2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hydrophilicity and selectivity

Article abstract of DOI:10.1016/j.bmc.2018.04.012

2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective leads for antimalarial drug development targeting the plasmepsins. Here we report the lead optimization studies with the aim to reduce inhibitor lipophilicity and increase selectivity

Full text of DOI:10.1016/j.bmc.2018.04.012

Accepted Manuscript
2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved hy-
drophilicity and selectivity
Dace Rasina, Georgijs Stakanovs, Oleksandr V. Borysov, Teodors Pantelejevs,
Raitis Bobrovs, Iveta Kanepe-Lapsa, Kaspars Tars, Kristaps Jaudzems, Aigars
Jirgensons
PII:
S0968-0896(18)30401-2
https://doi.org/10.1016/j.bmc.2018.04.012
BMC 14295
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Revised Date:
Accepted Date:
28 February 2018
29 March 2018
3 April 2018
Please cite this article as: Rasina, D., Stakanovs, G., Borysov, O.V., Pantelejevs, T., Bobrovs, R., Kanepe-Lapsa,
I., Tars, K., Jaudzems, K., Jirgensons, A., 2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved
hydrophilicity and selectivity, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.04.012
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2-Aminoquinazolin-4(3H)-one based plasmepsin inhibitors with improved
hydrophilicity and selectivity
Dace Rasina†, Georgijs Stakanovs†, Oleksandr V. Borysov†, Teodors Pantelejevs†,
Raitis Bobrovs†, Iveta Kanepe-Lapsa†, Kaspars Tars‡, Kristaps Jaudzems,† Aigars
Jirgensons†*
† Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga LV-1006, Latvia
‡ Biomedical Research and Study Centre, Ratsupites 1, Riga LV-1067, Latvia
Abstract: 2-Aminoquinazolin-4(3H)-ones were previously discovered as perspective
leads for antimalarial drug development targeting the plasmepsins. Here we report the
lead optimization studies with the aim to reduce inhibitor lipophilicity and increase
selectivity versus the human aspartic protease Cathepsin D. Exploiting the solvent
exposed area of the enzyme provides an option to install polar groups (R1) the 5-
position of 2-aminoquinazolin-4(3H)-one to inhibitors such as carboxylic acid without
scarifying enzymatic potency. Moreover, introduction of R1 substituents increased
selectivity factors of compounds in this series up to 100-fold for Plm II, IV vs CatD
inhibition. The introduction of flap pocket substituent (R2) at 7-postion of 2-
aminoquinazolin-4(3H)-one allows to remove Ph group from THF ring without
notably impairing Plm inhibitory potency. Based on these findings, inhibitors were
developed, which show Plm II and IV inhibitory potency in low nanomolar range and
remarkable selectivity against Cathepsin D along with decreased lipophilicity and
increased solubility.
Key words: Plasmepsins; Malaria; Plasmodium Falciparum; Cathepsin D; Inhibitors;
2-Aminoquinazolin-4(3H)-ones
1. Introduction
Malaria is a life threatening infectious disease caused by Plasmodium parasites, of
which Plasmodium falciparum is the most lethal [1]. The treatment and prophylaxis
of Malaria has been improved over the recent decades, considerably reducing the
infection cases and mortality [2]. However, these efforts are in danger due to an
alarming spread of drug-resistant malaria [3-5]. To address this problem, new drugs
targeting the life cycle of the parasite by yet unexploited mechanisms of action are
required [2,3]. Malarial aspartic proteases – plasmepsins (Plms) have been explored

