The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic "readers" and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.
The bromodomain and extra-terminal (BET) family proteins, consisting of BRD2, BRD3, BRD4, and testis-specific BRDT members, are epigenetic "readers" and play a key role in the regulation of gene transcription. BET proteins are considered to be attractive therapeutic targets for cancer and other human diseases. Recently, heterobifunctional small-molecule BET degraders have been designed based upon the proteolysis targeting chimera (PROTAC) concept to induce BET protein degradation. Herein, we present our design, synthesis, and evaluation of a new class of PROTAC BET degraders. One of the most promising compounds, 23, effectively degrades BRD4 protein at concentrations as low as 30 pM in the RS4;11 leukemia cell line, achieves an IC50 value of 51 pM in inhibition of RS4;11 cell growth and induces rapid tumor regression in vivo against RS4;11 xenograft tumors. These data establish that compound 23 (BETd-260/ZBC260) is a highly potent and efficacious BET degrader.
Bromodomain-containing
proteins are epigenetic “readers”.
By binding to acetylated lysine residues on the histone tails, bromodomain-containing
proteins play a key role in regulation of gene transcription.[1] Among bromodomain-containing proteins, the bromodomain
and extra-terminal domain (BET) family of proteins, consisting of
BRD2, BRD3, BRD4, and testis-specific BRDT members, have emerged as
exciting new therapeutic targets for cancer and other human diseases.[2−6] The discovery of 1 ((+)-JQ-1) as the first potent and
selective BET inhibitor (Figure ) has greatly promoted investigations of BET proteins
as new therapeutic targets in humancancers and other diseases.[7] Several BET inhibitors such as 2 (OTX015) and 3 (I-BET762) (Figure ) have been advanced into clinical development.[8−13] Recently, preliminary clinical data have provided an important clinical
proof-of-concept that BET inhibition has therapeutic potential for
the treatment of certain forms of humancancer, including NUT (nuclear
protein in testis) midline carcinoma, multiple myeloma, and acute
myeloid leukemia (AML).[7,10,11,14] Preclinical studies have also suggested
that BET inhibitors may have therapeutic potential for the treatment
of other humancancers, as well as other human diseases and conditions.[15−19]
Figure 1
Chemical
structures of representative BET inhibitors 1, 2, 3, 7, 8 and
three representative previously reported BET degraders 4, 5, and 6.
Chemical
structures of representative BET inhibitors 1, 2, 3, 7, 8 and
three representative previously reported BET degraders 4, 5, and 6.Small-molecule BET inhibitors are designed to bind the BET
bromodomains
and to block the interaction of BET proteins with acetylated lysine
residues on histone tails to regulate gene transcription. In addition
to small-molecule BET inhibitors, a new approach has recently been
developed to target BET proteins for degradation based upon the proteolysis
targeting chimera (PROTAC) concept.[20] In
this approach, a heterobifunctional (chimeric) molecule is designed
to contain a BET inhibitor, which binds to BET proteins, another small-molecule
ligand, which binds to an E3 ubiquitin ligase complex, and a linker
to tether these two ligands together.[21,22] A number of
BET degraders have been reported, including 4 (dBET1),[23]5 (ARV-771),[24]6 (ARV-825),[25] and
MZ1[26] (Figure ). Compounds 4 and 6 were designed using 1 or 2, two closely
related BET inhibitors, and thalidomide, which is a ligand for cereblon,
a component of the Cullin4A ubiquitin ligase complex.[23,25] In comparison, BET degrader 5 was designed using 1 for the BET inhibitor portion and a ligand for the von Hippel–Lindau
E3 ubiquitin ligase.[24] These BET degraders
have been shown to efficiently induce BET protein degradation and
to be more potent in inhibition of cancer cell growth and in induction
of apoptosis than their corresponding BET inhibitors. Compound 4 is more effective in inhibition of tumor growth in vivo
than 1 in an acute leukemia model in mice,[23] and compound 5 achieves effective
inhibition of tumor growth in a castration-resistance prostate cancer
xenograft model in mice.[24] Collectively,
these studies provide evidence that small-molecule degraders of BET
proteins may have a promising therapeutic potential for the treatment
of humancancers and potentially other diseases and conditions.Recently, our laboratory reported the discovery of azacarbazoles
as a new class of potent and selective BET bromodomain inhibitors.[27] In the present study, we report the discovery
of a new class of small-molecule BET degraders designed based upon
our azacarbazole-based BET inhibitors and thalidomide/lenalidomide
as ligands for cereblon/Cullin4A. Through extensive optimization of
the linker region, we have obtained a series of highly potent BET
degraders. Among these, compound 23 (BETd-260) is capable
of inducing degradation of BRD2, BRD3, and BRD4 proteins at 30–100
pM in the RS4;11 leukemia cells. Compound 23 achieves
an IC50 value of 51 pM in inhibition of the RS4;11 cell
growth and induces rapid tumor regression of the RS4;11 xenograft
tumors with no signs of toxicity in mice. Compound 23 is a highly potent and efficacious BET degrader and warrants extensive
evaluation for the treatment of humancancers and other diseases.
Results
and Discussion
Starting from our previously reported BET
inhibitor 7(27) (Figure ), we performed further optimization
for this class
of BET inhibitors and identified 8 (HJB97) as a high-affinity
BET inhibitor. In our FP-based competitive binding assays, 8 binds to BRD2, BRD3, and BRD4 with high affinities (Ki < 1 nM) and is >10 times more potent than 1 or 2 (Table ). Compound 8 also potently inhibits cell
growth
in RS4;11 and MOLM-13 acute leukemia cell lines, which are known to
be sensitive to BET inhibitors. Hence, compound 8 is
a potent BET inhibitor and was employed in our design of BET degraders.
Table 1
Binding Affinities of BET Inhibitors
IC50 values were obtained
from three separate experiments.
IC50 values were obtained
from three separate experiments.Upon the basis of the cocrystal structure of BRD4BD2 complexed
with 7 (PDB code 4Z93), we modeled the structure of BRD4BD2
complexed with 8 (Figure ). Our modeled structure showed that the 2-carboxamide
group attached to the [6,5,6] tricyclic system in 8 is
exposed to solvent, making it a suitable site for tethering to thalidomide/lenalidomide
for the design of potential PROTAC degraders of BET proteins (Figure ). Accordingly, we
designed and synthesized 9 as a potential BET degrader
using 8 for the BET inhibitor portion, thalidomide as
the cereblon ligand, and the same linker that was used in 4. In a cell growth assay, the BET degrader 9 has an
IC50 value of 4.3 nM in the RS4;11 acute leukemia cell
line and is 6 times more potent than the corresponding BET inhibitor 8 (Table ).
Western blotting analysis showed that 9, at concentrations
as low as 3–10 nM, is effective in decreasing the level of
BRD2, BRD3, and BRD4 proteins in the RS4;11 cells whereas the BET
inhibitor 8 at both 100 and 300 nM fails to decrease
the level of BRD2–4 proteins (Figure ). Two previously reported BET degraders, 4 and 6, also effectively decrease the level
of BRD2–4 proteins in the RS4;11 cell line (Figure ). Consistent with its testis-specific
expression, BRDT protein was not detected in the RS4;11 cells. We
concluded that 9 is a promising BET degrader for further
optimization.
Figure 2
(A) Cocrystal structure of BRD4 BD2 complexed with 7 (green, PDB code 4Z93). (B) Modeled structure of BRD4 BD2 complexed with 8 (yellow). Water molecules are shown as red spheres. Hydrogen
bonds
are depicted in dashed lines. Residues in BRD4 BD2 close to the proposed
linkage site of 8 are labeled in red.
Table 2
Optimization of Linker Length and
Composition
IC50 values were obtained
from three independent experiments.
Figure 3
Western blotting analysis of BRD2, BRD3, and BRD4 proteins
in RS4;11
cells treated with BET degraders 4, 6, and 9 and BET inhibitor 8. RS4;11 cells were treated
for 3 h with each individual compound at indicated concentrations,
and proteins were probed by specific antibodies. GAPDH was used as
the loading control.
