Rational design of phosphoinositide 3-kinase inhibitors that exhibit selectivity over the phosphoinositide 3-kinase isoform

Article abstract of DOI:10.1021/jm2007084

Of the four class I phosphoinositide 3-kinase (PI3K) isoforms, PI3K has justly received the most attention for its potential in cancer therapy. Herein we report our successful approaches to achieve PI3K vs PI3K selectivity for two chemical series. In the thienopyrimidine series of inhibitors, we propose that select ligands achieve selectivity derived from a hydrogen bonding interaction with Arg770 of PI3K that is not attained with the corresponding Lys777 of PI3K. In the benzoxepin series of inhibitors, the selectivity observed can be rationalized by the difference in electrostatic potential between the two isoforms in a given region rather than any specific interaction.

Full text of DOI:10.1021/jm2007084

Article
pubs.acs.org/jmc
Rational Design of Phosphoinositide 3-Kinase α Inhibitors That
Exhibit Selectivity over the Phosphoinositide 3-Kinase β Isoform†
Timothy P. Heffron,*,‡ BinQing Wei,‡ Alan Olivero,‡ Steven T. Staben,‡ Vickie Tsui,‡ Steven Do,‡
Jennafer Dotson,‡ Adrian J. Folkes,§ Paul Goldsmith,§ Richard Goldsmith,‡ Janet Gunzner,‡
John Lesnick,‡ Cristina Lewis,‡ Simon Mathieu,‡ Jim Nonomiya,‡ Stephen Shuttleworth,§
Daniel P. Sutherlin,‡ Nan Chi Wan,§ Shumei Wang,‡ Christian Wiesmann,‡ and Bing-Yan Zhu‡
‡Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United States
§Piramed Pharma, 957 Buckingham Avenue, Slough, Berks SL1 4NL, United Kingdom
S
* Supporting Information
ABSTRACT: Of the four class I phosphoinositide 3-kinase
(PI3K) isoforms, PI3Kα has justly received the most attention
for its potential in cancer therapy. Herein we report our
successful approaches to achieve PI3Kα vs PI3Kβ selectivity
for two chemical series. In the thienopyrimidine series of
inhibitors, we propose that select ligands achieve selectivity
derived from a hydrogen bonding interaction with Arg770 of
PI3Kα that is not attained with the corresponding Lys777 of
PI3Kβ. In the benzoxepin series of inhibitors, the selectivity
observed can be rationalized by the difference in electrostatic
potential between the two isoforms in a given region rather
than any specific interaction.
over PI3Kβ. This particular selectivity profile was of interest
because of the potential role of PI3Kβ in insulin signaling.9
Because of this potential critical role, it is reasonable to
speculate whether or not the tolerability of PI3Kα inhibitors for
the treatment of cancer might be improved if they have
selectivity over PI3Kβ. However, at the onset of our studies it
was unclear whether reduced PI3Kβ inhibition would
compromise efficacy. Taken together, compounds with high
PI3Kβ/PI3Kα selectivity were of interest to us as tools for
studying the implication on efficacy and safety. At the onset of
this effort, designing PI3K inhibitors that displayed selectivity
over PI3Kβ presented a challenge because no crystal structure
of PI3Kβ had been reported.10 On the other hand, cocrystal
structures containing PI3K inhibitors have been available for
PI3Kγ for several years.11 More recently, crystal structures of
PI3Kδ12 (cocrystallized with inhibitors) and PI3Kα13 have been
reported.14
To guide our attempts at achieving PI3Kα vs PI3Kβ
selectivity, we utilized a public crystal structure of PI3Kα15
along with a homology model of PI3Kβ developed from a
PI3Kδ crystal structure.16 While each of the class I PI3Ks
exhibits a high degree of homology within the active site of the
enzyme, there are differences in primary sequence among
residues that are predicted to reside on the periphery of the
active site in a solvent exposed region (Figure 1). These
INTRODUCTION
■
The phosphoinositide 3-kinases (PI3Ks) are attractive targets
for the design of small molecule inhibitors because of the
frequent occurrence of aberrant signaling of this pathway in
several different disease states. Within the PI3 kinase family,
there are four class I PI3 kinase isoforms (α, β, δ, and γ).1
PI3Kβ has been targeted for thrombosis prevention or
treatment, and small molecules that display selectivity for this
isoform have been reported.1−3 PI3Kδ and PI3Kγ have been
identified as targets for inflammatory, autoimmune, and
respiratory indications.4 Accordingly, compounds that display
modest to high levels of isoform selectivity have been reported
for the inhibition of PI3Kδ, PI3Kγ, or both.1,5 For its promise
in the treatment of cancer, inhibition of PI3Kα has received
considerable attention.6 However, despite the significant effort
to identify inhibitors of PI3Kα, there are only few reports of
PI3Kα inhibitors that either specifically inhibit this isoform or
achieve selectivity relative to any of the other isoforms.1,7
We previously reported our efforts to identify PI3Kα
inhibitors including clinical candidate 1 (GDC-0941).8 Each
of our previous disclosures culminated with molecules that are
highly selective relative to other kinases but inhibit each of the
four class I PI3K isoforms with minimal selectivity (Table 1).
As a continuation of this work, we became interested in
studying the biological effects of inhibitors of PI3Kα that had
differential selectivity profiles relative to one or more of the
other isoforms. Among the selectivity profiles we were
interested in evaluating was inhibition of PI3Kα with selectivity
Received: June 2, 2011
Published: October 10, 2011
© 2011 American Chemical Society
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Table 1. Class I PI3K IC50s of Key Compounds
IC50 (nM)
compd
PI3Kα
PI3Kβ
PI3Kδ
PI3Kγ
1
2
3
4
5
3
3
4
7
5
33
12
86
31
46
3
16
5
66
16
15
8
11
2
5
We discuss below our results in gaining selectivity for PI3Kα
relative to PI3Kβ on two distinct scaffolds. With each of the
two scaffolds studied, the selectivity observed can be explained
using our homology model of PI3Kβ.17 These studies lend
support to the use of our homology model of PI3Kβ for
compound design as well as provide opportunity to evaluate the
desirability of this selectivity profile.
RESULTS AND DISCUSSION
■
We first prepared compounds that achieve PI3Kβ/PI3Kα
selectivity fortuitously when evaluating analogues of 1. 1 is just
modestly selective for PI3Kα relative to PI3Kβ (Table 2).
However, modification of the central core of the thienopyr-
imidine to the isomeric thiophene (6, Table 2) resulted in
significantly improved PI3Kβ/PI3Kα selectivity as a result of
notable reduction in PI3Kβ inhibition.
A cocrystal structure of 1 in PI3Kγ shows that a sulfonamide
oxygen achieves a hydrogen bond with the backbone NH of
Ala805 (PI3Kγ).8a Our models suggest that this interaction is
likely maintained in the other PI3K isoforms with 1 hydrogen
bonding with Ser773 backbone NH in PI3Kα (Figure 2a) and
Asp780 backbone NH in PI3Kβ (Figure 2b). In addition, our
model of 1 in PI3Kα suggests the other sulfonamide oxygen is
capable of hydrogen bonding with Arg770 (Figure 2a).
However, according to our model, this is unlikely in PI3Kβ
in which Arg770 is now Lys777 (Figure 2b). This suggested
Figure 1. Cocrystal structure of 1 in PI3Kγ (PDB code 3DBS).
Residues nearest the active site that are different between PI3Kα and
at least one other isoform are highlighted.
differences presented an opportunity using our thienopyrimi-
dine scaffold to achieve the desired selectivity. In addition, there
is the potential for disparate conformational dynamics within
the P-loop (Met804-Pro810, PI3Kγ numbering).
Table 2. Comparison of PI3Kα and PI3Kβ Potency for 1 and Related Analogues
IC50 (nM)
compd
PI3Kα
PI3Kβ
IC50(PI3Kβ)/IC50(PI3Kα)
1
6
7
3
3
3
33
332
194
11
111
65
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portion of the molecule that is directed toward the residue
differences (R1) is essential to the selectivity observed, as
compounds without substitution of the thiophene have minimal
selectivity. Having identified that the indazole does not
contribute to PI3Kβ selectivity, we limited additional studies
to the aminopyrimidine motif (R2) that had previously been
identified to have superior potency and pharmacokinetic
properties.18 That the sulfonamide oxygens are essential to
achieving high PI3Kβ/PI3Kα selectivity is demonstrated with
piperazine 11 and piperidine 12. A comparison of 11 and 12
illustrates that a more basic center at the distal end of the ring
erodes selectivity. Piperidinesulfonylamide 13 has potency and
selectivity comparable to those of 8, indicating that the
remaining basic center does not impact either. The sulfone
analogue 14 exhibits reduced selectivity when compared to
compound 8 because of a modest drop in PI3Kβ potency. The
position of the sulfonamide oxygens is critical, as demonstrated
with compound 15 which had the lowest selectivity of any
sulfone or sulfonamide containing analogue. Nevertheless, the
20-fold selectivity of compound 15 is still significant relative to
the unsubstituted entry 10. Entry 16 illustrates that a carbonyl
functionality can also achieve good levels of selectivity. Finally,
an aromatic ring could replace the heterocyclic ring present in
the other examples. Arylsulfone 17 proved to be potent and
had reasonable selectivity; however, it had poor solubility that
prevented it from advancing further, as even at the higher
concentrations required in cellular assays the compound
precipitated over time. In general, the selectivity achieved is
unique to the PI3Kβ isoform, as consistently low levels of
selectivity (<10-fold) for PI3Kα were observed relative to
PI3Kδ and PI3Kγ.
Having identified an approach to selectivity for PI3Kα over
PI3Kβ, we were interested in whether we could apply the
hypothesis to achieve the same type of selectivity on a distinct
scaffold. Recently we disclosed initial efforts on a class of
benzoxepins that are potent PI3Kα inhibitors.8e A cocrystal
structure of a representative benzoxepin, 21, with PI3Kγ is
shown in Figure 3a. For the sake of comparison, Figure 3b
shows an overlay of benzoxepin 21 and thienopyrimidine 1.
From this overlay it is apparent that substitution of the benzene
ring of the benzoxepin offers a different vector compared with
substitution of the thienopyrimidine class of compounds.
An initial SAR scan compared R1 and R2 substitution of
thienobenzoxepins. An analysis of the specific substitution
revealed that R1 functionality generally led to increased
likelihood of PI3Kβ/PI3Kα selectivity when compared to R2
substitution (Figure 4). This result was unanticipated because it
seemed that R2 substitution would be able to grant access to
Arg770 and Ser773 that we exploited for selectivity in the
thienopyrimidine series (Figure 2). The apparent predisposal
for the desired selectivity led us to prioritize our efforts to R1
substitution to improve selectivity over PI3Kβ.
Revisiting the PI3Kα and PI3Kβ homology models we
employed earlier, we recognized that in the vicinity of the R1
position of the benzoxepin core there were a number of
residues that differed between PI3Kα and PI3Kβ (see Figure
1). Importantly, there are several acidic residues in the area of
PI3Kβ where R1 substitution is positioned. This is in contrast to
the same region of PI3Kα, in which there are no acidic residues.
Anticipating that many of the solvent exposed residues would
likely be flexible, making specific hydrogen bonding interactions
difficult to achieve, we viewed the relevant region of the
enzyme as an electrostatic potential map.19 This model
Figure 2. (a) Model of 1 in PI3Kα. Hydrogen bonds with Ser773
backbone NH and Arg770 are illustrated. (b) Model of 1 in PI3Kβ,
illustrating hydrogen bond with Asp780 backbone NH but suboptimal
distance to Lys777. (c) Model of 6 in PI3Kα. Hydrogen bonds with
Ser773 backbone NH and Arg770 are illustrated. (d) Model of 6 in
PI3Kβ, illustrating suboptimal hydrogen bonding distances to Asp780
backbone NH and Lys777. (e) Overlay of 1 (green), 6 (magenta), and
7 (lavender), with morpholine and indazole rigidly superimposed.
loss of a hydrogen bond is a possible explanation for the 10-fold
reduced potency against that isoform. Upon modification of the
central core to the isomeric thiophene 6, the sulfonamide
oxygens are positioned further away from the key backbone
NH (Figure 2c,d). Still, our model suggests that 6 is able to
achieve the two hydrogen bonds in PI3Kα (Figure 2c), while in
PI3Kβ 6 is no longer able to achieve either of these hydrogen
bonds. It is possible that this explanation accounts for why 6 is
100-fold less potent toward PI3Kβ relative to PI3Kα (Figure
2d). Additionally, compound 7, in which the thiophene has
been replaced by a furan, should display the piperazine
sulfonylamide with a vector much closer to compound 6 than
to 1 (Figure 2e). In the end, furanopyrimidine 7 achieves
potency and selectivity similar to those of compound 6.
Encouraged by the selectivity achieved with compounds 6
and 7, we extended our studies to further evaluate contributions
to isoform selectivity using the isomeric thiophene core of 6.
Table 3 highlights the SAR describing PI3Kβ/PI3Kα selectivity.
A comparison of compounds 6 and 8 demonstrates that the
indazole moiety contributes minimally to PI3Kβ selectivity and
that the aminopyrimidine group improves potency against both
PI3Kα and PI3Kβ isoforms. Entries 9 and 10 illustrate that the
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Table 3. Class I PI3K Isoform Selectivity for Isomeric Thienopyrimidines
suggested that simply incorporating highly electron rich or even
lipophilic functionality should result in improved selectivity for
PI3Kα relative to PI3Kβ. On the other hand, incorporation of a
positively charged functionality should lead to reduced
selectivity. Figure 5a depicts a model of benzoxepin 21
bound to PI3Kα, highlighting the electrostatic potential in
the region accessible through substitution of the core at R1.
Figure 5b depicts the electrostatic potential map of PI3Kβ in
the same area with the same compound included for reference.
Table 4 highlights that the electrostatic potential maps of
PI3Kα and PI3Kβ derived from a PI3Kα crystal structure and
our PI3Kβ homology model are indeed predictive of selectivity.
Entry 18 established that the benzoxepin core without
substitution of the benzene ring has good PI3Kα potency
and <20-fold selectivity relative to PI3Kβ. Compound 19 shows
the impact of an uncharged R1 substituent on PI3Kβ/PI3Kα
selectivity. In this case, the level of selectivity is increased to 82-
fold. We found that many uncharged groups at R1 resulted in
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Figure 5. Poisson−Boltzmann electrostatic potentials plotted on the
solvent accessible surfaces of PI3Kα crystal structure (PDB code
3HIZ) (a) and PI3Kβ homology model (b). Compound 21 is depicted
for reference.
Figure 3. (a) Cocrystal structure of benzoxepin 21 in PI3Kγ (PDB
code 3R7R). (b) Overlay of 1 in the cocrystal structure of 21 in PI3Kγ.
similarly enhanced PI3Kβ/PI3Kα selectivity. As further
examples, nitrile 20, acetamide 21, and amides 22 and 23 all
achieve greater than 50-fold selectivity for PI3Kα relative to
PI3Kβ. However, incorporating a basic center diminishes
selectivity as a result of increased affinity for PI3Kβ (24).
Moreover, as the electrostatic potential map predicts, exact
positioning of a positive charge is not essential to diminish
PI3Kβ/PI3Kα selectivity as examples 24 and 25 demonstrate.
When a negatively charged functionality was incorporated (26),
the selectivity increased to nearly 100-fold. However, because
the negative charge in 26 prevented the molecule from
effectively permeating a cell membrane, this compound was
not studied further. In contrast to the carboxylic acid in 26, the
related analogue 27 incorporated a basic amine that essentially
negated the impact of R1 substitution, as the potency and
selectivity are comparable to those of the unsubstituted 18.
Substitution of R2 also resulted in improvement in PI3Kβ/
PI3Kα selectivity (compounds 28−30), similar to how the
same functionality improved selectivity at R1 (compare
compounds 20 and 30 and compare 23 and 29).
the thienopyrimidine series, benzoxepins 18−34 also generally
display low levels of selectivity for PI3Kα relative to PI3Kδ and
PI3Kγ.
A study including 6 was consistent with the apparent
importance of PI3Kβ inhibition to inhibit proliferation of a
cancer cell that has lost phosphatase and tensin homologue
(PTEN) function.20 Evaluation of compounds with enhanced
PI3Kα to PI3Kβ enzyme selectivity in cell proliferation assays
showed a qualitative trend for a lesser effect on decreasing
proliferation in the PC3 cell line (PTEN-null) relative to
decreasing proliferation in the MCF7.1 cell line (PI3Kα
mutant) (Table 5). Specifically, in the thienopyrimidine series
of molecules, the weakly selective 1 had close to equipotent
activity in both cell lines, a trend that was also observed with
the weakly selective benzoxepin 31. Conversely, thienopyr-
imidines 6 and 13, which displayed high levels of PI3Kβ/PI3Kα
selectivity, were more potent in the MCF7.1 than the PC3
proliferation assay, with the same behavior observed with the
structurally distinct benzoxepin 32.
