afforded the desired boronic acid 9
(Scheme 2).
a
Reagents and conditions: (a) PdCl2(PPh3)2, CuI, i-Pr2NH, MW, 100
°C, 20 min.
Another strategy used to create diverse analogues for SAR
elucidation was the development of microwave-assisted carbon-
ylation reactions for the one-pot synthesis of carboxylic acids,
carboxylic esters, and amides. Using Mo(CO)6 as a carbonyl
source, the bromo group of 6 and the chloro group in
introduced substituents were successfully replaced via a
carbonyl-insertion reaction where the use of water, an alcohol,
or an amine led to the formation of carboxylic acids, esters, or
amides, respectively (Scheme 5). Interestingly, we were able to
synthesize aromatic ketones via the Heck reaction of aromatic
chlorides with butyl vinyl ether in the presence of PdCl2(dppf)
using DIPEA and DMF at 180 °C under microwave irradiation
conditions (Scheme 5).32−34
Scheme 2. Synthesis of Boronic Acid 9 for Reverse Suzuki
Couplings
a
a
Reagents and conditions: (a) (i) NiCl2(dppf), dppp, 1,3-bis-
(diphenylphosphino)propane, pinacol borane, diisopropylethylamine,
dioxane, MW, 160 °C, 15 min; (ii) aq HCl (1 M), 30 min, RT; (b)
Ar−Br, Pd(PPh3)4, Cs2CO3, DME, MW, 140 °C, 15 min.
Aromatic aminations were successfully carried out via direct
amine-halogen replacement (Br-thiophene, Cl-phenyl) using
cyclic secondary amines, where in the case of 6, ethyl 2-
oxocyclohexanecarboxylate was used in the presence of CuI and
Cs2CO3 in piperidine at 150 °C under microwave irradiation
conditions, and in the case of chlorophenyl analogues,
tBuO−Na+ was used in the presence of Pd2(dba)3 and XPhos
in DME at 150 °C also under microwave irradiation conditions
(Scheme 6).
In addition to the Suzuki reaction, we were able to directly
carry out aryl−aryl couplings using 6 and iodoaromatics in one
pot using bis(pinacolato)diboron in the presence of
PdCl2(dppf) in Cs2CO3/DME at 140 °C under microwave
irradiation conditions. Moreover, we were able to selectively
activate the H-thiophene site carrying out a carbon−carbon
(aromatic−thiophene) coupling reaction while leaving the
bromo group intact. This was achieved by reacting 6 with
iodobenze using PdCl2(PPh3)2 in combination with AgNO3
and KF in DMSO at 120 °C under microwave irradiation
conditions, affording 12 (Scheme 3).31
Acylations of aniline-containing thienopyranones were
carried out using acyl chlorides in pyridine at room temperature
(Scheme 7).
Another carbon−carbon coupling strategy used was the
Sonogashira reaction where bromo intermediate 6 was reacted
with aromatic alkynes in the presence of PdCl2(PPh3)2 and CuI
For SAR purposes, we wanted to explore the importance of
the carbonyl group of the thienopyranone and chromenone
systems. Transforming the carbonyl group into a thiocarbonyl
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a
Scheme 5. Synthesis of Carboxylic Acids, Esters, Amides,
And Ketones via Carbonyl Insertion and Heck Reactions
Scheme 8. Thienopyranthione Synthesis
a
a
Reagents and conditions: (a) For Z = O, NR1: palladacycle,
−
Mo(CO)6, HO-R or HNR1R2, (tBu)PH+BF4 , DBU and/or imidazole,
MW, THF (125 °C, 6 min or 175 °C, 15 min) or DMSO (125 °C, 6
min). For Z = CH2, R = H: (i) butyl vinyl ether, PdCl2(dppf), DIPEA,
DMF, MW, 180 °C, 6 min; (ii) aq HCl.
a
Reagents and conditions: (a) Lawesson’s reagent, toluene, MW, 130
°C, 20 min.
a
Scheme 6. Aromatic Thienopyranone Amination Reactions
afford phenol-diketo intermediate 24, which upon treatment
with CuCl2 in EtOH at 140 °C under microwave irradiation
conditions afforded the desried tetrahydro-2H-pyran-4-yl
analogue 25 (Scheme 9).
RESULTS AND DISCUSSION
■
The initial substitution of the thiophene for the phenyl core in
1 resulted in compound 26. To our surprise, we found that
compound 26 not only exhibited a similar or better PI3K and
mTOR enzymatic inhibition profile than 1, but it was also
slightly more water soluble, cell permeable, and potent when
tested in a cell proliferation assay using PC3 cells while
downregulating the PI3K pathway as illustrated by inhibiting
pAKT-S473 levels (Table 1). While compound 26 exhibited
slightly increased inhibition against PI3K-α and PI3K-γ in
comparison with 1, for the PI3K-β and PI3K-δ isoforms, this
compound has an increased potency of approximately 2-fold
and 4-fold, respectively. Additionally, mTOR is a clinically
validated target in cancer treatments and 26 demonstrated
almost 2-fold improvement in inhibiting mTOR kinase activity
versus 1. Evaluation of the ability of 26 to block the PI3K
pathway and decrease the pAKT activation in PC3 cells by 50%
versus 1 demonstrated over a 2-fold improvement in activity.
Lastly, as shown in Table 1, compound 26 showed an almost 2-
fold improvement versus 1 in the inhibition of prostate cancer
cell proliferation (PC3 cell line).
a
Reagents and conditions: (a) ethyl 2-oxocyclohexanecarboxylate,
CuI, Cs2CO3, piperidine, MW, 150 °C, 15 min; (b) tBuO−Na+,
Pd2(dba)3, morpholine, DME, MW, 150 °C, 15 min.
Scheme 7. Acylation of Aniline-Containing
Thienopyranones
a
With the above results providing the validation of the
thienopyranone core as a viable chemotype for PI3K inhibition,
we synthesized 19 diverse analogues (26−44) to obtain an
early SAR profile on the new scaffold. As we focused our efforts
toward PI3K-α selective inhibition, we pursued a structure-
based design approach, developing a predictive in silico
molecular modeling model for PI3K-α. It has been established
that the PI3K-α and PI3K-γ isozymes display a significant
protein sequence homology (∼35%) and kinase domain
homology (∼44%).35 To accomplish this, we started with the
available crystal structure of PI3K-γ cocrystallized with 1 (PDB
code: 1E7V), used MOE (Computational Chemistry Group
Inc.) to superimpose the human PI3K-α protein sequence from
the Swiss Prot protein sequence database (http://www.expasy.
ch/sprot) (UniProtKB/Swiss-Prot entry no. P42336, PK3CA_-
HUMAN) over the 3D coordinates of PI3K-γ removing both
a
Reagents and conditions: (a) acyl chloride, pyridine, 0 °C → RT.
group was accomplishing by treating compounds 1and 43 with
Lawesson’s reagent in toluene at 130 °C under microwave
irradiation conditions, affording the desired thienopyranthione
and chromenthione derivatives (Scheme 8).
To assess the influence of the nitrogen atom in the
morpholine group, a tetrahydro-2H-pyran-4-yl group was
incorporated in a direct analogue of 1. For this biphenylketone,
22 was used to alkylate pyran-containing methyl ester 23 to
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a
Scheme 9. Tetrahydro-2H-pyran-4-yl Analogue of 1
a
Reagents and conditions: (a) LDA, THF, 0 °C → RT; (b) CuCl2, EtOH, MW, 140 °C, 15 min.