(A) Cocrystal structure of BRD4BD2 complexed with 7 (green, PDB code 4Z93). (B) Modeled structure of BRD4BD2 complexed with 8 (yellow). Water molecules are shown as red spheres. Hydrogen
bonds
are depicted in dashed lines. Residues in BRD4BD2 close to the proposed
linkage site of 8 are labeled in red.Western blotting analysis of BRD2, BRD3, and BRD4 proteins
in RS4;11
cells treated with BET degraders 4, 6, and 9 and BET inhibitor 8. RS4;11 cells were treated
for 3 h with each individual compound at indicated concentrations,
and proteins were probed by specific antibodies. GAPDH was used as
the loading control.IC50 values were obtained
from three independent experiments.We next explored the length and the composition of
the linker in 9. Replacing the oxygen atom in 9 with an amino
group resulted in 10, whose cell growth inhibitory activity
is 2 times better than that of 9. Conversion of the amide
group (-CO-NH-) in 9 to an ethylene group (-CH2-CH2-) generated 11, which has an IC50 value of 0.48 nM in inhibition of cell growth in the RS4;11
cell line and is thus 10 times more potent than 9. Similar
conversion of the amide group in 10 to an ethylene group
yielded 12 which, with an IC50 value of 0.20
nM in inhibition of RS4;11 cell growth, is 10 times more potent than 10. These data demonstrate that both the length and the composition
of the linker have a considerable influence on the cellular potencies
of the resulting BET degraders.We next sought to determine
the optimal linker length in 12 for cellular potencies
by shortening the linker by one
methylene group progressively, which resulted in compounds 13–17 (Table ). Compound 13 with a linker one methylene
group shorter than that in 12 is 3 times less potent
than 12. However, compound 14 with two methylene
groups shorter than 12 in the linker achieves an IC50 value of 0.14 nM in inhibition of RS4;11 cell growth and
is slightly more potent than 12. Shortening the linker
in 14 by one additional methylene group resulted in 15, which is ∼3 times less potent than 14 in inhibition of RS4;11 cell growth, and further shortening the
linker in 15 by one methylene or ethylene group yielded 16 or 17, respectively, which is 5 and 10 times
less potent than 15, respectively, in inhibition of RS4;11
cell growth. These data demonstrate that for achieving the most potent
cell growth inhibition activity, an optimal linker length should comprise
-(CH2)4–7NH- in these BET degraders,
as is shown in compounds 12–15. When
the linker becomes too short as in, for examples, compounds 16 and 17, the cellular potency is greatly decreased.To investigate whether the SAR results obtained in the RS4;11 cell
line are valid in a different cell line, we evaluated this series
of compounds together with 4 and 6 as control
compounds for their cell growth inhibitory activity in the MOLM-13leukemia cell line, which harbors a mixed lineage leukemia protein
1 (MLL1) fusion gene and is also responsive to BET inhibitors in our
previous study.[27] The data obtained are
summarized in Table . In general, the IC50 values obtained for all these BET
degraders in the MOLM-13 cell line are 5–10 times higher than
those obtained in the RS4;11 cell line. Compounds 6 and 4 have IC50 values of 18.2 nM and 657 nM, respectively,
in inhibition of MOLM-13 cell growth and are 5.5 and 8.3 times less
potent than those in the RS4;11 cell line. We obtained a very similar
SAR result for BET degraders 9–17 in the MOLM-13 cell line when compared to that in the RS4;11 cell
line. Compounds 12 and 14 are two of the
most potent BET degraders in this series with IC50 values
of 1.2 nM and 2.1 nM, respectively, in inhibition of MOLM-13 cell
growth.Having determined the optimal linker length, we next
performed
further modifications of the linker composition in compound 14 (Table ). Replacement of the NH group in 14 with a methylene
group yielded 18, which with an IC50 value
of 0.17 nM in inhibition of RS4;11 cell growth is as potent as 14. Replacement of one methylene group in 18 with
an oxygen atom generated 19, which has an IC50 value of 0.42 nM in inhibition of RS4;11 cell growth and is 2 times
less potent than 18. Shortening the linker in 18 by one methylene group resulted in 20 which, with an
IC50 value of 0.14 nM in inhibition of RS4;11 cell growth,
is equipotent to 14. Thus, modifications of the linker
compositions identified compounds 18 and 20 as two very potent BET degraders with subnanomolar IC50 values in inhibition of RS4;11 cell growth.
Table 3
Further
Optimization of the Linker
and Phthalimide Moiety
IC50 values were obtained
from three independent experiments.
IC50 values were obtained
from three independent experiments.In all of the above synthesized compounds, we employed
thalidomide
as the ligand for cereblon. Lenalidomide has been developed as a second
generation thalidomide analogue for the treatment of multiple myeloma
and myelodysplastic syndromes.[28] Although
thalidomide and lenalidomide have similar binding affinities to cereblon,[29] they may have different cell permeability and/or
pharmacokinetic profiles, and thus we investigated the effect of replacing
thalidomide with lenalidomide in three potent BET degraders 18, 19, and 20. This effort resulted
in 21, 22, and 23, which achieve
IC50 values of 0.037 nM, 0.90 nM, and 0.051 nM, respectively,
in inhibition of RS4;11 cell growth. Hence, 21 and 23 are 2–3 times more potent than 18 and 20. Interestingly, in contrast to 21 and 23, compound 22 has an IC50 value
of 0.9 nM in inhibition of RS4;11 cell growth and is thus 2 times
less potent than 19. To further confirm the significance
of the linker length, we synthesized compounds 24 and 25 with one methylene or ethylene group shorter than that
in 23. Compounds 24 and 25 have
IC50 values of 0.98 nM and 9.6 nM in inhibition of RS4;11
cell growth, respectively, and are therefore 19 and 188 times less
potent than 23.To further investigate the mechanism
of action for this class of
BET degraders, we have designed two control compounds based upon 23. It has been shown that installation of a methyl group
on the amino group in the lenalidomide moiety blocks the binding of
thalidomide analogues to cereblon.[25,29] Accordingly,
we installed a methyl group on the amino group in the lenalidomide
moiety, which resulted in 26 (Table ). Compound 26 has an IC50 value of 35.1 nM in inhibition of RS4;11 cell growth and
is therefore >600 times less potent than 23, but the
IC50 is similar to that obtained for the BET inhibitor 8. Furthermore, 26 fails to induce degradation
of BRD2, BRD3, and BRD4 proteins in the RS4;11 cell line, indicating
that it inhibits cell growth by acting not as a BET degrader but as
a BET inhibitor (Figure S1 in Supporting Information). We synthesized compound 27 based upon a less potent
BET inhibitor 28, which binds to BRD2, BRD3, and BRD4
proteins with affinities of >100 times less potent than that of 8 (Table ).
Compound 27 has an IC50 value of >1000
nM
and is thus >10 000 and >400 times less potent than 23 in RS4;11 and MOLM13 cell growth inhibition assays, respectively
(Table ). These data
indicate that in order to achieve potent cell growth inhibition, a
BET degrader must be able to bind to both BET proteins and to the
ubiquitin ligase complex, which is consistent with their PROTAC design
and the expected mechanism of action.
Table 4
Investigation
of the Effect of BET
Protein and Cereblon Binding on Cellular Potencies of BET Degraders
IC50 values were obtained
from three independent experiments.
IC50 values were obtained
from three independent experiments.We examined the ability of four representative BET
degraders (21 and 23–25) to induce BET
degradation in the RS4;11 cell line, and the data are shown in Figure . All these four
compounds can effectively and potently induce degradation of BET proteins
in a dose-dependent manner after a 3 h treatment, and their potencies
in reducing the levels of BRD2–4 proteins correlate well with
their cellular potencies in inhibition of cell growth in the RS4;11
cell line. At concentrations as low as 10 nM, 25 effectively
decreases the level of BRD3 and BRD4 proteins but is less potent and
effective in decreasing in the level of BRD2 protein. Compound 24 is very effective in reducing the level of BRD2 and BRD4
at concentrations as low as 3 nM and the level of BRD3 at 1 nM. The
two most potent BET degraders, 21 and 23, are highly effective in decreasing the level of BRD2 and BRD4 proteins
at concentrations as low as 0.3 nM and decreasing the level of BRD3
at 0.1 nM with a 3 h treatment. Therefore, 21 and 23 are extremely potent degraders of BET proteins.
Figure 4
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins in RS4;11
cells treated with compounds 21 and 23–25. RS4;11 cells were treated for 3 h with individual compounds
at indicated concentrations, and proteins were probed by specific
antibodies. GAPDH was used as the loading control.
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins in RS4;11
cells treated with compounds 21 and 23–25. RS4;11 cells were treated for 3 h with individual compounds
at indicated concentrations, and proteins were probed by specific
antibodies. GAPDH was used as the loading control.We further investigated the potency of compound 23 in inducing BET protein degradation in the RS4;11 cells
with a 24
h treatment, with compound 8 included as a control (Figure ). Compound 23 is capable of effectively reducing the level of BRD2 and
BRD3 proteins at concentrations as low as 0.1 nM and the level of
BRD4 protein at a concentration of 0.03 nM. In comparison, the BET
inhibitor 8 has no effect on the level of BRD2–4
proteins at all concentrations tested (30–1000 nM).
Figure 5
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins, as
well as c-Myc and PARP in RS4;11 cells treated with BET degrader 23 and BET inhibitor 8. RS4;11 cells were treated
for 24 h with individual compounds at indicated concentrations, and
proteins were probed by specific antibodies. GAPDH was used as the
loading control.
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins, as
well as c-Myc and PARP in RS4;11 cells treated with BET degrader 23 and BET inhibitor 8. RS4;11 cells were treated
for 24 h with individual compounds at indicated concentrations, and
proteins were probed by specific antibodies. GAPDH was used as the
loading control.We examined the effects
of the BET degrader 23 and
the BET inhibitor 8 in the RS4;11 cell line on the level
of c-Myc (Figure ),
a protein known to be down-regulated by both BET inhibitors and degraders.[25,30] Consistent with its extremely high potency in inducing BET protein
degradation, 23 effectively down-regulates the level
of c-Myc at concentrations as low as 0.1 nM. In comparison, the BET
inhibitor 8 can also effectively down-regulate the level
of c-Myc but at concentrations of 300–1000 nM in the RS4;11
cell line. Hence, the BET degrader 23 is >1000 times
more potent than the BET inhibitor 8 in down-regulation
of c-Myc protein in the RS4;11 cell line.The mechanism of action
of BET protein degradation by 23 was further investigated
(Figure ). Addition
of the BET inhibitor 8 effectively
blocks the degradation of BRD2, BRD3, and BRD4 proteins induced by 23 (Figure A), further confirming that the degradation of BET proteins by 23 requires its binding to BET proteins. Similarly, addition
of lenalidomide also effectively blocks the degradation induced by 23 for all three BET proteins (Figure B), clearly indicating that degradation of
BET proteins by 23 is cereblon-dependent. The proteasome
inhibitor MG-132 and the NEDD8-activating enzyme (NAE) inhibitor MLN4924
also completely block the degradation of BET proteins by 23 (Figure C), indicating
that BET protein degradation by 23 depends upon proteasome
and NAE. These mechanistic data constitute clear evidence that 23 is a bona fide and highly potent BET degrader.
