Design and synthesis of a biaryl series as inhibitors for the bromodomains of CBP/P300

Article abstract of DOI:10.1016/j.bmcl.2017.11.025

A novel, potent, and orally bioavailable inhibitor of the bromodomain of CBP, compound 35 (GNE-207), has been identified through SAR investigations focused on optimizing al bicyclic heteroarene to replace the aniline present in the published GNE-272 series. Compound 35 has excellent CBP potency (CBP IC₅₀ = 1 nM, MYC EC₅₀ = 18 nM), a selectively index of >2500-fold against BRD4(1), and exhibits a good pharmacokinetic profile.

Full text of DOI:10.1016/j.bmcl.2017.11.025

Accepted Manuscript
Design and Synthesis of A Biaryl Series As Inhibitors for the Bromodomains
of CBP/P300
Kwong Wah Lai, F. Anthony Romero, Vickie Tsui, Maureen H. Beresini,
Gladys de Leon Boenig, Sarah M. Bronner, Kevin Chen, Zhongguo Chen, Edna
F. Choo, Terry D. Crawford, Patrick Cyr, Susan Kaufman, Yingjie Li, Jiangpeng
Liao, Wenfeng Liu, Justin Ly, Jeremy Murray, Weichao Shen, John Wai, Fei
Wang, Caicai Zhu, Xiaoyu Zhu, Steven Magnuson
PII:
S0960-894X(17)31113-7
https://doi.org/10.1016/j.bmcl.2017.11.025
BMCL 25431
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
1 September 2017
7 November 2017
13 November 2017
Please cite this article as: Lai, K.W., Romero, F.A., Tsui, V., Beresini, M.H., de Leon Boenig, G., Bronner, S.M.,
Chen, K., Chen, Z., Choo, E.F., Crawford, T.D., Cyr, P., Kaufman, S., Li, Y., Liao, J., Liu, W., Ly, J., Murray, J.,
Shen, W., Wai, J., Wang, F., Zhu, C., Zhu, X., Magnuson, S., Design and Synthesis of A Biaryl Series As Inhibitors
for the Bromodomains of CBP/P300, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/
1
0.1016/j.bmcl.2017.11.025
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Design and Synthesis of A Biaryl Series As Inhibitors for the Bromodomains of
CBP/P300
a†
b†
b
b
Kwong Wah Lai, F. Anthony Romero, Vickie Tsui, * Maureen H. Beresini,
b
b
a
a
Gladys de Leon Boenig, Sarah M. Bronner, Kevin Chen, Zhongguo Chen, Edna F.
b
b
b
b
a
a
Choo, Terry D. Crawford, Patrick Cyr, Susan Kaufman, Yingjie Li, Jiangpeng Liao,
a
b
b
a
a
a
Wenfeng Liu, Justin Ly, Jeremy Murray, Weichao Shen, John Wai, Fei Wang,
a
a
b
Caicai Zhu, Xiaoyu Zhu, Steven Magnuson *
a
WuXi AppTec Co., Ltd. 288 Fute Zhong Road, Waigaoqiao Free Trade Zone,
Shanghai 200131, People’s Republic of China.
b
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, United
States.
1

Abstract
A novel, potent, and orally bioavailable inhibitor of the bromodomain of CBP,
compound 35 (GNE-207), has been identified through SAR investigations focused on
optimizing al bicyclic heteroarene to replace the aniline present in the published GNE-
2
1
72 series. Compound 35 has excellent CBP potency (CBP IC50 = 1 nM, MYC EC50
=
8 nM), a selectively index of >2500-fold against BRD4(1), and exhibits a good
pharmacokinetic profile.
Keywords: Bromodomain, CBP inhibitor, volume of distribution, half-life
2

