Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of the MDM2-p53 interaction

Article abstract of DOI:10.1016/j.ejmech.2016.11.029

Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-MDM2. An extensive SAR study was performed based on our four-point pharmacophore model

Full text of DOI:10.1016/j.ejmech.2016.11.029

Accepted Manuscript
Rational design and synthesis of 1,5-disubstituted tetrazoles as potent inhibitors of
the MDM2-p53 interaction
Ewa Surmiak, Constantinos G. Neochoritis, Bogdan Musielak, Aleksandra Twarda-
Clapa, Katarzyna Kurpiewska, Grzegorz Dubin, Carlos Camacho, Tad A. Holak,
Alexander Dömling
PII:
S0223-5234(16)30967-9
10.1016/j.ejmech.2016.11.029
EJMECH 9062
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 August 2016
Revised Date: 10 November 2016
Accepted Date: 12 November 2016
Please cite this article as: E. Surmiak, C.G. Neochoritis, B. Musielak, A. Twarda-Clapa, K. Kurpiewska,
G. Dubin, C. Camacho, T.A. Holak, A. Dömling, Rational design and synthesis of 1,5-disubstituted
tetrazoles as potent inhibitors of the MDM2-p53 interaction, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.11.029.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Rational design and synthesis of 1,5-disubstituted tetrazoles as potent
inhibitors of the MDM2-p53 interaction
a
b
a
Ewa Surmiak , Constantinos G. Neochoritis , Bogdan Musielak , Aleksandra Twarda-Clapa
c,d
a
c,d
e
a,d
,
Katarzyna Kurpiewska , Grzegorz Dubin , Carlos Camacho, Tad A. Holak
,
*,b
Alexander Dömling
a
Department of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Krakow, Poland
b
Department of Pharmacy, Drug Design Department, University of Groningen, 9713AV
Groningen, The Netherlands
c
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University,
Gronostajowa 7, 30-387 Krakow, Poland
d
Malopolska Centre of Biotechnology, Jagiellonian University, Gronostajowa 7a, 30-387
Krakow, Poland
e Department of Computational and Systems Biology, University of Pittsburgh, 3501 Fifth
Avenue, Biomedical Science Tower 3, Pittsburgh, PA 15260, USA
Abstract
Using the computational pharmacophore-based ANCHOR.QUERY platform a new scaffold
was discovered. Potent compounds evolved inhibiting the protein-protein interaction p53-
MDM2. An extensive SAR study was performed based on our four-point pharmacophore
model, yielding derivatives with affinity to MDM2 in the nanomolar range. Their binding
affinity with MDM2 was evaluated using both fluorescence polarization (FP) assay and 2D-
NMR-HSQC experiments.
1
. Introduction
The tumor suppressor p53 protein, ‘the guardian of the genome’, has an overarching role in
protecting the organism from cancer. From the time of its discovery in l979, it has become
one of the most frequently researched proteins. Its regulation complexity, variety of roles and
importance in cancer makes it one of the best known, but still not fully understood,
challenging protein.
Nearly all human cancers have either mutated the p53 itself (50% all cancers) or
1
compromised the effectiveness of the p53 pathway. The latter group of tumors retains the
1

ACCEPTED MANUSCRIPT
wild type p53 (wt-p53) but its pathway is inactivated by negative regulators, mainly the
MDM2 and MDMX proteins. Therefore, a low-molecular-weight antagonist capable to
disrupt the MDM2/p53 interaction can reactivate p53 and inhibit or reverse tumor
2
,3
formation.
On the other hand, in tumors containing the wild type p53 blocking the interaction with
MDM2/MDMX by utilizing small molecule inhibitors, should provide an efficient, non-
2
genotoxic alternative for anticancer therapy. As far as the latter strategy is concerned, in the
4
last ten years tremendous progress has been observed since the initial demonstration of the
5
efficacy of the early inhibitor Nutlin-3. This resulted in the development of many small
molecule MDM2 inhibitors of different scaffolds, which several also entered into clinical
4
,6
evaluations. However, the discovery of new p53/MDM2/MDMX scaffolds is still of high
6
-10
interest due to insufficient PKPD properties currently seen in clinical trials.
MDM2/p53 interaction relies on the steric complementarity of the MDM2 cleft and the
hydrophobic face of the p53 helix, in particular a triad of p53 amino acids: Phe19, Trp23 and
1
1,12
Leu26, which is inserted deeply inside the binding pocket of MDM2.
Such interactions
define a three-finger-pharmacophore-model which characterizes the vast majority of the
1
3
currently available small-molecule MDM2 inhibitors. Nevertheless, certain new approaches,
1
4-17
as the four-finger-model, were recently proposed.
We have previously described highly potent p53-MDM2 antagonists addressing a novel
15
pocket formed by the often disordered N-terminus of MDM2. The scaffold consisted of an
α-aminoacylamide which can be conveniently formed in one synthetic step from commercial
1
8
or easily accessible starting materials using multicomponent reaction chemistry. In order to
change the physicochemical properties of the previously reported compound series, in this
study we used our pharmacophore-based virtual screening ANCHOR.QUERY platform to
1
9
discover alternative scaffolds. Among others, 1,5-disubstituted-1H-tetrazoles appeared as
promising scaffolds. Here we report the discovery, synthesis, optimization of the targeted
library of novel substituted tetrazoles based on the four-point pharmacophore model and
initial SAR for this class of compounds.
2

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2
. Results and discussion
2
. 1. Virtual Screening (VS)-based tetrazole scaffold discovery
The discovery of the current inhibitors was based on the recent four-point pharmacophore
1
5
model experimentally shown by co-crystallization by Bista el al. The main features of the
model are presented in Figure 1. The structure of novel inhibitor contains the 6-chloroindole-
2
-carboxylic acid which was used as an ‘anchor’ in order to mimic the Trp23 amino acid and
constrain the position of other substituents. Three additional binding sites were defined, that is
Phe19, Leu26 and the induced Leu26 subpocket, enlarged by the Tyr100 ‘open’ position.
Such model was evaluated using the open access pharmacophore based virtual screening
1
9
platform ANCHOR.QUERY software (http://anchorquery.csb.pitt.edu) (Figure 1A).
Thus, we uploaded the receptor of PDB ID 4MDN and we deleted the crystallographic
1
5
waters as Scorpion analysis declares them as less important. When using our recently
described α-aminoacylamide 1 with K of 600 nM as a template in ANCHOR.QUERY
i
(Figure 1B), the program automatically proposes a useful pharmacophore consisting of 1
aromatic, 2 aliphatic and 1 anchor pharmacophores (Figure 1C). However, we changed the
character of the initially hydrophobic site on top of Ar1 to an aromatic character. Querying
this model against a virtual library of ~2 billion compounds stored in ANCHOR.QUERY
resulted in a high ranking α-aminomethyl tetrazole scaffold which attracted our immediate
attention due to its drug-like features and ease of synthesis (Figure 1D). The scaffold was
predicted such that the Trp23 subpocket was occupied by an indole analogue, the Phe19
subpocket by an aliphatic substituent whereas the Leu26 subpocket by a substituted phenyl
group (Figure 1E). Finally, the induced Leu26 subpocket was occupied by a benzyloxy group.
The tetrazole residue occupied the central upper part of the pocket and served as a hub
providing appropriate substitution vectors to access the three subpockets (Figure 1).
3

ACCEPTED MANUSCRIPT
Figure 1. VS-based discovery of tetrazoles as potent p53 MDM2 antagonists. (A) Schematic
of the VS method for discovering new scaffolds based on template 1 from PDB ID 4MDN;
(B) 2D structure of 1 and imposed pharmacophore characters; (C) ANCHOR.QUERY derived
3
D four point pharmacophore model of 1: anchor in yellow, aromatic in pink, hydrophobe in
green; (D) one-pot MCR synthesis of tetrazole scaffold; (E) stereoview of alignment of small
molecule 1 (red lines) – MDM2 complex (grey lines) 4MDN with a predicted tetrazole
derivative (no 9 in ranking, blue sticks), hydrogen bond to backbone Leu54 is indicated with
yellow dotted line.
4

ACCEPTED MANUSCRIPT
2
.2. Synthesis of 1,5-disubstituted-1H-tetrazoles
The desired scaffold 2 can be easily obtained by an Ugi-tetrazole reaction (UT-4CR)
involving the anchoring 6-chloro-indole carboxaldehyde 4, the appropriate aliphatic amines
and isocyanides as well as TMSN (Scheme 1).
3
Scheme 1. Retrosynthetic scheme for the tetrazole derivatives.
Aldehyde 4 was synthesised from 6-chloro-indole derivative using the Vilsmeier-Haack
2
0
formylation reaction. To explore SAR of the Phe19 pocket we used commercially available,
bulky aliphatic amines. To explore Ile26 and induced pockets we probed m- and p-substituted
anilines and benzylamines with different degrees of rotational flexibility. The isocyanides 8,
1
2 and 17 were prepared by dehydration of the corresponding formamides (7, 11, 16
21
respectively) with POCl . In the case of isocyanides 8a-d, the required formamides 7a-d
3
were prepared by refluxing of the corresponding anilines 6a-d or 4-chlorobenzylamine 6e
2
2
with formic acid. The isocyanides 12a-e were prepared by formylation of hydroxyanilines
2
2
23
9
a-b, followed by the Williamson ether synthesis with various benzyl halides affording the
formamides 11a-e suitable for the isocyanide preparation. For the isocyanides 17a-c, the first
2
4
step was the amino group protection of 4-hydroxybenzylamine 13. Next, the Williamson
ether synthesis followed by deprotection resulted in the substituted benzylamine
hydrochlorides 15a-c. Finally, the latter can be easily formylated by refluxing in ethyl formate
2
5
with the presence of trimethylamine. All details describing synthesis of the required
precursors are shown in Schemes 2-4.
5