as potential drug targets for decades, [6-11], nevertheless, so far no plasmepsin
inhibitor has been developed to a clinical candidate.
Out of the 10 plasmepsins encoded in the Plasmodium falciparum genome, the
most attention has been paid to the digestive plasmepsins (Plm I, II, IV and HAP),
which are involved in the processing of haemoglobin to amino acids. However, their
inhibition may not be sufficient to kill the parasite as the haemoglobin digestion by
plasmepsins has redundant mechanisms [11-14]. On the other hand, certain digestive
plasmepsin inhibitors show parasite growth inhibition in red blood cells (RBC) at
nanomolar concentration [15,16] which is likely achieved by (co)-targeting the non-
digestive plasmepsins. Of these, only Plm V, IX and X subtypes are expressed in the
blood stages of the parasite. The subtype Plm V [17,18] is more distant from Plm I, II,
IV, while Plm IX and X share high sequence homology. It has been recently shown
that Plm IX is involved in merozoite invasion of RBC while Plm X is involved in
both, the invasion and the egress of merozoites [19,20]. Moreover, inhibitors of Plm
IX and Plm X were demonstrated which showed antiplasmodial activity in RBC
assays. These findings reinforce the importance of plasmepsins as targets for
antimalarial drug development. However, production of recombinant Plm IX and X
has only been possible in higher eukaryotic protein expression systems, such as insect
or mammalian cells [19,20]. For this reason, the more easily accessible digestive
plasmepsins [21-23] are still useful as model proteins for inhibitor development.
In the last decade, a notable focus has been on the search for non-peptidomimetic
plasmepsin inhibitors [10,24-28]. These inhibitors are more drug-like and also show
an improved selectivity profile against human aspartic proteases (e.g. Cathepsin D) as
compared to peptidomimetic inhibitors. Recently, we reported 2-aminoquinazolin-
4(3H)-ones 1 as non-peptidomimetic Plm I, II, IV inhibitors (Figure 1) [29].

Figure 1. Crucial binding interactions of inhibitors 1 with Plm II according to X-
ray of the complex (PDB ID 4z22) and proposed SAR study for evaluating
substituents at positions R1-3 in the follow-up inhibitor series 2. The pockets are
labeled according to protease nomenclature.
According to the X-ray structure of the complex of 1a and Plm II, these inhibitors
bind to the open-flap conformation of the enzyme. The major interactions involve the
2-aminoquinazolinone N1 and 2-amino groups, which form hydrogen bond
interactions with the catalytic aspartates, the Ph-THF moiety, which targets the S1’
sub-pocket and the 7-Ph substituent, which makes hydrophobic and stacking
interactions with residues in the flap-pocket. The inhibitor 1b bearing n-pentylphenyl
group as flap-pocket substituent showed similar inhibitory potency against subtypes
Plm I,II,IV while inhibitor 1c with phenylpropylphenyl substituent turned out to be
selective Plm IV inhibitor. Both compounds 1b,c inhibited Plasmodium growth at low
micromolar concentrations. However, due to their high lipophilicity the compounds
were poorly water-soluble, which limited their optimization. This prompted us to
generate a follow-up series of inhibitors 2 aimed at reducing lipophilicity without
sacrificing potency.
2. Results and discussion
2.1. SAR studies
Our strategy for reducing the inhibitor lipophilicity was to install hydrophilic
substituents in positions that are facing polar protein surfaces or are solvent-exposed.
Guided by the co-crystal structure of 1a and Plm II (Figure 1), we modified the
position R1 facing solvent exposed area. For 2-aminoquinazolinones bearing R1
substituents, two sets of compounds were tested – one with Ph group at THF, and one
with unsubstituted THF (compounds 3a,b and 2a-g, respectively, Table 1).
Substitution of THF (analogues 3a,b vs 2a-g) improved only Plm I and Plm IV
inhibition, while the potency against Plm II remained very similar. This is opposite to
the previous observation that the addition of Ph group at THF improved Plm II
inhibition five-fold (inhibitors 2h vs 1a, Table 1).
Compounds 2a-g with various R1 substituents showed a flat SAR as could be
expected by their situation in an area exposed to the solvent. Also, compounds 3a,b

showed very similar activity to the parent compound 1a suggesting that the R1 group
does not form specific interactions with the protein. This is in line with results from
molecular docking, which show that the R1 substituents may only form hydrogen
bond interactions with surface exposed backbone amide and side chain hydroxyl
groups of Val78 and Tyr192 (Figure 2) or with solvent molecules. Minor differences
in the compound activities could be attributed to positioning and electron donor
properties of functional groups in R1, where groups with better electron donor (H-
bond acceptor) properties are favoured. The slightly higher activities against Plm II
over Plm I, IV and CatD can be explained by a Tyr192Phe substitution in the latter,
which abolishes interactions with the THF oxygen and the propionate groups of 2b
and 3b. Importantly, selectivity factors of compounds in this series for Plm II, IV vs
CatD inhibition was increased up to 100-fold indicating that the selectivity against
CatD can be improved by introduction of R1 substituents.
Figure 2. Docked poses of compounds 2a, 3a (A) and 2b, 3b (B). Protein residues
are shown with thin sticks and labelled. Dashed red lines indicate hydrogen bonds
with aspartates and Val78 or Tyr192. Docking was performed on the crystal structure
of Plm II – 1a complex (PDB ID 4Z22) using Schrödinger Glide software.
Table 1. SAR of R1 Substituents in Inhibitors 1a, 2a-h and 3a,b
PlmI,
PlmII,
PlmIV,
CatD,
PSA
(Å2)c
Compd.
2a
R1
clogPc
LiPEd
IC50 (M)a IC50 (M)a IC50 (M)a IC50 (M)a
3.50 ± 0.04 0.33 ± 0.02 1.4 ± 0.2 ~100
3.6
113.3
2.3

