Microwave heated flow synthesis of spiro-oxindole dihydroquinazolinone based IRAP inhibitors

Article abstract of DOI:10.1021/op500237k

A fast and convenient synthetic route towards spiro-oxindole dihydroquinazolinones as novel and drug-like insulin-regulated aminopeptidase (IRAP) inhibitors is reported. The synthesis is performed using a MW heated continuous flow system employing 200 mm × 3 mm _i MW absorbing silicon carbide (SiC) or MW transparent borosilicate tubular reactors. A three-component MW-flow reaction to build up the spiro compounds (9 examples, 40-87% yield), using the SiC reactor, as well as a Suzuki-Miyaura cross-coupling reaction (71%), employing the borosilicate reactor, are presented with residence times down to 168 s. The continuous MW-flow routes provide a smooth and scalable synthetic methodology towards this class of IRAP inhibitors.

Full text of DOI:10.1021/op500237k

Communication
pubs.acs.org/OPRD
Microwave Heated Flow Synthesis of Spiro-oxindole
Dihydroquinazolinone Based IRAP Inhibitors
Karin Engen,† Jonas Savmarker,‡ Ulrika Rosenstrom,† Johan Wannberg,† Thomas Lundback,§
̈
̈
̈
Annika Jenmalm-Jensen,§ and Mats Larhed*,∥
‡
†Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Department of Medicinal Chemistry, Beijer Laboratory,
and ∥Department of Medicinal Chemistry, Science for Life Laboratory, BMC, Uppsala University, P.O. Box 574, SE-751 23 Uppsala,
Sweden
§Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology,
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Tomtebodavagen 23A, SE-171 65 Solna, Sweden
̈
S
* Supporting Information
design is the tubular, easy access, 200 mm reactor consisting of
ABSTRACT: A fast and convenient synthetic route
towards spiro-oxindole dihydroquinazolinones as novel
and drug-like insulin-regulated aminopeptidase (IRAP)
inhibitors is reported. The synthesis is performed using a
MW heated continuous flow system employing 200 mm ×
3 mm Øi MW absorbing silicon carbide (SiC) or MW
transparent borosilicate tubular reactors. A three-compo-
nent MW-flow reaction to build up the spiro compounds
(9 examples, 40−87% yield), using the SiC reactor, as well
as a Suzuki−Miyaura cross-coupling reaction (71%),
employing the borosilicate reactor, are presented with
residence times down to 168 s. The continuous MW-flow
routes provide a smooth and scalable synthetic method-
ology towards this class of IRAP inhibitors.
disposable borosilicate glass, or wear and chemically resistant
SiC.24,27−29
Inhibition of insulin-regulated aminopeptidase (IRAP), a
single-spanning transmembrane zinc−metallopeptidase, might
become a future pharmacotherapy against cognitive disor-
ders.30−35 Previously, our division has reported promising
constrained macrocycles derived from Ang IV as potent IRAP
inhibitors.36,37 However, these inhibitors are peptidic in
character and thus not likely to be able to enter the brain.
To reveal a new starting point for development of IRAP
inhibitors, a small molecule-based library of 10 500 compounds
were screened for their inhibitory activity against the enzymatic
activity of IRAP. After hit confirmation, the spiro-oxindole
dihydroquinazolinone 1a (Figure 1) with an IC50 value of 1.5
μM was identified as a representative member of a new class of
IRAP inhibitors.
INTRODUCTION
■
Almost 30 years ago, microwave-assisted organic synthesis was
introduced as a new way of promoting organic reactions.1−4
This technique has since then gone through rapid development
and is nowadays an established heating method both in
academia and industry for quick small-scale synthesis of a wide
range of compounds,5 including drug-like compounds,6
peptides,7 and polymers.8 The general advantages with
microwave (MW) heating over conventional heating are rapid
and uniform in situ heating, reduced reaction times, and in
many cases higher reaction yields.9
During the last two decades, lab-scale continuous flow (CF)
synthesis has enjoyed considerable progress as well.10,11 In this
field, a small reaction volume is heated in a continuous manner
instead of a large volume as in batch chemistry. Thus, this
organic synthesis methodology is very beneficial from a safety
perspective for the processing of explosive reagents, toxic
materials, and gas releasing reactions.10,11 Most commonly, the
heating method for CF has been conductive heating. The
problems featured with this heating methodology are time
requirements both regarding heating as well as cooling. In order
to facilitate CF synthesis in a standard hood and to provide on-
the-fly temperature control, we and others have developed
purpose-built small foot-print MW-flow instrumentation for
organic synthesis.4,12−27 One of the key features with our
Figure 1. Structure of the hit compound 1a.
Two and three-component syntheses of structurally similar
compounds to 1a have been published, involving both
Brønstedt and Lewis acid-catalyzed reactions.38−42 Commonly,
the reactions involve reflux for several hours. We aimed to
improve the synthetic methodology for use in a drug discovery
project towards the development of novel drug-like IRAP
inhibitors. Herein, we present a fast three-component MW-
Special Issue: Continuous Processes 14
Received: July 21, 2014
© XXXX American Chemical Society
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Scheme 1. MW synthesis of compound 1a
Table 1. Three-component scaffold formation of spiro-oxindole dihydroquinazolinone 1a
entry
2a (equiv)
3 (equiv)
4a (equiv)
flow (mL/min)
temp (°C)
reactor
yield
1
2
3
4
5
6
7
8
1
1
1
1
1
1
1
1
1
1
1
200
200
120
120
120
120
120
160
borosilicate
borosilicate
borosilicate
SiC
40%
43%
8%
1
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
1
1
1
1
55%
65%
74%
76%
82%
1
1.5
1.2
1.5
1.5
SiC
1.2
1.5
1.5
SiC
SiC
SiC
heated CF synthesis of a series of spiro-oxindole dihydroqui-
nazolinone analogues of 1a.
concentration as with the borosilicate reactor. Temperatures
ranging from 100 to 240 °C were investigated, using 20 °C
stepwise increasing increments. The collected reaction mixtures
were analyzed by LC-MS, which showed that a reaction
temperature of 120−160 °C was most beneficial. Lower
temperatures resulted in poor product formation, while
temperatures above 160 °C afforded product degradation.