Cyclic AMP response element binding protein, binding protein (CREBBP, CBP)
and the highly homologous paralog E1A-associated protein of 300 kDa (EP300, P300)
1‒5
are co-activators of many different transcription factors. Both CBP and P300 possess
several structured regions, which include the histone acetyltransferase (HAT) domain
that acetylates both histone and non-histone proteins, and an epigenetic ‘‘reader’’
6
bromodomain (BRD) that binds acetylated lysine (KAc). CBP/P300 bind to chromatin
via their BRD, and once associated with chromatin this complex will recruit additional
7
transcriptional machinery to modulate gene expression. SiteMap analysis predicts the
BRD of CBP to be a druggable target and garners increased interest from design and
8
selectivity perspectives. In fact, CBP and P300 have been implicated as potential
9‒12
oncology targets.
The BRD of CBP/P300 (hereafter together referred to as “CBP”)
13
regulates MYC, a transcription factor and oncogene widely expressed in human
cancer, which suggests a potential therapeutic strategy for targeting multiple myeloma
and other lymphoid malignancies. In addition, we have described the ability of CBP
bromodomain inhibitors to impair Treg differentiation and suppressive function, and this
14
activity could constitute a novel small molecule approach for cancer immunotherapy.
Given these combined activities, the CBP BRD has emerged as an especially
interesting new therapeutic target. To help unravel the biology and therapeutic potential
of CBP, several research groups have discovered various chemical probes that inhibit
15‒23
the BRD of CBP.
In a previous account, we reported the co-crystal structure of the screening hit 1
16
with the CBP BRD. The amide of 1 binds the KAc binding site, with the carbonyl
making a canonical hydrogen bond interaction to Asn1168 and a water-mediated
3

hydrogen bond to Tyr1125. The pyrazolopiperidine (PP) core is sandwiched between
the gatekeeper (Val1174) and Leu1120, while the phenyl ring makes van der Waals
interactions with the LPF shelf (Leu1109, Pro1110, Phe1111). Of particular interest was
the aniline N-H that makes water-mediated interactions with the backbone of Pro1110
and the sidechain of Gln1113 (Figure 1A). Despite this structural role, the aniline carries
a potential liability as a toxicophore via phase I oxidation that could lead to a highly
electrophilic quinone imine metabolite. We recently reported on a highly potent and
24
selective tetrahydroquinoline (THQ) series that was derived from 1. In parallel to the
development of this THQ series, we embarked on an alternate strategy to replace the
aniline with a bicyclic heteroarene. Here we describe the structure-based design of 35
(
(
GNE-207), a potent and selective CBP inhibitor that exhibits a good pharmacokinetic
PK) profile.
We initially explored structure-activity relationships (SAR) on 2 containing a
cyclopropylmethyl (c-Pr) moiety. The cyclopropylmethyl was derived from our earlier
16
studies on the SAR of 1 in our progress towards the development of GNE-272. It was
found that the cyclopropylmethyl substituent binds in the ZA loop region and improves
CBP potency. To observe whether we could simply replace the aniline of 2, we made
compound 3, which contained a phenyl substituent directly attached to the PP core.
This compound retained CBP potency compared to 2 and maintained good ligand
efficiency (LE). Encouraged by this result, we then attempted to increase the van der
Waals interaction of 3 with Pro1110 by replacing the phenyl with an indole. Compound 4
had an 8-fold increase in CBP inhibitory potency. We hypothesized that the indole was
important for stacking with Pro1110. To confirm this, we obtained a co-crystal structure
4

of 4 with the bromodomain of CBP (Figure 1B). As expected, the amide carbonyl makes
a direct hydrogen bond to Asn1168 and water-mediated hydrogen bonds to Tyr1125. In
addition, the indole A ring makes van der Waals interactions with Cβ and Cγ of Pro1110
while the B ring pi-stacks with the amide plane formed by the carbonyl from Leu1109
25
and the nitrogen from Pro1110. Meanwhile, the sidechain of Gln1113 rotated out to
face solvent. Together these structural observations account for the potency gain
observed and confirm the hypothesis that the water molecule ligating the aniline N-H on
1
and the receptor can be displaced.
Table 1. Initial Replacements of the Aniline
1
2
a
Cmpd
R
R
CBP IC50 (µM) LE (CBP)
1
H
0.74
0.15
0.24
0.45
0.42
0.42
2
3
CH c-Pr
2
CH c-Pr
2
5

