Catalyst-Free and Scalable Process for Synthesis of Novel MAP4K4 Inhibitor DMX-5804 and Its Glyco-Conjugates

Article abstract of DOI:10.1021/acs.oprd.1c00130

DMX-5804 is a potent and selective mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) inhibitor, which is currently under evaluation for the treatment of myocardial infarction. Here, we report the process development of scalable and practica

Full text of DOI:10.1021/acs.oprd.1c00130

pubs.acs.org/OPRD
Article
Catalyst-Free and Scalable Process for Synthesis of Novel MAP4K4
Inhibitor DMX-5804 and Its Glyco-Conjugates
Venkateswararao Eeda* and Vibhudutta Awasthi*
Cite This: Org. Process Res. Dev. 2021, 25, 1658−1663
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: DMX-5804 is a potent and selective mitogen-activated protein kinase kinase kinase kinase-4 (MAP4K4) inhibitor,
which is currently under evaluation for the treatment of myocardial infarction. Here, we report the process development of scalable
and practical synthesis of DMX-5804. Process optimization resulted in the following: (1) removal of transition metals from the
process and reduced duration of reactions, (2) a streamlined process with significantly improved yields using low-cost raw materials,
(3) importantly, microwave-assisted reactions were removed, (4) column purification avoided throughout the process, and (5)
crystallized products showed over 95% purity in each step.
KEYWORDS: process development, MAP4K4 inhibitor, DMX-5804, glyco-conjugates
human stem cell-derived cardiomyocytes as a platform for target
validation and compound development, the ICL group
identified two potent inhibitors of MAP4K4 (Figure 1) viz.,
INTRODUCTION
■
Mitogen-activated protein kinase kinase kinase kinase-4
(MAP4K4), also known as HGK (hematopoietic progenitor
kinase/germinal center kinase-like kinase) or NIK (Nck
interacting kinase, the mouse ortholog), is a serine/threonine
kinase. It plays an essential role in signal transduction by
modulating gene transcription in the nucleus in response to
changes in the cellular environment. It belongs to the
mammalian family of Ste20 protein kinases, with a molecular
target of TGFβ-activated kinase-1 (TAK1 or MAP3K7) which is
a key activator of c-Jun N-terminal kinase (JNK) pathway.
Recent work on MAP4K4 has projected it as a novel and
promising drug development target in cancer.1,2 Because of its
relatively high expression in the brain and the crucial role of
downstream p38 MAPK and JNK signaling in apoptosis, a few
studies have also reported the beneficial role of MAP4K4
inhibition in neuronal recovery post-injury.3,4 Our interest in
MAP4K4 signaling originates from its recently reported
participation in myocardial injury.5 Myocardial MAP4K4 is
activated in end-stage heart failure regardless of cause, that is,
dilation, hypertrophism, ischemia, and anthracycline-induced
cardiomyopathy.6 Moreover, MAP4K4 also plays a defining role
in pathologies that are precursors to myocardial infarction; for
example, MAP4K4 expression and/or activity is increased in the
aortas of mice and humans with atherosclerosis, and reduction of
endothelial MAP4K4 expression ameliorated atherosclerotic
lesion development and inflammatory signaling in a mouse
model.7
Figure 1. Selective small-molecule inhibitors of MAP4K4.
5,7-diphenyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one
(F1386-0303, IC50 34 nm) and 5-(4-(2-methoxyethoxy)-
phenyl)-7-phenyl-3,4a,7,7a-tetrahydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (DMX-5804, IC50 3 nm), the latter showing
significant reduction of infarct size in a mouse model.8 DMX-
5804 also exhibited better bioavailability in vivo as compared to
F1386-0303.
The discovery synthesis of DMX-5804 reported by the ICL
group was straightforward in terms of bond construction
Given that MAP4K4 was only recently recognized for its
regulatory role in myocardial injury and tissue recovery, research
on the development of its small-molecule inhibitors is relatively
nascent. Researchers from Imperial College of London (ICL,
London, UK) demonstrated that oxidative stress-induced
cardiomyocyte death requires MAP4K4 activation and its
pharmacologic inhibition by a novel molecule DMX-5804
reduces cardiomyocyte death both in vitro and in vivo.8 Using
Received: April 15, 2021
Published: July 8, 2021
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663
© 2021 American Chemical Society
1658

Organic Process Research & Development
pubs.acs.org/OPRD
Article
strategy.8 However, there is significant room for improvement in
reaction conditions and process optimization to enable large-
scale production. Gram to kilogram quantities of this new
chemical entity will be needed for pre-clinical and clinical
studies. In this article, we describe a process development that
resulted in an easy, practical, and scalable procedure for the
synthesis of crystalline DMX-5804 in excellent yields.
