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Discovery and SAR Study of Boronic Acid-Based Selective PDE3B

Inhibitors from a Novel DNA-Encoded Library
Ann M. Rowley,* Gang Yao,* Logan Andrews, Aaron Bedermann, Ross Biddulph, Ryan Bingham,
Jennifer J. Brady, Rachel Buxton, Ted Cecconie, Rona Cooper, Adam Csakai, Enoch N. Gao,
Melissa C. Grenier-Davies, Meghan Lawler, Yiqian Lian, Justyna Macina, Colin Macphee,
Lisa Marcaurelle, John Martin, Patricia McCormick, Rekha Pindoria, Martin Rauch, Warren Rocque,
Yingnian Shen, Lisa M. Shewchuk, Michael Squire, Will Stebbeds, Westley Tear, Xin Wang, Paris Ward,
and Shouhua Xiao
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sı Supporting Information

ABSTRACT: Human genetic evidence shows that PDE3B is

associated with metabolic and dyslipidemia phenotypes. A number
of PDE3 family selective inhibitors have been approved by the
FDA for various indications; however, given the undesirable
proarrhythmic effects in the heart, selectivity for PDE3B inhibition
over closely related family members (such as PDE3A; 48%
identity) is a critical consideration for development of PDE3B
therapeutics. Selectivity for PDE3B over PDE3A may be achieved
in a variety of ways, including properties intrinsic to the compound
or tissue-selective targeting. The high (>95%) active site homology
between PDE3A and B represents a massive obstacle for obtaining
selectivity at the active site; however, utilization of libraries with
high molecular diversity in high throughput screens may uncover
selective chemical matter. Herein, we employed a DNA-encoded library screen to identify PDE3B-selective inhibitors and identified
potent and selective boronic acid compounds bound at the active site.

■ INTRODUCTION
Cyclic nucleotide phosphodiesterases (PDEs) are a super-
family of enzymes that catalyze the hydrolysis of the
phosphodiester bonds of specific second messenger substrates
such as 3′,5′-cyclic adenosine monophosphate (cAMP) and
3′,5′-cyclic guanosine monophosphate (cGMP). They play a
critical role in the indirect regulation of many intracellular
metabolism pathways. 11 broad families of PDE isoenzymes
(PDE1-11) have been reported thus far in the literature with
each PDE family consisting of distinct genes that in turn
generate more than 100 PDE isoforms. PDEs generally exist in
the dimeric form, and each monomer is composed of three
distinct domains: a catalytic, an N-terminal, and a C-terminal
domain. The catalytic domain consists of approximately 350
conserved amino acids and can selectively hydrolyze cyclic
nucleotides in different ways. Phosphodiesterase 3 (PDE3)
family members are dual specificity PDEs that hydrolyze both
cAMP and cGMP with Km values in the submillimolar range
(Km cAMP = 0.2 mM, Km cGMP = 0.1 mM). There are two
PDE3 isoforms: PDE3A and PDE3B. PDE3A is mostly found
in cardiac tissue, platelets, and vascular smooth muscle cells,
while PDE3B is prevalently expressed in peripheral blood T-
cells, hepatocytes, and adipose tissue.1−4
Genome wide association studies from 23andMe and others
have identified associations of PDE3B with lipid phenotypes. A
rare stop-gain variant in the catalytic site of PDE3B (p.R783X,
rs150090666) is associated with reduced risk of high
triglyceride, low HDL, high LDL, high cholesterol, as well
obesity5 (Supporting Information, Figure S1). Literature
studies suggest that selective PDE3B inhibition would
effectively stimulate lipolysis and metabolism while avoiding
cardiovascular risks associated with PDE3A inhibition.6
Homology between PDE3A and PDE3B is high (identities:
48% positive substitutions: 67%) and increases significantly
(∼95% identity) within 15 Å in the active site pocket,
presenting strong challenges in the development of PDE3
Received: October 5, 2023
Revised: January 11, 2024
Accepted: January 19, 2024

© XXXX American Chemical Society https://doi.org/10.1021/acs.jmedchem.3c01562

A J. Med. Chem. XXXX, XXX, XXX−XXX
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Figure 1. Known PDE3 inhibitors.7−10

Figure 2. Structure of the aminobenzoic acid-based DEL resulting in PDE3B-selective chemical series.

Figure 3. (a) Three-dimensional view of the DEL selection output. (b) Copy number vs cycle 3 BBs for the boronic acid series. Green: enriched for
PDE3B only; red: enriched for both PDE3A and PDE3B.

isozyme-selective inhibitors. Very few selective PDE3B
inhibitors have been reported to date, and those that have
exhibited low potency. Some known PDE3 inhibitors are
shown in Figure 1.7−10 To identify novel PDE3B-selective
inhibitors, we screened the GSK collection of more than one
trillion DNA-encoded small molecules.
The DNA-encoded library (DEL) technology is an ultra-
high throughput screening technology which is capable of
screening millions to trillions of DNA-encoded molecules in a
single tube via affinity selection. In recent years, the DEL
B https://doi.org/10.1021/acs.jmedchem.3c01562
technology has become a popular platform to generate novel
small molecule hits for various therapeutic targets due to its
high throughput and low cost nature. DELs can be prepared
using a split and pool synthesis strategy which allows very large
libraries (>109 DEL molecules per library) to be generated in a
few steps. Each DEL molecule contains a covalently attached
unique DNA tag that serves as an identifier for the small
molecule. Affinity selections of a pool of DELs against the
immobilized target proteins, PCR amplification, and sequenc-
ing of the DNA tags of the enriched DEL molecules afford
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Table 1. Biochemical Data for Off-DNA Compounds, Varying Three Different Vectors
a
All compounds were tested in the assays n ≥ 2. Potency values quoted are mean of n’s. bPotency measurements for RF-MS PDE3B and PDE3A
listed in Table S3. cOn a single test occasion, the compound was inactive (pIC50 < 4.0).
putative binders with rich structure−activity data (on-DNA
SAR). Due to the facileness of affinity selection, multiple
selection conditions can be performed in parallel enabling
further triage of hits based on desired selectivities or binding
modes (ortho or allosteric binders etc.).11 The DEL
technology represents a unique approach to finding a selective
binder for PDE3B that has not been profiled previously and
therefore we sought to employ this technique to try to find
molecules with high selectivity.
■ RESULTS AND DISCUSSION
In an effort to identify novel selective PDE3B small molecule
inhibitors, we performed a parallel selection of both human
His-tagged PDE3B (511-1112) and human His-tagged PDE3A
(511-1141) against 1.9 trillion DNA-encoded small molecules
from the GSK collection (106 libraries in pool). DEL
molecules that were enriched against PDE3B were clustered
and analyzed. Clusters with >50% of members showing
PDE3B specific enrichment over PDE3A were prioritized for
further evaluation. Representative compounds from these
clusters were designed and synthesized without their DNA
tag, to confirm potency and selectivity in immobilized metal
ion affinity for phosphochemical (IMAP) and/or RapidFire
mass spectrometry (RF-MS) activity assays.12 Several chemical
series were identified to be PDE3B inhibitors but only one
series from a four-cycle library, defined by a boronic acid
functional group, showed PDE3B selectivity over PDE3A. As
shown in Figure 2, this particular four-cycle boronic acid
containing DEL was constructed utilizing 810 amines or amino
acids at cycle 1. In cycle 2, two 3-nitrobenzoic acid derivatives
were installed via amide bond formation and then
subsequently reduced and then in cycle 3, the aniline, as
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shown in Figure 2, was capped with 1178 amine building
blocks (BBs) (carboxylic acids, amino acids, and sulfonyl
chlorides). Half of the pool of DEL molecules were further
oxidized with N-hydroxypiperidine in cycle 4 to give the
corresponding phenol derivatives, resulting in a total of 3.8
million unique DNA-encoded compounds (Figure 2).
The initial off-DNA boronic acid molecules from one of the
PDE3B-selective clusters were designed based on the most
enriched disynthon combination of cycle 2 BB 3-borono-5-
nitrobenzoic acid and cycle 3 BB 2-methoxy-3,5-dimethylben-
zoic acid (Figure 3a). Compounds 1 and 2 exhibited a pIC50
value of 6.7 and 6.5, respectively, when tested against full-
length (FL) PDE3B (M1-E1112)/PDE3A(M1-Q1141) in the
IMAP assay; they also showed ∼3-fold selectivity for PDE3B
over PDE3A (Table 1). Substitution of the boronic acid
moiety with a hydroxy group resulted in a significant loss of
activity (Table 1, compound 3, IMAP PDE3B pIC50 = 4.4),
indicating the critical importance of the boronic acid group for
maintaining PDE3B inhibition. This result was consistent with
the selection results, in which neither the corresponding
phenol nor the desboronic acid compounds from the DEL
were enriched. Further analysis of the on-DNA SAR of the
boronic acid series revealed that certain cycle 3 BBs might
confer greater PDE3B/A IMAP selectivity. When examining a
plot of the enriched boronic acid DEL molecules (copy
number) versus cycle 3 BBs, it was observed that certain BB3
derived series had a higher percentage of PDE3B-specific
members than those derived from 2-methoxy-3,5-dimethyl-
benzoic acid (Figure 3b). Therefore, we targeted a compound
derived from one of the more selective cycle 3 BBs: 4,7-
dimethoxyquinoline-2-carboxylic acid and synthesized com-
pound 4, Table 1. Compound 4 exhibited moderate PDE3B
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Scheme 1. Representative Synthesis of Boronic Acid Analogues Exemplified by Compound 5
Scheme 2. Representative Synthesis of Boronic Acid Analogues in Table 2
activity (IMAP pIC50 = 6.5) and much improved PDE3B/A
IMAP selectivity (>30-fold).
The on-DNA SAR of cycle 1 BBs illustrated in the Spotfire
cube view (Figure 3a) indicated that broad variation at this
position was tolerated. Indeed, both the (R)- and (S)-2-amino-
2-cyclohexylacetic acid-derived hits (compounds 4 and 5
IMAP PDE3B pIC50 of 6.5 and 7.1, respectively) were active
PDE3B inhibitors with the R-isomer (4), showing greater
PDE3B/A IMAP selectivity than the S-isomer (5) (37-fold vs
12-fold). This finding indicated that the cycle 1 portion of the
molecule may be amenable to further optimization to improve
the PDE3B/A IMAP selectivity. In addition to the PDE3B
IMAP assay, the off-DNA hits were further evaluated in a RF-
MS assay using the native substrate cAMP. Both compounds 4
and 5 showed PDE3B/A RF-MS selectivity with the R-isomer
(4) displaying better PDE3B/A selectivity compared to the S-
isomer (5) (78-fold vs 26-fold), in agreement with the IMAP
assay data.
A representative synthesis of the boronic acid analogues
described above is illustrated in Scheme 1. Starting with the
(S)-2-amino-2-cyclohexyl-N-methylacetamide, coupling to 3-
(N-Boc-amino)-5-carboxyphenylboronic acid under HATU-
mediated conditions afforded the corresponding amide
intermediate. Removal of the Boc protecting group with HCl
and reaction with the requisite carboxylic acids under TCFH-
mediated coupling conditions provided the desired compound
4 and its related analogues.
A representative synthesis of the boronic acid analogues
described above is illustrated in Scheme 2. Starting with 1-
methyl 3-amino-5-boronobenzoate, coupling to the requisite
isoquinoline carboxylic acid under TCFH-mediated coupling
conditions provided the intermediate in good yield. Following
hydrolysis of the methyl ester, subsequent HATU-mediated
coupling was amenable to a range of amines allowing for rapid
SAR exploration. The IMAP and RF-MS results are shown in
Table 2.
Complete removal of the amide group resulted in a
significant loss of activity (IMAP pIC50 = 5.0 vs 6.5,
compounds 6 and 4, respectively) and selectivity (1.4-fold vs
37-fold IMAP PDE3B/A selectivity for 6 and 4, respectively)
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highlighting the importance of this moiety. Less drastic
truncations of 4 to small polar groups such as the primary
amide (7), methyl ester (9), and methyl amide (10) all showed
restoration of some of the PDE3B activity (IMAP pIC50 = 5.7
for all three). Encouragingly, these compounds all showed no
activity against PDE3A, improving upon the PDE3B/A
selectivity (>51, >52, and >44-fold IMAP selectivity for 7, 9,
and 10, respectively). This reduction in PDE3A activity was
confirmed by the orthogonal RF-MS assay (Table S1), with
only 7 showing any measurable activity (RF-MS PDE3A pIC50
= 4.1). Interestingly, the carboxylic acid analogue 8 showed
similar PDE3B activity to compounds 7, 9, and 10 but
displayed higher inhibition of PDE3A (IMAP pIC50 = 5.1),
resulting in a significant reduction in the PDE3B/A selectivity
(3-fold selective). Swapping a primary methyl amine to a
secondary dimethyl amine, as exemplified by analogues 10 and
11, resulted in a significant loss in PDE3B/A selectivity (>44
vs 23-fold, respectively). Further increasing the size of the
amide group substituent (12, IMAP pIC50 = 6.0) modestly
improved the activity but resulted in a decrease in PDE3B/A
selectivity (15-fold selective). N-methylation of the amide
adjacent to the isoquinoline ring (R2�Me, compound 13)
eliminated PDE3B activity. Reducing the size of the α-
substituent and replacing the methyl amide with a primary
amide on compounds 4 and 5 resulted in 14 and 15. Again the
(S)-enantiomer (15) showed higher PDE3B potency than the
(R)-enantiomer 14 (IMAP pIC50 = 6.5 vs 5.7 for 15 and 14,
respectively), although both showed significant reduction in
selectivity compared to compounds 7, 9, and 10. This
selectivity was regained when the primary amide in 14 was
replaced with a methoxymethyl (17). Compound 17
demonstrated submicromolar activity (IMAP pIC50 = 6.3)
against PDE3B and high PDE3B/A selectivity (>190-fold
selective IMAP, 50-fold selective RF-MS). Substitution at the
α-position appears critical for the selectivity, with 18 (>300-
fold selective) showing significant PDE3B/A selectivity
compared to its β-substituted matched pair 19 (14-fold
selective). Cyclization of the ether reduced the PDE3B/A
selectivity (20 and 21). Increasing the steric bulk of the amide
substituent and adding a chiral center (R1), from an isopentane
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Table 2. Potency and Selectivity SAR Table Examining Variation of the Left-Hand Portion of the Molecule While Maintaining
the Quinoline Moiety

a
All compounds were tested in the assays n ≥ 2. Potency values quoted are mean of n’s. bPotency measurements for RF-MS PDE3B and PDE3A
listed in Table S4. cCompound does not contain carbonyl attachment. dOn a singled test occasion, the compound was inactive (pIC50 < 4.0). eOn a
twoe test occasion, the compound was inactive (pIC50 < 4.0). fOn a threef test occasion, the compound was inactive (pIC50 < 4.0).