The product mixture was collected at 120 °C affording 55%
isolated yield (entry 4, Table 1). A flow rate of 0.5 mL/min was
used since lower flow (0.25 mL/min) furnished plugging of the
system, and higher flow rate (1 mL/min) resulted in less
product formation. In order to provide a more direct
comparison between the two reactor materials, a reaction
using the same conditions as with SiC was performed using the
corresponding borosilicate reactor (entry 3, Table 1), furnish-
ing poor conversion and only 8% isolated yield. Furthermore,
to investigate a lower flow rate and a higher temperature and to
evaluate whether the reported advantages noted in other
studies regarding more gentle heating using MWs could be
observed, a reaction at 200 °C with a flow rate of 0.5 mL/min
was performed (entry 2, Table 1). The reaction furnished 43%
yield, an outcome comparable with the 40% yield obtained at 1
mL/min (entry 1, Table 1). Noticeable is that the commonly
described advantages4,5,9 with in situ MWs vs wall heating
regarding higher temperatures, providing shorter reaction
times, and cleaner reactions were not observed in this case.
In contrast, employing the SiC reactor enabled lower reaction
temperatures, less complex reaction mixtures, and hence, easier
purification. We will continue to investigate plausible
explanations for the unexpected results observed by the
different mode of heating, but it is obvious to us that flow
profiles and heat gradients in the reaction mixture will differ
between the two reactors (SiC and borosilicate). The positive
outcome with the SiC reactor is in line with the conclusions by
Kappe; e.g., the positive effects of MW processing should be
considered as an effect of rapid heat exchange, something that
could be reproduced in small scale CF chemistry by wall
heating using reactor materials such as SiC.29
RESULTS AND DISCUSSION
■
We have previously developed a three-component batch
synthesis of IRAP inhibitor 1a from 5-bromo-1-methylisatin
2a, isatoic anhydride 3, and p-toluidine 4a using a 50:50
toluene−acetic acid solvent system. Thus, 5-bromoisatin was
first N-methylated to afford the starting material 2a using a
standard batch procedure. After a few test reactions, pure acetic
acid was selected as the solvent as it facilitated the yield and the
purification since 1a precipitated out upon cooling to room
temperature (72% yield). We next decided to change from
conventional reflux heating of 2 h to MW heating and sealed
vessels to reduce the reaction time. When heated to 150 °C
with a dedicated MW batch instrument, the reaction was
completed in 10 min, resulting in 75% yield of 1a.
The first MW-flow attempt was thereafter to synthesize 1a
using a MW transparent 200 mm × 3 mm Øi tubular
borosilicate glass reactor in our purpose-built MW-flow
applicator.24 We started by employing similar parameters as
previously developed for the MW batch synthesis. Hence, 2a, 3,
and 4a were used in a 1:1:1 relationship using acetic acid as
solvent with a concentration of 0.05 M (Scheme 1). A flow rate
of 1 mL/min was selected with a planned process temperature
range between 140 and 220 °C. This initial temperature screen
indicated that a temperature of 200 °C gave the best result and
collection for 5 min (1 mL/min) was performed, providing an
isolated yield of 40% of the desired compound 1a (entry 1,
Table 1). However, it was noted that the conversion was not
complete and that the starting material 2a still remained in the
processed reaction mixture together with uncharacterized
byproducts.
In parallel with this work, Konda et al. developed MW heated
CF synthesis using a different kind of reactor, a MW absorbing
200 mm × 3 mm Øi silicon carbide (SiC) tubular reactor, with
very promising results.28 Thus, we decided to investigate this
reactor for the multicomponent reaction. First of all, the
reactants were used with the same stoichiometry and
Both in the previously performed batch reactions and in the
flow synthesis of 1a, it was noted that remaining 5-bromo-N-
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Table 2. Synthesis of spiro-oxindole dihydroquinazolinones
methylisatin (2a) was observed in the mixture after the reaction
was performed. To investigate whether this could be avoided,
the nucleophilic p-toluidine (4a) was used in excess, affording a
reaction yield of 65% with the SiC reactor (entry 5, Table 1).
Next, using both 3 and 4a in excess (1.2 equiv) but with
otherwise identical conditions (120 °C, 0.5 mL/min) furnished
74% isolated yield of 1a (entry 6, Table 1). Surprisingly, using
1.5 equiv of 3 and 4a, respectively, resulted in similar yield
(entry 7, Table 1); however, increasing the temperature to 160
°C provided 1a in 82% yield (entry 8, Table 1). Hence, using
1.5 equiv of 3 and 4a, respectively, at 160 °C with a flow rate of
0.5 mL/min through the SiC reactor provided full conversion
of 2a and the highest isolated yield of 1a. The total residence
time for the three-component synthesis of 1a was only 168 s
(Table 1, entry 8).