4
CH c-Pr
0.031
0.42
2
a
All IC50 values are reported as means of values from at least two
determinations. TR-FRET assay with the isolated CBP bromodomain.
Figure 1. Co-crystal structures of (A) 1 in the CBP bromodomain with the ligand carbon
atoms shown in yellow and the protein carbon atoms shown in green (2.3 Å resolution,
PDB code 5KTU) and (B) 4 in the CBP bromodomain with the ligand carbon atoms
shown in orange and the protein carbon atoms shown in cyan (1.4 Å resolution, PDB
code 6ALC). Specific residues and structural features are displayed and labeled, and
the water-mediated hydrogen bond network between 1 and Gln1113 is depicted as grey
dashed lines.
6

Table 2. Heterocyclic Derivatives That Explore the LPF Shelf
a
a
b
Cmpd
Ar
CBP IC50 (µM)
BRD4 IC50 (µM)
Selectivity
39
5
0.12
4.7
6
7
0.043
0.079
11
256
>20
>253
8
9
0.081
0.03
0.13
>20
14
>247
467
1
1
0
1
14
108
0.09
7
>20
>222

1
2
0.19
>20
>105
a
All IC50 values are reported as means of values from at least two
determinations. TR-FRET assay with the isolated CBP or BRD4(1) bromodomain.
b
Selectivity is defined by [BRD4(1) IC50 (µM) / CBP IC50 (µM)].
We sought to improve the affinity of 4 and evaluate additional bicycles (Table 2).
24
During our parallel work on the THQ series we had discovered that a tetrahydropyran
(
THP) capped pyrazole appeared beneficial for potency, and thus we continued
exploring bicycles with the THP in place. Additionally, at this stage, we started
monitoring selectivity against bromodomain 1 of the bromodomain-containing protein 4
(
BRD4(1)). Inhibition of the bromodomain and extra terminal (BET) family
bromodomains has an overlapping phenotype with CBP and we were particularly
focused on BRD4(1) (our surrogate for BET bromodomains) as the off-target. The
indole 5 displayed an approximately 4-fold decrease in potency compared to 4, but the
reverse indole 6 gained back the CBP potency and showed improved BRD4(1)
selectivity compared to 5. We hypothesize that this SAR may result from a slight shift of
the PP core due to the THP, which may affect the shape complementarity of the indole
with CBP and BRD4(1). Compounds 7 and 8 were synthesized to evaluate whether an
interaction between the ring carbonyl and Arg1173 can be made. Unfortunately, 7 and 8
were 2-fold less potent biochemically compared with indole 6, indicating that the
carbonyl may be too bulky to directly interact with the guanidine group of Arg1173. Next,
8

we envisioned that the more lipophilic isoquinoline could be a suitable chemotype to
replace the indole since the weakly basic nitrogen atom on isoquinoline might be able to
electronically interact with Arg1173 without the extra bulk offered by 7 and 8. Moreover,
the isoquinoline ring system could offer a vector to explore substitutions at the C3
position as a means of gaining potency. We were pleased to observe that isoquinoline 9
indeed increased CBP potency (4-fold) and further improved selectivity against BRD4(1)
to >400-fold. Intrigued by these results, we evaluated other nitrogen-containing bicycles
(
10–12) and none appeared to match the potency of 9. We chose to further examine
SAR on 9.
Table 3. Effect of Substitution on the Isoquinoline Ring of 9
CBP IC50
BRD4 IC50
b
Cmpd
R
Selectivity
a
a
(
µM)
(µM)
9
H
0.03
14
14
15
467
333
395
1
1
3
4
4-Cl
4-Et
0.042
0.038
9

1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
4-CH OH
0.081
0.023
0.18
>20
13
>247
565
2
4-OMe
4-CN
5-Cl
>20
>20
7.7
8.5
>20
11
>111
>222
592
0.09
5-Me
5-Et
0.013
0.031
0.065
0.027
0.28
274
5-CH OH
2
5-OMe
5-CN
6-Cl
>308
407
>20
19
>71
0.17
112
6-Me
6-Et
0.058
0.024
0.069
0.069
0.65
12
207
7.8
>20
8.9
>20
325
6-CH OH
2
6-OMe
6-CN
>290
129
>31
a
All IC50 values are reported as means of values from at least two
determinations. TR-FRET assay with the isolated CBP or BRD4(1) bromodomain.
b
Selectivity is defined by [BRD4(1) IC50 (µM) / CBP IC50 (µM)].
We systematically explored whether substitution off the isoquinoline ring of 9
could improve CBP potency and maintain selectivity against BRD4(1). Based upon
models, it was anticipated that the addition of small substituents to the 4-, 5- and 6-
1
0

