A combination of flow and batch mode processes for the efficient preparation of mGlu2/3 receptor negative allosteric modulators (NAMs)

Article abstract of DOI:10.1016/j.tet.2018.03.068

Benzodiazepinones are privileged scaffolds with activity against multiple therapeutically relevant biological targets. In support of our ongoing studies around allosteric modulators of metabotropic glutamate receptors (mGlus) we required the multigram synthesis of a β-ketoester key intermediate. We report the continuous flow synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate and its transformation to potent mGlu_2/3 negative allosteric modulators (NAMs) in batch mode.

Full text of DOI:10.1016/j.tet.2018.03.068

Accepted Manuscript
A combination of flow and batch mode processes for the efficient preparation of
mGlu
2/3
receptor negative allosteric modulators (NAMs)
Raveendra Panickar Dhanya, Ananda Herath, Douglas J. Sheffler, Nicholas D.P.
Cosford
PII:
S0040-4020(18)30351-X
10.1016/j.tet.2018.03.068
TET 29408
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 24 October 2017
Revised Date: 26 March 2018
Accepted Date: 28 March 2018
Please cite this article as: Dhanya RP, Herath A, Sheffler DJ, Cosford NDP, A combination of flow and
batch mode processes for the efficient preparation of mGlu
receptor negative allosteric modulators
2/3
(NAMs), Tetrahedron (2018), doi: 10.1016/j.tet.2018.03.068.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
A Combination of Flow and Batch Mode Processes for the Efficient
Preparation of mGlu2/3 Receptor Negative Allosteric Modulators (NAMs)
Raveendra Panickar Dhanya, Ananda Herath, Douglas J. Sheffler, and Nicholas D. P. Cosford*
Cancer Metabolism and Signaling Networks Program, NCI-Designated Cancer Center, Sanford Burnham Prebys Medical
Discovery Institute, 10901 N. Torrey Pines Rd., La Jolla, CA 92037, USA
ABSTRACT
Benzodiazepinones are privileged scaffolds with activity against multiple therapeutically relevant
biological targets. In support of our ongoing studies around allosteric modulators of metabotropic glutamate
receptors (mGlus) we required the multigram synthesis of a β-ketoester key intermediate. We report the
continuous flow synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate and its transformation to
potent mGlu2/3 negative allosteric modulators (NAMs) in batch mode.
Keywords: flow chemistry, microreactor, multistep synthesis, β-ketoester, metabotropic glutamate receptor,
benzodiazepinone.
* Corresponding author.
E-mail address: ncosford@sbpdiscovery.org (Nicholas D. P. Cosford)
1

INTRODUCTION
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The amino acid glutamate is the predominant excitatory neurotransmitter, acting at neurons both
peripherally as well as in the central nervous system (CNS). Glutamate mediates fast synaptic transmission
through ionotropic glutamate receptors, ligand-gated ion channels that include α-amino-3-hydroxy-5-
methyl-4-isoxazolepropionic acid (AMPA), kainate, and N-methyl-D-aspartate (NMDA) receptor
subtypes.[1] It also exerts a modulatory role via the family of G protein-coupled receptors known as
metabotropic glutamate (mGlu) receptors. There are eight mGlu receptor subtypes that are grouped
according to their respective second messenger systems and sequence homology. The Group I mGlus
(mGlu1 and mGlu5) are positively coupled to Ca2+ signaling, while Groups II (mGlu2, mGlu3) and Group III
(mGlu4, mGlu6, mGlu7 and mGlu8) negatively regulate the activity of adenylyl cyclase via coupling to Gi/o
proteins.[2] Several orthosteric (competitive) agonists and antagonists that are selective for Group II mGlu
receptors have been reported. These compounds are analogues of glutamate that bind to the endogenous
glutamate binding site in the extracellular “venus fly trap” (VFT) domain of the receptor.[3, 4] On the other
hand, efforts by our group and others have focused on the discovery and optimization of small molecule
allosteric modulators that bind non-competitively to mGlu receptors. Over the past several years selective
positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) of mGlu2 and/or mGlu3
have been reported.[5-7] This interest was prompted by the concept that such compounds may have
improved therapeutic properties by subtly modulating the activity of malfunctioning receptor signaling
pathways in concert with the endogenous neurotransmitter (glutamate). Furthermore, high sequence
homology in the mGlu2 and mGlu3 glutamate binding sites limits the possibility of selective orthosteric
ligands. Thus, the improved selectivity of allosteric modulators may offer the benefit of an improved side-
effect profile, as well as superior drug-like properties compared with competitive agonists and antagonists.
Importantly, allosteric modulators of mGlu2 and/or mGlu3 receptors have significant potential as drugs for
the treatment of various CNS disorders such as schizophrenia, anxiety, drug dependence, cognitive
dysfunction or depression that are caused by aberrant glutamatergic transmission [6-13]
.
2