Figure 6
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins after
a 2 h pretreatment with 8 (A), lenalidomide (B), or a
proteasome inhibitor MG-132 or a NEDD8-activating enzyme (NAE) inhibitor
MLN4924 (C), followed by a 3 h treatment with 23 at 1
nM in RS4;11 cells.
Western blotting
analysis of BRD2, BRD3, and BRD4 proteins after
a 2 h pretreatment with 8 (A), lenalidomide (B), or a
proteasome inhibitor MG-132 or a NEDD8-activating enzyme (NAE) inhibitor
MLN4924 (C), followed by a 3 h treatment with 23 at 1
nM in RS4;11 cells.We employed flow cytometry
analysis to investigate the ability
of the BET degrader 23 and the BET inhibitor 8 to induce cell cycle arrest and apoptosis in the RS4;11 and MOLM-13
cell lines (Figure ). Both compounds were found to effectively induce cell cycle arrest
in a dose-dependent manner in both cell lines, but they have very
different potencies. While the BET inhibitor 8 is effective
at 30 nM in the RS4;11 cell line in inducing cell cycle arrest, the
BET degrader 23 has a strong effect at concentrations
as low as 0.3 nM. In the MOLM-13 cell line, 8 is effective
at 30–100 nM in inducing cell cycle arrest while 23 is very effective at 1–3 nM. We also observed a sharp contrast
in their ability to induce apoptosis. While the BET degrader 23 induces robust apoptosis in both RS4;11 and MOLM-13 cell
lines at 3–10 nM concentrations with a 24 h treatment, the
BET inhibitor 8 is ineffective in both cell lines at
concentrations as high as 300 nM.
Figure 7
Induction of cell cycle arrest and apoptosis
by BET degrader 23 and BET inhibitor 8 in
the RS4;11 and MOLM-13
cell lines by flow cytometry analysis.
Induction of cell cycle arrest and apoptosis
by BET degrader 23 and BET inhibitor 8 in
the RS4;11 and MOLM-13
cell lines by flow cytometry analysis.We tested the antitumor activity of 23 in the
RS4;11
xenograft model in mice, and the results are shown in Figure . Dosing of the mice bearing
RS4;11 xenograft tumors with 5 mg/kg of 23 intravenously
every other day, three times a week for 3 weeks, achieved rapid tumor
regression with a maximum of >90% regression observed. There was
no
animal weight loss or other signs of toxicity in mice treated with
compound 23, and the animal weight gained in the vehicle
control group of mice was attributed largely to rapid tumor growth.
Therefore, the in vivo data firmly establish that 23 has
highly efficacious antitumor activity at a well-tolerated dose-schedule
in mice.
Figure 8
BET degrader 23 induces complete tumor regression
in the xenograft model of human RS4;11 tumor cells with minimal body
weight change.
BET degrader 23 induces complete tumor regression
in the xenograft model of humanRS4;11 tumor cells with minimal body
weight change.To gain a better understanding
of the strong antitumor activity
of 23 and its mechanism of action in vivo, we performed
a pharmacodynamics (PD) analysis in SCIDmice bearing the RS4;11 xenograft
tumors (Figure ).
In this experiment, mice bearing one or two RS4;11 xenograft tumors
were administered a single dose of 23 at 5 mg/kg intravenously
and were sacrificed at 1, 3, 6, and 24 h time-points after the treatment.
Mice treated with vehicle control were sacrificed at the 6 h time
point. Western blotting analysis was performed to probe the level
of BRD2, BRD3, and BRD4 proteins, as well as c-Myc, caspase-3, and
PARP proteins in the tumor tissue. Our PD data (Figure ) clearly show that a single dose of 23 was highly effective, dramatically reducing the level of
BRD2, BRD3, and BRD4 proteins in the RS4;11 tumor tissue, starting
from 1 h with the effect persisting for >24 h. The level of c-Myc
was strongly down-regulated, with the effect persisting for at least
6 h. Robust cleavage of PARP and caspase-3 was observed, starting
from the 3 h time-point and with a peak effect at the 6 h time point,
indicating strong apoptosis induction by 23. Therefore,
our PD analysis clearly demonstrates that a single dose of 23 is sufficient to induce near complete degradation of BRD2, BRD3,
and BRD4 proteins for >24 h, accompanied by robust cleavage of
PARP
and caspase-3, and strong down-regulation of c-Myc protein.
Figure 9
Pharmacodynamic
analysis of compound 23 in RS4;11
xenograft tumor tissue. SCID mice bearing RS4;11 tumors were treated
with a single intravenous dose of 23 at 5 mg/kg. Mice
were sacrificed at 1, 3, 6, and 24 h time-points after administration
with compound 23 or at 6 h with vehicle control, and
tumors were harvested from mice for Western blotting analysis of BRD2,
BRD3, BRD4, c-Myc, PARP and cleaved PARP (Cl PARP), and caspase-3
and cleaved caspase-3 (Cl caspase-3). Actin was used as the loading
control. Two mice were used for each time-point with each mouse bearing
either one or two tumors.
Pharmacodynamic
analysis of compound 23 inRS4;11
xenograft tumor tissue. SCIDmice bearing RS4;11 tumors were treated
with a single intravenous dose of 23 at 5 mg/kg. Mice
were sacrificed at 1, 3, 6, and 24 h time-points after administration
with compound 23 or at 6 h with vehicle control, and
tumors were harvested from mice for Western blotting analysis of BRD2,
BRD3, BRD4, c-Myc, PARP and cleaved PARP (Cl PARP), and caspase-3
and cleaved caspase-3 (Cl caspase-3). Actin was used as the loading
control. Two mice were used for each time-point with each mouse bearing
either one or two tumors.We analyzed concentrations of 23 in plasma and
also
in RS4;11 tumors in the same mice used in the PD experiment (Table ). Our data showed
that despite its relatively large size (MW = 798.8), 23 can effectively penetrate the RS4;11 xenograft tumor tissue and
has a concentration of 166.3, 98.5, and 35.8 ng/g in the tumor at
1, 3, and 6 h time-points, respectively, exceeding the drug concentrations
needed for effective BET degradation shown in our in vitro experiments
(Figures and 5). The drug concentration, however, was undetectable
after 24 h. Together with the PD data, our PK data indicate that a
single dose of 23 at 5 mg/kg achieves a sufficient exposure
in the tumor tissue for effective induction of BET protein degradation
for over 24 h.
Table 5
Analysis of Concentrations of Compound 23 in Plasma and RS4;11 Tumor Tissuea
concn
in plasma (ng/mL)
concn
in tumor (ng/g)
time-point (h)
individual
mice
mean
individual tumor
mean
SD
1
422
362
392
135.5
123
240.5
166.3
64.5
3
58
76.1
67.1
86
89
120.5
98.5
19.1
6
5.5
7.4
6.4
39.4
31.9
36.2
35.8
3.8
24
<1
<1
<1
<1
<1
<1
<1
Mice bearing RS4;11 xenograft
tumors were administered with a single dose of compound 23 at 5 mg/kg intravenously. Mice were sacrificed at 1, 3, 6, and 24
h time-points after administration with compound 23,
and plasma and tumor tissue were collected for analysis. Two mice
were used for each time-point with each mouse bearing either one or
two tumors (three tumors for each time point).
Mice bearing RS4;11 xenograft
tumors were administered with a single dose of compound 23 at 5 mg/kg intravenously. Mice were sacrificed at 1, 3, 6, and 24
h time-points after administration with compound 23,
and plasma and tumor tissue were collected for analysis. Two mice
were used for each time-point with each mouse bearing either one or
two tumors (three tumors for each time point).We also evaluated the metabolism
of compound 23 in
mouse liver microsomes. The major metabolite was proposed to be mono-
or dihydroxylated product occurring in the alkyl chain in the linker.
In addition, cleavage of the C(sp3)–C(sp2) bond between the linker and cereblon binding ligand and N-deethylation
of the pyrazole moiety are other alternative metabolic pathways (for
detailed information, see Figure S5).
Chemistry
The synthesis of compounds 8 and 28 is
outlined in Scheme . Briefly, 3-cyclopropyl-3-oxopropanenitrile (29) and ethylhydrazine oxalate (30) were heated in EtOH
to generate 31. Compound 32 was prepared
as described, and intermediate 33 was obtained from 32 in four steps.[31] Coupling of 31 with 33 followed by hydrolysis of the methyl
ester produced the key intermediate 34. Amidation of 34 with MeNH2 in the presence of 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium
3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) at room temperature
in dimethylformamide (DMF) gave the target compound 8. Compound 36 was formed by dehalogenation of 33 in the presence of Pd/C and H2, followed by
hydrolysis. Acyl chloride formation followed by amidation of 36 generated 28.
