Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP Inhibitors

Article abstract of DOI:10.1002/open.201900344

Insulin-regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non-peptide IRAP inhibitors derived from a spiro-oxindole dihydroquinazolinone screening hit (pIC₅₀ 5.8). The compounds were synthesized either by a simple microwave (MW)-promoted three-component reaction, or by a two-step one-pot procedure. For decoration of the oxindole ring system, rapid MW-assisted Suzuki-Miyaura cross-couplings (1 min) were performed. A small improvement of potency (pIC₅₀ 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S-configuration of the spiro-oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

Full text of DOI:10.1002/open.201900344

DOI: 10.1002/open.201900344
Full Papers
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Synthesis, Evaluation and Proposed Binding Pose of
Substituted Spiro-Oxindole Dihydroquinazolinones as IRAP
Inhibitors
Karin Engen,[a] Sudarsana Reddy Vanga,[b] Thomas Lundbäck,[c, d] Faith Agalo,[a] Vivek Konda,[a]
Annika Jenmalm Jensen,[c] Johan Åqvist,[b] Hugo Gutiérrez-de-Terán,[e] Mathias Hallberg,[f]
Mats Larhed,[g] and Ulrika Rosenström*[a]
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Insulin-regulated aminopeptidase (IRAP) is a new potential
macromolecular target for drugs aimed for treatment of
cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV)
improves the memory and learning in rats. The majority of the
known IRAP inhibitors are peptidic in character and suffer from
poor pharmacokinetic properties. Herein, we present a series of
small non-peptide IRAP inhibitors derived from a spiro-oxindole
dihydroquinazolinone screening hit (pIC50 5.8). The compounds
were synthesized either by a simple microwave (MW)-promoted
three-component reaction, or by a two-step one-pot procedure.
For decoration of the oxindole ring system, rapid MW-assisted
Suzuki-Miyaura cross-couplings (1 min) were performed. A small
improvement of potency (pIC50 6.6 for the most potent
compound) and an increased solubility could be achieved. As
deduced from computational modelling and MD simulations it
is proposed that the S-configuration of the spiro-oxindole
dihydroquinazolinones accounts for the inhibition of IRAP.
1. Introduction
Insulin-regulated aminopeptidase (IRAP, oxytocinase, EC
3.4.11.3) is an enzyme abundantly expressed in areas of the
brain associated with cognition, including amygdala, hippo-
campus and cerebral cortex. Interference with the amino-
peptidase activity, such as after administration of the endoge-
nous hexapeptide angiotensin IV (Ang IV), has demonstrated
that cognitive enhancement in experimental models can be
accomplished.[1–4] Mechanistically IRAP has the unique ability to
cleave the N-terminal peptide bonds of macrocyclic peptidic
regulators of cognition, such as oxytocin and vasopressin.[5–8]
The interest in IRAP as a potential target for treatment of
cognitive disorders has grown during the last decade. However,
IRAP also mediates several important physiological and patho-
logical activities not only those associated to cognition. It has
also an impact on parameters related to immunology, i.e.
antigen processing and trafficking of T-cells receptors; glucose
metabolism, i.e. trafficking of glucose transporters; and fibrosis,
i.e. regulation of fibrotic responses in heart and kidney.[9–15]
Hence, efficient inhibitors of IRAP could serve as a potential
new class of cognitive enhancers in the future, but in addition
conceivably, also act as agents useful for e.g. regulation of
immune responses. Highly potent peptidic and pseudo-peptidic
IRAP inhibitors with selectivity for IRAP, as opposed to other
aminopeptidases, have been identified.[16–20] Our group synthe-
sized both linear and constrained Ang IV analogues[21–23] and
macrocycles mimicking the N-terminal of oxytocin and obtained
a series of inhibitors with high potency and stability.[24,25] One of
the compounds, HA08 (see Figure 1), was recently proven to
alter dendritic spine density in rat hippocampal primary
cultures.[26] However, HA08 and structurally related analogues
are still peptidic in character and are foreseen to suffer from
[a] K. Engen, Dr. F. Agalo, V. Konda, Dr. U. Rosenström
Department of Medicinal Chemistry
Uppsala University
SE-751 23 Uppsala, SWEDEN
E-mail: ulrika.rosenstrom@ilk.uu.se
[b] Dr. S. R. Vanga, Prof. J. Åqvist
Department of Cell and Molecular Biology
Uppsala University
SE-751 23 Uppsala, SWEDEN
[c] Dr. T. Lundbäck, Dr. A. J. Jensen
Chemical Biology Consortium Sweden, Science for Life Laboratory, Depart-
ment of Medical Biochemistry and Biophysics
Karolinska Institutet
SE-171 65 Solna, SWEDEN
[d] Dr. T. Lundbäck
Mechanistic Biology & Profiling, Discovery Sciences, R&D
AstraZeneca
SE-431 83 Göteborg, SWEDEN
[e] Dr. H. Gutiérrez-de-Terán
Science for Life Laboratory, Department of Cell and Molecular Biology
Uppsala University
SE-751 23 Uppsala, SWEDEN
[f] Prof. Dr. M. Hallberg
The Beijer Laboratory, Department of Pharmaceutical Biosciences
Uppsala University
SE-751 23 Uppsala, SWEDEN
[g] Prof. Dr. M. Larhed
Science for Life Laboratory, Department of Medicinal Chemistry
Uppsala University
SE-751 23 Uppsala, SWEDEN
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/open.201900344
© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This
is an open access article under the terms of the Creative Commons Attri-
bution Non-Commercial NoDerivs License, which permits use and distribu-
tion in any medium, provided the original work is properly cited, the use is
non-commercial and no modifications or adaptations are made.
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Table 1. Evaluation of compounds 1–24 as IRAP inhibitors.
1
2
3
4
5
6
7
a
Cmpd
R1
R5
R7
pIC50
8
9
5.8�0.2 (21)[b]
4.8�0.02 (2)[c]
Inactive (2)[d]
1
Figure 1. Two examples of known IRAP inhibitors.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
2
3
4
5
6
4.0�0.4 (4)[b]
Inactive (3)[b]
4.9�0.03 (3)[b]
5.7�0.03 (3)[b]
6.0�0.06 (3)[b]
low bioavailability and a limited ability to pass the blood-brain-
barrier. The first published drug-like class of IRAP inhibitors,
encompassing a benzopyran scaffold (e.g. HFI-419), has been
reported to exhibit a dose-dependent improvement of visual
recognition task in rats. Administration in lateral ventricles also
improved spatial working memory.[27] Furthermore, it was
recently proposed that these IRAP inhibitors facilitate memory
by increasing hippocampal dendritic spine density via a GLUT4-
mediated mechanism.[28] Altogether, these benzopyran-based
molecules represent the first generation of non-peptidic IRAP
inhibitors that elicit effects in in vivo experimental models.
Unfortunately, this class of inhibitors seems to be associated
with a relatively short half-life and high plasma clearance.[29]
The promising in vivo results encouraged us to commence a
screening campaign applying an IRAP activity assay based on
natural expression of the target in Chinese Hamster Ovary
(CHO) cells to identify novel drug-like IRAP inhibitors. A
compound library of approximately 10 500 compounds was
screened and a limited number of hit families were identified.[30]
The basic structure activity relationship (SAR) of an arylsulfona-
mide-based class of inhibitors from this screen was recently
reported, and we could also demonstrate that these com-
pounds can increase the number of mushroom-shaped den-
5.8�0.08 (5)[b]
4.5�0.01 (2)[c]
Inactive (2)[d]
7
8
<3.9 (3)[b]
9
4.8�0.3 (3)[b]
4.8�0.2 (3)[b]
<3.9 (3)[b]
Inactive (3)[b]
Inactive (4)[b]
Inactive (3)[b]
<3.9 (3)[b]
10
11
12
13
14
15
16
17
18
19
20
21
<3.9 (3)[b]
Inactive (3)[b]
4.2�0.4 (4)[b]
4.1�0.4 (3)[b]
<3.9 (3)[b]
Inactive (4)[b]
22
23
24
Inactive (3)[b]
4.6�0.3 (4)[b]
<3.9 (3)[b]
dritic spines,
a
morphology associated with memory
enhancement.[31–33]
[a] Reported pIC50 values represent the mean � standard deviation of best-
fit values (the number of independent test occasions is provided within
brackets). No values below 3.9 are reported as this was the highest
compound concentration tested. [b] Evaluated on IRAP from CHO cells. [c]
Evaluated on human membrane bound IRAP overexpressed in HEK293
cells. [d] Evaluated on human aminopeptidase N overexpressed in HEK293
cells.
Herein, we report the synthesis and initial SAR of a new
class of small-molecule IRAP inhibitors, comprising a spiro-
oxindole dihydroquinazolinone scaffold, originating from a hit
compound (1) exhibiting a pIC50 value of 5.8.[30] Compound 1 is
relatively lipophilic with a measured logD of 3.4 and suffers
from poor solubility and metabolic stability (Tables 1 and 2).
Our aims in this study were to gain a better understanding of
the SAR around this scaffold, define the mechanism of IRAP
inhibition and use this knowledge to improve the properties of
compound 1.
of analogs in a smooth and scalable way using microwave (MW)
heated continuous flow synthesis (1–3, 23, 26–29, 31).[40,41] Here
we show that these spiro-compounds can be obtained utilizing
MW heated batch chemistry in a rapid and simple way. By
mixing isatoic anhydride, the appropriate aniline and the
appropriate isatin in acetic acid and heat the reaction in a
sealed vial using MW irradiation for 10 min, the desired
compounds (4–7, 9, 24–25) could be obtained in moderate to
excellent yields (53–93%, Scheme 1). By utilizing this method,
compounds with variations in position 1, 5 and 7 could be
investigated (see Table 1 for nomenclature). Evaluation of the
2. Results and Discussion
Two and three-component reactions of structurally similar
compounds have been published, typically requiring reflux for
several hours.[34–39] We aimed to improve the synthetic proce-
dures for this type of scaffold and have previously demon-
strated that compound 1 can be synthesized alongside a series
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Table 2. Evaluation of selected compounds regarding lipophilicity, solubility, in vitro metabolic stability and plasma protein binding.
1
2
3
[b]
Cmpd
LogD7.4
Sol.[a]
HLM Clint
HLM
Rat hep.
Rat hep.