Scheme 3. Representative Synthesis of Right-Hand SAR Analogues

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Table 3. Potency and Selectivity Assay Results for Right-Hand SAR Analogues

a
All compounds were tested in the assays n ≥ 2. Potency values quoted are mean of n’s. bPotency measurements for RF-MS PDE3B and PDE3A
listed in Table S5. cOn a singlec test occasion, the compound was inactive (pIC50 < 4.0). dOn a twod test occasion, the compound was inactive
(pIC50 < 4.0). eOn a threee test occasion, the compound was inactive (pIC50 < 4.0). fOn a single test occasion, the compound was highly active
(pIC50 > 8.77).

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Figure 4. (a) X-ray crystal structure of boronic compound 23 binding to PDE3B (PDB 8SYC). (b) Overlay of crystal structure of 23 in PDE3B
(green protein, green VDM surface, and orange ligand, PDB 8SYC) with an aligned crystal structure of DNMDP in PDE3A (purple protein, purple
VDW surface, and cyan ligand PDB 7KWE). Structures superposed with MOE2020.0901. (c) Docking of ICOS compound (IMAP PDE3B pIC50
7.16, IMAP PDE3A pIC50 6.05, cyan ligand) (structure shown in Figure 1)8 to the cocrystal structure of Table 2, compound 23 with PDE3B (gray
protein, gray VDM surface, and orange ligand, PDB 8SYC).