A series of different analogues to the hit IRAP inhibitor 1a
was then synthesized utilizing the developed MW−SiC−CF
conditions from entry 8 in Table 1. A variety of anilines and
isatins were used to afford compounds 1b−i in 40−87% yield
(Table 2). We also intended to use aliphatic amines; however,
this did not work under these conditions probably due to
protonation. Likewise to the preparation of 1a from 2a, the
additional isatins 2b,c were prepared by N-methylation in batch
mode before participating in the multicomponent reaction.
Biological testing of the spiro-oxindole dihydroquinazoli-
nones 1a−i indicated that the bromine is preferred in the 5-
position, since removal of it (1g) decreased the activity more
than 10-fold, and switching of the bromine from the 5 to the 7-
position (1h) gave an inactive compound. In an attempt to
further look into the structure−activity-relationship (SAR), the
ability to perform a Suzuki−Miyaura cross-coupling reaction on
1a using MW-CF was investigated.43,44 The reaction conditions
were first optimized using MW batch chemistry with the aim to
reduce dehalogenation and hydrolysis of the aryl bromide 1a.
Different Pd-catalyst (Pd(PPh3)4, Pd(tBu3)2, PdCl2(dppf)),
bases (DBU, NaOH, KOH), solvent systems (MeCN, DMF,
DMA, and methyl ethyl ketone with adducts of H2O),
temperatures (160 °C, 180 °C, 200 °C), and 4-tolylboronic
acid in excess (1.5−3 equiv) were evaluated.45 From these
investigations, it was observed that minimization of water
content and slight excess of base were necessary to avoid
hydrolysis of the aminal part of 1a. Dehalogenation was not
highly dependent on concentration, temperature, or Pd-source.
However, it was affected by solvent and base. The following
reaction parameters were found to be highly suitable for the
Suzuki−Miyaura cross coupling; 1a (0.1 mmol, 1 equiv),
boronic acid (2 equiv), DBU (1.5 equiv), PdCl2(dppf) (2 mol
%), and MeCN with 2% H2O (5 mL). These MW-batch
conditions, using p-tolylboronic acid (5) as an arylating agent
and 180 °C for 1 min provided an isolated yield of 93% of 1j.
Next, we transferred the cross-coupling to the flow system
going back to the 3 mm Øi borosilicate reactor and aiming to
produce a library of biphenyl compounds in a CF process. A
temperature screen was performed ranging from 160 to 220 °C
with 20 °C increasing increments at a flow of 0.5 mL/min,
which showed that the highest temperature gave full conversion
of 1a. The flow rate was thereafter investigated, and the
productivities at 0.75, 1.0, 1.5, and 2.0 mL/min were analyzed,
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respectively. However, when increasing the flow rate, full
conversion of 1a could not be obtained. Product mixture was
thus collected for 5 min with a flow rate of 0.5 mL/min at 220
°C, which furnished a residence time of 168 s and a 71%
isolated yield of 1j (Scheme 2). Compared to the related work
by Konda et al., no issues with clogging or reactor rupture were
observed during these experiments.28
was determined using a calibrated external IR sensor. An Optris
CT sensor with a LT22 sensing head was used for the SiC tube
reactor, while the sensor used for the borosilicate glass tube
reactor was an Optris CSmicro 3 M sensor. For a schematic
picture of the MW-flow system, see the Supporting
Information. Commercially available starting materials and
1
solvents were used without further purification. H and 13C
NMR spectra were recorded at 25 °C and at 400 and 100 MHz,
respectively. Chemical shift are reported in parts per million
with the solvent residual peak as internal standard (CDCl3 δH
7.26, CDCl3 δC 77.16; CD3OD δH 3.31, CD3OD δC 49.0;
(CD3)2SO δH 2.50, (CD3)2SO δC 39.52). When using a
mixture of CD3OD and CDCl3 as solvent for NMR, the
reference peak was set to CD3OD. The batch microwave
experiments were conducted using a Biotage Smith Synthesizer
single mode cavity and sealed 2−5 mL process vials. Analytical
thin-layer chromatography was performed on silica gel 60 F-
254 plates and visualized with UV-light. Flash column
chromatography was performed on silica gel 60 (40−63 μM).
Analytical HPLC-UV/MS was performed on a Dionex Ultimate
3000 HPLC system with a Bruker Amazon SL ion trap mass
spectrometer and detection by UV (DAD) and MS (ESI+),
using a Phenomenex Kinetex C18 column (50 × 3.0 mm, 2.6
μM particle size, 100 Å pore size) and a flow rate of 1.5 mL/
min. A gradient of H2O/MeCN/0.05% HCOOH was used.
High-resolution MS (HRMS) data was produced using an ES-
TOF instrument.
General Procedure for N-Methylation of Isatins (2a−
c). To a suspension of the appropriate isatin (1 equiv) and
Cs2CO3 (2 equiv) in dry MeCN (0.05 M isatin) was added MeI
(1.1 equiv). The reaction mixture was stirred for 7 h at ambient
temperature. Subsequently the reaction mixture was concen-
trated under reduced pressure, dissolved in EtOAc and washed
with water and brine, dried over MgSO4, filtered, and
concentrated. The product was used in the next step without
further purification.
5-Bromo-1-methylindolone-2,3-dione (2a). The com-
pound was synthesized according to the general procedure
from 5-bromo-isatin (1.00 g, 4.42 mmol), Cs2CO3 (2.90 g, 8.90
mmol), and MeI (0.31 mL, 4.98 mmol) to give 2a as a red solid
(1.05 g, 98%).46 1H NMR (400 MHz, DMSO-d6) δ 7.85 (dd, J
= 8.4, 2.1 Hz, 1H), 7.69 (d, J = 2.1 Hz, 1H), 7.12 (d, J = 8.4 Hz,
1H), 3.12 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 182.6,
158.2, 150.7, 140.2, 126.9, 119.6, 115.3, 113.1, 26.5.