PPB[d]
Plasma stab[e]
[c]
t 1=
Clint
t 1=
2
2
1
7
3.4�0.1
2.7�0.1
4.9�0.1
3.7�0.1
3.8�0.2
2.6�0.1
3.0�0.1
3.7�0.1
2.4�0.01
1.6�0.1
0.2–1.4[f]
1.8�0.3
0.2�0.1g
0.3–3.5[f]
0.1–1.3[f]
20�1.2
4–16[f]
>300[h]
>300[h]
>300[h]
>300[h]
>300[h]
>300[h]
>300[h]
>300[h]
>300[h]
81�10
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
<2.3[h]
8.6�1.0g
135�11
114�4
5.2�0.4
6.1�0.2
<2.3[g,h]
4.3�0.5
9.8�2
106�11
100�5
102�25
101�7
99�3
4
5
6
7
8
9
15
32_1
32_2
36
37
38
39
40
>300[h]
0.25–3.2
1.9�0.5
1.5�0.3
5.5�0.4
4.1�0.8
1.9�0.4
14�0.6
40�4
>300[g,h]
168�20[h]
84�14
<2.3[g,h]
4.2�0.5[h]
8.5�1.6
5.5�1.4[h]
2.8�0.06[h]
7.6�0.6
5.2�0.9
97�15
98�3
132�39[h]
248�5[h]
91�8
2.4–7.5[f]
58�16
814�63
105�7
99�1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
136�22
95�2
All data are reported as the average and standard deviation from three independent test occasions. [a] Dried DMSO solubility in aqueous phosphate buffer
(PBS) at pH 7.4; μM. [b] Human liver microsome intrinsic clearance; μL/min/mg. [c] Rat hepatocytes intrinsic clearance; μL/min/106 cells. [d] Human protein
plasma binding; % free. [e] Human plasma stability; % compound remaining after 18 h incubation. [f] High-throughput solubility assays are associated with
large variability, especially below 10 μM range. When the standard deviation is higher than half the mean value, the entire range is given. [g] An outlier value
was observed with higher solubility amongst the three replicates. The higher value was removed. Also in the metabolic assays when an outlier with lower
clearance or longer half-life was detected among the three replicates, this value was removed. [h] A higher uncertainty is associated with these values as
compound dilutions resulted in non-linear responses.
vealed that the few differences between the two orthologue
IRAP enzymes (sequence ID=88%, data not shown) are not
located in the binding site residues. As described below, a key
driver in the further optimization has thus been to improve on
compound solubility to get a better separation between
observed potencies and solubilities to improve on these
comparisons. Both compounds were shown to be completely
inactive on the closely related APN under identical assay
conditions, i.e. demonstrating selectivity for IRAP (Table 1).
As the previous results indicated that an electron-with-
drawing and/or lipophilic substituent in the 5-position was
beneficial for inhibition, we performed Suzuki-Miyaura cross
couplings on 1 to incorporate aryl and vinyl groups (11–22,
Scheme 2), in order to further explore the SAR of lipophilic
substituents at this position. The reaction conditions were
optimized in order to increase reaction speed and reduce
dehalogenation and hydrolysis of the aminal part. Different Pd-
catalysts, bases, solvent systems, temperatures and equivalents
of boronic acid were investigated. It could be concluded that
dehalogenation was affected by solvent and base, but not by
concentration, temperature or Pd-source.
Scheme 1. Improved batch synthesis of the spiro-structure employing aryl-
amines.
synthesized compounds revealed that a substituent in the 5-
position of the oxindole moiety was crucial for inhibition.
Removal of the bromine (2) resulted in loss of activity and
displacement to the 7-position (3) resulted in an inactive
compound.
Exchange of the bromine for the other halogens fluorine (4),
chlorine (5) and iodine (6) resulted in slightly increased activity
from fluorine to iodine. Substitution of the bromine with a nitro
group (7) retained activity, whilst reduction to the amine (8)
reduced the activity dramatically. A methoxy (9) or methyl-
group (10) instead of the original bromine also resulted in loss
of activity by tenfold.
Minimization of the water content and slight excess of base
was necessary to avoid hydrolysis.[41] The reaction optimization
resulted in a swift and simple MW-procedure to obtain the
desired compounds in only a few minutes (Scheme 2).
Since the screen was performed on IRAP derived from CHO-
cells, we also wanted to confirm inhibitory activity on the
human orthologue (hIRAP) as well as to investigate selectivity
against the closely related aminopeptidase N (APN). The hit
compound 1 and the equipotent analog 7 were both evaluated
in this respect (Table 1). Both compounds lost approximately 10
to 15-fold activity on hIRAP compared to IRAP from CHO-cells,
meaning the potency approached a concentration where visual
precipitation was observed (>50 μM). Sequence analysis re-
Scheme 2. Suzuki-Miyaura cross couplings to introduce different aryl,
heteroaryl and vinyl substituents.
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Introduction of substituted aryls (11–17), heteroaryls (18–
20) or vinyl groups (21–22) rendered inactive or compounds
with poor activity compared to 1. In summary, introduction of
these kind of lipophilic substituents in the 5-position seemed to
be detrimental for activity, indicating that the electron-with-
drawing properties of previous substituents (1, 4–10) rendered
the active compounds. Regarding the N-substituent of the
oxindole moiety, removal of the methyl group (23) decreased
the activity 30-fold, and removal of both the methyl and the
bromine (24) decreased the activity even more. Since the
compounds did not show any improvement in potency on IRAP
from CHO-cells, none of the compounds were evaluated on the
human orthologue.
Table 3. Evaluation of compounds 25–41 as IRAP-inhibitors.
1
2
3
4
5
6
7
8
9
[a]
Cmpd
R3
pIC50
25
26
5.2�0.2 (3)[b]
5.2�0.4 (3)[b]
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
27
28
29
5.3�0.1 (3)[b]
5.7�0.3 (3)[b]
5.5�0.2 (3)[b]
Since no potency or DMPK improvements (Table 2) were
observed when substituting the oxindole part of 1, we next
investigated the p-tolyl moiety (Table 3). Removal of the entire
moiety as in 25 resulted in a small drop of activity. Removal of
the methyl group (26) or exchange to a triflouromethyl (27)
also gave a slight activity loss. Substitution with a larger tert-
butyl group (28) or iodine (29) provided compounds with the
same activity as the hit compound (1). Similar activities were
also observed when introducing a benzyloxy substituent (30) or
an oxazole ring in the meta position (31). When we incorpo-
rated a carbon linker between the scaffold and the aryl group,
hence used aliphatic amines, we had to modify the reaction
conditions for the synthesis. Instead of using acetic acid as both
solvent and catalyst, we used ethanol as solvent applying 5%
AcOH as catalyst to obtain the intermediate A when reacting
isatoic anhydride with the appropriate amine (Scheme 3). A
quick solvent evaporation followed by dissolution in EtOH and
this time adding 1% TFA as catalyst to perform the final MW-
promoted ring closure reaction gave compounds 30, 32–41 in
53–82% yield over two steps (Scheme 3). Introduction of a one-
carbon spacer between the scaffold and the aromatic ring (32)
resulted in the first compound with higher inhibitory capacity
than the hit compound (Table 3). However, also this compound
lost activity on human IRAP, i.e. in agreement with observations
made for 1 and 7. Despite the reduced potency on human IRAP,
32 was separated into the pure enantiomers by chiral super-
critical fluid chromatography (SFC) and these were evaluated
for their inhibitory capacities. The most active enantiomer (32_
1) displayed a pIC50-value of 6.7, whereas the other enantiomer
(32_2) was more than two orders of magnitude less potent
(4.1). No substantial differences between the enantiomers were
observed regarding solubility, in vitro metabolic stability or
plasma protein binding (Table 2). Additional SAR-investigation
in this position revealed that elongation of the linker with one
additional carbon (33) reduced the activity somewhat, but still
rendered a better inhibitor than the parent compound. A
saturated cyclohexyl (34) also moderately increased the inhib-
ition compared to 1. Substitution with heteroaryls such as 2-
pyridyl (35), 3-pyridyl (36) or furyl (37), in an attempt to increase
the solubility compared to 32, unfortunately provided com-
pounds with lower potency. Moreover, despite being more
polar (see Table 2), these compounds also lost activity on
human IRAP. Introduction of a saturated carbon chain (38)
furnished the most active compound in this series so far. Again,
30
31
5.5�0.3 (3)[b]
5.7�0.3 (3)[b]
6.4�0.2 (5)[b]
5.3�0.01 (2)[c]
Inactive (2)[d]
6.7�0.05 (4)[b]
4.1�0.2 (4)[b]
32
32_1
32_2
1st eluted enantiomer
2nd eluted enantiomer
33
34
35
6.2�0.07 (3)[b]
6.2�0.1 (3)[b]
5.2�0.2 (3)[b]
5.7�0.06 (3)[b]
4.5�0.05 (2)[c]
Inactive (1)[d]
6.2�0.07 (3)[b]
4.9�0.1 (2)[c]
Inactive (1)[d]
36
37
38
39
6.6�0.05 (3)[b]
5.5�0.02 (3)[b]
4.3�0.07 (2)[c]
Inactive (1)[d]
5.6�0.08 (3)[b]
4.4�0.04 (2)[c]
Inactive (1)[d]
40
41
4.7�0.1 (3)[b]
[a] pIC50 =negative log of the IC50 in molar. Values represent the mean �
standard deviation of best-fit values (the number of independent test
occasions is provided within brackets). No values below 3.9 are reported as
this was the highest compound concentration tested. [b] Evaluated on
IRAP from Chinese hamster ovary cells. [c] Evaluated on human membrane
bound IRAP overexpressed in HEK293 cells. [d] Evaluated on human
aminopeptidase N overexpressed in HEK293 cells.
Scheme 3. Modified two step synthesis of the spiro-structure employing
aliphatic amines.
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since we had seen that the compounds lost activity on human
IRAP, we next tried to synthesize more soluble derivatives.
However, introduction of heteroatoms in the chains (39–41)
decreased the inhibition compared to 38, indicating that a
lipophilic substituent in this part of the molecule is favorable
but not necessary. Despite improved solubility (Table 2), these
compounds lost activity on human IRAP, while retaining
selectivity for IRAP versus APN. It should be emphasized that
compound 40 is equipotent with the hit compound (1), but
shows considerably better solubility, indicating that it is
possible to improve this property while maintaining activity.
Unfortunately, all compounds synthesized in order to improve
solubility still suffered from poor in vitro metabolic stability
properties in both human liver microsomes and rat hepatocytes.
The plasma protein binding is an issue with compound 1 and
most of the tested compounds, with the exception of
compound 40 which has a large free fraction.
We were interested in better understanding the mode of
action of these compounds and whether the observed SAR
could be rationalized in terms of their interaction with IRAP.
This prompted us to perform docking studies of selected
compounds in an attempt to discover their putative binding
site. Ligands 1, 3, 12, 24, 32, 38 and 40 were selected for
docking studies on the basis of their measured potencies in the
enzymatic assay. An initial analysis of the protein surface area,
based on the available IRAP crystal structures, (open and closed
conformations), allowed the identification of three potential
sites for small molecule binding, herein referred to as H
(Histidine-cluster), A (allosteric site), and Z (Zinc-site), depicted
in Supporting Information Figure S1.
ligands in their S configuration, with the only exception of
compound 12, found a conserved binding mode on the Z-site
(see Supporting Information Figure S1). While being in the
vicinity of the Zn2+, the ligand is not coordinating the ion,
having the N-Me at 4 Å distance to the ion. In this pose the
oxindole moiety makes a hydrogen bond to Tyr549 while the
bromine is coordinated to Arg439. The second ring is making a
π-π stacking interaction with Phe544, which is only possible
when considering the S stereoisomer. Similar docking explora-
tion was performed on the closed conformation of IRAP crystal
structure (PDB ID: 5MJ6).[42] The results from the docking in this
“closed” conformation of IRAP did not show any conserved
binding pose that could explain the SAR of the series.