to a 1-methoxy-3-methylbutan-2-yl, improved the PDE3B/A
selectivity, as demonstrated by the comparison of compounds
22 and 17 (IMAP pIC50 = 14 vs >190-fold selective,
respectively). Compounds 23−26 derived from reactions
with secondary amines (namely, N-methylbenzyl amine, 3-
cyclopropoxyazetidine, pyrrolidine, and 2,3-dihydro-1H-
pyrrolo[3,4-c]pyridine, respectively) demonstrated comparable
PDE3B activity (IMAP pIC50 ranging from 6.1 to 6.7) to
compound 18 but with reduced PDE3B/A selectivity. Overall,
compound 18 demonstrated the best combination of IMAP
PDE3B activity and PDE3B/A selectivity (IMAP pIC50 = 6.5,
>300-fold selective).
With the left-hand amide portion of the molecule optimized,
we turned our attention to the quinoline moiety with the goal
of further improving potency and PDE3B/A selectivity. A
representative synthesis of these analogs is exemplified in
Scheme 3. Monoaryl systems generally gave lower activity and
minimal selectivity (Table 3, compounds 2, 28−31) with the
trisubstituted phenyl derivates showing the greatest potency
(Table 3, compounds 2 and 29). In contrast to the quinoline
derivatives (Table 3, compounds 4 and 5), no appreciable
selectivity difference was observed between the (R)- and the
(S)-enantiomers (Table 3, compounds 29 vs 2). Analogues
containing biaryl 6,6 fused ring systems showed variable
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PDE3B activity with compounds 35 and 47 being the most
potent. Compared to compound 5, both showed ∼10-fold
improvement in potency, albeit at the expense of PDE3B/A
selectivity (0.74-fold selective for both 35 and 47 vs 12-fold
selective for compound 5). Both the 4- and 7-methoxy groups
on the quinoline ring appeared to be required for selectivity.
Removing the 7-methoxy group on the quinoline ring resulted
in a significant loss of selectivity (1.6-fold selective for 32 vs 12
for 5). Moving the 7-methoxy group to the 6 position (33)
resulted in both lower activity and complete loss of selectivity.
Removing both the methoxy groups on the quinoline (37)
improved the PDE3A activity more than PDE3B activity and
again resulted in a complete loss of selectivity. Moving or
removing the nitrogen on the quinoline resulted in a significant
loss of PDE3B activity (38, 39, 44−46 vs 37), highlighting the
importance of this nitrogen atom. Interestingly, other 6, 6-
nitrogen containing heterocyclic ring systems 40−43 and the
quinoxaline 47 were connected at a different position of the
ring compared to the quinolines and still maintained PDE3B
activity even without the nitrogen at the 1 position. This
observation implies that a different binding pose may be
operative here. Overall, all the 6,6 fused ring systems explored
the 4,7-dimethoxyquinoline system and still provided the best
PDE3B/A selectivity (up to >300 fold, IMAP). While we were
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able to improve the PDE3B activity with other 6,6 fused ring
systems, no improvement in PDE3B/A selectivity was
observed. The SAR around both the left-hand amide and the
quinoline region of the molecule implies that both parts of the
molecule have a synergistic effect in the high PDE3B/A
selectivity observed for compound 18.
To gain structural insights, several efforts were undertaken
to obtain cocrystallographic structures. We were successful in
crystallizing compound 23 with human PDE3B (Figure 4a).
The structure confirmed that the boronic acid of compound 23
binds at the active site of PDE3B. As shown in Figure 4a, the
boronic acid group occupied the metal subdomain and formed
potential key interactions with the two magnesium metal ions
as well as a network of water molecules. In addition, the
boronic acid is also shown to form likely hydrogen bond (HB)
interactions with the two conserved aspartic acids (i.e., Asp822
and Asp937). These extensive metal-chelating interactions
serve as an anchor point for the binding of compound 23 to
PDE3B, consistent with the observation that the boronic acid
group is essential for activity. The dimethoxyquinoline sits in
the hydrophobic site between the “hydrophobic clamp” formed
by Phe991 and aliphatic amino acid Ile955. In contrast to
cAMP or cGMP, compound 23 is not within the typical HB
distance to the highly conserved Gln988. Instead, the side
chain of Gln988 flipped out and moved away to accommodate
the 4-methoxy group of the quinoline moiety. The 7-methoxy
group extended into a small pocket between Try736, Trp951,
and Gly940, which has not been observed in other PDE3B
inhibitors such as the aryl dihydropyridazinones.8 A trapped
water molecule was observed at the bottom of the pocket
which potentially could be replaced with a polar group to
increase the potency of the PDE3B inhibitor. The active
binding conformation has the quinoline and center phenyl ring
coplanar, which may be a result of the intramolecular HB
between the amide hydrogen and the nitrogen on the
quinoline ring system. This would explain the complete loss
of activity of compound 13 as methylation of the amide
nitrogen would result in the loss of this intramolecular HB and
make the coplanar conformation highly energetically unfavor-
able. Losing the nitrogen in the quinoline ring likely had the
same effect (44, 45, and 46). Finally, the left-hand amide
group is seen occupying the hydrophobic cavity at the entrance
of the PDE3B catalytic site, consistent with the DEL
information wherein this position was attached to the DNA
tag and assumed to be solvent exposed.
To understand the origin of the selectivity of the boronic
acid compounds, we overlaid the crystal structure of Table 1,
compound 23 in PDE3B with an aligned crystal structure of
DNMDP (Figure 1) in PDE3A (Figure 4c).13 In contrast to
the PDE3A inhibitor DNMDP (Figure 1) and other reported
nonselective PDE3B inhibitors, 23 does not appear to form
any polar interactions with the highly conserved Gln988. The
4-methoxy group of the quinoline moiety instead displaces the
Gln988 and occupies the small pocket made available by the
displacement of Gln988. The major polar interactions between
23 and PDE3B are derived from the boronic acid HB
interactions in the metal-binding site instead. Close examina-
tion of the hydrophobic binding site indicates the pocket
between Try736, Trp951, and Gly940 are smaller in PDE3A
than PDE3B due to the movement of the side chains (i.e.,
Gly953 in PDE3A and Gly940 in PDE3B) and therefore steric
clash may occur with the 7-methoxy group of the quinoline
moiety in PDE3A but not in PDE3B. However, Gln988 in
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PDE3A can also be displaced in the same way as in PDE3B,
giving rise to questions that may only be answered by the
cocrystal structure of compound 23 with PDE3A which we
were unable to obtain. This hypothesis could explain why both
4- and 7-methoxy groups on the quinoline were required for
higher PDE3B/A selectivity.
The difference between PDE3A and PDE3B at the solvent-
exposed region where the left-hand amide moiety resides is still
more subtle. Some side chain movement (i.e., Thr855 in
PDE3A and Thr829 in PDE3B) could be responsible for the
improved selectivity for some compounds over others (e.g., 23
vs 24) but the structural impact of this part of the ligand
molecules are less obvious from the crystal structure. It is
conceivable that in the FL enzyme, polypeptide chains in the
noncatalytic domain, which have lower homology between
PDE3A and PDE3B, interact with the binding site in this
solvent-exposed region and thus are contributing to the
specificity. Overall, both the SAR and structural information
suggested that cooperative interactions between the left-hand
amide and the quinoline region of the boronic acids with the
enzyme are required to achieve high PDE3B/A selectivity.
Conceivably one way to maximize this cooperation is to
rigidify the ligand molecule which will make it easier to control
the relative geometry of the substituents on the two ends of the
molecule and systematically explore the two binding sites. In
addition to improving PDE3B/A selectivity, preorganization of
the compound into the bound conformation could also
increase the binding affinity by decreasing ligand strain in
the bound conformation or an entropic effect. Further
chemistry efforts would be required to test this hypothesis
which is beyond the scope of this paper.
In order to gain insights into how an active site binder could
exhibit selectivity, we computationally docked the ICOS
compound (Figure 1),7 which showed ∼12× PDE3B/A
selectivity (PDE3B/3A pIC50 = 7.15/6.05) in our IMAP
assay (Supporting Information) to the cocrystal structure of
compound 23, with PDE3B (Figure 4b, right). In this docking
model, the 7-methoxy group of compound 23 occupied the
same binding pocket as the hydroxy ethyl group of the ICOS
compound.8 In contrast, the nonselective PDE3B inhibitor
DNMDP (Figure 1)13 does not occupy this pocket. This
observation further suggests that binding in this region of the
protein may play a role in driving PDE3B/PBE3A selectivity.
Finally, it was of interest to us to explore the phenotypic
activity of our top selective compound (Table 3, compound
18) as well as our top active compound (Table 3, compound
35). Compound 18 showed selectivity far superior to
previously characterized compounds Merck 8a8 (reported:
pIC50 = 10, selectivity 6) and ICOS7 (IMAP PDE3B pIC50 =
7.16, selectivity = 12). In order to determine how potency of
these compounds translated from a biochemical assay to a
cellular assay context, we investigated the compounds in
adipocyte lipolysis cellular assay. The adipocyte lipolysis assay
examines PDE-mediated hydrolysis of cAMP to 5′-AMP,
which results in decreased lipolysis. In this assay, inhibition of
PDE activity leads to a reduction in lipolysis and increase in
glycerol levels. PDE3B expression is significantly enriched in
adipocytes compared to PDE3A, allowing us to interrogate
PDE3B activity in a cellular context. Upon testing compound
18, at a single 10 μM dose, we saw limited but significant
activity, whereas Merck 8a8 demonstrated a larger response,
consistent with its higher biochemical potency (Supporting
Information, Figure S2). Lack of demonstration of substantial
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cellular activity for compound 18 could be due to differences
in the bioactive conformation of the compound, inadequate
cell penetration, or lack of biochemical potency for PDE3B
compared to Merck8a. Compound 35 demonstrated a
response in the adipocyte assay similar to that of Merck 8a8
and ICOS,7 which is an indication that a more biochemically
potent compound is needed for a more significant cellular
response.
Importantly, our rank ordering of potency held over other
compounds profiled simultaneously, suggesting that the
activity is not an off-target effect. While rather preliminary,
this data led us to hypothesize that upon further investigation
of this series, perhaps one could improve potency while
maintaining selectivity, enough to show meaningful selectivity
data in the adipocyte lipolysis cellular assay.
■ CONCLUSIONS
Employing a screen of PDE3B with greater diversity of
chemical matter than previous efforts outlined in the
literature,7−9 such as the DELs used here, demonstrates that
the PDE3B active site remains a viable target. We identified a
small molecule bound in the active site capable of achieving
selectivity for PDE3A/B, where activity was mediated by a
boronic acid group. Activity of the compound was confirmed
in cells, but selectivity was not as apparent as in the
biochemical assays. We encountered challenges with engineer-
ing better biochemical potency at the expense of selectivity. In
addition, we were unable to engineer away the boronic acid
group, without compromising compound potency. The
boronic acid is, in general, a challenge for development of an
orally bioavailable drug.
Despite these challenges, PDE3B remains a compelling
therapeutic target, supported by human genetics. The
development of oral small molecule therapeutics for PDE3B
may represent an attractive alternative to antibody therapy,
such as that used for blockage of PCSK9 to achieve an
improved lipid profile with cardiovascular benefit. Our data