It was interesting to note that substitution of R3 with an
amide resulted in diminished PI3Kβ/PI3Kα selectivity. For
example, compound 31 has just 10-fold selectivity whereas the
analogue without the amide at R3 (compound 22) has 87-fold
selectivity. Moving the position of the N,N-dimethylamide
from R3 to R4 resulted in a notable gain in selectivity (32). A
similar trend was observed with N-methylpiperazineamides 33
and 34. In this case, moving the amide to R4 restores the
selectivity found in the simpler analogue 22. A cocrystal
structure of compound 33 with PI3Kγ (Figure 6) shows that
the piperazineamide carbonyl achieves a hydrogen bond with
Ser806. A potential explanation for this reduction in selectivity
is that, despite being conserved among the four isoforms, the
conformation of Ser806 as part of the flexible G-loop is such
that a hydrogen bond to it is more beneficial for PI3Kβ potency
than for PI3Kα and so results in reduced selectivity. Similar to
In addition to having high levels of PI3Kβ/PI3Kα selectivity,
both series of molecules generally exhibit a very high level of
selectivity over other kinases as well. In a panel of 101 kinases
at Upstate, only one was inhibited by >80% by 6 when tested at
1 μM (CAMK2D). To represent the benzoxepin series of
compounds, 21, 22, and 33 were each evaluated in a panel of
51 kinases provided by Invitrogen’s SelectScreen service. In
each case no kinases were inhibited by >50% at 1 μM test
compound.
Of the compounds that displayed significant PI3Kβ/PI3Kα
selectivity, 6, 7, and 32 were evaluated in rodent pharmaco-
kinetics studies. Compounds 7 and 32 each had a clearance rate
greater than liver blood flow in rats (data not shown). On the
other hand, compound 6 had attractive pharmacokinetic
properties in rats as well as mice and so was selected for
further study in in vivo efficacy models.20
Figure 4. Correlation of the position of substitution on the benzoxepin core and level of PI3Kβ/PI3Kα selectivity.
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Table 4
the reaction with dimethyl carbonate. After deprotection of the
amino group, chlorosulfonyl isocyanate was used to convert the
amine (37) to a urea which then underwent cyclization in basic
methanolic medium. The resulting pyrimidinedione was
converted to 38 by the action of POCl3 followed by
incorporation of morpholine. Formylation of 38 led to a
mixture of the aldehyde 39 and hemiacetal 40. This mixture
CHEMISTRY
■
The synthesis of the furan analogue 7 (Scheme 1) commenced
with furan-3-carboxylic acid which was converted to the
requisite amino group by Curtius rearrangement to give the
Boc-protected amine 36. A methyl ester was introduced at the
2-position of the furan by lithiation followed by quenching of
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CONCLUSION
■
Herein we have described two distinct approaches to achieving
selectivity for PI3Kβ relative to PI3Kα. We suggest that the
PI3Kβ/PI3Kα selectivity achieved in the thienopyrimidine
series of inhibitors results from hydrogen bonding interactions
between the ligand and Arg770 of PI3Kα that is not attained
with the corresponding Lys777 of PI3Kβ. In the benzoxepin
series of inhibitors, we postulate that the selectivity attained is a
result of the difference in electrostatic potential between the
two isoforms in a given region rather than any specific
interaction or even the difference between one specific residue
between the two enzyme isoforms. The approach to selectivity
utilized in the benzoxepin series has the potential to be general
and applicable to additional scaffolds as long as they contain the
potential to occupy the critical space depicted in Figure 5. Each
of the two approaches described lends credibility to the use of
our PI3Kβ homology model. The approaches to gaining
selectivity presented here should aid in the design of additional
compounds that achieve selectivity either for or against PI3Kβ.
Our studies of the impact of the selectivity over PI3Kβ in
comparison to the pan-inhibitors previously reported are
ongoing.
EXPERIMENTAL SECTION
Chemistry. All solvents and reagents were used as obtained. H
Figure 6. A cocrystal structure of PI3Kγ and 33 (PDB code 3T8M)
shows a hydrogen bond between the piperazineamide carbonyl and
Ser806.
■
1
NMR spectra were recorded with a Bruker Avance DPX400
spectrometer or a Varian Inova 400 NMR spectrometer and
referenced to tetramethylsilane. Chemical shifts are expressed as δ
units using tetramethylsilane as the external standard (in NMR
description, s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
and br, broad peak). All final compounds were purified to >95%
chemical purity, as assayed by HPLC (Waters Acquity UPLC column,
21 mm × 50 mm, 1.7 μm) with a gradient of 0−90% acetonitrile
(containing 0.038% TFA) in 0.1% aqueous TFA, with UV detection at
254 and 210 nm and with CAD detection with an ESA Corona
detector.
smoothly underwent reductive amination to give 41 as a single
product. Finally, Suzuki coupling yielded furanopyrimidine 7.21
A general synthetic approach to thienopyrimidine analogues
(Scheme 2) followed the course of the synthesis of 1 reported
earlier.8a The commercially available amino ester 42 was
cyclized to the pyrimidinedione in molten urea. This dione was
subsequently converted to the dichloropyrimidine, and then
morpholine was incorporated to yield 43. At this stage, the
intermediate could be converted to the benzylic alcohol and
subsequently to the chloride. This chloride enabled smooth
conversion to interemediate amines and led to desired products
after Suzuki coupling. Alternatively, iodine could be introduced
on the thiophene intermediate 43 and sequential Suzuki
coupling yielded 17.22
A representative synthesis of benzoxepins began with
alkylation of 3-bromophenol with ethyl 4-bromobutanoate
(Scheme 3). The ester of the resulting ether was saponified and
subjected to acidic conditions to facilitate acylation. Ketone 45
was then homologated to the β-chlorovinylaldehyde using the
Vilsmeier reagent and subsequently cyclized to the thiophene.
Saponification of the resulting ester afforded acid 46. Amide
coupling provided the versatile intermediate 47. This
intermediate was utilized in the synthesis of numerous final
compounds.23
Compounds 1−4 have been described previously.8
tert-Butyl Furan-3-ylcarbamate (36). 3-Furoic acid (5.60 g, 1.0
equiv) was dissolved in tert-butanol (200 mL) and treated with
triethylamine (10 mL, 1.4 equiv) and diphenylphosphorylazide (12
mL, 1.1 equiv). The mixture was heated at reflux for 18 h. The reaction
mixture was cooled to room temperature, then concentrated to 50 mL
and poured into saturated aqueous NaHCO3. The mixture was stirred
at 0 °C for 2 h. Solid was collected by filtration and dried under high
vacuum. The crude reaction mixture was purified by flash
1
chromatography to yield 36 (6.95 g, 76%). H NMR (CDCl3, 400
MHz) δ 7.71 (br s, 1H), 7.27 (m, 1H), 6.27 (br s, 1H), 6.20 (br s,
1H), 1.50 (s, 9H). MS (ESI): m/z (M + H)+ 184.1.
Methyl 3-Aminofuran-2-carboxylate (37). To a solution of
tert-butyl furan-3-ylcarbamate (1.7 g, 1.0 equiv) in THF (50 mL) at
−30 °C was added TMEDA (1.75 mL, 1.3 equiv) followed by 1.6 M
solution of n-butyllithium (8.4 mL, 2.25 equiv, 1.6 M in hexanes). The
reaction mixture was allowed to warm to 0 °C and stirred for 1 h
before being cooled back to −30 °C. Dimethyl carbonate (2.4 mL, 3.0
equiv) was quickly added before the reaction mixture was allowed to
Table 5
IC50 (nM)
EC50 (μM)
MCF7.1
compd
PI3Kα
PI3Kβ
PC3
IC50(PI3Kβ)/IC50(PI3Kα)
EC50(PC3)/ EC50(MCF7.1)
1
3
3
1
2
3
33
332
75
0.39
0.39
0.11
0.4
0.34
0.97
0.41
0.5
11
111
75
0.9
2.5
3.7
1.3
6.4
6
13
31
32
22
10
204
0.11
0.72
69
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a
Scheme 1
a
Reagents and conditions: (a) DPPA, Et3N, t-BuOH; (b) n-BuLi, TMEDA, CO(OMe)2; (c) TFA, CH2Cl2; (d) ClO2SNCO, CH2Cl2; (e) 1.5 M
NaOH in MeOH; (f) POCl3, i-Pr2NEtN; (g) morpholine, MeOH; (h) n-BuLi, THF, −78 °C, DMF; (i) piperazinemethanesulfonamide HCl,
CH(OMe)3, DCE; (j) NaBH(OAc)3; (k) indazole-4-boronic acid pinacol ester, PdCl2(PPh3)2, Na2CO3, toluene/EtOH/water (4:2:1).
a
Scheme 2
a
Reagents and conditions: (a) ClSO2NC, CH2Cl2, aq NaOH, aq HCl; (b) POCl3, CH3CN, reflux, 24 h; (c) morpholine, MeOH, 1 h; (d) n-BuLi,
THF, −78 °C, DMF; (e) NaBH4, MeOH; (f) SOCl2, CH2Cl2; (g) HNRR′, i-Pr2NEt, THF; (h) 2-aminopyrimidine-5-boronic acid pinacol ester,
PdCl2(PPh3)2, 1 M aq KOAc, CH3CN, microwave, 150 °C, 15 min; (i) HCl, MeOH; (j) MeSO2Cl, Et3N, THF; (k) n-BuLi, THF, −78 °C, I2; (l) 3-
SO2Me phenylboronic acid, PdCl2(PPh3)2, 1 M aq Na2CO3, CH3CN, microwave, 100 °C, 10 min.
warm to room temperature for 1 h. The reaction mixture was
quenched with 2 M HCl, followed by addition of saturated aqueous
NaCl. The mixture was extracted with ethyl acetate. The combined
organic extracts were dried with Na2SO4 and concentrated. The crude
reaction mixture was purified by flash chromatography to yield methyl
3-(tert-butoxycarbonylamino)furan-2-carboxylate (1.14 g, 51%). 1H
NMR (400 MHz, CDCl3) δ 8.18 (br s, 1H), 7.38 (d, J = 1.9 Hz, 1H),
7.25 (br s, 1H), 3.92 (s, 3H), 1.52 (s, 9H). MS (ESI): m/z (M + H)+
242.1. This intermediate (1.14 g, 1.0 equiv) was dissolved in
dichloromethane (8 mL) and treated with trifluoroacetic acid (5
mL). The reaction mixture was stirred at room temperature for 3 h
and was then concentrated. Residue was dissolved in dichloromethane
and washed with saturated aqueous NaHCO3. The organic layer was
dried (Na2SO4) and concentrated. The mixture was extracted with
ethyl acetate. The combined organic extracts were dried with Na2SO4
and concentrated. The crude reaction mixture was purified by flash
The mixture was slowly warmed to room temperature and stirred for
40 min. The reaction was concentrated. To the residue was added 6 N
HCl (3.5 mL), and the mixture was heated to 100 °C for 20 min. The
reaction mixture was allowed to cool to room temperature and was
neutralized with saturated aqueous NaHCO3. Solid was collected by
filtration to yield methyl 3-ureidofuran-2-carboxylate as a beige solid
which was used in the next reaction without further purification (MS
(ESI): m/z (M + H)+ 185.1). The crude intermediate was suspended
in methanol (6 mL) and treated with 1.5 M NaOH (1.5 mL). The
reaction mixture was heated to reflux for 90 min. The reaction mixture
was allowed to cool to room temperature and was acidified with 6 N
HCl to pH 3. The mixture was concentrated. Methanol was added to
the residue and the solid was filtered and dried at 95 °C under high
vacuum for 24 h to yield furo[3,2-d]pyrimidine-2,4-diol (90 mg, 84%
1
yield over two steps). H NMR (300 MHz, DMSO) δ 11.22 (br s,
1H), 11.05 (br s, 1H), 8.01 (d, J = 2.0 Hz, 1H), 6.52 (d, J = 2.0 Hz,
1H). MS (ESI): m/z (M + H)+ 153.0. This intermediate (39 mg, 1.0
equiv) was dissolved in POCl3 (1.8 mL). The mixture was cooled to
−40 °C, and N,N-diisopropylethylamine (0.45 mL) was slowly added.
The reaction mixture was then heated to reflux for 48 h, then cooled to
room temperature. The reaction mixture was poured into ice−water.
The mixture was extracted with ethyl acetate. The combined organic
1
chromatography to yield 37 (574 mg, 86%). H NMR (400 MHz,
CDCl3) δ 7.26 (m, 1H), 6.13 (d, J = 2.0 Hz, 1H), 4.57 (br s, 2H), 3.89
(s, 3H). MS (ESI): m/z (M + H)+ 142.0.
2-Chloro-4-morpholinofuro[3,2-d]pyrimidine (38). To a sol-
ution of 37 (100 mg, 1.0 equiv) in dichloromethane (3 mL) at −78 °C
was added chlorosulfonyl isocyanate (0.09 mL, 1.4 equiv) dropwise.
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a
sodium carbonate (36 mg, 3.5 equiv). The vial was sealed and heated
with stirring in the microwave to 150 °C for 15 min. The crude
reaction mixture was concentrated and purified by reverse phase
HPLC to afford 7 (42 mg, 88% yield). MS (ESI): m/z (M + H)+
498.2.
Scheme 3
4-(2-Chlorothieno[2,3-d]pyrimidin-4-yl)morpholine (43). A 5
L reaction vial equipped with a mechanical stirrer, internal temperature
probe, and a nitrogen bubbler was charged with 42 (95 g) and CH2Cl2
(2.85 L), and the mixture was cooled to −60 °C. Chlorosulfonyl
isocyanate was added at a rate such that the internal temperature
remained at −60 to −55 °C. After completion of addition the mixture
was allowed to warm to ambient temperature. The reaction was
monitored for complete consumption of starting material by LC/MS.
The reaction mixture was concentrated to dryness and the solid
residue transferred back to the 5 L reaction vial by water (1.8 L). This
mixture was heated at 75 °C for 1 h, then cooled to 30 °C. Next 10 M
aqueous NaOH (200 mL) was added and this mixture was heated at
85 °C for 20 min before cooling to room temperature. The mixture
was then acidified to pH 1 by the addition of concentrated HCl. The
mixture was then stirred for 18 h at ambient temperature with a
precipitate forming. This solid material was collected by vacuum
filtration and the filter cake washed with water. The solid material was
then dried in a vacuum oven at 55 °C for 24 h to afford thieno[2,3-
a
Reagents and conditions: (a) ethyl 4-bromobutanoate, K2CO3, cat.
NaI, acetone; (b) LiOH, H2O, THF; (c) PPA, toluene; (d) POCl3,
DMF; (e) K2CO3, methyl thioglycolate, DMF; (f) LiOH, H2O, THF;
(g) SOCl2; (h) N-methyl-2-chloroaniline, NEt3, DMAP, CH2Cl2; (i)
aryl boronate, Pd(PPh3)4, 1 M aq Na2CO3, DMF; (j) CuCN, DMF;
(k) NH2COCH3, Cs2CO3, Xantphos, Pd2(dba)3, dioxane; (l)
Mo(CO)6, MeOH, THF, amine, Herrmann’s catalyst, DBU.
1
d]pyrimidine-2,4(1H,3H)-dione as an off white solid (80 g, 79%). H
NMR (DMSO-d6, 400 MHz) δ 7.08 (d, J = 5.6 Hz, 1H), 7.12 (d, J =
5.6 Hz, 1H). ESI-MS: m/z = 169 [M + H]+. This intermediate (16.8 g,
0.1 mol) was added to POCl3 (100 mL), and the mixture was heated
at reflux overnight. After POCl3 was removed under reduce pressure
an aqueous NaHCO3 solution was added and extracted with EtOAc
(300 mL × 2). The organic phase was dried over Na2SO4 and filtered.
The filtrate was concentrated to give 2,4-dichlorothieno[2,3-d]-
layers were washed with saturated aqueous NaHCO3, dried (Na2SO4),
and concentrated to yield 2,4-dichlorofuro[3,2-d]pyrimidine (23 mg,
48%). 1H NMR (400 MHz, CDCl3) δ 8.10−8.08 (m, 1H), 7.04−7.02
(m, 1H). MS (ESI): m/z (M + H)+ 153.0. This intermediate (23 mg,
1.0 equiv) was suspended in methanol (1.7 mL) and treated with
morpholine (0.09 mL, 4.0 equiv). The reaction mixture was stirred at
room temperature for 2 h before being quenched with saturated
aqueous NaHCO3. The mixture was extracted with dichloromethane.