ACCEPTED MANUSCRIPT
Scheme 2. A general scheme for preparation of isocyanides 8a-e, based on anilines and 4-
a
chlorobenzylamine.
a
Reagents and conditions: (a) cmpds 6a-e (1.0 equiv.), formic acid (excess), reflux, 16 h, 80-99%; (b) cmpds 7a-
e (1.0 equiv.), POCl (1.0 equiv.), Et N (4.0 equiv.), DCM 0 ºC-rt, 3 h, 75-88%.
3
3
Scheme 3. A general scheme for preparation of isocyanides 12a-e, based on
a
benzyloxyanilines.
a
Reagents and conditions: (a) cmpds 9a or 9b (1.0 equiv.), formic acid (excess), reflux, 16 h, 72-95%; (b) cmpds
1
0a or 10b (1.0 equiv.), benzyl bromide (1.1 equiv.), potassium carbonate (1.5 equiv.), acetonitrile, reflux, 16 h,
0-83%; (c) cmpds 11a-e (1.0 equiv.), POCl (1.0 equiv.), Et N (4.0 equiv.), DCM, 0 ºC-rt, 3 h, 81-94%.
7
3
3
6

ACCEPTED MANUSCRIPT
Scheme 4. A general scheme for preparation of isocyanides 17a-c, based on N-
a
(benzyloxybenzyl) formamides.
+
-
NH Cl
NH2
NHBoc
3
NHCHO
POCl3, Et N
NC
4
1
. R Br, K
2
CO
3
(
2
Boc) O, NaHCO3
MeCN
HCO
2
Et
3
Et N
MeOH
2. HCl(aq)
3
DCM
OR4
OR4
OR4
17a-c
OH
OH
13
14
15a-c
16a-c
NC
NC
NC
O
O
O
F
Cl
Br
17c 93%
17a 56%
17b 98%
a
Reagents and conditions: (a) Cmpd 13 (1.0 equiv.), Boc O (1.1 equiv.), NaHCO (2.5 equiv.), methanol, rt, 16
2
3
h, 99%; (b) (i). cmpd 14 (1.0 equiv.), benzyl bromide (1.1 equiv.), potassium carbonate (1.5 equiv.), acetonitrile,
reflux, 16 h, (ii). 2 M HCl in MeOH, rt, 16 h; (c) cmpds 15a-c (1.0 equiv.), Et N (4.0 equiv.), ethyl
3
formate/MeOH (4:1, excess), reflux, 16 h 82-94%; (d) cmpds 16a-c (1.0 equiv.), POCl (1.0 equiv.), Et N (4.0
3
3
equiv.), DCM, 0 ºC-rt, 3 h 56-98%.
It was possible to confirm the structure of the isocyanide 12a by a single crystal X-ray
analysis revealing a coplanarity between the two phenyl groups with the isocyanide group
(Figure 2).
Figure 2. Molecular geometry observed in the crystal structures of 12a, showing the atom
labelling scheme. Displacement ellipsoids of non-hydrogen atoms are drawn at the 50%
probability level. H atoms are presented as small spheres with an arbitrary radius.
7

ACCEPTED MANUSCRIPT
Next, we proceeded in the UT-4CR and the subsequent carboxylic acid ethyl ester
hydrolysis (Scheme 5). After some optimization, the MCRs were performed under microwave
o
irradiation at 120 C for 40 min (we found that they also proceed after refluxing in sealed
tubes for 2-3 days) yielding the tetrazole esters 3. To the contrary, the reaction did not
proceed at room temperature, even after several days of stirring with an already pre-formed
Schiff base. The yields of the products did not significantly vary depending on the nature of
the isocyanide or amine. It is noteworthy, that sometimes when methanol was used as a
solvent, transterification occurred, whereas this was not the case when 2,2,2-trifluoroethanol
(TFE) was used. The hydrolysis was performed in an ethanol/water solution (1:1) with excess
of LiOH (10.0 equiv.) affording the target acids 2. The compounds are presented in Table 1.
a
Scheme 5. The general synthetic scheme for the tetrazole derivatives.
a
Reagents and conditions: (a) 6-chloro-3-formyl-1H-indole-2-carboxylate 4 (1.0 equiv.), amine 5 (1.0 equiv.),
isocyanide 8, 12, 17 (1.0 equiv.), TMSN (1.0 equiv.), 2,2,2-trifluoroethanol (1 mL), 120 ºC, 40 min, MW, 38-
3
7
4%; (b) UT-4CR product 3 (1.0 equiv.), LiOH (10.0 equiv.), EtOH/H O (1:1, 4 mL), reflux, 24-72 h, 10-79%.
2
8

ACCEPTED MANUSCRIPT
Table 1. Yields and activities of tetrazole-based inhibitors of p53-MDM2/X interaction.
K [µM] for
i
Yield (%)
Compound
Ester/Acid
Amine
Isocyanide
[a]
MDM2
1
2
(R )
(R )
Ester 3
Acid 2
Ester 3
Acid 2
3
3
3
.1/2.1
.2/2.2
.3/2.3
cyclopentyl
cyclohexyl
cycloheptyl
51
71
60
69
n.a.
4.38
8
a
65
62
n.a.
4.89
n.a.
117
10.87
8b
0.17
(44)
3
3
3
.4/2.4
.5/2.5
.6/2.6
cyclohexylmethyl
amyl
74
45
58
32
33
53
(
46 )
n.a.
0.29
0.64
8
c
44
cyclohexylmethyl
(
55)
3
3
.7/2.7
.8/2.8
amyl
74
56
52
37
n.a.
0.10
0.52
8d
cyclohexylmethyl
n.a.
n.a.
3
.9/2.9
amyl
43
55
0.65
8e
3
3
.10/2.10
.11/2.11
4-chlorobenzyl
44
47
21
53
n.a.
n.a.
0.17
0.06
cyclohexylmethyl
3
.12/2.12
amyl
45
59
n.a.
0.08
3
3
3
.13/2.13
.14/2.14
.15/2.15
cyclopentyl
cyclohexyl
cycloheptyl
12a
40
46
32
46
63
67
n.a.
n.a.
n.a.
15.70
2.47
0.99
1.06
9.52
3
.16/2.16
cyclopentyl
57
62
12b
(
16.17)
3
3
.17/2.17
.18/2.18
cyclohexyl
cycloheptyl
41
30
63
63
n.a
n.a
1.93
1.53
9

ACCEPTED MANUSCRIPT
3.19/2.19
3.20/2.20
3.21/2.21
3.22/2.22
3.23/2.23
1-adamantyl
10
57
66
72
59
21
21
67
57
23
n.a.
n.a.
n.a.
n.a.
n.a.
1.64
0.23
0.17
0.20
36.57
1
2c
cyclohexylmethyl
cyclobutyl
amyl
12d
cyclohexylmethyl
3
.24/2.24
amyl
62
53
n.a.
n.a.
0.97
0.68
1
2e
3
3
.25/2.25
.26/2.26
cyclohexylmethyl
amyl
48
64
29
35
n.a.
0.86
0.02
17a
26.08
3
.27/2.27
cyclohexylmethyl
52
25
(
6.99)
0
.05
3
3
.28/2.28
.29/2.29
amyl
41
52
25
56
18.14
n.a.
(
4.29)
17b
cyclohexylmethyl
0.13
3
.30/2.30
amyl
46
60
n.a.
0.03
17c
3
3
.31/2.31
.32/2.32
cyclohexylmethyl
4-chlorobenzyl
57
51
47
79
n.a.
n.a.
0.07
0.09
3
.33/2.33
amyl
O
52
67
n.a.
0.12
Br
[a]
data in parentheses refers to MDMX protein whenever the compound showed any activity (all compounds
were tested against MDM2 and MDMX). n.a. – no activity against MDM2 protein. K values were calculated
i
based on fluorescence polarization binding assay (see experimental section and supporting information).
1
0

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The purity and identity of all intermediates and final compounds were confirmed by various
methods (LC-MS, NMR, HRMS, elemental analysis and IR). In addition, the structure and
identity of the described scaffold was confirmed by a single crystal X-ray analysis of
compound 3.26 (Figure 3). The structure demonstrates that the ring planes of substituents at
positions 1 and 5 are almost coplanar, being constrained by tetrazole geometry and are
oriented vertically to the plane of the tetrazole ring.
Figure 3. Crystal structure of compound 3.26 (racemic form) showing the asymmetric unit. A
single water molecule has co-crystallised with the compound. Displacement ellipsoids of non-
hydrogen atoms are shown at the 30% probability level. H atoms are presented as small
spheres with an arbitrary radius.
2
.3. Biophysical Screening and Structure-Activity Relationship Studies
Two complementary assays based on independent physicochemical principles, HSQC
NMR and fluorescence polarisation (FP) were used to exclude false positive hits.
Fluorescence polarization (FP) assay was employed to determine the inhibitory affinities (Ki)
2
6,27
of tetrazole derivatives against MDM2 and MDMX as previously described.
are presented in Table 1.
The results
Most of the obtained tetrazoles of a general structure of 2 are active towards MDM2, with
affinity below 1 µM. Surprisingly, some of the ester precursors (3) were also weakly active in
the tested system (3.4, 3.6, 3.16, 3.27 and 3.28). Compound 2.27 exhibited the best affinity
(20 nM), followed by compounds 2.28-2.33. This is consistent with the design expectations
1
1