show that the active site should not be ruled out as a path for
obtaining selectivity. Minimization of on-target toxicity in the
heart may also be achieved by selective tissue targeting. For
example, potent but nonselective PDE3A/B compounds could
be delivered to fat deposits by engineering lipid nanoparticles
functionalized to target white adipose vasculature, resulting in
the subsequent uptake by the surrounding adipose tissue and
initiation of lipolysis. However, at this time, the technology for
selective adipose tissue-targeting requires further maturation
before it can be used to robustly deliver small molecule
payloads in the clinic. We look forward to this research
informing future efforts in these areas to further explore the
impact of a PDE3B novel therapeutic.
■ EXPERIMENTAL SECTION
IMAP TR-FRET Activity Assay. This protocol describes a TR-
FRET-based IMAP assay to measure inhibition of cAMP hydrolysis
by PDE3. The immobilized metal ion affinity for phosphochemical
(IMAP) TR-FRET assay is a homogeneous, antibody-free, and
sensitive assay provided in kit form by Molecular Devices. The PDE
reaction is performed using a fluorescent labeled substrate (FAM-
cAMP). IMAP assays are based on the binding of phosphate to
immobilized metal coordination complexes on nanoparticles. A
terbium (Tb) donor enables a fluorescent energy transfer to occur
when FAM-AMP product is present�FAM-AMP and Tb donor both
bind to the nanoparticle, bringing the Tb donor in close proximity to
the fluorescein acceptor on the product and enabling FRET.
I https://doi.org/10.1021/acs.jmedchem.3c01562
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Following the hydrolysis of the FAM-cAMP substrate by PDE3, the
FAM-AMP product is generated. When the product is bound to the
IMAP nanoparticle (and Tb is also bound), there is a high signal in
the 520 nm channel, and the ratio of 520:490 nm channels is also
high. When PDE3B activity is inhibited, no product is formed, and a
decrease in the signal at 520 nm and the ratio 520:490 nm are
observed as there is no longer any FAM-AMP in close proximity to
the Tb-donor.
A TR-FRET IMAP assay (Molecular Devices R8160) was used to
measure the inhibition of cAMP hydrolysis by PDE3A or PDE3B.
Test compounds were serially diluted 1:4 in DMSO as an 11-point
curve with a top final assay concentration of 100 μM (40 nL/well in a
384 well white low volume assay plate (Greiner Bio-One, 782075).
Columns 6 and 18 contained DMSO or 1 mM Trequinsin (10 μM
final assay concentration), respectively, and served as the high
(uninhibited) and low (inhibited) controls, respectively. Assays were
performed using either FL PDE3B (1.6 nM) with 130 nM FAM-
cAMP substrate (Molecular Devices, R7506) and a 120 min reaction
time or FL PDE3A (1.6 nM) with 50 nM FAM-cAMP substrate and a
45 min reaction time. Enzymes and substrates were prepared in assay
buffer [50 mM Tris−HCl pH7.5 (Gibco), 8.3 mM MgCl2 (Sigma),
1.7 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetra
acetic acid (EGTA) (Sigma), 0.05% BSA (Sigma), and 0.01%
Pluronic F127 (Sigma)].
Following the defined reaction time, the IMAP quench reagent was
dispensed to the plate and allowed to equilibrate for a minimum of 3
h, with assay plates covered to protect from light, prior to reading on
the PHERAstar plate reader (BMG Labtech Ltd.). Plates were read at
an excitation of 337 nm and a dual emission of 520 and 490 nm.
RapidFire MS/MS Activity Assay. To determine compound
inhibitory potencies for FL PDE3B and 3A, a biochemical assay
measuring the formation of 5′-AMP product by the RapidFire-MS/
MS system was applied. 200 nL of test compound stocks (in 100%
DMSO) were prestamped into 384-well polypropylene microtiter
plates (781280, Greiner Bio One) after being serial diluted 1:3 (one
volume stock adds into 2 volumes of 100% DMSO) from top
concentration of 10 mM for a 11-step concentration series. A 10 μL of
total assay volume in an assay buffer consisted of 50 mM Tris−HCl,
pH 7.5, 8.3 mM MgCl2, 1.7 mM EGTA (BM-151, Boston
BioProducts), supplemented with 0.1 mg/mL bovine serum albumin
(BSA) (A7030, Sigma), 0.0018% (V/V) ultrapure Tween-20 (359T-
A,G-Biosciences), and 2% (V/V) DMSO, freshly prepared 5.0 μL of
substrate solution of adenosine 3′,5′-cyclic monophosphate (3′5′-
cyclic AMP) (A6885, Sigma) at 2× apparent Km level, or 450 nM and
270 nM, for FL PDE3B (GST-Tev-10H-PDE3B (M1-E1112)-FLAG)
and 3A (GST-Tev-10H-PDE3A (M1-Q1141)-FLAG), respectively)
and then 5.0 μL of enzyme solution (2.4 and 0.54 nM, for FL PDE3B
and 3A, respectively) were added. The enzyme reaction was allowed
to proceed at room temperature (22 °C) for 50 min. The reaction was
quenched with the addition of 40 μL of quench solution containing
0.13% (V/V) trifluoracetic acid (TFA) (302031, Sigma) and 0.1 μM
15
N5-Mono-AMP (900382, Aldrich). As 0 or 100% inhibition
controls, on each assay plate, columns 6 and 18 contain only
DMSO and column 18 with 40 μL of preadded quenching solution.
After being centrifuged at 2000 rpm for >10 min, the quenched
reaction mixture was analyzed on a RapidFire/MS/MS system by
detecting the formation of a 5′-AMP product. With eluent A of 0.1%
(V/V) TFA in 100% (V/V) water and eluent B of 0.5% (V/V) TFA
in 50% (V/V)/50% (V/V) acetonitrile/water, the mixture was first
desalted by a graphite-C Hypercarb type D (C18) (G9203-80106,
Agilent) separation matrix cartridge on a RapidFire 300 LC system
(Agilent Technologies) and then analyzed by a coupled Agilent
6495C triple quadruple mass spectrometer (Agilent Technologies)
with electrospray ionization (ESI) source in the multireaction
monitoring mode. For the ESI source, the curtain gas (GF) is 20
L/min; NEB gas 25 psi; ion spray voltage (CAP) 4000 V; spray
temperature (ST) 400 °C; spray flow (SF) 12 L/min; NOZ 500 V;
HP RF 150 V; LP RF 60 V; and Delta EMV 200 V14
Fragments of enzyme reaction product 5′-mono-AMP, internal
standard (IS) 15N5-5′-AMP, and substrate 3′-5′ cAMP were
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
monitored by the transition of 348.1 m/z → 136.1 m/z (Q1 → Q3),
353.1 m/z → 141.1 m/z, and 330.1 m/z → 136.1 m/z, respectively,
and with 50 ms dwell time, and using an instrument-specific fragment
voltage (FV), collision energy (CE) + 20 V, and collision cell exit
potential (CAV) + 5 V. Calibration curves (12 points 1:2 serial
diluted from top concentration up to 1.0 μM) were set up using a 5′-
AMP product standard (01930, Fluka) dissolved in the same assay
buffer. Data were acquired and processed with Analyst for Windows
(version 1.6.2, build 8489). The mass spectrometry detection peak
areas for both product and substrate fragments were normalized using
the peak area of the internal standard. Product concentrations were
determined with normalized peak area from product calibration
curves. On each assay plate, column 6 contains only DMSO and
column18 with predispensed TFA as high (zero inhibition) and low
(100% inhibition) controls, respectively. Data were fit to the following
equation (as described for the IMAP assays) to determine IC50. In the
case of IC50s ≫ [Enzyme], a simple four parameter fit was used, with
data normalized to high and low controls, where y is the % of
normalized enzyme activity (or inhibition), x is the concentration of
inhibitor, and s is the Hill slope factor. The mean values and standard
deviation of pIC50 values from at least 2−3 replicates were reported.
General Chemistry Experimental. Solvents and reagents were
purchased from commercial suppliers and used as received. Reactions
were monitored by liquid chromatography−mass spectrometry (LC−
MS). The LC−MS analysis was performed on one of the following:
(1) Thermo Scientific LC−MS instrument equipped with a
Kinetex XB-C18 column (2.1 × 50 mm, 1.7 μm packing
diameter) and a Thermo Scientific LTQ XL linear ion trap
mass spectrometer (positive ion). Analytes were detected as a
summed UV wavelength of 210−400 nm. The liquid-phase
method with 0.6 mL/min flow rate used gradient elution with
the mobiles phases as (A) water containing 0.1% volume/
volume (v/v) formic acid and (B) acetonitrile containing 0.1%
v/v formic acid.
(2) Thermo Scientific LC−MS instrument equipped with a
Kinetex C8 column (2.1 × 30 mm, 1.7 μm packing diameter)
and a Thermo Scientific LCQ fleet ion trap mass spectrometer
(positive ion). Analytes were detected as a summed UV
wavelength of 200−400 nm. The liquid-phase method with
0.65 mL/min flow rate used gradient elution with the mobiles
phases as (A) water containing 0.1% v/v formic acid and (B)
acetonitrile containing 0.1% v/v formic acid and 0.1% v/v
water.
(3) Waters Acquity UPLC instrument equipped with a CSH C19
column (2.1 × 300 mm, 1.8 μm packing diameter) and a
Waters electrospray ionization mass spectrometer (positive
and negative ion). The liquid-phase method used gradient
elution with the mobiles phases as (A) water containing 0.1%
v/v formic acid and (B) acetonitrile containing 0.1% v/v
formic acid.
(4) Waters Acquity UPLC instrument equipped with a CSH C18
column (2.1 × 30 mm, 1.7 μm packing diameter) and a Waters
electrospray ionization mass spectrometer (positive and
negative ion). The liquid-phase method used gradient elution
with the mobile phases as (A) water containing 0.1% v/v
formic acid and (B) acetonitrile containing 0.1% v/v formic
acid or (A) 95:5 water containing 0.1% NH4OH/acetonitrile
(pH = 9.4) and (B) acetonitrile.
The NMR spectra were recorded at ambient temperature (unless
otherwise stated) using standard pulse methods on any of the
following spectrometers and signal frequencies: Bruker Ascend 400
(1H = 400 MHz), Bruker AVANCE 400 (1H = 400 MHz), or Bruker
DPX400 (1H = 400 MHz). Chemical shifts are referenced to the
residual solvent peak and are reported in parts per million. Coupling
constants are quoted to the nearest 0.1 Hz, and multiplicities are given
by the following abbreviations and combinations thereof: s (singlet),
d (doublet), t (triplet), dd (doublet of doublets), dt (doublet of
triplets), dquin (doublet of quintets), m (multiplet), and br (broad).
The purifications were performed on one of the following:
J https://doi.org/10.1021/acs.jmedchem.3c01562
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(1) Flash column chromatography was performed using a
Teledyne Isco CombiFlash RF Flash chromatography system
with SNAP silica cartridges.
(2) Prep HPLC was performed using a Agilent 1290 Infinity II
HPLC/MS, with an Agilent 1290 Infinity II Mass Directed
Automated Purification System, while scanning at wavelengths
215 and 254 nm. A liquid-phase method was used on a Water
Xselect C18 column (100 mm × 19 mm, 5 μm packing
diameter, 40 mL/min flow rate). Gradient elution at ambient
temperature was used with the mobile phases as (A) H2O
containing 0.1% (v/v) formic acid and (B) acetonitrile
containing 0.1% (v/v) formic acid.
(3) Prep HPLC was performed using a Gilson Prep HPLC system.
Analytes were detected scanning for specific wavelengths
between 200 and 400 nm. A liquid-phase method was used on
a Water Xselect CSH C18 column (100 mm × 19 mm, 5 μm
packing diameter, 40 mL/min flow rate). Gradient elution at
ambient temperature was used with the mobile phases as (A)
H2O containing 0.1% (v/v) formic acid and (B) acetonitrile
containing 0.1% (v/v) formic acid.
(4) Teledyne ISCO AccqPrep HP150 equipped with a CSH
XSELECT 30 × 75 Prep HPLC column (30 mm, 75 mm, 5
μm). The liquid-phase method used gradient elution with
mobile phases as (A) water containing 0.1% v/v formic acid
and (B) acetonitrile containing 0.1% v/v formic acid.
The purity of the synthesized compounds was determined by LC-
MS analysis. All compounds for biological testing were more than
95% pure unless otherwise stated.
General Procedure for TCFH Amide Couplings. A 4 mL vial
was charged with a magnetic stir bar and carboxylic acid (0.165
mmoL, 1.1 equiv). To this vial, a 0.6 mL aliquot of a 0.25 M stock
solution of (S)-3-borono-5-((1-cyclohexyl-2-(methylamino)-2-
oxoethyl)carbamoyl)benzenaminium chloride (55 mg, 0.15 mmol)
in NMP was added. Next, a 0.2 mL aliquot of a 2.625 M stock
solution of N-ethyl-N-isopropan-2-amine (68 mg, 0.525 mmol, 3.5
equiv) in NMP was added. Lastly, a 0.2 mL aliquot of a 0.9 M stock
solution of N-(chloro(dimethylamino)methylene)-N-methylmethana-