We selected a 3D grid with a 10 Å radius at the ATP binding
site and proceeded to dock 1 and the 19 synthesized
thienopyranone analogues (26−44) using two of the previously
established pharmacophoric constrains, namely the H-bond
with Val851, and the spatial location of the carbonyl group. Out
of all the docking engines we tested, we found that only FlexX
(BioSolveIT GmbH) could accurately dock all the thienopyr-
anone compounds where the FlexX docking scores for the
docked compounds also correlated with approximately 80%
accuracy the thienopyranone analogues that exhibited PI3K-α
enzymatic selectivity.
Table 1. Comparison of IC50 Values (nM) and Cellular
Activity for 26 versus 1
fold
improvement
a
category
enzyme
1
26
PI3K class 1A
PI3K class 1A
PI3K class 1A
PI3K class 1B
p110-α
p110-β
p110-δ
p110-γ
mTOR
356
297
378
784
1570
610
1.2
1.9
4.1
1.1
1.7
736
3224
1775
1060
PI3K super family
enzyme
pathway inhibition
IC50 (μM)
pAKT
1.3
0.55
2.4
cell proliferation
IC50 (nM)
PC3
cells
7500−12100
4100
1.8−3.0
As shown in Figures 2 and 3, like in the case of 1, new
fragments introduced at the 3 position of the thienopyranone
skeleton (a) are placed in the same space in PI3K that the
phenyl group of 1 occupies, (b) are oriented and projected
toward solvent exposure, and (c) can potentially engage in
additional H-bond interactions with amino acid residues
located in the outer region of PI3K’s ATP catalytic site and
gain PI3K binding affinity. As it is shown in Table 2, the lack of
substituents at the 3 position of the thienopyranone skeleton
(33) as well as changing the phenyl group for nonaromatic
cyclic substituents (17, 18) (data not shown) resulted in
virtually a complete loss of affinity toward all PI3K isozymes.
a
Value for 1 divided by the value obtained for 26.
water molecules and 1 to create a PI3K-α homology model. In
our work, we use the PI3K-α numbering when referring to
amino acid residues (e.g., Val882 (PI3K-γ) → Val851 (PI3K-
α), Lys833 (PI3K-γ) → Lys802 (PI3K-α)). It is worth noting
that as we focused on the ATP catalytic site for in silico
docking, many of the residues differ between these two
isoforms (e.g., PI3K-γ → PI3K-α: Thr866 → His855, Ile881 →
Val850, Lys802 → Arg770, Ala805 → Ser773).36
Figure 2. PI3K-α homology docking model (derived from PDB code 1E7V) showing 1and 26 superimposed.
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To assess the impact of inserting aliphatic groups between
the phenyl group and the thienopyrane core in 26, two
analogues containing a para-methoxyphenyl group with a
methylene (45) and alkynyl (92) linker were made and found
that, in the case of 45, a 3-fold loss against PI3K-δ was observed
while exhibiting almost a 2-fold increase in potency against
PI3K-α and PI3K-β, and in the case of 92, activity against PIK3-
αα was practically the same, with a dramatic loss against PI3K-δ
affinity (note: PI3K-β data not available).
Electron withdrawing groups were introduced at the para
position of the phenyl ring in 26, whereupon it was observed
that inclusion of a nitrile group (46) was detrimental for PI3K-
α affinity (IC50 = 4.6 μM) followed by a trifluoromethyl group
(49) (IC50 = 551 nM). However, in the case of Cl (47) and
carboxylic acid (69), the affinity toward PI3K-α increased (IC50
= 112 and 80 nM, respectively).
Figure 3. 2D depiction of 26 at the PI3K-α active site (derived from
PDB code 1E7V).
The PI3K-α model suggests that when 26 is accommodated
at the ATP binding site, substituents at the 3 and 4 positions on
the phenyl ring can be tolerated, particularly position 4, as
substituents are projected toward solvent exposure. To probe
the extended space from the ATP pocket while searching for
additional interactions, we synthesized 3- and 4-substituted
amide analogues containing a pyridyl moiety and found that all
analogues (110, 111, 113, 115, 117) exhibited higher potency
against PI3K-α compared to 26 where the trend was that the 3-
subsituted amides (115, 117) were more potent than the 4-
substituted ones (110, 111, 113).
We then proceeded to explore the PI3K inhibition profile of
several reverse-amide analogues and discovered that while the
3-substituted reverse amide analogues (124, 125) suffered a
great loss of PI3K-α affinity (IC50 = 24 μM and 14 μM,
respectively), the 4-substituted reverse amide analogues (119,
126) were quite active, with compound 126 being the most
potent PI3K-α analogue made in this series (IC50 = 14 nM). A
molecular docking analysis of compound 126 indicates that, in
addition to the required H-bond interaction with Val851, the
phenyl group engages in four hydrophobic interactions with
Met772, Trp780, Ile800, and Met992, while the pyridyl group
forms two hydrophobic π-stacking interactions with Trp780
and His855 (Figure 5). We rationalize that the high PI3K-α
affinity exhibited by 126 can be attributed to the combination
of all these interactions at the catalytic site.
To investigate the impact of the carbonyl group of the
chromenone scaffold, 1 was treated with Lawesson’s reagent
under microwave irradiation conditions and converted into the
corresponding thione 20 and found to lose PI3K affinity against
all isoforms (Figure 6). This observation was confirmed with
the synthesis of 21, which also exhibited loss of PI3K enzymatic
affinity (data not shown).
Moreover, to also assess the role of the amino group within
the morpholino moiety in 1, compound 25 was made where a
tetrahydro pyranyl group was present. This change was found
to be detrimental for PI3K-α activity, as only a 44% inhibition
at 10 μM was detected.
Replacing the phenyl group with furan and thiophene groups,
the latter a common phenyl isostere, gave analogues with
opposite activity profiles. When the phenyl group was replaced
with 2-furan (29), this compound exhibited almost a 3-fold
improved affinity for PI3K-β while losing affinity toward the
other 3 PI3K isozymes, particularly against PI3K-δ. On the
other hand, when 3-furan (42) was introduced, activity against
all PI3K isoforms was greatly reduced. An opposite trend was
observed in the case of the introduction of the thiophene
group, as when the phenyl group was replaced with 2-
thiophene (37), loss in activity for all PI3K isoforms was
observed. However, when 3-thiophene (41) was introduced, an
unexpected 3-fold activity and selectivity toward PI3K-α was
observed.
When 3-pyridyl (27) and 4-pyridyl (28) were used instead of
the phenyl group, the former analogue retained similar PI3K-α
affinity like 26 while the latter analogue exhibited a 3-fold
affinity toward PI3K-α, yet both still exhibit PI3K-α selectivity.
Interestingly, when 2-indolyl (35) was used to replace the
phenyl ring of 26, an almost 4-fold improvement in PI3K-α
affinity was found, although a 5-fold affinity increase toward
PI3K-γ was also observed. On the other hand, when the indolyl
moiety was connected to main scaffold at indolyl’s 5 position
(40), pan-PI3K inhibition was achieved (161−667 nM range)
with slight preference for PI3K-α and PI3K-β.