Reaction conditions: (a) TEA,
toluene, reflux; (b) tert-butyl bromoacetate, KI,
KHCO3, DMF, 60 °C; (c) TFA, rt; (d) N-Boc-1,4-butanediamine, HATU, DIPEA, DMF, rt; (e) TFA, DCM, rt; (f)
HATU, DIPEA, DMF, rt.The synthetic route
to compound 10 is shown in Scheme . Compound 43 was obtained by
treatment of 3-nitrophthalic anhydride
(42) with benzyl bromide, followed by reduction of the
nitro group with stannous chloride dihydrate and alkylation with tert-butyl bromoacetate. Removal of the benzyl groups of 43 followed by amide condensation resulted in 44. Compound 10 was prepared from 44 by the
same procedure as was used for the synthesis of 9.
Reaction conditions:
(a) TsOH·H2O, BnOH, 100 °C; BnBr, NaHCO3, DMF, 100 °C;
(b) SnCl2·2H2O, EtOAc, 50 °C; (c) tert-butyl bromoacetate, DIPEA, DMF, 90 °C; (d) Pd/C,
H2, EtOH, rt; (e) 38, pyridine, 110 °C;
(f) TFA, rt; (g) N-Boc-1,4-butanediamine, HATU, DIPEA,
DMF, rt; (h) TFA, DCM, rt; (i) HATU, DIPEA, DMF, rt.As shown in Scheme , compound 11 was synthesized starting from the
intermediate 39. Alkylation of the hydroxyl group of 39 with
7-bromo-1-heptanol gave 46. Compound 47 was
obtained by conversion of the hydroxyl group to an amine group in 46 through three steps. Condensation of 47 with 34 using previously described methods afforded compound 11.
Reaction
conditions: (a) 7-bromoheptan-1-ol,
NaHCO3, KI, DMF, 80 °C; (b) MsCl, TEA, DCM; (c) NaN3, DMF, 80 °C; (d) PPh3, THF/H2O,
rt; (e) HATU, DIPEA, DMF, rt.Compounds 12–17 were synthesized
according to the route shown in Scheme . Compound 49 was prepared from 48 by the strategy used in the preparation of 39. Compounds 50a–f were formed by
substitution reaction of 49 with different length of
mono-Boc protected alkyl diamines. Boc-deprotection with TFA in DCM
afforded the intermediates 51a–f,
which were subsequently condensed with 34 to generate
compounds 12–17.
Reaction conditions:
(a) NaOAc,
AcOH, reflux; (b) DIPEA, DMF, 80 °C; (c) TFA, DCM, rt; (d) HATU,
DIPEA, DMF, rt.The synthesis of compounds 18 and 20 is
shown in Scheme .
Sonogashira coupling of 53, which was prepared in a manner
similar to that used to produce 49, with different terminal
alkyne-containing linker substrates gave 54a,b. Reduction of the alkyne group in 54a,b resulted in 55a,b, whose Boc group was
removed in TFA/DCM to form 56a,b. Compounds 18 and 20 were obtained by condensation of 56a,b with 34 using the same method
as was used for 9.
Reaction conditions: (a) NaOAc,
AcOH; (b) tert-butyl pent-4-yn-1-ylcarbamate or tert-butyl hex-5-yn-1-ylcarbamate, Pd(PPh3)2Cl2, CuI, DMF/TEA, 80 °C; (c) Pd/C, H2, EtOH, rt; (d) TFA, DCM, rt; (e) HATU, DIPEA, DMF, rt.Synthesis of compounds 19 and 22 is shown
in Scheme . Bromination
of 57 with N-bromosuccinimide (NBS)
and benzoyl peroxide (BPO) gave 58, which was heated
with 38 and triethylamine (TEA) in MeCN to generate 59. Compounds 19 and 22 were prepared
starting from 53 and 59, respectively, in
the same way as 53 was converted to 18 and 20.
Reaction conditions: (a) NBS,
BPO, benzene, reflux; (b) TEA, MeCN, reflux; (c) tert-butyl (2-(prop-2-yn-1-yloxy)ethyl)carbamate, Pd(PPh3)2Cl2, CuI, DMF/TEA, 80 °C; (d) Pd/C, H2, EtOH, rt; (e) TFA, DCM, rt; (f) HATU, DIPEA, DMF, rt.Starting from 59, compounds 21, 23–25, and 27 were
prepared
using the procedure outlined in Scheme , which is similar to the procedure used in the preparation
of 22 from 59.
Reaction conditions: (a) tert-butyl prop-2-yn-1-ylcarbamate or tert-butyl but-3-yn-1-ylcarbamate or tert-butyl pent-4-yn-1-ylcarbamate
or tert-butyl hex-5-yn-1-ylcarbamate, Pd(PPh3)2Cl2, CuI, DMF/TEA, 80 °C; (b)
Pd/C, H2, EtOH, rt; (c) TFA, DCM, rt; (d) HATU, DIPEA,
DMF, rt.Finally, the synthesis of compound 26 is outlined
in Scheme . Methylation
of 59 with MeI yielded 63. Compound 26 was prepared from 63 by a process similar
to that used for the synthesis of 18 and 20 from 53.
Upon the basis of a novel, azacarbazole-containing potent BET inhibitor
and thalidomide/lenalidomide, we have designed a new class of PROTAC
small-molecule degraders of BET proteins. Through extensive optimization
of the linker length and composition, we have obtained a number of
highly potent small-molecule BET protein degraders as exemplified
by compound 23. Compound 23 effectively
induces degradation of the BET proteins BRD2, BRD3, and BRD4 at concentrations
as low as 0.1–0.3 nM with a 3 h treatment in the RS4;11 acute
leukemia cell line and is capable of inducing degradation of BRD4
protein at concentrations as low as 30 pM with a 24 h treatment. Compound 23 achieves an IC50 value of 51 pM and 2.3 nM in
inhibition of cell growth in the RS4;11 and MOLM-13 acute leukemia
cell lines, respectively. Our mechanistic investigation firmly establishes
that compound 23 is a bona fide PROTAC BET degrader,
whose activity requires that 23 binds to BET proteins
and the cereblon/Cullin4A E3 ubiquitin ligase complex. Compound 23 effectively induces both cell cycle arrest and apoptosis
in RS4;11 and MOLM-13 acute leukemia cell lines at subnanomolar to
low nanomolar concentrations. Significantly, 23 achieves
>90% tumor regression in the RS4;11 xenograft model in mice at
a well-tolerated
dose-schedule. Our PD analysis showed that a single, intravenous dose
of 23 is capable of inducing profound degradation of
BET proteins in the tumor tissue with effect persisting for >24
h,
accompanied by robust cleavage of PARP and caspase-3 and strong down-regulation
of c-Myc. In addition to its potent anticancer activity against acute
leukemia cells, our recent study also showed that compound 23 is very potent and efficacious against triple-negative human breast
cancer in vitro and in vivo.[32] Collectively,
our data demonstrate that 23 is a highly potent, efficacious,
and promising BET degrader and warrants further evaluation as a potential
new therapy for the treatment of human acute leukemia and other types
of humancancer.
Experimental Section
Chemistry.
General Experiment and Information
Unless
otherwise noted, all purchased reagents were used as received without
further purification. 1HNMR and 13CNMR spectra
were recorded on a Bruker Advance 400 or 300 MHz spectrometer. 1HNMR spectra were reported in parts per million (ppm) downfield
from tetramethylsilane (TMS). All 13CNMR spectra were
reported in ppm and obtained with 1H decoupling. In reported
spectral data, the format (δ) chemical shift (multiplicity, J values in Hz, integration) was used with the following
abbreviations: s = singlet, d = doublet, t = triplet, q = quartet,
m = multiplet. MS analyses were carried out with a Waters UPLC–mass
spectrometer. The final compounds were all purified by C18 reverse
phase preparative HPLC column with solvent A (0.1% TFA in H2O) and solvent B (0.1% TFA in MeCN) as eluents. The purity of all
the final compounds was confirmed to be >95% purity by UPLC–MS
or UPLC.
Ethylhydrazine oxalate
(30, 41.0 g, 0.27 mmol, 1.2 equiv) in 200 mL of EtOH
was added to a stirred solution of 3-cyclopropyl-3-oxopropanenitrile
(29, 25.0 g, 0.23 mol, 1.0 equiv). The resulting solution
was heated to reflux for 12 h. After cooling to room temperature,
most of the solvent was evaporated. Workup was performed with EtOAc
and saturated brine solution. The combined organic layer was dried
over anhydrous Na2SO4. After filtration and
concentration, the residue was purified by flash column chromatography
with hexane/EtOAc to afford the desired compound 31 as
a slightly yellow solid (22.6 g, 65% yield). 1HNMR (400
MHz, CDCl3) 5.07 (s, 1H), 3.82 (q, J =
7.2 Hz, 2H), 3.55 (s, 2H, NH2), 1.78–1.71 (m, 1H),
1.28 (t, J = 7.2 Hz, 3H), 0.80–0.75 (m, 2H),
0.57–0.54 (m, 2H). UPLC–MS calculated for C8H14N3 [M + H]+: 152.12, found 152.09.