minium hexafluorophosphate(V) (51 mg, 0.18 mmol, 1.2 equiv) in
NMP was added. The reaction mixture was then allowed to stir at
room temperature for 18 h. Compounds were purified via reverse-
phase chromatography to afford the desired amide products.
General Procedure for HATU Amide Couplings. A 4 mL vial
was charged with a magnetic stir bar and carboxylic acid (0.087 mmol,
1 equiv). To this vial was added a solution of HATU (0.095 mmol,
0.2 M in DMF, 1.09 equiv) and DIPEA 0.379 mmol, 4.33 equiv). The
resulting mixture was allowed to stir at room temperature for 5 min.
After this time, a solution of amine (0.094 mmol, 1.08 equiv) was
added, and the reaction was allowed to stir overnight. After this time,
the reaction was concentrated to dryness. Compounds were purified
via reverse-phase chromatography to afford the desired amide
products.
General Procedure for DMTMM Amide Coupling. To a 4 mL
vial charged with a magnetic stir bar and amine (0.125 mmol, 1.25
equiv) was added sodium 3-borono-5-(4,7-dimethoxyquinoline-2-
carboxamido)benzoate (compound 8, 176 mM stock solution in
NMP, 0.100 mmol, 1.0 equiv), DMTMM (687 mM stock solution in
NMP, 0.120 mmol, 1.2 equiv), and N-methylmorpholine (1.5 M
solution in NMP). The reaction mixture was allowed to stir at room
temperature for 18 h. Compounds were purified by reverse-phase
chromatography to afford the desired amide products.
tert-Butyl (1-(Ethylcarbamoyl)azepan-3-yl)carbamate (Inter-
mediate A). To a vial charged with a magnetic stir bar and tert-
butyl azepan-3-ylcarbamate (610.0 mg, 2.84 mmol) was added DCM
(6.1 mL). The solution was chilled in an ice bath, then to the solution
was added ethyl isocyanate (225 μL, 2.84 mmol, 1 equiv) dropwise.
The reaction mixture was allowed to stir at room temperature for 20
h. The product was purified by normal-phase chromatography to
afford the desired product (595 mg, 73% yield). LC−MS m/z: 186.07
[M + H-Boc]+. 1H NMR (methanol-d4, 400 MHz): δ 6.6−6.8 (m,
1H), 3.4−3.8 (m, 3H), 3.1−3.3 (m, 3H), 3.0−3.1 (m, 1H), 1.7−2.0
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
(m, 3H), 1.6−1.7 (m, 1H), 1.47 (s, 9H), 1.3−1.4 (m, 2H), 1.17 (t,
3H, J = 1.0 Hz).
(3-((tert-Butoxycarbonyl)amino)-5-((1-(ethylcarbamoyl)azepan-
3-yl)carbamoyl)phenyl)boronic Acid (Intermediate B). To a vial
charged with a magnetic stir bar and tert-butyl (1-(ethylcarbamoyl)-
azepan-3-yl)carbamate (Intermediate A, 36 mg, 126 μmol) was
added DCM (630 μL). The solution was chilled in an ice bath, then
to the solution was added TFA (19 μL, 252 μmol, 2 equiv) dropwise.
The reaction mixture was allowed to stir at room temperature for 18
h, then concentrated to afford the crude oil. The crude product was
dissolved in DMF (630 μL) and used directly. To a separate glass vial
charged with 3-borono-5-((tert-butoxycarbonyl)amino)benzoic acid
(35 mg, 126 μmol, 1 equiv) and HATU (48 mg, 126 μmol, 1 equiv)
was added DMF (630 μL) followed by DIPEA (66 μL, 378 μmol, 3
equiv). The premix solution was allowed to react at room temperature
for 15 min, then the intermediate amine (126 μmol, 1 equiv) was
added. The reaction mixture was allowed to stir at room temperature
for 2 h, then concentrated to afford the crude product. A portion of
the crude product was purified by reverse-phase chromatography to
afford the desired product. LC−MS m/z: 449.18 [M + H]+. 1H NMR
(methanol-d4, 400 MHz): δ ppm 7.82 (s, 1H), 7.73 (br s, 1H), 7.68
(br s, 1H), 7.58 (br s, 1H), 3.87 (br s, 1H), 3.50 (br dd, 2H, J = 3.1,
14.9 Hz), 3.2−3.3 (m, 1H), 3.2−3.2 (m, 3H), 1.9−2.0 (m, 1H), 1.7−
1.8 (m, 2H), 1.4−1.7 (m, 2H), 1.43 (s, 9H), 1.2−1.4 (m, 1H), 1.09
(t, 3H, J = 7.2 Hz).
(3-Amino-5-((1-(ethylcarbamoyl)azepan-3-yl)carbamoyl)-
phenyl)boronic Acid (Intermediate C). To a vial charged with a
magnetic stir bar and tert-butyl (1-(ethylcarbamoyl)azepan-3-yl)-
carbamate (Intermediate A, 386 mg, 1.35 mmol) was added DCM
(6.8 mL). The solution was chilled in an ice bath, then to the solution
was added TFA (313 μL, 4.07 mmol, 3 equiv) dropwise. The reaction
mixture was allowed to stir at room temperature for 18 h, then
concentrated to afford the crude product as a yellow oil. The crude
product was dissolved in DMF (6.8 mL) and used directly. To a
separate glass vial charged with 3-((tert-butoxycarbonyl)amino)-5-
hydroxybenzoic acid (343 mg, 1.35 mmol, 1 equiv) and HATU (515
mg, 1.35 mmol, 1 equiv) was added DMF (6.8 mL) followed by
DIPEA (708 μL, 4.07 mmol, 3 equiv). The premix solution was
allowed to react at room temperature for 15 min, then the
intermediate amine (1.35 mmol, 1 equiv) was added. The reaction
mixture was allowed to stir at room temperature for 2 h, then
concentrated to afford the crude product. A portion of the crude
product was purified by reverse-phase chromatography to afford the
desired product. LC−MS m/z: 421.15 [M + H]+. 1H NMR
(methanol-d4, 400 MHz): δ ppm 7.1−7.2 (m, 1H), 7.0−7.1 (m,
1H), 6.7−6.8 (m, 1H), 3.8−3.9 (m, 1H), 3.3−3.6 (m, 4H), 3.0−3.2
(m, 4H), 2.9−3.0 (m, 1H), 1.5−2.0 (m, 5H), 1.4−1.4 (m, 2H), 1.3−
1.4 (m, 9H), 1.2−1.3 (m, 2H), 1.0−1.1 (m, 3H).
(3-((1-(Ethylcarbamoyl)azepan-3-yl)carbamoyl)-5-(2-methoxy-
3,5-dimethylbenzamido)phenyl)boronic Acid (1). To a vial charged
with a magnetic stir bar and (3-((tert-butoxycarbonyl)amino)-5-((1-
(ethylcarbamoyl)azepan-3-yl)carbamoyl)phenyl)boronic acid (Inter-
mediate B, 170 mg, 0.379 mmol) was added DCM (3 mL) followed
by TFA (0.58 mL, 7.58 mmol, 20 equiv). The reaction mixture was
allowed to stir at room temperature for 2 h, then concentrated to
afford the crude product, which was dissolved in DMF (2 mL) and
used directly. To the solution was added 2-methoxy-3,5-dimethyl-
benzoic acid (50 mg, 0.277 mmol, 1 equiv) followed by DIPEA (0.19
mL, 1.11 mmol, 4 equiv) and HATU (106 mg, 0.277 mmol, 1.4
equiv). The reaction mixture was allowed to stir at room temperature
for 3 h, then concentrated to afford the crude product. The crude
product was purified by reverse-phase chromatography to afford the
desired product (83 mg, 59% yield). LC−MS m/z: 511.18 [M + H]+.
1
H NMR DMSO-d6: δ ppm 10.2−10.3 (s, 1H), 8.4−8.5 (d, 1H),
8.1−8.2 (d, 3H), 7.9−8.0 (s, 1H), 7.2−7.3 (d, 2H), 6.6−6.7 (br s,
1H), 3.9−4.0 (br s, 1H), 3.7−3.8 (s, 3H), 3.5−3.7 (m, 7H), 3.1−3.2
(m, 4H), 2.2−2.3 (d, 6H), 1.7−1.9 (m, 3H), 1.5−1.6 (m, 2H), 1.2−
1.4 (m, 1H), 1.0−1.1 (t, 3H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(2-methoxy-3,5-dimethylbenzamido)phenyl)boronic Acid (2).
K https://doi.org/10.1021/acs.jmedchem.3c01562
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Compound 2 was prepared according to the HATU amide coupling
procedure. LC−MS m/z: 496.25 [M + H]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.18−7.84 (m, 3H), 7.42 (s, 1H), 7.22 (s, 1H),
4.35 (d, 1H, J = 8.3 Hz), 3.81 (s, 3H), 2.76 (s, 3H), 2.33 (s, 6H),
1.90−1.67 (m, 6H), 1.37−1.06 (m, 5H).
N-Ethyl-3-(3-hydroxy-5-(2-methoxy-3,5-dimethylbenzamido)-
benzamido)azepane-1-carboxamide (3). To a vial charged with a
magnetic stir bar and tert-butyl (3-((1-(ethylcarbamoyl)azepan-3-
yl)carbamoyl)-5-hydroxyphenyl)carbamate (Intermediate C, 214 mg,
0.509 mmol) was added DCM (4 mL) followed by TFA (0.78 mL,
10.2 mmol, 20 equiv). The reaction mixture was allowed to stir at
room temperature for 2 h, then concentrated to afford the crude
product, which was dissolved in DMF (2 mL) and used directly. To
the solution was added 2-methoxy-3,5-dimethylbenzoic acid (70 mg,
0.388 mmol, 1 equiv) followed by DIPEA (0.27 mL, 1.55 mmol, 4
equiv) and HATU (148 mg, 0.388 mmol, 1.3 equiv). The reaction
mixture was allowed to stir at room temperature for 3 h, then
concentrated to afford the crude product. The crude product was
purified by reverse-phase chromatography to afford the desired
product (65 mg, 35% yield). LC−MS m/z: 483.13 [M + H]+. 1H
NMR DMSO-d6: δ ppm 10.2−10.3 (s, 1H), 9.6−9.8 (s, 1H), 8.4−8.5
(d, 1H), 7.5−7.6 (m, 2H), 7.1−7.2 (s, 2H), 6.9−7.0 (s, 1H), 6.6−6.7
(s, 1H), 3.9−4.0 (br s, 1H), 3.7−3.8 (s, 3H), 3.4−3.6 (m, 8H), 3.0−
3.3 (m, 4H), 2.2−2.3 (d, 6H), 1.7−1.9 (m, 3H), 1.4−1.7 (m, 2H),
1.2−1.4 (m, 1H), 1−1.1 (t, 3H).
2,2,2-Trifluoroacetic Acid Compound with (R)-(3-((1-Cyclohexyl-
2-(methylamino)-2-oxoethyl)carbamoyl)-5-(4,7-dimethoxyquino-
line-2-carboxamido)phenyl)boronic Acid (1:1) (4). To a solution of
(R)-(3-((tert-butoxycarbonyl)amino)-5-((1-cyclohexyl-2-(methylami-
no)-2-oxoethyl)carbamoyl)phenyl)boronic acid (28 mg, 0.065 mmol)
in dioxane (1 mL) was added 4 N HCl in dioxane (1 mL). The
reaction mixture was stirred at room temperature for 18 h, diluted
with ether (10 mL). The reaction mixture was centrifuged, the
supernatant was decanted, and the solid was dried and used for the
next step directly. The above prepared amine was dissolved in a
mixture of acetonitrile (2 mL)/DMF (0.5 mL), and 4,7-dimethox-
yquinoline-2-carboxylic acid (19.59 mg, 0.084 mmol), 1-methyl-1H-
imidazole (26.5 mg, 0.323 mmol), and TCFH (46.5 mg, 0.084 mmol)
were added successively. The reaction mixture was stirred at room
temperature for 18 h, concentrated to dryness, and the crude was
purified by reverse-phase chromatography to afford the desired
product (17 mg, 38% yield). LC−MS m/z: 549.2 [M + H]. 1H NMR
DMSO-d6: δ ppm 10.76 (s, 1H), 8.41 (s, 1H), 8.37 (s, 1H), 7.98−
8.20 (m, 4H), 7.62 (s, 1H), 7.59 (d, J = 2.5 Hz, 1H), 7.34 (dd, J =
9.19, 2.5 Hz, 1H), 4.31−4.38 (m, 1H), 4.16 (s, 3H), 3.97 (s, 3H),
2.62 (d, J = 4 Hz, 3H), 1.67−1.82 (m, 4H), 1.54−1.67 (m, 2H),
1.13−1.25 (m, 3H), 0.96−1.12 (m, 2H).
2,2,2-Trifluoroacetic Acid Compound with (S)-(3-((1-Cyclohexyl-
2-(methylamino)-2-oxoethyl)carbamoyl)-5-(4,7-dimethoxyquino-
line-2-carboxamido)phenyl)boronic Acid (1:1) (5). To a solution of
(S)-(3-((tert-butoxycarbonyl)amino)-5-((1-cyclohexyl-2-(methylami-
no)-2-oxoethyl)carbamoyl)phenyl)boronic acid (28 mg, 0.065 mmol)
in dioxane (1 mL) was added 4 N HCl in dioxane (1 mL). The
reaction mixture was stirred at room temperature for 18 h, diluted
with ether (10 mL). The reaction mixture was centrifuged, the
supernatant was decanted, and the solid was dried and used for the
next step directly. The above prepared amine was dissolved in a
mixture of acetonitrile (2 mL)/DMF (0.5 mL), and 4,7-dimethox-
yquinoline-2-carboxylic acid (19.59 mg, 0.084 mmol), 1-methyl-1H-
imidazole (26.5 mg, 0.323 mmol), and TCFH (46.5 mg, 0.084 mmol)
were added successively. The reaction mixture was stirred at room
temperature for 18 h, concentrated to dryness, and the crude was
purified by reverse-phase chromatography to afford the desired
product (21 mg, 47% yield). LC−MS m/z: 549.3 [M + H]+. 1H NMR
(400 MHz, DMSO-d6): δ ppm 10.76 (s, 1H), 8.41 (s, 1H), 8.37 (s,
1H), 8.13 (d, J = 9.13 Hz, 1H), 8.10−8.05 (m, 3H), 7.62 (s, 1H),
7.58 (d, J = 2.38 Hz, 1H), 7.34, (dd, J = 9.26, 1.5 Hz, 1H), 4.31−4.38
(m, 1H), 4.16 (s, 3H), 3.97 (s, 3H), 2.62 (d, J = 4.5 Hz, 3H), 1.67−
1.82 (m, 4H), 1.54−1.67 (m, 2H), 1.13−1.27 (m, 3H), 0.96−1.13
(2H).
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
(3-(4,7-Dimethoxyquinoline-2-carboxamido)phenyl)boronic
Acid (6). In a vial, (3-aminophenyl)boronic acid (18 mg, 0.13 mmol)
was stirred in ACN (1 mL) and DMF (0.25 mL). To the solution,
4,7-dimethoxyquinoline-2-carboxylic acid (30 mg, 0.13 mmol) was
added followed by N-methylimidazole (52 μL, 0.64 mmol), then
TCFH (54 mg, 0.19 mmol). The reaction was stirred at RT for 1 h,
then volatiles were removed in vacuo. The resulting mixture was
purified via reverse-phase chromatography to afford the desired
product (28 mg, 58%). LC−MS m/z: [353.2]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.14−8.22 (m, 1H) 8.04−8.10 (m, 1H) 7.89−7.98
(m, 1H) 7.68−7.74 (m, 1H) 7.53−7.60 (m, 1H) 7.38−7.47 (m, 2H)
7.25−7.33 (m, 1H) 4.20 (s, 3H) 4.01 (s, 3H).
(3-Carbamoyl-5-(4,7-dimethoxyquinoline-2-carboxamido)-
phenyl)boronic Acid (7). Compound 7 was prepared according to the
general procedure for DMTMM amide coupling (hydrochloride salt
of amine used). LC−MS m/z: 396.31 [M + H]+. 1H NMR (400
MHz, MeOD-d4): δ ppm 8.37−8.44 (m, 1H) 8.24−8.32 (m, 1H)
8.14−8.22 (m, 1H) 7.90−8.09 (m, 1H) 7.66−7.75 (m, 1H) 7.53−
7.58 (m, 1H) 7.25−7.32 (m, 1H) 4.19 (s, 3H) 3.94−4.07 (m, 3H).
Sodium 3-Borono-5-(4,7-dimethoxyquinoline-2-carboxamido)-
benzoate (8). To a 250 mL round-bottomed flask charged with a
magnetic stir bar, 4,7-dimethyoxyquinoline-2-carboxylic acid (2.00 g,