1-Methylindoline-2,3-dione (2b). The compound was
synthesized according to the general procedure from isatin
(2.00 g, 13.59 mmol), Cs2CO3 (8.90 g, 27.32 mmol), and MeI
(0.95 mL, 15.26 mmol) to give 2b as a red solid (1.99 g,
91%).46 1H NMR (400 MHz, CDCl3) δ 7.58 (td, J = 7.8, 1.3
Hz, 1H), 7.53 (ddd, J = 7.4, 1.4, 0.7 Hz, 1H), 7.09 (td, J = 7.5,
0.8 Hz, 1H), 6.88 (dt, J = 7.9, 0.7 Hz, 1H), 3.21 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 183.4, 158.3, 151.5, 138.5, 125.2,
123.9, 117.4, 110.1, 26.3.
Scheme 2. MW-heated continuous flow Suzuki−Miyaura
cross-coupling reaction
Disappointingly, elongated biaryl compound 1j furnished no
IRAP inhibiting activity. This results indicated that substitution
of the bromine in the 5-position is not permitted, and the idea
of producing Suzuki-Miyaura based IRAP-inhibitor library by
MW-CF methodology was ultimately abandoned. In the near
future, other parts of the molecule will be investigated to gain
further knowledge about the SAR for this class of IRAP
inhibitors.
CONCLUSION
■
As a result of a screen of 10 500 compound a new type of spiro-
oxindole dihydroquinazolinone based IRAP inhibitors (1a) was
identified. Hence, a fast, productive, and scalable synthetic
method for this class of structures was needed. Thus, we
developed a synthetic route employing MW-heated CF
synthesis with a MW-absorbent 200 mm × 3 mm Øi SiC
reactor to smoothly produce 1a in 82% isolated yield. This
method was compatible with different kinds of anilines and
isatines, delivering nine test compounds (1a−i) in moderate to
high yields. Further, we also showed that derivatization of 1a by
Suzuki−Miyaura cross-coupling was possible using the MW-
flow setup. This time a MW-transparent borosilicate reactor
was used providing an isolated yield of 71% of the cross-
coupling product 1j. While it may be debatable if the synthetic
procedures described within this report is a better route to the
target compounds 1a−j, or not, we wish to inform the reader of
our work, which allows an alternative approach to be
considered. The utilized MW-CF system is limited to lab-
scale, but the technique as such is scalable. We will continue to
investigate the flow and temperature profiles of the reaction
mixture in the CF tube reactors, as well as various technical
modifications/additions in order to disrupt a laminar flow
profile.
7-Bromo-1-methylindoline-2,3-dione (2c). The com-
pound was synthesized according to the general procedure
from 7-bromo-isatin (1.00 g, 4.24 mmol), Cs2CO3 (2.90 g, 8.90
mmol), and MeI (0.31 mL, 4.98 mmol) to give 2c as a red solid
(973 mg, 92%).47 1H NMR (400 MHz, CDCl3) δ 7.70 (dd, J =
1.3, 8.1 Hz, 1H), 7.56 (dd, J = 1.3, 7.3 Hz, 1H), 6.99 (ddd, J =
8.1, 7.3, 0.6 Hz, 1H), 3.65 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 182.6, 158.7, 148.4, 143.9, 125.2, 124.6, 120.6, 104.5,
29.9.
EXPERIMENTAL SECTION
■
General Information. The MW generator, reactor,
applicator, and cavity are part of the ArrheniusOne system by
WaveCraft AB. This system operates in a nonresonant mode
and features a straight tube borosilicate glass or Si/C reactor.
The pump used was a Shimadzu LC-10AD HPLC pump.
WaveCraft adjustable back-pressure regulator ranging from 10
to 150 bar was used to regulate the pressure. The temperature
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General Procedure for the Optimization or the Three-
Component Formation of 1a. A 0.05 M solution of 2a (1
equiv), p-toluidine (1−1.5 equiv), and isatoic anhydride (1−1.5
equiv) in AcOH was stirred at ambient temperature for
approximately 30 min to afford all reactants in solution, and the
reaction mixture was subsequently filtered through a 25 mm
syringe filter with 0.45 μM PTFE membrane to removed
remaining particles. The reaction mixture was then pumped
through the 200 mm × 3 mm Øi borosilicate or SiC reactor at a
flow rate of 0.25, 0.5, and 1 mL/min and at temperatures of
100−240 °C with intervals of 20 °C. The wash-out volume was
3 mL.
chromatography using 20% EtOAc in toluene followed by a
second column using 40% EtOAc in pentane as eluent to afford
1c as a light orange solid (211 mg, 40%). 1H NMR (400 MHz,
CD3OD) δ 7.85 (dd, J = 7.8, 1.6 Hz, 1H), 7.65 (d, J = 2.0 Hz,
1H), 7.48 (d, J = 8.3 Hz, 2H), 7.41 (dd, J = 8.4, 2.0 Hz, 1H),
7.37−7.30 (m, 1H), 7.11 (d, J = 8.2 Hz, 2H), 6.86 (td, J = 7.6,
1.1 Hz, 1H), 6.69 (dd, J = 8.1, 1.0 Hz, 1H), 6.62 (d, J = 8.4 Hz,
1H), 3.01 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 173.6,
165.3, 145.8, 142.3, 141.1, 135.1, 134.8, 130.3, 129.2, 128.9,
128.7, 126.5, 119.9, 116.4, 115.0, 114.9, 111.1, 110.6, 78.0, 26.4.