To check the stability of this binding mode, obtained in the
“open” conformation of IRAP, we performed MD simulations of
the complex with ligand 38, one of the most potent
compounds in the series. The average root-mean-square
deviation (RMSD) along all replica simulations is 0.44�0.01 Å
for protein backbone and 2.26�0.5 Å for the ligand. This
indeed results in a refined position for the ligand, which is
shown in Figure 2. While this refined pose is mostly consistent
with the initial docking, the main difference is located in the
translation of the ligand by approximately additional 1.5 Å away
from Zn2+, allowing a water molecule to insert in between
Tyr549 and the oxygen from the oxindole (Figure 2). As the zinc
ion is a critical component of substrate recognition and
processing during the catalytic cycle, we wondered if the space
between the ligand and the Zn2+ ion could simultaneously
accommodate the small L-Leu-pNA substrate used in our assays,
and whether such simultaneous binding would retain discrim-
ination between S and R stereoisomers. Superposition of the
IRAP-substrate complex with the IRAP-38 equilibrated pose
(Figure 2b) shows how the substrate is predicted to bind,
placing the Leucine and para-nitrophenyl groups in equivalent
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
In the open conformation of IRAP (PDB ID: 4PJ6), independ-
ent docking of the two stereoisomers on the H- and A-sites did
not yield any conserved binding mode that was compatible
with the SAR extracted from Tables 1 and 3. Conversely, all the
Figure 2. a) Binding mode of ligand 38 (green sticks) in IRAP obtained from a representative snapshot from the MD simulation. b) The same snapshot, with
the superposition of substrate L-Leu-pNA (orange sticks) as docked to IRAP, in two different points of view. Zn2+ and water molecules are shown as grey and
red spheres, respectively, and bromine atoms are represented in magenta. Key residues are shown in wheat colour sticks
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positions to the Val1 and Tyr2 sidechains of Ang IV.[26] As
described below this model of simultaneous binding of
inhibitor 38 and substrate L-Leu-pNA prompted further exper-
imental verification of the mechanism of action, i.e. to what
extent compound binding is competitive, non-competitive or
uncompetitive with substrate binding.
1
2
3
4
5
6
We next examined whether the proposed binding pose
could also explain the SAR observed for this series. The
orientation of the 5-position of the oxindole moiety, i.e. the one
holding the bromine in the original hit compound 1, is in line
with the high propensity observed for the interactions of
halogens with arginine,[43] explaining why the replacement of
the halogenated or electron withdrawing groups in active
compounds 4–7 by either aryl (11–17), heteroaryl (18–20) or
vinyl groups (21–22) rendered inactive or weakly active
compounds (Table 1). This is also in line with the inability of
compound 12, carrying the bulkiest substituent at this position,
to find this binding mode. Encouragingly, the ranking of the
compounds based on their pIC50 values is reproduced by the
tendency of the scoring function, with the exception of ligand
32 (Supporting Information Table 1).
In order to complement the docking studies and to better
understand whether the substrate can be simultaneously
accommodated in the active site, we performed a set of
additional experiments. First, enzyme kinetics experiments
revealed that compound 32 act as an uncompetitive inhibitor
to L-Leu-pNA (Figure 3), suggesting it does not bind at a
mutually exclusive binding site, i.e. in the immediate vicinity of
the catalytic Zn2+ ion. This is in contrast to previously published
IRAP inhibitors. Although these data are in line with the
simultaneous binding model as presented in Figure 2b, the
uncommon observation of an uncompetitive inhibition pattern
prompted us to look back at previously established data in our
labs as to the reversible nature of the interaction. We have
previously reported data from jump-dilution experiments with
compound 1,[30] demonstrating a fully reversible behavior. To
further corroborate this finding, we had also performed
inhibition experiments in which the incubation time for the
enzymatic reaction was allowed to extend beyond completion.
Such over incubation is expected to result in a significant loss
of sensitivity to reversible inhibitors,[44] and indeed we observed
a right-shifting of the concentration responses over time
(Supporting Information Figure S2). Taken together, these data
verify the inability of 1 to irreversibly inactivate IRAP and
demonstrates the ability of a representative compound to bind
simultaneously as the substrate to IRAP.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Figure 3. a) IRAP reaction rates as a function of substrate concentration for
compound 32 at concentrations between 39 nM and 150 μM. The solid lines
represent the best fit to a model for uncompetitive inhibition, resulting in a
K
m value of 0.67�0.07 mM, a Vmax of 5.3�0.3 mAbs/min and a Ki value of
0.54�0.06 μM. b) Best-fit Km (■) and Vmax (*) values when the data were
instead fitted to a Michaelis-Menten model for each compound concen-
tration with no assumption of the inhibition mode.
The tested ligands (1, 7, 32, 36–37, and 39–40) demon-
strated specificity for IRAP over human APN as reported in
Tables 1 and 3. Accordingly, attempts to dock these ligands in
the corresponding site in APN (PDB ID: 4FYQ) did not yield any
binding poses, as it also occurred with docking on the closed
conformation of IRAP. The specificity of the binding orientation
discussed above for the IRAP-open conformation is illustrated
by superposition of the obtained complex in this structure, with
the APN and IRAP-closed crystal structures (Figure 4). The figure
illustrates how the selectivity for IRAP-open conformation might
come from the GAMEN loop, which in the IRAP-closed
Figure 4. Binding mode of ligand core (cyan color sticks), in IRAP super-
imposed with APN and IRAP-closed conformation. The GAMEN loop of APN
(PDB ID: 4FYQ) and IRAP-closed conformation (PDB ID: 5MJ6) is represented
in red, and green respectively, as opposed to the equivalent loop in IRAP
(PDB ID: 4PJ6). IRAP is represented in grey color surface and Zn2+ is shown
as light blue sphere.
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Purification was performed on column chromatography using silica
gel (60–120 mesh size), and for test compounds, as small amount
of the final product was purified using reversed-phase high-
performance liquid chromatography (RP-HPLC) (UV-triggered
(254 nm) fraction collection with a Dionex UltiMate 3000 HPLC
system, using an Macherey-Nagel Nucleodur C18 column (21×
125 mm, 5 μm particle size), and H2O/CH3CN/0.1% CF3COOH as
eluent in a gradient (10 mL/min over 15–20 min)). High resolution
molecular masses (HRMS) were determined on a mass spectrometer
equipped with an ESI source and 7-T hybrid linear ion trap (LTQ).
Nuclear magnetic resonance (NMR) spectra were recorded at
400 MHz for 1H and 101 MHz for 13C. Chemical shifts (δ) are
reported in ppm with the residual solvent peak as internal standard
(1H, CDCl3 7.26 ppm, CD3OD 3.31 ppm, DMSO-d6 2.50 ppm; 13C,
CDCl3 77.16 ppm, CD3OD 49.00 ppm, DMSO-d6 39.52 ppm). Cou-
pling constants J are reported in hertz (Hz). All final compounds
were �95% pure as determined by HPLC (UV at 254 nm) and NMR.
conformation adopts a specific, induced fit conformation similar
to APN, and in both cases clearly distinct to the IRAP-open
structure, and in analogy to our recent findings on IRAP specific
cyclic peptide inhibitors.[26]
1
2
3
4
5
6
7
8
9
3. Conclusions
We have prepared and evaluated a new class of IRAP inhibitors
synthesized by
reaction, or by
a
a
simple MW-promoted three-component
two-step-one-pot procedure. Additional
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
structural modifications have been introduced by a rapid
Suzuki-Miyaura cross coupling reaction to incorporate various
aryl-, heteroaryl- and vinyl-groups in only a few minutes. This
spiro-oxindole based class of compounds is one of the few
published non-peptidic IRAP inhibitors with sub-μM affinity.
They are also selective towards the closely related APN. The
computational modeling of these series of compounds suggests
that the S-configuration of the substituted spiro-oxindole
dihydroquinazolinones accounts for the inhibitory capacity
within the series. The interactions with the binding cavity in
IRAP are thus stereoselective, and the corresponding R-config-
uration is postulated to be essentially inactive in all cases.
Furthermore, the proposed binding mode is compatible with
the simultaneous binding of L-Leu-pNA, in agreement with the
uncompetitive IRAP inhibition here determined for a represen-
tative compound. As such, this class of compounds represents
an interesting starting point for further developments towards
in vivo candidates. Necessary developments include further
improvements in potency, as this is still considerably lower than
for the peptide-based inhibitors. The compounds also suffer
from poor in vitro metabolic stability and most of them have
poor solubility, in line with observations for other published
small-molecule based IRAP inhibitors.[29] Importantly, compound
40 shows that it is possible to increase the solubility substan-
tially and still maintain a potency comparable to 1. Efforts to
improve the pharmacokinetic properties are ongoing with the
aim to transform these IRAP inhibitors into valuable research
tools and to enable assessment of their potential as future
cognitive enhancers.
Synthesis, spectral data and analysis of compounds 1–3, 17, 23, 26–
29 and 31 have already been published.[41]
Synthesis of Compounds 4–9, 24–25
General Procedure: N-methylated isatin (1 equiv), the appropriate
aniline (1.2 equiv) and isatoic anhydride (1.2 equiv) were mixed in
acetic acid (0.1 M based on isatin) and heated by microwave
°
irradiation at 150 C for 10 min (fixed hold time) in a sealed vial. The
reaction mixture was cooled to room temperature upon which the
product precipitate from the solution. The crude products were
filtered and purified by recrystallization from EtOH.
5-Fluoro-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (4). 5-Fluoro-1-methylindoline-2,3-dione (200 mg,
1.12 mmol), isatoic anhydride (220 mg, 1.34 mmol), p-toluidine
(120 mg, 1.34 mmol) and AcOH (11 mL). White powder (300 mg,
1
66%). H NMR (400 MHz, DMSO-d6) δ 7.68 (dd, J=1.5, 7.8 Hz, 1H),
7.63–7.57 (m, 2H), 7.32 (ddd, J=1.6, 7.3, 8.0 Hz, 1H), 7.12 (ddd, J=
2.7, 8.6, 9.4 Hz, 1H), 7.04–6.99 (m, 2H), 6.92–6.82 (m, 3H), 6.78 (ddd,
J=1.0, 7.4, 8.2 Hz, 1H), 6.69 (dd, J=1.0, 8.1 Hz, 1H), 3.02 (s, 3H), 2.18
(s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.9, 163.40, 158.4 (d, JC-F
=
241 Hz), 145.8, 139.4 (d, JC-F =1.8 Hz), 137.1, 135.1, 133.8, 129.3,
128.4 (d, JC-F =7.9 Hz), 127.5, 117.9, 117.3 (d, JC-F =24.3 Hz), 114.5,
114.2 (d, JC-F =25.2 Hz), 114.1, 110.1 (d, JC-F =7.6 Hz), 76.2, 26.1, 20.6.