8.58 mmol, 1.0 equiv) and BOP (4.16 g, 9.43 mmol, 1.1 equiv) were
added NMP (20 mL). To the solution was added DIPEA (4.49 mL,
25.7 mmol, 3.0 equiv), and the solution was allowed to stir at room
temperature for 10 min. Separately, to a 40 mL reaction vial charged
with (3-amino-5-(methoxycarbonyl)phenyl)boronic acid (1.67 g, 8.58
mmol, 1.0 equiv) was added NMP (10 mL), and the solution was
sonicated to aid dissolution. Following the 10 min preactivation, the
amine solution was added to the reaction mixture via a syringe over 5
min, and the resulting reaction mixture was allowed to react at room
temperature for 1 h. The reaction mixture was then slowly diluted
with half-saturated aqueous ammonium chloride solution (250 mL),
and the resulting suspension was stirred at room temperature for 10
min. The suspended solids were isolated via filtration, washed with
half-saturated sodium chloride, and dried. The crude solid was
dispersed into a bilayer of ethyl acetate (150 mL) and water (75 mL),
and the resulting suspended solid was isolated via filtration and
washed with ethyl acetate and dried to afford the acid intermediate as
a light tan solid (2.63 g, 6.41 mmol, 74.8% yield, LC−MS m/z 411.2
[M + H]+). The crude product was transferred to a 250 mL round-
bottomed flask and dissolved in acetonitrile (60 mL) and water (20
mL). The solution was treated with 6 N aqueous sodium hydroxide
(4.29 mL, 25.7 mmol, 4.0 equiv), and the reaction mixture was
allowed to react at room temperature for 1.5 h. The solution was
further diluted with 40 mL water, and the reaction mixture was
allowed to continue to react at room temperature overnight. The
mixture was concentrated under reduced pressure to remove the
majority of acetonitrile, then treated with 1 N aqueous hydrochloric
acid to adjust the pH to 6. The suspended solid was isolated via
filtration and triturated with 2-propanol and dried to afford the
desired product (2.35 g, 5.34 mmol, 62.3%). LC−MS m/z: 397.2 [M
+ H]+. 1H NMR (400 MHz, DMSO-d6): δ ppm 10.6−10.8 (m, 1H),
9.3−9.9 (m, 2H), 8.5 (s, 1H), 8.3 (s, 1H), 8.2 (s, 1H), 8.1 (d, J = 9.26
Hz, 1H), 7.6−7.7 (m, 2H), 7.3 (dd, J = 9.13, 2.13 Hz, 1H) 4.2 (s, 3H)
4.0 (s, 3H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-
(methoxycarbonyl)phenyl)boronic Acid (9). In a vial 4,7-dimethox-
yquinoline-2-carboxylic acid (500 mg, 2.1 mmol) was stirred in NMP
(24 mL) with BOP (1.04 g, 2.4 mmol) and DIPEA (1.1 mL, 6.4
mmol), the reaction was stirred for 5 min, then (3-amino-5-
(methoxycarbonyl)phenyl)boronic acid (420 mg, 2.1 mmol) was
added in NMP (12 mL). The reaction was stirred for 1H, then half-
saturated ammonium chloride (75 mL) was added slowly, and the
reaction was filtered. The precipitate was then purified via reverse-
phase chromatography to afford the desired product (15.2 mg, 1.7%).
LC−MS m/z: 411.2 [M + H]+. 1H NMR (400 MHz, MeOD-d4): δ
ppm 8.59−8.65 (m, 1H) 8.29−8.35 (m, 1H) 8.17−8.22 (m, 1H)
8.07−8.13 (m, 1H) 7.69−7.76 (m, 1H) 7.56−7.60 (m,1H) 7.26−7.36
(m, 1H) 4.18−4.24 (m, 3H) 4.02 (s, 3H) 3.91−3.97 (m, 3H).
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(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-
(methylcarbamoyl)phenyl)boronic Acid (10). Compound 10 was
prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 410.28 [M + H]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.31−8.41 (m, 1H) 8.18−8.25 (m, 1H) 8.09−8.17
(m, 1H) 7.82−8.01 (m, 1H) 7.62−7.67 (m, 1H) 7.51−7.56 (m, 1H)
7.23−7.29 (m, 1H) 4.16 (s, 3H) 3.94−4.05 (m, 3H) 2.90−3.00 (m,
3H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-
(dimethylcarbamoyl)phenyl)boronic Acid (11). In a vial, 3-borono-
5-(4,7-dimethoxyquinoline-2-carboxamido)benzoic acid (100 mg,
0.25 mmol), 40% dimethylamine in water (64 μL, 0.51 mmol), and
HATU (140 mg, 0.38 mmol) were stirred in DCM (2 mL) with DMF
(0.5 mL), and DIPEA (176 μL, 1.010 mmol) was added. The reaction
was stirred at RT for 19 h. Volatiles were removed in vacuo, then the
subsequent reaction mixture was taken up in DCM and saturated
sodium bicarbonate. The layers were separated, and the aqueous layer
was extracted the DCM three times. The organic layers were
combined, and volatiles were removed in vacuo. The precipitate was
then purified via reverse-phase chromatography to afford the desired
product (36 mg, 32%). LC−MS m/z: 424.3 [M + H]+. 1H NMR (400
MHz, MeOD-d4): δ ppm 8.30−8.36 (m, 1H) 8.11−8.16 (m, 1H)
8.05−8.09 (m, 1H) 7.97−8.01 (m, 1H) 7.63−7.68 (m, 1H) 7.51−
7.57 (m, 1H) 7.43−7.50 (m, 1H) 4.38 (s, 3H) 4.02−4.11 (m, 3H)
3.04−3.18 (m, 6H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-
(isopropylcarbamoyl)phenyl)boronic Acid (12). In a vial, 4,7-
dimethoxyquinoline-2-carboxylic acid (53 mg, 0.23 mmol) and
HATU (130 mg, 0.34 mmol) were stirred in DMF (1.1 mL), and
DIPEA (160 μL, 0.90 mmol) was added. The reaction was stirred at
RT for 15 min, then (3-amino-5-(isopropylcarbamoyl)phenyl)boronic
acid (50 mg, 0.225 mmol) was added in DMF (1.1 mL), and the
reaction was stirred at RT for 1 h. All volatiles were removed in vacuo.
The reaction mixture was then purified via reverse-phase chromatog-
raphy to afford the desired product. LC−MS m/z: 438.3 [M + H]+.
1
H NMR (400 MHz, MeOD-d4): δ ppm 8.28−8.35 (m, 1H) 8.21−
8.26 (m, 1H) 8.13−8.18 (m, 1H) 7.81−7.87 (m, 1H) 7.64−7.69 (m,
1H) 7.53−7.57 (m, 1H) 7.25−7.30 (m, 1H) 4.21−4.30 (m, 1H) 4.17
(s, 3H) 4.00 (s, 3H) 1.29 (d, J = 6.6 Hz, 6H).
(3-(4,7-Dimethoxy-N-methylquinoline-2-carboxamido)-5-
(isopropylcarbamoyl)phenyl)boronic Acid (13). A vial containing N-
(3-bromo-5-(isopropylcarbamoyl)phenyl)-4,7-dimethoxy-N-methyl-
quinoline-2-carboxamide (35 mg, 0.072 mmol), Pd(dppf)Cl2 (5 mg,
0.006 mmol), B2Pin2 (20 mg, 0.079 mmol), potassium acetate (21
mg, 0.214 mmol), 1,4-dioxane (0.75 mL), and water (0.010 mL) was
sparged with nitrogen gas for 1 min and heated at 80 °C for 2 h. After
this time, the reaction was allowed to cool to room temperature. LC−
MS shows two desired products (in the boronic acid form as well as
the boronic ester form). The reaction was concentrated to dryness
and then purified via reverse-phase chromatography to afford the
desired product (5 mg, 15%). LC−MS m/z: 452.25 [M + H]+. 1H
NMR (400 MHz, MeOD-d4): δ ppm 8.1−8.3 (s, 1H), 7.8−8.0 (m,
1H), 7.6−7.8 (m, 2H), 7.3−7.5 (s, 1H), 7.0−7.2 (s, 2H), 6.7−6.9 (s,
1H), 4.0−4.2 (br s, 1H), 3.8−4.0 (m, 6H), 3.5−3.7 (s, 3H), 1.1−1.4
(m, 8H).
(R)-(3-((1-Amino-3-methyl-1-oxobutan-2-yl)carbamoyl)-5-(4,7-
dimethoxyquinoline-2-carboxamido)phenyl)boronic Acid (14).
Compound 14 was prepared according to the general procedure for
DMTMM amide coupling. LC−MS m/z: 495.24 [M + H]+. 1H NMR
(400 MHz, MeOD-d4): δ ppm 8.34−8.44 (m, 1H) 8.22−8.31 (m,
1H) 8.12−8.18 (m, 1H) 7.85−8.06 (m, 1H) 7.64−7.69 (m, 1H)
7.53−7.58 (m, 1H) 7.23−7.30 (m, 1H) 4.38−4.49 (m, 1H) 4.17 (s,
3H) 3.95−4.05 (m, 3H) 2.15−2.30 (m, 1H) 0.94−1.18 (m, 6H).
(S)-(3-((1-Amino-3-methyl-1-oxobutan-2-yl)carbamoyl)-5-(4,7-
dimethoxyquinoline-2-carboxamido)phenyl)boronic Acid (15).
Compound 15 was prepared according to the general procedure for
DMTMM amide coupling. LC−MS m/z: 495.2 [M + H]+. 1H NMR
(700 MHz, DMSO-d6): δ ppm 10.76 (s, 1H) 8.44 (s, 1H) 8.38 (d, J =
1.29 Hz, 1H) 8.25 (s, 2H) 8.06 (s,1H) 8.12 (d, J = 9.46 Hz, 1H) 7.91
(d, J = 9.04 Hz, 1H) 7.61 (s, 1H) 7.58 (d, J = 2.58 Hz, 1H) 7.53 (s,
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
1H) 7.33 (dd, J = 9.04, 2.58 Hz, 1H) 7.15 (s, 1H) 4.34−4.39 (m, 1H)
4.15 (s, 3H) 3.97 (s, 3H) 2.08−2.15 (m, 1H) 0.95 (dd, J = 6.45, 5.16
Hz, 6H).
(R)-(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((1-(methyla-
mino)-1-oxopropan-2-yl)carbamoyl)phenyl)boronic Acid (16).
Compound 16 was prepared according to the general procedure for
DMTMM amide coupling. LC−MS m/z: 481.24 [M + H]+. 1H NMR
(400 MHz, MeOD-d4): δ ppm 8.33−8.41 (m, 1H) 8.21−8.31 (m,
1H) 8.11−8.18 (m, 1H) 7.88−8.08 (m, 1H) 7.62−7.69 (m, 1H)
7.50−7.56 (m, 1H) 7.22−7.31 (m, 1H) 4.51−4.63 (m, 1H) 4.17 (s,
3H) 3.95−4.06 (m, 3H) 2.74−2.81 (m, 3H) 1.43−1.56 (m, 3H).
(R)-(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((1-methoxy-
3-methylbutan-2-yl)carbamoyl)phenyl)boronic Acid (17). Com-
pound 17 was prepared according to the general procedure for
DMTMM amide coupling. LC−MS m/z: 496.29 [M + H]+. 1H NMR
(400 MHz, MeOD-d4): δ ppm 8.30−8.36 (m, 1H) 8.19−8.26 (m,
1H) 8.12−8.17 (m, 1H) 7.82−8.04 (m, 1H) 7.63−7.69 (m, 1H)
7.51−7.57 (m, 1H) 7.24−7.30 (m, 1H) 4.17 (s, 3H) 4.04−4.12 (m,
1H) 3.96−4.03 (m, 3H) 3.53−3.63 (m, 2H) 3.37−3.42 (m, 3H)
1.95−2.07 (m, 1H) 0.98−1.08 (m, 6H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((1-methoxypro-
pan-2-yl)carbamoyl)phenyl)boronic Acid (18). Compound 18 was
prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 468.27 [M + H]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.28−8.34 (m, 1H) 8.12−8.27 (m, 2H) 7.80−8.03
(m, 1H) 7.63−7.68 (m, 1H) 7.52−7.57 (m, 1H) 7.24−7.30 (m, 1H)
4.30−4.43 (m, 1H) 4.17 (s, 3H) 3.95−4.07 (m, 3H) 3.50−3.57 (m,
1H) 3.39−3.49 (m, 4H) 1.22−1.35 (m, 3H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((2-
methoxypropyl)carbamoyl)phenyl)boronic Acid (19). Compound
19 was prepared according to the general procedure for DMTMM
amide coupling. LC−MS m/z: 468.27 [M + H]+. 1H NMR (400
MHz, MeOD-d4): δ ppm 8.32−8.38 (m, 1H) 8.12−8.28 (m, 2H)
7.81−8.04 (m, 1H) 7.63−7.68 (m, 1H) 7.51−7.56 (m, 1H) 7.23−
7.30 (m, 1H) 4.17 (s, 3H) 3.96−4.05 (m, 3H) 3.57−3.67 (m, 1H)
3.39−3.52 (m, 5H) 1.18−1.26 (m, 3H).
(S)-(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((tetrahydro-
2H-pyran-3-yl)carbamoyl)phenyl)boronic Acid (20). Compound 20
was prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 480.34 [M + H]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.28−8.36 (m, 1H) 8.19−8.27 (m, 1H) 8.06−8.17
(m, 1H) 7.80−8.01 (m, 1H) 7.62−7.67 (m, 1H) 7.49−7.57 (m, 1H)
7.20−7.32 (m, 1H) 4.17 (s, 3H) 4.04−4.12 (m, 1H) 3.93−4.02 (m,
4H) 3.80−3.89 (m, 1H) 3.45−3.56 (m, 1H) 3.34−3.42 (m, 1H)
1.66−2.12 (m, 4H). 93% pure by LCMS.
(R)-(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-((tetrahydro-
2H-pyran-3-yl)carbamoyl)phenyl)boronic Acid (21). Compound 21
was prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 480.34 [M + H]+. 1H NMR (400 MHz,
MeOD-d4): δ ppm 8.28−8.34 (m, 1H) 8.21−8.27 (m, 1H) 8.12−8.18
(m, 1H) 7.80−8.00 (m, 1H) 7.63−7.68 (m, 1H) 7.51−7.57 (m, 1H)
7.23−7.30 (m, 1H) 4.17 (s, 3H) 4.04−4.12 (m, 1H) 3.94−4.02 (m,
4H) 3.80−3.88 (m, 1H) 3.45−3.55 (m, 1H) 3.34−3.43 (m, 1H)
1.66−2.13 (m, 4H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-
(isobutylcarbamoyl)phenyl)boronic Acid (22). Compound 22 was
prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 452.2 [M + H]+. 1H NMR (600 MHz,
DMSO-d6): δ ppm 10.72 (s, 1H) 8.40 (t, J = 5.87 Hz, 1H) 8.38 (t, J =
2.02 Hz, 1H) 8.31−8.33 (m, 1H) 8.20 (s, 2H) 8.12 (d, J = 9.17 Hz,
1H) 8.00 (s, 1H) 7.60 (s, 1H)7.57 (d, J = 2.57 Hz, 1H) 7.33 (dd, J =
9.17, 2.57 Hz, 1H) 4.15 (s, 3H) 3.97 (s, 3H) 3.07−3.14 (m, 2H) 1.88
(dquin, J = 13.48, 6.81, 6.81, 6.81, 6.81 Hz, 1H) 0.92 (d, J = 6.60 Hz,
6H).
(3-(Benzyl(methyl)carbamoyl)-5-(4,7-dimethoxyquinoline-2-
carboxamido)phenyl)boronic Acid (23). In a vial, 3-borono-5-(4,7-
dimethoxyquinoline-2-carboxamido)benzoic acid (100 mg, 0.25
mmol), N-methyl-1-phenylmethanamine (65 μL, 0.51 mmol), and
HATU (140 mg, 0.38 mmol) were stirred in DCM (2.0 mL) with
DMF (0.5 mL), and DIPEA (180 μL, 1.0 mmol) was added. The
reaction was stirred at RT for 16 h, then additional N-methyl-1-
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phenylmethanamine (65 μL, 0.51 mmol), HATU (140 mg, 0.38
mmol), and DIPEA (180 mL, 1.0 mmol) were added, and the
reaction was stirred for an additional 2 h. All volatiles were removed
in vacuo, then taken up in brine and DCM, the aqueous layer was
extracted with DCM three times, then the organic layers were
combined, and all volatiles were removed in vacuo. The reaction
mixture was then purified via reverse-phase chromatography to afford
the desired product (64 mg, 49%). LC−MS m/z: 500.3 [M + H]+. 1H
NMR (400 MHz, methanol-d4): δ ppm 8.1−8.3 (m, 3H) 7.7−7.8 (m,
1H) 7.5−7.6 (m, 2H) 7.2−7.5 (m, 6H) 4.6−4.8 (m, 2H) 4.3 (br s,
3H) 4.0−4.1 (m, 3H) 2.9−3.0 (m, 3H). 94% pure by LCMS.
(3-(Benzyl(methyl)carbamoyl)-5-(4,7-dimethoxyquinoline-2-
carboxamido)phenyl)boronic Acid (23). In a vial, 3-borono-5-(4,7-