HRMS (ES) m/z calcd. for C23H15BrF3N3O2: [M + H]+
502.0365, found: 502.0378.
General Procedure for Three-Component Formation
of 1a−h. A 0.05 M solution of the appropriate N-methylated
isatin 2a−c (1 equiv), the appropriate aniline (1.5 equiv), and
isatoic anhydride (1.5 equiv) in AcOH was stirred at ambient
temperature for 30 min to afford all reactants in solution, and
the reaction mixture was subsequently filtered through a 25 mm
syringe filter with 0.45 μM PTFE membrane to removed
remaining particles. The reaction mixture was pumped through
the 200 mm × 3 mm Øi SiC reactor at a flow of 0.5 mL/min
and at a temperature of 160 °C. The obtained product was
either purified by suspension in hot EtOH, cooling to ambient
temperature and filtration, or by flash column chromatography.
5-Bromo-1-methyl-3′-(p-tolyl)-1′H-spiro[indoline-
3,2′-quinazoline]-2,4′(3′H)-dione (1a). The product was
synthesized according to the general procedure, and a reaction
mixture volume of 21.0 mL was collected. The reaction mixture
was left for 14 h for the product to precipitate out. The crude
product was filtered and subsequently suspended in hot EtOH,
cooled to ambient temperature and filtered, and 1a was
obtained as a beige solid (286 mg, 82%). 1H NMR (400 MHz,
DMSO-d6) δ 7.82 (d, J = 2.0 Hz, 1H), 7.68 (dd, J = 7.7, 1.5 Hz,
1H), 7.62 (s, 1H), 7.46 (dd, J = 8.4, 2.1 Hz, 1H), 7.32 (ddd, J =
8.0, 7.3, 1.6 Hz, 1H), 7.07−6.99 (m, 2H), 6.90−6.82 (m, 3H),
6.79 (td, J = 7.6, 1.1 Hz, 1H), 6.69 (dd, J = 8.1, 1.0 Hz, 1H),
3.01 (s, 3H), 2.19 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
174.0, 163.8, 146.2, 142.9, 137.6, 135.5, 134.2, 134.1, 129.7,
129.4, 129.3, 127.9, 118.4, 115.0, 114.96, 114.94, 114.6, 111.5,
76.5, 26.5, 21.0. HRMS (ES) m/z calcd. for C23H18BrN3O2: [M
+ H]+ 448.0661, found: 448.0662.
5-Bromo-1-methyl-3′-phenyl-1′H-spiro[indoline-3,2′-
quinazoline]-2,4′(3′H)-dione (1b). The product was synthe-
sized according to the general procedure, and a reaction
mixture volume of 21.0 mL was collected. The reaction mixture
was left for 14 h for the product to precipitate out. The crude
product was filtered and subsequently suspended in hot EtOH,
cooled to ambient temperature, and filtered, and 1b was
obtained as a yellow solid (389 mg, 85%). 1H NMR (400 MHz,
CD3OD) δ 7.85 (dd, J = 7.8, 1.5 Hz, 1H), 7.64 (d, J = 2.0 Hz,
1H), 7.37 (dd, J = 8.3, 2.0 Hz, 1H), 7.32 (ddd, J = 8.1, 7.3, 1.6
Hz, 1H), 7.24−7.14 (m, 3H), 6.98−6.91 (m, 2H), 6.85 (td, J =
7.6, 1.0 Hz, 1H), 6.70−6.65 (m, 1H), 6.57 (d, J = 8.3 Hz, 1H),
2.99 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 174.0, 165.4,
145.9, 142.4, 137.5, 134.9, 134.4, 129.6, 129.5, 129.1, 128.9,
128.7, 119.7, 116.2, 115.1, 114.9, 110.9, 77.3, 26.3. HRMS (ES)
m/z calcd. for C22H16BrN3O2: [M + H]+ 434.0493, found:
434.0504.
5-Bromo-3′-(4-(tert-Butyl)phenyl)-1-methyl-1′H-spiro-
[indoline-3,2′-quinazoline]-2,4′(3′H)-dione (1d). The
product was synthesized according to the general procedure,
and a reaction mixture volume of 21.0 mL was collected. The
crude product was purified by flash column chromatography
using 20% EtOAc in toluene followed by a second column
using 40% EtOAc in pentane as eluent to afford 1d as a light
pink solid (404 mg, 78%). 1H NMR (400 MHz, CDCl3) δ 8.00
(ddd, J = 7.8, 1.5, 0.5 Hz, 1H), 7.58 (dd, J = 2.0, 0.4 Hz, 1H),
7.39 (ddd, J = 8.4, 2.0, 0.5 Hz, 1H), 7.37−7.33 (m, 1H), 7.23−
7.16 (m, 2H), 7.02−6.95 (m, 1H), 6.84 (d, J = 8.1 Hz, 1H),
6.65 (ddd, J = 8.0, 1.0, 0.5 Hz, 1H), 6.53 (d, J = 8.3 Hz, 1H),
2.97 (s, 3H), 1.21 (s, 9H). 13C NMR (101 MHz, CDCl3) δ
172.6, 164.1, 151.5, 144.0, 141.8, 134.29, 134.27, 134.1, 129.6,
129.2, 128.6, 128.4, 126.1, 120.0, 116.7, 116.1, 115.4, 110.4,
76.6, 34.7, 31.3, 26.4. HRMS (ES) m/z calcd. for
C26H24BrN3O2: [M + H]+ 490.1135, found: 490.1130.