positions of the isoquinoline system could favorably enhance potency. Interestingly, a
significant electronic effect was noted (Table 3). CBP potency remained largely
unaffected when the 4-, 5- and 6-positions of the isoquinoline were substituted with
weakly electron-withdrawing or electron-donating groups (F, Cl, Me, Et and OMe).
However, when a strongly electron withdrawing cyano group was introduced, a
significant reduction in potency was observed. There are multiple hypotheses for the
drop in potency observed in cyano analogs (17, 23 and 29) based on modeling studies.
25
The cyano group could potentially weaken the aromatic-proline interaction formed
between the isoquinoline and Pro1110. Alternatively, the cyano group could be too polar
for the Leu1109 sidechain in the case of 4- or 5-substitutions (17 and 23) or for the Cβ
and Cγ on Gln1113 in the case of the 6-sustitution (29).
Table 4. Heterocyclic Derivatives That Explore the LPF Shelf
b
BRD4
MYC EC50
LM Clhep
CBP IC50
a
Cmpd
Ar
IC50
(µM)
M / R / H
a
(
µM)
a
(
µM)
1
1

3
3
0
1
0.0012
0.0028
2.5
3.3
0.032
0.11
20 / 18 / 8.3
51 / 22 / 16
3
3
2
3
0.0033
0.0033
2.8
6.7
0.22
38 / 22 / 13
36 / 20 / 11
0.076
3
3
4
0.0024
0.0012
3.8
3.2
0.28
32 / 45 / 11
36 / 15 / 9.3
5
0.018
a
All IC50 and EC50 values are reported as means of values from at least two
determinations. TR-FRET assay with the isolated CBP or BRD4(1) bromodomain. MYC
b
expression in MV-4-11 cells. Mouse (M), rat (R), human (H) liver microsome-predicted
hepatic clearance (mL/min/kg).
In order to increase interaction within the LPF shelf we introduced heterocycles
to the 3-position of the isoquinoline linker (Table 4). Introduction of an N-methyl-
pyrazole to 9 led to 30-fold enhancement in CBP potency compared to the parent 9. At
1
2

this point we started monitoring liver microsome stability and 30 exhibited good
metabolic stability in various species. Next, we incorporated a functional group (e.g.,
CHF and CN) onto the distal pyrazole ring in an attempt to further interact with
2
Arg1173. Unfortunately, there was a 2- to 3-fold decrease in CBP potency (31–34)
compared to 30. The binding mode of 30 was elucidated by crystallography (Figure 2).
The PP core resides in KAc-binding pocket as expected, with the carbonyl interacting
directly with Asn1168 and via a water molecule with Tyr1125. As predicted during the
compound design process, the nitrogen atom of the isoquinoline ring engages in
hydrogen bond interaction with Arg1173. Also, as in the structure with 4, another
compound without the aniline, the sidechain of Gln1113 is rotated and faces the solvent
(
Figure 2A). One face of the isoquinoline and N-methylpyrazole resides on the lipophilic
shelf and makes van der Waals interactions with Leu1109 and Pro1110, while the other
face is mostly solvent-exposed (Figure 2B).
1
3

Figure 2. Co-crystal structure of 30 in the CBP bromodomain (1.6 Å resolution,
PDB code: 6ALB) displaying the ligand in green and CBP ribbons in brown. The
hydrogen bond between Arg1173 and nitrogen of isoquinoline is shown as grey dashed
lines. (A) Residues discussed in the text are shown as sticks and labeled, and
conserved water molecules are shown as red spheres. (B) The solvent-accessible
surface area of the binding site is shown as in grey.
It was at this time during the work on the herterocyclic modification of 30 that we
had identified GNE-781 (36), a highly potent and selective lead compound in the
24
aforementioned THQ series (Table 5). A concern for GNE-781 was the short half-life
that it exhibited, which was in part due to the low volume of distribution. Introduction of
26
basicity has been shown to increase half-life. In order to test this hypothesis, we
obtained mouse PK for 30 (containing a weakly basic amine), and we were pleased to
observe that the volume of distribution increased as well as the half-life, albeit by a
small amount, relative to 36. To explore the possibility of increasing the half-life further,
we anticipated that replacement of the distal pyrazole with an additional weakly basic
meta-linked pyridine would improve the volume of distribution while retaining the
25,26
desirable conformation of the molecule.
Furthermore, introduction of an electron-
withdrawing amide group could potentially increase the metabolic stability of the
pyridine ring and, based on modeling, the 6’-position of the pyridine is solvent exposed
and can accommodate the amide (Figure 3). Consistent with our modeling hypothesis,
pyridine 35 had comparable CBP and MYC potencies relative to GNE-781, and
exhibited ~2500-fold selectivity against BRD4. Though 35 has one additional hydrogen-
bond donor compared with 30, its permeability was maintained, which could be
1
4