During our early SAR assessment, a common trend was
found where analogues containing electron donating groups on
the phenyl ring of 26 (with the exception of 3-amino analogue
34) all exhibited higher affinity toward all 4 PI3K isozymes and,
particularly, PI3K-α selectivity (compounds 30, 31, 32, 34, 38,
39, 43, 44). Of particular note was compound 43, as it was the
first compound of the series to exhibit selective PI3K-α
inhibition (IC50 = 34 nM) along with some of the most potent
inhibition of the other isoforms, defining it as a much more
potent pan-PI3K inhibitor than 1. The PI3K-α computational
docking model indicates that 43 is expected to engage an
additional H-bond interaction with Thr856 which explains the
high potency of this analogue (Figure 4).
One unforeseen discovery was when the 4-N-methylamino
analogue 55 was tested and compared against its 4-N,N-
dimethylamino relative 32, the latter dimethylated aniline
analogue exhibited PI3K-α selectivity with an IC50 value of 95
nM, while the former monomethylated analogue exhibited both
PI3K-α and PI3K-δ selectivity (IC50 = 56 and 26 nM,
respectively).
Although we focused our efforts on PI3K-α inhibition, this
compound series afforded three PI3K-δ selective analogues,
namely 51, 57, and 58 (see Table 3). The most clinically
advanced δ-isoform selective PI3K inhibitor CAL-101 (128)
(recently renamed GS-1101)37 is included in Table 3 for
comparison. A comparison of δ selectivity is shown in Table 4
and demonstrates better selectivity against the γ isoform than
128 for all three compounds but less selectivity against α and β
isoforms.
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Table 2. Pan-PI3K Inhibition Profile of 5-Morpholino-7H-thieno[3,2-b]pyran-7-one Analogues (IC50 Values in nM)
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Table 2. continued
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Table 2. continued
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Table 2. continued
Figure 4. PI3K-α homology docking model (derived from PDB code 1E7V) of compound 43.
Although it is not clear why these analogues exhibit such
great affinity toward PI3K-δ, it should not be a surprise that 51
and 57 exhibit similar PI3K inhibition profile as they only differ
from each other by one methylene unit on the ester
component. On the other hand, compound 58 is structurally
distinct, having a pyrimidine group instead of the phenyl group
as well as having a N-methyl-piperazine moiety whose sp3-based
N-methyl group is prone to act as a hydrogen-bond acceptor
potentially, establishing a yet-to-be-determined additional key
interaction resulting in the observed acute PI3K-δ affinity.
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Figure 5. Binding mode of 126 docked in the catalytic site of the PI3K-α homology model (derived from PDB code 1E7V). Coloring of surface area
based on formal charges (blue = positive, red = negative, white = neutral).
Figure 6. PI3K profile of 1 and its thione analogue 20.
Biochemical Analysis of Potency of Compounds. To
ascertain the relative potency of the different thienopyranone
derivatives in cell-based assays, we turned to several PI3K
activated human tumor cell lines, PC3, H1299, and 786−0.
The inhibition of the PI3K pathway by compounds 43 (pan-
PI3K inhibitor) and 51 (δ-PI3K selective) was demonstrated in
PC3 (prostate cancer) cells by the decreased phosphorylation
of the key pathway element AKT (pAKT) as shown in Table 5.
These data show potent pathway inhibition for both
compounds at basal levels with no stimulation (PBS) or with
the growth factor stimulation by VEGF or Bv8 (Bombina
variegata peptide 8). Bv8 stimulation of angiogenesis is thought
to be a resistance mechanism that bypasses VEGF and thus
induce tumors to be refractory to anti-VEGF therapies.38,39
Thus, inhibition of angiogenic stimulation via this mechanism
(blocking Bv8 signaling through AKT) might represent a new
approach to inhibit VEGF-resistant angiogenesis and is
accomplished by both the pan and δ selective inhibition of
PI3K. Also, potent inhibition of AKT activation under insulin-
like growth factor (IGF) stimulation conditions was noted. In
all cases, the pan-inhibitor 43 showed more potency than the δ-
selective compound 51. Inhibition of cellular proliferation by
the pan-PI3K inhibitors 32 and 43 versus 1 is shown in Table 6.
These results demonstrate the dramatic increase in potency
across prostate (PC3), lung (H1299), renal cell (786-O), and
breast cancer (BT-474) cell lines for compounds with the new
thienopyranone scaffold versus 1.
Cancer Stem Cell Inhibitory Activity. Considerable
evidence implicates PTEN, PI3K, and the GSK3β/β-catenin
pathway in the control of breast cancer stem cell proliferation
and expansion.40,41 Hence, we tested our PI3K inhibitors for
the capacity to block a well-studied putative cancer stem cell
phenotype, adhesion independent growth of mammospheres in
MCF7 cells. The evaluation of two pan-PI3K inhibiting
compounds (43 and 119) for their ability to inhibit breast
cancer mammosphere formation is shown in Figure 7. SF1126,
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Table 3. PI3K-δ Selective Inhibitors (IC50 Values in nM) Including 128 (CAL-101)
the clinical stage prodrug of 1, was included and found to be
inferior to the new thienopyranone compounds in the
inhibition of mammosphere related growth in vitro. The
approved anticancer kinase inhibitor lapatinib was also included
as an internal control. We evaluated each compound at 1 and
10 μM concentrations. These results show that the new
compounds are statistically more potent toward cancer stem
cells than lapatinib, the EGFR (ErbB2 and ErbB1) kinase
inhibitor shown clinically to decrease breast cancer stem cells in
biopsies.42 The treatment of MCF7 mammosphere cultures
with different inhibitors in vitro resulted in the following
results: SF1126 (10 μM), 36% of control; Lapatinib (1 μM),
67% of control; compd 43 (10 μM), 12.4% of control; compd
43 (1 μM), 39.9% of control; compd 119 (10 μM), 13.7% of
control, and compound 119 (1 μM), 41.2% of control. The
thienopyranone compounds 43 and 119 were noted to be 2-
fold more potent compared to lapatinib in blocking mammo-
sphere formation at equimolar concentrations in vitro.
In summary, we have successfully identified and made a
thiophene isostere of the well-known chromenone system
found in 1. This new 5-morpholino-7H-thieno[3,2-b]pyran-7-
one compound series afforded analogues with PI3K-α
selectivity as predicted by our PI3K-α homology model.
Additionally, in some cases, analogues displayed an unexpected
affinity toward other PI3K isozymes, such as compounds 51,
57, and 58 (PI3K-δ selectivity). Cell-based assays demonstrated
that these compounds potently inhibit the downstream target
of PI3K, pAKT, confirming their inhibitory modulation of the
PI3K cell signaling cascade. Also, cell-based assays demon-
Table 4. Comparison of New Compounds with 128 for δ
Selectivity
a
isoform selectivity multiples of PI3K-δ versus
compd
PI3K-α
PI3K-β
PI3K-γ
51
26
33
65
300
24
43
45
60
36
57
58
19
128
328
225
a
Divide PI3K-isoform IC50 by the IC50 for PI3K-δ isoform
Table 5. Concentrations (μM) of Compounds 43 and 51
Required to Inhibit pAKT to Half-Maximal Levels
Stimulated by Bv8, VEGF, and IGF in PC3 (Prostate
Cancer) Cell Line as Described in the Experimental Section
stimulant
43
51
PBS
Bv8
0.229
0.564
0.1164
0.1523
0.888
0.9406
0.3317
2.117
VEGF
IGF
Table 6. Cancer Cell Proliferation IC50 (μM)
PC3
H1299
RCC 786-O
BT-474
1
12.1
2.0
11.1
3.0
18.1
7.2
20.7
4.3
32
43
1.6
0.9
3.3
3.8
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Figure 7. Breast cancer mammosphere assay: The number of mammospheres formed from MCF7 cells plated in treated 96-well plates is plotted on
Y-axis versus treatment on X-axis. As indicated by the horizontal bar, all treated groups were significantly decreased relative to control untreated cells
p < 0.001. Other statistical comparisons included: SF1126 (10 μM) to compound 43 (10 μM), p = 3.3 × 10−5; compound 43 (1 μM) to Lapatanib
(1 uM), p = 0.00022; compound 43 (1 μM) to SF1126 (10 μM), p = 0.19948; compound 43 (10 μM) to Lapatinib (1 μM), p = 8.10 × 10−7.