The combined organic layers were dried (Na2SO4) and concentrated
to yield 38 (14 mg, 48%). 1H NMR (400 MHz, CDCl3) δ 7.74 (d, J =
2.2 Hz, 1H), 6.79 (d, J = 2.2 Hz, 1H), 4.10−3.96 (m, 4H), 3.83 (dd, J
= 6.2, 3.6 Hz, 4H). MS (ESI): m/z (M + H)+ 240.0.
2-Chloro-4-morpholinofuro[3,2-d]pyrimidine-6-carbalde-
hyde (39). To a solution of 38 (40 mg, 1.0 equiv) in THF (1.7 mL)
at −78 °C was added 1.6 M solution of n-butyllithium (0.14 mL, 1.3
equiv, 1.6 M in hexanes). The reaction mixture was stirred at −78 °C
for 30 min. DMF (0.05 mL, 4.0 equiv) was added, and reaction
mixture was allowed to slowly warm to room temperature and stirred
for 90 min. The reaction was quenched with water, and the aqueous
layer was extracted with dichloromethane. The combined organic
layers were dried (Na2SO4) and concentrated. The crude reaction
mixture was purified by flash chromatography to yield 39 (22 mg,
1
pyrimidine (17.1 g, 84% yield). H NMR (DMSO-d6, 400 MHz) δ
8.07 (d, J = 6.0 Hz, 1H), 7.52 (d, J = 6.0 Hz, 1H). ESI-MS: m/z = 204
[M + H]+. To a solution of the dichloropyrimidine intermediate (10.2
g, 50 mmol) in EtOH (100 mL), morpholine (6.1 g, 70 mmol) was
added dropwise. The mixture was stirred at room temperature
overnight. TLC showed that the reaction was completed. The reaction
mixture was filtered and washed with a small amount of EtOH to give
43 (11.0 g, 85% yield). 1H NMR (DMSO-d6, 400 MHz) δ 7.78
(s,1H), 5.07 (s, 2H), 3.86 (t, J = 4.8 Hz, 4H), 3.72 (t, J = 4.8 Hz, 4H).
ESI-MS: m/z = 255.8 [M + H]+ .
2-Chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbalde-
hyde. To a solution of 4-(2-chlorothieno[2,3-d]pyrimidin-4-yl)-
morpholine (25.5 g, 0.1 mol) in THF (200 mL) was added n-BuLi
(64 mL, 0.16 mol) at −78 °C. The mixture was stirred for 2 h at −78
°C under N2, and DMF (28 g, 0.4 mol) was added dropwsie. The
reaction mixture was stirred for an additional hour at −78 °C, and the
reaction was quenched by 0.5 M HCl. The mixture was extracted with
CH2Cl2 (300 mL × 3), and the organic layer was dried over Na2SO4
and concentrated. The residue was purified by column chromatog-
raphy (CH2Cl2/EtOAc = 4:1) to give the desired product (21 g, 75%).
1H NMR (400 MHz, CDCl3) δ 10.00 (s, 1H), 8.01 (s, 1H), 4.08−4.03
1
50%). H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 7.48 (s, 1H),
4.19−4.00 (m, 4H), 3.92−3.81 (m, 4H). MS (ESI): m/z (M + H)+
268.0.
2-Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)-4-
morpholinofuro[3,2-d]pyrimidine (41). 39 (65 mg) was dissolved
in 1,2-dichloroethane (9.7 mL) and treated with the hydrochloride salt
of 1-methanesulfonylpiperazine (69 mg), sodium acetate (28 mg), and
trimethyl orthoformate (0.27 mL). The reaction mixture was stirred at
room temperature for 12 h. Sodium triacetoxyborohydride (62 mg)
was added, and reaction mixture was stirred at room temperature for 8
h. The reaction mixture was quenched with saturated aqueous
NaHCO3 and extracted with dichloromethane. The combined organic
layers were dried (Na2SO4) and concentrated. The crude reaction
mixture was purified by flash chromatography to yield 41 (70 mg,
(m, 4H), 3.91−3.86 (m, 4H). ESI-MS: m/z = 283.8 [M + H]+
.
(2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-
methanol. To a solution of 2-chloro-4-morpholinothieno[2,3-d]-
pyrimidine-6-carbaldehyde (21 g, 0.07 mol) in MeOH (200 mL),
NaBH4 (4.1 g, 0.11 mol) was added at 0 °C. The mixture was stirred
for 1 h. The mixture was quenched by 0.1 M HCl and extracted with
CH2Cl2. It was dried and concentrated to give the crude product,
which was used for the next step without further purification (18 g,
1
84%). H NMR (DMSO-d6, 400 MHz) δ 7.48 (s,1H), 5.74 (s, 1H),
4.68 (s, 1H), 3.83 (t, J = 4.8 Hz, 4H), 3.70 (t, J = 4.8 Hz, 4H). ESI-
1
MS: m/z = 285.8 [M + H]+
68%). H NMR (400 MHz, CDCl3) δ 6.63 (s, 1H), 4.04−3.97 (m,
.
4H), 3.87−3.80 (m, 4H), 3.73 (s, 2H), 3.32−3.25 (m, 4H), 2.80 (s,
4-(2-Chloro-6-(chloromethyl)thieno[2,3-d]pyrimidin-4-yl)-
morpholine. To a solution of (2-chloro-4-morpholinothieno[2,3-
d]pyrimidin-6-yl)methanol (18 g, 0.06 mol) in CH2Cl2 (400 mL),
SOCl2 (17 g, 0.15 mol) was added dropwise at 0 °C. The mixture was
stirred for 30 min, concentrated, and dissolved in CH2Cl2 (200 mL). It
was washed with aqueous NaHCO3, concentrated, and purified by
column chromatography (EtOAc/hexanes = 1:3) to give the desired
3H), 2.70−2.63 (m, 4H). MS (ESI): m/z (M + H)+ 416.1.
2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)-
methyl)-4-morpholinofuro[3,2-d]pyrimidine (7). 41 (40 mg, 1.0
equiv) was dissolved in toluene/ethanol/water (4:2:1, 1.6 mL) and
treated with 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-inda-
zole (59 mg, 2.5 equiv), PdCl2(PPh3)2 (6.8 mg, 0.10 equiv), and
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product (16 g, 89%). 1H NMR (CDCl3, 400 MHz) δ 7.81(s, 1H), 5.09
(s, 2H), 3.87−3.85 (m, 4H), 3.74−3.73 (m, 4H). ESI-MS: m/z =
diluted with CH2Cl2 (50 mL), washed with water, and dried over
Na2SO4. The solvent was removed under reduced pressure, and the
residue was purified by HPLC to give the desired product (41 mg,
14% yield). 1H NMR (CDCl3, 400 MHz) δ 8.98 (s, 1H), 8.28 (d, J =
0.8 Hz, 1H), 7.55−7.26 (m, 4H), 3.72 (t, J = 4.8 Hz, 4H), 3.72 (t, J =
4.8 Hz, 4H). ESI-MS: m/z = 337.8 [M + H]+.
308.3 [M + H]+
.
tert-Butyl 4-((2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-
6-yl)methyl)piperazine-1-carboxylate. To a solution of 4-(2-
chloro-6-(chloromethyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (1.5
g, 5.0 mmol) in THF (50 mL) was added tert-butyl piperazine-1-
carboxylate (1.86 g, 10 mmol) at room temperature. i-Pr2NEt (1.3 g,
10 mmol) was added dropwise after 20 min, and the mixture was
stirred at 70 °C overnight. The mixture was concentrated and purified
by silica gel chromatography (CH2Cl2/MeOH = 40:1) to give the
5-(4-Morpholinothieno[2,3-d]pyrimidin-2-yl)pyrimidin-2-
amine (10). To a solution of 43 (0.256 g) and 5-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine (263 mg) in acetonitrile
(2.0 mL) was added 1 M aqueous sodium carbonate (1 mL). The
reaction mixture was sealed with a rubber septum and degassed by
purging nitrogen through the mixture via a syringe. Next bis-
(triphenyphosphine)palladium dichloride (35 mg) was added under
a nitrogen flush and the reaction vessel was sealed. The reaction
mixture was heated on a Biotage Emrys Optimizer microwave at 160
°C for 10 min and then cooled to room temperature. The reaction
mixture was diluted with EtOAc and neutralized by titration with
concentrated HCl. A dark solid was removed from this reaction
mixture by vacuum filtration through a small bed of Celite. The
filtration pad was then flushed with EtOAc and water. The filtrate was
transferred to a separatory funnel, and the organic layer was separated
from the aqueous. The organic layer was dried (Na2SO4), the solvent
was removed in vacuo, and the residue was purified by RP-HPLC to
desired product (1.4 g, 65%). ESI-MS: m/z = 454.1 [M + H]+
.
4-(2-Chloro-6-(piperazin-1-ylmethyl)thieno[2,3-d]pyrimidin-
4-yl)morpholine. To a solution of tert-butyl 4-((2-chloro-4-
morpholinothieno[2,3-d]pyrimidin-6-yl)methyl)piperazine-1-carboxy-
late (1.4 g, 3.2 mmol) in MeOH (20 mL) was added MeOH/HCl (4
M, 20 mL) at 0 °C. The reaction mixture was stirred for 2 h at room
temperature. TLC showed the reaction was completed. The mixture
was concentrated to afford the desired product as HCl salt, which was
used for the next step without further purification (1.04 g, 87% yield).
ESI-MS: m/z = 354.1 [M + H]+
4-(2-Chloro-6-((4-(methyls.ulfonyl)piperazin-1-yl)methyl)-
thieno[2,3-d]pyrimidin-4-yl)morpholine. To a solution of 4-(2-
chloro-6-(piperazin-1-ylmethyl)thieno[2,3-d]pyrimidin-4-yl)-
morpholine (1.4 g, 3.2 mmol) in THF (80 mL) was added Et3N (1.29
g, 12.8 mmol) and MeSO2Cl (0.9 g, 8 mmol) at 0 °C. The reaction
mixture was stirred for 12 h at room temperature. The mixture was
quenched with ice−water and extracted with CH2Cl2 (50 mL × 3).
The combined organic layers were dried over Na2SO4 and
concentrated to give the crude product which was used for the next
step without further purification (0.65 g, 42% yield). 1H NMR
(DMSO-d6, 400 MHz) δ 7.77 (s, 1H), 4.49 (s, 2H), 3.97−3.67 (m,
6H), 3.56−3.40 (m, 6H), 3.07−2.99 (m, 4H), 2.35 (s, 3 H). ESI-MS:
1
give 10 (66 mg, 17% yield). H NMR (CDCl3, 400 MHz) δ 9.30 (s,
2H), 7.34 (d, J = 6.1 Hz, 1H), 7.26 (d, J = 6.1 Hz, 1H), 4.01−3.96 (m,
4H), 3.90−3.86 (m, 4H). ESI-MS: MH + 1 = 315.1.
5-(6-((4-Methylpiperazin-1-yl)methyl)-4-morpholinothieno-
[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine (11). To a solution of
4-(2-chloro-6-(chloromethyl)thieno[2,3-d]pyrimidin-4-yl)morpholine
(1.5 g, 5.0 mmol) in CH2Cl2 (10 mL) was added 1-methylpiperazine
(0.5 g, 5.0 mmol) at room temperature. i-Pr2NEt (0.78 g, 6.0 mmol)
was added dropwise after 20 min, and the mixture was stirred at 40 °C
for 3 h. LCMS showed that the reaction was completed. The mixture
was concentrated and purified by silica gel (CH2Cl2/MeOH = 40:1) to
give 4-(2-chloro-6-((4-methylpiperazin-1-yl)methyl)thieno[2,3-d]-
pyrimidin-4-yl)morpholine (1 g, 83% yield). ESI-MS: m/z = 367.9
[M + H]+. 11 was prepared as described for 10 from 4-(2-chloro-6-
((4-methylpiperazin-1-yl)methyl)thieno[2,3-d ]pyrimidin-4-yl)-
morpholine (15% yield). 1H NMR (CDCl3, 400 MHz) δ 9.31 (s, 2H),
8.16 (s, 1H), 4.7 (s, 2H), 4.02−3.78 (m, 8H), 3.62−3.48 (m, 8H),
3.56 (t, J = 9.6 Hz, 4H), 3.33−3.11 (m, 8H), 2.81 (s, 3H). LCMS
(ESI): m/z = 427.1 [M + H]+.
m/z = 432.0 [M + H]+
.
4-(2-(1H-Indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)-
methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (6). 4-(2-
Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[2,3-d]-
pyrimidin-4-yl)morpholine (412 mg) was combined with 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indazole (327 mg),
PdCl2(PPh3)2 (33 mg), 1 M aqueous sodium carbonate (3 mL),
and acetonitrile (3 mL). The mixture was heated to 140 °C in a
microwave reactor for 20 min. The contents were extracted with
EtOAc, concentrated, then purified by reverse phase HPLC to yield 6
1
Diethyl (2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-6-
yl)methylphosphonate. A solution of 4-(2-chloro-6-
(chloromethyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (5.0 g, 16.5
mmol) in P(OEt)3 (30 mL) was heated to 150 °C for 5 h. LCMS
showed that the reaction was completed. The mixture was
concentrated and purified by silica gel chromatography (CH2Cl2/
EtOAc = 1:2) to give the desired product (6.0 g, 95% yield). 1H NMR
(CDCl3, 400 MHz) δ 7.22 (s, 1H), 4.12 (q, J = 7.6 Hz, 4H), 3.93 (t, J
= 4.4 Hz, 4H), 3.82 (t, J = 4.4 Hz, 4H), 3.34 (d, J = 22.0 Hz, 2H), 1.31
(t, J = 7.6 Hz, 6H).
(193 mg, 39% yield). H NMR (CDCl3, 400 MHz) δ 2.67 (m, 4H),
2.81 (s, 3H), 3.30 (m, 4H), 3.83 (s, 2H), 3.92−3.94 (m, 4H), 3.98−
4.00 (m, 4H), 7.17 (s,1H), 7.50 (t, J = 7.8 Hz, 1H), 7.59 (d, J = 8.3 Hz,
1H), 8.31 (d, J = 7.0 Hz, 1H), 10.12 (br s,1H). ESI-MS: m/z = 514.10
[M + H]+.
5-(6-((4-(Methylsulfonyl)piperazin-1-yl)methyl)-4-
morpholinothieno[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine
(8). 4-(2-Chloro-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno-
[2,3-d]pyrimidin-4-yl)morpholine (150 mg, 0.35 mmol), 5-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)pyrimidin-2-amine (144 mg, 0.7
mmol), Cs2CO3 (228 mg, 0.7 mmol), and dioxane/H2O (3 mL/1 mL)
were combined in a microwave tube. The mixture was purged with N2
for 20 min. Then Pd(dppf)Cl2 (51 mg, 0.07 mmol) was added. The
reaction mixture was heated to 120 °C for 20 min under microwave
irradiation. It was diluted with CH2Cl2 (50 mL), washed with water,
and dried over Na2SO4. The solvent was removed by underduced
pressure, and the residue was purified by HPLC to give the desired
tert-Butyl 4-((2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-
6-yl)methylene)piperidine-1-carboxylate. To a mixture of so-
dium hydride (746 mg, 31 mmol) in THF at 0 °C under N2 was added
diethyl (2-chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-
methylphosphonate (4.20 g, 10 mmol) in THF. The mixture was
stirred for 30 min at 0 °C, and tert-butyl 4-oxopiperidine-1-carboxylate
(2.0 g, 12 mmol) in THF was added. The mixture was stirred at room
temperature for 3 h. TLC showed the reaction was completed. The
mixture was filtered through Celite. The filtrate was concentrated and
purified by chromatography to give the desired product (4.42 g, 93%
1
product (28 mg, 16%). H NMR (CDCl3, 400 MHz) δ 9.12 (s, 2H),
7.77 (s, 1H), 7.31 (s, 2H), 4.49 (s, 2H), 3.97−3.77 (m, 8H), 3.37−
3.35 (m, 8H), 2.99 (s, 3H). LCMS (ESI): m/z = 491.0 [M + H]+.