associated with the intra-molecular hydrogen bond between amide N-H and the nitrogen
atom of the distal pyridine. The methyl urea on GNE-781 was evaluated on compound
3
5 and similar potency and selectivity were observed for this compound. However, the
compound had low permeability and minimal oral exposure and thus we chose to further
evaluate compound 35.
Figure 3. Compound 35 modeled into the CBP bromodomain (see Supplemental
Information on methods).
Compound 35 exhibited moderate clearance in PK, with acceptable oral
bioavailability. Evaluation using the BROMOscan technology platform (Figure 4)
revealed the exquisite selectivity of 35. Additionally, when tested (full details in
1
5

Supporting Information) in a Cerep off-target screening panel (10 µM, 43 receptors), 35
did not inhibit any target at >40%. It was at this point in our discovery program that
GNE-781 became the compound we focused our attention on due to its exquisite
potency, selectivity and PK profile. While compound 35 did have an improved half-life
compared to GNE-781, the selectivity for CBP over BRD4 was not as good as the
selectivity that GNE-781 possessed. Not only did GNE-781 have exquisite potency and
selectivity, it was also found to be non-CNS penetrant, an attribute that we wanted in
our molecules. Further safety evaluation and brain penetration of 35 was never
pursued, however, compound 35 became an instrumental in vitro tool representing a
distinct chemical series.
1
6

Figure 4. Broad Bromodomain Profile of 35. K values obtained for 35 across a broad
d
panel of bromodomains as assayed with the BROMOscan platform; the assay is based
on competition between compound and affinity resin for binding to soluble DNA-tagged
bromodomains. Bromodomains in black were included in the screen and those in grey
were not. The two large red circles indicate K
d
values of <1 nM and small red circles
values are > 3µM. For
indicate K values of 1–3 µM. If not marked by a circle then K
d
d
details see Supporting Information.
Table 5. Overall Profile of 30, 35 and 36
1
7

Cmpd
36 (GNE-781)
30
35 (GNE-207)
O
N
F
N
H
Structure
N
N
F
N
O
N
N
CBP IC50
0
.9
1.0
1.0
a
(
nM)
BRD4 IC50
5
,100
2,500
2080
32
3,100
2580
18
a
(
nM)
b
Selectivity
5,670
MYC EC50
6
.6
a
(
nM)
MDCK Papp.
-6
A : B (x 10
19
10
12
c
cm/s)
PPB
9
4 / 94 / 84
85 / 76 / 70
20 / 18 / 8
<21 / 14 / <6
90 / 96 / 95
36 / 15 / 9
<21 / 10 / <6
d
(
%) M/R/H
LM Clhep
(
mL/min/kg)
35 / 18 / 10
e
M/R/H
Hep Clhep
mL/min/kg)
<
21 / <10 / <6
(
1
8

f
M/R/H
kSol (µM)
CL
g
95
92
11
66
18
(
mL/min/kg)
5.2
h
V
ss (L/kg)
0.6
1.4
73
1.3
1.6
67
2.8
2
T
1/2 (h)
F (%)
50
a
All IC50 and EC50 values are reported as means of values from at least two
determinations. TR-FRET assay with the isolated CBP or BRD4(1) bromodomain. MYC
b
expression in MV-4-11 cells. Selectivity is defined by [BRD4(1) IC50 (µM) / CBP IC50
c
‒6
‒1 d
(
µM)]. MDCK cell line; apical-to-basolateral; units = × 10 cm s . Mouse (M), rat (R)
e
and human (H) plasma protein binding. Mouse (M), rat (R) and human (H) liver
f
microsome-predicted hepatic clearance. Mouse (M), rat (R) and human (H) hepatocyte-
g
predicted hepatic clearance. Kinetic solubility was measured at pH 7.4 in PBS buffer.
h
‒1
Mouse PK was performed in CD-1 mice; compounds were dosed iv (1 mg kg ) as
−1
PEG400/H
2
O (35/65) solution and po (5 mg kg ) as an aqueous suspension with 0.2%
Tween 80.
Scheme 1. Synthesis of 2
1
9