Figure 8. Kinase selectivity panel of 43 (232 kinases offered and screened by Reaction Biology Corp). Colors indicate % activity remaining testing at
10 μM going from complete inhibition (red) to no inhibition (blue).
strated potent cell growth inhibitory activity for these novel
compounds. Moreover, we present evidence that PI3K
inhibitors can potently inhibit MCF7 cancer stem cell
proliferation in an in vitro model. In other studies, we have
confirmed the kinome specificity of compound 43 in a panel of
232 kinases (single dose at 10 μM), finding that only three
other kinases are almost completely or completed inhibited:
mTOR (IC50 = 280 nM), PIM-1 (IC50 = 43 nM), and DNA-PK
(IC50 = 9 nM) (Figure 8, red). Incidentally, besides PIM-1, the
kinase profile of 43 is similar to Novartis’ clinical stage PI3K
inhibitor BEZ235, which also inhibits PI3K, mTOR, and DNA-
PK.43,44
Additionally, we have demonstrated favorable PK and in vivo
efficacy of compound 43 in mouse xenograft tumor models and
performed initial PK determinations (results to be published
elsewhere).45,46 The exploration of this new compound series is
under development to optimize the current hits for PI3K pan
and isoform-selective inhibition and in vivo drug like activity.
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yl-1,3,2-dioxaborolane (1.10 mL, 7.59 mmol). The reaction mixture
was magnetically stirred and heated via microwave irradiation for 15
min at 160 °C. Upon cooling to room temperature, the reaction was
transferred to a separatory funnel, diluted with dichloromethane (100
mL), and washed three times with saturated aqueous ammonium
chloride solution (100 mL). The dichloromethane layer was dried over
magnesium sulfate, filtered, and concentrated in vacuo. The resulting
brown foam was stirred in aqueous hydrochloric acid (1.0M, 100 mL)
for 30 min, frozen, and lyophilized to give 5-morpholino-7-oxo-7H-
thieno[3,2-b]pyran-3-ylboronic acid (9) (1.51 g, 4.15 mmol) as a
brown solid. MS (ESI): m/z (M + H)+ 283.
2-Morpholinoethyl 4-(5-morpholino-7-oxo-7H-thieno[3,2-
b]pyran-3-yl)benzoate (11). A 2 mL microwave vial was charged
with a magnetic stirring bar, compound 10 (90 mg, 249 μmol),
compound 6 (79 mg, 249 μmol), bis(pinacolato)diboron (70 mg, 274
μmol), cesium carbonate (325 mg, 997 μmol), dichloro[1,1′-
bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane ad-
duct (6 mg, 7 μmol), and 1,2-dimethoxyethane (1 mL). The reaction
mixture was sealed, and the reaction mixture was magnetically stirred
and heated under microwave irradiation at 140 °C for 15 min to give
desired product 11. MS (ESI): m/z (M + H)+ 471.
3-Bromo-5-morpholino-2-phenyl-7H-thieno[3,2-b]pyran-7-
one (12). A 2 mL conical microwave vial was charged with a magnetic
stirring bar, compound 6 (50 mg, 158 μmol), phenyl iodide (27 mg,
132 μmol), potassium fluoride (11.5 mg, 198 μmol), silver nitrate (34
mg, 198 μmol), anhydrous dimethyl sulfoxide (1 mL), and
dichlorobis(triphenylphosphine)palladium(II) (4.6 mg, 6.6 μmol).
The reaction mixture was sealed, and the reaction mixture was
magnetically stirred and heated under microwave irradiation at 120 °C
for 15 min to give desired product 12. MS (ESI): m/z (M + H)+ 392.
5-Morpholino-3-(piperidin-1-yl)-7H-thieno[3,2-b]pyran-7-
one (17). A 0.5 mL microwave vial was charged with a magnetic
stirring bar, compound 6 (50 mg, 158 μmol), cesium carbonate (103
mg, 316 μmol), copper(I) iodide (3 mg, 31 μmol), ethyl 2-
oxocyclohexanecarboxylate (10 μL, 63 μmol), and piperidine (200
μL). The reaction mixture was sealed, and the reaction mixture was
magnetically stirred and heated via microwave irradiation at 150 °C for
15 min to give desired product 17. MS (ESI): m/z (M + H)+ 321.
5-Morpholino-3-(4-morpholinophenyl)-7H-thieno[3,2-b]-
pyran-7-one (18). A 2 mL conical microwave vial was charged with a
magnetic stirring bar, compound 42 (80 mg, 230 μmol), sodium tert-
butoxide (31 mg, 322 μmol), tris(dibenzylideneacetone)-
dipalladium(0) (3 mg, 3.5 μmol), 2-dicyclohexylphosphino-2′,4′,6′-
triisopropylbiphenyl (8.8 mg, 21 μmol), dimethoxyethane (0.5 mL),
and morpholine (24 μL, 276 μmol). The reaction mixture was sealed,
and the reaction mixture was magnetically stirred and heated via
microwave irradiation at 150 °C for 15 min to give desired product 18.
MS (ESI): m/z (M + H)+ 399.
2-Morpholino-8-phenyl-4H-chromene-4-thione (20). A 2 mL
conical microwave vial was charged with a magnetic stirring bar, 2-
morpholino-8-phenyl-4H-chromen-4-one (1) (150 mg, 488 μmol),
Lawesson’s reagent (118 mg, 293 μmol), and toluene (2 mL). The
reaction mixture was sealed, and the reaction mixture was magnetically
stirred and heated via microwave irradiation at 130 °C for 20 min. The
final mixture was poured onto water (approximately 30 mL), extracted
with dichloromethane (3 × 5 mL), the combined extracts dried over
anhydrous magnesium sulfate, filtered, and concentrated to dryness.
Purification via column chromatography (silica gel; hexanes/ethyl
acetate (1:1), then 100% ethyl acetate) afforded pure thione 20 (128
mg, 81% yield). 1H NMR (DMSO-d6): 8.50 (dd, J = 8.0, 1.7 Hz, 1H),
7.69−7.45 (m, 7H), 6.90 (s, 1H), 3.67 (t, J = 4.9 Hz, 4H), 3.50 (t, J =
4.9 Hz, 4H). MS (ESI): m/z (M + H)+ 324.
EXPERIMENTAL SECTION
■
1
Chemistry. All solvents and reagents were used as obtained. H
NMR spectra were recorded on a JEOL ECX 400 NMR spectrometer
and referenced to tetramethylsilane. Chemical shifts are expressed as δ
units using tetramethylsilane as internal standard (in NMR
description: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broad signal; J, joules; Hz, hertz). Microwave irradiation synthesis
was carried out using a Biotage Initiator 60 microwave synthesizer.