0.6 ± 0.1
11.5 ± 1
1.0 ± 0.2
0.28 ± 0.02 0.50 ± 0.08
20.0 ± 1.2
>100
4.8
2.8
4.3
111.2
114.1
111.4
1.5
2.4
1.9
3a
2b
3b
0.50 ± 0.06
0.40 ± 0.03
6.0 ± 0.5
0.6 ± 0.1
11.0 ± 0.8
5.0 ± 0.5
3.0 ± 0.2
2.0 ± 0.5
1.00 ± 0.06
0.48 ± 0.04
0.82 ± 0.08
2.4 ± 0.2
1.5 ± 0.1
1.0 ± 0.3
>100
71 ± 2
93 ± 2
3.5
3.5
3.4
77.3
77.6
93.4
2.1
2.3
2.6
2c
2d
2e
3.4 ± 0.2
0.60 ± 0.08
2.5 ± 0.6
>100
2.8
3.6
117.8
116.1
2.8
2.2
2f
1.0 ± 0.4
6.0 ± 0.3
0.50 ± 0.02
2.3 ± 0.1
1.5 ± 0.1
98 ± 2
2g
2hb
1ab
1.30 ± 0.06
53.4 ± 0.8
13.8 ± 0.6
2.7
4.4
65.7
67.5
3.1
1.9
H
1.30 ± 0.06 0.57 ± 0.02 0.60 ± 0.03
aPlm I,II,IV and CatD inhibitory activity was determined by enzymatic FRET assay in triplicate experiments; bData
c
d
from ref. [29]. 7; LipE calculated using IC50 values of
Plm IV inhibition .
Next, we explored the possibility of removing the Ph substituent from the THF
ring. Docking and molecular dynamics (MD) simulations of inhibitors containing the
flap pocket substituent and either Ph-substituted THF or unsubstituted THF group
showed that the presence of Ph substituent in this position affects inhibitor mobility in
the binding pocket. During the MD simulation, compounds 1a-c were
conformationally locked and exhibited high rigidity, while compounds 2h, 4a-b
lacking the Ph substituent were more flexible (Fig. 3A). In the case of
phenylpropylphenyl as flap pocket substituent, the Ph-THF group additionally caused
a slight misalignment of the inhibitor (1c) with respect to the binding pose of
compound 4b (Fig. 3B). Based on these data we hypothesized that the benefit of the
Ph-THF group in compounds containing a flap pocket substituent may be diminished
due to a larger entropic penalty associated with constraining the ligand flexibility
and/or due to ligand inability to maintain an optimal binding pose, when both terminal

groups are locked in hydrophobic pockets (flap pocket and S1’ sub-pocket).
Experimentally it was found that compound 4a lacking the Ph substituent at THF
exhibits a very similar inhibitory potency against Plms I,II and IV and CatD compared
to the parent compound 1b (Table 2). Phenylpropylphenyl group, previously found as
Plm IV subtype selectivity inducing group, was added as R2 substituent (compound
4b). Again, this compound lacking Ph substituent at THF ring did not reduce the
activity against Plm IV compared to the parent compound 1c, however, the subtype
selectivity against Plm I, II was decreased. Attachment of n-octyl chain directly to the
7-position of quinazolinone scaffold also led to potent Plms I,II and IV inhibitors 4c
and 5 irrespective of the substitution at THF ring. However, the introduction of this
flap pocket substituent significantly reduced selectivity against CatD.
The results from SAR studies of compound series 2 and 4 suggested to introduce
ionisable substituent at 5-position and to remove the phenyl substituent from THF
group which would lead to increased selectivity and hydrophilicity (Table 1). At the
same time, addition of flap-pocket substituents at 7-position of quinazolinone lacking
Ph group at THF ring is beneficial to achieve high inhibitory potency against
plasmepsins (compounds 4a-c, Table 2). Based on these considerations,
quinazolinone derivatives 6a-c and 7a,b were prepared bearing the carboxyethyl and
carboxyphenyl groups at the 5-position (Table 3). Compound 6a containing
pentylphenyl group as a flap-pocket substituent and carboxyethyl group at the 5-
position showed high inhibitory potency against all Plm subtypes. In the case of
carboxyphenyl analogue 7a, the potency was high only against Plms II and IV
subtypes. Notably, selectivity for these compounds 6a,7a against CatD was retained at
very high level. Introduction of phenylpropylphenyl as flap-pocket substituent
induced remarkable Plm IV subtype selectivity only in the case of carboxyphenyl
group containing analogue 7b. In the case of carboxyethyl substituent (compound 6b)
the potency was relatively high against all Plms subtypes. Introduction of n-octyl
group at 7-position and carboxyethyl group at 5-position of quinazolinone led to
potent inhibitor 6c for Plm I,II,IV subtypes, however the selectivity against CatD was
lost.