ACCEPTED MANUSCRIPT
where derivatives with the elongated isocyanide moiety (based on 17a-c) should target an
additional, induced subpocket. Reducing the length of this moiety, utilizing substituted phenyl
isocyanides (isocyanides: 8a-d and 12a-12e, tetrazoles: 2.1-2.9 and 2.13-2.26), results in
slight decrease in affinities compared to the most active compounds. The inhibitors based on
the 4-chlorobenzyl isocyanide (8e) 2.10-2.12 exhibited better affinities than the corresponding
derivatives based on 4-chlorophenyl isocyanide (8b) 2.4-2.5. It seems that introducing
additional flexibility at this part of the molecule is essential for tighter fit and better binding to
MDM2. Noteworthy, even compounds which served as examples of the three-point
pharmacophore model (2.4-2.12) exhibited high affinities, comparable to their four-point
analogues (2.13-2.26).
No clear trend in affinity of compounds was observed comparing the different amine part
of tested molecules. However other trends are clearly noted. Compounds having no halogen
atom at the isocyanide-derived part (2.1-2.3, 2.13-2.18) are generally less active compared to
halogen containing derivatives. Among different halogens (e.g. F: 2.27-2.28, Cl: 2.29-2.30,
Br: 2.31-2.33), fluorine-substituted compounds showed best activities, with the most potent
compound 2.27, although the affinities of chlorine and bromine substituted compounds were
only slightly less. Incorporation of two halogens on both ortho positions of the elongated part
of the scaffold (2.23-2.24) significantly lowered the activity, with the worst binding
compound 2.23. A similar pattern was observed in the phenyl isocyanide-based inhibitors.
Exchanging the 4-chlorophenyl (2.4-2.5) group for the 3-chloro-4-fluorophenyl (2.8-2.9)
decreased the affinity. The presence of the isopropyl group lead to slightly better binding
(compounds 2.6-2.7 compared to 2.4-2.5).
Interestingly, selected compounds (3.4, 3.4, 3.6, 3.16, 3.27, 2.4, 2.28) demonstrated activity
against MDMX in parallel to MDM2. Nevertheless, affinities towards MDMX were around
1
000-fold lower compared to those against MDM2.
Compound 2.27 was chosen as a model for further characterization due to its high affinity
1
15
towards MDM2. H- N HSQC titration was used as a second orthogonal screening
2
8,29
system.
Strongest ligand-induced perturbations were observed in cross peaks
corresponding to residues Val88, Leu82, Gly12 and Thr49 (Figure 4). These included
doubling of cross peaks (two distinct sets of resonances originating from free and ligand-
bound protein); a behaviour which is characteristic for “slow” chemical exchange, for ligands
characterized by K lower than micromolar. Perturbations in resonances of residues within
D
the expected binding cleft advocates for the binding mode of 2.27 comparable to that
1
2

ACCEPTED MANUSCRIPT
predicted in our modelling whereas the “slow” exchange compares sub-micromolar affinity of
the compound.
1
15
Figure 4. NMR spectra for the H- N HSQC-based titration experiment of MDM2 with 2.27.
Red: reference MDM2 alone; blue: molar ratio protein/ligand 1:0.5; green: overtitrated
MDM2 (the ratio protein/ligand 1:2). Enlarged fragments show resonance peak doubling.
3
. Conclusions
A novel 1,5-disubstituted tetrazole scaffold targeting a clinically relevant p53/MDM2
interaction has been identified using our virtual screening platform approach
ANCHOR.QUERY. A focused library of more than 60 compounds was synthetized using
convergent 2-step multicomponent reaction chemistry. FP-monitored SAR analysis allowed
for the fast optimization of the scaffold up to low nM potency. Compound (2.27) resulted as
the most potent in the series, which inhibited the MDM2/p53 interaction with a K of 20 nM,
i
improving the affinity of the templated α-aminoacylamide 1 (K = 600 nM). The affinity and
i
the rough binding mode were confirmed using independent assay (2D NMR). Due to
1
3

ACCEPTED MANUSCRIPT
generally satisfactory ADMET properties of tetrazoles our compounds could serve as an
excellent starting point for further development of pharmaceutically relevant MDM2/p53
interaction inhibitors.
4
4
4
. Experimental section
.1. General methods
.1.1. Virtual Screening. The receptor and small molecule 1 (derived from PDB ID 4MDN)
were uploaded into ANCHOR.QUERY (http://anchorquery.csb.pitt.edu/). The pharmacophore
character of the benzylic phenyl group was changed in ANCHOR.QUERY from hydrophobe
to aromatic. Under the filters tab > hit reduction, the maximum hits per molecules were set to
1
. The maximum total hits was set to 200 and ranking was done according to lowest
molecular weight. The query yielded 134 compounds, several based on the tetrazole scaffold.
The hit compounds are energy optimized and such results were further visually inspected.
4
.1.2. Synthesis and analysis. All syntheses were performed using general procedures
summarized in Schemes 1-5 and as described below in details. Reagents were obtained from
commercial suppliers (Sigma Aldrich, ABCR, Acros and AK Scientific) and used without
further purification unless otherwise noted. All microwave irradiation reactions were carried
out in a Biotage Initiator™ Microwave Synthesizer.
Nuclear magnetic resonance spectra were recorded on a Bruker Avance 500 or 600
1
13
spectrometers { H NMR (500 MHz; 600 MHz), C NMR (126 MHz; 151 MHz)}. Chemical
1
shifts for H NMR were reported as δ values and coupling constants were in hertz (Hz). The
following abbreviations were used for spin multiplicity: s = singlet, br s = broad singlet, d =
doublet, t = triplet, q = quartet, quin = quintet, dd = double of doublets, ddd = double doublet
1
3
of doublets, m = multiplet. Chemical shifts for C NMR were reported in δ relative to the
solvent peak. Thin layer chromatography was performed on Fluka precoated silica gel plates
(0.20 mm thick, particle size 25 µm). Flash chromatography was performed on a Teledyne
ISCO Combiflash Rf, using RediSep Rf Normal-phase Silica Flash Columns (Silica Gel 60 Å,
®
®
2
30-400 mesh) and on a Reveleris X2 Flash Chromatography, using Grace Reveleris Silica
flash cartridges (12 grams). Elemental analysis was performed on a Vario Micro Cube
apparatus. Electrospray ionization mass spectra (ESI-MS) were recorded on a Waters
Investigator Semi-prep 15 SFC-MS instrument or on Shimadzu LCMS-2020 apparatus. The
1
4

ACCEPTED MANUSCRIPT
LC-MS measurements were performed on Shimadzu LCMS-2020 apparatus (column:
KINETEX C18 5 µM, 100A, 100 x 4.6 mm; method time: 10 min; solvents gradients A: 0-10
min 80% MeCN, 20% H O with negative ionisation for derivatives of tetrazole-carboxylic
2
acids; B: 0-10 min 75% MeCN, 25% MeOH with negative ionisation for amides and positive
for derivatives of tetrazole-esters). Melting points were determined with a Ascon-M5
apparatus. Melting points are reported uncorrected.
4
.1.3. Protein expression and purification. N-terminal domain of human MDM2 (residues 1-
1
18) was cloned into pET-20 vector (Novagen) and expressed in Escherichia coli BL21(DE3)
2
6
as described previously. In brief, cells were grown at 37 °C and induced with 1 mM IPTG at
OD600 of 0.8 and grown for additional 5 h at 37 °C. Cells were collected by centrifugation and
lysed by sonication. Inclusion bodies were collected by centrifugation, washed with PBS
containing 0.05% Triton-X100 and subsequently solubilized in 6 M guanidine hydrochloride
in 100 mM Tris-HCl, pH 8.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol. The
protein was dialyzed against 4 M guanidine hydrochloride, pH 3.5 supplemented with 10 mM
β-mercaptoethanol. Following, the protein was refolded by dropwise addition into 10 mM
Tris-HCl, pH 7.0, containing 1 mM EDTA and 10 mM β-mercaptoethanol and incubating
overnight at 4 °C. Ammonium sulphate was added to the final concentration of 1.5 M and the
refolded protein was recovered on Butyl Sepharose 4 Fast Flow (GE Healthcare). The protein
was eluted using 100 mM Tris-HCl, pH 7.2, containing 5 mM β-mercaptoethanol and further
purified by gel filtration on HiLoad 16/60 Superdex75 (GE Healthcare) in 50 mM phosphate
buffer pH 7.4 containing 150 mM NaCl and 5 mM DTT.
N-terminal domain of human MDMX (residues 1-134) was cloned into pET-46Ek/LIC vector
(Novagen). Cells were grown at 37 °C and induced with 0.5 mM IPTG at OD600 nm of 0.6.
The recombinant protein expression was carried for 12 h at 20 °C. The protein was purified
under native conditions using Ni-NTA Agarose (GE Healthcare). Preparation was polished by
gel filtration on HiLoad 16/60 Superdex75 (GE Healthcare).
4
.1.4. Fluorescence polarization binding assay. Fluorescence polarization experiments were
2
6
performed as previously reported by Czarna et al. using Tecan InfinitePro F200 plate reader
with the 485 nm excitation and 535 nm emission filters. The fluorescence intensities, parallel
and perpendicular to the plane of excitation, were measured in Corning black 96-well NBS
assay plates at room temperature. Fluorescence polarization values were expressed in
millipolarization units (mP). All the experiments were performed in duplicates and plates
1
5