5-Bromo-3′-(4-iodophenyl)-1-methyl-1′H-spiro-
[indoline-3,2′-quinazoline]-2,4′(3′H)-dione (1e). The
product was synthesized according to the general procedure,
and a reaction mixture volume of 8.0 mL was collected. The
crude product was purified by flash column chromatography
using 20% EtOAc in toluene as eluent to afford 1e as an off-
white solid (195 mg, 87%). 1H NMR (400 MHz, DMSO-d6) δ
7.85 (d, J = 2.0 Hz, 1H), 7.71−7.65 (m, 2H), 7.63−7.58 (m,
2H), 7.50 (dd, J = 8.4, 2.1 Hz, 1H), 7.34 (ddd, J = 8.0, 7.3, 1.6
Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.79 (ddd, J = 7.5, 5.8, 1.2
Hz, 3H), 6.69 (dd, J = 8.2, 1.0 Hz, 1H), 3.03 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 173.3, 163.3, 145.8, 142.5, 137.8,
137.6, 134.0, 133.9, 131.5, 129.1, 128.5, 127.5, 118.1, 114.7,
114.3, 111.3, 94.3, 75.9, 26.2. HRMS (ES) m/z calcd. for
C22H15BrIN3O2: [M + H]+ 559.9459, found: 559.9471.
5-Bromo-1-methyl-3′-(3-(oxazol-5-yl)phenyl)-1′H-
spiro[indoline-3,2′-quinazoline]-2,4′(3′H)-dione (1f). The
product was synthesized according to the general procedure,
and a reaction mixture volume of 15.0 mL was collected. The
reaction mixture was left for 14 h for the product to precipitate
out. The crude product was filtered and subsequently
suspended in hot EtOH, cooled to ambient temperature, and
filtered, and 1f was obtained as a brown solid (230 mg, 61%).
1H NMR (400 MHz, DMSO-d6) δ 8.43 (s, 1H), 7.93 (d, J = 2.1
Hz, 1H), 7.74−7.69 (m, 2H), 7.62 (s, 1H), 7.53 (dt, J = 7.9, 1.2
Hz, 1H), 7.44 (dd, J = 8.4, 2.1 Hz, 1H), 7.40−7.31 (m, 3H),
6.96 (d, J = 7.7 Hz, 1H), 6.84 (d, J = 8.4 Hz, 1H), 6.80 (dd, J =
7.6, 1.0 Hz, 1H), 6.72 (dd, J = 8.2, 1.0 Hz, 1H), 3.02 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ 173.5, 163.3, 152.1, 149.5,
145.8, 142.5, 138.4, 134.0, 133.8, 129.8, 129.3, 128.4, 128.0,
127.6, 123.5, 122.4, 118.2, 114.6, 114.4, 114.3, 111.1, 76.1, 26.1.
HRMS (ES) m/z calcd. for C25H17BrN4O3: [M + H]+
501.0544, found: 501.0562.
5-Bromo-1-methyl-3′-(4-(trifluoromethyl)phenyl)-1′H-
spiro[indoline-3,2′-quinazoline]-2,4′(3′H)-dione (1c).
The product was synthesized according to the general
procedure, and a reaction mixture volume of 21.0 mL was
collected. The crude product was purified by flash column
E
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1-Methyl-3′-(p-tolyl)-1′H-spiro[indoline-3,2′-quinazo-
line]-2,4′(3′H)-dione (1g). The product was synthesized
according to the general procedure, and a reaction mixture
volume of 21.0 mL was collected. The reaction mixture was left
for 14 h for the product to precipitate out. The crude product
was filtered and subsequently suspended in hot EtOH, cooled
to ambient temperature, and filtered, and 1g was obtained as a
Hz, 1H), 7.27−7.22 (m, 2H), 7.01−6.96 (m, 2H), 6.92 (m,
3H), 6.77 (ddd, J = 7.8, 7.3, 1.1 Hz, 1H), 6.70 (ddd, J = 8.1, 1.1,
0.5 Hz, 1H), 3.06 (s, 3H), 2.33 (s, 3H), 2.15 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 174.1, 163.6, 146.0, 142.4, 136.9,
136.46, 136.47, 135.4, 134.9, 133.6, 129.5, 129.2, 128.8, 127.5,
127.4, 126.1, 124.2, 117.8, 114.7, 114.1, 109.4, 76.2, 26.1, 20.6,
20.5. HRMS (ES) m/z calcd. for C30H25N3O2: [M + H]+
460.2003, found: 460.2025.