Compared to the discovery synthesis method, the reported
method eliminates the need for transition-metal catalysts,
anhydrous solvents, commercially unavailable borate inter-
mediate, and the microwave-assisted reaction step. Moreover,
our method is not dependent on column purification of
intermediates and high-performance liquid chromatography
(HPLC) purification of the final product which are required
steps in the discovery method. The new method affords
quantitative yields of DMX-5804 in a reproducible manner and
provides an easy three-step route to create a novel library of
compounds of the MAP4K4 inhibitor class.
d]pyrimidine (1) in 33% yield.8 The chloropyrimidine
derivative was converted to 5-iodo-7-phenyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (2) by refluxing in acetic acid in
the presence of sodium acetate for 15 h, reportedly with 97%
yield. Compound 2 was coupled with 2-(4-(2-methoxyethoxy)-
phenyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolan by using micro-
wave-assisted Suzuki−Miyaura cross-coupling reaction at 120
°C for 3 h in the presence of Pd(dppf)Cl2. The resultant DMX-
5804 was reported at an 18% yield. The overall yield of this
discovery method was reported as 22%. Importantly, all stages
required purification by preparative HPLC.
In our lab, we tried to reproduce this route to synthesize
DMX-5804; however, we faced several obstacles in the process.
First, we could not find any commercial source for 2-phenyl-1,
3,2-dioxoborinone, and no reference was available to make this
intermediate. To overcome this issue, we performed the stage 1
reaction with phenylboronic acid as a substitute for phenyl
dioxoborinone and used copper(II) acetate monohydrate in
DMF at 60 °C. However, this strategy yielded only trace
amounts of the product even after 24 h. Arnold et al. reported the
same reaction for the synthesis of 1 using phenylboronic acid in
dichloromethane, but the reaction duration was 12 d at room
temperature, which is time-consuming and not meant for
commercial production purposes.9 With trace amounts of 1 in
hand, we proceeded further to synthesize 2 using the conditions
reported in the discovery method. This reaction proceeded
smoothly and afforded excellent yields of 2 (>90% yield).
However, the microwave reaction in stage iii (Scheme 1) to
synthesize DMX-5804 was again challenging. We observed
significant charring, and the desired compound was in trace
amounts (<5%). We tried this reaction without microwave
assistance, but no product formation occurred. It is noteworthy
that we used a conventionally available kitchen microwave for
this reaction, which might have significant bearing on the way
the reaction proceeded. Regardless, the reaction appears to
require substantial energy input to drive this reaction forward.
Based on these experiences, we decided to establish a de novo
synthetic route for DMX-5804.
RESULTS AND DISCUSSION
■
Discovery Synthetic Route to DMX-5804.8 Synthesis of
DMX-5804 via the discovery route is shown in Scheme 1. The
a
Scheme 1. Discovery Route for Synthesis of DMX-5804
a
Reagents and conditions: (i) phenylboronic acid, cupric acetate,
dimethylformamide, 60 °C, 15−33%; (ii) Na-acetate, acetic acid, 100
°C, 15 h; (iii) 2-(4-(2-methoxyethoxy)phenyl-4,4,5,5-tetramethyl-
1,3,2-dioxaborolan, Pd(dppf)Cl2, potassium carbonate, microwave
reactor, 120 °C, 18% yield.
De Novo Synthesis of DMX-5804. The discovery synthetic
route is capable of supplying medicinal chemists with quantities
of DMX-5804 sufficient for initial in vitro and in vivo studies;
however, it is apparent that this route needs significant
modifications to obtain meaningful amounts of DMX-5804 in
a reproducible manner. We identified the following drawbacks of
the discovery method, which make it very challenging, if not
impossible, to translate to a commercial scale: (a) the
researchers used Chan−Lam-like amination with excessive
copper(II) acetate monohydrate and 2-phenyl-1, 3,2-dioxobor-
inone (commercially not available and no synthetic scheme
reported) at 60 °C for 24 h. Following the required work-up, the
crude compound was purified by reverse-phase preparative
HPLC-MS to afford 4-chloro-5-iodo-7-phenyl-7H-pyrrolo[2,3-
a
Scheme 2. New Strategy for Synthesis of DMX-5804
a
Reagents and conditions: (i) NaHCO3, DMF, 50 °C (ii) malononitrile, KOH, methanol reflux; (iii) 95% formic acid, reflux.