ACCEPTED MANUSCRIPT
were read 15 min after mixing of all assay components. For each assay, new protein stocks
were thawed and the protein concentrations were determined using Bradford method. Assay
buffer contained 50 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA and 5% DMSO.
Competition binding assays were performed using 10 nM fluorescent P2 peptide (5’-FAM-
LTFEHYWAQLTS) and protein concentration equivalent to f =0.8. Tested compounds,
0
dissolved in DMSO were evaluated at serial dilutions. Nutlin 3 (Cayman Chemicals) and
peptide Z (SQETFSDLWKLLPEN) served as positive controls for MDM2 and MDMX,
respectively. Inhibition curves were fitted using Excel program to obtain IC and K values.
5
0
i
2
7
All calculations were done according to Huang et al.
1
15
15
4
.1.5. H- N HSQC binding assay. Uniform N isotope labeling was achieved by expression
1
5
of the protein in the M9 minimal media containing NH Cl as the sole nitrogen source. 10%
4
(
v/v) of D O was added to the samples to provide lock signal. All the spectra were recorded at
2
1
15
3
00K using a Bruker Avance 600 MHz spectrometer. H- N heteronuclear correlations were
i
obtained using the SOFAST-HMQC pulse sequence. Assignment of the amide groups of
ii-iii
MDM2 was obtained as previously reported.
4
.1.6. Crystal structure determination. Single crystals of 3.26 and 12a were obtained by slow
crystallization from mixture of ethanol and water. Diffraction data were collected at 120 K on
SuperNova (Rigaku Oxford Diffraction) four circle diffractometer with a microfocus Mo Kα
(0.71069 Å) radiation source and a graphite monochromator equipped with a CryoJet HT
cryostat system (Oxford Instruments). The crystal structure was solved using a direct method
iv
implemented in SUPERFLIP software. The crystals belonged to space group P-1 as
suggested by the CrysAlisPro diffraction data processing software (Oxford Diffraction). The
v
structure was refined by a full-matrix least squares technique using SHELXL-97.
Calculations were performed using WinGX (ver. 2013.2) integrated system.
All non-hydrogen atoms were refined anisotropically to ensure the convergence of the
refinement process. All hydrogen atoms joined to carbon atoms were positioned with an
idealized geometry, and refined using a riding model. The O- and N-bound hydrogen atoms
were located from difference Fourier map and refined freely.
The disorder for the pentane moiety was modelled by splitting atoms with the highest
anisotropic displacement parameters (ADPs) into two components. To ensure the convergence
of the refinement some distances of disordered fragments were restrained, and the ADPs of
the disordered atoms were also restrained to have similar values.
1
6

ACCEPTED MANUSCRIPT
4
4
6
.2. Synthetic procedures and analytical data.
.2.1. 6-Chloro-3-formyl-1H-indole-2-carboxylic acid ethyl ester 4
-Chloro-1H-indole-2-carboxylic acid ethyl ester (5.0 g, 22.3 mmol) and DMF (20 mL) were
placed in round-bottom flask equipped with CaCl tube. Then, POCl (2.49 mL, 26.8 mmol)
2
3
was added dropwise and reaction mixture was heated overnight (16 h) at 50 ºC. Afterwards,
the reaction was cooled to rt, quenched with saturated NaHCO solution and extracted with
3
ethyl acetate. Organic layer was collected, washed with water and brine, dried over anhydrous
MgSO and evaporated. The crude product was washed with diethyl ether giving compound 4
4
2
0
as a light yellow solid in 92% (5.15 g) yield.
1
mp: 243-244 ºC; NMR: H (600 MHz, DMSO-d ): δ 12.90 (s, 1H), 10.58 (s, 1H), 8.22 (d, J
6
=
8.6 Hz, 1H), 7.56 (d, J = 1.5 Hz, 1H), 7.32 (dd, J = 8.6, 1.9 Hz, 1H), 4.46 (q, J = 7.1 Hz,
1
3
2
1
1
H), 1.40 (t, J = 7.1 Hz, 3H); C (151 MHz, DMSO-d ): δ 187.5, 159.9, 136.1, 133.5, 130.4,
6
-1
24.0, 123.9, 123.4, 118.2, 112.6, 62.0, 14.1; IR (ATR) [cm ]: 3154, 2992, 2908, 1726,
639, 1571, 1532, 1433, 1300, 1221, 1195, 1100, 1163, 1030, 916, 854, 779, 698; Elemental
analysis: Calcd for C H ClNO : C, 57.27; H, 4.01; N, 5.57, found: C, 57.51; H, 4.26; N,
1
2
10
3
-
5
.50; LC-MS (DAD/ESI): t = 2.97 min, Calcd for C H ClNO (m/z): [M-H] 250.03,
R 12 10 3
-
-
-
[
M+2-H] 250.02, found: [M-H] 250.05, [M+2-H] 252.05.
t
4
.2.2. Butyl 4-hydroxybenzylcarbamate 14
4
-Hydroxybenzylamine 13 (4.00 g, 32.5 mmol), (Boc) O (7.80 g, 35.8 mmol) and NaHCO3
2
(3.41 g, 81.2 mmol) were refluxed in methanol overnight (16 h). Afterwards, the reaction was
cooled to rt and methanol was evaporated. To the resulting slurry, water and ethyl acetate
were added and the aqueous phase was extracted with ethyl acetate. Organic layers were
collected, washed with water and brine, dried over anhydrous MgSO and evaporated, giving
4
crude product as brown oil. The crude product was precipitated and washed with diethyl
ether/petroleum ether (approx. 1:1) giving compound 14 as light brown solid with 99% (7.17
2
4
g) yield.
mp: 87 ºC; NMR: H (600 MHz, CDCl ): δ 7.10 (d, J = 7.3 Hz, 2H), 6.77 (dt, J = 8.6, 2.0
1
3
1
3
Hz, 2H), 4.86 (s, 1H), 4.22 (s, 2H), 1.46 (s, 9H); C (151 MHz, CDCl ): δ 156.3, 155.5,
3
-1
1
30.5, 129.0, 115.6, 79.9, 44.3, 28.6; IR (ATR) [cm ]: 3340, 3153, 2974, 1673, 1615, 1598,
549, 1517, 1448, 1367, 1292, 1257, 1227, 1160; Elemental analysis: Calcd for C H NO :
1
1
2
17
3
C, 64.55; H, 7.67; N, 6.27, found: C, 64.69; H, 7.50; N, 6.09; LC-MS (DAD/ESI): t = 5.31
R
-
-
min, Calcd for C H NO (m/z): [M-H] 222.26, found: [M-H] 222.10.
1
2
17
3
1
7

ACCEPTED MANUSCRIPT
4
.2.3. General synthetic procedures for formylation and analytical data
Method A
The corresponding aniline or benzylamine 6a-e or 9a-b and formic acid (excess,
approximately 5 mL for 10 mmol of aniline) were placed together in a round-bottom flask
equipped with CaCl tube and refluxed overnight (16 h). Next, formic acid was evaporated
2
and the resulting solid (except for compound 7c) was washed with diethyl ether giving crude
product (7b-e or 10a-b; yields 72-99%).
Method B
The corresponding benzylamine hydrochloride salts 15a-c (1.0 equiv.), Et N (4.0 equiv.),
3
ethyl formate and methanol (4:1 ratio) were refluxed overnight (16 h). After the reaction was
completed, the products were poured into saturated NH Cl solution, which was then extracted
4
with ethyl acetate. Organic layers were collected, washed with water and brine, dried over
anhydrous MgSO and evaporated. The resulting solid was washed with diethyl ether giving
4
crude products (16a-c; yields 82-94%).
4
.2.3.1. N-(3-phenoxyphenyl)formamide 7a
To a stirred solution of 3-(phenoxy)aniline 6a (1.0 equiv.) in ethylformate (3 mL),
trimethylamine (1.0 equiv) was added. The reaction mixture was refluxed for 2 days and the
resulting mixture was extracted with ethyl acetate. Organic layers were collected, washed
with water and brine, dried over anhydrous MgSO and evaporated giving the product 7a as
4
white solid in 80% yield.
1
NMR: mixture of two rotamers H (600 MHz, CDCl ): δ 8.58 (d, J = 10 Hz, 1H), 8.36 (s,
3
1
3
1
H), 7.50 (d, J = 5 Hz, 2H), 7.36-7.30 (m, 4H), 7.12-6.98 (m, 12H), ; C (151 MHz, CDCl ):
3
δ 162.8, 159.0, 130.1, 130.0, 123.8, 123.5, 123.0, 123.5, 122.0, 121.5, 119.8, 119.0, 118.8.
4
.2.3.2. N-(4-Chlorophenyl)formamide 7b
Method A: 4-chloroaniline 6b (3.3 g, 25.9 mmol). Crude product was recrystallized from
CHCl giving 7b as light gray crystals in 90% (3.61 g) yield.
3
vi
1
mp: 101 ºC (lit. 100-102ºC) ; NMR: mixture of two rotamers H (600 MHz, CDCl ): δ (main
3
rotamer) 10.31 (s, 1H), 8.28 (d, J = 1.8 Hz, 1H), 7.61 (dt, J = 8.9, 2.0 Hz, 2H), 7.42-7.33 (m,
1
3
2
1
1
H); C (151 MHz, CDCl ): δ 162.5, 159.7, 137.4, 137.2, 129.2, 128.8, 127.4, 127.1, 120.7,
3
-1
19.0; IR (ATR) [cm ]: 3258, 3193, 3122, 3061, 2896, 1686, 1668, 1608, 1543, 1490, 1398,
312, 1255, 1172, 1088, 1012, 830, 769; Elemental analysis: Calcd for C H ClNO: C, 54.04;
7
6
H, 3.89; N, 9.00, found: C, 54.06; H, 4.07; N, 8.98; LC-MS (DAD/ESI): t = 2.86 min, Calcd
R
1
8