1
yellow solid (289 mg, 75%). H NMR (400 MHz, CD3OD) δ
Enzymatic Assay. An enzymatic assay measuring IRAP
activity was applied for screening purposes as well as follow-up
dose−response characterization. Membrane preparations from
Chinese hamster ovary (CHO) cells are used as a source of
enzyme activity. The extent of enzymatic activity in each sample
was quantified in a microtiter based screening assay using the
peptide like substrate L-leucine-p-nitroanilide (L-Leu-pNA),
which upon IRAP mediated cleavage produces p-nitroanilide
which absorbs at 405 nm. The screen (reported elsewhere) was
conducted in a 384-well format using transparent microplates,
whereas follow-up dose−response experiments were conducted
in the equivalent transparent 96-well plate (Nunc, product
number: 269620). In this format the assay volume was 200 μL,
and the buffer was 50 mM Tris-HCl, 150 mM NaCl, and 0.1
mM phenylmethanesulfonylfluoride (PMSF) at pH 7.4. The
assays were conducted in the presence of a final concentration
of 1 mM L-Leu-pNA and CHO cell membranes from a total of
50.000−200.000 cells per well. The CHO membrane
preparation was performed as described in Demaegt et al.,
2004, with the lysis of washed cells being done by means of
ultrasonication followed by multiple strokes (>20) with a
Dounze homogenizer and an ultracentrifugation procedure for
pelleting and washing the membranes.48 The protocol for
running the dose−response assay started with a serial dilution
of the compound stock solutions at 10 mM in pure DMSO by a
factor of 1/3 in columns 1 through to 11 of the U-formed 96-
well plates. Column 12 was reserved for controls meaning the
equivalent amount of DMSO was added to the wells A12−D12,
whereas a 10 mM stock solution of 6 (Figure 2) was placed in
7.84 (dd, J = 7.8, 1.5 Hz, 1H), 7.51 (ddd, J = 7.5, 1.3, 0.6 Hz,
1H), 7.30 (ddd, J = 8.1, 7.3, 1.6 Hz, 1H), 7.25 (td, J = 7.8, 1.3
Hz, 1H), 7.02 (td, J = 7.6, 1.0 Hz, 1H), 6.97−6.91 (m, 2H),
6.87−6.75 (m, 3H), 6.70−6.63 (m, 2H), 3.02 (s, 3H), 2.19 (s,
3H). 13C NMR (101 MHz, CD3OD) δ 174.5, 165.8, 146.1,
143.2, 138.5, 135.0, 134.6, 131.6, 129.9, 128.6, 127.7, 126.0,
123.9, 119.5, 115.4, 114.8, 109.3, 77.4, 26.3, 21.1. HRMS (ES)
m/z calcd. for C23H19N3O2: [M + H]+ 370.1554, found:
370.1556.
7-Bromo-1-methyl-3′-(p-tolyl)-1′H-spiro[indoline-
3,2′-quinazoline]-2,4′(3′H)-dione (1h). The product was
synthesized according to the general procedure, and a reaction
mixture volume of 13.0 mL was collected. The reaction mixture
was left for 14 h for the product to precipitate out. The crude
product was filtered and subsequently suspended in hot EtOH,
cooled to ambient temperature, and filtered, and 1h was
obtained as a yellow solid (215 mg, 74%). 1H NMR (400 MHz,
DMSO-d6) δ 7.71−7.66 (m, 2H), 7.61 (s, 1H), 7.44 (dd, J =
8.2, 1.2 Hz, 1H), 7.33 (dddd, J = 8.1, 7.3, 1.6, 0.8 Hz, 1H),
7.06−7.00 (m, 2H), 6.96 (dd, J = 8.2, 7.3 Hz, 1H), 6.84−6.75
(m, 3H), 6.68 (ddd, J = 8.2, 1.1, 0.5 Hz, 1H), 2.19 (s, 3H). 13
C
NMR (101 MHz, DMSO-d6) δ 174.6, 163.5, 145.8, 145.7,
140.3, 137.2, 136.2, 135.1, 135.1, 133.9, 130.2, 129.4, 127.6,
126.1, 124.7, 118.1, 114.5, 114.2, 101.7, 75.6, 29.5, 20.6. HRMS
(ES) m/z calcd. for C23H18BrN3O2: [M + H]+ 448.0667, found:
448.0661.
5-Bromo-3′-(p-tolyl)-1′H-spiro[indoline-3,2′-quinazo-
line]-2,4′(3′H)-dione (1i). The product was synthesized
according to the general procedure, and a reaction mixture
volume of 21.0 mL was collected. The crude product was
purified by flash column chromatography using 40% EtOAc in
1
hexane to afford 1i as a grey solid (354 mg, 78%). H NMR
(400 MHz, CD3OD) δ 7.85 (dd, J = 7.8, 1.5 Hz, 1H), 7.54 (d, J
= 2.0 Hz, 1H), 7.29 (ddd, J = 8.1, 7.3, 1.6 Hz, 1H), 7.25 (dd, J =
8.3, 2.0 Hz, 1H), 6.97 (dt, J = 7.3, 0.9 Hz, 2H), 6.90−6.81 (m,
3H), 6.63 (dd, J = 8.1, 0.9 Hz, 1H), 6.51 (d, J = 8.3 Hz, 1H),
2.19 (s, 3H). 13C NMR (101 MHz, CD3OD) δ 175.4, 164.9,
145.0, 140.0, 138.3, 134.5, 134.3, 133.9, 131.8, 130.0, 129.8,
128.9, 128.5, 119.6, 115.3, 115.3, 114.6, 112.2, 77.5, 21.0.
HRMS (ES) m/z calcd. for C22H16BrN3O2: [M + H]+
434.0509, found: 434.0504.