We recently initiated a programme focused on the design, synthesis and in vitro and in vivo
ACCEPTED MANUSCRIPT
evaluation of mGlu2/3 negative allosteric modulators (NAMs). One component of our research involved the
investigation of a series of mGlu2/3 NAMs exemplified by the benzodiazepine derivative MNI-137 (1a).[14]
In order to investigate the structure-activity relationships (SAR) around this compound, we required large
amounts of a common intermediate that could be converted to multiple analogues. We identified tert-butyl
3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6) as such an intermediate that would provide access to
analogues in which various substituents could be introduced into the fused aryl ring. Keeping in mind the
objective of generating multi-gram amounts of intermediate, we elected to investigate the continuous flow
(microfluidic) synthesis of this key heterocyclic β-ketoester derivative.
Over the past few years, multistep microfluidic chip-based processes have emerged as attractive methods
for the large scale preparation of numerous organic molecules.[15, 16] Advantages include enhanced reagent
mixing, optimal heat transfer, small reaction volumes, precise reaction times, and the ability to conduct
multistep reactions in a single, unbroken microreactor sequence. We have previously described the
development of automated flow chemistry methods to rapidly access complex, drug-like heterocycles from
readily available precursors. Thus, we have reported the continuous flow syntheses of bis-substituted 1,2,4-
oxadiazoles,[17] functionalized imidazo[1,2-a] heterocycles,[18, 19] pyrrole-3-carboxylic acid derivatives,[20]
23]
2-(1H-indol-3-yl)thiazoles[21] and 5-(thiazol-2-yl)-3,4-dihydropyrimidin-2(1H)-one derivatives.[22,
Herein we describe the flow synthesis of the key intermediate tert-butyl 3-(2-cyanopyridin-4-yl)-3-
oxopropanoate and its utility in the preparation of mGlu2/3 receptor NAMs that are analogues of compound
1a.[14]
Figure 1. The prototypical benzodiazepine-derived mGlu2/3 receptor negative allosteric modulator MNI-137 (1a).
3

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RESULTS AND DISCUSSION
Scheme 1. The batch synthesis of 2-cyanoisonicotinate (4) and tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate
(6). i) mCPBA, CH2Cl2, 25 ºC, 16 h; ii) TMSCN, Et3N, MeCN, 80 ºC, 2 h; iii) LDA, THF, -78 ºC, 4 h.
As noted above, the key intermediate required for the preparation of the target benzodiazepinone
derivatives is a heterocyclic β-ketoester derivative with potentially sensitive functionality. The reported
batch mode synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6) is shown in Scheme 1. The
procedure involves an initial synthesis of methyl 2-cyanoisonicotinate (4), followed by the use of this
intermediate in the preparation of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6). Thus, methyl 4-
carboxypyridine (2) is treated with mCPBA in methylene chloride to provide methyl 4-carboxypyridine N-
oxide (3). This material is then treated with cyanotrimethylsilane in acetonitrile to afford methyl 2-
cyanoisonicotinate (4). Intermediate 4 is then subjected to base-mediated condensation with tert-
butylacetate (5) to furnish tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6).
Scheme 2. The batch synthesis of 2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl derivatives. i) Toluene,
∆, 1 h; ii) TFA, CH2Cl2, 0 ºC, 7 h.
The batch mode preparation of benzodiazepinone derivatives is performed as shown in Scheme 2.
Thus, condensation of mono N-Boc protected o-phenelenediamines 7 with tert-butyl β-ketoesters 6 in
boiling toluene provides the corresponding β-ketoamide derivatives 8. Upon TFA-mediated Boc-
4