ACCEPTED MANUSCRIPT
-
-
-
-
for C H ClNO (m/z): [M-H] 154.01, [M+2-H] 156.00, found: [M-H] 154.05, [M+2-H]
7
6
1
56.00.
4
.2.3.3. N-(4-Isopropylphenyl)formamide 7c
Method A: 4-isopropylaniline 6c (3.0 mL g, 21.9 mmol). Product 7c was obtained as brown
vii
liquid in 99% (3.52 g) yield.
1
NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.83 (d, J = 10.8 Hz,
3
1
H), 8.64 (d, J = 11.4 Hz, 1H), 8.32 (d, J = 1.9 Hz, 1H), 8.04 (s, 1H), 7.47 (dt, J = 8.5, 1.9
Hz, 2H), 7.20 (dt, J = 8.5, 1.8 Hz, 2H), 7.17 (dt, J = 8.4, 1.8 Hz, 2H), 7.03 (dt, J = 8.5, 1.9
1
3
Hz, 2H), 2.93-2.82 (m, 2H), 1.22 (d, J = 6.9 Hz, 6H), 1.24 (d, J = 6.9 Hz, 6H); C (151
MHz, CDCl ): δ 163.3, 159.5, 146.3, 145.6, 134.7, 134.5, 127.7, 120.7, 120.3, 119.2, 33.7,
3
-1
3
3.6, 24.0; IR (ATR) [cm ]: 3268, 3114, 3053, 2960, 2870, 1683, 1610, 1520, 1412, 1313,
-
8
32; LC-MS (DAD/ESI): t = 5.68 min, Calcd for C H NO (m/z): [M-H] 162.09, found:
R
10 13
-
[
M-H] 162.10.
4
.2.3.4. N-(3-Chloro-4-fluorophenyl)formamide 7d
Method A: 3-chloro-4-fluoroaniline 6d (3.2 g, 21.8 mmol). Crude product was recrystallized
from CHCl giving compound 7d as colorless crystals in 93% (3.52 g) yield.
3
viii
1
mp: 96 ºC (lit. 94-95 ºC) ; NMR: mixture of two rotamers H (600 MHz, DMSO-d ): δ
6
(main rotamer) 10.39 (s, 1H), 8.29 (d, J = 1.4 Hz, 1H), 7.90 (dd, J = 6.8, 2.6 Hz, 1H), 7.47
1
3
(
ddd, J = 9.0, 4.3, 2.6 Hz, 1H), 7.38 (t, J = 9.1 Hz, 1H); C (151 MHz, DMSO-d ): δ 162.8,
6
1
1
2
59.9, 154.5, 154.1, 152.9, 152.4, 135.9, 135.5, 135.5, 120.6, 119.5, 119.4, 119.3, 119.2,
-1
19.0, 117.90, 117.85, 117.6, 117.4, 117.2, 117.0; IR (ATR) [cm ]: 3256, 3203, 3143, 3077,
690, 2863, 1674, 1609, 1559, 1496, 1403, 1319, 1254, 1164, 1061, 867, 819, 754, 712;
Elemental analysis: Calcd for C H ClFNO: C, 48.44; H, 2.90; N, 8.07, found: C, 48.63; H,
7
5
-
3
.00; N, 8.08; LC-MS (DAD/ESI): t = 2.80 min, Calcd for C H ClFNO (m/z): [M-H]
R 7 5
-
-
-
1
72.00, [M+2-H] 174.00, found: [M-H] 172.00, [M+2-H] 174.00.
4
.2.3.5. N-(4-Chlorobenzyl)formamide 7e
Method A: 4-chlorobenzylamine 6e (4.0 mL g, 32.9 mmol). Crude product was recrystallized
from cyclohexane giving compound 7e as colorless crystals in 86% (4.79 g) yield.
1
mp: 142 ºC; NMR: H (600 MHz, DMSO-d ): δ 8.36 (s, 1H), 7.48-7.40 (m, 4H), 3.89 (s, 2H);
6
1
3
-1
C (151 MHz, DMSO-d ): δ 165.5, 137.0, 132.1, 130.1, 128.3, 42.5; IR (ATR) [cm ]: 2892,
6
2
783, 2708, 2635, 1624, 1574, 1497, 1442, 1370, 1342, 1153, 1100, 1010, 910, 818, 771;
1
9

ACCEPTED MANUSCRIPT
Elemental analysis: Calcd for C H ClFNO*H O: C, 51.21; H, 5.37; N, 7.47, found: C, 51.17;
7
5
2
H, 5.19; N, 7.22.
4
.2.3.6. N-(4-Hydroxyphenyl)formamide 10a
Method A: 4-hydroxyaniline 9a (2.0 g, 18.3 mmol). Crude product was recrystallized from
CHCl /cyclohexane giving compound 10a as light brown solid in 95% (2.39 g) yield.
3
vi
1
mp: 137 ºC (lit. 135-137 ºC) ; NMR: mixture of two rotamers (approx. 1:3) H (600 MHz,
DMSO-d ): δ (main rotamer) 9.88 (s, 1H), 9.22 (s, 1H), 8.15 (d, J = 2.0 Hz, 1H), 7.37 (dt, J =
6
1
3
8
1
2
.8., 2.2 Hz, 2H), 6.70 (dt, J = 8.8, 2.2 Hz, 2H); C (151 MHz, DMSO-d ): δ 162.6, 158.8,
6
-1
54.2, 153.5, 130.0, 129.7, 120.8, 120.2, 115.8, 115.2; IR (ATR) [cm ]: 3305, 3094, 2979,
883, 2798, 2677, 2611, 1657, 1558, 1507, 1400, 1379, 1252, 1170, 1107, 828; Elemental
analysis: Calcd for C H NO : C, 61.30; H, 10.21; N, 5.14, found: C, 61.10; H, 5.43; N, 10.14;
7
7
2
-
-
LC-MS (DAD/ESI): t 5.16 min, Calcd for C H NO (m/z): [M-H] 136.13, found: [M-H]
R
7
7
2
1
36.10.
4
.2.3.7. N-(3-Hydroxyphenyl)formamide 10b
Method A: 3-hydroxyaniline 9b (3.2 g, 21.8 mmol). Crude product was recrystallized from
CHCl giving compound 10b as light green solid in 72% (3.64 g) yield.
3
ix
1
mp: 112 ºC (lit. 135-137 ºC) ; NMR: mixture of two rotamers (approx. 1:3) H (600 MHz,
DMSO-d ): δ (main rotamer) 10.04 (s, 1H), 9.41 (s, 1H), 8.22 (d, J = 1.9 Hz, 1H), 7.17 (t, J =
6
1
3
2
.1 Hz, 1H), 7.07 (t, J = 8.1 Hz, 1H), 6.47 (ddd, J = 8.1, 2.3, 0.7 Hz, 1H); C (151 MHz,
DMSO-d ): δ 162.4, 159.5, 158.3, 157.7, 139.5, 130.2, 129.6, 110.8, 110.8, 109.9, 108.0,
6
-1
1
06.4, 104.7; IR (ATR) [cm ]: 3327, 3131, 1644, 1598, 1614, 1545, 1488, 1402, 1332, 1293,
200, 935, 783, 717; Elemental analysis: Calcd for C H N O: C, 61.30; H, 10.21; N, 5.14,
1
7
7
2
found: C, 61.31; H, 5.14; N, 10.21; LC-MS (DAD/ESI): t = 5.14 min, Calcd for C H NO
2
R
7
7
-
-
(
m/z): [M-H] 136.13, found: [M-H] 136.05.
4
.2.3.8. N-4-[(2-Fluorobenzyl)oxy]benzylformamide 16a
Method B: Hydrochloride 15a (1.4 g, 5.2 mmol), Et N (2.92 mL, 21.0 mmol). Crude product
3
was recrystallized from EtOH/H O giving compound 16c as colourless solid in 82% (1.12 g)
2
yield.
1
mp: 202-203 ºC; NMR: mixture of two rotamers H (600 MHz, CDCl ): δ (main rotamer)
3
8
.24 (s, 1H), 7.49 (td, J = 7.5, 1.3 Hz, 1H), 7.34-7.29 (m, 1H), 7.22 (d, J = 8.6 Hz, 2H), 7.19-
.14 (m, 1H), 7.11-7.06 (m, 1H), 6.95 (d, J = 8.6 Hz, 2H), 5.83 (s, 1H), 5.12 (s, 2H), 4.42 (d,
7
2
0