Figure 2. Structure of the inhibitor used as a control for enzymatic
activity.
wells E12−H12 serving as a control representing 100%
inhibition of the enzymatic activity. The DMSO solutions
were then diluted with assay buffer and transferred to the
transparent assay plates in triplicates (50 μL to each well both
for samples and controls), followed by addition of diluted
homogenized CHO cell membranes (50 μL) and substrate
(100 μL) to initiate the enzymatic reactions. The plates were
covered with lids and then incubated at room temperature until
a statistically significant difference (6−12 h) was observed
between the controls (Z′ values above 0.8). Care was taken to
ensure the readings were taken at a time point when the
absorbance signal still increased linearly with time. Raw data
were then imported into Microsoft Excel for a conversion to %
inhibition data based on the controls on each plate. Following
averaging of data from the triplicate samples the curves were
fitted to a four-parameter dose response model within XLfit
1-Methyl-3′,5-di-p-tolyl-1′H-spiro[indoline-3,2′-quina-
zoline]-2,4′(3′H)-dione (1j). A 0.02 M solution of 1a (90 mg,
0.2 mmol), p-tolylboronic acid (54 mg, 0.4 mmol), DBU (60
μL, 0.4 mmol), and PdCl2(dppf) (2 mol %) in 2% H2O in
MeCN was mixed and filtered through a 25 mm syringe filter
with 0.45 μM PTFE membrane to remove the remaining
particles. The reaction mixture was pumped through the 200
mm × 3 mm Øi borosilicate reactor at a flow of 0.5 mL/min
and a temperature of 220 °C. The crude product was
concentrated and purified by flash column chromatography
using 0.5−2.5% MeOH in DCM as eluent to afford 1j as a
1
white solid (92 mg, 71%). H NMR (400 MHz, DMSO-d6) δ
7.91 (dd, J = 2.0, 0.5 Hz, 1H), 7.69 (dd, J = 7.8, 1.6 Hz, 1H),
7.63 (s, 1H), 7.58−7.49 (m, 3H), 7.31 (ddd, J = 8.1, 7.3, 1.6
F
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Organic Process Research & Development
Communication
(22) Bowman, M. D.; Holcomb, J. L.; Kormos, C. M.; Leadbeater, N.
E.; Williams, V. A. Org. Process Res. Dev. 2008, 12, 41.
(model 205) to obtain best-fit values for the IC50 value, Hill
slope, and the upper and lower limits of the dose−response
curve.
(23) Morschhauser, R.; Krull, M.; Kayser, C.; Boberski, C.; Bierbaum,
̈
R.; Puschner, P. A.; Glasnov, T. N.; Kappe, C. O. Green Process Synth.
̈
2012, 1, 281.
ASSOCIATED CONTENT
* Supporting Information
Copies of spectra and chromatograms. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
̈
(24) Ohrngren, P.; Fardost, A.; Russo, F.; Schanche, J.-S.; Fagrell, M.;
S
Larhed, M. Org. Process Res. Dev. 2012, 16, 1053.
(25) Rydfjord, J.; Svensson, F.; Trejos, A.; Sjoberg, P. J. R.; Skold, C.;
̈
̈
Savmarker, J.; Odell, L. R.; Larhed, M. Chem.Eur. J. 2013, 19, 13803.
̈
(26) Rydfjord, J.; Svensson, F.; Fagrell, M.; Savmarker, J.; Thulin, M.;
Larhed, M. Beilstein J. Org. Chem. 2013, 9, 2079.
̈
AUTHOR INFORMATION
Corresponding Author
*Tel.: +46 18 471 4667. Fax: +46 18 471 4474. E-mail: mats.
larhed@orgfarm.uu.se.
■
(27) Sauks, J. M.; Mallik, D.; Lawryshyn, Y.; Bender, T.; Organ, M.
Org. Process Res. Dev. 2013, DOI: 10.1021/op400026g.
(28) Konda, V.; Rydfjord, J.; Savmarker, J.; Larhed, M. Org. Process
̈
Res. Dev. 2014, DOI: 10.1021/op50017989.
(29) Kappe, C. O. Acc. Chem. Res. 2013, 46, 1579.
(30) Braszko, J.; Kupryszewski, G.; Witczuk, B.; Wisn
Neuroscience 1988, 27, 777.
Notes
́
iewski, K.
The authors declare no competing financial interest.
(31) Andersson, H.; Hallberg, M. Int. J. Hypertension 2012, 2012,
789671.
(32) Harding, J. W.; Cook, V. I.; Miller-Wing, A. V.; Hanesworth, J.
M.; Sardinia, M. F.; Hall, K. L.; Stobb, J. W.; Swanson, G. N.;
Coleman, J. K. M.; Wright, J. W.; Harding, E. C. Brain Res. 1992, 583,
340.
(33) Swanson, G. N.; Hanesworth, J. M.; Sardinia, M. F.; Coleman, J.
K.; Wright, J. W.; Hall, K. L.; Miller-Wing, A. V.; Stobb, J. W.; Cook,
V. I.; Harding, E. C. Regul. Pept. 1992, 40, 409.
ACKNOWLEDGMENTS
■
Funding for the work at Chemical Biology Consortium Sweden
was provided by the Swedish Research Council and Karolinska
Institutet (A.J.J. and T.L). We thank the Knut and Alice
Wallenberg Foundation for financial support. We also want to
express our gratitude to Magnus Fagrell and Wavecraft AB for
their help with the fabrication of the SiC reactors.
(34) Albiston, A. L.; McDowall, S. G.; Matsacos, D.; Sim, P.; Clune,
E.; Mustafa, T.; Lee, J.; Mendelsohn, F. A.; Simpson, R. J.; Connolly,
L. M.; Chai, S. Y. J. Biol. Chem. 2001, 276, 48623.
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G.; Dupont, A.; Michotte, Y.; Vanderheyden, P. Heart Fail. Rev. 2008,
13, 321.
ABBREVIATIONS
MW, microwave; CF, continuous flow; SiC, silicon carbide;
IRAP, insulin-regulated aminopeptidase; Ang IV, Angiotensin
IV
■
(36) Andersson, H.; Demaegdt, H.; Vauquelin, G.; Lindeberg, G.;
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