Analytical data: UV absorbance (254 and 214 nm), evaporative light
scattering (Alltech ELSD 2000), and mass spectra were measured with
a Shimadzu LCMS-2010 spectrometer using an ESI source coupled to
a Shimadzu HPLC-2010 system operating in reverse mode with a
YMC CombiScreen ODS-AQ column with a particle size of 5 μm, a
pore size of 12 nm, 50 mm long with a 4.6 mm I.D., using solvent A
(water with 0.1% TFA), solvent B (acetonitrile with 0.1% TFA), and a
flow rate of 3 mL/min starting with a mixture of 95% A and 5% B.
Solvent B is linearly ramped to 95% at 5.0 min, held at 95% until 6.0
min, then linearly ramped back down to 5% at 6.5 min. Compound
purification: Pure desired products were isolated from crude mixtures
using the Shimadzu HPLC-MS 2010 system in preparative mode. The
preparative column is a YMC CombiPrep ODS-AQ, with a particle
size of 5 μm and a pore size of 12 nm, 50 mm long and 20 mm I.D. A
flow rate of 25 mL/min starting with a mixture of 95% A and 5% B.
Solvent B is linearly ramped to 95% at 25.0 min, held at 95% until 30.0
min, then linearly ramped back down to 5% at 2 min. An MRA
(Rheodyne mass rate attenuator) splitter was employed to split a small
part of the eluant stream, typically 0.1%, to the detectors, while the rest
was sent to the Gilson 215 liquid handler. A Shimadzu LC-10ADvp
pump was used to pump 3 mL/min of methanol through the MRA to
deliver the sample to the UV−vis, ELS, and MS detectors. The system
was run in mass-triggered mode to collect fractions containing desired
product based on molecular mass. Purity of all biologically evaluated
compounds was greater than 95%.
1-(4-Bromo-3-hydroxythiophen-2-yl)-3-morpholinopro-
pane-1,3-dione (5). An oven-dried 100 mL round-bottom flask was
charged with a magnetic stirring bar, tetrahydrofuran (5 mL), and
lithium diisopropylamide (2.0 M in hexanes, 6.7 mL, 13.5 mmol). The
reaction mixture was magnetically stirred and cooled to 0 °C under
argon gas. A solution of N-acetylmorpholine (6.75 mmol) in
tetrahydrofuran (1 mL) was added, and the reaction was stirred at 0
°C for 1 h. A solution of methyl 4-bromo-3-hydroxythiophene-2-
carboxylate (4.22 mmol) in tetrahydrofuran (2 mL) was added
dropwise over 5 min, and the reaction was allowed to warm to room
temperature overnight. The reaction was quenched by adding aqueous
10% HCl (20 mL). The resulting solution was transferred to a
separatory funnel, diluted with water (80 mL), and extracted three
times with dichloromethane (100 mL). The organic layers were
combined, dried over magnesium sulfate, filtered, and concentrated in
vacuo to give desired product as a brown solid, and it was used without
further purification.
3-Bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (6). An
oven-dried 100 mL round-bottom flask was charged with a magnetic
stirring bar, 5 (1.95g, 5.83 mmol) and dissolved in dichloromethane
(30 mL) under magnetic stirring. Trifluoromethanesulfonic anhydride
(2.45 mL, 14.6 mmol) was added portionwise over 2 min, and the
reaction was stirred at room temperature. After stirring overnight, the
reaction was concentrated in vacuo and redissolved in methanol (10
mL). After stirring for 4 h, the reaction was concentrated in vacuo and
diluted with aqueous 5% sodium bicarbonate solution (100 mL). The
reaction was transferred to a separatory funnel and extracted three
times with dichloromethane (100 mL). The organic layers were
combined, dried over magnesium sulfate, filtered, and concentrated in
vacuo to afford 6 as a brown solid (1.52g, 4.81 mmol, 82%). MS (ESI):
m/z (M + H)+ 316.
5-Morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-ylboronic
Acid (9). A 20 mL microwave vial was charged with a magnetic stirring
bar, compound 6 (1.20 g, 3.80 mmol), 1,3-bis(diphenylphosphino)-
propane (156 mg, 0.38 mmol), 1,3-bis(diphenylphosphino)propane
nickel(II) chloride (205 mg, 0.38 mmol), dioxane (8 mL),
diisopropylethylamine (1.98 mL, 11.39 mmol), and 4,4,5,5-tetrameth-
3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-5-morpholino-7H-
thieno[3,2-b]pyran-7-thione (21). Compound 21 was prepared
from compound 43 under the same reaction conditions described for
1
compound 20. H NMR (DMSO-d6): 8.07 (s, 1H), 7.22−7.19 (m,
2H), 6.98 (dd, J = 8.0, 0.8 Hz, 1H), 5.53 (s, 1H), 4.28 (s, 4H), 3.71 (t,
J = 5.0 Hz, 4H), 3.43 (t, J = 4.9 Hz, 4H). MS (ESI): m/z (M + H)+
388.
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1-(2-Hydroxybiphenyl-3-yl)-3-(tetrahydro-2H-pyran-4-yl)-
propane-1,3-dione (24). An oven-dried 100 mL round-bottom flask
was charged with a magnetic stirring bar, tetrahydrofuran (10 mL),
and lithium diisopropylamide (2.0 M in hexanes, 6.9 mL, 13.8 mmol).
The mixture was magnetically stirred and cooled to 0 °C under argon
gas. 1-(2-Hydroxybiphenyl-3-yl)ethanone (0.92 g, 4.34 mmol) was
added, and the reaction was stirred at 0 °C for 1 h. A solution of
methyl tetrahydro-2H-pyran-4-carboxylate (1.0 g, 6.94 mmol) in
tetrahydrofuran (2 mL) was added dropwise over 5 min, and the
reaction was allowed to warm to room temperature overnight. The
reaction was quenched by the addition of aqueous 10% hydrochloric
acid solution (20 mL) until pH = 3. The resulting solution was
transferred to a separatory funnel, diluted with water (80 mL), and
extracted three times with dichloromethane (100 mL). The organic
layers were combined, dried over magnesium sulfate, filtered, and
concentrated in vacuo to give crude 1-(2-hydroxybiphenyl-3-yl)-3-
(tetrahydro-2H-pyran-4-yl)propane-1,3-dione as a brown oil. The
crude product was used as is without further purification.
8-Phenyl-2-(tetrahydro-2H-pyran-4-yl)-4H-chromen-4-one
(25). A 5 mL microwave vial was charged with a magnetic stirring bar,
crude 24 (320 mg, 1 mmol), ethanol (3 mL), and copper(II) chloride
(13 mg, 0.1 mmol). The reaction vial was sealed, the reaction mixture
magnetically stirred and heated via microwave irradiation at 140 °C for
10 min. The resulting solution was concentrated in vacuo and purified
by high-pressure liquid chromatography to give desired product 25. 1H
NMR (CD3OD): 8.12 (dd, J = 8.1, 1.6 Hz, 1H), 7.78 (dd, J = 7.5, 1.6
Hz, 1H), 7.64−7.40 (m, 6H), 6.29 (s, 1H), 3.97 (dd, J = 11.4, 2.6 Hz,
2H), 3.48 (dt, J = 11.6, 2.3 Hz, 2H), 2.89 (tt, J = 11.5, 4.0 Hz, 1H),
1.89−1.63 (m, 4H). MS (ESI): m/z (M + H)+ 307.