dimethoxyquinoline-2-carboxamido)benzoic acid (100 mg, 0.25
mmol), N-methyl-1-phenylmethanamine (65 μL, 0.51 mmol), and
HATU (140 mg, 0.38 mmol) were stirred in DCM (2.0 mL) with
DMF (0.5 mL), and DIPEA (180 μL, 1.0 mmol) was added. The
reaction was stirred at RT for 16 h, then additional N-methyl-1-
phenylmethanamine (65 μL, 0.51 mmol), HATU (140 mg, 0.38
mmol), and DIPEA (180 mL, 1.0 mmol) were added, and the
reaction was stirred for an additional 2 h. All volatiles were removed
in vacuo, then taken up in brine and DCM, the aqueous layer was
extracted with DCM three times, then the organic layers were
combined, and all volatiles were removed in vacuo. The reaction
mixture was then purified via reverse-phase chromatography to afford
the desired product (64 mg, 49%). LC−MS m/z: 500.3 [M + H]+. 1H
(400 MHz, MeOD-d4): δ ppm 8.08−8.27 (m, 3H) 7.74−7.83 (m,
1H) 7.47−7.62 (m, 2H) 7.19−7.45 (m, 6H) 4.59−4.83 (m, 2H) 4.26
(br s, 3H) 3.97−4.07 (m, 3H) 2.94−3.10 (m, 3H). 94% pure by LC−
MS.
(3-(3-Cyclopropoxyazetidine-1-carbonyl)-5-(4,7-dimethoxyqui-
noline-2-carboxamido)phenyl)boronic Acid (24). Compound 24
was prepared according to the general procedure for DMTMM amide
coupling. LC−MS m/z: 492.2 [M + H]+. 1H NMR1H NMR (700
MHz, DMSO-d6): δ ppm 10.73−10.86 (m,7.33, 1H) 4.49−4.55 (m,
1H), 4.43−4.49 (m, 1H), 4.27−4.32 (m, 1H), 4.23−4.27 (m, 1H),
4.14 (s, 3H) 3.97 (s, 3H) 3.88−3.93 (m, 1H) 3.36 (dt, J = 6.02, 3.01
Hz, 1H) 0.41−0.60 (m, 4H).
(3-(4,7-Dimethoxyquinoline-2-carboxamido)-5-(pyrrolidine-1-
carbonyl)phenyl)boronic Acid (25). Compound 25 was prepared
according to the general procedure for DMTMM amide. LC−MS m/
z: 514.3 [M + H]+. 1H NMR (700 MHz, DMSO-d6): δ ppm 10.71−
10.84 (m, 1H) 8.19 (m, 2H) 8.19−8.28 (m, 3H) 8.10−8.19 (m, 2H)
7.63 (s, 1H) 7.54−7.61 (m, 2H) 7.44 (m, 6H) 7.18−7.44 (m, 6H)
4.50−4.76 (m, 2H) 4.14 (s, 3H) 3.96 (s, 3H) 3.18−3.26 (m, 2H)
1.02−1.18 (m, 3H). 93% pure by LC−MS.
(3-(2,3-Dihydro-1H-pyrrolo[3,4-c]pyridine-2-carbonyl)-5-(4,7-di-
methoxyquinoline-2-carboxamido)phenyl)boronic Acid (26). Com-
pound 26 was prepared according to the general procedure for
DMTMM amide. LC−MS m/z: 499.2 [M + H]+. 1H NMR (700
MHz, DMSO-d6): δ ppm 10.82 (d, J = 2.58 Hz, 1H) 8.55−8.69 (m,
1H) 8.51 (dd, J = 17.85, 4.95 Hz, 1H) 8.34 (d, J = 1.29 Hz, 1H) 8.28
(br d, J = 8.17 Hz, 1H) 8.26 (br s, 2H) 8.12 (d, J = 9.03 Hz, 1H) 7.81
(d, J = 1.29 Hz, 1H) 7.59 (s, 1H) 7.57 (d, J = 2.15 Hz, 1H) 7.38−7.53
(m, 1H) 7.33 (dd, J = 9.04, 2.58 Hz, 1H) 4.88−5.01 (m, 4H) 4.14 (s,
3H) 3.96 (s, 3H). 91% pure by LC−MS.
(R)-(3-((tert-Butoxycarbonyl)amino)-5-((1-cyclohexyl-2-(methyl-
amino)-2-oxoethyl)carbamoyl)phenyl)boronic Acid (Intermediate
D). To a glass vial charged with 3-borono-5-((tert-butoxycarbonyl)-
amino)benzoic acid (528 mg, 1.88 mmol, 1 equiv) and HATU (786
mg, 2.07 mmol, 1.1 equiv) was added DMF (4.7 mL) followed by
DIPEA (982 μL, 5.64 mmol, 3 equiv). The premix solution was
allowed to react at room temperature for 15 min, then a solution of
(R)-2-amino-2-cyclohexyl-N-methylacetamide (320 mg, 1.88 mmol, 1
equiv) in DMF (4.7 mL) was added. The reaction mixture was
allowed to stir at room temperature for 2 h, then concentrated to
afford the crude product. The crude oil was purified by reverse-phase
chromatography to afford the desired product. LC−MS m/z: 434.09
[M + H]+. 1H NMR (MeOD-d4, 400 MHz): δ ppm 7.81 (s, 1H), 7.71
(br s, 1H), 7.63 (br s, 1H), 4.2−4.3 (m, 1H), 2.6−2.7 (m, 3H), 1.5−
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
1.8 (m, 8H), 1.4−1.5 (m, 1H), 1.43 (s, 9H), 1.34 (s, 1H), 1.0−1.2
(m, 7H).
(R)-(3-(6-Chloro-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-
carboxamido)-5-((1-cyclohexyl-2-(methylamino)-2-oxoethyl)-
carbamoyl)phenyl)boronic Acid (27). To a solution of (R)-(3-
amino-5-((1-cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
phenyl)boronic acid (59 mg, 0.18 mmol), in DCM (0.4 mL) and
ACN (0.4 mL), 6-chloro-3-oxo-3,4-dihydro-2H-benzo[b][1,4]-
oxazine-8-carboxylic acid (40 mg, 0.18 mmol) and 1-methyl-1H-
imidazole (98 μL, 1.2 mmol) were added, followed by TCFH (99 mg,
0.351 mmol). The reaction was stirred at RT for 1 h, then all volatiles
were removed in vacuo. The reaction mixture was purified via reverse-
phase chromatography to afford the desired product (64 mg, 49%).
LC−MS m/z: 543.2 [M + H]+. 1H NMR (400 MHz, methanol-d4): δ
ppm 8.1−8.3 (m, 2H), 8.1 (s, 1H), 7.8 (s, 1H), 7.5 (d, J = 2.25 Hz,
1H), 7.1 (d, J = 2.38 Hz, 1H), 4.8 (s, 2H), 4.3−4.4 (m, 1H), 2.8 (d, J
= 4.00 Hz, 3H), 1.8−1.9 (m, 2H), 1.7−1.8 (m, 2H), 1.6−1.7 (m,
2H), 1.2−1.4 (m, 5H), 1.0−1.2 (m, 3H).
(R)-(3-(6-Chloro-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-8-
carboxamido)-5-((1-cyclohexyl-2-(methylamino)-2-oxoethyl)-
carbamoyl)phenyl)boronic Acid (27). To a solution of (R)-(3-
amino-5-((1-cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
phenyl)boronic acid (59 mg, 0.18 mmol), in DCM (0.4 mL) and
ACN (0.4 mL), 6-chloro-3-oxo-3,4-dihydro-2H-benzo[b][1,4]-
oxazine-8-carboxylic acid (40 mg, 0.18 mmol) and 1-methyl-1H-
imidazole (98 μL, 1.2 mmol) were added, followed by TCFH (99 mg,
0.351 mmol). The reaction was stirred at RT for 1 h, then all volatiles
were removed in vacuo. The reaction mixture was purified via reverse-
phase chromatography to afford the desired product (64 mg, 49%).
LC−MS m/z: 543.2 [M + H]+. 1H NMR (400 MHz, MeOD-d4): δ
ppm 1.00−1.19 (m, 3H) 1.22−1.38 (m, 5H) 1.63−1.73 (m, 2H)
1.75−1.82 (m, 2H) 1.83−1.91 (m, 2H) 2.76 (d, J = 4.00 Hz, 3H)
4.30−4.38 (m, 1H) 4.80 (s, 2H) 7.08 (d, J = 2.38 Hz, 1H) 7.45 (d, J
= 2.25 Hz, 1H) 7.84 (s, 1H) 8.05 (s, 1H) 8.12−8.28 (m, 2H). 100%
pure by LC−MS.
(3-(((R)-1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(2-(2,6-dimethylphenoxy)propanamido)phenyl)boronic Acid
(28). To a solution of (R)-(3-amino-5-((1-cyclohexyl-2-(methylami-
no)-2-oxoethyl)carbamoyl)phenyl)boronic acid (30 mg, 0.09 mmol),
in DCM (0.2 mL) and ACN (0.2 mL), and 2-(2,6-dimethylphenoxy)-
propanoic acid (17.5 mg, 0.090 mmol) and 1-methyl-1H-imidazole
(50 μL, 0.6 mmol) were added, followed by TCFH (51 mg, 0.18
mmol). The reaction was stirred at RT for 2 h, then water was added.
The aqueous layer was extracted with DCM three times, the organic
layers were combined, washed with brine, the dried over magnesium
sulfate, filtered, and volatiles removed in vacuo. The reaction mixture
was purified via reverse-phase chromatography to afford the desired
product (11 mg, 25%). LC−MS m/z: 510.3 [M + H1H NMR (400
MHz, MeOD-d4): δ ppm 1.04−1.15 (m, 2H) 1.17−1.36 (m, 5H)
1.55 (d, J = 6.63 Hz, 3H) 1.64−1.71 (m, 2H) 1.73−1.82 (m, 2H)
1.82−1.91 (m, 2H) 2.32 (s, 6H) 2.75 (s, 3H) 3.21 (dd, J = 14.76,
7.25 Hz, 1H) 4.33 (d, J = 8.38 Hz, 1H) 4.55 (dd, J = 13.38, 6.63 Hz,
1H) 6.94 (t, J = 7.50 Hz, 1H) 7.04 (d, J = 7.63 Hz, 2H) 7.83 (s, 1H)
8.07 (d, J = 14.13 Hz, 2H).
(R)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(2-methoxy-3,5-dimethylbenzamido)phenyl)boronic Acid (29).
Compound 29 was prepared according to the HATU amide coupling
procedure to give the desired product (7.5 mg, 17%). LC−MS m/z:
496.25 [M + H]+. 1H NMR (400 MHz, MeOD-d4): δ ppm 8.18−7.84
(m, 3H), 7.42 (s, 1H), 7.22 (s, 1H), 4.35 (d, 1H, J = 8.3 Hz), 3.81 (s,
3H), 2.76 (s, 3H), 2.33 (s, 6H), 1.90−1.67 (m, 6H), 1.37−1.06 (m,
5H).
(R)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(4-methoxypicolinamido)phenyl)boronic Acid (30). To a solution
of (R)-(3-amino-5-((1-cyclohexyl-2-(methylamino)-2-oxoethyl)-
carbamoyl)phenyl)boronic acid (50 mg, 0.15 mmol), 4-methoxypi-
colinic acid (23 mg, 0.15 mmol), 1-methyl-1H-imidazole (84 μL, 1.1
mmol) in DCM (0.4 mL), and acetonitrile (0.4 mL) was added
TCFH (84 mg, 0.300 mmol). The reaction was stirred for 2 h at room
temperature, then water was added, and the aqueous layer was
extracted with DCM three times. The organic layers were combined,
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washed with brine, dried over magnesium sulfate, filtered, and
volatiles removed in vacuo. The reaction mixture purified via reverse-
phase chromatography to afford the desired product (8.6 mg, 12%).
LC−MS m/z: 469.3 [M + H]+. 1H NMR (400 MHz, MeOD-d4): δ
ppm 1.04−1.17 (m, 2H) 1.20−1.38 (m, 4H) 1.63−1.73 (m, 2H)
1.74−1.82 (m, 2H) 1.83−1.92 (m, 2H) 2.77 (s, 3H) 4.07 (s, 3H)
4.34 (d, J = 8.51 Hz, 1H) 7.35 (d, J = 4.88 Hz, 1H) 7.87 (s, 1H) 7.96
(s, 1H) 8.20 (s, 1H) 8.28 (s, 1H) 8.60 (d, J = 5.63 Hz, 1H).
(R)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(5-methoxy-2-methylpyrimidine-4-carboxamido)phenyl)boronic
Acid, 2,2,2-Trifluoroacetic Acid Compound (1:1) (31). To a solution
of (R)-(3-((tert-butoxycarbonyl)amino)-5-((1-cyclohexyl-2-(methyla-
mino)-2-oxoethyl)carbamoyl)phenyl)boronic acid (28 mg, 0.065
mmol) in dioxane (1 mL) was added 4 N HCl in dioxane (1 mL).
The reaction mixture was stirred at room temperature for 18 h, then
diluted with ether (10 mL). The reaction mixture was centrifuged, the
supernatant was decanted, and the resulting solid was dried to afford
the crude intermediate. The intermediate was dissolved in a mixture
of acetonitrile (2 mL)/DMF (0.5 mL), and 5-methoxy-2-methylpyr-
imidine-4-carboxylic acid (16.30 mg, 0.097 mmol), 1-methyl-1H-
imidazole (26.5 mg, 0.323 mmol), and TCFH (53.6 mg, 0.097 mmol)
were added successively. The reaction mixture was stirred at room
temperature for 18 h, concentrated to dryness, and the crude was
purified by reverse-phase chromatography to afford the desired
product (18 mg, 44% yield). LC−MS m/z: 484.17 [M + H]+. 1H
NMR (400 MHz, DMSO-d6): δ ppm 10.63 (s, 1H), 8.71 (s, 1H),
8.17−8.13 (m, 2H), 8.11−7.97 (m, 3H), 4.4−4.18 (m, 1H), 3.94 (s,
3H), 2.62 (s, 3H), 2.6 (d, J = 8.0 Hz, 3H), 1.80−1.65 (m, 4H), 1.65−
1.51 (m, 2H), 1.24−1.10 (m, 3H), 1.10−0.95 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(4-methoxyquinoline-2-carboxamido)phenyl)boronic Acid (32).
Compound 32 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 519.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.77 (s, 1H) 8.41 (t, 1H, J = 1.9 Hz) 8.38
(s, 1H) 8.25 (d, 2H, J = 7.3 Hz) 8.21 (d, 1H, J = 8.2 Hz) 8.08−8.03
(m, 3H) 7.91 (t, 1H, J = 7.9 Hz) 7.74−7.71 (m, 2H) 4.35 (t, 1H, J =
8.4 Hz) 4.19 (s, 3H) 2.62 (d, 3H, J = 2.0 Hz) 1.80−1.75 (m, 2H)
1.74−1.69 (m, 2H) 1.64−1.56 (m, 2H) 1.22−1.11 (m, 3H) 1.11−
0.98 (m, 2H). 87% pure by LCMS.
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(4,6-dimethoxyquinoline-2-carboxamido)phenyl)boronic Acid
(33). Compound 33 was prepared according to the general procedure
for TCFH couplings. LC−MS m/z: 549.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.70 (s, 1H) 8.40 (s, 1H) 8.37 (s, 1H)
8.23 (s, 2H) 8.12 (d, 2H, J = 9.0 Hz) 8.07−8.05 (m, 2H) 8.03 (d, 1H,
J = 8.6 Hz) 7.70 (s, 1H) 7.55−7.53 (m, 1H) 7.50 (d, 1H, J = 3.0 Hz)
4.35−4.33 (m, 1H) 4.18 (s, 3H) 3.95 (s, 3H) 2.63−2.62 (m, 3H)
2.53−2.52 (m, 3H) 1.80−1.75 (m, 2H) 1.73−1.69 (m, 2H) 1.17−
1.14 (m, 3H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(6-methoxyquinoline-2-carboxamido)phenyl)boronic Acid (34).
Compound 34 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 519.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.7 (s, 1H) 8.5 (d, J = 8.2 Hz, 1H) 8.40
(m, 2H) 8.2 (m, 3H) 8.2 (d, J = 9.5 Hz, 1H) 8.0−8.1 (m, 3H) 7.5−
7.6 (m, 1H) 7.5 (d, J = 3.0 Hz, 1H) 4.0 (s, 3H) 2.6 (m, 3H) 2.5 (m,
2H) 1.8 (m, 2H) 1.7 (m, 2H) 1.5−1.6 (m, 3H) 1.1−1.2 (m, 3H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(4-methylquinoline-2-carboxamido)phenyl)boronic Acid (35).
Compound 35 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 503.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.77 (s, 1H) 8.40 (d, 2H, J = 10.9 Hz)
8.27 (d, 1H, J = 7.8 Hz) 8.25−8.23 (m, 3H) 8.14 (s, 1H) 8.08−8.03
(m, 3H) 7.94−7.91 (m, 1H) 7.79 (t, 1H, J = 7.8 Hz) 4.34 (t, 1H, J =
8.4 Hz) 2.84 (d, 3H, J = 0.9 Hz) 2.63−2.62 (m, 3H) 2.53−2.52 (m,
1H) 1.80−1.75 (m, 2H) 1.74−1.69 (m, 2H) 1.61−1.57 (m, 1H)
1.22−1.11 (m, 3H) 1.09−0.98 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(5-methyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-
7-carboxamido)phenyl)boronic Acid (36). Compound 36 was
prepared according to the general procedure for TCFH couplings.
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
LC−MS m/z: 537.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6): δ
ppm 11.74 (s, 1H) 11.44 (s, 1H) 10.32 (s, 1H) 8.20 (s, 2H) 8.12 (d,
1H, J = 2.2 Hz) 8.09 (s, 1H) 8.06−8.03 (m, 1H) 8.00 (s, 1H) 7.96 (d,