4-(2-(1H-Indazol-4-yl)thieno[2,3-d]pyrimidin-4-yl)-
morpholine (9). 4-(2-Chlorothieno[2,3-d]pyrimidin-4-yl)-
morpholine (255 mg, 1 mmol), 4-(4,4,5,5-tetramethyl-1,3,2-dioxabor-
olan-2-yl)-1H-indazole (366 mg, 1.5 mmol), Cs2CO3 (652 mg, 2
mmol), and dioxane/H2O (3 mL/1 mL) were combined in a
microwave tube. It was bubbled with N2 for 20 min. Pd(dppf)Cl2 (146
mg, 0.2 mmol) was added, and the mixture was heated under
microwave irradiation to 120 °C for 30 min. The reaction mixture was
1
yield). H NMR (400 MHz, CDCl3) δ: 7.03 (s, 1H), 6.40 (s, 1H),
3.94−3.92 (m, 4H), 3.84−3.82 (m, 4H), 3.53−3.48 (m, 4H), 2.65−
2.60 (m, 2H), 2.40−2.35 (m, 2H), 1.49 (s, 9H). LCMS (ESI): m/z =
451.1 [M + H]+.
4-(2-Chloro-6-(piperidin-4-ylidenemethyl)thieno[2,3-d]-
pyrimidin-4-yl)morpholine. To a solution of tert-butyl 4-((2-
chloro-4-morpholinothieno[2,3-d ]pyrimidin-6-yl)methylene)-
piperidine-1-carboxylate (4.42 g, 9.4 mmol) in methanol (10 mL) was
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added HCl/MeOH, and the reaction mixture was stirred at room
temperature for 4 h. LCMS showed the reaction was completed.
Solvent was removed under reduced pressure to give the product
tert-butyl ester (1.01 g) and sodium thiomethoxide (635 mg) was
heated to 80 °C in DMF (10 mL). After 4 h, the reaction mixture was
diluted with water, extracted with ethyl acetate, dried over MgSO4,
filtered, and concentrated to give 4-methylsulfanylpiperidine-1-
carboxylic acid tert-butyl ester (600 mg). This intermediate was
dissolved in chloroform (15 mL), and m-CPBA (1.46 g) was added.
After being stirred for 48 h, the reaction mixture was diluted with
CH2Cl2, washed with saturated aqueous NaHCO3, dried over MgSO4,
filtered, and concentrated to afford 4-methanesulfonylpiperidine-1-
carboxylic acid tert-butyl ester (505 mg). Treatment of this
intermediate with HCl in CH2Cl2 and MeOH yielded crude 4-
methanesulfonylpiperidine as its HCl salt. This intermediate (252 mg)
was added to 2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbal-
dehyde (250 mg) in 1,2-DCE (5 mL). Subsequently, triethyl
orthoformate (330 μL) was added. After the mixture was stirred for
6 h, Na(OAc)3BH (530 mg) was added. The mixture was stirred
overnight. The reaction was quenched by pouring onto water. The
aqueous layer was extracted with dichloromethane, and the combined
organics were dried over Na2SO4 and concentrated. This intermediate
was reacted with 2-amino-5-boronic acid according to a similar Suzuki
coupling procedure as described for compound 10 to afford 14 (12%
1
without further purification (3.5 g, 96% yield). H NMR (DMSO-d6,
400 MHz) δ 9.18 (s, 2H), 7.58 (s, 1H), 6.51 (s, 1H), 3.67 (t, J = 4.4
Hz, 4H), 3.73 (t, J = 4.4 Hz, 4H), 3.15 (m, 4H), 2.81 (d, J = 6.4 Hz,
2H), 2.50 (d, J = 6.4 Hz, 2H).
4-(2-Chloro-6-(piperidin-4-ylmethyl)thieno[2,3-d]pyrimidin-
4-yl)morpholine. A mixture of 4-(2-chloro-6-(piperidin-4-
ylidenemethyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (3.4 g, 9.7
mmol) and Pd/C (170 mg) in methanol was stirred at room
temperature under H2 (1 atm) for 4 h. LCMS showed that the
reaction was completed. Solvent was removed under reduced pressure
1
to give the product without further purification (3.2 g, 90% yield). H
NMR (CD3OD, 400 MHz) δ 7.31 (s, 1H), 3.95 (t, J = 4.4 Hz, 4H),
3.80 (t, J = 4.4 Hz, 4H), 3.40−3.20 (m, 2H), 3.01−2.90 (m, 4H),
1.97−1.93 (m, 3H), 1.53−1.43 (m, 2H). LCMS (ESI): m/z = 352.9
[M + H]+
.
4-(2-Chloro-6-((1-(2,2,2-trifluoroethyl)piperidin-4-yl)-
methyl)thieno[2,3-d]pyrimidin-4-yl)morpholine. To a solution
of 4-(2-chloro-6-(piperidin-4-ylmethyl)thieno[2,3-d]pyrimidin-4-yl)-
morpholine (0.6 g, 1.4 mmol) and i-Pr2NEt (0.54 g, 4.2 mmol) in
CH2Cl2 (20 mL) was added 2,2,2-trifluoroacetic anhydride (0.5 g, 2.8
mmol) at room temperature. The resulting mixture was stirred at
room temperature overnight. The mixture was purified by column
chromatography (petroleum ether/ethyl acetate, 5:1 to 2:1) to give
crude 1-(4-((2-chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-
1
yield over two steps). H NMR (400 MHz, CDCl3) δ 1.88−2.00 (m,
2H), 2.04−2.20 (m, 4H), 2.83−2.86 (m, 4H), 3.13−3.20 (m, 2H),
3.81 (s, 2H), 3.88−3.90 (m, 4H), 3.92−3.96 (m, 4H), 5.25 (br s, 2H),
7.18 (s, 1H), 9.37 (s, 1H). LCMS (ESI): m/z = 490.34 [M + H]+.
N-((2-(2-Aminopyrimidin-5-yl)-4-morpholinothieno[2,3-d]-
pyrimidin-6-yl)methyl)-N-methylmethanesulfonamide. To a
solution of 2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbalde-
hyde (100 mg., 0.350 mmol) in anhydrous THF (0.500 mL) was
added methylamine as a 2.0 M solution in THF (0.700 mL, 2.0 equiv).
This reaction mixture was stirred overnight at room temperature under
nitrogen. The reaction mixture was concentrated in vacuo, and the
resulting solid residue was taken into MeOH/THF, 1/1 (0.500 mL).
Next, NaBH3 was added as a solid in several aliquots. The resulting
slurry was stirred at room temperature for 2 h. The reaction mixture
was quenched with a minimum of dilute aqueous HCl. The reaction
mixture was then diluted with excess EtOAc and washed with water
(×1) and saline (×1). The organic was dried (Na2SO4), and the
solvent was removed in vacuo. The crude product was carried forward
1
methyl)piperidin-1-yl)-2,2,2-trifluoroethanone. H NMR (400 MHz,
CDCl3) δ 6.96 (s, 1H), 4.57−4.54 (m, 1H), 3.92−3.89 (m, 4H),
3.82−3.85 (m, 4H), 3.14−3.07 (m, 1H), 2.82−2.74 (m, 3H), 1.95−
1.83 (m, 3H), 1.38−1.21 (m, 3H). LCMS (ESI): M + H+ = 449.1. To
a solution of this intermediate intermediate (600 mg, 1.34 mmol) in
THF was added BH3/CH3SCH3 (10 M, 20 mL) dropwise at 0 °C.
The mixture was stirred at room temperature for 2 h. MeOH (75 mL)
was added dropwise to the mixture at −15 °C. Solvent was removed
under reduced pressure and the crude product was purified by column
chromatography (hexanes/EtOAc = 5:1 to 3:1) to afford 4-(2-chloro-
6-((1- (2,2,2-trifluoroethyl)piperidin-4-yl)methyl)thieno[2,3-d ]-
1
pyrimidin-4-yl)morpholine (250 mg, 41% yield over two steps). H
NMR (400 MHz, CDCl3) δ 6.92 (s, 1H), 3.92−3.89 (m, 4H), 3.85−
3.82 (m, 4H), 2.93−2.90 (m, 4H), 2.77−2.75 (d, 2H), 2.36−2.31 (m,
2H), 1.71−1.61 (m, 2H), 1.61−1.57 (m, 1H), 1.40−1.34 (m, 2H).
LCMS (ESI): M + H+ = 435.1.
5-(4-Morpholino-6-((1-(2,2,2-trifluoroethyl)piperidin-4-yl)-
methyl)thieno[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine (12).
12 was prepared as described for 10 from 4-(2-chloro-6-((1-(2,2,2-
trifluoroethyl)piperidin-4-yl)methyl)thieno [2,3-d] pyrimidin-4-yl)-
morpholine (14% yield). 1H NMR (400 MHz, CDCl3) δ 9.26 (s,
2H), 6.94 (s, 1H), 5.30−5.29 (d, 2H), 3.94−3.91 (m, 4H), 3.91−3.86
(m, 4H), 2.99−2.92 (m, 4H), 2.81−2.79 (m, 2H), 2.36−2.30 (m, 2H),
1.74−1.70 (m, 2H), 1.42−1.39 (m, 2H), 1.42 (s, 1H). LCMS (ESI):
m/z = 494.1 [M + H]+.
1
with out purification. H NMR (400 MHz, CDCl3) δ 7.17 (s, 1H),
3.98 (s, 2H), 3.95−3.90 (m, 4H), 3.86−3.81 (m, 4H), 2.51 (s, 3H).
LCMS (ESI): m/z = 299.0 [M + H]+. The crude amine was dissolved
in methylene chloride (5 mL), and the mixture was cooled to 0 °C
under nitrogen. Methanesulfonyl chloride (130 μL) was added, and
the mixture was stirred for 12 h at room temperature. The mixture was
diluted with water and 1 M HCl, and the aqueous layer was extracted
with CH2Cl2. The sample was dried over MgSO4 and concentrated.
Purification was by silica gel chromatography (0−5% MeOH in
1
CH2Cl2) to give the title compound (108 mg, 84% yield). H NMR
(400 MHz, C6D6) δ 6.91 (s, 1H), 4.00 (s, 2H), 3.45−3.38 (m, 4H),
3.35−3.28 (m, 4H), 2.44 (s, 3H), 2.18 (s, 3H). LCMS (ESI): m/z =
377.0 [M + H]+
.
5-(6-((1-(Methylsulfonyl)piperidin-4-yl)methyl)-4-
morpholinothieno[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine
(13). 4-(2-Chloro-6-(piperidin-4-ylmethyl)thieno[2,3-d]pyrimidin-4-
yl)morpholine (100 mg) was dissolved in CH2Cl2 (15 mL) and cooled
to 0 °C. Triethylamine (270 μL) and methanesulfonyl chloride (36
μL) were added, and the mixture was allowed to warm to room
temperature and stirred for 2 h. The reaction was quenched with
saturated aqueous NaHCO3, and the aqueous layer was extracted with
ethyl acetate. The combined organics were washed with brine, dried
over Na2SO4, filtered, and concentrated. The crude product was
utilized in a Suzuki coupling according to the procedure described for
N-((2-(2-Aminopyrimidin-5-yl)-4-morpholinothieno[2,3-d]-
pyrimidin-6-yl)methyl)-N-methylmethanesulfonamide (15).
15 was prepared as described for 9 from N-((2-(2-aminopyrimidin-
5-yl)-4-morpholinothieno[2,3-d]pyrimidin-6-yl)methyl)-N-methylme-
thanesulfonamide (9% yield). 1H NMR (400 MHz, DMSO) δ 9.10 (s,
2H), 7.61 (s, 1H), 4.51 (s, 2H), 3.97−3.87 (m, 4H), 3.82−3.68 (m,
4H), 3.00 (s, 3H), 2.88 (s, 3H). LCMS (ESI): m/z = 436.5 [M + H]+.
4-((2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-
methyl)-N,N-dimethylpiperazine-1-carboxamide. To a solution
of 2-chloro-4-morpholinothieno[2,3-d]pyrimidine-6-carbaldehyde
(200 mg) in 1,2-dichlorethane (10 mL) was added piperazine-1-
carboxylic acid dimethylamide hydrochloride (291 mg) and triethyl
orthoformate (390 μL). The mixture was stirred for 2 h at room
temperature. Na(OAc)3BH (674 mg) was added, and the resulting
reaction mixture was stirred for 16 h at room temperature. The
reaction was quenched with water, and the aqueous layer was extracted
with CH2Cl2. The combined organics were dried over Na2SO4,
filtered, and concentrated. The crude compound was purified by silica
1
10 to yield 13 (6 mg, 5% yield over two steps). H NMR (400 MHz,
DMSO-d6) δ 9.08 (s, 2H), 7.37 (s, 1H), 7.07 (s, 2H), 3.92−3.88 (m,
4H), 3.80−3.75 (m, 4H), 3.60−3.50 (m, 2H), 2.85 (app q, J = 12, 4
Hz), 2.83 (s, 3H), 2.69 (app t, J = 11.2 Hz), 1.75 (app t, J = 11.1 Hz,
3H), 1.33−1.20 (m, 2H). LCMS (ESI): m/z = 490.2 [M + H]+.
5-(6-((4-(Methylsulfonyl)piperidin-1-yl)methyl)-4-
morpholinothieno[2,3-d]pyrimidin-2-yl)pyrimidin-2-amine
(14). A mixture of 4-methanesulfonyloxypiperidine-1-carboxylic acid
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Journal of Medicinal Chemistry
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gel chromatography (4% MeOH in CH2Cl2) to yield the desired
compound (0.12 g, 27% yield).
CH2Cl2, and treated with a solution of 2-chloro-N-methylaniline (59
mg), Et3N (0.11 mL), and a trace amount of DMAP. The mixture was
allowed to warm to room temperature to stir overnight. The mixture
was diluted with saturated NaHCO3 and extracted with CH2Cl2. The
combined extracts were concentrated and the residue was purified by
flash column chromatography (24 g SiO2, 10−50% EtOAc in hexanes)
4-((2-(2-Aminopyrimidin-5-yl)-4-morpholinothieno[2,3-d]-
pyrimidin-6-yl)methyl)-N,N-dimethylpiperazine-1-carboxa-
mide (16). 4-((2-Chloro-4-morpholinothieno[2,3-d]pyrimidin-6-yl)-
methyl)-N,N-dimethylpiperazine-1-carboxamide was reacted with 2-
amino-5-boronic acid according to a similar Suzuki coupling procedure
1
to give 18 as a colorless solid (58 mg, 41% yield). H NMR (500
1
as described for compound 11 to afford 16 (47% yield). H NMR
MHz, DMSO) δ 7.69−7.60 (m, 2H), 7.57−7.48 (m, 2H), 7.41 (br d, J
= 8.1 Hz, 1H), 7.23−7.18 (m, 1H), 7.06−7.01 (m, 1H), 6.98 (d, J =
8.1 Hz, 1H), 6.61 (br s, 1H), 4.17 (t, J = 5.0 Hz, 2H), 3.27 s, 3H), 2.95
(br s, 2H). LCMS: (M)+ 370.1.
(400 MHz, CDCl3) δ 2.55 (m, 4H), 2.84 (s, 6H), 3.32 (m, 4H), 3.79
(s, 2H), 3.89−3.91 (m, 4H), 3.95−3.97 (m, 4H), 5.24 (br s, 2H), 7.14
(s, 1H), 9.30 (s, 2H). MS (ESI+): (M + H)+ 484.31.
4-(2-Chloro-5-iodothieno[2,3-d]pyrimidin-4-yl)morpholine.
4-(2-Chlorothieno[2,3-d]pyrimidin-4-yl)morpholine (500 mg) in
THF (50 mL) was cooled to −78 °C. n-BuLi (2 mL, 2.5 M in
hexane) was added, and the reaction mixture was warmed to −40 °C
and stirred for 50 min. The mixture was then recooled to −78 °C, and
a solution of I2 (1000 mg) in THF (30 mL) was added. The reaction
mixture was warmed to room temperature and after 30 min was
quenched with water and diluted with CH2Cl2. The organic layer was
separated, washed with saturated aqueous Na2SO3 and brine, then
dried over Na2SO4, filtered, and concentrated to yield the desired
8-Bromo-3,4-dihydrobenzo[b]oxepin-5(2H)-one (45). Solid
3-bromophenol (10.0 g, 58 mmol) was added portionwise to a stirred
suspension of K2CO3 in acetone (100 mL) at room temperature.