1659
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663

Organic Process Research & Development
pubs.acs.org/OPRD
Article
requirement of transition-metal catalysts, (b) high cost of
boronate intermediate, (c) microwave-assisted reaction, (d)
need of preparative HPLC for purification of intermediates and
the final product, (e) poor yields with reproducibility challenges
in cross-coupling reactions, and (f) safety concerns in discarding
metallic complexes.
Because of the high cost and inaccessibility of boronates in
stage 1, we sought to start synthesis from readily available and
inexpensive starting materials. As shown in Scheme 2, we
identified substituted 2-bromo-1-(4-(2-methoxyethoxy)-
phenyl)ethan-1-one (3a) as a suitable starting material for
DMX-5804. It was reacted with aniline in DMF at room
temperature in the presence of a mild base for 3 h to produce 1-
(4-(2-methoxyethoxy)phenyl)-2-(phenylamino)ethan-1-one
(4) in quantitative yields (90%). The product was recrystallized
in isopropyl alcohol. In the next step, a pyrrole derivative (5) was
synthesized via Knoevenagel condensation of malononitrile. We
screened diisopropylethylamine, trimethylamine, KOH, and
potassium-tert-butoxide as bases for this reaction. The reaction
with KOH provided >95% of 5, with 80−86% isolated yield; the
other bases yielded approximately 70% conversion.
With 2-amino-pyrrole-3-carbonitrile derivative (5) in hand,
we turned our focus to the assembly of the pyrimidine ring in the
structure of DMX-5804. Compound 5 was refluxed in 95%
formic acid for 8−10 h. After cooling, the reaction mixture was
poured in ice-water to precipitate the crude compound. The
crude compound was dissolved in a mixture of chloroform/
methanol (1:5), decolorized with activated charcoal, and
recrystallized from chloroform/methanol mixture to obtain
DMX-5804 with 99% purity as analyzed by HPLC (Figure 2). It
First, we attempted base-catalyzed O-alkylation in a variety of
conditions (Table 1). Initially, we performed the reaction in
acetonitrile using potassium carbonate at 60 °C for 12 h, which
resulted in only trace amounts of DMX-5804 but approximately
20% compound 7 (pyrimidine O-alkylation) and unreacted
starting material. We also screened various solvents combined
with different bases to get DMX-5804 but failed. The reaction
involving potassium carbonate in DMF resulted in the
quantitative consumption of the starting material. However,
DMX-5804 was obtained only in small amounts (5−7%),
whereas compound 7 was isolated as the major product (88%).
To check the stability of compound 7, we refluxed it in dioxane−
HCl for 6 h. Compound 7 was stable in acidic conditions, and its
solubility in polar solvents was significantly better than that of
DMX-5804. Also, its HPLC retention time was identical to that
of DMX-5804 (data not shown).
In order to shift alkylation to the desired phenolic site, we
changed the reaction conditions to acidic by using boron
trifluoride diethyl etherate as a Lewis acid (entries 9−11 of
Table 1). We found that in the presence of boron trifluoride
diethyl etherate (BF3OEt2) in dichloromethane, the reaction
resulted in phenolic alkylation (entry 9). Hypothesizing that the
relatively low separated yields of this reaction are due to the
insolubility of phenolic derivative in dichloromethane, we
decided to perform this reaction in neat conditions with
BF3OEt2. This modification produced satisfactory yields of
DMX-5804 (entry 10). Furthermore, we added an excess of
methyl glycol as a solvent as well as a reactant in the presence of
BF3OEt2 to obtain quantitative yields of DMX-5804 in good
time (entry 11). It is worth mentioning that in these acidic
conditions, the other regio-isomer (pyrimidine alkylated
compound 7) was completely absent. In addition to the
discovery compound DMX-5804, we employed this route for
the synthesis of ribose- and glucose-conjugated analogues.
These sugar analogues were particularly interesting because of
their increased solubility in an aqueous medium compared to
virtually insoluble DMX-5804. More work is needed to study the
mechanistic basis of preferential phenolic alkylation in the
presence of BF3OEt2. Previously, Coyle et al., have reported the
use of BF3OEt2 for phenolic alkylation.10 It has been recently
reported that BF3OEt2 promotes the formation of carbocation
with aliphatic −OH groups by coordination of the oxygen atom,
followed by the transfer of the alkyl moiety.11,12 Thus, a
possibility of activation of aliphatic −OH in methoxyethanol by
BF3OEt2 exists. Simultaneously, Lewis acid BF3OEt2 can also
deactivate the pyrimidinone ring by preventing keto→enol
conversion, secondary to its interaction with −NH group.