ACCEPTED MANUSCRIPT
1
3
J = 5.5 Hz, 2H); C (151 MHz, CDCl ): δ 161.3, 160.9, 159.6, 158.1, 130.1, 129.9, 129.8,
3
1
6
1
29.7, 129.7, 129.7, 129.3, 128.4, 124.3, 124.3, 124.0, 124.0, 111.5, 115.3, 115.3, 115.1, 63.8,
-1
3.7, 41.7; IR (ATR) [cm ]: 3272, 3041, 2880, 1653, 1631, 1539, 1515, 1490, 1456, 1386,
306, 1254, 1231, 1174, 1056, 814, 799, 784, 750; Elemental analysis: Calcd for
C H FNO : C, 69.49; H, 5.44;, N, 5.40, found: C, 70.17; H; 5.43, N, 5.15.
15
14
2
4
.2.3.9. N-4-[(2-Chlorobenzyl)oxy]benzylformamide 16b
Method B: Hydrochloride 15b (1.2 g, 4.2 mmol), Et N (2.35 mL, 16.9 mmol). Crude product
3
was recrystallized from EtOH/H O giving compound 16b as colourless solid in 94% (1.09 g)
2
yield.
1
mp: 111-112 ºC; NMR: mixture of two rotamers H (600 MHz, CDCl ): δ (main rotamer)
3
8
.24 (s, 1H), 7.55-7.52 (m, 1H), 7.41-7.38 (m, 1H), 7.30-7.25 (m, 2H), 7.23 (d, J = 8.6 Hz,
1
3
2
H), 6.96 (dt, J = 8.5, 2.0 Hz, 2H), 5.83 (s, 1H), 5.16 (s, 2H), 4.43 (d, J = 5.6 Hz, 2H); C
(
151 MHz, CDCl ): δ 161.0, 158.2, 134.7, 132.7, 130.3, 129.6, 129.4, 129.2, 128.9, 128.6,
3
-1
1
27.1, 115.3, 67.3, 41.9; IR (ATR) [cm ]: 3281, 3023, 2874, 1648, 1515, 1443, 1382, 1351,
305, 1254, 1219, 1181, 1126, 1045, 1036, 810, 752, 693; Elemental analysis: Calcd for
1
C H ClNO : C, 65.34; H, 5.12; N, 5.08, found: C, 65.97; H, 5.22; N, 4.94; LC-MS
15
14
2
-
-
(
DAD/ESI): t = 2.81 min, Calcd for C H ClNO (m/z): [M-H] 274.06, [M+2-H] 276.06,
R 15 14 2
-
-
found: [M-H] 274.05, [M+2-H] 276.05.
4
.2.3.10. N-4-[(2-Bromobenzyl)oxy]benzylformamide 16c
Method B: Hydrochloride 15c (2.0 g, 6.1 mmol), Et N (1.71 mL, 12.2 mmol). Crude product
3
was recrystallized from EtOH/H O giving compound 16c as colourless crystals in 89% (1.74
2
g) yield.
1
mp: 120 ºC; NMR: mixture of two rotamers H (600 MHz, CDCl ): δ (main rotamer) 8.25 (s,
3
1
H), 7.59 (dd, J = 8.0, 0.8 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.33 (td, J = 7.6, 0.8 Hz, 1H),
.23 (d, J = 8.6 Hz, 2H), 7.19 (t, J = 6.8 Hz, 1H), 6.95 (d, J = 8.6 Hz, 2H), 5.18 (s, 2H), 4.43
7
1
3
(
d, J = 5.5 Hz, 2H); C (151 MHz, CDCl ): δ 161.0, 158.2, 136.3, 132.8, 130.3, 129.4, 129.0,
3
-
1
1
1
28.6, 127.7, 122.5, 115.5, 115.3, 69.6, 41.9 IR (ATR) [cm ]: 3263, 3035, 2887, 1645, 1514,
383, 1253, 1217, 1026, 752; Elemental analysis: Calcd for C H BrNO : C, 56.27; H, 4.41;
1
5
14
2
N, 4.37, found: C, 56.54; H, 4.78; N, 4.36; LC-MS (DAD/ESI): t = 2.84 min, Calcd for
R
-
-
-
-
C H BrNO (m/z): [M-H] 318.01, [M+2-H] 320.01, found: [M-H] 318.05, [M+2-H]
15
14
2
3
20.05.
2
1

ACCEPTED MANUSCRIPT
4
.2.4. General synthetic procedures for Williamson ethers synthesis and analytical data
Method A
The corresponding formamides 10a or 10b (1.0 equiv.), benzyl halide (1.1 equiv.) and K CO3
2
(1.5 equiv.) in acetonitrile were refluxed overnight (16 h). After the reactions were completed,
water was added and the resulting mixture was extracted with ethyl acetate. Organic layers
were collected, washed with water and brine, dried over anhydrous MgSO and evaporated.
4
The resulting solid was washed with petroleum ether/diethyl ether (approx. 1:1) giving the
crude product (11c-e; yields 71-83%).
Method B
Tert-butyl 4-hydroxybenzylcarbamate 14 (1.0 equiv.), benzyl halide (1.1 equiv.), K CO (1.5
2
3
equiv.) and acetonitrile were refluxed overnight (16 h). After the reaction was completed,
water was added and the resulting mixture was extracted with ethyl acetate. Organic layers
were collected, washed with water and brine, dried over anhydrous MgSO and evaporated
4
giving the intermediate products as white solids. This solid was stirred overnight at rt in
excess of 2 M HCl solution in MeOH (approx. 30 mL). After this, the solvent was evaporated
and the resulting solid was washed with diethyl ether giving the crude product (15a-c; yields
8
2-94%).
4
.2.4.1. N-(4-benzyloxyphenyl)formamide 11a
To a stirred solution of 4-(benzyloxy)aniline 10a (1.0 equiv.) in ethylformate (3 mL),
trimethylamine (1.0 equiv) was added. The reaction mixture was refluxed for 2 days and the
resulting mixture was extracted with ethyl acetate. Organic layers were collected, washed
with water and brine, dried over anhydrous MgSO and evaporated giving the product as
4
x
white solid in 70% yield.
1
NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.50 (d, J = 10.0 Hz,
3
1
H), 8.27 (s, 1H), 7.43-7.30 (m, 12H), 7.02-6.90 (m, 6H), 5.03 (s, 4H).
4
.2.4.2. N-(3-benzyloxyphenyl)formamide 11b
To a stirred solution of 3-(benzyloxy)aniline 10b (1.0 equiv.) in ethylformate (3 mL),
catalytic amount of pTsOH monohydrate was added. The reaction mixture was stirred at rt for
3
days and the resulting mixture was extracted with ethyl acetate. Organic layers were
collected, washed with water and brine, dried over anhydrous MgSO and evaporated giving
4
x
the product 11b as white solid in 80% yield.
2
2

ACCEPTED MANUSCRIPT
1
NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.67 (d, J = 10.0 Hz,
3
1
H), 8.32 (s, 1H), 7.97-7.67 (m, 22H), 5.05 (s, 2H), 5.03 (s, 2H).
4
.2.4.3. N-(4-(2-Bromobenzyloxy)phenyl)formamide 11c
Method A: Formamide 10a (0.9 g, 6.6 mmol), 2-bromobenzyl bromide (1.80 g, 7.1 mmol),
K CO (2.64 g, 9.8 mmol). Crude product was recrystallized from EtOH/H O giving
2
3
2
compound 11c as colourless needles in 80% (1.61 g) yield.
1
mp: 87 ºC; NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.52 (d, J
3
=
11.5 Hz, 1H), 8.33 (d, J = 1.5 Hz, 1H), 7.63 (d, J = 11.1 Hz, 1H), 7.61-7.57 (m, 2H), 7.53
(d, J = 7.7 Hz, 1H), 7.46 (dt, J = 9.1, 2.1 Hz, 2H), 7.33 (qd, J = 7.5, 1.1 Hz, 2H), 7.22-7.17
(m, 2H), 7.14 (s, 1H), 7.04 (dt, J = 8.9, 2.1 Hz, 2H), 6.98 (dt, J = 9.8, 2.3 Hz, 2H), 6.95 (dt, J
1
3
=
9.0, 2.1 Hz, 2H), 5.13 (s, 2H), 5.12 (s, 2H); C (151 MHz, CDCl ): δ 162.9, 158.9, 156.6,
3
1
1
2
55.7, 136.3, 136.0, 132.9, 132.8, 130.4, 130.0, 129.6, 129.4, 129.0, 129.0, 127.8, 127.7,
-1
22.5, 122.5, 121.9, 121.9, 121.7, 116.1, 115.5, 69.9, 69.8; IR (ATR) [cm ]: 3222, 3178,
887, 1660, 1537, 1508, 1391, 1252, 1025, 813, 759; Elemental analysis: Calcd for
C H BrNO : C, 54.92; H, 3.95; N, 4.58, found: C, 55.06; H, 3.87; N, 4.53; LC-MS
14
12
2
-
-
(
DAD/ESI): t = 2.89 min, Calcd for C H BrNO (m/z): [M-H] 304.00, [M+2-H] 306.00,
R 14 12 2
-
-
found: [M-H] 304.05, [M+2-H] 306.05.
4
.2.4.4. N-(4-(2,6-Dichlorobenzyloxy)phenyl)formamide 11d
Method A: Formamide 10a (0.6 g, 4.4 mmol), 2,6-dichlorobenzyl bromide (1.16 g, 4.8 mmol),
K CO (0.91 g, 6.6 mmol). Crude product was recrystallized from EtOH/H O giving
2
3
2
compound 11d as colourless crystals in 71% (0.92 g) yield.
1
mp: 132 ºC; NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.53 (d, J
3
=
11.4 Hz, 1H), 8.34 (d, J = 1.6 Hz, 1H), 7.70 (d, J = 11.1 Hz, 1H), 7.48 (dt, J = 8.9, 2.1 Hz,
2
5
1
3
7
5
H), 7.39-7.35 (m, 4H), 7.28-7.22 (m, 2H), 7.19 (s, 1H), 7.09- 6.98 (m, 6H), 5.27 (s, 2H),
1
3
.26 (s, 2H); C (151 MHz, CDCl ): δ 162.9, 128.9, 157.0, 156.2, 137.1, 137.1, 132.3, 132.0,
3
-1
30.7, 130.6, 130.2, 128.7, 128.6, 121.9, 121.8, 116.4, 115.7, 65.8, 65.8; IR (ATR) [cm ]:
235, 3193, 3117, 3053, 2953, 2896, 1650, 1602, 1507, 1437, 1406, 1225, 1151, 1010, 835,
69, 704; Elemental analysis: Calcd for C H Cl NO : C, 56.78; H, 3.74; N, 4.73, found: C,
14
11
2
2
6.62; H, 4.00; N, 4.65; LC-MS (DAD/ESI): t = 2.90 min, Calcd for C H Cl NO (m/z):
R
14 11
2
2
[
M-H]- 294.01, [M+2-H]- 296.01, found: [M-H]- 294.05, [M+2-H]- 296.00.
2
3