O
S
H
N
a, b
c
N
d
N
H
N
Boc
N
N
HN
Boc
Boc
37
38
39
H
N
N
N
e
N
N
N
HN
O
HN
O
4
0
2
Reagents and conditions: (a) isothiocyanatobenzene, KOtBu, THF, rt; (b) CH
3
I, 40 °C,
9
7%; (c) hydrazine monohydrate, EtOH, 85 °C, 97%; (d) i. HCl, EtOAc, rt, ii. Ac
2
O, TEA,
DCM, rt, 98%; (e) (bromomethyl)cyclopropane, Cs CO , DMF, 100 °C, 27%.
2
3
The synthesis of 2 is depicted in Scheme 1. Treatment of piperidone 37 with 1-
isothiocyanato-3-methylbenzene followed by alkylation with methyliodide produced 38.
Cyclization of 38 with hydrazine afforded Boc protected pyrazolopiperidine 39.
Deprotection of the Boc group followed by acetylation yielded acetamide 40. Final N-
alkylation of 40 with (bromomethyl)cyclopropane produced 2.
Scheme 2. Synthesis of 3 and 4
2
0

Reagents and conditions: (a) (bromomethyl)cyclopropane, K
HCl, EtOAc, rt, ii. Ac O, TEA, DMF, rt, 99%; (c) phenylboronic acid, Pd(dppf)Cl
Na CO , dioxane, H O, 100 °C, 10%; (d) 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)-1H-indole, Pd(PPh , Cs CO , dioxane, H O, 100 °C, 16%.
2
CO
3
, DMF, 80 °C; (b) i.
2
2
,
2
3
2
3
)
4
2
3
2
Compounds 3 and 4 were prepared according to Scheme 2. The synthesis of 41 has
27
been described in the literature. N-alkylation of 41 with (bromomethyl)cyclopropane
produced 42. Boc deprotection and N-acetylation gave acetamide 43. A palladium-
catalyzed Suzuki strategy was utilized to couple the iodo-PP core with the boronic acid
or boronate ester to produce 3 and 4.
Scheme 3. Synthesis of Compounds 5‒12, 13, 18 and 24
2
1

Reagents and conditions: (a) i. Ar–B(OH)
5 mol %), K PO (2.5 equiv), dioxane/H O, 60 °C; (b) i. (1-(tert-butoxycarbonyl)-1H-
indol-3-yl)boronic acid, XPhos pre-catalyst (G2) (4 mol %), Na CO (2.5 equiv),
THF/H O, 60 °C, ii. TFA, DCM, rt; (c) i. B Pin , XPhos pre-catalyst (G2) (3 mol %),
XPhos (6 mol %), KOAc (3 equiv), dioxane, 80 °C, ii. Ar–Br, XPhos pre-catalyst (G2) (4
mol %), K PO (2.5 equiv), dioxane/H O, 90 °C (d) i. B Pin , Pd(dppf)Cl (10 mol %),
KOAc (3 equiv), dioxane, 80 °C, ii. Ar–Br, Pd(dppf)Cl (10 mol %), K PO (3 equiv),
dioxane/H O, 80 °C.
2
, XPhos pre-catalyst (G2) (5 mol %), XPhos
(
3
4
2
2
3
2
2
2
3
4
2
2
2
2
2
3
4
2
The general synthetic route used for the preparation of compounds listed in Table 1 and
chloro-isoquinolines 13, 18 and 24 is depicted in Scheme 3. Synthesis of 44 has been
24
described in the literature. Bromide 44 was treated with a heteroarene boronic acid
2
2

and XPhos pre-catalyst (G2) to give 5, 6, 10 and 11. Alternatively, the bromo-PP core
4 was first treated with bis(pinacolato)diboran and XPhos pre-catalyst to give the
4
corresponding boronate ester (structure not shown), which was immediately cross-
coupled with the heteroaryl bromides under Suzuki conditions to give 7‒9, 12, 13, 18
28
and 24.
Scheme 4. Synthesis of Compounds 14‒17, 19‒23 and 25‒29
2
3