MAP4K4 Inhibition. We submitted DMX-5804, ribose-
analogue 9, and regio-isomer 7 to examine their MAP4K4
inhibitory activity in a cell-free assay using recombinant
MAP4K4 enzyme and myelin basic protein (MBP) as its
substrate. The results of the assay are given in Figure 3. We
found that the concentration for 50% inhibition of MAP4K4
(IC50) of DMX-5804 was 205.9 nM. Compound 7, where the
alkoxy group was O-conjugated in pyrimidine ring, showed no
inhibitory activity in the concentration range tested. However,
ribose-analogue 9 showed inhibitory activity (IC50 = 255.8 nM)
comparable to that of DMX-5804. These data demonstrate that
it is possible to modify the phenolic group for targeting purposes
and/or to alter the pharmacokinetic and pharmacodynamic
profiles of this class.
Figure 2. HPLC profile of DMX-5804.
is noteworthy that the reaction mixture and conditions did not
impact the stability of DMX-5804. This observation is in
contrast to the discovery method where significant charring and
loss of the final product were found.
General New Route for Synthesis of DMX-5804 and Its
Analogues. After obtaining reproducible yields of DMX-5804
from Scheme 2 reaction, we focused our attention on
generalizing this scheme to synthesize novel analogues of
DMX-5804. We modified O-alkoxy functionality because
modification in any other place in the structure of DMX-5804
has been reported detrimental to the purported MAP4K4
inhibitory activity. Here, we replaced compound 3a (of Scheme
2) with 2-bromo-1-(4-methoxyphenyl)ethan-1-one (3b in
Scheme 3) to obtain a compound with a phenolic functional
group (6) which is hypothesized to serve as an intermediate for
the synthesis of DMX-5804 or its analogues (Scheme 3).
1660
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663

Organic Process Research & Development
pubs.acs.org/OPRD
Article
a
Scheme 3. General Scheme for Synthesis of DMX-5804 Analogues
a
Reagents and conditions: reagents and conditions: (i) NaHCO3, DMF, 50 °C (ii) malononitrile, KOH, MeOH reflux; (iii) 95% formic acid,
reflux; boron tribromide in dichloromethane (iv) base, 1-bromo-2-methoxyethane, appropriate solvent; (v) BF3OEt2, 2-methoxyethan-1-ol, at 60−
70 °C.
Table 1. Reaction Conditions for Base-Catalyzed and Acid-Catalyzed Synthesis of DMX-5804
a
entry
solvent
base/acid
time (h)
temp (°C)
DMX-5804 (%)
7 (%)
1
2
3
4
5
6
7
8
acetonitrile
acetone
DMF
DMF
DMSO
DMF
THF
DMF
DCM
K2CO3
K2CO3
K2CO3
CsCO3
K2CO3
TEA
NaH
NaH
BF3OEt2
BF3OEt2
BF3OEt2
12
15
12
12
15
15
5
8
reflux
reflux
80
80
60
80
10
10
40
trace
Trace
5−7
20
<10
88
70
55
60
20
15
10
trace
trace
trace
trace
25−30
60−70
80−85
9
10
11
10−12
10−12
6−8
60
70−80
methyl glycol
a
For entries 1−8, 1-bromo-2-methoxyethane was used as a reactant; for entries 9−10, 2-methoxyethan-1-ol was used as a reactant (Scheme 3).
Figure 3. Biological MAP4K4 inhibitory activity of DMX-5804, compound 7, and compound 9.
assisted reaction step, which is very conducive to commercial-
CONCLUSIONS
■
scale synthesis of DMX-5804. A potential limitation of this
method is the sequential use of both acidic and basic reaction
steps, which makes it difficult to choose an appropriate
protection strategy for the synthesis of certain analogues.
However, this limitation does not affect the synthesis of DMX-
5804.
The new route described above (Scheme 2) to synthesize DMX-
5804 was effectively used for a large-scale manufacturing process
which generated API grade (>99.99 by HPLC area %) in
excellent (80−85%) yield. In the optimized route, 1-(4-(2-
methoxyethoxy)phenyl)-2-(phenylamino)ethan-1-one (1)
eliminated the need of using a transition-metal catalyst and
expensive borates. In addition, this intermediate made it easy to
access various DMX-5804 analogues. In comparison, the initial
discovery route generated DMX-5804 in only a 22% yield. It is
worth noting that this three-step sequence did not require
purification steps using column chromatography. The general
scheme reported here takes 6−8 h to complete and also
eliminates the use of anhydrous solvents and the microwave-
EXPERIMENTAL SECTION
■
General Information. All reagents and solvents were
purchased from commercial suppliers and used without further
1
purification. H and 13C NMR spectra were recorded on a
Varian spectrometer in suitable deuterated solvents. The
solvents and measurement frequency used are indicated for
1661
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663

Organic Process Research & Development
pubs.acs.org/OPRD
Article
each experiment. The signal of the residual deuterated solvent
relative to tetramethylsilane was used as the internal reference.