5-Morpholino-3-phenyl-7H-thieno[3,2-b]pyran-7-one (26). A
2 mL conical microwave vial was charged with a magnetic stirring bar,
3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (6) (50 mg,
0.16 mmol), phenylboronic acid (29 mg, 0.24 mmol), cesium
carbonate (103 mg, 0.32 mmol), tetrakis(triphenylphosphine)-
palladium(0) (9 mg, 0.008 mmol), and dimethoxyethane (1 mL).
The reaction mixture was magnetically stirred and heated via
microwave irradiation for 15 min at 130 °C. Upon cooling to room
temperature, the reaction was concentrated in vacuo and purified using
high-pressure liquid chromatography to give 5-morpholino-3-phenyl-
7H-thieno[3,2-b]pyran-7-one (26) (10 mg, 3.2 μmol) as a white solid.
1H NMR (DMSO-d6): 8.18 (s, 1H), 7.72 (dd, J = 8.5, 1.3 Hz, 2H),
raphy to afford desired product (85). 1H NMR (DMSO-d6): 10.20 (s,
1H), 8.14 (t, J = 1.8 Hz, 1H), 7.55 (s, 1H), 5.52 (s, 1H), 3.96 (s, 3H),
3.88 (s, 4H), 3.45 (s, 4H). MS (ESI): m/z (M − H)− 392.
5-Morpholino-3-(1H-pyrrol-2-yl)-7H-thieno[3,2-b]pyran-7-
one (87). A 20 mL vial was charged with a magnetic stirring bar, Boc-
protected compound 65 (50 mg, 124 μmol), and hydrochloric acid
(4.0 M in dioxane, 10 mL). The reaction mixture was magnetically
stirred overnight at room temperature, concentrated in vacuo, and
purified via reverse phase high-pressure liquid chromatography to give
1
desired product 87. H NMR (CDCl3): 8.96 (s, 1H), 7.55 (s, 1H),
6.93 (s, 1H), 6.53 (s, 1H), 6.33 (s, 1H), 5.48 (s, 1H), 3.85 (s, 4H),
3.49 (s, 4H). MS (ESI): m/z (M + H)+ 303.
5-Morpholino-3-(phenylethynyl)-7H-thieno[3,2-b]pyran-7-
one (88). A microwave vial was charged with a magnetic stirring bar,
compound 6 (50 mg, 158.1 μmol), phenylacetylene (32.0 mg, 316.2
μmol), trans-dichlorobis(triphenylphosphine)palladium(II) (5.6 mg,
7.9 μmol), copper(I) iodide (1.6 mg, 7.9 μmol), and diisopropylamine
(1.0 mL). The mixture was magnetically stirred and heated via
microwave irradiation at 100 °C for 20 min. The mixture was cooled
to room temperature and then concentrated in vacuo, resulting in a
brown solid. The solid was dissolved in methanol (1.0 mL) and loaded
onto an Isolute functionalized silica column (PE-AX/SCX-2). The
column was washed with methanol and 7N methanolic ammonia. The
methanolic ammonia fractions were combined and concentrated in
vacuo. The resulting solid was purified using preparative high-pressure
liquid chromatography to give desired product 88 (24.0 mg, 71.1
1
μmol) as an orange solid. H NMR (DMSO-d6): 8.31 (s, 1H), 7.59−
7.57 (m, 2H), 7.48−7.44 (m, 3H), 5.53 (s, 1H), 3.74 (t, J = 4.9 Hz,
4H), 3.49 (t, J = 4.9 Hz, 4H). MS (ESI): m/z (M + H)+ 338.
5-Morpholino-7-oxo-N-phenyl-7H-thieno[3,2-b]pyran-3-car-
boxamide (97). A 2 mL microwave vial was charged with a magnetic
stirring bar, compound 6 (100 mg, 316 μmol), molybdenumhex-
acarbonyl (42 mg, 160 μmol), trans-di(μ-acetato)bis[o-(di-o-tolyl-
phosphino)benzyl]dipalladium(II) (4.6 mg, 4.9 μmol), tri-tert-
butylphosphonium tetrafluoroborate (2.8 mg, 12.0 μmol), tetrahy-
drofuran (500 μL), aniline (45 μL, 483 μmol), and 1,8-
diazabicyclo[5.4.0]undec-7-ene (32 μL, 210 μmol). The vial was
immediately sealed, magnetically stirred, and heated via microwave
irradiation at 125 °C for 6 min. The mixture was cooled to room
temperature and loaded onto an Isolute functionalized silica column
(PE-AX/SCX-2). The column was washed with methanol, and the
product was eluted with 7N methanolic ammonia. The methanolic
ammonia fractions were combined and concentrated in vacuo. The
resulting solid was purified using preparative high-pressure liquid
chromatography to give desired product 97 (3.88 mg, 10.9 μmol) as a
yellow solid. 1H NMR (CDCl3): 8.24 (s, 1H), 8.18 (s, 1H), 7.64−7.20
(m, 5H), 5.46 (s, 1H), 3.82 (s, 4H), 3.46 (s, 4H). MS (ESI): m/z (M
+ H)+ 357.
7.51 (dd, J = 8.1, 7.0 Hz, 2H), 7.42 (d, J = 7.4 Hz, 1H), 5.54 (s, 1H),
3.71 (t, J = 5.0 Hz, 4H), 3.43 (t, J = 4.9 Hz, 4H). MS (ESI): m/z (M +
H)+ 314.
5-Morpholino-3-(pyridin-3-yl)-7H-thieno[3,2-b]pyran-7-one
(27). A 2 mL conical microwave vial was charged with a magnetic
stirring bar, 3-bromo-5-morpholino-7H-thieno[3,2-b]pyran-7-one (6)
(50 mg, 0.16 mmol), 3-pyridylboronic acid (29 mg, 0.24 mmol),
cesium carbonate (103 mg, 0.32 mmol), dichloro[1,1′-bis-
(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct
(9 mg, 0.008 mmol), and dimethoxyethane (1 mL). The reaction
mixture was magnetically stirred and heated via microwave irradiation
for 15 min at 180 °C. Upon cooling to room temperature, the reaction
was concentrated in vacuo and purified using high-pressure liquid
chromatography to give 5-morpholino-3-(pyridin-3-yl)-7H-thieno[3,2-
b]pyran-7-one (27) (6 mg, 1.9 μmol) as a white solid. 1H NMR
(DMSO-d6): 8.94 (dd, J = 2.3, 0.9 Hz, 1H), 8.61 (dd, J = 4.9, 1.6 Hz,
1H), 8.33 (s, 1H), 8.14 (ddd, J = 7.8, 2.3, 1.6 Hz, 1H), 7.55 (ddd, J =
7.8, 4.9, 0.9 Hz, 1H), 5.56 (s, 1H), 3.71 (t, J = 4.9 Hz, 4H), 3.42 (t, J =
5.0 Hz, 4H). MS (ESI): m/z (M + H)+ 315.
Methyl 3-Hydroxy-4-(5-morpholino-7-oxo-7H-thieno[3,2-b]-
pyran-3-yl)thiophene-2-carboxylate (85). A 2 mL conical micro-
wave vial was charged with a magnetic stirring bar, compound 9 (57
mg, 0.16 mmol), cesium carbonate (102 mg, 0.31 mmol), methyl 4-
bromo-3-hydroxythiophene-2-carboxylate (56 mg, 0.24 mmol),
tetrakis(triphenylphosphine)palladium(0) (9 mg, 0.08 mmol), and
dimethoxyethane (0.5 mL). The reaction mixture was magnetically
stirred and heated via microwave irradiation for 15 min at 140 °C.