deprotection the intermediate β-ketoamides undergo in-situ cyclization to yield the desired 1,3-dihydro-
ACCEPTED MANUSCRIPT
benzo[b][1,4]diazepin-2-ones derivatives 1.
Scheme 3. Microfluidic synthesis of methyl 2-cyanoisonicotinate (4).
CO2Me
10 mL
1.0 mL/min
95oC
N
2
1M, ACN
CO2Me
5 mL
0.2 mL/min
mCPBA
150oC
1.0 mL/min
N
O
1M, ACN
CO2Me
3
, 8 g/h
TMSCN/Et3N (1:2.5)
1M, ACN
0.2 mL/min
N
CN
4
The batch synthesis of methyl 2-cyanoisonicotinate (4) involves two different solvents, a non-polar
haloalkane (dichloromethane) in the first step and a polar aprotic solvent (acetonitrile) in the second step
(Scheme 1). For the flow synthesis we determined that it was possible for the N-oxide formation to proceed
in acetonitrile, thus avoiding the need for solvent change part way through the process. Thus, as shown in
Scheme 3, methyl 4-carboxypyridine (2) was dissolved in MeCN as a 1 M solution. Similarly, mCPBA was
also dissolved in MeCN at a concentration of 1M, and each of these solutions was pumped at a rate of 1
mL/minute initially through a T-mixer at ambient temperature and subsequently into a 10 mL flow reactor
o
held at 95 C. All high temperature flow reactions were carried out under an optimal pressure of 8 bar
controlled by the back pressure regulator (BPR). The acetonitrile solution exiting the reactor contained the
methyl 4-carboxypyridine N-oxide (3) generated during the flow process at a rate of 8g/h. This material
was pumped into a second T-mixer and combined with a mixture of cyanotrimethylsilane and triethylamine
(1:2.5) as a 1M solution in acetonitrile with both streams flowing into the T-mixer at a rate of 0.2
o
mL/minute. The combined reaction mixture was then pumped through a 5 mL reactor held at 150 C to
form the desired methyl 2-cyanoisonicotinate (4). This material was collected and used in a second flow
process as shown in Scheme 4. For the next step we established that THF was a suitable solvent to use in
5

the flow process and that a 2 M solution was a viable concentration for both the reactants (4 and 5) and the
ACCEPTED MANUSCRIPT
lithium amide base (LDA). Optimization of the LDA step involved testing multiple temperatures from -70
o
o
oC through 0 C to establish that -30 C was ideal for the flow process. As illustrated in Scheme 4, a 2M
solution of tert-butylacetate (5) and a 2M solution of LDA are both pumped at a rate of 0.2 mL/minute into
o
a T-mixer that then flows the reaction mixture into a cooler module held at –30 C. A 1 M solution of
methyl 2-cyanoisonicotinate (4) in THF is pumped (0.3 mL/min) via a T-mixer to combine with the
solution of lithium 1-(tert-butoxy)ethen-1-olate exiting the cooler module. A solution of the key
intermediate tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6) is collected and for use in the
preparation of the target benzodaizepinone derivatives and described in Scheme 2 and the Experimental.
Scheme 4. Microfluidic synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6).
CO2Me
O
N
CN
OtBu
4
5
1M, THF
2 M, THF
0.3 mL/min
0.2 mL/min
-30 oC
O
O
NC
OtBu
LDA, 2M, THF
0.2 mL/min
N
6
To illustrate the utility of the flow/batch synthetic procedure we prepared a series of
benzodiazepinone analogues using the optimized chemistry and evaluated them in vitro against mGlu2 or
mGlu3 receptors. The cellular assay used for in vitro testing employs HEK cells that express either mGlu2
or mGlu3 in addition to G protein-coupled inwardly rectifiying potassium (GIRK) channels. These
specifically transfected cells are employed in an assay that utilizes thallium flux, as previously described,[24]
and the results for compounds 1a-1g are shown in Table 1. Thus, the prototypical benzodiazepinone
mGlu2/3 NAM 1a (MNI-137) was determined to have IC50 values in this assay of 84 nM (mGlu2) and 135
nM (mGlu3), respectively (Table 1, entry 1). Replacement of the bromine atom at R1 in 1a with fluorine, as
6