All spectra are reported in supplemental data as follows:
chemical shift δ in parts per million (multiplicity, coupling
constant J in hertz, and number of protons). The resonance
multiplicity is described as singlet (s), doublet (d), triplet (t),
quartet (q), multiplet (m), doublet of doublets (dd), doublet of
triplets (dt), or triplet of doublets (td). Product purity was
determined by HPLC. For reaction monitoring, analytical thin-
layer chromatography (TLC) was performed on Merck Silica gel
60 F254 strips and visualization was accomplished by irradiation
with UV light (254 or 366 nm).
HPLC. HPLC was performed using an Azura P 6.1L HPLC
system (Knauer, Berlin, Germany) with a UV-1 detector set at λ
= 254 nm. The samples were separated on a Sonoma C18, 10
μm, 4.6 × 250 mm using water-acetonitrile gradient 5−95% at
1.5 mL/min.
2-Bromo-1-(4-methoxyphenyl)ethan-1-one (3). To a sol-
ution of 4-hydroxyacetophenone (25 g) in dimethylformamide,
potassium carbonate (38 g) and 1-bromo-2-methoxyethane (30
g) were added. The reaction mixture was heated to 90 °C for
12−20 h and monitored by TLC. After completion of the
reaction, the mixture was cooled to room temperature, poured
into ice-water, and extracted with ethyl acetate. The organic
extract was dried and evaporated. The residue was purified by
flash column using hexanes/ethyl acetate (1:9). The obtained 1-
(4-(2-methoxyethoxy)phenyl)ethan-1-one (71%) intermediate
was brominated in diethyl ether to get 3 (90%). The conditions
of this bromination reaction using bromine have been previously
reported.13
1-(4-(2-Methoxyethoxy)phenyl)-2-(phenylamino)ethan-1-
one (4). A round-bottom flask fitted with a magnetic stir bar was
charged with aniline (1.07 mol), sodium bicarbonate (1.28
mol), and N,N′-dimethylformamide (DMF, 300 mL). The
slurry was stirred while adding 2-bromo-1-(4-(2-
methoxyethoxy)phenyl)ethan-1-one (1.18 mol, dissolved in
DMF) dropwise. The mixture was heated to 50 °C and stirred
for 5 h. The reaction mixture was cooled to room temperature
and poured on to ice-water to obtain a precipitate which was
filtered and recrystallized from isopropyl alcohol as a light yellow
solid (92% yield). 1H NMR (400 MHz, CDCl3): δ 7.98 (d, J =
8.7 Hz, 2H), 7.23 (m, 2H), 7.00 (d, J = 8.7 Hz, 2H), 6.75 (d, J =
7.7 Hz, 3H), 4.57 (s, 2H), 4.20 (d, J = 4.5 Hz, 2H), 3.78 (d, J =
4.5 Hz, 2H), and 3.46 (s, 3H).
methane/ethanol (1:2) to afford DMX-5804 as a white solid
(80−85% yield). 1H NMR (400 MHz, CDCl3): δ 12.30 (s, 1H),
7.94 (s, 1H), 7.79 (d, J = 8.6 Hz, 2H), 7.63 (d, J = 7.5 Hz, 2H),
7.53 (t, J = 7.8 Hz, 2H), 7.41 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.6
Hz, 2H), 4.17 (t, J = 4.7 Hz, 2H), 3.77 (t, J = 4.7 Hz, 2H), and
3.46 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 160.74,
158.09, 148.33, 143.07, 137.26, 129.79, 129.44, 127.67, 125.73,
124.80, 121.78, 121.41, 114.59, 106.35, 71.07, 67.30, and 59.20;
mp 158−160 °C;
5-(4-(2-Hydroxy)phenyl)-7-phenyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (6). To a cold (−30 °C)
solution of 5-(4-methoxyphenyl)-7-phenyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (300 mg) in dichloromethane
was added BBr3 (Scheme 3). The temperature of the reaction
mixture was raised to room temperature by stirring for 1 h. The
solvent was evaporated and to the residue was added saturated
ice-cold NaHCO3 solution. The precipitate was filtered to
1
obtain the title compound 6 (286 mg, 100% yield). H NMR
(400 MHz, DMSO-d6): δ 12.05 (s, 1H), 9.34 (s, 1H), 7.92 (d, J
= 2.99 Hz, 1H), 7.77 (d, J = 8.59 Hz, 2H), 7.74 (d, J = 7.55 Hz,
2H), 7.61 (s, 1H), 7.53 (t, J = 7.83 Hz, 2H), 7.39 (t, J = 7.39 Hz,
1H), and 6.75 (d, J = 8.59 Hz, 2H); 13C NMR (101 MHz,
DMSO-d6): δ 159.04, 156.62, 148.09, 144.61, 137.72, 130.02,
129.58, 127.43, 124.91, 124.59, 121.53, 120.67, 115.23, and
106.43.