Compound 32, Prepared in Three Steps Following
a Published Method[32]
Compound 32 (13.16 g, 40 mmol, 1.0 equiv) and ethyl cyanoformate (19.76
mL, 0.2 mol, 5.0 equiv) were added to a round-bottom flask at room
temperature. A 4.0 M hydrogen chloride solution in dioxane (90 mL)
was added, and the reaction mixture was refluxed at 82 °C for
12 h. The reaction was then cooled to room temperature, and the solvents
were removed on a rotary evaporator. To this crude mixture, 10% NaOHaqueous solution (120 mL) and EtOH (100 mL) were added, and the solution
was heated to reflux for 6 h. The volatile components were then removed
on a rotary evaporator, and the aqueous residue was acidified with
2 Naqueous HCl. The product was allowed to precipitate at 0 °C.
Filtration of the mixture furnished pure 7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylic acid
as a yellow solid (11.34 g, 80% yield). UPLC–MS calculated
for C17H15N4O5 [M + H]+: 355.10, found 355.45.Concentrated sulfuric acid (15
mL) was added to a solution of 7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylic
acid (11.34 g, 32 mmol) in EtOH (300 mL) in a round-bottom flask.
The mixture was heated to reflux for 12 h. The volatile components
were removed on a rotary evaporator. Then 400 mL of EtOAc was added.
The product was allowed to precipitate. Filtration of the mixture
furnished pure methyl 7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylate
as a solid (10.61 g, 90% yield). 1HNMR (300 MHz, CD3OD) δ (ppm) 7.86 (s, 1H), 7.39 (s, 1H), 4.07 (s, 3H),
3.93 (s, 3H), 2.34 (s, 3H), 2.17 (s, 3H).Methyl 7-(3,5-dimethylisoxazol-4-yl)-4-hydroxy-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylate
(10.61 g, 28.8 mmol) and POCl3 (200 mL) were stirred in
a round-bottom flask at 90 °C for 12 h, then cooled to room temperature,
and the volatile components were removed on a rotary evaporator. EtOAc
(200 mL) was added, and the pH was adjusted to 8 using aqueous NaOH
solution. Filtration of the mixture furnished compound 33 as a yellow solid (7.80 g, 70% yield). 1HNMR (400 MHz,
DMSO-d6) δ (ppm) 7.81 (s, 1H), 7.55
(s, 1H), 3.92 (s, 3H), 3.90 (s, 3H), 2.33 (s, 3H), 2.12 (s, 3H). UPLC–MS
calculated for C18H16ClN4O4 [M + H]+: 387.09, found 387.18.Pd2(dba)3 (183 mg, 0.2 mmol, 0.1 equiv),
racemic BINAP (249 mg, 0.4 mmol, 0.2 equiv), and K3PO4 (1.70 g, 8.0 mmol, 4.0 equiv) were added sequentially to
a solution of compound 33 (774 mg, 2.0 mmol, 1.0 equiv)
in toluene (25 mL). The solution was purged and refilled with nitrogen
three times before compound 31 (604 mg, 4.0 mmol, 2.0
equiv) was added. The solution was purged and refilled with nitrogen
again. The resulting solution was heated to 110 °C and stirred
for 12 h. After cooling to room temperature, the solution was filtered
through Celite and concentrated. The residue was purified by flash
column chromatography with DCM/MeOH to afford methyl 4-((3-cyclopropyl-1-ethyl-1H-pyrazol-5-yl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylate
as a yellow solid (702 mg, 70% yield). 1HNMR (400 MHz,
DMSO-d6) 12.15 (s, 1H), 12.05 (s, 1H),
8.06 (s, 1H), 7.56 (s, 1H), 7.22 (s, 1H), 3.99 (q, J = 7.2 Hz, 2H), 3.79 (s, 3H), 3.13 (s, 3H), 2.24 (s, 3H), 2.05 (s,
3H), 1.78–1.71 (m, 1H), 1.14 (t, J = 7.2 Hz,
3H), 0.80–0.75 (m, 2H), 0.57–0.54 (m, 2H).LiOH
(101 mg, 4.2 mmol, 3.0 equiv) was added to a solution of methyl
4-((3-cyclopropyl-1-ethyl-n class="Chemical">1H-pyrazol-5-yl)amino)-7-(3,5-dimethylisoxazol-4-yl)-6-methoxy-9H-pyrimido[4,5-b]indole-2-carboxylate
(702 mg, 1.4 mmol, 1.0 equiv) in THF (20 mL). The solution was stirred
at room temperature for 12 h. After concentration, the residue was
purified by preparative HPLC to afford the desired compound 34 as a yellow solid (545 mg, 80% yield).
HATU (27 mg,
0.07 mmol, 1.4 equiv), n class="Chemical">DIPEA (27 μL, 0.15 mmol,
3.0 equiv), and methylamine (2.0 M in THF 0.1 mL, 0.20 mmol, 4.0 equiv)
were added sequentially to a stirred solution of compound 34 (24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was
stirred at room temperature for 2 h. Preparative HPLC afforded the
pure product (8) as a slightly yellow solid (17 mg, 68%
yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 12.17 (s, 1H), 9.27 (s, 1H), 8.21 (s, 1H), 7.43 (s,
1H), 7.34 (s, 1H), 5.92 (s, 1H), 3.96 (q, J = 7.2
Hz, 2H), 3.81 (s, 3H), 2.81 (d, J = 4.8 Hz, 3H),
2.30 (s, 3H), 2.09 (s, 3H), 1.92–1.85 (m, 1H), 1.32 (t, J = 7.2 Hz, 3H), 0.87–0.82 (m, 2H), 0.64–0.60
(m, 2H). UPLC–MS calculated for C26H29N8O3 [M + H]+: 501.24, found 501.22.
Purity, 99.0%.
In a round-bottom flask, 3-nitrophthalic anhydride 42 (5.79 g, 30 mmol, 1.0 equiv) and p-toluenesulfonic
acid monohydrate (571 mg, 3 mmol, 0.1 equiv) were mixed in benzyl
alcohol (20 mL). The mixture was heated to 100 °C and stirred
for 12 h. After cooling to room temperature, benzyl bromide (7.1 mL,
45 mmol, 1.5 equiv), KI (498 mg, 3 mmol, 0.1 equiv), KHCO3 (9.0 g, 90 mmol, 3.0 equiv), and DMF (25 mL) were added. The mixture
was heated to 100 °C and stirred for 6 h. After cooling to room
temperature, the solvent was evaporated and the mixture was then poured
into H2O (300 mL). The solution was extracted with EtOAc.
The combined organic layers were washed with brine and dried over
anhydrous Na2SO4. After filtration and evaporation,
the crude residue was purified by flash column chromatography with
hexane/EtOAc to give the intermediate dibenzyl 3-nitrophthalate as
a slightly yellow solid (9.4 g, 80% yield).In a round-bottom
flask, dibenzyl 3-nitrophthalate (9.4 g, 24 mmol, 1.0 equiv) was dissolved
inEtOAc (100 mL). Then stannous chloride dihydrate (11.3 g, 50 mmol,
2.08 equiv) was added portionwise to the reaction mixture. The resulting
reaction mixture was heated to 50 °C and stirred for 12 h. Then
NaOH (aq) was added to the reaction mixture to quench the reaction.
The reaction mixture was filtered through Celite and washed with EtOAc.