in 1b, resulted in an approximate 3-fold loss of potency at mGlu and a >20-fold loss of potency at mGlu
ACCEPTED MANUSCRIPT
2
3
(Table 1, entry 2). On the other hand, replacement of bromine with trifluoromethyl, as in 1c, led to
retension of potency at mGlu2 (IC50 = 74 nM) and a 3-fold loss of potency at mGlu3 (IC50 = 388 nM)
compared with 1a (Table 1, entry 3). Interestingly, replacement of bromine with the much larger halogen
iodine, as in 1d, resulted in a 2-fold increase in potency at mGlu2 (IC50 = 35 nM) and retension of potency
at mGlu3 (IC50 = 159 nM) compared with 1a (Table 1, entry 4). Another way to improve potency was noted
when the hydrogen at R2 was replaced with a methyl substituent, as in compound 1e. As shown in Table 1,
entry 5, addition of the R2 methyl in 1e increases potency at both mGlu2 (IC50 = 33 nM) and at mGlu3 (IC50
= 173 nM) compared with 1c (entry 3). Introduction of the highly electron withdrawing nitrile substituent at
R1, as in 1f, resulted in a 5-fold loss of potency at mGlu2 (IC50 = 467 nM) and a 24-fold loss of potency at
mGlu3 (IC50 = 3314 nM) compared with 1a (Table 1, entry 6). Finally, replacement of the bromine atom at
R1 in 1a with a bulky arylalkynyl substituent, as in 1g, resulted in largest loss of potency at mGlu2 (14-fold)
and a 9-fold loss of potency at mGlu3 (Table 1, entry 7). Overall, even within this relatively small library of
analogues, a distinctive and useful structure-activity relationship (SAR) emerged to form the basis for the
design of additional potent analogues.
Table 1. In vitro on-target potency data for mGlu2/3 receptor NAMsa
Entry
ID
1a
1b
1c
1d
1e
1f
R1
Br
F
R2
H
mGlu2 IC50 µM
0.084 ± 0.012
0.310 ± 0.081
0.074 ± 0.011
0.035 ± 0.008
0.033 ± 0.007
0.467 ± 0.041
mGlu3 IC50 µM
0.135 ± 0.043
2.915 ± 1.286
0.388 ± 0.113
0.159 ± 0.050
0.173 ± 0.029
3.314 ± 1.382
1
2
3
4
5
6
H
CF3
I
H
H
CF3
CN
CH3
H
7

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7
1g
1.200 ± 0.256
1.265 ± 0.409
amGlu2 and mGlu3 NAM IC50 µM data represent the ± SEM for at least three independent experiments performed in triplicate in either mGlu2 or mGlu3 GIRK
thallium flux assays as previously described[24] and utilizing a 1 hour incubation with test compound prior to assay.
8

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CONCLUSION
In summary, we have developed an efficient procedure for the preparation of mGlu2/3
NAMs (1) using a combination of flow and batch chemistry. These benzodiazepine derivatives
are constructed rapidly and in high yield from inexpensive, readily available starting materials.
The new process provides access to diversely substituted analogues in quantities (150-350 mg)
sufficient for both initial in vitro testing, as well as further evaluation, including assessment in
vivo. This is an important consideration when expanding a lead series using medicinal chemistry
in an active drug discovery program. The further characterization of these and additional
analogues in this series will be described in detail in future disclosures.
EXPERIMENTAL
Unless otherwise noted, all solvents and other reagents are commercially available and used
without further purification. Purity and characterization of compounds were established by a
combination of liquid chromatography-mass spectroscopy (LC-MS) and NMR analytical
techniques and was >95% for all compounds. Silica gel column chromatography was carried out
using prepacked silica cartridges from RediSep (ISCO Ltd.) and eluted using an Isco Companion
1
system. Melting points were reordered on a MEL-TEMP® apparatus and are uncorrected. H-
and 13C-NMR spectra were obtained on a Jeol 400 spectrometer at 400 MHz and 100 MHz,
respectively. Chemical shifts are reported in δ (ppm) relative to residual solvent peaks or TMS as
internal standards. Coupling constants are reported in Hz. High-resolution ESI-TOF mass spectra
were acquired from the Mass Spectrometry Core at The Sanford-Burnham Medical Research
Institute (Orlando, Florida). HPLC-MS analyses were performed on a Shimadzu 2010EV LCMS
using the following conditions: Kromisil C18 column (reverse phase, 4.6 mm × 50 mm); a linear
9