Base-Catalyzed Alkylation. To a solution of compound 6
(25 g) in DMF (150 mL) was added 1-bromo-2-methoxyethane,
or any alkyl halide. After stirring at 80−90 °C for 12−15 h, the
reaction mixture was cooled to room temperature and poured in
crushed ice-water mixture. After stirring for 1 h, the precipitate
was collected and recrystallized from DCM/ethanol (1:2).
4-(4-(2-Methoxyethoxy)-7-phenyl-7H-pyrrolo[2,3-d]-
pyrimidin-5-yl)phenol (Regio-Isomer 7). A crystalline white
solid obtained in 80% yield. 1H NMR (400 MHz, DMSO-d6): δ
9.37 (s, 1H), 8.16 (s, 1H), 7.74 (m, 4H), 7.63 (s, 1H), 7.54 (d, J
= 7.9 Hz, 2H), 7.41 (d, J = 7.4 Hz, 1H), 6.76 (d, J = 8.6 Hz, 2H),
4.16 (t, J = 5.7 Hz, 2H), 3.58 (d, J = 5.2 Hz, 2H), and 3.24 (s,
3H). 13C NMR (101 MHz, DMSO-d6): δ 158.01, 156.68,
147.78, 147.36, 137.55, 130.16, 129.64, 127.48, 124.82, 124.45,
121.42, 121.18, 115.17, 105.60, 69.83, 58.55, and 45.67; mp
221−224 °C.
Acidic O-Alkylation. To a mixture of compound 6 (25 g)
and BF3·OEt2 (50 mL) was added a solution of an appropriate
substrate (2-methoxyethan-1-ol, β-D-glucose pentaacetate, or 1-
O-acetyl-2,3,5-tri-O-benzoyl-beta-D-ribofuranose). In one con-
dition, the solutions of substrate were prepared in dichloro-
methane, whereas in another condition, the substrates were
added solid to the reaction mixture (neat). After stirring at 25 °C
for 18 h, the reaction mixture was evaporated. The residue was
washed with saturated NaHCO3 (50 mL) and extracted with
chloroform (50 mL × 2). The combined organic phase was dried
over anhydrous MgSO4, concentrated, and crystallized from
ethanol.
2-Amino-4-(4-(2-methoxyethoxy)phenyl)-1-phenyl-1H-
pyrrole-3-carbonitrile (5). A round-bottom flask fitted with a
magnetic stir bar was charged with 4 (0.31 mol), potassium
hydroxide (0.94 mol) dissolved in water (50 mL), malononitrile
(0.34 mol), and methanol (450 mL). The mixture was heated to
80 °C for 4 h. Afterward, the reaction mixture was cooled and
evaporated to 200 mL. The precipitate was filtered and
recrystallized in cold isopropyl alcohol to obtain 2-amino-4-
(4-(2-methoxyethoxy)phenyl)-1-phenyl-1H-pyrrole-3-carboni-
1
trile 5 as a brown solid (88% yield). H NMR (400 MHz,
7-Phenyl-5-(4-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-
(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)phenyl)-3,7-
dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (8). 1H NMR (400
MHz, DMSO-d6): δ (d, J = 3.6 Hz, 1H), 7.95 (d, J = 3.7 Hz, 3H),
7.75 (m, 3H), 7.55 (t, J = 5.28 Hz, 2H), 7.41 (t, J = 7.42 Hz, 1H),
7.04 (dd, J = 2.94 Hz, 2H), 4.88 (d, J = 7.39 Hz, 1H), 3.96 (br s,
5H), 3.70 (dd, J = 5.28 Hz, 1H), 3.47 (dd, J = 5.86 Hz, 1H), 3.34
(m, 1H), 3.26 (m, 2H), and 3.17 (t, J = 3.17 Hz, 1H); mp 149−
152 °C.