Sodium iodide (NaI, 1.0 g) was added, followed by ethyl 4-
bromobutyrate (9.2 mL, 64 mmol). The reaction mixture was heated
at 80 °C overnight, cooled to room temperature, diluted with water,
and extracted with ethyl acetate to give crude ethyl 4-(3-
bromophenoxy)butanoate that was used in the next saponification
without purification. The above crude residue was taken up in 100 mL
of THF and 50 mL of water and treated with lithium hydroxide LiOH
(hydrate, 4.9 g). The whole was heated at 50 °C for 2 days. The
mixture was cooled to room temperature and acidified to pH 1 with 2
N HCl. The aqueous layer was extracted with ethyl acetate. The
combined organics were washed with brine and dried over sodium
sulfate to give crude 4-(3-bromophenoxy)butanoic acid as a sticky
1
compound (650 mg, 90% yield). H NMR (400 MHz, CDCl3) δ 7.53
(s, 1H), 3.94−3.82 (m, 4H). MS (ESI+): (M + H)+ 382.2.
5-(6-(3-(Methylsulfonyl)phenyl)-4-morpholinothieno[2,3-d]-
pyrimidin-2-yl)pyrimidin-2-amine (17). 2-Chloro-6-iodo-4-
morpholinothieno[2,3-d]pyrimidine (525 mg), 3-(methylsulfonyl)-
phenylboronic acid (303 mg), and bis(triphenylphosphine)palladium-
(II) dichloride (96 mg) in 1 M Na2CO3 aqueous solution (4.2 mL)
and acetonitrile (9 mL) were heated to 100 °C in a sealed microwave
reactor for 30 min. Upon completion, the mixture was extracted with
dichloromethane (3 × 15 mL). The combined organic layers were
dried with sodium sulfate, filtered, and concentrated. The crude
mixture was purified by flash chromatography to yield 4-(2-chloro-6-
(3-(methylsulfonyl)phenyl)thieno[2,3-d]pyrimidin-4-yl)morpholine
which was used in the next step (MS (ESI+): (M + H)+ 410.0). 17 was
prepared as described for 10 from 4-(2-chloro-6-(3-(methylsulfonyl)-
phenyl)thieno[2,3-d]pyrimidin-4-yl)morpholine (7% yield over two
1
solid. H NMR (DMSO-d6, 500 MHz) 7.24 (m, 1H), 7.13 (m, 1H),
7.11 (m, 1H), 6.95 (m, 1H), 3.99 (m, 2H), 2.37 (m, 2H), 1.94 (m,
2H). To a stirred suspension of polyphosphoric acid (PPA, ∼60 g)
and Celite (∼40 g) in 100 mL of toluene were added crude 4-(3-
bromophenoxy)butanoic acid (∼58 mmol) in one portion and 10 mL
of toluene rinse. The resultant suspension was heated at 110 °C for 5
h. The toluene was decanted through a plug of Celite, and the
remaining slurry was washed repeatedly with toluene and ethyl acetate.
The eluent was concentrated and purified by flash column
chromatography (4:1 hexane/EtOAc) to give 45 (7 g, ∼50% yield
1
over two steps). H NMR (DMSO-d6, 500 MHz) δ 7.55 (d, J = 8.5
1
steps). H NMR (400 MHz, CDCl3) δ 9.13 (s, 2H), 8.29−8.25 (m,
Hz, 1H), 7.37 (d, J = 1.5 Hz, 1H), 7.35 (dd, J = 8.5, 1.5 Hz, 1H), 4.24
(t, J = 6.5 Hz, 2H), 2.79 (t, J = 7.0 Hz, 2H), 2.14 (m, 2H).
1H), 8.22−8.18 (m, 1H), 8.15 (s, 1H), 7.95−7.91 (m, 1H), 7.77 (t, J
= 8 Hz, 1H), 7.24 (br s, 2H), 4.06−3.98 (m, 4H), 3.86−3.78 (m, 4H).
MS (ESI+): (M + H)+ 469.1.
Methyl 8-Bromo-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-
2-carboxylate (46). Phosphorus oxychloride (1.88 mL, 20.8 mmol)
was added dropwise to DMF (5 mL) at 0 °C. After 30 min a solution
of 8-bromo-3,4-dihydrobenzo[b]oxepin-5(2H)-one (2.0 g, 8. 3 mmol)
in 8 mL of DMF was added dropwise. The reaction mixture was
allowed to reach room temperature and was stirred for 2 h, then
poured slowly over rapidly stirred ice−water. The aqueous layer was
extracted with ethyl acetate and the combined organics were washed
with brine, dried over sodium sulfate, and concentrated to give a crude
residue which was used in the next step without purification. The
residue was dissolved in 10 mL of DMF and treated sequentially with
potassium carbonate (2.20 g, 16.6 mmol) and methyl thioglycolate
(0.83 mL). The whole was heated at 50 °C overnight, cooled to room
temperature, and diluted with water. The aqueous layer was extracted
with ethyl acetate. The combined organics were washed with brine,
dried over sodium sulfate, and concentrated. The crude residue was
purified by flash column chromatography (20−50% ethyl acetate in
4,5-Dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxylic
Acid. POCl3 (4.66 mL, 51.2 mmol) was added dropwise to 12 mL of
DMF at 0 °C. After 30 min, 3,4-dihydrobenzo[b]oxepin-5(2H)-one
(3.32 g, 20.5 mmol) was added as a solution in 15 mL of DMF. The
solution was stirred, warming to room temperature over 2 h. The
mixture was poured over ice−water, and the aqueous layer was
extracted with EtOAc. The combined organics were washed with
brine, dried over sodium sulfate, and concentrated. The residue was
taken up in DMF (20 mL) and treated sequentially with potassium
carbonate (5.66 g, 41 mmol) and then methyl thioglycolate (2.05 mL,
22.6 mmol). The mixture was heated at 50 °C overnight. Water was
added and the mixture extracted with EtOAc. The combined organics
were washed with brine, dried over sodium sulfate, and concentrated.
Purification by flash column chromatography gave 4.22 g of the methyl
ester as a light yellow solid. Then 3.0 g of the methyl ester (11.5
mmol) was taken up in 20 mL of THF and 10 mL of water and treated
with LiOH (monohydrate, 970 mg, 2 equiv). The mixture was heated
at 50 °C for 3 h. The solution was acidified with 2 N HCl and
extracted with EtOAc and the combined organics were washed with
brine, dried over sodium sulfate, and concentrated to give the acid as a
colorless solid (2.64 g, 73% yield over two steps). 1H NMR (500
MHz, DMSO) δ 13.11 (br s, 1H), 7.79−7.64 (m, 1H), 7.60 (s, 1H),
7.27 (ddd, J = 8.1, 7.3, 1.6 Hz, 1H), 7.11 (ddd, J = 7.9, 7.3, 1.3 Hz,
1H), 7.04 (dd, J = 8.1, 1.2 Hz, 1H), 4.31−4.26 (t, J = 5.2 Hz, 2H), 3.20
(t, J = 5.2 Hz, 2H).
1
hexanes) to give 46 (2.20 g, 78% yield) as a colorless solid. H NMR
(DMSO-d6, 500 MHz) δ 7.70 (s, 1H), 7.67 (d, J = 8.5 Hz, 1H), 7.31−
7.28 (m, 2H), 4.32 (t, J = 5.0 Hz, 2H), 3.84 (s, 3H), 3.21 (t, J = 5.0
Hz, 2H).
8-Bromo-N-(2-chlorophenyl)-N-methyl-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-2-carboxamide (47). A solution of 46
(2.0 g, 5.9 mmol) was suspended in 5 mL of THF and 5 mL of water.
LiOH (monohydrate, 620 mg) was added in one portion. The mixture
was heated at 50 °C for 3 h. The mixture was cooled to room
temperature and acidified with 2 N HCl. The mixture was extracted
with EtOAc and the combined organics were washed with brine and
dried over sodium sulfate. The crude residue was dissolved in 7 mL of
SOCl2 and heated at 80 °C for 2 h. The mixture was cooled to room
temperature and concentrated. The crude yellow residue was diluted
N-(2-Chlorophenyl)-N-methyl-4,5-dihydrobenzo[b]thieno-
[2,3-d]oxepine-2-carboxamide (18). A solution of the carboxylic
acid (94 mg) was heated with 2 mL of SOCl2 at 80 °C for 2 h. The
mixture was concentrated by rotoevaporation, dissolved in 2 mL of
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Journal of Medicinal Chemistry
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with CH2Cl2 (10 mL) and cooled to 0°C. A solution of 2-chloro-N-
methyaniline (963 mg) and Et3N (1.64 mL) in 5 mL of CH2Cl2 was
added over a few minutes. The mixture was allowed to warm to room
temperature and was stirred overnight. The mixture was diluted with
saturated NaHCO3 and extracted with CH2Cl2. The combined extracts
were concentrated and the residue was purified by flash column
chromatography (80 g SiO2, 10−50% EtOAc in hexanes) to give 47 as
(6.59 g, 69% yield). 1H NMR (400 MHz, CDCl3) δ 7.56 (dd, J = 1.6,
7.2 Hz, 2H), 7.48−7.42 (m, 2H), 7.35−7.30 (m, 3H), 6.72 (s, 1H),
4.18 (t, J = 5.2 Hz, 2H), 3.83 (s, 3H), 3.33 (s, 3H), 2.99 (t, J = 5.2 Hz,
2H). MS (ESI+): (M + H)+ 428.0.
2-[(2-Chlorophenyl)methylcarbamonyl]-4,5-dihydro-6-oxa-
1-thiabenzo[e]azulene-8-carboxylic Acid. To a solution of
methyl 2-((2-chlorophenyl)(methyl)carbamoyl)-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-8-carboxylate (6.59 g, 15.4 mmol) in THF (25
mL) and water (25 mL) was added LiOH monohydrate (1.29 g). After
the solution was heated at 50 °C for 2 h, it was cooled to room
temperature and acidified with 2 N HCl. The precipitate was separated
1
a colorless solid (2.10 g, 79% over three steps.). H NMR (500 MHz,
DMSO) δ 7.70−7.60 (m, 2H), 7.56−7.47 (m, 2H), 7.35 (br d, J = 8.2
Hz, 1H), 7.26−7.18 (overlapping m, 2H), 6.56 (br s, 1H), 4.19 (br t, J
= 4.8 Hz, 2H), 3.31 (s, 3H), 2.94 (br s, 2H). MS (ESI+): (M + H)+
449.9.
1
and dried to give the desired product (6.06 g, 95% yield). H NMR
(400 MHz, CDCl3) δ 7.62 (dd, J = 1.2, 8.0 Hz, 2H), 7.48−7.44 (m,
2H), 7.37−7.30 (m, 3H), 6.75 (s, 1H), 4.19 (t, J = 5.2 Hz, 2H), 3.33
(s, 3H), 3.00 (t, J = 5.2 Hz, 2H). MS (ESI+): (M + H)+ 414.0.
2-((2-Chlorophenyl)(methyl)carbamoyl)-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-8-carbonyl Chloride. A solution of 2-
[(2-chlorophenyl)methylcarbamonyl]-4,5-dihydro-6-oxa-1-thiabenzo-
[e]azulene-8-carboxylic acid (200 mg, 0.24 mmol) in SOCl2 (4 mL)
was heated at 80 °C for 2 h. It was concentrated to give the crude acid
chloride, which used in the next step without further purification.
N2-(2-Chlorophenyl)-N8-ethyl-N2-methyl-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-2,8-dicarboxamide (23). A solution of
2-[(2-chloro-phenyl)methylcarbamonyl]-4,5-dihydro-6-oxa-1-
thiabenzo[e]azulene-8-carboxylic acid (200 mg, 0.24 mmol) in SOCl2
(4 mL) was heated at 80 °C for 2 h. It was concentrated to give the
crude acid chloride. The crude acid chloride was taken up in THF (20
mL) and added to a solution of CH3CH2NH2 (42 mg, 0.92 mmol)
and pyridine (0.2 mL) in THF (5 mL). After the mixture was stirred at
room temperature overnight, it was concentrated, dissolved in EtOAc,
washed with water and brine, dried over Na2SO4, and concentrated to
give the desired product (119 mg, 27% yield over two steps). 1H NMR
(400 MHz, DMSO-d6) δ 7.53−7.47 (m, 2H), 7.42−7.33 (m, 2H), 6.73
(s, 1H), 6.03 (s, 1H), 4.23 (t, J = 4.8 Hz, 2H), 3.50−3.42 (m, 2H),
3.38 (s, 3H), 3.03 (t, J = 4.8 Hz, 2H), 1.27−1.21 (m, 3H). MS (ESI+):
(M + H)+ 441.0.
8-Bromomethyl-4,5-dihydro-6-oxa-1-thiabenzo[e]azuene-2-
carboxylic Acid (2-Chlorophenyl)methylamide. A mixture of 2-
[(2-chlorophenyl)methylcarbamonyl]-4,5-dihydro-6-oxa-1-thiabenzo-
[e]azulene-8-carboxylic acid methyl ester (1 g, 2.3 mmol) and lithium
aluminum hydride in dry THF (30 mL) was stirred at 0 °C under
nitrogen for 30 min and monitored by LCMS. The reaction mixture
was quenched by the slow addition of water with stirring and warmed
to room temperature. The aqueous layer was extracted with EtOAc,
and the combined organics were dried over anhydrous Na2SO4,
filtered, and concentrated. The crude product was recrystallized from
MeOH to give crude 8-hydromethyl-4,5-dihydro-6-oxa-1-thiabenzo-
[e]azuene-2-carboxylic acid (2-chlorophenyl)methylamide as a yellow
solid (LCMS (ESI+): (M + H)+ 399.9). To a solution of this
intermediate (1.25 g, 3.13 mmol) in THF (20 mL) was added a
solution of PBr3 (0.88 mL) in CH2Cl2 (20 mL) dropwise. The
resulting mixture was stirred at room temperature overnight. The
reaction mixture was quenched with water and extracted with EtOAc.
The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated under vacuum to give a yellow oil. The crude product
was recrystallized from MeOH to give the titled product as a yellow
solid (1.4 g, 48% yield over two steps). 1H NMR (CDCl3, 400 MHz) δ
7.51 (d, J = 7.6 Hz, 1H), 7.41−7.33 (m, 4H), 6.98 (dd, J = 2.4, 4.0 Hz,
2H), 6.73 (s, 1H), 4.38 (s, 2H), 4.21 (t, J = 5.2 Hz, 2H), 3.37 (s, 3H),
3.00 (t, J = 5.2 Hz, 2H). LCMS (ESI+): (M + H)+ 461.8.
N-(2-Chlorophenyl)-N-methyl-8-(1H-pyrazol-5-yl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide (19). A
solution of 47 (200 mg, 0.47 mmol) in MeCN (1.5 mL) and water
(1.5 mL) was treated with 3-pyrazoleboronic acid (65 mg) and
potassium acetate (150 mg). The mixture was purged with nitrogen
and then charged with Pd(PPh3)4 (41 mg). The mixture was heated by
microwave irradiation at 140 °C for 20 min. The mixture was diluted
with water and extracted with ethyl acetate. Purification by reverse
1
phase HPLC gave 19 as a colorless solid (194 mg, 83% yield). H
NMR (400 MHz, DMSO) δ 13.30, 12.93 (br s, 0.2 + 0.8H, pyrazole
tautomer NHs), 7.76−7.34 (overlapping m, 8H), 6.73 (s, 1H), 6.59
(br s, 1H), 4.20 (br s, 2H), 3.30 (s, 3H, obstructed by water), 2.97 (br
s, 2H). LCMS: (M)+ 436.1.
N-(2-Chlorophenyl)-8-cyano-N-methyl-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carboxamide (20). A solution of 47 (50
mg, 0.11 mmol) in 1 mL of DMF was treated with CuCN (30 mg, 3
equiv). The mixture was heated by microwave irradiation at 250 °C for
20 min. Saturated ammonium chloride was added and the mixture
extracted with ethyl acetate. The combined extracts were filtered over
Celite, concentrated and the residue was purified by reverse phase
HPLC to give 20 (16 mg, 36% yield). 1H NMR (500 MHz, DMSO) δ
7.66 (br s, 2H), 7.62−7.45 (overlapping m, 4H), 6.65 (br s, 1H), 4.23
(br s, 2H), 3.29 (br s, 3H), 3.01 (br s, 2H). LCMS: (M)+ 395.1.
8-Acetamido-N-(2-chlorophenyl)-N-methyl-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide (21). A
solution of 47 (100 mg, 0.22 mmol), cesium carbonate (158 mg,
2.2 equiv), acetamide (20 mg, 1.5 equiv), and xantphos (13 mg, 0.10
equiv) in 2 mL of dioxane was sparged with nitrogen. Pd2dba3 (10 mg,
0.05 equiv) was added and the mixture heated at 100 °C in a sealed
tube for 8 h. Water was added and the mixture extracted with EtOAc.