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gradient from 10% acetonitrile and 90% water to 95% acetonitrile and 5% water over 4.5 min;
flow rate of 1 mL/min; UV photodiode array detection from 200 to 300 nm. Continuous flow
(microreactor) experiments were carried out using a Vapourtec R Series Flow Chemistry System.
Flow synthesis of methyl 2-cyanoisonicotinate (4).
A solution of methyl 4-carboxypyridine (1M in MeCN) and a solution of mCPBA (1M in
o
MeCN) were flowed via a T-mixer into a 10 mL flow reactor at 95 C at a rate of 1.0 mL per
minute (Scheme 3). The solution of methyl 4-carboxypyridine N-oxide exiting the first reactor
was combined with TMSCN/NEt3 (1:2.5, 1M in MeCN) via a second T-mixer. This solution was
o
flowed into a 5 mL reactor at 150 C at a rate of 0.2 mL per minute. The solution exiting the
reactor was collected and the solvent evaporated to provide methyl 2-cyanoisonicotinate (4).
Flow synthesis of tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6).
A solution of tert-butyl acetate (2M in THF) was flowed into a cooled flow reactor at a
rate of 0.2 mL per minute and a stream of LDA (2M in THF) is introduced to the same flow
reactor, also at a rate of 0.2 mL per minute. During this process the temperature of the flow
o
reactor is held at –30 C to allow formation of the anion. A solution of methyl 2-
cyanoisonicotinate (1M in THF) is flowed into the stream containing the tert-butyl acetate anion
at a rate of 0.3 mL per minute. The solution exiting the reactor was collected and the solvent
evaporated to furnish tert-butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (6). The residue was
mixed with excess water and extracted three times with dichloromethane. The combined organic
layers were washed with brine, dried over magnesium sulfate, filtered, and concentrated and
residue was purified via automated flash chromatography (SiO2) to afford the desired product
(CombiFlash® Rf column). The solvent gradient was 90% hexane to 50% ethyl acetate over 15
min at a flow rate of 15 mL/min. The purified tert-butyl 3-(2-cyanopyridin-4-yl)-3-
10

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oxopropanoate (6) appeared as a mixture of keto-enol forms in a 1:3 ratio as a pale yellow solid
(8 g, 66 % yield ). 1H NMR keto form (CDCl3): δ 8.96 (d, J = 5.0 Hz, 1H), 8.18 (s, 1H), 7.82 (m,
13
1H), 3.90 (s, 2H), 1.41 (s, 9H). C NMR keto form (100 MHz, CDCl3): δ 190.5, 165.2, 152.5,
1
143.2, 135.4, 126.2, 122.6, 116.6, 83.4, 47.2, 27.9. H NMR enol form (CDCl3): δ 8.77 (d, J =
4.6 Hz, 1H), 7.96 (s, 1H), 7.78 (m, 1H), 5.71 (s, 1H), 1.59 (s, 9H). 13C NMR keto form (100
MHz, CDCl3): δ 172.0, 165.1, 151.8, 142.8, 134.8, 124.6, 122.7, 116.9, 93.1, 82.8, 28.3. LCMS
ESI m/z: 247.00 [M + H]+. HRMS (ESI) m/z calcd for C13H14N2O3 [M + H]+: 247.1001. Found:
247.1072.
General procedure for the batch synthesis of benzodiazepinones.
4-(8-Fluoro-2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)picolinonitrile (1b).
tert-Butyl (2-amino-4-fluorophenyl)carbamate[25] (0.226 g, 1 mmol) and tert-butyl 3-(2-
cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) were dissolved in toluene (5 mL) and
heated at reflux for 1h. The reaction mixture was cooled and the excess solvent was removed
under reduced pressure. The crude intermediate was taken up in CH2Cl2 and TFA (1.14 g, 10
mmol) was added dropwise to it at 0 ºC. The resulting reaction mixture was stirred at rt for 7 h.
The crude mixture was washed with sodium bicarbonate solution and the aqueous layer extracted
with CH2Cl2. The combined organic layers were washed with water, then brine, and dried over
Na2SO4. Evaporation of the solvent followed by column chromatography using hexanes:ethyl
acetate (60:40) yielded the title compound. Yellow solid (0.186 g, 66%). 1H NMR (DMSO-d6): δ
10.74 (s, 1H), 8.91 (d, J = 5.0 Hz, 1H), 8.55 (s, 1H), 8.26-8.24 (m, 1H), 7.30-7.23 (m, 3H), 3.62
13
(s, 2H). C NMR(, DMSO-d6): δ 165.7, 159.3, 156.4, 152.2, 145.1, 139.6, 133.6, 127.0, 126.5,
11