O
O
O
N
N
N
c
d − f
N
N
N
N
N
N
N
N
N
O
O
O
OH
1
2
2
5 (4-CH OH)
45 (4-CH=CH2)
46 (5-CH=CH2)
47 (6-CH=CH2)
2
14 (4-Et)
20 (5-Et)
26 (6-Et)
1 (5-CH OH)
2
7 (6-CH OH)
2
b
O
O
O
N
N
N
N
N
N
a
g
N
N
N
N
N
N
O
O
O
Me
4 Cl
OMe
6
5
1
2
9 (5-Me)
5 (6-Me)
13 (4-Cl)
18 (5-Cl)
24 (6-Cl)
16 (4-OMe)
22 (5-OMe)
28 (6-OMe)
h
i-Pr
O
i-Pr
i-Pr
N
P
N
N
OSO2Me
Pd
N
NH2
O
CN
17 (4-CN)
23 (5-CN)
29 (6-CN)
t-BuXPhos pre-catalyst(G3)
Reagents and conditions: (a) MeBF
adamantylphosphine (1 equiv), Cs CO
tetramethyl-2-vinyl-1,3,2-dioxaborolane, XPhos pre-catalyst (G2) (10 mol %), XPhos (10
mol %), Na CO (2 equiv), THF/H O, 60 °C, 51–91%; (c) 10% Pd/C (5 mol %), H (15
psi), MeOH, rt, 3–13%. (d) OsO , THF/H O, rt; (e) NaIO , THF/H O, rt; (f) NaBH
3
K (1 equiv), Pd(OAc)
2
(1 equiv), butyl-di-1-
2
3
2
(1 equiv), PhMe/H O, 80 °C, 8–9%. (b) 4,4,5,5-
2
3
2
2
4
2
4
2
4
,
2
4

MeOH, 0 °C, 3–7% three steps. (g) MeOH (5 equiv), t-BuXPhos pre-catalyst (G3) (20
mol %), t-BuXPhos (20 mol %), t-BuONa (1.4 equiv), dioxane, 50 °C, 7–9%. (h)
4
K Fe(CN)
6 2
⋅3H O (0.5 equiv), t-BuXPhos pre-catalyst (G3) (10 mol %), t-BuXPhos (10
2
mol %), KOAc (3 equiv), dioxane/H O, 120 °C, 7–10%.
As outlined in Scheme 4, chloro-substituted isoquinoline (13, 18 and 24) served as the
key intermediate toward the syntheses of methyl, ethyl, hydroxyl, methoxy and cyano
variants. Briefly, Suzuki cross-coupling of 13 and 18 with the methyl Molander salt led to
1
9 and 25. Cross-coupling of 13, 18 and 24 with vinyl boronate afforded 45, 46 and 47,
which upon hydrogenation produced the ethyl analogs 14, 20 and 26, respectively. The
vinyl intermediates were transformed to the hydroxymethyls (15, 21 and 27) by a
sequence of dihydroxylation, oxidative cleavage and reduction. To convert chloride (13,
1
8 and 24) to the corresponding methoxyl analogs (16, 22 and 28), a MeOH and t-
29
BuXPhos pre-catalyst combination was used. Palladium-catalyzed cyanation of 13, 18
30
and 24 led to 17, 23 and 29, respectively.
Scheme 5. Synthesis of Compounds 30 and 35
2
5