Concentration and purification by reverse phase HPLC gave 21 (53
1
mg, 56% yield). H NMR (500 MHz, DMSO) δ 10.03 (s, 1H), 7.65
(m, 2H), 7.52 (m, 2H), 7.36 (m, 2H), 7.18 (m, 1H), 6.50 (br s, 1H),
4.15 (br s, 2H), 3.27 (s, 3H), 2.91 (br s, 2H), 2.03 (s, 3H). LCMS:
(M)+ 427.1.
Methyl 2-((2-Chlorophenyl)(methyl)carbamoyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-8-carboxylate (22).
Under ambient atmosphere, a solution of 47 (100 mg, 0.22 mmol)
in 2 mL of THF was treated sequentially with molybdenum
hexacarbonyl (58 mg, 0.22 mmol), methylamine (2.0 M in THF,
0.33 mL, 0.66 mmol), and Hermann’s catalyst (31 mg, 15 mol %).
DBU (0.02 mL, 0.11 mmol) was added and the vial quickly sealed.
The mixture was heated by microwave irradiation at 150 °C for 20
min. The mixture was diluted with EtOAc and filtered over Celite.
Concentration and purification by flash column chromatography and
then reverse-phase HPLC gave 22 (25 mg, 26% yield). 1H NMR (500
MHz, DMSO) δ 8.42 (br q, 1H), 7.70−7.60 (m, 2H), 7.57−7.50 (m,
2H), 7.49 (s, 1H), 7.43 (s, 1H), 6.56 (br s, 1H), 4.20 (br s, 2H), 3.28
(s, 3H), 2.97 (br s, 2H), 2.75 (d, J = 4.5 Hz, 3H). LCMS: (M)+ 427.1.
Methyl 2-((2-Chlorophenyl)(methyl)carbamoyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-8-carboxylate. A mix-
ture of 8-bromo-N-(2-chlorophenyl)-N-methyl-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carboxamide (10 g, 0.022 mol), Pd(OAc)2
(2.47 g, 0.011 mmol), dppf (10 g, 0.018 mol), and triethylamine
(4.45 g, 0.044 mol) in DMF (50 mL) and MeOH (100 mL) was
stirred under CO atmosphere (50 psi) at 70 °C for 2 days. After it was
filtered and concentrated, the crude product was purified by column
chromatography (hexanes/EtOAc = 5:1) to give the desired product
N-(2-Chlorophenyl)-N-methyl-8-((methylamino)methyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide (24). To
a solution of CH3NH2 (0.25 mL) in EtOH was added Et3N (0.5 mL)
and 8-bromomethyl-4,5-dihydro-6-oxa-1-thiabenzo[e]azuene-2-carbox-
ylic acid (2-chlorophenyl)methylamide (250 mg, 0.55 mmol). The
reaction mixture was stirred at 60 °C for 2 h. The reaction mixture was
added to water, and the aqueous layer was extracted with EtOAc (20
mL × 3). The sample was dried over Na2SO4, concentrated, and
purified by silica gel flash chromatography (CH2Cl2/MeOH = 10:1) to
1
give 24 as a light yellow solid (96 mg, 43%). H NMR (CDCl3, 400
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Journal of Medicinal Chemistry
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MHz) δ 9.73 (br, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.37−7.29 (m, 4H),
7.15 (d, J = 8.0 Hz, 1H), 7.02 (s, 1H), 6.82 (s, 1H), 4.12 (t, J = 4.8 Hz,
2H), 3.90 (s, 2H), 3.31 (s, 3H), 2.94 (t, J = 4.8 Hz, 2H), 2.45 (s, 3H).
LCMS (ESI+): (M + H)+ 412.9.
1H), 4.28−4.19 (m, 2H), 3.25 (s, 3H), 2.98−2.90 (m, 2H). LCMS
(ESI+): (M + H)+ 471.1.
8-(3-(Aminomethyl)phenyl)-N-(2-chlorophenyl)-N-methyl-
4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide
(27). A solution of N-(2-chlorophenyl)-8-(3-cyanophenyl)-N-methyl-
4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide (300 mg,
0.63 mmol), Raney Ni (1 g), and NH3·H2O (2.5 mL) in MeOH
(50 mL) was stirred at 30 °C under hydrogen atmosphere (50 psi).
The reaction mixture was filtered through Celite, concentrated,
washed with water, and purified by preparative TLC to afford 27 (34
mg, 11% yield). 1H NMR (DMSO-d6, 400 MHz) δ 8.38 (s, 2H), 7.87
(s, 1H), 7.71−7.65 (m, 3H), 7.34−7.41 (m, 6H), 7.35 (s, 1H), 6.59 (s,
1H), 4.22 (s, 2H), 4.07 (s, 2H), 3.33 (s, 3H), 2.98 (s, 2H). LCMS
(ESI+): (M + H)+ 475.1.
N8-(2-Aminoethyl)-N 2-(2-chlorophenyl)-N 2-methyl-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2,8-dicarboxamide
(25). 25 was prepared according to the procedure described for 23
from 2-((2-chlorophenyl)(methyl)carbamoyl)-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-8- carbonyl chloride (30% yield). 1H NMR
(400 MHz, CDCl3) δ 8.30−8.10 (m, 3H), 7.45−7.19 (m, 5H), 6.30 (s,
1H), 3.92 (s, 2H), 3.57−3.10 (m, 9H), 2.70−2.59 (m, 2H). MS (ESI
+): (M + H)+ 456.0.
(3-Bromophenyl)acetic Acid Methyl Ester. A solution of (3-
bromophenyl)acetic acid (5 g, 23.3 mmol) and concentrated H2SO4
(2 mL) in CH3OH (100 mL) was stirred at reflux for 3 h. The solution
was cooled, concentrated, and diluted with water and EtOAc. The
organic layer was washed with water and dried to give (3-
bromophenyl)acetic acid methyl ester (5.1 g, 96%). 1H NMR
(DMSO-d6, 400 MHz) δ 7.47 (s, 1H), 7.40 (m, 1H), 7.25 (m, 2H),
3.68 (s, 2H), 3.58 (s, 3H). ESI-MS, m/z (M + H+): 228.
N2-(2-Chlorophenyl)-N2-methyl-4,5-dihydrobenzo[b]thieno-
[2,3-d]oxepine-2,9-dicarboxamide (28). To a mixture of 30 (120
mg) and K2CO3 (49 mg) in dry DMSO (2 mL) was added 33% H2O2
(40 μL). The reaction mixture stirred overnight at room temperature.
Ice was added to the resulting solution, and the solid formed was
collected, washed with water, and dried to afford 30 (119 mg, 95%
1
yield). H NMR (CDCl3, 400 MHz) δ 7.99 (d, J = 1.81 Hz, 1H),
[3-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)phenyl]-
acetic Acid Methyl Ester. A mixture of (3-bromophenyl)acetic acid
methyl ester (2.0 g, 8.7 mmol), 4,4,5,5,4′,4′,5′,5′-octamethyl[2,2′]bi-
[[1,3,2]dioxaborolanyl] (3.33 g, 13.1 mmol), KOAc (2.56 g, 26.1
mmol), and Pd(dppf)Cl2 (400 mg) in DMSO (40 mL) was stirred at
80 °C for 4 h. The cooled solution was poured into water and
extracted with EtOAc. The organic layer was washed with water, dried,
concentrated, and purified by column to afford the crude product [3-
(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)phenyl]acetic acid methyl
ester (1.6 g). 1H NMR (DMSO-d6, 400 MHz) δ 7.56 (s, 1H), 7.54 (d,
J = 1.2 Hz, 1H), 7.3 (m, 2H), 3.7 (s, 2H), 3.6 (s, 3H), 1.28 (s, 12H).
Methyl 2-(3-(2-((2-Chlorophenyl)(methyl)carbamoyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepin-8-yl)phenyl)acetate. A
mixture of 8-bromo-N-(2-chlorophenyl)-N-methyl-4,5-dihydrobenzo-
[b]thieno[2,3-d]voxepine-2-carboxamide (500 mg, 1.11 mmol), [3-
(4,4,5,5-tetramethyl[1, 3, 2]dioxaborolan-2-yl)phenyl]acetic acid
methyl ester (615 mg, 2.23 mmol), sodium carbonate (460 mg, 3.33
mmol), and Pd(dppf)Cl2 (40 mg) in CH3CN (4 mL) was heated
under microwave irradiation at 100 °C for 50 min. The mixture was
filtered through Celite, concentrated, and purified by preparative
HPLC to afford the product methyl 2-(3-(2-((2-chlorophenyl)-
(methyl)carbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepin-8-yl)-
phenyl)acetate (200 mg, 35% yield). 1H NMR (DMSO-d6, 400 MHz)
δ 7.65−7.63 (m, 2H), 7.62−7.48 (m, 5H), 7.47−7.32 (m, 2H), 7.24−
7.22 (m, 2H), 6.57 (s, 1H), 4.19 (t, J = 4.8 Hz, 2H), 3.72 (s, 2H), 3.59
(s, 3H), 3.25 (s, 3H), 2.95(s, 2H). LCMS (ESI+): (M + H)+ 518.2.
2-(3-(2-((2-Chlorophenyl)(methyl)carbamoyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepin-8-yl)phenyl)acetic Acid
(26). A mixture of methyl 2-(3-(2-((2-chlorophenyl)(methyl)-
carbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepin-8-yl)phenyl)-
acetate (100 mg, 2.4 mmol) and LiOH (16.2 mg, 0.39 mmol) in THF/
H2O (10 mL/10 mL) was stirred at 50 °C for 2 h. After the solution
was cooled, it was adjusted to pH 2 with HCl (aqueous, 6 N). The
resulting solution was concentrated and extracted with EtOAc. The
combined organic layer was washed with water and concentrated to
afford the product (90 mg, 92% yield). 1H NMR (CDCl3, 400 MHz) δ
7.47−7.41 (m, 4H), 7.34−7.29 (m, 4H), 7.19 (s, 1H), 7.15−7.13 (m,
2H), 6.71 (s, 1H), 4.20 (t, J = 5.2 Hz, 2H), 3.64 (s, 2H), 3.33 (s, 3H),
2.98 (t, J = 5.2 Hz, 2H). LCMS (ESI+): (M + H)+ 501.7.
7.60−7.55 (m, 2H), 7.45−7.39 (m, 3H), 7.03 (d, J = 8.44 Hz, 1H),
6.71 (s, 1H), 5.75 (br s, 2H-NH2), 4.28 (t, J = 5.06 Hz, 2H), 3.42 (s,
3H), 3.04 (t, J = 5.02, 2H). LCMS (ESI+): (M + H)+ 413.14.
N2-(2-Chlorophenyl)-N9-ethyl-N2-methyl-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-2,9-dicarboxamide (29). A mixture of
28 (70 mg), TFA (37.5 μL), triethylsilane (80 μL), and acetaldehyde
(28 μL) in acetonitrile was stirred at room temperature overnight and
then was concentrated. Purification on silica yielded 29 (34 mg, 46%
1
yield). H NMR (CDCl3, 400 MHz) δ 7.81 (m, 1H), 7.47−7.44 (m,
2H), 7.36−7.29 (m, 3H), 6.91 (d, J = 8.41 Hz, 1H), 6.59 (s, 1H), 5.94
(m, 1H), 4.17 (t, J = 5.10 Hz, 2H), 3.43 (m, 2H), 3.32 (s, 3H), 2.93 (t,
J = 5.07 Hz, 2H), 1.21 (t, J = 7.28, 3H). LCMS (ESI+): (M + H)+
482.15.
3-Bromo-4-(2-thiophen-3-ylethoxy)benzonitrile. To a solu-
tion of 3-bromo-4-hydroxybenzonitrile (8.50 g) in THF (200 mL)
were added 2-(3-thienyl)ethanol (4.42 mL) and triphenylphosphine
(11.80 g). The mixture was cooled to 0 °C, and diethylazodicarbox-
ylate (7.09 mL) was added dropwise. The mixture was stirred at room
temperature for 16 h. The reaction mixture was concentrated and the
residue redissolved in diethyl ether (100 mL). The mixture was stirred
for 10 min at 0 °C and then filtered. The filtrate was washed with
saturated aqueous sodium carbonate (80 mL), 1 M HCl (80 mL), and
brine, dried over MgSO4, concentrated, then purified on silica gel to
afford the desired compound (11.7 g, 95% yield).
4,5-Dihydro-6-oxa-1-thiabenzo[e]azulene-9-carbonitrile. To
a solution of 3-bromo-4-(2-thiophen-3-ylethoxy)benzonitrile (3.08 g)
in DMF (20 mL) was added palladium acetate (224 mg), PPh3 (525
mg), and K2CO3 (2.76 g), and the mixture was heated at 90 °C for 16
h. After cooling to room temperature, the mixture was diluted with
ethyl acetate (60 mL) and filtered. The filtrate was washed with brine,
dried over MgSO4, concentrated, and purified by silica gel
chromatography to afford the title compound (1.75 g, 77% yield).
2-Bromo-4,5-dihydro-6-oxa-1-thiabenzo[e]azulene-9-car-
bonitrile. To a solution of 4,5-dihydro-6-oxa-1-thiabenzo[e]azulene-
9-carbonitrile (4.58 g) in CH2Cl2 (30 mL) and acetic acid (30 mL)
was added N-bromosuccinimide (3.95 g), and the mixture was stirred
for 16 h at room temperature. Water (100 mL) was added and the
solid collected by filtration and air-dried to give the title compound
(3.0 g, 49% yield).
N-(2-Chlorophenyl)-8-(3-cyanophenyl)-N-methyl-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide. A mix-
ture of 8-bromo-N-(2-chlorophenyl)-N-methyl-4,5-dihydrobenzo[b]-
thieno [2,3-d]oxepine-2-carboxamide (500 mg, 1.11 mmol), 3-
cyanophenylboronic acid (200 mg, 1.25 mmol), sodium carbonate
(400 mg, 3 mmol), and Pd(PPh3)2Cl2(100 mg) in CH3CN (4 mL)
was heated under microwave irradiation at 100 °C for 40 min. The
mixture was filtered through Celite, concentrated, and purified by
9-Cyano-4,5-dihydro-6-oxa-1-thiabenzo[e]azulene-2-car-
boxylic Acid. To a solution of 2-bromo-4,5-dihydro-6-oxa-1-
thiabenzo[e]azulene-9-carbonitrile (1.0 g) in THF (25 mL) at −78
°C was added n-BuLi (1.44 mL), and the mixture was allowed to warm
to −10 °C over 1 h. The mixture was then recooled to −78 °C, and
carbon dioxide was bubbled through the solution for 15 min. The
mixture was then allowed to warm to room temperature over 4 h. The
reaction was quenched with water (5 mL) and the product extracted
into saturated aqueous Na2CO3 (30 mL). The aqueous layer was
washed with ethyl acetate (30 mL) and then acidified to pH 2−3 with
1
column to afford the product (400 mg, 69% yield). H NMR (CDCl3,
400 MHz) δ 8.18 (s, 1H), 8.05 (d, J = 8.4 Hz, 1H), 7.85 (d, J = 8.0 Hz,
1H), 7.70−7.59 (m, 3H), 7.58−7.42 (m, 4H), 7.40 (s, 1H), 6.65 (s,
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1
2 M HCl. The product was then extracted into ethyl acetate and the
organic layers were dried over MgSO4 and concentrated to give the
title compound (0.43 g, 48% yield).
the product (23.0 g, 89% yield). H NMR (DMSO-d6, 400 MHz) δ
7.74−7.69 (m, 2H), 7.58−7.47 (m, 4H), 6.79 (s, 1H), 4.23 (t, J = 4.8
Hz, 2H), 3.83 (s, 3H), 3.31 (s, 3H), 3.01 (br, 5H, CH2), 2.94 (s, 3H).
2-((2-Chloro-4-(dimethylcarbamoyl)phenyl)(methyl)-
carbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-8-car-
boxylic Acid. Methyl 2-((2-chloro-4-(dimethylcarbamoyl)phenyl)-
(methyl)carbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-8-car-
boxylate (25.0 g, 50.1 mmol) was dissolved in THF (50 mL) and
water (50 mL). The solution was treated with LiOH·H2O (5.26 g,
125.3 mmol). The reaction mixture was stirred at room temperature
for 2 h. THF was removed under reduced pressure, and the mixture
was acidified by HCl (2 N). The resulting precipitate was filtered,
washed with water, and dried to give desired product (20.00 g, 82%
yield). 1H NMR (CDCl3, 400 MHz) δ 7.66−7.60 (m, 3H), 7.46−7.42
(m, 3H), 6.99 (s, 1H), 4.23 (t, J = 5.0 Hz, 2H), 3.39 (s, 3H), 3.14 (s,
3H), 3.07 (t, J = 5.0 Hz, 2H), 3.02 (s, 3H).