Upon cooling to room temperature, the reaction was concentrated in
vacuo and the residue purified via high-pressure liquid chromatog-
Phenyl 5-Morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-car-
boxylate (105). A 2 mL conical microwave vial was charged with a
magnetic stirring bar, compound 6 (50 mg, 158 μmol), phenol (22
mg, 237 μmol), molybdenum hexacarbonyl (42 mg, 158 μmol), trans-
di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium(II) (4
mg, 4.7 μmol), tri-tert-butylphosphonium tetrafluoroborate (2.8 mg,
9.5 μmol), tetrahydrofuran (0.5 mL), and 1,8-diazabicyclo[5.4.0]-
undec-7-ene (71 μL, 474 μmol). The reaction mixture was sealed, and
the reaction mixture was magnetically stirred and heated via
microwave irradiation at 125 °C for 6 min to give ester 105. MS
(ESI): m/z (M + H)+ 358.
5-Morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-carboxylic
Acid (106). A 2 mL conical microwave vial was charged with a
magnetic stirring bar, compound 6 (50 mg, 158 μmol), molybdenum
hexacarbonyl (42 mg, 158 μmol), trans-di(μ-acetato)bis[o-(di-o-tolyl-
phosphino)benzyl]dipalladium(II) (2.0 mg, 2.3 μmol), tri-tert-
butylphosphonium tetrafluoroborate (1.4 mg, 4.7 μmol), dimethyl
sulfoxide (0.5 mL), water (2.8 μL, 158 μmol), and 1,8-
diazabicyclo[5.4.0]undec-7-ene (16 μL, 106 μmol). The reaction
mixture was sealed, and the reaction mixture was magnetically stirred
and heated via microwave irradiation at 125 °C for 6 min to give the
desired carboxylic acid 106. MS (ESI): m/z (M − H)− 280.
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Journal of Medicinal Chemistry
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3-Acetyl-5-morpholino-7H-thieno[3,2-b]pyran-7-one (107).
A 2 mL conical microwave vial was charged with a magnetic stirring
bar, compound 6 (100 mg, 316 μmol), dimethylformamide (0.5 mL),
N,N-diisopropylethylamine (165 μL, 949 μmol), butyl vinyl ether (124
μL, 949 μmol), and dichloro[1,1′-bis(diphenylphosphino)ferrocene]-
palladium(II) dichloromethane adduct (10 mg, 12.6 μmol). The
reaction mixture was sealed, and the reaction mixture was magnetically
stirred and heated via microwave irradiation at 180 °C for 15 min.
After cooling to room temperature, the reaction was charged with
aqueous hydrochloric acid (1.0 M, 2 mL) and stirred at room
temperature for 2 h to give desired ketone 107. MS (ESI): m/z (M +
H)+ 280.
N-Benzyl-4-(5-morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-
yl)benzamide (108). A 2 mL conical microwave vial was charged
with a magnetic stirring bar, compound 42 (80 mg, 230 μmol), trans-
di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium(II) (6
mg, 7 μmol), tri-tert-butylphosphonium tetrafluoroborate (4 mg, 14
μmol), molybdenumhexacarbonyl (61 mg, 230 μmol), tetrahydrofuran
(500 μL), benzylamine (75 μL, 690 μmol), and 1,8-
diazabicyclo[5.4.0]undec-7-ene (103 μL, 690 μmol). The reaction
mixture was magnetically stirred and heated via microwave irradiation
for 15 min at 170 °C. Upon cooling to room temperature, the reaction
was concentrated in vacuo and purified using high-pressure liquid
chromatography to give desired product 108 as a white solid. 1H NMR
(DMSO-d6): 9.14 (t, J = 5.7 Hz, 1H), 8.30 (s, 1H), 8.04 (d, J = 6.0 Hz,
2H), 7.84 (d, J = 6.0 Hz, 2H), 7.34 (d, J = 6.0 Hz, 4H), 7.30−7.18 (m,
1H), 5.56 (s, 1H), 4.51 (d, J = 6.5 Hz, 2H), 3.72 (t, J = 4.4 Hz, 4H),
3.45 (s, 4H). MS (ESI): m/z (M + H)+ 447.
Butyl 3-(5-Morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)-
benzoate (118). A 2 mL microwave vial was charged with a
magnetic stirring bar, compound 61 (80 mg, 230 μmol), imidazole (47
mg, 690 μmol), molybdenum hexacarbonyl (60 mg, 230umol), trans-
di(μ-acetato)bis[o-(di-o-tolyl-phosphino)benzyl]dipalladium(II) (6.5
mg, 6.9 μmol), tri-tert-butylphosphonium tetrafluoroborate (4.0 mg,
13.9 μmol), tetrahydrofuran (500 μL), n-butanol (63 μL, 690 μmol),
and 1,8-diazabicyclo[5.4.0]undec-7-ene (103 μL, 690 μmol). The vial
was immediately sealed, and the reaction mixture was magnetically
stirred and heated via microwave irradiation at 170 °C for 15 min in a
microwave reactor to give desired butyl ester 118. MS (ESI): m/z (M
+ H)+ 414.
nm). Sigmoidal curves were drawn for the dose−responses, and IC50
values were calculated using the software package GraphPad Prism4
(GraphPad Software, Inc., San Diego, CA), and from these graphs the
concentration required to decrease proliferation by 50% was
calculated.
pAKT Status in Prostate Cancer Cells (PC3). PC3 cells were
obtained from the American Type Culture Collection (ATCC,
Manassas, VA, catalogue no. CRL-1435). Two million cells from the
prostate cancer line PC3 were placed into 6 cm culture dishes and
allowed to grow in complete RPMI 1640 media (Invitrogen, Carlsbad,
CA, catalogue no. 22400−105) with 10% fetal bovine serum
(Invitrogen, Carlsbad, CA, catalogue no. 10438−026). After this
time period, the cells were serum starved for 5 h, followed by
application of the test compound. Test compound was added as a
DMSO solution such that the final DMSO concentration in the cell
media was less than or equal to 0.2% by volume. After 30 min of
exposure, the growth factor stimulant, human IGF-1 (PeproTech, Inc.,
Rocky Hill, NJ, catalogue no. 100−11), was added to a final
concentration of 0.1 μg/mL in each well. After 30 min of IGF-1
exposure, cells were removed from the media and cell lysates were
prepared using RIPA Lysis buffer (Upstate, Lake Placid, NY, catalogue
no. 20−188) and kept on ice. The pAKT serine 473 level was
measured in duplicate samples of the cell lysates using commercially
available assays such as the Pathscan Sandwich ELISA kit for Ser473
pAKT (Cell Signaling, Danvers, MA, catalogue no. 7160). A
SpectraMax Plus spectrophotometric plate reader (Molecular Devices,
Sunnydale, CA) was used to measure the optical density signal for
pAKT at 450 nm (OD450 nm). The pAKT OD450 nm readings were
normalized by total protein amount in the cell lysates determined by
standard methods.