HRMS (ES) m/z calcd. for C23H19FN3O2: [M+H]+ 388.1461, found:
388.1449.
5-Chloro-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazo-
line]-2,4’(3’H)-dione
(5).
5-Chloro-1-methylindoline-2,3-dione
(200 mg, 1.02 mmol), isatoic anhydride (110 mg, 1.23 mmol), p-
toluidine (110 mg, 1.23 mmol) and AcOH (10 mL). White powder
(270 mg, 65%). 1H NMR (400 MHz, DMSO-d6) δ 7.73 (dd, J=0.4,
2.2 Hz, 1H), 7.68 (dd, J=1.6, 7.7 Hz, 1H), 7.63–7.61 (m, 1H), 7.36–
7.30 (m, 2H), 7.04–7.00 (m, 2H), 6.91 (dd, J=0.4, 8.4 Hz, 1H), 6.85 (d,
J=7.9 Hz, 2H), 6.78 (ddd, J=.1, 7.3, 7.8 Hz, 1H), 6.69 (ddd, J=0.5,
1.1, 8.1 Hz, 1H), 3.02 (s, 3H), 2.19 (s, 3H). 13C NMR (101 MHz, DMSO-
d6) δ 173.8, 163.6, 145.9, 142.2, 137.3, 135.2, 133.9, 130.9, 129.5,
128.7, 127.6, 127.0, 126.4, 118.2, 114.6, 114.3, 110.7, 76.2, 26.2, 20.7.
HRMS (ES) m/z calcd. for C23H19ClN3O2: [M+H]+ 404.1166, found:
404.1170.
Experimental Section
General Information
Microwave heating was performed in a Biotage single-mode
microwave reactor producing controlled irradiation at 2450 MHz
with a power of 0–400 W. The reaction temperature was deter-
mined and controlled using the built-in online IR-sensor. Microwave
mediated reactions were performed in septum sealed Biotage vials.
Analytical TLC was performed on silica gel 60 F-254 plates and
visualized with UV light (λ=254 nm). Analytical HPLC/ESI-MS was
performed on a Dionex UltiMate 3000 HPLC system with a Bruker
amazon SL ion trap mass spectrometer and detection by UV (diode
array detector, 214, 254 and 280 nm) and electrospray ionization
(ESI) MS using a Phenomenex Kinetex C18 column (50×3.0 mm,
2.6 μm particle size, 100 Å pore size) with gradients of H2O/CH3CN/
0.05% HCOOH as mobile phase at a flow rate of 1.5 mL/min.
5-Iodo-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (6). 5-Iodo-1-methylindoline-2,3-dione (147 mg,
0.51 mmol), p-toluidine (57 mg, 0.61 mmol), isatoic anhydride
(98 mg, 0.60 mmol) and AcOH (5 mL). White powder (165 mg,
1
67%). H NMR (400 MHz, DMSO-d6) δ 7.92 (d, J=1.8 Hz, 1H), 7.68
(dd, J=1.5, 7.8 Hz, 1H), 7.63–7.60 (m, 2H), 7.32 (ddd, J=1.6, 7.3,
8.1 Hz, 1H), 7.02 (d, J=8.2 Hz, 2H), 6.83 (d, J=7.9 Hz, 2H), 6.80–6.76
(m, 1H), 6.73 (d, J=8.2 Hz, 1H), 6.68 (dd, J=1.0, 8.1 Hz, 1H), 3.00 (s,
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3H), 2.19 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.3, 163.4, 145.7,
142.9, 139.4, 137.1, 135.1, 134.4, 133.7, 129.3, 129.0, 127.5, 118.0,
114.5, 114.1, 111.5, 85.7, 75.9, 26.0, 20.6. HRMS (ES) m/z calcd. for
C23H19IN3O2: [M+H]+ 496.0522, found: 496.0525.
1.0, 7.2, 8.2 Hz, 1H), 6.60 (dd, J=0.9, 8.1 Hz, 1H), 3.07 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 173.9, 163.7, 146.5, 142.9, 133.6, 133.6,
131.0, 127.7, 127.0, 117.6, 114.6, 114.2, 114.0, 111.2, 70.8, 26.1.
HRMS (ES) m/z calcd. for C16H13N3O2Br: [M+H]+ 358.0191, found:
358.0189.
1
2
3
4
5
6
7
8
9
1-Methyl-5-nitro-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (7). 1-Methyl-5-nitroindoline-2,3-dione (50 mg,
0.24 mmol), isatoic anhydride (42 mg, 0.26 mmol), p-touidine
(28 mg, 0.26 mmol) and AcOH (2.4 mL). Off-white powder (56 mg,
Synthesis of Compounds 10–16, 18–22
1
General procedure Suzuki-Miyaura cross-coupling reaction. The
appropriate boronic acid/ester (0.45 mmol; for compound 10
56%). H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J=2.3 Hz, 1H), 8.24
(dd, J=2.4, 8.7 Hz, 1H), 7.72 (dd, J=1.6, 7.8 Hz, 1H), 7.70 (s, 1H),
7.36 (ddd, J=1.6, 7.3, 8.1 Hz, 1H), 7.14 (d, J=8.7 Hz, 1H), 7.03–6.99
(m, 2H), 6.88–6.85 (m, 2H), 6.83 (ddd, J=1.1, 7.3, 7.8 Hz, 1H), 6.71
(dd, J=1.1, 8.1 Hz, 1H), 3.11 (s, 3H), 2.16 (s, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 174.5, 163.2, 149.0, 145.5, 142.9, 137.4, 134.8, 134.0,
129.5, 127.9, 127.8, 127.6, 121.7, 118.4, 114.5, 114.3, 109.7, 75.7,
26.5, 20.6. HRMS (ES) m/z calcd. for C23H19N4O2: [M+H]+ 415.1406,
found: 415.1403.
2.24 mmol), compound
1 (0.22 mmol), DBU (0.34 mmol) and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
PdCl2(dppf) (0.006 mmol, 2.5 mol%) were added to a sealed vial,
which was evacuated and backfilled with nitrogen. MeCN (9.8 mL)
and H2O (0.2 mL) were added and the mixture was heated by
°
microwave irradiation for 1 min at 180 C (fixed hold time). The
crude product was concentrated under reduced pressure and
purified by silica flash column chromatography.
1,5-Dimethyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (10). Methylboronic acid. White solid (26 mg, 30%).
1H NMR (400 MHz, DMSO-d6) δ 7.67 (dd, J=1.6, 7.8 Hz, 1H), 7.55 (s,
1H), 7.45 (d, J=1.7 Hz, 1H), 7.30 (ddd, J=1.6, 7.3, 8.2 Hz, 1H), 7.07
(ddd, J=0.9, 1.8, 7.9 Hz, 1H), 6.98 (dd, J=0.8, 8.4 Hz, 2H), 6.80 (d,
J=8.4 Hz, 2H), 6.76 (t, J=7.3 Hz, 2H), 6.68 (dd, J=1.0, 8.1 Hz, 1H),
2.98 (s, 3H), 2.23 (s, 3H), 2.17 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 173.8, 163.7, 146.1, 140.8, 136.9, 135.4, 133.7, 132.0, 131.1, 129.2,
127.5, 126.9, 126.7, 117.8, 114.5, 114.1, 108.8, 76.3, 26.0, 20.6. HRMS
(ES) m/z calcd. for C24H22N3O2: [M+H]+ 384.1712, found: 384.1729.
5-Amino-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (8). Compound 7 (20 mg, 0.05 mmol) was dissolved
in 10 mL EtOH and 7 mL MeOH and Pd/C (10 mol%) were added.
The reaction mixture was degassed and flushed with nitrogen three
times, and finally filled with H2-gas. The mixture was stirred for 6 h
at ambient temperature, and was subsequently filtered through
celite and concentrated in vacou to afford a light yellow powder. 1H
NMR (400 MHz, DMSO-d6) δ 7.68 (dt, J=7.9, 1.7 Hz, 1H), 7.59 (s, 1H),
7.36–7.27 (m, 2H), 7.00 (d, J=8.3 Hz, 2H), 6.94 (d, J=8.3 Hz, 1H),
6.85–6.73 (m, 4H), 6.69 (dd, J=8.2, 1.3 Hz, 1H), 2.97 (s, 3H), 2.18 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 173.7, 163.9, 146.4, 137.5,
135.5, 134.3, 129.7, 129.3, 128.6, 128.0, 118.4, 114.8, 114.6, 117.9,
110.2, 76.8, 26.5, 21.0. HRMS (ES) m/z calcd. for C23H21N4O2: [M+H]+
385.1665, found: 385.1676.
5-(4-Fluorophenyl)-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (11). 4-Fluorophenylboronic acid.
1
Light yellow solid (95.7 mg, 93%). H NMR (400 MHz, CDCl3) δ 8.03
(dd, J=1.6, 7.8 Hz, 1H), 7.69 (dd, J=0.5, 1.9 Hz, 1H), 7.45–7.38 (m,
3H), 7.35 (ddd, J=1.6, 7.3, 8.0 Hz, 1H), 7.15–7.08 (m, 2H), 6.99–6.94
(m, 3H), 6.89 (d, J=7.8 Hz, 2H), 6.71 (dd, J=0.5, 8.2 Hz, 1H), 6.65
(ddd, J=0.5, 1.1, 8.0 Hz, 1H), 4.61 (s, 1H), 3.08 (s, 3H), 2.21 (s, 3H).
13C NMR (101 MHz, CDCl3) δ 173.5, 162.6 (d, JC-F =247.6 Hz), 161.4,
156.3, 144.1, 142.3, 138.1, 136.3 (d, JC-F =3.2 Hz), 136.0, 135.0, 134.0,
129.9, 129.8, 129.1, 128.4 (d, JC-F =8.5 Hz), 128.1, 124.3, 120.6, 116.9,
116.0 (d, JC-F =21.5 Hz), 115.0, 109.1, 76.7, 26.4, 21.2. HRMS (ES) m/z
calcd. for C29H23FN3O2: [M+H]+ 464.1774, found: 464.1767.