N-(2-Chlorophenyl)-9-cyano-N-methyl-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carboxamide (30). To a solution of 9-
cyano-4,5-dihydro-6-oxa-1-thiabenzo[e]azulene-2-carboxylic acid (300
mg) in CH2Cl2 (10 mL) was added DMF (1 drop) and oxalyl chloride
(0.165 mL), and the mixture was stirred for 30 min at room
temperature. The mixture was concentrated, and the residue was
dissolved in acetonitrile (10 mL). To this solution were added 2-
chloro-N-methylaniline (0.165 mL) and K2CO3 (308 mg), and the
mixture was stirred for 16 h at room temperature. The mixture was
partitioned between water (40 mL) and ethyl acetate (30 mL). The
organic layer was dried over Na2SO4, concentrated, and purified on
1
silica to afford 30 (149 mg, 34% yield). H NMR (CDCl3, 400 MHz)
δ 7.72 (d, J = 1.65 Hz, 1H), 7.57 (m, 1H), 7.47−7.38 (m, 4 H), 7.03
(d, J = 8.4 Hz, 1H), 6.92 (s, 1H), 4.29 (t, J = 5.02 Hz, 2 H), 3.41 (s,
3H), 3.08 (t, J = 5.00 Hz, 2H). LCMS (ESI+): (M + H)+ 395.
4-Amino-3-chloro-N,N-dimethylbenzamide. i-Pr2NEt (116
mL, 699.5 mmol) was added to a suspension of dimethylamine
hydrochloride (28.51 g, 349.6 mmol) in THF (600 mL). The mixture
was stirred at room temperature for 0.5 h. Then 4-amino-3-
chlorobenzoic acid (30.00 g, 174.8 mmol) and HATU (86.41 g,
227.2 mmol) were added to the above suspension separately. The
reaction mixture was stirred for an additional 1.5 h. It was
concentrated, and the residue was partitioned between EtOAc (300
mL) and water (150 mL). The organic phase was washed with water,
dried over Na2SO4, and concentrated under vacuum. The crude
product was purified by silica gel chromatography (hexanes/EtOAc =
1:1) to afford the pure product (34.72 g, 89% yield). 1H NMR
(CDCl3, 400 MHz) δ 7.39 (s, 1H), 7.19 (d, J = 8.4 Hz, 1H), 6.74 (d, J
= 8.4 Hz, 1H), 4.24 (br, 2H), 3.05 (s, 6H).
N2-(2-Chloro-4-(dimethylcarbamoyl)phenyl)-N2,N8-dimeth-
yl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2,8-dicarboxa-
mide (31). To a suspension of H2NMe·HCl (5.02 g, 74.4 mmol) in
THF (180 mL), i-Pr2NEt (16.83 g, 130.2 mmol) was added. The
mixture was stirred at room temperature for 0.5 h. Then 2-((2-chloro-
4-(dimethylcarbamoyl)phenyl)(methyl)carbamoyl)-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-8-carboxylic acid (9.00 g, 18.6 mmol) and
HATU (21.22 g, 55.8 mmol) were added to the above suspension
separately. The reaction mixture was stirred at room temperature for 2
h. Solvent was removed, and the resulting mixture was dissolved in
CH2Cl2 (200 mL), washed with HCl (2 N, 200 mL) and water (100
mL), and dried over Na2SO4. The crude product was purified by silica
gel chromatography (CH2Cl2/MeOH = 10:1) to give the product
1
(3.70 g, 40% yield). H NMR (CDCl3, 400 MHz) δ 7.58 (s, 1H),
7.42−7.29 (m, 4H), 6.92 (s, 1H,), 6.34 (d, J = 4.0 Hz, 1H), 4.20 (t, J =
5.0 Hz, 2H), 3.37 (s, 3H), 3.12 (s, 3H), 3.04−2.98 (m, 8H, CH2).
LCMS (ESI+): (M + H)+ 498.2.
8-Bromo-N-(2-chloro-4-(dimethylcarbamoyl)phenyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide. A solu-
tion of 8-bromo-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carbox-
ylic acid (25.00 g, 76.9 mmol) in SOCl2 (200 mL) was heated at 90−
100 °C for 3 h. It was concentrated to give the crude acid chloride. A
suspension of the acid chloride in THF (600 mL) was treated with a
solution of 4-amino-3-chloro-N,N-dimethylbenzamide (18.23 g, 92.3
mmol) and pyridine (14 mL) in THF (200 mL) at 0 °C. The mixture
was warmed to room temperature and stirred overnight. After it was
concentrated, water (200 mL) was added to the mixture. The resulting
precipitate was collected by filtration, washed with water, and dried to
3-Amino-4-chloro-N,N-dimethylbenzamide. 3-Amino-4-chlor-
obenzoic acid (3.11 g), dimethylamine hydrochloride (2.96 g),
N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)uronium hexofluoro-
phosphate (8.09 g), and N,N-diisopropylethylamine (12.6 mL) were
combined in 50 mL of anhydrous tetrahydrofuran, and the mixture was
allowed to stir for 12 h. The reaction mixture was partitioned with
saturated sodium bicarbonate and ethyl acetate. The organic layer was
concentrated and then purified by silica gel chromatography on a
Teledyne ISCO 140 g functionalized amine column to give the desired
1
compound (3.5 g, 97% yield). H NMR (400 MHz, CDCl3) δ 7.26−
1
7.21 (d, J = 8.1 Hz, 1H), 6.84−6.79 (d, J = 1.7 Hz, 1H), 6.72−6.66
(dd, J = 8.2, 1.7 Hz, 1H), 4.40−3.90 (s, 2H), 3.07 (s, 3H), 2.97 (s,
3H). LCMS (ESI+): (M + H)+ = 199.0
give the desired product (29.88 g, 77% yield). H NMR (DMSO-d6,
400 MHz) δ 10.14 (s, 1H), 7.86 (s, 1H), 7.67−7.57 (m, 3H), 7.40−
7.25 (m, 3H), 4.32 (t, J = 5.2 Hz, 2H), 3.21 (t, J = 5.2 Hz, 2H), 2.95
(s, 3H), 2.92 (s, 3H).
8-Bromo-N-(2-chloro-5-(dimethylcarbamoyl)phenyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide. 8-
Bromo-4,5-dihydrothieno[3,2-d][1]benzoxepine-2-carboxylic acid
(816 mg) was suspended in thionyl chloride (10 mL) and heated at
85 °C with a Vigreux condensation column attached for 2 h. The
mixture was cooled to room temperature and concentrated. Then 35
mL of anhydrous THF was added and the mixture was cooled to 0 °C.
3-Amino4-chloro-N,N-dimethylbenzamide (548.3 mg) in 5 mL of
anhydrous THF and pyridine (0.609 mL) were added. The mixture
was allowed to warm to room temperature and was stirred for 12 h.
The reaction mixture was concentrated and purified by silica gel
chromatography (0−100% ethyl acetate in heptanes) to give the
8-Bromo-N-(2-chloro-4-(dimethylcarbamoyl)phenyl)-N-
methyl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxa-
mide. To solution of 8-bromo-N-(2-chloro-4-(dimethylcarbamoyl)-
phenyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide
(29.0 g, 57.3 mmol) and Cs2CO3 (37.40 g, 114.7 mmol) in DMF (600
mL) was slowly added CH3I (30 mL, 479.8 mmol). The reaction
mixture was stirred at room temperature overnight. DMF (about 200
mL) was removed under reduced pressure, and water (100 mL) was
added to the mixture.. The resulting precipitate was filtered, washed
with water, and dried to give the desired product (29.50 g, 99% yield).
1H NMR (DMSO-d6, 400 MHz) δ 7.72−7.68 (m, 2H), 7.52−7.49 (m,
1
desired product (1.09 g, 86% yield). H NMR (400 MHz, CDCl3) δ
1H), 7.30−7.20 (m, 3H), 6.76 (s, 1H), 4.19 (s, 2H), 3.29 (s, 3H), 3.00
(s, 3H), 2.96 (s, 2H), 2.93 (s, 3H).
8.68−8.49 (br s, 1H), 7.65−7.59 (d, J = 8.5 Hz, 1H), 7.51−7.6 (d,
1H), 7.49−7.46 (br s, 1H), 7.28−7.25 (m, 2H), 7.25−7.23 (d, J = 1.7
Hz, 1H), 7.23−7.21 (br s, 1H), 4.42−4.34 (t, J = 5.1 Hz, 2H), 3.33−
3.23 (t, J = 5.1 Hz, 2H), 3.14−3.08 (br s, 6H). LCMS (ESI+): (M +
H)+ = 506.1.
8-Bromo-N-(2-chloro-5-(dimethylcarbamoyl)phenyl)-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide. 8-
Bromo-N- [2-chloro-5-(dimethylcarbamoyl)phenyl]-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxamide (1.09 g) was
dissolved in DMF (50 mL), and cesium carbonate (843 mg) was
added followed by methyl iodide (0.15 mL). The mixture was stirred
Methyl 2-((2-Chloro-4-(dimethylcarbamoyl)phenyl)(methyl)-
carbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-8-car-
b o x y l a t e . A m i x t u r e o f 8 - b r o m o - N - ( 2 - c h l o r o - 4 -
(dimethylcarbamoyl)phenyl)-N-methyl-4,5-dihydrobenzo[b]thieno-
[2,3-d]oxepine-2-carboxamide (27.0 g, 51.9 mmol), dppf (23.20 g,
41.9 mmol), Pd(OAc)2 (5.8 g, 25.8 mmol), and Et3N (27 mL) in
DMF (270 mL) and MeOH (400 mL) was stirred under a CO
atmosphere (50 psi) at 70 °C for 2 days. The reaction mixture was
filtered, and the filtrate was concentrated. The crude product was
purified by silica gel chromatography (hexanes/EtOAc = 1:1) to give
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at room temperature overnight. The mixture was diluted with water,
and the aqueous layer was extracted with ethyl acetate. The sample
was purified by silica gel chromatography (25−100% ethyl acetate in
heptanes) and concentrated to give the desired compound (1.12 g,
89% yield). 1H NMR (400 MHz, CDCl3) δ 7.61−7.54 (d, J = 8.2 Hz,
1H), 7.49−7.43 (dd, J = 8.2, 1.9 Hz, 1H), 7.41−7.37 (d, J = 1.9 Hz,
1H), 7.25−7.22 (d, J = 3.6 Hz, 1H), 7.17−7.12 (d, J = 2.0 Hz, 1H),
7.10−7.02 (dd, J = 8.5, 2.0 Hz, 1H), 6.93−6.87 (s, 1H), 4.25−4.16 (t, J
= 5.2 Hz, 2H), 3.44−3.34 (s, 3H), 3.09−3.05 (s, 3H), 3.03−2.99 (m, J
was washed with water and dried to give the desired product (0.58 g,
57% yield). LCMS (ESI+): (M + H)+ 491.8.
8-Bromo-N-(2-chloro-4-(4-methylpiperazine-1-carbonyl)-
phenyl)-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-
2-carboxamide. To a mixture of 1-methylpiperazine (0.32 g, 3.2
mmol), i-Pr2NEt (1.2 mL, 6.9 mmol), and HATU (0.79 g, 2.08 mmol)
in THF (20 mL) was added 4-(8-bromo-N-methyl-4,5-dihydrobenzo-
[b]thieno[2,3-d]oxepine-2-carboxamido)-3-chlorobenzoic acid (0.79 g,
1.6 mmol) under nitrogen at room temperature. The reaction mixture
was stirred overnight, diluted with water, and extracted with EtOAc.
The organic layer was dried over Na2SO4, concentrated in vacuo, and
purified by silica gel flash column chromatography (hexanes/EtOAc =
2:1) to give the titled product (0.82 g, 89% yield). LCMS (ESI+): (M
+ H)+ 573.9.
= 2.9 Hz, 3H), 2.88−2.84 (br s, 3H). LCMS (ESI+): (M + H)+
=
520.0.
M e t h y l 2 - ( 2 - C h l o r o - 5 - ( d i m e t h y l c a b a m o y l ) -
phenylcarbamoyl)-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-
8-carboxylate (32). 32 was prepared in analogy to 22 (33% yield).
1H NMR (400 MHz, DMSO) δ 8.56−8.37 (d, J = 4.6 Hz, 1H), 7.79−
7.64 (m, 2H), 7.61−7.37 (m, J = 36.2, 11.3 Hz, 4H), 6.78−6.64 (s,
1H), 4.23−4.16 (t, J = 4.0 Hz, 2H), 3.31−3.27 (s, 3H), 3.03−2.98 (d, J
= 5.9 Hz, 5H), 2.98−2.95 (s, 3H), 2.89−2.85 (s, 3H), 2.78−2.73 (d, J
= 4.5 Hz, 3H). LCMS (ESI+): (M + H)+ = 498.9
N2-(2-Chloro-4-(4-methylpiperazine-1-carbonyl)phenyl)-
N2,N8-dimethyl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2,8-
dicarboxamide (33). A suspension of 8-bromo-N-(2-chloro-4-(4-
methylpiperazine-1-carbonyl)phenyl)-N-methyl-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carboxamide (450 mg, 0.78 mmol), Pd(OAc)2
(9 mg, 0.039 mmol), Xantphos (45 mg, 0.078 mmol), MeNH2·HCl
(79 mg, 1.15 mmol), and Na2CO3 (240 mg, 2.3 mmol) in toluene (10
mL) was heated at 80 °C under an atmosphere of CO (balloon)
overnight. Then it was filtered, concentrated, and purified by silica gel
flash column chromatography (hexanes/EtOAc = 1:3) to give the
Methyl 4-Amino-3-chlorobenzoate. SOCl2 (42.7 mL, 585
mmol) was slowly added into MeOH (300 mL) at 0 °C, and the
solution was stirred for 1 h at 0 °C. Then 4-amino-3-chlorobenzoic
acid (20.0 g, 117 mmol) was added into the above solution in one
portion at 0 °C. After addition, it was allowed to reach room
temperature and stirred for 2 days. The reaction mixture was
concentrated to remove the solvent and the remaining SOCl2.
Water (200 mL) was added to the residue, and NaHCO3 (11.80 g, 140
mmol) was added in one portion. The mixture was stirred at room
temperature for half an hour, and it was extracted with EtOAc (150
mL × 2). The combined organic phases were dried over Na2SO4,
filtered, and concentrated to give the desired product (20.45 g, 94%
1
titled product (162 mg, 38% yield). H NMR (CDCl3, 400 MHz) δ
7.57 (s, 1H), 7.43−7.31 (m, 5H), 6.97 (s, 1H), 6.35 (q, J = 4.8 Hz,
1H), 4.24 (t, J = 5.2 Hz, 2H), 3.80 (s, 2H), 3.45 (s, 2H), 3.39 (s, 3H),
3.07 (t, J = 5.2 Hz, 2H), 2.99 (d, J = 4.8 Hz, 3H), 2.52 (s, 2H), 2.32 (s,
5H). LCMS (ESI+): (M + H)+ 553.0.
Methyl 3-Amino-4-chlorobenzoate. SOCl2 (107 mL, 1.47
mol) was slowly added to MeOH (800 mL) at 0 °C, and the solution
was stirred for 1 h at 0 °C. Then 3-amino-4-chlorobenzoic acid (50.0
g, 0.29 mmol) was added to the above solution in one portion at 0 °C.
It was allowed to reach room temperature and stirred for 2 days. The
reaction mixture was concentrated to remove the solvent and
remaining SOCl2. Water (200 mL) was added to the residue and
basified with saturated NaHCO3 solution to pH > 7, and the resulting
precipitate was filtered and dried to give the desired product (49 g,
1
yield). H NMR (CDCl3, 400 MHz) δ 7.95 (s, 1H), 7.76−7.73 (m,
1H), 6.72 (d, J = 8.4 Hz, 1H), 4.48 (s, 2H), 3.84 (s, 3H). LCMS (ESI
+): (M + H)+ 185.8.
Methyl 4-(8-Bromo-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-3-chlorobenzoate. 8-Bromo-4,5-
dihydrobenzo[b]thieno[2,3-d]oxepine-2-carboxylic acid (25.0 g, 77
mmol) was dissolved in SOCl2 (300 mL). After the reaction mixture
was heated at reflux for 4 h it was concentrated to remove the
remaining SOCl2 to give the crude 8-bromo-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carbonyl chloride. NaH (6.15 g, 154 mmol,
60% in mineral oil) was slowly added to the solution of methyl 4-
amino-3-chlorobenzoate (25.55 g, 154 mmol) in THF (200 mL). The
suspension of the above acid chloride (25.42 g, 77 mmol) in THF
(300 mL) was added into the above solution at 0 °C. After addition,
the reaction mixture was stirred at room temperature overnight.