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123.8, 117.2, 115.1 (d, J = 23.0 Hz), 113.4 (d, J = 23.0 Hz), 40.1. LCMS ESI m/z: 281.00 [M +
H]+. HRMS (ESI) m/z calcd for C15H9FN4O [M + H]+: 281.0762. Found: 281.0834.
4-(8-Bromo-2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)picolinonitrile (1a).
tert-Butyl (2-amino-4-bromophenyl)carbamate[25] (0.286 g, 1 mmol), tert-butyl 3-(2-
cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol) were
1
processed according to the general procedure. Yellow solid (0.267 g, 78%), mp: 238-240 ºC. H
NMR (DMSO-d6) δ 10.77 (s, 1H), 8.89 (d, J = 5.0 Hz, 1H), 8.51 (s, 1H), 8.24 (dd, J = 1.0 Hz,
5.0 Hz, 1H), 7.41-7.36 (m, 3H), 3.61 (s, 2H); 13C NMR (DMSO-d6) δ 165.7, 155.6, 152.2, 145.2,
137.7, 133.5, 131.7, 130.0, 127.1, 126.5, 125.1, 124.2, 119.5, 117.3, 40.2. LCMS ESI m/z:
341.00 [M + H]+. HRMS (ESI) m/z calcd for C15H9BrN4O [M+H]+ 340.9959, found: 341.0092.
4-(2-Oxo-8-(trifluoromethyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)picolinonitrile
(1c).
tert-Butyl (2-amino-4-(trifluoromethyl)phenyl)carbamate[25] (0.276 g, 1 mmol), tert-butyl 3-(2-
cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol) were
1
processed according to the general procedure. Yellow solid (0.261 g, 79%), mp: 196-198 ºC. H
NMR (DMSO-d6) δ 10.93 (s, 1H), 8.95 (d, J = 5.5 Hz, 1H), 8.53 (s, 1H), 8.26-8.24 (m, 1H),
7.64-7.53 (m, 3H), 3.71 (s, 2H); 13C NMR (DMSO-d6) δ 166.0, 157.6, 152.3, 145.0, 141.2,
12