5-Methoxy-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazo-
line]-2,4’(3’H)-dione (9). 5-Methoxy-1-methylindoline-2,3-dione
(60 mg, 0.31 mmol), isatoic anhydride (62 mg, 0.38 mmol), p-
toluidine (41 mg, 0.38 mmol) and AcOH (3 mL). Off-white powder
(117 mg, 93%). 1H NMR (400 MHz, DMSO-d6) δ 7.67 (dd, J=1.6,
7.8 Hz, 1H), 7.55 (s, 1H), 7.30 (ddd, J=1.6, 7.3, 8.4 Hz, 1H), 7.26 (d,
J=2.4 Hz, 1H), 7.00 (dd, J=1.0, 8.5 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H),
6.81 (d, J=2.5 Hz, 1H), 6.79 (s, 1H), 6.76 (td, J=1.1, 7.6 Hz, 1H), 6.69
(dd, J=1.0, 8.2 Hz, 1H), 3.70 (s, 3H), 2.99 (s, 3H), 2.17 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 173.8, 163.7, 155.6, 146.1, 136.9, 136.5,
135.4, 133.7, 129.2, 127.9, 127.5, 117.8, 115.9, 114.6, 114.1, 112.8,
109.7, 76.5, 55.8, 26.0, 20.6. HRMS (ES) m/z calcd. for C24H22N3O3: [M
+H]+ 400.1661, found: 400.1658.
1-Methyl-5-(4-phenoxyphenyl)-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (12). 4-Phenoxyphenylboronic acid.
Light yellow solid (117 mg, 96%). 1H NMR (400 MHz, DMSO-d6)
δ 7.93 (d, J=1.9 Hz, 1H), 7.69 (dd, J=1.6, 7.8 Hz, 1H), 7.67–7.63 (m,
3H), 7.56 (dd, J=1.9, 8.2 Hz, 1H), 7.42 (m, 2H), 7.31 (ddd, J=1.6, 7.3,
8.1 Hz, 1H), 7.17 (tt, J=1.1, 7.4 Hz, 1H), 7.08–7.04 (m, 4H), 6.99 (d,
J=8.3 Hz, 2H), 6.94 (d, J=8.2 Hz, 1H), 6.91 (m, 2H), 6.77 (dd, J=1.0,
3’-(p-Tolyl)-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-dione
(24). Isatin (147 mg, 1.0 mmol), isatoic anhydride (163 mg,
1.0 mmol), p-toluidine (107 mg, 1.0 mmol) and AcOH (10 mL). Off-
7.6 Hz, 1H), 6.70 (dd, J=1.0, 8.2 Hz, 1H), 3.06 (s, 3H), 2.15 (s, 3H). 13
C
1
NMR (101 MHz, DMSO-d6) δ 174.1, 163.6, 156.5, 156.20, 146.0, 142.4,
136.9, 136.1, 135.4, 134.5, 134.3, 133.6, 130.1, 129.2, 128.9, 127.9,
127.5, 124.2, 119.1, 118.9, 117.8, 117.1, 114.7, 114.1, 109.5, 76.2,
26.1, 20.6. HRMS (ES) m/z calcd. for C35H28N3O3: [M+H]+ 538.2131,
found: 538.2143.
white powder (252 mg, 71%). H NMR (400 MHz, DMSO-d6) δ 10.38
(s, 1H), 7.66 (dd, J=7.8, 1.6 Hz, 1H), 7.58 (s, 1H), 7.54 (dd, J=7.5,
1.2 Hz, 1H), 7.30 (ddd, J=8.5, 7.3, 1.6 Hz, 1H), 7.16 (td, J=7.7,
1.3 Hz, 1H), 7.01 (d, J=8.1 Hz, 2H), 6.94 (td, J=7.5, 1.0 Hz, 1H), 6.86
(d, J=8.1 Hz, 2H), 6.75 (td, J=7.5, 1.0 Hz, 1H), 6.70 (dd, J=8.0,
1.0 Hz, 1H), 6.65 (d, J=7.7 Hz, 1H), 2.18 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 175.5, 163.6, 146.1, 141.6, 136.8, 135.4, 133.6, 130.8, 129.2,
127.5, 127.4, 126.4, 122.1, 117.6, 114.6, 114.0, 110.1, 76.4, 20.5.
HRMS (ES) m/z calcd. for C22H18N3O2: [M+H]+ 356.1399, found:
356.1412
N-(3-(1-Methyl-2,4’-dioxo-3’-(p-tolyl)-3’,4’-dihydro-1’H-spiro[indo-
line-3,2’-quinazolin]-5-yl)phenyl)acetamide (13). 3-Acetamidophe-
nylboronic acid. Light yellow solid (89 mg, 80%). 1H NMR (400 MHz,
DMSO-d6) δ 10.00 (s, 1H), 7.85 (d, J=2.0 Hz, 2H), 7.70 (dd, J=1.6,
7.7 Hz, 1H), 7.67 (s, 1H), 7.57 (ddd, J=1.1, 2.1, 8.0 Hz, 1H), 7.52 (dd,
J=1.9, 8.2 Hz, 1H), 7.36 (m, 1H), 7.34–7.26 (m, 2H), 6.98 (dd, J=8.1,
9.2 Hz, 3H), 6.87 (d, J=8.5 Hz, 2H), 6.78 (td, J=1.1, 7.5 Hz, 1H), 6.71
(dd, J=1.0, 8.1 Hz, 1H), 3.06 (s, 3H), 2.15 (s, 3H), 2.06 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 174.1, 168.4, 163.6, 146.0, 142.7, 139.94,
139.82, 137.0, 135.3, 134.9, 133.7, 129.35, 129.26, 129.1, 127.49,
127.46, 124.4, 120.9, 117.8, 117.7, 116.8, 114.5, 114.1, 109.5, 76.2,
5-Bromo-1-methyl-1’H-spiro[indoline-3,2’-quinazoline]-2,4’(3’H)-
dione
(25).
5-bromo-1-methylindoline-2,3-dione
(202 mg,
0.84 mmol), isatoic ahydride (140 mg, 0.86 mmol), NH4OAc (70 mg,
0.91 mmol) and AcOH (10 mL). Beige powder (301 mg, 45%). 1H
NMR (400 MHz, DMSO-d6) δ 8.33 (s, 1H), 7.65–7.61 (m, 3H), 7.29 (s,
1H), 7.25 (ddd, J=1.6, 7.2, 8.1 Hz, 1H), 7.06 (m, 1H), 6.72 (ddd, J=
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1
26.1, 24.1, 20.5. HRMS (ES) m/z calcd. for C31H27N4O3: [M+H]+
solid (43 mg, 43%). H NMR (400 MHz, DMSO-d6) δ 7.94 (dd, J=0.5,
2.0 Hz, 1H), 7.70 (dd, J=1.6, 7.7 Hz, 1H), 7.65 (s, 1H), 7.53 (dd, J=
2.0, 8.2 Hz, 1H), 7.51 (dd, J=1.2, 3.6 Hz, 1H), 7.49 (dd, J=1.1, 5.1 Hz,
1H), 7.32 (ddd, J=1.6, 7.2, 8.1 Hz, 1H), 7.11 (dd, J=3.6, 5.1 Hz, 1H),
6.99 (d, J=8.5 Hz, 2H), 6.91 (dd, J=0.5, 8.2 Hz, 1H), 6.88 (d, J=
8.1 Hz, 2H), 6.78 (ddd, J=1.1, 7.3, 8.3 Hz, 1H), 6.70 (ddd, J=0.5, 1.1,
8.2 Hz, 1H), 3.05 (s, 3H), 2.15 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
δ 174.0, 163.6, 145.9, 142.8, 142.5, 137.0, 135.3, 133.7, 129.3, 128.9,
128.6, 128.0, 127.6, 127.5, 125.1, 123.3, 117.9, 114.6, 114.1, 109.6,
76.1, 26.1, 20.5. HRMS (ES) m/z calcd. for C27H22N3O2S: [M+H]+
452.1433, found: 452.1439.
1
2
3
4
5
6
7
8
9
503.2083, found: 503.2078.
4-(1-Methyl-2,4’-dioxo-3’-(p-tolyl)-3’,4’-dihydro-1’H-spiro[indoline-
3,2’-quinazolin]-5-yl)benzonitrile (14). 4-Cyanophenylboronic acid.
Light beige solid (83 mg, 79%). 1H NMR (400 MHz, CDCl3) δ 8.02
(ddd, J=0.5, 1.6, 7.8 Hz, 1H), 7.74 (dd, J=0.5, 1.9 Hz, 1H), 7.71 (dm,
J=8.7 Hz, 2H), 7.56 (dm, J=8.7 Hz, 2H), 7.50 (dd, J=1.9, 8.2 Hz, 1H),
7.35 (ddd, J=1.6, 7.3, 8.0 Hz, 1H), 6.99–6.93 (m, 3H), 6.87 (d, J=
7.8 Hz, 2H), 6.76 (dd, J=0.5, 8.2 Hz, 1H), 6.65 (ddd, J=0.5, 1.1,
8.0 Hz, 1H), 3.09 (s, 3H), 2.20 (s, 3H). 13C NMR (101 MHz, CDCl3)
δ 173.5, 164.2, 144.4, 144.0, 143.6, 138.3, 134.9, 134.7, 134.1, 132.9,
130.4, 129.9, 129.2, 129.0, 128.5, 127.3, 124.5, 120.8, 118.9, 116.8,
115.0, 111.2, 109.4, 76.6, 26.5, 21.2. HRMS (ES) m/z calcd. for
C30H22N4O2Na: [M+H]+ 493.1640, found: 493.1645.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
1-Methyl-3’-(p-tolyl)-5-vinyl-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (21). 2,4,6-Trivinylcyclotriboroxane pyridine com-
1
plex. Beige solid (84 mg, 95%). H NMR (400 MHz, DMSO-d6) δ 7.82
(d, J=1.8 Hz, 1H), 7.68 (dd, J=1.6, 7.7 Hz, 1H), 7.59 (s, 1H), 7.35–
7.28 (m, 2H), 6.98 (dd, J=0.9, 8.2 Hz, 2H), 6.86–6.81 (m, 3H), 6.77
(ddd, J=1.1, 7.3, 7.8 Hz, 1H), 6.71–6.62 (m, 2H), 5.79 (dd, J=0.9,
17.7 Hz, 1H), 5.16 (dd, J=0.9, 10.9 Hz, 1H), 3.02 (s, 3H), 2.16 (s, 3H).
13C NMR (101 MHz, DMSO-d6) δ 174.1, 163.7, 146.0, 142.8, 137.0,
136.0, 135.3, 133.7, 132.4, 129.3, 127.5, 127.2, 123.7, 117.9, 114.6,
114.1, 113.1, 109.0, 76.2, 26.1, 20.6. HRMS (ES) m/z calcd. for
C25H22N3O2: [M+H]+ 396.1712, found: 396.1723.