Solvent was removed and the residue was washed with water and dried
to give the desired product (15.5 g, 49% yield). LCMS (ESI+): (M +
H)+ 506.0.
1
91% yield). H NMR (CDCl3, 400 MHz) δ 7.44−7.26 (m, 3H), 4.18
(br, 2H), 3.88 (s, 3H).
Methyl 3-(8-Bromo-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-4-chlorobenzoate. A solution of 8-
bromo-4,5-dihydro-6-oxa-1-thiabenzo[e]azulene-2-carboxylic acid (25
g) in SOCl2 (200 mL) was heated at 80°C for 3 h. It was concentrated
to give the crude acid chloride. A suspension of the crude acid chloride
(∼0.077 mol) from the above in THF (1000 mL) at 0 °C was treated
with a solution of 3-amino-4-chlorobenzoic acid methyl ester (15.7 g,
1.1 equiv) and pyridine (30 mL) in THF (100 mL). The mixture was
allowed to reach room temperature overnight. The volume of reaction
mixture was reduced by 50% under reduced pressure, and it was
diluted with water. The resulting precipitate was filtered and washed
with water and Et2O. The filter cake was dried to a constant weight
under vacuum to give the desired product (32.9 g, 87% yield). LCMS:
(ESI, m/z) = 494 [M + H]+.
Methyl 4-(8-Bromo-N-methyl-4,5-dihydrobenzo[b]thieno-
[2,3-d]oxepine-2-carboxamido)-3-chlorobenzoate. To a solu-
tion of methyl 4-(8-bromo-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-3-chlorobenzoate (18.16 g, 37 mmol) and
Cs2CO3 (24.01 g, 74 mmol) in DMF (500 mL) was added CH3I (23
mL, 368 mmol). The reaction mixture was stirred at room temperature
overnight. DMF was removed under reduced pressure, and water (200
mL) was added to the mixture. The resulting precipitate was filtered,
washed with water, and dried to give the desired product (15.0 g, 80%
Methyl 3-(8-Bromo-N-methyl-4,5-dihydrobenzo[b]thieno-
[2,3-d]oxepine-2-carboxamido)-4-chlorobenzoate. To a solu-
tion of methyl 3-(8-bromo-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-4-chlorobenzoate (32 g, 65 mmol) and
Cs2CO3 (42.4 g, 130 mmol) in DMF (500 mL) was added CH3I
(12 mL, 195 mmol). The reaction mixture was stirred at room
temperature overnight. DMF was removed under reduced pressure,
and water was added to the mixture. The resulting precipitate was
filtered, washed with water, and dried to give the desired product (31
1
yield). H NMR (CDCl3, 400 MHz) δ 8.18 (d, J = 1.6 Hz, 1H), 7.99
(dd, J1 = 1.6 Hz, J2 = 8.0 Hz, 1H), 7.43−7.08 (m, 4H), 6.73 (s, 1H),
4.21 (t, J = 5.2 Hz, 2H), 3.96 (s, 3H), 3.39 (s, 3H), 2.99 (t, J = 5.2 Hz,
2H). LCMS (ESI+): (M + H)+ 506.0.
1
4-(8-Bromo-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-3-chlorobenzoic Acid. To the suspen-
sion of methyl 4-(8-bromo-N-methyl-4,5-dihydrobenzo[b]thieno [2,3-
d]oxepine-2-carboxamido)-3-chlorobenzoate (1.0 g, 1.97 mmol) in
THF (8 mL) and H2O (8 mL) was added LiOH·H2O (166 mg, 3.96
mmol). The reaction mixture was stirred at room temperature
overnight. It was acidified with HCl (2 N) to pH 2−3 and
concentrated to remove most of the solvent. The resulting precipitate
g, 94% yield). H NMR (CDCl3, 400 MHz) δ 8.07−7.08 (m, 6H),
6.81 (s, 1H), 4.22 (t, J = 5.2 Hz, 2H), 3.94 (s, 3H), 3.40 (s, 3H), 3.01
(t, J = 5.2 Hz, 2H).
3-(8-Bromo-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-d]-
oxepine-2-carboxamido)-4-chlorobenzoic Acid. To a suspen-
sion of methyl 3-(8-bromo-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-
d]oxepine-2-carboxamido)-4-chlorobenzoate (31 g, 61 mmol) in THF
(200 mL) and H2O (100 mL) was added LiOH·H2O (6.42 g, 153
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Journal of Medicinal Chemistry
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mmol). The reaction mixture was stirred at room temperature
overnight. Then it was acidified with HCl (2 N) to pH 2−3 and
concentrated. The resulting precipitate was washed with water and
dried to give the desired product (27.3 g, 91% yield). LC-MS: (ESI,
m/z) = 494 [M + H]+.
respectively, prior to the addition of CellTiter-Glo reagent (Promega)
and reading of luminescence using an Analyst plate reader. For
antiproliferative assays, a cytostatic agent such as aphidicolin and a
cytotoxic agent such as staurosporine were included as controls.
Dose−response curves were fit to a four-parameter equation, and
relative EC50 values were calculated using the Assay Explorer software.
All cellular EC50 values represent geometric mean values of a
minimum of two determinations, and these assays generally produced
results within 3-fold of the reported mean.
8-Bromo-N-(2-chloro-5-(4-methylpiperazine-1-carbonyl)-
phenyl)-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-
2-carboxamide. To a mixture of 1-methylpiperazine (1.22 g, 12.18
mmol), i-Pr2NEt (3 mL), and HATU (2.78 g, 7.31 mmol) in THF (60
mL) was added 3-(8-bromo-N-methyl-4,5-dihydrobenzo[b]thieno[2,3-
d]oxepine-2-carboxamido)-4-chlorobenzoic acid (3.0 g, 6.09 mmol)
under nitrogen at room temperature. The reaction mixture was stirred
overnight, diluted with water, and extracted with EtOAc. The organic
layer was dried over Na2SO4 and concentrated in vacuo to give the
ASSOCIATED CONTENT
* Supporting Information
■
S
A homology model of human PI3Kβ in PDB format and the
sequence alignment of human PI3Kβ and mouse PI3Kδ that
was used for model building. This material is available free of
charge via the Internet at http://pubs.acs.org.
1
titled product (3.3 g, 94% yield). H NMR (DMSO-d6, 400 MHz) δ
7.74−7.21 (m, 6H), 6.67 (s, 1H), 4.18 (t, J = 4.4 Hz, 2H), 3.31−2.12
(m, 16H). LCMS: (ESI, m/z) = 574.0 [M + H]+.
N2-(2-Chloro-5-(4-methylpiperazine-1-carbonyl)phenyl)-
N2,N8-dimethyl-4,5-dihydrobenzo[b]thieno[2,3-d]oxepine-2,8-
dicarboxamide (34). A suspension of 8-bromo-N-(2-chloro-5-(4-
methylpiperazine-1-carbonyl)phenyl)-N-methyl-4,5-dihydrobenzo[b]-
thieno[2,3-d]oxepine-2-carboxamide (500 mg, 0.87 mmol), Pd(OAc)2
(10 mg, 0.0435 mmol), Xantphos (50 mg, 0.087 mmol), MeNH2·HCl
(88 mg, 1.30 mmol), and Na2CO3 (277 mg, 2.61 mmol) in toluene
(10 mL) was heated at 80 °C under CO (1 atm) overnight. It was
filtered and concentrated, and the crude product was purified by silica
gel flash column chromatography (CH2Cl2/MeOH = 40:1 to 20: 1) to
give 33 (149 mg, 31%). 1H NMR (CDCl3, 400 MHz) δ 7.60−6.94 (m,
7H), 6.23 (s, 1H), 4.23 (t, J = 4.8 Hz, 2H), 3.79−2.09 (m, 19H).
LCMS: (ESI, m/z) = 553 [M + H]+.
Accession Codes
†The coordinates of 33 in PI3Kγ have been deposited with
PDB code 3T8M.
AUTHOR INFORMATION
Corresponding Author
*Phone: (650) 467-3214. Fax: (650) 225-2061. E-mail:
theffron@gene.com.
■
ACKNOWLEDGMENTS
■
The authors thank Mengling Wong, Chris Hamman, Michael
Hayes, Steve Huhn, and Wen Chiu for compound purification
and determination of purity by HPLC, mass spectrometry, and
1H NMR. The authors also thank Krista K. Bowman, Alberto
Estevez, Kyle Mortara, and Jiansheng Wu for technical
assistance of protein expression and purification, and Jeremy
Murray for depositing structures to the PDB.
Characterization of Biochemical and Cellular Activity in
Vitro. Enzymatic activity of the class I PI3K isoforms was measured
using a fluorescence polarization assay that monitors formation of the
product 3,4,5-inositol triphosphate molecule (PIP3), as it competes
with fluorescently labeled PIP3 for binding to the GRP-1 pleckstrin
homology domain protein. An increase in phosphatidylinositide 3-
phosphate product results in a decrease in fluorescence polarization
signal as the labeled fluorophore is displaced from the GRP-1 protein
binding site. Class I PI3K isoforms were purchased from Perkin-Elmer
or were expressed and purified as heterodimeric recombinant proteins.
Tetramethylrhodamine-labeled PIP3 (TAMRA-PIP3), di-C8-PIP2,
and PIP3 detection reagents were purchased from Echelon
Biosciences. PI3K isoforms were assayed under initial rate conditions
in the presence of 10 mM Tris (pH 7.5), 25 μM ATP, 9.75 μM PIP2,
5% glycerol, 4 mM MgCl2, 50 mM NaCl, 0.05% (v/v) Chaps, 1 mM
dithiothreitol, 2% (v/v) DMSO at the following concentrations for
each isoform: PI3Kα,β at 60 ng/mL; PI3Kγ at 8 ng/mL; PI3Kδ at 45
ng/mL. After assay for 30 min at 25 °C, reactions were terminated
with a final concentration of 9 mM EDTA, 4.5 nM TAMRA-PIP3, and
4.2 μg/mL GRP-1 detector protein before reading fluorescence
polarization on an Envision plate reader. IC50 values were calculated
from the fit of the dose−response curves to a four-parameter equation.
Apparent Ki values were determined at a fixed concentration of ATP
near the measured Km for ATP for each isoenzyme and were calculated
by fitting of the dose−response curves to an equation for tight-binding
competitive inhibition. All IC50 and apparent Ki values represent
geometric mean values of a minimum of three determinations. These
assays generally produced results within 2-fold of the reported mean.
Antiproliferative cellular assays were conducted using PC3 and
MCF7.1 human tumor cell lines provided by the ATCC or Genentech
Research Laboratories, respectively. MCF7.1 is an in vivo selected line
developed at Genentech and originally derived from the parental
human MCF7 breast cancer cell line (ATCC, Manassas, VA). Cell
lines were cultured in RPMI supplemented with 10% fetal bovine
serum, 100 units/mL penicillin, 100 μg/mL streptomycin, 10 mM
HEPES, and 2 mM glutamine at 37 °C under 5% CO2. MCF7.1 cells
or PC3 cells were seeded in 384-well plates in medium at 1000 or
3000 cells/well, respectively, and incubated overnight prior to the
addition of compounds to a final DMSO concentration of 0.5% v/v.
MCF7.1 cells and PC3 cells were incubated for 3 and 4 days,
ABBREVIATIONS USED
■
PI3K, phosphoinositide 3-kinase; PDB, Protein Data Bank;
PTEN, phosphatase and tensin homologue; SAR, structure−
activity relationship; CAMK2D, calcium/calmodulin-dependent
protein kinase type II δ; Boc, tert-butyl carbamate; DPPA,
diphenylphosphorylazide; TMEDA, tetramethylethylenedi-
amine; TFA, trifluoroacetic acid; DMF, dimethylformamide;
DMSO, dimethylsulfoxide; DCE, 1,2-dichloroethane; THF,
tetrahydrofuran; PPA, polyphosphoric acid; DMAP, 4-dime-
thylaminopyridine; DBU, diazobicyloundecane; m-CPBA, 3-
chloroperoxybenzoic acid; HATU, N,N,N′,N′-tetramethyl-O-(7-
azabenzotiazol-1-yl)uronium hexafluorophosphate; PIP3, 3,4,5-
inositol triphosphate
REFERENCES
■
(1) Recent reviews that discuss the isoform selectivity of PI3K
inhibitors include the following: (a) Marone, R.; Cmiljanovic, V.;
Giese, B.; Wymann, M. P. Targeting Phosphoinositide 3-Kinase:
Moving towards Therapy. Biochim. Biophys. Acta 2008, 1784, 159−
185. (b) Knight, Z. A.; Shokat, K. M. Chemically Targetting the PI3K
Family. Biochem. Soc. Trans. 2007, 35, 245−249. (c) Ward, S. G.;
Finan, P. Isoform-Specific Phosphoinositide 3-Kinase Inhibitors as
Therapeutic Agents. Curr. Opin. Pharmacol. 2003, 3, 426−434.
(d) Sundstrom, T. J.; Anderson, A. C.; Wright, D. L. Inhibitors of
Phosphoinositide-3-Kinase: A Structure-Based Approach to Under-
standing Potency and Selectivity. Org. Biomol. Chem. 2009, 7, 840−
850.
(2) Jackson, S. P.; Schoenwaelder, S. M.; Goncalves, I.; Nesbitt, W.
S.; Yap, C. L.; Wright, C. E.; Kenche, V.; Anderson, K. E.; Dopheide, S.
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7831
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PI3Kα Mutations. Science 2007, 318, 1744−1748.
(14) For a comparative analysis of the binding sites of the four class I
PI3Ks see the following: (a) Amzel, L. M.; Huang, C.-H.; Mandelker,
D.; Lengauer, C.; Gabelli, S. B.; Vogelstein, B. Structural Comparisons
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669. (b) Sabbah, D. A.; Vennerstrom, J. L.; Zhong, H. Docking Studies
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(15) To predict binding poses by docking, a PI3Kα H1042R mutant
structure (PDB code 3HIZ) was chosen over the only currently
available wildtype structure (PDB code 2RD0). This is because the
latter is considered to suffer more significantly from artifacts of crystal
packing. Docking was performed using GLIDE (Schrodinger, LLC),
̈
and the conformations of three mobile side chains (R770, K802,
D933) were allowed to move. The docked pose of GDC-941 was
consistent with the reported structure of this inhibitor with mouse
PI3Kδ (PDB code 2WXP).
(23) The synthetic details of the remaining compounds in Table 4
are found in the Supporting Information and the following: Do, S.;
Goldsmith, R.; Heffron, T.; Kolesnikov, A.; Staben, S.; Olivero, A. G.;
Siu, M.; Sutherlin, D. P.; Zhu, B.-Y.; Goldsmith, P.; Bayliss, T.; Folkes,
A.; Pegg, N. Preparation of Benzopyran and Benzoxepin as PI3K
Inhibitors. US2009/0247567 A1, 2009.
(16) The sequences of the kinase domains of the four PI3K isoforms
from multiple species were aligned using PSI-BLAST (NCBI). A
mouse PI3Kδ structure (PDB code 2WXP) was chosen as the
template because among all available structures, it shares with human
PI3Kβ the highest level of sequence homology both overall and
around the active site. In addition, residues 1−114 at the amino
terminus, which is expected to be far removed from the active site
according to the known structures, were omitted in modeling. Finally,
sequence alignment was manually refined (sequence identity of 52%;
see Supporting Information) before a model of human PI3Kβ in
complex with GDC-941 (from the template) was built using PRIME
(Schrodinger, LLC) with standard parameters.
̈
(17) A comparison of our homology model of PI3Kβ with the
recently reported 3.3 Å cocrystal structure of PI3Kβ and 1 (ref 10)
shows that the homology model correctly predicted the confirmation
of the P-loop (Lys771-Ser775, numbering from PDB code 2Y3A),
validating the choice of PI3Kδ as the homology template for the
binding pocket.
(18) The aminopyrimidine group also adds mTOR inhibition and in
many cases improved in vivo Cl. For details see refs 8b and 8c.
(19) Electrostatic potential maps were computed using a Poisson−
Boltzmann (PB) equation solver APBS Tools2 plugin for PyMOL
(14a-c). Calculation was performed in nonlinear PB mode with these
standard parameters: protein dielectric set as 2 and solvent dielectric as
78; atomic charges and radii assigned according to the AMBER force
field; 0.150 M concentration for the +1 and −1 ion species; solvent
radius of 1.4 Å; system temperature of 310 K. The molecule surface
was computed using the option “similar to cubic spline, but with 7th
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