CDCl3): δ 7.54 (m, 4H), 7.41 (m, 4H), 6.95 (d, J = 8.7 Hz, 2H),
4.14 (d, J = 4.6 Hz, 2H), 3.76 (d, J = 4.6 Hz, 2H), and 3.45 (s,
3H).
5-(4-(2-Methoxyethoxy)phenyl)-7-phenyl-3,7-dihydro-4H-
pyrrolo[2,3-d]pyrimidin-4-one (DMX-5804). Compound 5
(0.22 mol) was dissolved in 85% formic acid (200 mL) and
refluxed for 8−10 h. After cooling to room temperature, the
mixture was poured onto ice-water to obtain a precipitate. The
precipitate was filtered, dried, and recrystallized from dichloro-
1662
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663

Organic Process Research & Development
pubs.acs.org/OPRD
Article
(3) Larhammar, M.; Huntwork-Rodriguez, S.; Rudhard, Y.; Sengupta-
Ghosh, A.; Lewcock, J. W. The Ste20 Family Kinases MAP4K4,
MINK1, and TNIK Converge to Regulate Stress-Induced JNK
Signaling in Neurons. J. Neurosci. 2017, 37, 11074−11084.
(4) Wu, C.; Watts, M. E.; Rubin, L. L. MAP4K4 Activation Mediates
Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis. Cell
Rep. 2019, 26, 1143.
(5) Xie, M.; Zhang, D.; Wang, S. C.; Santo, M.; Wang, X. Z.; Pocius, J.
S.; Taffet, G. E.; Michael, L. H.; Entman, M. L.; Mann, D. L.; Tan, T.-
H.; Schneider, M. D. The Protein Kinase MAP4K4 Is Activated in
Failing Human Hearts and Mediates Cardiomyocyte Apoptosis in
Experimental Models, in vitro and in vivo. Circualtion 2007, 116,
II_420.
(6) Golforoush, P.; Schneider, M. D. Intensive care for human hearts
in pluripotent stem cell models. npj Regener. Med. 2020, 5, 4.
(7) Roth Flach, R. J.; Skoura, A.; Matevossian, A.; Danai, L. V.; Zheng,
W.; Cortes, C.; Bhattacharya, S. K.; Aouadi, M.; Hagan, N.; Yawe, J. C.;
Vangala, P.; Menendez, L. G.; Cooper, M. P.; Fitzgibbons, T. P.;
Buckbinder, L.; Czech, M. P. Endothelial protein kinase MAP4K4
promotes vascular inflammation and atherosclerosis. Nat. Commun.
2015, 6, 8995.
(8) Fiedler, L. R.; Chapman, K.; Xie, M.; Maifoshie, E.; Jenkins, M.;
Golforoush, P. A.; Bellahcene, M.; Noseda, M.; Faust, D.; Jarvis, A.;
Newton, G.; Paiva, M. A.; Harada, M.; Stuckey, D. J.; Song, W.; Habib,
J.; Narasimhan, P.; Aqil, R.; Sanmugalingam, D.; Yan, R.; Pavanello, L.;
Sano, M.; Wang, S. C.; Sampson, R. D.; Kanayaganam, S.; Taffet, G. E.;
Michael, L. H.; Entman, M. L.; Tan, T.-H.; Harding, S. E.; Low, C. M.
R.; Tralau-Stewart, C.; Perrior, T.; Schneider, M. D. MAP4K4
Inhibition Promotes Survival of Human Stem Cell-Derived Cardio-
myocytes and Reduces Infarct Size In Vivo. Cell Stem Cell 2019, 24,
579−591.
5-(4-((3,4-Dihydroxy-5-(hydroxymethyl)tetrahydrofuran-
2-yl)oxy)phenyl)-7-phenyl-3,7-dihydro-4H-pyrrolo[2,3-d]-
pyrimidin-4-one (9). 1H NMR (400 MHz, DMSO-d6): δ 12.11
(d, J = 3.6 Hz, 1H), 7.95 (d, J = 3.7 Hz, 1H), 7.89 (d, J = 8.7 Hz,
2H), 7.76 (d, J = 7.4 Hz, 2H), 7.71 (s, 1H), 7.54 (t, J = 7.9 Hz,
2H), 7.41 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 8.8 Hz, 2H), 5.48 (s,
1H), 5.31 (s, 1H), 4.98 (s, 1H), 4.69 (s, 1H), 4.02 (s, 2H), 3.89
(m, 1H), 3.55 (dd, J = 5.0 Hz, 1H), and 3.37 (m, 1H); mp 155−
158 °C.