5-(2-Ethylphenyl)-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-qui-
nazoline]-2,4’(3’H)-dione (15). 2-Ethylphenylboronic acid. Off-white
solid (68 mg, 64%). 1H NMR (400 MHz, CD3OD including CDCl3)
δ 7.85 (dd, J=7.9, 1.6 Hz, 1H), 7.42 (d, J=1.8 Hz, 1H), 7.37 (s, 1H),
7.29 (ddd, J=8.8, 7.3, 1.6 Hz, 1H), 7.27–7.23 (m, 2H), 7.18 (dd, J=
7.7, 2.6 Hz, 1H), 7.16 (ddd, J=8.0, 1.7, 0.6 Hz, 1H), 7.04–7.00 (m, 1H),
6.96 (d, J=8.0 Hz, 2H), 6.84 (d, J=8.0 Hz, 2H), 6.81 (ddd, J=8.4, 7.3,
1.6 Hz, 1H), 6.70 (d, J=8.1 Hz, 1H), 6.64 (dd, J=8.2, 1.1 Hz, 1H), 3.07
(s, 3H), 2.41 (q, J=7.5 Hz, 2H), 2.20 (s, 3H), 0.97 (t, J=7.5, 3H). 13C
NMR (101 MHz, CD3OD including CDCl3) δ 174.4, 165.3, 145.4, 141.9,
141.7, 140.5, 138.2, 137.8, 135.0, 134.3, 132.1, 130.0, 129.8, 128.9,
128.6, 128.0, 127.1, 126.8, 125.9, 119.6, 115.7, 114.6, 108.7, 77.1,
26.3, 21.1, 15.6. HRMS (ES) m/z calcd. for C31H28N3O2: [M+H]+
474.2182, found: 474.2194.
(E)-1-Methyl-5-styryl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quinazo-
line]-2,4’(3’H)-dione (22). E-Styrylboronic acid. Light pink solid
(61 mg, 58%). 1H NMR (400 MHz, CD3OD and CDCl3) δ 7.86 (ddd, J=
0.5, 1.6, 7.8 Hz, 1H), 7.74 (d, J=1.7 Hz, 1H), 7.46 (m, 2H), 7.38 (dd,
J=1.8, 8.2 Hz, 1H), 7.35–7.29 (m, 3H), 7.23 (m, 1H), 7.00 (d, J=
3.3 Hz, 2H), 6.96 (m, 2H), 6.87–6.81 (m, 3H), 6.68 (ddd, J=0.5, 1.1,
8.1 Hz, 1H), 6.67 (d, J=8.2 Hz, 1H), 3.03 (s, 3H), 2.18 (s, 3H). 13C NMR
(101 MHz, CD3OD and CDCl3) δ 174.6, 165.8, 146.1, 142.6, 138.6,
137.5, 135.0, 134.7, 133.8, 130.1, 130.0, 129.1, 128.7, 128.4, 128.4,
128.1, 127.9, 126.8, 123.7, 119.5, 115.4, 114.8, 109.5, 77.5, 26.4, 21.1.
HRMS (ES) m/z calcd. for C31H26N3O2: [M+H]+ 472.2025, found:
472.2037.
5-(2-Methoxyphenyl)-1-methyl-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (16). 2-Methoxyphenylboronic acid.
1
Off-white solid (96 mg, 91%). H NMR (400 MHz, DMSO-d6) δ 7.72
(dd, J=0.4, 1.8 Hz, 1H), 7.68 (dd, J=1.5, 7.8 Hz, 1H), 7.62 (s, 1H),
7.36 (dd, J=1.8, 8.1 Hz, 1H), 7.33 (m, 1H), 7.30 (m, 1H), 7.21 (dd, J=
1.7, 7.5 Hz, 1H), 7.10 (dd, J=1.1, 8.4 Hz, 1H), 7.04–7.00 (m, 3H), 6.91
(dd, J=0.4, 8.1 Hz, 1H), 6.87 (d, J=8.3 Hz, 2H), 6.77 (ddd, J=1.0, 7.4,
8.2 Hz, 1H), 6.69 (dd, J=0.9, 8.3 Hz, 1H), 3.77 (s, 3H), 3.04 (s, 3H),
2.18 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 173.9, 163.7, 156.0,
146.1, 142.0, 137.0, 135.3, 133.7, 132.8, 131.7, 130.1, 129.2, 128.9,
128.9, 127.5, 127.0, 126.5, 120.9, 117.9, 114.6, 114.2, 111.9, 108.8,
76.4, 55.6, 26.1, 20.6. HRMS (ES) m/z calcd. for C30H26N3O3: [M+H]+
476.1974, found: 476.1970.
Synthesis of Compounds 30, 32–41
General Procedure – Step 1: A mixture of isatoic anhydride
(1.2 mmol) and the appropriate amine (1.2 mmol) in a solution of
°
5% AcOH in EtOH (10 mL) was heated by MW irradiation at 150 C
for 10 min (fixed hold time) in a sealed vial. The solvent was
removed under reduced pressure and the intermediate used in the
next step without further purification.
1-Methyl-5-(pyridin-4-yl)-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-quina-
zoline]-2,4’(3’H)-dione (18). 4-Pyridinylboronic acid. Yellow solid
1
General Procedure – Step 2: 5-Br-N-Me-isatin (0.48 mmol) was
dissolved in 1% TFA in EtOH (5 mL) and the product from step 1
(0.48 mmol) was added. The mixture was heated by MW irradiation
(70 mg, 70%). H NMR (400 MHz, CDCl3) δ 8.64 (dd, J=4.4, 1.7 Hz,
2H), 8.05 (dd, J=7.7, 1.5 Hz, 1H), 7.80 (dd, J=2.0, 0.6 Hz, 1H), 7.56
(dd, J=8.2, 1.9 Hz, 1H), 7.39–7.34 (m, 3H), 7.01–6.95 (m, 3H), 6.88 (d,
J=7.8 Hz, 2H), 6.77 (dd, J=8.2, 0.5 Hz, 1H), 6.67 (ddd, J=8.0, 1.1,
0.6 Hz, 1H), 4.63 (s, 1H), 3.10 (s, 3H), 2.21 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 173.6, 164.1, 150.6, 147.1, 144.0, 143.9, 138.2, 135.0, 134.1,
133.6, 130.2, 129.9, 129.2, 128.5, 124.3, 121.1, 120.8, 117.0, 115.0,
109.4, 76.5, 26.5, 21.2. HRMS (ES) m/z calcd. for C28H23N4O2: [M+H]+
447.1821, found: 447.1813.
°
at 150 C for 40 min (fixed hold time). The mixture was allowed to
cool to ambient temperature whereupon the product precipitates
from the solution. The precipitate was filtered off and purified by
silica flash column chromatography.
3’-(4-(Benzyloxy)phenyl)-5-bromo-1-methyl-1’H-spiro[indoline-
3,2’-quinazoline]-2,4’(3’H)-dione (30). 4-Benzyloxyaniline. White
1
powder (180 mg, 79%). H NMR (400 MHz, DMSO-d6) δ 7.82 (d, J=
1-Methyl-5-(quinolin-8-yl)-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-qui-
nazoline]-2,4’(3’H)-dione (19). 8-Quinolinylboronic acid. Beige solid
2.0 Hz, 1H), 7.68 (dd, J=1.6, 7.8 Hz, 1H), 7.61 (s, 1H), 7.48 (dd, J=
2.1, 8.3 Hz, 1H), 7.41–7.30 (m, 6H), 6.91–6.82 (m, 5H), 6.78 (ddd, J=
1.1, 7.3, 8.3 Hz, 1H), 6.68 (dd, J=1.0, 8.3 Hz, 1H), 4.99 (s, 2H), 3.00 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 173.6, 163.5, 157.5, 145.8,
142.5, 136.7, 133.8, 133.7, 130.4, 129.1, 128.9, 128.4, 127.93, 127.86,
127.6, 118.0, 114.8, 114.6, 114.5, 114.2, 111.2, 76.2, 69.3, 26.1. HRMS
(ES) m/z calcd. for C29H23BrN3O3: [M+H]+ 540.0923, found:
540.0926.
1
(87 mg, 79%). H NMR (400 MHz, CDCl3) δ 8.92 (dd, J=4.2, 1.8 Hz,
1H), 8.22 (dd, J=8.3, 1.8 Hz, 1H), 7.99 (dd, J=7.8, 1.6 Hz, 1H), 7.94
(d, J=1.7 Hz, 1H), 7.83 (dt, J=7.3, 3.7 Hz, 1H), 7.63–7.57 (m, 3H),
7.44 (dd, J=8.3, 4.2 Hz, 1H), 7.31 (ddd, J=8.0, 7.3, 1.6 Hz, 1H), 7.06–
6.99 (m, 4H), 6.89 (td, J=7.6, 1.1 Hz, 1H), 6.75 (d, J=8.1 Hz, 1H),
6.62 (dd, J=8.0, 1.0 Hz, 1H), 3.09 (s, 3H), 2.22 (s, 3H). HRMS (ES) m/z
calcd. for C32H25N4O2: [M+H]+ 497.1978, found: 497.1980.
3’-Benzyl-5-bromo-1-methyl-1’H-spiro[indoline-3,2’-quinazoline]-
1-Methyl-5-(thiophen-2-yl)-3’-(p-tolyl)-1’H-spiro[indoline-3,2’-qui-
nazoline]-2,4’(3’H)-dione (20). 2-Thienylboronic acid. Light beige
1
2,4’(3’H)-dione (32). Benzylamine. White powder (130 mg, 53%). H
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NMR (400 MHz, DMSO-d6) δ 7.76 (dd, J=6.7, 1.1, 1H), 7.63 (dd, J=
8.4, 2.1, 1H), 7.44 (s, 1H), 7.39 (d, J=2.0, 1H), 7.27 (ddd, J=8.3, 7.1,
1.5, 1H), 7.17–7.12 (m, 3H), 6.97 (d, J=8.4, 1H), 6.82–6.76 (m, 3H),
6.62 (d, J=8.1, 1H), 4.43–4.31 (m, 2H), 2.78 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 173.1, 163.9, 145.8, 143.3, 137.0, 134.3, 133.7,
129.1, 128.0, 127.9, 127.8, 127.7, 127.3, 118.3, 114.8, 114.6, 114.3,
111.6, 74.6, 45.7, 26.03. HRMS (ES) m/z calcd. for C23H19BrN3O2: [M+
H]+ 448.0661, found: 448.0669.
(ddd, J=8.1, 1.1, 0.5 Hz, 1H), 6.23 (dd, J=3.2, 1.8 Hz, 1H), 5.80 (dd,
J=3.2, 0.9 Hz, 1H), 4.58 (dd, J=15.8, 0.8 Hz, 1H), 4.21 (d, J=15.9 Hz,
1H), 2.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 172.4, 163.1, 149.7,
145.5, 143.3, 142.3, 134.1, 133.6, 128.4, 127.7, 127.5, 118.1, 114.6,
114.5, 114.1, 111.4, 110.3, 108.6, 74.3, 38.3, 26.0. HRMS (ES) m/z
calcd. for C21H16N3O3BrNa: [M+H]+ 460.0273, found: 460.0287.