The filtrate was extracted with EtOAc and brine. The combined organic
layer was dried over anhydrous Na2SO4. After
filtration and evaporation, the crude residue was purified by flash
column chromatography with hexane/EtOAc to give dibenzyl 3-aminophthalate
as a slightly yellow solid (7.8 g, 90% yield). 1HNMR (400
MHz, CDCl3) δ (ppm) 7.45–7.36 (m, 10H), 7.22
(t, J = 8.4 Hz, 1H), 6.96 (d, J =
7.2 Hz, 1H), 6.76 (d, J = 7.6 Hz, 1H), 5.36 (s, 2H,
NH2), 5.26 (s, 2H), 5.10 (s, 2H).In a round-bottom
flask, dibenzyl 3-aminophthalate (2.0 g, 5.54
mmol, 1.0 equiv) and KI (100 mg, 0.56 mmol, 0.1 equiv) were combined
with anhydrous DMF (10 mL). tert-Butyl bromoacetate
(2.4 mL, 16.6 mmol, 3.0 equiv) and DIPEA (4.8 mL, 27.7 mmol, 5.0 equiv)
were added to the reaction mixture which was then heated to 90 °C
and stirred for 12 h. After cooling to room temperature, most of the
solvent was evaporated and the residue was purified by column chromatography
with hexane/EtOAc to give compound 43 as a slightly yellow
solid (1.05 g, 40% yield). 1HNMR (400 MHz, CDCl3) δ (ppm) 7.40–7.25 (m, 11H), 6.87 (d, J = 7.2 Hz, 1H), 6.65 (d, J = 8.4 Hz, 1H), 5.20 (s,
2H), 4.95 (s, 2H), 3.88 (d, J = 4.8 Hz, 2H), 1.52
(s, 9H); 13CNMR (100 MHz, CDCl3) δ (ppm)
169.16, 168.86, 167.61, 148.44, 135.78, 135.66, 135.42, 132.82, 128.72,
128.62, 128.58, 128.40, 128.22, 128.16, 116.54, 113.89, 111.32, 82.38,
67.32, 66.97, 46.07, 28.27.In a round-bottom flask, compound 43 (1.0 g, 2.1 mmol)
was dissolved in EtOH (20 mL). 100 mg of Pd/C (10 wt %) was added
under N2. The flask was purged and refilled with H2 three times. Then the reaction mixture was stirred at room
temperature under 1 atm of H2. Once the starting material
disappeared, as judged by TLC, the mixture was filtered through Celite
and washed with EtOH. After evaporation of the solvent, 3-aminopiperidine-2,6-dione
hydrochloride 38 (380 mg, 2.31 mmol, 1.1 equiv) and pyridine
(20 mL) were added. The reaction mixture was heated to 110 °C
and stirred overnight. After cooling to room temperature, the solvent
was evaporated as much as possible and the residue was poured into
H2O. After extraction three times with EtOAc, the combined
organic layer was washed with brine and dried over anhydrous Na2SO4. After filtration and evaporation, the crude
residue was purified by flash column chromatography with DCM/MeOH
to give compound 44 as a yellow solid (325 mg, 40% yield).TFA (2.0 mL) was added to compound 44, obtained as
described above. The intermediate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine
was obtained by evaporation of the solvent without further purification. 1HNMR (400 MHz, DMSO-d6) δ
(ppm) 12.91 (s, 1H, COOH), 11.10 (s, 1H, NH), 7.59 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 6.8 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 6.86 (t, J = 5.6 Hz, 1H,
NH), 5.08 (dd, J = 13.2 Hz, J =
5.6 Hz, 1H), 4.12 (d, J = 5.2 Hz, 2H), 2.94–2.85
(m, 1H), 2.63–2.49 (m, 2H), 2.09–2.07 (m, 1H); 13CNMR (100 MHz, DMSO-d6) δ
(ppm) 173.28, 171.90, 170.52, 169.26, 167.75, 146.28, 136.60, 132.48,
118.18, 111.54, 110.11, 60.22, 49.07, 31.46, 22.61. UPLC–MS
calculated for C15H14N3O6 [M + 1]+: 332.09, found 332.00.Following the procedure
for the synthesis of compound 41, compound 45 (250 mg, 0.76 mmol) was synthesized from
the intermediate (2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)glycine.HATU (27 mg, 0.07 mmol, 1.4 equiv), DIPEA (27 μL, 0.15 mmol,
3.0 equiv), and compound 45 (28 mg, 0.07 mmol, 1.4 equiv)
were added sequentially to a stirred solution of compound 34 (24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was
stirred at room temperature for 2 h. Preparative HPLC purification
afforded the pure product (10) as a slightly yellow solid
(32 mg, 75% yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 12.32 (s, 1H), 11.09 (s, 1H), 9.41 (s,
1H), 8.26 (t, J = 5.6 Hz, 1H), 8.19 (t, J = 5.6 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.48 (s,
1H), 7.41 (s, 1H), 7.03 (d, J = 6.8 Hz, 1H), 6.87
(d, J = 8.8 Hz, 1H), 5.94 (s, 1H), 5.07 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H), 3.97 (q, J = 7.2 Hz, 2H), 3.94 (s, 2H), 3.82 (s, 3H), 3.29–3.26
(m, 2H), 3.15–3.12 (m, 2H), 2.93–2.84 (m, 1H), 2.61–2.45
(m, 2H), 2.30 (s, 3H), 2.10 (s, 3H), 2.05–2.01 (m, 1H), 1.92–1.85
(m, 1H), 1.51–1.45 (m, 4H), 1.31 (t, J = 7.2
Hz, 3H), 0.87–0.82 (m, 2H), 0.64–0.60 (m, 2H). UPLC–MS
calculated for C44H47N12O8 [M + H]+: 871.36, found 871.39. Purity, 91.4%.
KI (33 mg, 0.2 mmol, 0.1 equiv), NaHCO3 (336 mg, 4.0 mmol, 2.0 equiv), and 7-bromo-1-heptanol (468 mg, 2.4
mmol, 1.2 equiv) were added sequentially to a solution of compound 39 (548 mg, 2.0 mmol, 1.0 equiv) in DMF (10 mL). The resulting
solution was heated to 60 °C and stirred for 12 h. After cooling
to room temperature, the solution was filtered through Celite and
concentrated. The residue was purified by preparative HPLC to afford 46 as a colorless solid (589 mg, 76% yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 11.10
(s, 1H), 7.80 (t, J = 7.80 Hz, 1H), 7.51 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H),
5.09 (dd, J = 12.8 Hz, J = 5.2 Hz,
1H), 4.20 (t, J = 6.4 Hz, 3H), 3.39 (t, J = 6.8 Hz, 2H), 2.90–2.86 (m, 1H), 2.62–2.50 (m, 2H),
2.08–2.02 (m, 1H), 1.78–1.72 (m, 2H), 1.48–1.29
(m, 8H); 13CNMR (100 MHz, DMSO-d6) δ (ppm) 173.24, 170.42, 167.32, 165.77, 156.50, 137.47,
133.72, 120.23, 116.69, 115.57, 69.27, 61.16, 49.22, 32.94, 31.43,
29.07, 28.89, 25.94, 25.82, 22.48. UPLC–MS calculated for C20H25N2O6 [M + H]+: 389.17, found 389.18.MsCl (0.14 mL, 1.70 mmol, 1.2 equiv)
and TEA (0.30 mL, 2.13 mmol, 1.5 equiv) were added sequentially to
a solution of compound 46 (589 mg, 1.42 mmol, 1.0 equiv)
inDCM (10 mL) at 0 °C. The resulting solution was stirred at
0 °C for 2 h. Then the solvent was evaporated to afford the crude
residue. Sodium azide (277 mg, 4.25 mmol, 3.0 equiv) was added to
the above residue in DMF (6 mL). The solution was stirred at 70 °C
for 2 h and then filtered through Celite. The residue was purified
by preparative HPLC to afford the desired product as a white solid
(386 mg, 66% yield in two steps). 10% Pd/C (30 mg) was added to the
above compound in EtOH (10 mL). The flask was purged and refilled
with H2 three times. The solution was stirred at room temperature
under H2 for 12 h. Filtration through Celite removed the
Pd/C, and then the solution was concentrated and the residue was purified
by preparative HPLC to afford the desired compound 47 as a colorless oil (72 mg, 20% yield).HATU (27 mg, 0.07 mmol,
1.4 equiv), DIPEA (27 μL, 0.15 mmol,
3.0 equiv), and the amine 47 (27.1 mg, 0.07 mmol, 1.4
equiv) were added sequentially to a stirred solution of the intermediate 34 (24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution
was stirred at room temperature for 2 h. Preparative HPLC purification
afforded the pure product 11 as a slightly yellow solid
(19 mg, 45% yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 12.30 (s, 1H), 11.08 (s, 1H), 9.28 (s,
1H), 8.16 (t, J = 5.6 Hz, 1H), 7.79 (t, J = 8.0 Hz, 1H), 7.52 (s, 1H), 7.51 (d, J = 8.0 Hz,
1H), 7.43 (d, J = 8.0 Hz, 1H), 7.34 (s, 1H), 5.94
(s, 1H), 5.07 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H), 4.21 (t, J = 6.4 Hz, 1H), 3.94 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.29–3.24 (m, 2H),
2.91–2.82 (m, 1H), 2.59–2.44 (m, 2H), 2.30 (s, 3H),
2.10 (s, 3H), 2.05–1.99 (m, 1H), 1.91–1.84 (m, 1H),
1.81–1.74 (m, 2H), 1.53–1.47 (m, 4H), 1.41–1.30
(m, 7H), 0.86–0.81 (m, 2H), 0.64–0.60 (m, 2H). UPLC–MS
calculated for C45H49N10O8 [M + H]+: 857.37, found 857.35. Purity, 98.2%.
General
Procedure for Synthesis of Compounds 12–17
In a round-bottom flask, 3-fluorophthalic
anhydride 48 (6.64 g, 40 mmol, 1.0 equiv), 3-aminopiperidine-2,6-dione
hydrochloride 38 (6.58 g, 40 mmol, 1.0 equiv), and sodium
acetate (3.94 g, 48 mmol, 1.2 equiv) were mixed in AcOH (120 mL).