Concentrations of test compounds required to inhibit IGF
stimulated pAKT levels to 50% of maximum levels in PC3 cells
(termed DM50 for decreased maximum 50%) were calculated by
inputting the dose responses in the software package GraphPad
Prism4 (GraphPad Software, Inc., San Diego, CA). These values (in
μMolar units) are listed in Table 4.
Cancer Stem Cell Assay. A mammosphere cancer stem cell
inhibition assay was modified from that described in the literature47,48
using a subset of MCF7 breast cancer cells that possess the ability to
evade anchorage independent apoptosis and form mammospheres in
vitro. Commercially available MCF7 breast cancer cells were cultured
in MEM supplemented with 0.01 mg/mL of bovine insulin and 10%
FBS at 37 °C in humidified atmosphere (5% CO2). Floating cells in
the 2-day cultures were collected, washed, and resuspended in
DMEM-F12 (50:50) supplemented with 0.4% BSA, 5 μg/mL bovine
insulin, 20 ng/mL bFGF, and 10 ng/mL EGF. Cells were then passed
through a 40 μM sieve to remove any cell clusters and adjusted to a
density of 10000 cells/mL. Approximately 500 cells were plated in
quadruplicate in a sterile poly-HEMA-treated 96-well plate. Cells were
then treated with either control or test solutions consisting of the
kinase inhibitors at 1 or 10 μM. The numbers of mammospheres
(clusters of cells numbering more than 4 or 5 cells per cluster) were
then counted using a microscope after 5−7 days of incubation and 37
°C, 5% CO2.
N-(4-(5-Morpholino-7-oxo-7H-thieno[3,2-b]pyran-3-yl)-
phenyl)nicotinamide (119). A 40 mL vial was charged with a
magnetic stirring bar, compound 60 (200 mg, 609 μmol), and pyridine
(5 mL). The mixture was magnetically stirred and cooled to 0 °C in an
ice bath. Isonicotinoyl chloride hydrochloric acid (669 μmol) was
added, and the reaction was stirred at room temperature for 30 min.
The reaction was diluted with dichloromethane (15 mL) and washed
three times with saturated aqueous sodium bicarbonate solution (15
mL). The dichloromethane layer was dried over magnesium sulfate,
filtered, and concentrated in vacuo to give desired product 119 (94
1
mg, 217 μmol) as a yellow solid. H NMR (CD3OD): 9.11 (s, 1H),
8.78 (s, 1H), 8.40 (s, 1H), 7.99 (s, 1H), 7.89−7.70 (m, 6H), 5.60 (s,
1H), 3.56 (s, 4H), 3.31 (s, 4H). MS (ESI): m/z (M + H)+ 434.
Cell-Based Assays. Cell lines were obtained from the American
Type Culture Collection (ATCC, Manassas, VA, catalogue no. CRL-
1435). Two thousand cells from the cell lines were placed in 50 μL of
complete RPMI 1640 media (Invitrogen, Carlsbad, CA, catalogue no.
22400−105) with 10% fetal bovine serum (Invitrogen, Carlsbad, CA,
catalogue no. 10438−026) into each well of 96-well cell culture plates
in a hexaplicate sampling pattern. A 50 μL aliquot of test compound
(prepared from stock test compounds in DMSO diluted into media)
was added to the appropriate well such that the final concentrations of
test compound were 200, 40, 8, 1.6, 0.32, 0.064, 0.0128, 0.00256, and 0
μM, and the final DMSO concentration was less than or equal to 0.2%
. The plates were incubated for 72 h at 37 °C in an atmosphere of 5%
CO2. At the end of this time, a 10 μL aliquot of WST solution (Roche
Applied Science, Mannheim, Germany) was added into each of the
wells. Cells were exposed to the WST solution for 4 h. A SpectraMax
Plus spectrophotometric plate reader (Molecular Devices, Sunnyvale,
CA) was then used to measure the optical density at 450 nm (OD450
Biostatistical Methodology. The evaluation of difference in the
biochemical and cell-based assays was determined using Student t test.
Experiments were repeated three times.
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthetic methods used for analogues 28−84, 86, 89−96, 98−
104, 109−117, and 120−127. This material is available free of
charge via the Internet at http://pubs.acs.org.
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Journal of Medicinal Chemistry
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AUTHOR INFORMATION
Corresponding Author
*For G.A.M.: phone, (520) 626-1895; fax, (520) 626-0794; E-
mail, gmorales@bio5.org. For D.L.D.: phone, (858) 534-3355;
fax, (858) 822-0022; E-mail: ddurden@ucsd.edu.
■
Present Address
⊥For G.A.M.: BIO5 Institute (Oro Valley), The University of
Arizona, 1580 East Hanley Boulevard, Oro Valley, Arizona
85737, United States. For J.R.G.: SignalRx Pharmaceuticals,
12545 El Camino Real, San Diego, California 92130, United
States.
Notes
The authors declare no competing financial interest.
ABBREVIATIONS USED
■
P I 3 K , p h o s p h a t i d y l i n o s i t o l - 3 - k i n a s e ; P I P 2 ,
phosphatidylinositol(4, 5)-bisphosphate; PIP3,
phosphatidylinositol(3,4,5)-trisphosphate; PTEN, phosphatase
and tensin homologue; AKT, protein kinase B; ATP, adenosine
triphosphate; SAR, structure−activity relationship; PIK3CA,
phosphoinositide-3-kinase, catalytic, α polypeptide; PIM-1,
proviral insertion in murine lymphoma 1 kinase; PLK-1,
polo-like kinase 1; DNA-PK, DNA-dependent protein kinase;
mTOR, mammalian target of rapamycin; LDA, lithium
diisopropylamide; THF, tetrahydrofuran; DME, dimethoxy-
ethane; DCM, dichloromethane; RT, room temperature; MW,
microwave irradiation; dppf, 1,1′-bis(diphenylphosphino)-
ferrocene; dppp, 1,3-bis(diphenylphosphino)propane; Ar,
aromatic; DMSO, dimethyl sulfoxide; DIPEA, diisopropylethyl-
amine; DMF, dimethylformamide; DBU, 1,8-diazabicyclo-
[5.4.0]undec-7-ene; dba, dibenzylideneacetone; XPhos, 2-
dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl; EtOH,
ethanol; pAKT, phosphorylated AKT; PDB, Protein Data
Bank; compd, compound; PBS, phosphate buffered saline;
VEGF, vascular endothelial growth factor; Bv8, Bombina
variegata peptide 8; IGF, insulin-like growth factor; EGFR,
epidermal growth factor receptor; μM, micromolar; nM,
nanomolar; IC50, half maximal inhibitory concentration; PK,
pharmacokinetics; NMR, nuclear magnetic resonance; UV,
ultraviolet; ELSD, evaporative light scattering detector; HPLC,
high pressure liquid chromatography; LCMS, liquid chroma-
tography mass spectrometry; TFA, trifluoroacetic acid; mL,
milliliter; min, minute; MS, mass spectrometry; MRA, mass rate
attenuator; LC, liquid chromatography; ELS, evaporative light
scattering; ESI, electrospray ionization; mg, milligram; mmol,
millimole; μmol, micromole; g, gram; μL, microliter; ELISA,
enzyme-linked immunosorbent assay; DM50, decreased
maximum 50%; MEM, minimum essential medium; DMEM,
Dulbecco’s Modified Eagle’s Medium; BSA, bovine serum
albumin; bFGF, basic fibroblast growth factor; EGF, epidermal
growth factor; cmpd, compound
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