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133.6, 130.6, 129.2, 126.6, 125.3, 122.5, 119.2, 117.2, 39.5; LCMS ESI m/z: 332.00 [M + H]+.
HRMS (ESI) m/z calcd for C16H9F3N4O [M+H]+ 332.0729, found 332.0801.
4-(8-Iodo-2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-yl)picolinonitrile (1d).
tert-Butyl (2-amino-4-iodophenyl)carbamate[25] (0.334 g, 1 mmol), tert-butyl 3-(2-cyanopyridin-
4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol) were processed
1
according to the general procedure. White solid (0.352 g, 90%), mp: 253-255 ºC. H NMR
(DMSO-d6): δ 10.74 (s, 1H), 8.92 (d, J= 5.0 Hz, 1H), 8.54 (s, 1H), 8.25-8.23 (m, 1H), 7.58-7.56
(m, 2H), 7.24 (d, J = 8.2 Hz, 1H), 3.62 (s, 2H); 13C NMR (DMSO-d6): δ 165.7, 155.6, 152.2,
145.2, 138.1, 133.5, 132.8, 131.6, 130.1, 129.9, 126.5, 125.0, 117.2, 92.5, 40.1. LCMS ESI m/z:
389.00[M + H]+. HRMS (ESI) m/z calcd for C15H9IN4O [M+H]+ 388.9818, found 388.9892.
4-(7-Methyl-2-oxo-8-(trifluoromethyl)-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-
yl)picolinonitrile (1e).
tert-Butyl (2-amino-5-methyl-4-(trifluoromethyl)phenyl)carbamate[26] (0.290 g, 1 mmol), tert-
butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol)
were processed according to the general procedure. Yellow solid (0.248 g, 72%), mp: 228-230
1
ºC. H NMR (DMSO-d6) δ 10.83 (s, 1H), 8.93 (d, J = 5.0 Hz, 1H), 8.55 (s, 1H), 8.26-8.24 (m,
1H), 7.52 (brs, 2H), 3.75 (s, 2H), 2.45 (s, 3H); 13C NMR (DMSO-d6) δ 165.8, 157.4, 152.2,
13

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145.0, 140.8, 137.7, 133.6, 131.1, 128.2, 126.5, 125.6, 119.9, 117.2, 40.1, 17.9. LCMS ESI m/z:
345.00 [M + H]+. HRMS (ESI) m/z calcd for C17H11F3N4O [M+H]+ 345.0881; found 345.0956.
4-(2-Cyanopyridin-4-yl)-2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepine-8-carbonitrile (1f).
tert-Butyl (2-amino-4-cyanophenyl)carbamate[27] (0.233 g, 1 mmol), and tert-butyl 3-(2-
cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol) were
1
processed according to the general procedure. Yellow solid (0.227 g, 79%), mp: 213-215 ºC. H
NMR (DMSO-d6) δ: 10.83 (s, 1H), 8.93 (d, J = 5.0 Hz, 1H), 8.54 (s, 1H), 8.27-8.25 (m, 1H),
7.52-7.51 (m, 3H), 3.76 (s, 2H). 13C NMR (DMSO-d6) δ 165.9, 158.0, 152.3, 144.9, 141.8, 133.6,
131.0, 129.2, 127.6, 126.3, 123.7, 121.9, 118.2, 117.2, 109.3, 40.1. LCMS ESI m/z: 288.00 [M +
H]+. HRMS (ESI) m/z calcd for C16H9N5O [M+H]+ 288.0821; found 288.0878.
4-(8-((4-Fluorophenyl)ethynyl)-2-oxo-2,3-dihydro-1H-benzo[b][1,4]diazepin-4-
yl)picolinonitrile (1g).
tert-Butyl (2-amino-4-((4-fluorophenyl)ethynyl)phenyl)carbamate[28] (0.326 g, 1 mmol), tert-
butyl 3-(2-cyanopyridin-4-yl)-3-oxopropanoate (0.320 g, 1.3 mmol) and TFA (1.14 g, 10 mmol)
were processed according to the general procedure. Yellow solid (0.258 g, 68%), mp: 229-231
1
ºC. H NMR (DMSO-d6) δ 10.83 (s, 1H), 8.92 (d, J = 5.0 Hz, 1H), 8.56 (s, 1H), 8.26-8.25 (m,
13
1H), 7.65-7.61 (m, 2H), 7.50-7.25 (m, 5H), 3.64 (s, 2H); C NMR (DMSO-d6) δ 165.7, 155.7,
14

ACCEPTED MANUSCRIPT
152.3, 152.0, 145.2, 138.6, 133.9, 133.8, 130.7, 128.7, 127.2, 126.0, 125.2, 124.6, 121.0, 118.5,
117.3, 116.3, 115.0, 89.4, 88.3, 40.1. LCMS ESI m/z: 381.00 [M + H]+. HRMS (ESI) m/z calcd
for C23H13FN4O [M+H]+ 381.1065; found 381.1144.
ACKNOWLEDGEMENT
This work was supported by National Institutes of Health grant nos. MH106865 and
MH087989.
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