The resulting reaction mixture was heated to reflux at 140 °C
for 12 h. After cooling to room temperature, most of the AcOH was
evaporated and the residue was purified by flash column chromatography
with DCM/MeOH to obtain compound 49 as a slightly yellow
solid (9.7 g, 88% yield). UPLC–MS calculated for C13H10FN2O4 [M + H]+: 277.06,
found 277.02. 1HNMR (400 MHz, DMSO-d6) δ (ppm) 11.15 (s, 1H), 7.98–7.93 (m, 1H), 7.80–7.72
(m, 2H), 5.17 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 2.95–2.86 (m, 1H), 2.64–2.47 (m, 2H),
2.10–2.06 (m, 1H).Mono-Boc protected alkyl diamines
(1.13 mmol, 1.1 equiv) of different lengths were added to a stirred
solution of compound 49 (285 mg, 1.03 mmol, 1.0 equiv)
in DMF (6.0 mL) and DIPEA (0.36 mL, 2.06 mmol, 2.0 equiv). The reaction
mixture was stirred at 90 °C for 12 h. Then the mixture was cooled
to room temperature, poured into H2O, and extracted twice
with EtOAc. The combined organic layer was washed with brine, dried
over anhydrous Na2SO4. After filtration and
evaporation, the crude residue was purified by preparative HPLC to
give the intermediate (50a–f) which
was dissolved in DCM (4.0 mL) and TFA (2.0 mL). After stirring for
1 h, the solvent was evaporated to give the crude product 51a–f, which was used in the next step without further
purification.HATU (27 mg, 0.07 mmol, 1.4 equiv), n class="Chemical">DIPEA (27
μL, 0.15 mmol,
3.0 equiv), and the amine intermediate 51a–f (0.07 mmol, 1.4 equiv) were added sequentially to a stirred
solution of compound 34 (24 mg, 0.05 mmol, 1.0 equiv)
in DMF (2.0 mL). The solution was stirred at room temperature for
2 h. Preparative HPLC purification afforded the pure product 12–17.
In a round-bottom flask, 3-bromophthalic anhydride 52 (2.27 g, 10.0 mmol, 1.0 equiv), 3-aminopiperidine-2,6-dione
hydrochloride 38 (1.81 g, 11 mmol, 1.1 equiv), and sodium
acetate (0.98 g, 12 mmol, 1.2 equiv) were mixed inacetic acid (30
mL). The resulting reaction mixture was heated to reflux at 140 °C
for 12 h. After cooling to room temperature, most of the AcOH was
evaporated and the residue was purified by flash column chromatography
with DCM/MeOH to give compound 53 as a purple solid (2.70
g, 80% yield). 1HNMR (400 MHz, DMSO-d6) δ 11.15 (s, 1H), 8.06 (d, J =
8.0 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.78 (t, J = 7.6 Hz, 1H), 5.18 (dd, J = 12.8 Hz, J = 5.6 Hz, 1H), 2.95–2.86 (m, 1H), 2.65–2.48
(m, 2H), 2.11–2.05 (m, 1H); 13CNMR (100 MHz, DMSO-d6) δ 173.21, 170.18, 166.07, 165.65, 139.66,
136.73, 134.15, 129.20, 123.31, 118.11, 49.65, 31.39, 22.30. UPLC–MS
calculated for C13H10BrN2O4 [M + H]+: 336.98, found 336.95.In a round-bottom
flask, compound 53 (674 mg, 2.0 mmol, 1.0 equiv) and tert-butyl hex-5-yn-1-ylcarbamate (934 mg, 4.0 mmol, 2.0
equiv) were added to a solution of CuI (76 mg, 0.4 mmol, 0.2 equiv)
and Pd(PPh3)2Cl2 (140 mg, 0.2 mmol,
0.1 equiv) in DMF (10 mL). The solution was purged and refilled with
nitrogen three times. Then trimethylamine (5.0 mL) was added. The
solution was purged and refilled with nitrogen again. The mixture
was stirred at 70 °C for 3 h under Ar. The solution was cooled
to room temperature and filtered through Celite. The residue was purified
by flash column chromatography to yield compound 54b as
a slightly yellow solid (653 mg, 72% yield). UPLC–MS calculated
for C24H27N3NaO6 [M +
Na]+ = 476.18, found 476.18.10% Pd/C (60 mg) was
added to a solution of compound 54b (653 mg, 1.44 mmol,
1.0 equiv) in EtOH (10 mL) under N2. The solution was purged
and refilled with hydrogen three times.
The solution was stirred at room temperature for 12 h. After filtration,
the solvent was evaporated to give the crude residue 55b, which was dissolved in DCM (4.0 mL) and TFA (2.0 mL). The reaction
mixture was stirred at room temperature for 1 h. Preparative HPLC
purification afforded compound 56b as a colorless oil
(411 mg, 80% yield in two steps).HATU (27 mg, 0.07 mmol, 1.4
equiv), DIPEA (27 μL, 0.15 mmol,
3.0 equiv), and 56b (25 mg, 0.07 mmol, 1.4 equiv) were
added sequentially to a stirred solution of compound 34 (24 mg, 0.05 mmol, 1.0 equiv) in DMF (2.0 mL). The solution was
stirred at room temperature for 2 h. Preparative HPLC purification
afforded the pure product 18 as a slightly yellow solid
(29 mg, 70% yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 12.25 (s, 1H), 11.10 (s, 1H), 9.29 (s,
1H), 8.15 (t, J = 5.6 Hz, 1H), 7.78–7.69 (m,
3H), 7.58 (s, 1H), 7.37 (s, 1H), 5.94 (s, 1H), 5.12 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 3.94 (q, J = 7.2 Hz, 2H), 3.83 (s, 3H), 3.28–3.23 (m, 2H), 3.05 (t, J = 8.0 Hz, 2H), 2.93–2.83 (m, 1H), 2.61–2.49
(m, 2H), 2.30 (s, 3H), 2.10 (s, 3H), 2.07–2.03 (m, 1H), 1.89–1.82
(m, 1H), 1.66–1.60 (m, 2H), 1.53–1.47 (m, 2H), 1.36–1.27
(m, 7H), 0.84–0.79 (m, 2H), 0.63–0.59 (m, 2H). UPLC–MS
calculated for C44H47N10O7 [M + H]+: 827.36, found 827.28. Purity, 98.7%.
Following the procedure used in the synthesis
of compound 18 from the intermediate 53,
compound 20 was obtained with tert-butyl
pent-4-yn-1-ylcarbamate as the linker instead of tert-butyl hex-5-yn-1-ylcarbamate. 1HNMR (400 MHz, DMSO-d6) δ (ppm) 12.22 (s, 1H), 11.10 (s, 1H),
9.29 (s, 1H), 8.19 (t, J = 5.6 Hz, 1H), 7.76–7.71
(m, 3H), 7.54 (s, 1H), 7.35 (s, 1H), 5.93 (s, 1H), 5.12 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 3.95 (q, J = 7.2 Hz, 2H), 3.82 (s, 3H), 3.29–3.24 (m, 2H),
3.06 (t, J = 8.0 Hz, 2H), 2.92–2.83 (m, 1H),
2.61–2.55 (m, 2H), 2.30 (s, 3H), 2.10 (s, 3H), 2.05–2.00
(m, 1H), 1.90–1.85 (m, 1H), 1.67–1.61 (m, 2H), 1.57–1.52
(m, 2H), 1.41–1.35 (m, 2H), 1.31 (t, J = 7.2
Hz, 3H), 0.85–0.81 (m, 2H), 0.63–0.59 (m, 2H). UPLC–MS
calculated for C43H44N10O7 [M + H]+: 812.34, found 813.33. Purity, 95.3%.
General
Procedure for Synthesis of Compounds 21–25 and 27
NBS (17.09 g,
96 mmol, 1.2 equiv) and n class="Chemical">BPO (1.938 g, 8.0 mmol, 0.1 equiv) were added
to a stirred solution of methyl 3-bromo-2-methylbenzoate 57 (18.33 g, 80 mmol, 1.0 equiv) in benzene (150 mL). The
solution was heated at reflux for 6 h. After cooling to room temperature,
the solvent was evaporated and the residue was purified by flash column
chromatography with hexane/EtOAc to afford the intermediate 58 (22.17 g, 90% yield) as a slightly yellow solid.
Compound 38 (14.48 g, 88 mmol, 1.22 equiv) and TEA (13.38
mL, 96 mmol, 1.33 equiv) were added to a stirred solution of compound 58 (22.17 g, 72 mmol, 1.0 equiv) inMeCN (150 mL). The solution
was stirred at 80 °C for 12 h and then cooled to room temperature,
and most of the solvent was evaporated. EtOAc (200 mL) and H2O (200 mL) were added to the residue and the solution was filtered
to afford the crude product as a purple solid 59 (17.4
g, 75% yield). 1HNMR (400 MHz, DMSO-d6) δ (ppm) 11.00 (s, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 5.15 (dd, J = 13.2 Hz, J = 5.2 Hz, 1H), 4.42 (d, J = 17.6 Hz,
1H), 4.27 (d, J = 17.6 Hz, 1H), 2.96–2.87
(m, 1H), 2.62–2.42 (m, 2H), 2.07–2.00 (m, 1H); 13CNMR (100 MHz, DMSO-d6) δ
(ppm) 173.30, 171.32, 167.69, 142.58, 135.10, 134.41, 130.98, 122.96,
117.79, 52.22, 48.46, 31.66, 22.74. UPLC–MS calculated for
C13H12BrN2O3 [M + H]+: 323.00, found 322.90.Following the procedures used
to prepare compound 18 from the intermediate 53, compounds 21–25 and 27 with different alkynyl
chains were obtained as indicated in Schemes and 9.