1
2
3
4
5
6
7
8
9
5-Bromo-3’-butyl-1-methyl-1’H-spiro[indoline-3,2’-quinazoline]-
2,4’(3’H)-dione (38). Butylamine. Beige solid (290 mg, 70%). 1H
NMR (400 MHz, DMSO-d6) δ 7.74–7.69 (m, 2H), 7.66 (dd, J=1.6,
7.7 Hz, 1H), 7.42 (s, 1H), 7.26 (ddd, J=1.6, 7.2, 8.1 Hz, 1H), 7.15 (dd,
J=0.6, 8.2 Hz, 1H), 6.74 (td, J=1.1, 7.4 Hz, 1H), 6.59 (dd, J=1.0,
8.1 Hz, 1H), 3.12 (s, 3H), 3.07 (m, 1H), 2.78 (m, 1H), 1.31 (m, 1H), 1.19
(m, 1H), 1.04 (m, 2H), 0.65 (t, J=7.3 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 173.2, 163.1, 145.3, 142.9, 134.1, 133.3, 128.9, 128.2,
127.2, 117.9, 114.7, 114.4, 113.8, 111.7, 75.0, 42.5, 30.1, 26.2, 19.4,
13.3. HRMS (ES) m/z calcd. for C20H21N3O2Br: [M+H]+ 414.0817,
found: 414.0816.
5-Bromo-1-methyl-3’-phenethyl-1’H-spiro[indoline-3,2’-quinazo-
line]-2,4’(3’H)-dione (33). Phenylethylamine. Beige solid (380 mg,
1
82%). H NMR (400 MHz, DMSO-d6) δ 7.74 (dd, J=2.1, 8.4 Hz, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
7.71 (dd, J=1.6, 7.7 Hz, 1H), 7.68 (dd, J=0.4, 2.0 Hz, 1H), 7.47 (s,
1H), 7.28 (ddd, J=1.6, 7.3, 8.1 Hz, 1H), 7.25–7.19 (m, 2H), 7.18–7.13
(m, 2H), 6.88–6.84 (m, 2H), 6.77 (ddd, J=1.0, 7.2, 7.7 Hz, 1H), 6.62
(ddd, J=0.5, 1.1, 8.1 Hz, 1H), 3.39–3.33 (m, 1H), 3.11 (s, 3H), 2.79
(ddd, J=5.5, 10.5, 12.7 Hz, 1H), 2.70 (td, J=5.0, 10.8, 11.5 Hz, 1H),
2.66–2.58 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 173.1, 163.3,
145.4, 143.1, 138.5, 134.2, 133.5, 128.7, 128.5, 128.23, 128.21, 127.2,
126.3, 118.0, 114.8, 114.3, 113.9, 111.7, 75.2, 45.0, 34.3, 26.2. HRMS
(ES) m/z calcd. for C24H21N3O2Br: [M+H]+ 462.0817, found:
462.0826.
5-Bromo-3’-(2-methoxyethyl)-1-methyl-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (39). 2-Methoxyethylamine. Beige sol-
1
id (260 mg, 63%). H NMR (400 MHz, DMSO-d6) δ 7.73 (d, J=2.1 Hz,
1H), 7.69 (dd, J=8.4, 2.1 Hz, 1H), 7.66 (dd, J=7.8, 1.5 Hz, 1H), 7.41
(s, 1H), 7.26 (ddd, J=8.3, 7.4, 1.6 Hz, 1H), 7.10 (d, J=8.4 Hz, 1H),
6.75 (td, J=7.5, 1.0 Hz, 1H), 6.60 (dd, J=8.4, 2.1 Hz, 1H), 3.30–3.21
(m, 3H), 3.20–3.11 (m, 1H), 3.09 (s, 3H), 2.97 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 172.9, 163.6, 145.3, 143.4, 134.0, 133.4, 128.5,
128.4, 127.2, 117.9, 114.6, 114.5, 113.9, 111.5, 75.1, 69.9, 57.8, 42.6,
26.2. HRMS (ES) m/z calcd. for C19H19N3O3Br: [M+H]+ 416.0610,
found: 416.0609
5-Bromo-3’-(cyclohexylmethyl)-1-methyl-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (34). Cyclohexylmethylamine. Beige
1
solid (291 mg, 64%). H NMR (400 MHz, DMSO-d6) δ 7.74–7.70 (m,
2H), 7.66 (dd, J=1.6, 7.8 Hz, 1H), 7.42 (s, 1H), 7.26 (ddd, J=1.6, 7.3,
8.1 Hz, 1H), 7.14 (dd, J=2.0, 7.1 Hz, 1H), 6.75 (td, J=1.1, 7.5 Hz, 1H),
6.60 (dd, J=1.0, 8.1 Hz, 1H), 3.11 (s, 3H), 3.06 (dd, J=6.7, 13.8 Hz,
1H), 2.71 (dd, J=7.7, 13.8 Hz, 1H), 1.61–1.48 (m, 3H), 1.43–1.34 (m,
2H), 1.15–1.05 (m, 1H), 1.04–0.85 (m, 3H), 0.80–0.61 (m, 2H). 13C
NMR (101 MHz, DMSO-d6) δ 173.1, 163.8, 145.3, 143.0, 134.1, 133.3,
128.6, 128.6, 127.4, 117.9, 114.7, 114.5, 113.9, 111.6, 74.9, 48.4, 37.3,
30.6, 30.5, 26.2, 25.8, 25.6. HRMS (ES) m/z calcd. for C23H25N3O2Br: [M
+H]+ 454.1130, found: 454.1126.
5-Bromo-3’-(2-(dimethylamino)ethyl)-1-methyl-1’H-spiro[indoline-
3,2’-quinazoline]-2,4’(3’H)-dione (40). Dimethylethylenediamine.
1
Beige solid (240 mg, 56%). H NMR (400 MHz, DMSO-d6) δ 7.79 (d,
J=2.0 Hz, 1H), 7.74 (dd, J=8.4, 2.0 Hz, 1H), 7.70 (dd, J=7.8, 1.5 Hz,
1H), 7.59 (s, 1H), 7.32 (ddd, J=8.4, 7.4, 1.5 Hz, 1H), 7.19 (d, J=
8.4 Hz, 1H), 6.79 (td, J=7.5, 0.7 Hz, 1H), 6.63 (dd, J=8.2, 0.7 Hz, 1H),
3.55–3.43 (m, 1H), 3.14 (s, 3H), 3.08–2.98 (m, 3H), 2.73 (s, 6H). 13C
NMR (101 MHz, DMSO-d6) δ 172.4, 164.2, 145.4, 142.9, 134.5, 134.2,
128.5, 127.7, 127.3, 118.1, 115.1, 114.1, 113.3, 112.2, 75.6, 55.0, 43.1,
42.3, 38.2, 26.4. HRMS (ES) m/z calcd. for C20H22N4O2Br: [M+H]+
429.0926, found: 429.0909
5-Bromo-1-methyl-3’-(pyridin-2-ylmethyl)-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (35). 2-Picolylamine. Beige solid
(270 mg, 60%). 1H NMR (400 MHz, DMSO-d6) δ 8.41 (dt, J=1.3,
5.1 Hz, 1H), 7.90 (td, J=1.8, 7.7 Hz, 1H), 7.71 (dd, J=1.6, 7.8 Hz, 1H),
7.57 (dd, J=2.1, 8.4 Hz, 1H), 7.54 (s, 1H), 7.41–7.36 (m, 2H), 7.35 (d,
J=2.0 Hz, 1H), 7.32 (ddd, J=1.6, 7.3, 8.7 Hz, 1H), 7.04 (d, J=8.4 Hz,
1H), 6.79 (td, J=1.1, 7.5 Hz, 1H), 6.65 (dd, J=0.9, 8.1 Hz, 1H), 4.80
(d, J=16.2 Hz, 1H), 4.04 (d, J=16.2 Hz, 1H), 3.02 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 172.7, 163.7, 155.8, 146.4, 145.7, 143.1, 139.2,
134.2, 133.9, 128.5, 127.9, 127.6, 123.2, 122.8, 118.2, 114.5, 114.1,
113.9, 111.7, 75.2, 46.6, 26.2. HRMS (ES) m/z calcd. for C22H18N4O2Br:
[M+H]+ 449.0613, found: 449.0619.
5-Bromo-3’-(3-(dimethylamino)propyl)-1-methyl-1’H-spiro[indo-
line-3,2’-quinazoline]-2,4’(3’H)-dione
(41).
3-Dimeth-
1
ylaminopropylamine. Beige solid (230 mg, 52%). H NMR (400 MHz,
DMSO-d6) δ 7.83 (d, J=2.1 Hz, 1H), 7.73 (dd, J=8.4, 2.1 Hz, 1H), 7.68
(dd, J=7.8, 1.5 Hz, 1H), 7.51 (s, 1H), 7.29 (ddd, J=8.7, 7.3, 1.6 Hz,
1H), 7.18 (d, J=8.4 Hz, 1H), 6.76 (td, J=7.5, 1.0 Hz, 1H), 6.62 (dd, J=
8.1, 1.0 Hz, 1H), 3.27 (dt, J=14.3, 7.1 Hz, 1H), 2.95 (p, J=5.7 Hz, 2H),
2.81 (dt, J=13.9, 6.8 Hz, 1H), 2.69 (dd, J=11.5, 4.6 Hz, 6H), 1.64 (p,
J=7.3 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) δ 173.0, 164.0, 145.4,
142.9, 134.3, 133.8, 128.7, 128.1, 127.3, 118.0, 115.0, 113.9, 113.8,
112.0, 75.3, 54.4, 42.1, 41.9, 26.3, 23.5. HRMS (ES) m/z calcd. for
C21H24N4O2Br: [M+H]+ 443.1083, found: 443.1093
5-Bromo-1-methyl-3’-(pyridin-3-ylmethyl)-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (36). 3-Picolylamine. Beige solid
(271 mg, 60%). 1H NMR (400 MHz, DMSO-d6) δ 8.63 (dd, J=1.5,
5.4 Hz, 1H), 8.26 (d, J=2.1 Hz, 1H), 7.84 (dt, J=1.8, 8.2 Hz, 1H), 7.74
(dd, J=1.6, 7.8 Hz, 1H), 7.68–7.62 (m, 2H), 7.57 (s, 1H), 7.43 (d, J=
2.0 Hz, 1H), 7.32 (ddd, J=1.6, 7.3, 8.1 Hz, 1H), 7.09 (d, J=8.4 Hz, 1H),
6.80 (td, J=1.1, 7.4 Hz, 1H), 6.64 (dd, J=1.0, 8.2 Hz, 1H), 4.61 (d, J=
16.0 Hz, 1H), 4.24 (d, J=16.0 Hz, 1H), 2.96 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 172.6, 163.9, 158.4, 158.1, 145.6, 144.2, 142.9,
140.5, 135.7, 134.3, 134.0, 128.6, 127.7, 127.6, 125.1, 118.1, 114.6,
114.1, 113.6, 111.9, 75.0, 43.1, 26.1. HRMS (ES) m/z calcd. for
C22H18N4O2Br: [M+H]+ 449.0613, found: 449.0617.