MAP4K4 Inhibition Assay. MAP4K4 assay for inhibition
concentration (IC50) determination was performed using a kit
by BioAssay Systems Services (Hayward, CA). Briefly, MAP4K4
in assay buffer (40 ng in 10 μL) was incubated with varying
concentrations of test compounds (5 μL in dimethylsulfoxide)
for 15 min at 37 °C. The compounds were titrated from 900 nM
(final reaction concentration) in 3× steps. Compound dilutions
were made in assay buffer with 0.036% dimethylsulfoxide.
Reactions were initiated by adding 5 μL of the reaction mix (2.6
μg/μL MBP, 193 μM ATP; final reaction concentration of 13 μg
MBP, 48 μM ATP) and run at 37 °C for 20 min. These kinase
reactions were stopped with an ADP detection reagent and
fluorescence was measured at 530Ex/590Em. IC50 was computed
using GraphPad Prism Nonlinear Sigmoidal Dose-Response
fitting.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.1c00130.
■
sı
(9) Arnold, L. D.; Cesario, C.; Coate, H.; Crew, A. P.; Dong, H.;
Foreman, K.; Honda, A.; Laufer, R.; Li, A.-H.; Mulvihill, K. M.;
Mulvihill, M. J.; Nigro, A. I.; Panicker, B.; Steinig, A. G.; Sun, Y.; Weng,
Q.; Werner, D. S.; Wyle, M. J.; Zhang, T. 6,6-bicyclic ring substituted
heterobicyclic protein kinase inhibitors. U.S. Patent 8,101,613 B2,
2012.
1H NMR of 4, 5, 6, 7, 8, 9, and DMX-5804; 13C NMR of 7
and DMX-5804; mass spectra of 7, 8, 9, and DMX-5804;
and HPLC profile of compound 6 (PDF)
(10) Coyle, T.; Brumer, H.; Stubbs, K. A. An improved preparation of
some aryl alpha-L-arabinofuranosides for use as chromogenic
substrates for alpha-L-arabinofuranosidases. Can. J. Chem. 2015, 93,
1176−1180.
AUTHOR INFORMATION
Corresponding Authors
■
Venkateswararao Eeda − Department of Pharmaceutical
Sciences, University of Oklahoma Health Science Center,
Oklahoma City, Oklahoma 73117, United States;
Email: veeda@ouhsc.edu
Vibhudutta Awasthi − Department of Pharmaceutical Sciences,
University of Oklahoma Health Science Center, Oklahoma
City, Oklahoma 73117, United States; orcid.org/0000-
0002-3611-9009; Email: vawasthi@ouhsc.edu
(11) Jumbam, N. D.; Maganga, Y.; Masamba, W.; Mbunye, N. I.;
Mgoqi, E.; Mtwa, S. A New Alkylation of Aryl Alcohols by Boron
Trifluoride Etherate. Molecules 2019, 24, 3720.
(12) Estopiná-Durán, S.; Donnelly, L. J.; McLean, E. B.; Hockin, B.
̃
M.; Slawin, A. M. Z.; Taylor, J. E. Aryl Boronic Acid Catalysed
Dehydrative Substitution of Benzylic Alcohols for C-O Bond
Formation. Chemistry 2019, 25, 3950−3956.
(13) Yatabe, T. New Amide Compounds. International Patent
Number WO 9827108; World Intellectual Property Organization,
1998.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.1c00130
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was partially supported by funds from Sandra K and
David L Gilliland Chair in Nuclear Pharmacy to V.A.
■
REFERENCES
■
(1) Gao, X.; Gao, C.; Liu, G.; Hu, J. MAP4K4: an emerging
therapeutic target in cancer. Cell Biosci. 2016, 6, 56.
(2) Wright, J. H.; Wang, X.; Manning, G.; LaMere, B. J.; Le, P.; Zhu,
S.; Khatry, D.; Flanagan, P. M.; Buckley, S. D.; Whyte, D. B.; Howlett,
A. R.; Bischoff, J. R.; Lipson, K. E.; Jallal, B. The STE20 kinase HGK is
broadly expressed in human tumor cells and can modulate cellular
transformation, invasion, and adhesion. Mol. Cell. Biol. 2003, 23, 2068−
2082.
1663
https://doi.org/10.1021/acs.oprd.1c00130
Org. Process Res. Dev. 2021, 25, 1658−1663