10% Pd/C (20 mg)
was added to a stirred solution
of compound 33 (200 mg, 0.52 mmol, 1.0 equiv) in 10 mL
of DMF under N2. The flask was purged and refilled with
H2 three times. The solution was then stirred at 60 °C
for 12 h. After cooling to room temperature, the solution was filtered
through Celite. After concentration, the residue crude intermediate 35 was suspended in THF (30 mL), and LiOH (38 mg, 1.55 mmol,
3.0 equiv) was added. The resulting solution was stirred at room temperature
for 12 h. After concentration, the residue was purified by preparative
HPLC to afford the compound 36 as a yellow solid (52
mg, 30% yield for two steps). UPLC–MS calculated for C17H15N4O4 [M + H]+: 339.11, found 339.08.Compound 36 (150 mg, 0.44
mmol) was suspended in thionyl chloride (0.64 mL, 8.8 mmol), and two
drops of dry DMF were added. The mixture was stirred at reflux for
2 h. Excess thionyl chloride was removed in vacuo, and the resulting
solid was dried. The resulting acyl chloride was used without further
purification. Dry THF (15 mL) was added to the acyl chloride. The
mixture was cooled to 0 °C, and subsequently, methylamine hydrochloride
(45 mg, 0.67 mmol) was added. After stirring at room temperature for
2 h, the mixture was evaporated and purified by preparative HPLC to
get 61.8 mg of compound 28 with a yield of 40%. ESI-MS
calculated for C18H17N5O3 [M + H]+ = 52.13. Obtained: 352.17. 1HNMR
(400 MHz, MeOD-d4) δ 9.55 (s, 1H),
8.03 (s, 1H), 7.54 (s, 1H), 3.96 (s, 3H), 2.35 (s, 3H), 2.18 (s, 3H),
1.31 (t, J = 7.3 Hz, 3H). UPLC analysis (10 min from
10% to 100% (MeCN/H2O containing 0.1% CF3CO2H)): retention time, 2.70 min; peak area, 98.12%.
Determination
of Biochemical Binding Affinities to BET Proteins
Binding
affinities of BET inhibitors to BRD2 (BD1 and BD2 proteins),
BRD3 (BD1 and BD2 proteins), BRD4 (BD1 and BD2 proteins) were determined
using our established fluorescence-polarization binding assays as
described previously.[27]
Cell Growth
Inhibition, Apoptosis Analysis, and Western Blotting
The
human acute leukemiaRS4;11 cell line (CRL-1873) was purchased
from the American Type Culture Collection, and the human acute leukemiaMOLM-13 cell line was purchased from the DSMZ German cell bank (ACC554).
In all experiments, RS4;11 and MOLM-13humanleukemia cells were used
within three months of thawing fresh vials. RS4;11 and MOLM-13 cells
were cultured in RPMI 1640 media supplemented with 10% FBS and 1%
penicillin–streptomycin at 37 °C in a humidified atmosphere
containing 5% CO2 in air.In cell growth experiments,
cells were seeded in 96-well cell culture plates at a density of 10000−20000
cells/well in 100 μL of culture medium. Each compound tested
was serially diluted in the appropriate medium, and 100 μL of
the diluted solution containing the tested compound was added to the
appropriate wells of the cell plate. After addition of the tested
compound, the cells were incubated for 4 days at 37 °C in an
atmosphere of 5% CO2. Cell growth was evaluated by a lactate
dehydrogenase-based WST-8 assay (Dojindo Molecular Technologies) using
a Tecan Infinite M1000 multimode microplate reader (Tecan, Morrisville,
NC). The WST-8 reagent was added to the plate, incubated for at least
1 h, and read at 450 nm. The readings were normalized to the DMSO-treated
cells, and the IC50 was calculated by nonlinear regression
analysis using GraphPad Prism 6 software.Flow cytometry was
used to analyze effects of the drugs on cell
cycle (propidium iodide staining) and apoptosis (annexin V and propidium
iodide staining). Cell were treated with compounds at the indicated
concentrations for 24 h, collected, stained, and analyzed by flow
cytometry.For Western blot analysis, 2 × 106 cells/well were
treated with compounds at the indicated concentrations for various
times. Cells were collected and lysed inRIPA buffer containing protease
inhibitors. An amount of 20 μg of lysate was run in each lane
of a PAGE–SDS and blotted into PVDF membranes. Antibodies for
immunoblotting were BRD2, BRD3, and BRD4 purchased from Bethyl Laboratories
(Montgomery, TX, USA), c-Myc from Cell Signaling Technology (Danvers,
MA, USA), and GAPDH from Santa Cruz Biotechnologies (Dallas, TX, USA).
Efficacy and Pharmacodynamics Studies in the RS4;11 Xenograft
Model in Mice
All animal experiments were done under the
guidelines of the University of Michigan Committee for Use and Care
of Animals and using an approved animal protocol (PRO00005315, PI,
Shaomeng Wang).To develop xenograft tumors, 5 × 106 RS4;11 cells with 50% Matrigel were injected subcutaneously
on the dorsal side of severe combined immunodeficient (SCID) mice,
obtained from Charles River, one tumor per mouse. When tumors reached
∼100 mm3, mice were randomly assigned to treatment
and vehicle control groups. Animals were monitored daily for any signs
of toxicity and weighed 2–3 times per week during the treatment
and weighed at least weekly after the treatment ended. Tumor size
was measured 2–3 times per week by electronic calipers during
the treatment period and at least weekly after the treatment was ended.
Tumor volume was calculated as V = LW2/2, where L is the
length and W is the width of the tumor.For
pharmacodynamic analysis, resected RS4;11 xenograft tumor tissues
were ground into powder in liquid nitrogen and lysed in lysis buffer
(1% CHAPS, 150 mM NaCl, 20 mM Tris-HCl, 1 mM. EDTA, 1 mM EGTA, and
COMPLETE proteinase inhibitor (Roche)). Whole tumor lysates were separated
on 4–20% Novex gels. The separated proteins were transferred
to a polyvinylidene difluoride membrane for immunoblotting. The following
antibodies were used: rabbit polyclonal antibodies for BRD2 (A302-583A),
BRD3 (A302-368A), and BRD4 (A301-985A100) from Bethyl Laboratories;
c-Myc (D84C12), PARP (46D11), and caspase-3 (8G10) from Cell Signaling
Technology, and actin goat polyclonal antibody from Santa Cruz Biotechnology.
The secondary antibody used was horseradish peroxidase conjugated
goat anti-rabbit (Thermo Scientific). The BIO-RAD Clarity Western
Enhanced Chemiluminescence Substrates and HyBlot Chemiluminescence
film were used for signal development and detection using a SRX-101A
tabletop processor (Konica Minolta).
Determination of Drug Concentrations
in Plasma and RS4;11 Tumor
Tissue
Pharmacokinetics of compound 23 was determined
in female SCID C.B-17 mice bearing RS4;11 tumors following a single
intravenous dose of 5 mg/kg. Compound 23 was dissolved
in the vehicle containing 20% (v/v) polyethylene glycol 400, 6% (v/v)
Cremophor EL and 74% (v/v) PBS (20% PCP). Mice were sacrificed at
1, 3, 6, and 24 h after drug treatment and at 6 h after vehicle treatment,
followed by collection of blood samples (300 μL) and tumor samples.
Blood samples were centrifuged at 15 000 rpm for 10 min, then
the supernatant plasma was saved for analysis. Isolated tumor samples
were immediately frozen and ground with a mortar and pestle in liquid
nitrogen. All plasma and tumor samples were stored at −80 °C
prior to analysis.Plasma and tumor concentrations of compound 23 were determined by a LC–MS/MS method developed and
validated for this study. The LC–MS/MS method, consisting of
a Shimadzu HPLC system and chromatographic separation of tested compound,
was achieved using a Waters XBridge-C18 column (5 cm × 2.1 mm,
3.5 μm). An AB Sciex QTrap 5500 mass spectrometer equipped with
an electrospray ionization source (Applied Biosystems, Toronto, Canada)
in the positive-ion multiple reaction monitoring (MRM) mode was used
for detection. The mobile phases were 0.1% formic acid in purified
water (A) and 0.1% formic acid in acetonitrile (B). The gradient (B)
was held at 10% (0–0.3 min), increased to 95% at 0.7 min, then
held at isocratic 95% B for 2.3 min and then immediately stepped back
down to 10% for 2 min re-equilibration. The flow rate was set at 0.4
mL/min.
In Vitro Metabolite Identification in Mouse Liver Microsomes
Compound 23 (10 μM) was incubated with mouse
liver microsomes (MLM) and β-NADPH at 37 °C for 20 and
40 min. The reaction was quenched by adding a 3-fold volume of ice-cold
acetonitrile. The mixture was centrifuged at 15 000 rpm for
10 min, and the supernatant was saved under −80 °C for
analysis. The negative control samples were prepared by a similar
procedure without NADPH or using boiled microsomes. The general approach
for metabolite identification using AB Sciex QTrap 5500 mass spectrometer
involves the following steps: (1) Obtain a product ion spectrum of
the parent compound to establish fragmentation. (2) Interpret the
spectrum to identify major fragment ion and possible neutral loss.
(3) Collect spectra of samples using both established precursor ion
scan and neutral loss scan. EMS scan of both control and samples were
acquired also. (4) Run product ion scans and MRM scans for all possible
metabolite identified from step 3 plus expected metabolite. (5) Interpret
the spectrum of the metabolites and determine the structure with their
logical fragmentation pattern.
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Scheme 1
Scheme 2
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Scheme 7
Scheme 8
Scheme 9