ACCEPTED MANUSCRIPT
4
.2.4.5. N-3-[(4-Bromobenzyl)oxy]phenylformamide 11e
Method A: Formamide 10b (1.0 g, 7.29 mmol), 4-bromobenzyl bromide (2.0 g, 8.0 mmol),
K CO (1.51 g, 10.9 mmol). Crude product was recrystallized from EtOH/H O giving
2
3
2
compound 11e as colourless solid in 83% (1.85 g) yield.
mp: 138 ºC; NMR: mixture of two rotamers (approx. 1:1) H (600 MHz, CDCl ): δ 8.68 (d, J
1
3
=
=
11.4 Hz, 1H), 8.37 (s, 1H), 7.77 (d, J = 9.8 Hz, 1H), 7.51 (tt, J = 8.9, 2.0 Hz, 4H), 7.43 (t, J
2.2 Hz, 1H), 7.30 (dd, J = 8.3, 5.3 Hz, 4H), 7.26 (t, J = 8.3 Hz, 1H), 7.22 (t, J = 8.1 Hz,
1
H), 6.98 (dd, J = 8.0, 1.3 Hz, 1H), 6.77 (dd, J = 8.3, 2.2 Hz, 1H), 6.74 (dd, J = 8.3, 2.4 Hz,
1
3
1
H), 6.69 (dd, J = 7.9, 1.7 Hz, 1H), 6.67 (t, J = 2.1 Hz, 1H), 5.02 (s, 4H); C (151 MHz,
CDCl ): δ 162.3, 159.7, 159.2, 159.0, 138.2, 138.0, 135.9, 135.6, 132.0, 131.9, 130.9, 130.0,
3
1
29.3, 129.2, 122.3, 122.1, 112.4, 111.6, 111.5, 111.3, 106.9, 106.0, 69.5, 69.4; IR (ATR)
-1
[
cm ]: 3208, 3144, 3061, 3013, 2922, 1710, 1612, 1500, 1311, 1267, 1160, 1030, 1013, 849,
8
08, 767; Elemental analysis: Calcd for C H BrNO : C, 54.92; H, 3.95; N, 4.58; Found: C,
14 12 2
5
4.97; H, 3.60; N, 4.45; LC-MS (DAD/ESI): t = 2.90 min, Calcd for C H BrNO (m/z):
R
14 12
2
-
-
-
-
[
M-H] 304.00, [M+2-H] 306.00, found: [M-H] 304.00, [M+2-H] 306.00.
4
.2.4.6. 4-[(2-Fluorobenzyl)oxy]benzylamine hydrochloride 15a
Method B: Boc-protected amine 14 (2.0 g, 9.0 mmol), 2-fluorobenzyl bromide (1.19 mL, 9.9
mmol), K CO (1.69 g, 12.2 mmol). Crude product was recrystallized from H O giving
2
3
2
compound 15a as colourless solid in 67% (1.60 g) yield.
1
mp: 202-203 ºC (decomposition); NMR: H (600 MHz, DMSO-d ): δ 8.36 (s, 3H), 7.55 (t, J
6
=
6.9 Hz, 1H), 7.49-7.39 (m, 2H), 7.29-7.21 (m, 2H), 7.06 (d, J = 8.7 Hz, 2H), 5.16 (s, 2H),
1
3
3
.96 (s, 2H); C (151 MHz, DMSO-d ): δ 161.2, 159.6, 158.2, 130.7, 130.7, 130.6, 130.5,
6
-
1
30.5, 130.4, 126.5, 124.5, 124.5, 123.7, 115.5, 115.4, 114.7, 63.6, 63.6, 41.6; IR (ATR) [cm
1
]: 3311, 3085, 2918, 1610, 1587, 1516, 14902, 1455, 1382, 1244, 1233, 1174, 1052, 832,
7
51.
4
.2.4.7. 4-[(2-Chlorobenzyl)oxy]benzylamine hydrochloride 15b
Method B: Boc-protected amine 14 (2.0 g, 9.0 mmol), 2-chlorobenzyl bromide (1.01 mL, 9.9
mmol), K CO (1.69 g, 12.2 mmol). Crude product was recrystallized from H O giving
2
3
2
compound 15b as colourless solid in 57% (1.31 g) yield.
1
mp: 190-191 ºC (decomposition); NMR: H (600 MHz, DMSO-d ): δ 8.39 (s, 3H), 7.60-7.56
6
(m, 1H), 7.53-7.49 (m, 1H), 7.44 (d, J = 8.7 Hz, 2H), 7.42-7.36 (m, 2H), 7.06 (dt, J = 8.7, 1.8
1
3
Hz, 2H), 5.17 (s, 2H), 3.93 (s, 2H); C (151 MHz, DMSO-d ): δ 158.2, 134.2, 132.7, 130.6,
6
2
4

ACCEPTED MANUSCRIPT
-1
1
1
30.1, 129.9, 129.4, 127.4, 126.6, 114.7, 66.9, 41.6; IR (ATR) [cm ]: 3303, 3073, 2920,
608, 1513, 1448, 1380, 1295, 1250, 1175, 1048, 1037, 831, 744.
4
.2.4.8. 4-[(2-Bromobenzyl)oxy]benzylamine hydrochloride 15c
Method B: Boc-protected amine 14 (2.50 g, 11.2 mmol), 2-bromobenzyl bromide (3.07 g,
2.2 mmol), K CO (2.31 g, 16.8 mmol). Crude product was recrystallized from H O giving
1
2
3
2
compound 15c as colourless solid in 58% (2.13 g) yield.
1
mp: 199-200 ºC (decomposition); NMR: H (600 MHz, DMSO-d ): δ 8.32 (s, 3H), 7.68 (dd,
6
J = 8.0, 1.0 Hz, 1H), 7.57 (dd, J = 7.6, 1.5 Hz, 1H), 7.46-7.40 (m, 3H), 7.31 (td, J = 7.8, 1.7
1
3
Hz, 1H), 7.05 (dt, J = 8.7, 1.9 Hz, 2H), 5.14 (s, 2H), 3.94 (s, 2H); C (151 MHz, DMSO-d ):
6
-1
δ 158.2, 135.7, 132.7, 130.6, 130.2, 127.9, 126.6, 122.9, 114.8, 69.1, 41.7; IR (ATR) [cm ]:
3
308, 3067, 2916, 1607, 1514, 1437, 1376, 1296, 1249, 1175, 1046, 1028, 829, 743.
4
.2.5.General synthetic procedures for isocyanide synthesis and analytical data
The corresponding formamide 7a-e, 11a-e or 16a-c (1.0 equiv.), Et N (4 or 6 equiv.) and
3
DCM were put together in a round-bottom flask and cooled to 0 ºC. Then POCl was added
3
dropwise and the reaction was stirred at rt for 3-4 h. After the reaction was completed, the
products were poured into ice cold solution of NaHCO and left to reach rt. The precipitated
3
solid was filtered off and washed with DCM. The remaining water phase was extracted with
DCM. Organic layers were collected, washed with water, dried over anhydrous MgSO and
4
evaporated. The resulting oils were purified on silica pad with copious amount of DCM,
which was then collected and evaporated, giving the crude isocyanides (8a-e, 12a-e or 17a-c;
yields 56-98%), which were used further without purification.
4
.2.5.1.3-Phenoxyphenyl isocyanide 8a
Compound 7a (0.9 g, 4.2 mmol), POCl (0.37 mL, 3.8 mmol), Et N (2.75 mL, 20.0 mmol).
3
3
Product 8a was obtained as brown solid in 85% (0.7 g) yield (R : 0.70, petroleum
f
ether/EtOAc 3:1)
1
NMR: H (500 MHz, CDCl ): δ 7.39 (t, J = 5 Hz, 2H), 7.33 (d, J = 10 Hz, 2H), 7.19 (t, J =
3
1
3
1
0 Hz, 1H), 7.03 (d, J = 5 Hz, 2H), 6.95 (d, J = 10 Hz, 2H); C (125 MHz, CDCl ): δ 163.6,
3
1
58.5, 155.9, 130.3, 128.2, 124.8, 120.0, 118.8.
2
5