Chiral Separation of Compound 32
Analytical run. Supercritical fluid chromatography (SFC) analysis
was performed on a SFC system connected to a back pressure
regulator and a PDA detector. The sample was diluted to a
concentration of around 1 mg/mL and 10 μL was injected to a 5 μm
CHIRALPAK-ID, 4.6×150 mm (diameter×length) column held at
5-Bromo-3’-(furan-2-ylmethyl)-1-methyl-1’H-spiro[indoline-3,2’-
quinazoline]-2,4’(3’H)-dione (37). Furfurylamine. Beige solid
(277 mg, 63%). 1H NMR (400 MHz, DMSO-d6) δ 7.71 (dd, J=7.8,
1.6 Hz, 1H), 7.67 (dd, J=8.4, 2.1 Hz, 1H), 7.58 (d, J=2.0 Hz, 1H), 7.45
(s, 1H), 7.40 (dd, J=1.8, 0.8 Hz, 1H), 7.27 (ddd, J=8.1, 7.3, 1.6 Hz,
1H), 7.05 (d, J=8.4 Hz, 1H), 6.77 (ddd, J=7.7, 7.2, 1.1 Hz, 1H), 6.61
°
45 C. An isocratic condition of 35% MeOH in CO2 was applied at a
flow rate of 5 mL/min. The back pressure was set to 120 bar. The
PDA scanned from 220 to 400.
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Preparative run. The enantiomers of 32 were separated by
preparative SFC. The sample for chiral separation was prepared by
dissolving 20 mg in 600 μL MeOH and the preparative run was
performed by stacked injections on a SFC system connected to a
PDA detector. The column used was a 5 μm CHIRALPAK-ID, 10×
and Glu1002, which result in an overall neutral simulation
sphere.[26,32,33] During a 175 ps equilibration stage, the system was
slowly heated to the target temperature of 310 K while initial
positional restrains on all solute heavy atoms were gradually
released. The subsequent MD production phase consisted of 5
replica simulations of 2 ns each, with randomized initial velocities,
accounting for a total of 10 ns sampling trajectories. A time step of
1 fs was used, and no positional restraints were applied. Solvent
bonds and angles were constrained using the SHAKE algorithm.[43]
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250 mm (diameter×length) column held at 45 C. An isocratic
condition of 35% MeOH in CO2 was applied at a flow rate of 15 mL/
min. The back pressure was set to 120 bar. The PDA scanned from
220 to 400 and the enantiomers were collected in separated
fractions (with the aid of 2 mL/min of MeOH as make up solvent for
the collection) and pooled from each injection.
Biological Methods
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Molecular Modelling
Overexpression of Human IRAP and Aminopeptidase N
The crystal structure of human IRAP was retrieved from the protein
data bank (PDB code 4PJ6)[45] and prepared as previously described
for docking and molecular dynamics (MD) simulations, including
protonation and fixing sidechain rotamers.[26] The ligands 1, 3, 12,
24, 32, 38 and 40 were built in their most probable 3D
conformation using Maestro Molecular Modelling Interface version
10.3 (Schrödinger, LLC; NY). The LigPrep module was used to
generate separate versions for their S and R configuration.
Overexpression of human IRAP and aminopeptidase N (APN) was
achieved in Freestyle HEK293-F Cells (Invitrogen R790-07). The cells
were grown in 80 ml of Freestyle Expression Medium (Invitrogen
12338–018) in 250 ml Erlenmeyer flasks (Corning 431144.40). The
°
flasks were kept at 37 C, 8% CO2, 70% relative humidity and with
shaking at 130–135 rpm in an Infors Multitron incubator. The cells
were transfected with 1.1 μg IRAP overexpressing plasmid/106
cells[30] or 0.7 μg APN overexpressing plasmid[17] (kindly provided Dr.
Alexandros Nikolau at Vrije Universiteit Brussels, Brussels, Belgium)/
106 cells in the presence of 2 μg polyethyleneimine/106 cells
(Polyethyleneimine, linear, MW-25 000, Cat. No. 23966, Polyscien-
ces). Prior to overexpression, the plasmids were transfected and
propagated in Mach1 E.coli (Invitrogen C8620-03). The plasmid
DNA was prepared using QIAGEN Plasmid Plus Maxi Kit (12963).
48 h post-transfection, the cells were harvested by centrifugation at
130×g for 3 min. The cells were washed once with PBS and the cell
Docking and Molecular Dynamics Simulations
A thorough docking exploration was conducted using Glide[46] in
the extra-precision (XP) mode, performing independent explora-
tions for each isomeric form of the molecule on three areas of the
IRAP inner crevice, with cubic grids of 30 Å size on each axis,
centred at different points: One grid covered the so-called H-site,
with centre on the equivalent position of the binding site of the Cα
atom of His4 in Ang IV as previously determined.[26] The next area
was the Z-site (close to Zn2+ site centred at x=28.98, y=2.14, z=
76.40) and finally, we also explored a putative A-site (allosteric site,
away from Zn2+) with centre at x=30.97, y=À 1.41, z=59.52.
Docking on the Z and A sites was performed using ligand-based
core constraints (Core SMARTS: “c1cccc(NC2)c1 C23NCc4c(N3)
cccc4” ) while no core constraints were used in H-site docking
calculations. Ligands were ranked using XP-Glide scoring
function,[47] which can be written as
°
pellets were stored at À 20 C until used for membrane prepara-
tions.
Membrane Preparations
All experiments were based on membrane preparations from either
CHO cells or HEK293T suspension cells overexpressing human IRAP
or APN. Membrane preparation was achieved essentially as
described previously[52] using the following procedure. Washed cell
°
pellets were taken out of the À 20 C freezers and quickly thawed
and then placed on ice. Pellets were resuspended in ice-cold
50 mM Tris-HCl at pH 7.4 supplemented with 0.1 mM of phenyl-
methanesulfonyl fluoride (PMSF; Sigma-Aldrich 78830) and then
sonicated for 3×15 sec with 30 sec interruption time using a
Branson Digital sonifier 250. The sonicated cells were then trans-
ferred to a Dounce homogenizer and homogenized by a minimum
of 20 strokes while keeping everything on ice. The homogenate
GlideScoreXP¼Ecoul þ EvdW þ Ebind þ Epenalty
(1)
Where the favourable and non-favourable terms for binding are
grouped into Ebind and Epenalty respectively.
MD simulations of the IRAP-38 complex as determined in binding
site Z were performed using the Q[48] program and the OPLS-AA
force field there implemented.[49] Parameters for the Zn2+ ion and
ligand 38 were retrieved from the automatic parametrization
available in Macromodel, version 10.3 (Schrödinger LLC). Simula-
tions were done under spherical boundary conditions, with a
simulation sphere of 25 Å radius centred on the equivalent position
of the Cα atom of His4 in Ang IV.[26,32,33] This sphere was solvated
with TIP3P water molecules[50] and subjected to polarization and
radial constraints according to the surface constrained all-atom
solvent model[48,51] to mimic the properties of bulk water at the
sphere surface. Protein atoms outside the simulation sphere were
restrained to their initial positions and only interacted with the
system through their bonded terms. All ionizable residues within
20 Å of the Zn2+ ion were treated in their charged form, and from
those within the outer sphere layer the following residues were
also considered with their formal charge, respecting established
ionic pairs: Lys520, Lys726, Glu767, Asp773, Arg817, Glu818,
Arg820, Glu825, Arg858, Glu887, Lys890, Lys892, Glu895, Arg933,
°
was thereafter transferred to ice-cold tubes and centrifuged at 4 C
and 30.000×g for 30 min. The supernatant was discarded and the
membranes were resuspended in buffer and again homogenized
using the Dounce homogenizer (20 strokes) and thereafter
centrifuged as described above. This procedure was repeated twice
and the final membrane pellets from CHO cells were then kept
°
frozen at À 20 C until used. A slightly modified procedure was
applied for the HEK293T cell pellets, where the membrane
suspension from the second was aliquoted into separate vials and
°
kept frozen at À 80 C until the day of use. Prior to addition to the
microplates the thawed membrane suspensions were homogenized
in a Dounce homogenizer.
Concentration-Response Experiments in 96-Well Format.
Details for the assay used to evaluate the inhibitory activity of the
compounds have already been published.[30] In short:
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Concentration-response experiments were performed in transpar-
ent 96-well microtiter plates (Nunc, product no. 269620) using
membrane preparations from CHO-cells, known to abundantly
express IRAP and contain minimal contaminating APN activities.
The extent of enzymatic activity in each sample was quantified
using the peptide L-Leu-p-nitroanilid (L-Leu-pNA, Sigma-Aldrich,
product no. L9125), which upon IRAP-mediated cleavage of the N-
terminal amide bond produces p-nitroaniline that absorbs at
405 nm. The final assay volume was 200 μL in an assay buffer
consisting of 50 mM Tris-HCl, 150 mM NaCl and 0.1 mM phenyl-
methanesulfonylfluoride (PMSF) at pH 7.4. The assay was conducted
in the presence of a final concentration of 1 mM L-Leu-pNA.
Concentration-response experiments using the human orthologue
of IRAP or human APN were conducted using the identical set-up
as for the absorbance based CHO cell membranes assay. Aliquots of
membranes from HEK293 suspension cells overexpressing IRAP or
APN were thawed, suspended in assay buffer, homogenized using
the Dounce homogenizer and diluted with assay buffer.
is acknowledged. We thank Dr. Jonas Brånalt and the Wave 1
team at AstraZeneca R&D in Gothenburg for help with compound
registration and testing of preclinical properties. We thank Leif
Dahllund at SciLifeLab Drug Discovery and Development platform
for help with expression of human IRAP and APN. We thank
Camilo Persson for skillful assistance in the chemistry lab.
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Keywords: enzymes
·
inhibitors
·
insulin-regulated
aminopeptidases · preclinical profiling · spiro compounds
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The enzyme kinetics experiments were performed essentially as
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Compound Profiling
Preclinical profiling of the compounds was performed at AstraZe-
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metabolic stability assays based on rat hepatocytes and human
liver microsomes, respectively, a logD assay investigating partition-
ing between an aqueous solution and octanol, a solubility assay in
PBS at pH 7.4 and a plasma protein binding assay performed in
human serum and reporting both the fraction unbound and
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Acknowledgements
The work described herein was supported by Uppsala University,
the Swedish Research Council, the Kjell and Märta Beijer
Foundation, the Swedish Brain Foundation, and King Gustaf V:s
and Queen Victoria’s Foundation of Freemasons. Computing time
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