1H, J = 8.6 Hz) 7.14 (s, 1H) 4.31 (t, 1H, J = 8.6 Hz) 2.61 (d, 3H, J =
4.7 Hz) 2.54 (s, 3H) 1.77−1.74 (m, 2H) 1.71−1.68 (m, 2H) 1.63−
1.53 (m, 2H) 1.20−1.11 (m, 3H) 1.08−0.94 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(quinoline-2-carboxamido)phenyl)boronic Acid (37). Compound
37 was prepared according to the general procedure for TCFH
couplings. LC−MS m/z: 489.2 [M + H]+. 1H NMR (400 MHz,
DMSO-d6): δ ppm 10.81 (s, 1H) 8.66 (d, 1H, J = 8.6 Hz) 8.41 (d,
2H, J = 2.6 Hz) 8.28 (s, 1H) 8.27 (d, 1H, J = 3.0 Hz) 8.24 (s, 2H)
8.14 (d, 1H, J = 7.8 Hz) 8.08−8.04 (m, 3H) 7.94 (t, 1H, J = 7.9 Hz)
7.79−7.77 (m, 1H) 4.35 (t, 1H, J = 8.4 Hz) 3.41 (s, 1H) 2.53−2.52
(m, 3H) 1.80−1.75 (m, 2H) 1.69−1.73 (m, 2H) 1.63−1.47 (m, 2H)
1.21−1.16 (m, 2H) 1.16−1.15 (s, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(isoquinoline-3-carboxamido)phenyl)boronic Acid (38). Com-
pound 38 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 489.2 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.77 (s, 1H) 9.49 (s, 1H) 8.74−8.71 (m,
1H), 8.39 (d, 2H, J = 9.5 Hz) 8.33 (d, 1H, J = 7.7 Hz) 8.28 (d, 2H, J
= 7.7 Hz) 8.07−8.02 (m, 3H) 7.95−7.92 (m, 1H) 7.89−7.86 (m, 1H)
4.34 (t, 1H, J = 8.4 Hz) 2.63−2.61 (m, 3H) 2.53−2.52 (m, 1H)
1.80−1.76 (m, 2H) 1.74−1.69 (m, 3H) 1.64−1.57 (m, 2H) 1.22−
1.11 (m, 3H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(quinoline-3-carboxamido)phenyl)boronic Acid (39). Compound
39 was prepared according to the general procedure for TCFH
couplings. LC−MS m/z: 489.2 [M + H]+. 1H NMR (400 MHz,
DMSO-d6): δ ppm 10.70 (s, 1H) 9.40 (d, 1H, J = 2.2 Hz) 9.02 (d,
1H, J = 2.2 Hz) 8.31 (d, 2H, J = 6.0 Hz) 8.25−8.21 (m, 1H) 8.21−
8.12 (m, 3H) 8.08−8.06 (m, 2H) 8.01 (d, 1H, J = 9.0 Hz) 7.94−7.90
(m, 1H) 7.75 (t, 1H, J = 7.6 Hz) 4.35 (t, 1H, J = 8.4 Hz) 2.62 (d, 3H,
J = 4.8 Hz) 1.80−1.69 (m, 4H) 1.65−1.55 (m, 2H) 1.22−1.11 (m,
3H) 1.10−0.98 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(isoquinoline-1-carboxamido)phenyl)boronic Acid (40). Com-
pound 40 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 489.2 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.86 (s, 1H) 8.87 (d, 1H, J = 8.7 Hz) 8.64
(d, 1H, J = 5.6 Hz) 8.36 (s, 1H) 8.32 (t, 1H, J = 1.9 Hz) 8.23 (s, 2H)
8.13−8.08 (m, 2H) 8.07−8.03 (m, 3H) 7.89−7.86 (t, 1H, J = 7.8 Hz)
7.80−7.77 (m, 1H) 4.33 (t, 1H, J = 8.4 Hz) 2.62 (d, 3H, J = 4.7 Hz)
1.80−1.75 (m, 2H) 1.73−1.69 (m, 2H) 1.65−1.55 (m, 2H) 1.22−
1.10 (m, 3H) 1.09−0.98 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(1,5-naphthyridine-2-carboxamido)phenyl)boronic Acid (41).
Compound 41 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 490.2 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.85 (s, 1H) 9.16 (s, 1H) 8.69 (d, 1H, J =
8.6 Hz) 8.65 (d, 1H, J = 8.2 Hz) 8.49 (d, 1H, J = 8.6 Hz) 8.40 (s, 2H)
8.33−8.19 (m, 2H) 8.08 (s, 1H) 8.07 (d, 1H, J = 6.0 Hz) 8.04 (d, 1H,
J = 8.6 Hz) 7.97−7.95 (m, 1H) 4.35 (t, 1H, J = 8.6 Hz) 2.63−2.62
(m, 3H) 1.80−1.75 (m, 2H) 1.73−1.69 (m, 2H) 1.63−1.57 (m, 2H)
1.22−1.11 (m, 3H) 1.09−0.98 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(1,7-naphthyridine-2-carboxamido)phenyl)boronic Acid (42).
Compound 42 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 490.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.90 (s, 1H) 9.63 (s, 1H) 8.76 (d, 1H, J =
5.6 Hz) 8.73 (d, 1H, J = 8.6 Hz) 8.48 (d, 1H, J = 8.2 Hz) 8.41 (d, 2H,
J = 0.9 Hz) 8.25 (s, 2H) 8.09−8.06 (m, 3H) 8.04 (d, 1H, J = 9.0 Hz)
4.35 (s, 1H) 2.53−2.52 (m, 3H) 1.82−1.75 (m, 2H) 1.75−1.68 (m,
2H) 1.65−1.55 (m, 2H) 1.16−1.14 (m, 3H) 1.10−0.97 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(1,8-naphthyridine-2-carboxamido)phenyl)boronic Acid (43).
Compound 43 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 490.2 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.86 (s, 1H) 9.28−9.26 (m, 1H) 8.81−
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8.75 (m, 1H) 8.65 (d, 1H, J = 8.1 Hz) 8.47−8.34 (m, 3H) 8.10−8.02
(m, 3H) 7.83−7.76 (m, 1H) 4.36−4.29 (m, 1H) 2.64−2.61 (m, 3H)
1.82−1.76 (m, 2H) 1.75−1.69 (m, 2H) 1.67−1.54 (m, 2H) 1.24−
1.12 (m, 3H) 1.11−0.95 (m, 2H). 87% pure by LCMS.
(S)-(3-(2-Naphthamido)-5-((1-cyclohexyl-2-(methylamino)-2-
oxoethyl)carbamoyl)phenyl)boronic Acid (44). Compound 44 was
prepared according to the general procedure for TCFH couplings.
LC−MS m/z: 488.2 [M + H]+. 1H NMR (400 MHz, DMSO-d6): δ
ppm 10.5 (s, 1H) 8.6 (s, 1H) 8.3 (s, 2H) 8.2 (s, 2H) 8.1 (d, J = 9.9
Hz, 1H) 8.0−8.1 (m, 5H) 8.0 (d, J = 8.6 Hz, 1H) 7.6−7.7 (m, 2H)
4.3 (t, J = 8.4 Hz, 1H) 2.6 (m, 3H) 1.7−1.8 (m, 4H) 1.6 (m, 2H)
1.1−1.2 (m, 3H) 1.0−1.1 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(6-methoxy-2-naphthamido)phenyl)boronic Acid (45). Com-
pound 45 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 518.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.42 (s, 1H) 8.57 (s, 1H) 8.30−8.28 (m,
2H) 8.21 (s, 2H) 8.08−8.02 (m, 3H) 7.98 (t, 2H, J = 9.7 Hz) 7.94 (d,
1H, J = 8.6 Hz) 7.43 (d, 1H, J = 2.6 Hz) 7.29−7.27 (m, 1H) 4.34 (t,
1H, J = 8.6 Hz) 3.9 (s, 3H) 2.63−2.60 (m, 3H) 1.79−1.75 (m, 2H)
1.72−1.69 (m, 2H) 1.63−1.57 (m, 2H) 1.21−1.11 (m, 3H) 1.08−
0.98 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(7-methoxy-2-naphthamido)phenyl)boronic Acid (46). Com-
pound 46 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 518.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 10.47 (s, 1H) 8.52 (s, 1H) 8.30 (d, 2H, J =
0.9 Hz) 8.21 (s, 2H) 8.08−8.05 (m, 1H) 8.04 (s, 1H) 7.99−7.97 (m,
2H) 7.94 (d, 1H, J = 8.6 Hz) 7.91−7.89 (m, 1H) 7.47 (d, 1H, J = 2.6
Hz) 7.31−7.29 (m, 1H) 4.34 (t, 1H, J = 8.4 Hz) 3.92 (s, 3H) 2.63−
2.61 (m, 3H) 2.53−2.52 (m, 1H) 1.79−1.70 (m, 4H) 1.61−1.56 (m,
1H) 1.22−1.10 (m, 3H) 1.08−0.97 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(7-fluoroquinoxaline-5-carboxamido)phenyl)boronic Acid (47).
Compound 47 was prepared according to the general procedure for
TCFH couplings. LC−MS m/z: 508.3 [M + H]+. 1H NMR (400
MHz, DMSO-d6): δ ppm 11.59 (s, 1H) 9.14 (d, 1H, J = 1.7 Hz) 9.12
(d, 1H, J = 1.7 Hz) 8.32 (s, 1H) 8.31 (d, 1H, J = 9.3 Hz) 8.26 (s, 2H)
8.22 (d, 1H, J = 1.3 Hz) 8.16−8.13 (m, 1H) 8.07−8.04 (m, 3H) 4.33
(t, 1H, J = 8.4 Hz) 2.62−2.61 (m, 3H) 1.79−1.74 (m, 2H) 1.73−1.67
(m, 2H) 1.63−1.56 (m, 2H) 1.21−1.11 (m, 3H) 1.09−0.97 (m, 2H).
(S)-(3-((1-Cyclohexyl-2-(methylamino)-2-oxoethyl)carbamoyl)-
5-(8-methoxy-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-
carboxamido)phenyl)boronic Acid (48). To a vial containing 8-
methoxy-3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazine-6-carboxylic
acid (72 mg, 0.323 mmol) and HATU (123 mg, 0.324 mmol) were
added DMF (2 mL) and DIPEA (0.154 mL, 0.882 mmol). The
reaction was allowed to stir at room temperature for 5 min. After this
time, a solution of (S)-(3-amino-5-((1-cyclohexyl-2-(methylamino)-
2-oxoethyl)carbamoyl)phenyl)boronic acid (98 mg, 0.294 mmol) in
DMF (2 mL) was added, and the reaction was allowed to stir at room
temperature for 18 h. The reaction was concentrated to dryness, and
the compound was isolated via reverse-phase chromatography to
afford the desired product (93 mg, 59%). LC−MS m/z: 539.17 [M +
H]+. 1H NMR (400 MHz, DMSO-d6): δ ppm 10.87 (s, 1H,
exchangeable), 10.22 (s, 1H, exchangeable), 8.26−8.19 (m, 3H),
8.08−7.96 (m, 3H), 7.3−7.4 (s, 1H), 7.2−7.1 (s, 1H), 4.63 (s, 3H),
4.3−4.4 (m, 1H), 3.89 (m, 4H), 2.61−2.60 (m, 2H), 1.81−1.51 (m,
5H), 1.29−0.94 (m, 5H). One exchangeable proton not observed,
likely under solvent peak.
■
*
ASSOCIATED CONTENT
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c01562.
Experimental details, data for compound character-
ization, protein preparation, and in vitro data and
crystallography parameters (PDF)
Formula strings (CSV)
J. Med. Chem. XXXX, XXX, XXX−XXX
Journal of Medicinal Chemistry
Accession Codes
PDB ID Codes: 8SYC.
■ AUTHOR INFORMATION
Corresponding Authors
Ann M. Rowley − GSK, Collegeville, Pennsylvania 19426,
United States; orcid.org/0000-0002-3857-9447;
Email: Ann.X.Rowley@gsk.com
Gang Yao − GSK, Encoded Library Technologies, NCE
Molecular Discovery, Cambridge, Massachusetts 02140,
United States; Email: Gang.X.Yao@gsk.com
Authors
Logan Andrews − 23andMe Inc, Therapeutics, South San
Francisco, California 94080, United States; Present
Address: Animol Discovery, One Boston Place, Suite
3930, 201 Washington St., Boston, MA 02108, United
States; orcid.org/0000-0002-5092-4530
Aaron Bedermann − GSK, Collegeville, Pennsylvania 19426,
United States; Present Address: Janssen Pharmaceuticals,
1400 McKean Rd, Springhouse, PA 19477, United
States.; orcid.org/0009-0003-3168-2083
Ross Biddulph − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Ryan Bingham − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Jennifer J. Brady − 23andMe Inc, Therapeutics, South San
Francisco, California 94080, United States; orcid.org/
0009-0000-1794-0494
Rachel Buxton − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Ted Cecconie − GSK, Collegeville, Pennsylvania 19426,
United States
Rona Cooper − GSK, Collegeville, Pennsylvania 19426,
United States
Adam Csakai − GSK, Encoded Library Technologies, NCE
Molecular Discovery, Cambridge, Massachusetts 02140,
United States
Enoch N. Gao − GSK, Collegeville, Pennsylvania 19426,
United States
Melissa C. Grenier-Davies − GSK, Encoded Library
Technologies, NCE Molecular Discovery, Cambridge,
Massachusetts 02140, United States
Meghan Lawler − GSK, Encoded Library Technologies, NCE
Molecular Discovery, Cambridge, Massachusetts 02140,
United States; Present Address: Anagenex, 20 Maguire Rd.
Suite 302, Lexington, MA 02421, United States.
Yiqian Lian − GSK, Collegeville, Pennsylvania 19426, United
States; orcid.org/0000-0002-2163-0189
Justyna Macina − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Colin Macphee − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Lisa Marcaurelle − GSK, Encoded Library Technologies, NCE
Molecular Discovery, Cambridge, Massachusetts 02140,
United States
John Martin − GSK, Collegeville, Pennsylvania 19426, United
States
Patricia McCormick − GSK, Collegeville, Pennsylvania
19426, United States
Rekha Pindoria − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
P https://doi.org/10.1021/acs.jmedchem.3c01562
pubs.acs.org/jmc Article
Martin Rauch − GSK, Collegeville, Pennsylvania 19426,
United States
Warren Rocque − GSK, Collegeville, Pennsylvania 19426,
United States
Yingnian Shen − GSK, Collegeville, Pennsylvania 19426,
United States
Lisa M. Shewchuk − GSK, Collegeville, Pennsylvania 19426,
United States
Michael Squire − GSK, Collegeville, Pennsylvania 19426,
United States
Will Stebbeds − GSK Medicines Research Centre, Stevenage
SG1 2NY, U.K.
Westley Tear − GSK, Encoded Library Technologies, NCE
Molecular Discovery, Cambridge, Massachusetts 02140,
United States
Xin Wang − 23andMe Inc, Therapeutics, South San Francisco,
California 94080, United States; orcid.org/0000-0001-
7242-357X
Paris Ward − GSK, Collegeville, Pennsylvania 19426, United
States
Shouhua Xiao − 23andMe Inc, Therapeutics, South San
Francisco, California 94080, United States; Present
Address: RA Capital Management, 200 Berkeley St., 18th
Floor, Boston, MA 02116, United States.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.3c01562
Author Contributions
The manuscript was written through contributions of all
authors. These authors contributed equally: Logan Andrews,
Aaron Bedermann, Ross Biddulph, Ryan Bingham, Jennifer J.
Brady, Rachel Buxton, Ted Cecconie, Rona Cooper, Adam
Csakai, Enoch N. Gao, Melissa C. Grenier-Davies, Meghan
Lawler, Yiqian Lian, Justyna Macina, Colin Macphee, Lisa
Marcaurelle, John Martin, Patricia McCormick, Rekha
Pindoria, Martin Rauch, Warren Rocque, Yingnian Shen, Lisa
M. Shewchuk, Michael Squire, Will Stebbeds, Westley Tear,
Xin Wang, Paris Ward, and Shouhua Xiao.
Notes
The authors declare no competing financial interest.
■ ABBREVIATIONS
BOP, benzotriazol-1-yloxytris(dimethylamino)phosphonium
hexafluorophosphate; DIPEA, diisopropylethylamine;
DMTMM, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-mor-
pholinium chloride; HATU, hexafluorophosphate azabenzo-
triazole tetramethyl uronium; IMAP, immobilized metal
affinity for phosphochemicals; RF-MS, rapid fire mass
spectrometry; TCFH, chloro-N,N,N′,N′-tetramethylformami-
dinium hexafluorophosphate
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