ACCEPTED MANUSCRIPT
4
.2.5.2. 4-Chlorophenyl isocyanide 8b
Compound 7b (3.5 g, 22.5 mmol), POCl (2.10 mL, 22.5 mmol), Et N (12.55 mL, 90.0
3
3
mmol). Product 8b was obtained as brown solid in 82% (2.80 g) yield (R : 0.67, petroleum
f
xi
ether/EtOAc 3:1).
1
13
NMR: H (500 MHz, CDCl ): δ 7.38 (dt, J = 8.8, 2.1 Hz, 2H), 7.32 (d, J = 8.6 Hz, 2H); C
3
(
125 MHz, CDCl ): δ 165.7, 135.6, 129.9, 127.8.
3
4
.2.5.3. 4-Isopropylphenyl isocyanide 8c
Compound 7c (2.5 g, 15.3 mmol), POCl (1.43 mL, 15.3 mmol), Et N (8.55 mL, 61.3 mmol).
3
3
Product 8c was obtained as brown liquid in 77% (1.72 g) yield (R : 0.70, petroleum
f
xii
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.29 (d, J = 8.3 Hz, 2H), 7.23 (d, J = 8.4 Hz, 2H), 2.92
3
13
(
hept, J = 7.0 Hz, 1H), 6.69 (d, J = 7.0 Hz, 6H); C (125 MHz, CDCl ): δ 163.2 (t, J = 5.0
3 NC
Hz), 150.6, 127.5, 126.4, 34.1, 23.8.
4
.2.5.4. 4-Fluoro-3-chlorophenyl isocyanide 8d
Compound 7d (3.0 g, 17.3 mmol), POCl (1.61 mL, 17.3 mmol), Et N (9.64 mL, 69.1 mmol).
3
3
Product 8d was obtained as dark green solid in 88% (2.37 g) yield (R : 0.60, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.47 (dd, J = 6.2, 2.4 Hz, 1H), 7.32-7.27 (m, 1H), 7.18 (t, J
3
1
3
=
8.6 Hz, 1H); C (125 MHz, CDCl ): δ 166.1, 159.3, 157.3, 128.9, 126.6, 126.5, 117.7,
3
1
17.5.
4
.2.5.5. 4-Chlorobenzyl isocyanide 8e
Compound 7e (2.0 g, 11.8 mmol), POCl (1.09 mL, 11.8 mmol), Et N (6.58 mL, 17.2 mmol).
3
3
Product 8e was obtained as dark orange liquid in 75% (1.33 g) yield (R : 0.80, petroleum
f
xiii
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.37 (d, J = 8.5 Hz, 2H), 7.29 (d, J = 8.4 Hz, 2H), 4.62 (s,
3
1
3
2
H); C (125 MHz, CDCl ): δ 134.4, 130.9, 129.2, 128.1, 53.3, 45.0 (t, J = 7.2 Hz).
3
NC
4
.2.5.6. 4-(Benzyloxy)phenyl isocyanide 12a
Compound 11a (0.75 g, 3.3 mmol), POCl (0.3 mL, 3.2 mmol), Et N (2.29 mL, 16.5 mmol).
3
3
Product 12a was obtained as brown solid in 81% (0.552 g) yield (R : 0.77, petroleum
f
ether/EtOAc 3:1).
2
6

ACCEPTED MANUSCRIPT
1
NMR: H (500 MHz, CDCl ): δ 7.40-7.39 (m, 5 H), 7.29 (d, J = 10 Hz, 2H), 6.93 (d, J = 10
3
1
3
Hz, 2H), 5.06 (s, 2H); C (125 MHz, CDCl ): δ 162.9, 159.2, 128.9, 128.5, 128.0, 127.6,
3
1
15.7, 70.5.
4
.2.5.7. 3-(Benzyloxy)phenyl isocyanide 12b
Compound 11b (0.310 g, 1.36 mmol), POCl (0.13 mL, 1.4 mmol), Et N (0.97 mL, 6.7
3
3
mmol). Product 12b was obtained as brown solid in 86% (0.245 g) yield (R : 0.76, petroleum
f
xi
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.41-7.39 (m, 5 H), 7.38-7.35 (m, 1H), 7.29-7.25 (m, 1H),
3
7
.01-6.97 (m, 2H), 5.06 (s, 2H).
4
.2.5.8. 4-[(2-Bromobenzyl)oxy]phenyl isocyanide 12c
Compound 11c (1.50 g, 4.9 mmol), POCl (0.46 mL, 4.9 mmol), Et N (2.73 mL, 19.6 mmol).
3
3
Product 12c was obtained as light yellow solid in 92% (1.30 g) yield (R : 0.57, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.60 (d, J = 8.0 Hz, 1H), 7.74 (dd, J = 7.7, 0.6 Hz, 1H),
3
7
.34-7.31 (m, 3H), 7.22 (td, J = 7.6, 0.6 Hz, 1H), 6.96 (dt, J = 9.0, 1.9 Hz, 2H), 5.14 (s, 2H);
1
3
C (125 MHz, CDCl ): δ 163.0, 158.8, 135.4, 132.9, 129.7, 129.0, 128.0, 127.8, 122.5, 115.6,
3
6
9.8.
4
.2.5.9. 4-[(2,6-Dichlorobenzyl)oxy]phenyl isocyanide 12d
Compound 11d (0.8 g, 2.7 mmol), POCl (0.25 mL, 2.7 mmol), Et N (1.51 mL, 10.8 mmol).
3
3
Product 12d was obtained as light yellow solid in 93% (0.70 g) yield (R : 0.63, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.38 (d, J = 8.1 Hz, 2H), 7.35 (d, J = 8.9 Hz, 2H), 7.30-7.26
3
1
3
(
m, 1H), 7.00 (dt, J = 8.7, 1.9 Hz, 2H), 5.29 (s, 2H); C (125 MHz, CDCl ): δ 163.0, 159.2,
3
1
37.1, 131.5, 130.9, 128.7, 127.9, 115.7, 65.6.
4
.2.5.10. 3-[(4-bromobenzyl)oxy)]phenyl isocyanide 12e
Compound 11e (1.3 g, 4.3 mmol), POCl (0.39 mL, 4.3 mmol), Et N (2.37 mL, 17.0 mmol).
3
3
Product 12e was obtained as light yellow solid in 94% (1.15 g) yield (R : 0.77, petroleum
f
ether/EtOAc 3:1).
2
7

ACCEPTED MANUSCRIPT
1
NMR: H (500 MHz, CDCl ): δ 7.53 (d, J = 8.34 Hz, 2H), 7.32-7.27 (m, 3H), 7.01-6.97 (m,
3
1
3
2
H), 6.95 (s, 1H), 5.01 (s, 2H); C (125 MHz, CDCl ): δ 164.2, 159.0, 135.1, 132.0, 130.5,
3
1
29.2, 122.4, 119.4, 116.6, 112.9, 69.7.
4
.2.5.11. 4-[(2-Fluorobenzyl)oxy]benzyl isocyanide 17a
Compound 16a (1.0 g, 3.7 mmol), POCl (0.35 mL, 3.7 mmol), Et N (3.12 mL, 23.4 mmol).
3
3
Product 17a was obtained as light yellow solid in 56% (0.50 g) yield (R : 0.67, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.52 (t, J = 7.3 Hz, 1H), 7.35 (q, J = 6.8 Hz, 1H), 7.29 (d, J
3
=
8.5 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H), 7.12 (t, J = 8.8 Hz, 1H), 7.03 (d, J = 8.6 Hz, 2H),
1
3
5
1
.16 (s, 2H), 4.58 (s, 2H); C (125 MHz, CDCl ): δ 161.5, 158.6, 129.9, 129.9, 129.8, 129.7,
3
28.2, 125.1, 124.4, 124.4, 115.5, 115.4, 115.3, 63.9, 63.8, 45.1 (t, JNC = 7.2 Hz).
4
.2.5.12. 4-[(2-Chlorobenzyl)oxy]benzyl isocyanide 17b
Compound 16b (1.0 g, 3.6 mmol), POCl (0.34 mL, 3.6 mmol), Et N (2.03 mL, 14.6 mmol).
3
3
Product 17b was obtained as light yellow solid in 98% (0.92 g) yield (R : 0.65, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.53 (dd, J = 6.9, 2.1 Hz, 1H), 7.40 (dd, J = 7.0, 2.0 Hz, 1H),
3
1
3
7
1
.30-7.35 (m, 4H), 5.17 (s, 2H), 4.56 (s, 2H); C (125 MHz, CDCl ): δ 158.6, 134.5, 132.7,
3
29.5, 129.2, 128.9, 128.3, 127.1, 125.1, 115.4, 67.3, 45.1 (t, JNC = 6.8 Hz).
4
.2.5.13. 4-[(2-Bromobenzyl)oxy]benzyl isocyanide 17c
Compound 16c (1.5 g, 4.7 mmol), POCl (0.44 mL, 4.7 mmol), Et N (2.61 mL, 18.7 mmol).
3
3
Product 17c was obtained as the light yellow solid in 93% (1.32 g) yield (R : 0.85, petroleum
f
ether/EtOAc 3:1).
1
NMR: H (500 MHz, CDCl ): δ 7.60 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.6 Hz, 1H), 7.34 (t, J
3
=
7.5 Hz, 1H), 7.28 (d, J = 8.6 Hz, 2H), 7.20 (t, J = 7.1 Hz, 1H), 7.00 (d, J = 8.6 Hz, 2H),
13
5
.14 (s, 2H), 4.57 (s, 2H); C (125 MHz, CDCl ): δ 158.6, 136.0, 132.8, 129.5, 129.0, 128.3,
3
1
27.7, 125.2, 122.5, 115.4, 69.6, 45.2 (t, JNC = 7.0 Hz).
4
.2.6. General synthetic procedures for the U-T reaction and analytical data
Aldehyde 4 (1.0 mmol), amine 5 (1.0 mmol), isocyanide 8a-e, 12a-e or 17a-c (1.0 mmol),
TMSN (1.0 mmol) were dissolved in 2,2,2-trifluoroethanol (1.0 mL) and placed into a
3
microwave oven for 40 min at 120 ºC. After this time, the solvent was evaporated and the
2
8