Reagents and conditions: (a) Ar-Bpin, Pd(dppf)Cl
dioxane/H O, 90 °C, 81–92%; (b) 49, B Pin , Pd(dppf)Cl
DMF, 90 °C, 24%; (c) 50, B Pin , XPhos pre-catalyst (G2) (10 mol %), XPhos (10 mol
), KOAc (3 equiv), dioxane, 90 °C, 84%; (d) 44, Xphos pre-catalyst (G2) (10 mol %),
XPhos (10 mol %), Na CO (3 equiv), THF/H O, 60 °C, 10–21%.
2
(5 mol %), Na
2
CO
3
(3 equiv),
2
2
2
2
(5 mol %), KOAc (3 equiv),
2
2
%
2
3
2
Scheme 5 shows the synthesis of analogs 30 and 35. Commercially available 3-
bromo-8-chloroisoquinoline 48 was subjected to chemo-selective Suzuki coupling with
heteroaryl boronate esters to furnish 49 and 50. The resultant chlorides (49 and 50)
were converted to the corresponding boronate esters (51 and 52) that were
subsequently subjected to Suzuki coupling with PP-bromide 44 to afford 30 and 35,
respectively.
2
6

Scheme 6. Synthesis of Compounds 31‒34
Reagents and conditions: (a) methyl 2,2-dimethoxyacetate, TEA, MeOH, 80 °C, 84%;
(
b) H
Pin
dioxane, 80 °C; (e) 44, XPhos pre-catalyst (G2) (10 mol %), K
dioxane/H O, 90 °C, 28% from 56; (f) TBAF, THF, rt, 75%; (g) TEA, PhN(SO
DCM, rt, 29% from 57; (h) Ar–Bpin, Xphos pre-catalyst (G2) (10 mol %), XPhos (10 mol
), Na CO (3 equiv), THF/H O, 60 °C, 5–28%.
2
SO
, XPhos pre-catalyst (G2) (10 mol %), XPhos (10 mol %), KOAc (3 equiv),
PO (3 equiv),
4
, H
2
O, 0 °C–rt, 75%; (c) TIPS–Cl, 1H-imidazole, DMF, 0 °C–rt, 58%; (d)
B
2
2
3
4
2
2
CF
3
) ,
2
%
2
3
2
2
7

To expedite the distal heteroaryl SAR exploration, we also developed a route that
enabled late stage installation of a functional group to the 3-position of the isoquinoline
(
Scheme 6). Benzyl amine 53 was treated with methyl 2,2-dimethoxyacetate and
triethylamine in methanol to produce amide 54, which upon treatment with H SO
2
4
furnished isoquinolin-3(2H)-one 55. Treatment of 55 with TIPS–Cl and imidazole
afforded 56 which was transformed to the corresponding boronate ester and then
coupled with 44 under the similar Suzuki conditions to afford 57. Desilylation of 57 with
TBAF followed by triflation then subsequent Suzuki coupling with various heteroaryl
boronate esters to give desired analogs 31–34.
In summary, through structure-based design and optimization, a new class of
isoquinoline-type CBP inhibitors has been discovered. We systematically examined the
SAR on the C4, C5 and C6 positions of the isoquinoline. The introduction of the distal
heterocycle to the C3 position of isoquinoline proved to be essential to achieve single-
digit nM potency for CBP. The X-ray structure of compound 30 in complex with the CBP
BRD was obtained and illustrated the interaction of the nitrogen of the isoquinoline ring
with Arg1173. More strikingly, mouse PK results of selected inhibitors revealed that
combination of isoquinoline and pyridine, as exemplified by compound 35, can
complement each other in increasing volume of distribution and half-life.
Author Information
Corresponding author
*V.T. E-mail: vickie.tsui@aya.yale.edu
2
8

*S.M. E-mail: magnuson.steven@gene.com
†
These authors contributed equally.
Note
The author confirms that this article content has no conflicts of interest.
Acknowledgements
We thank Mengling Wong, Michael Hayes, and Amber Guillen for compound
purification. Baiwei Lin, Deven Wang, and Yutao Jiang are acknowleged for analytical
support. Grady Howes, Jan Seerveld, Hao Zheng, Ted Peters, and Gigi Yuen for help
with compound management and logistics are also recognized.
References
(
1)
Bannister AJ, Kouzarides T. The CBP Co-Activator Is a Histone cetyltransferase.
Nature 1996;384:641–643.
(
2) Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y. The
transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell
996;87:953−959.
3) Vo N, Goodman RH. CREB-binding protein and p300 in transcriptional
regulation. J. Biol. Chem. 2001;276:13505–13508.
1
(
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