Figure 3. Influence of the Ph substituent at the THF ring on bound inhibitor
flexibility and binding pose. (A) Root mean square fluctuations (RMSF) of inhibitor
atoms during a 70 ns MD simulation. The atom numbering used is shown on the
structure above the plot. (B) Comparison of docked poses of inhibitors 1c and 4b. The
protein active site cavity is mapped with electrostatic potential surface. Docking was
performed on the crystal structure of Plm II – 1a complex (PDB ID 4Z22) using
Schrödinger Glide software.
Table 2 SAR of R2 Substituents in Inhibitors 1b,c,4a-c,5
PlmI,
PlmII,
PlmIV,
CatD
PSA
(Å2)c
Compd.
R2
clogPc
LiPE
IC50 (M)a
IC50 (M)a
IC50 (M)a
IC50 (M)a

0.70 ± 0.04
0.30 ± 0.01
1.30 ± 0.08
3.2 ± 0.1
0.075 ± 0.004 0.092 ± 0.005 3.20 ± 0.16
4.4
6.2
5.4
7.7
4.0
5.8
71.4
68.0
68.8
66.3
69.5
68.9
2.6
0.8
1.6
-0.8
2.8
1.2
4a
1bb
4b
1cb
4c
0.15 ± 0.01
0.70 ± 0.03
10.0 ± 0.5
0.10 ± 0.01
0.10 ± 0.04
0.13 ± 0.03
5.0 ± 0.2
>100
6.0 ± 0.3
0.090 ± 0.004 0.093 ± 0.005 0.155 ± 0.007 2.70 ± 0.14
n-octyl
0.08 ± 0.02 0.088 ± 0.003 0.11 ± 0.02 0.40 ± 0.04
5
aPlm I,II,IV and CatD inhibitory activity was determined by enzymatic FRET assay in triplicate experiments; bData
from ref. [29] c 7; dLipE calculated using IC50 values of Plm
IV inhibition .
Table 3 SAR of R2 Substituents in Inhibitors 6a-c;7a,b
PlmI,
PlmII,
PlmIV,
CatD,
PSA
Comp.
6a
R2
clogPb
4.6
LiPEc
2.8
(Å2)b
IC50 (M)a
IC50 (M)a
IC50 (M)a
IC50 (M)a
0.050 ± 0.003 0.027 ± 0.002 0.042 ± 0.002
21.0 ± 1.1
29.0 ± 1.4
116.4
113.5
113.5
113.5
115.8
2.40 ± 0.12
0.74 ± 0.04
16.7 ± 0.8
0.023 ± 0.001 0.045 ± 0.002
5.3
2.0
7a
0.080 ± 0.004 0.030 ± 0.002 13.00 ± 0.65
5.5
2.0
6b
0.90 ± 0.05
0.027 ± 0.001
25.0 ± 1.2
6.3
1.2
7b
n-octyl
0.020 ± 0.001 0.022 ± 0.001 0.110 ± 0.006
0.43 ± 0.02
4.1
2.9
6c
aPlm I,II,IV and CatD inhibitory activity was determined by enzymatic FRET assay in triplicate experiments.
b
7; cLipE calculated using IC50 values of Plm IV inhibition .
The most notable effect on reducing lipophilicity and consequently improving LiPE
was achieved by removal of Ph substituent from THF group (e.g. compounds 2a vs,
3a, 2b vs, 3b, 2h vs 1a, Table 1 and 4a vs 1b, 4b vs 1c, Table 2). Introduction of
oxycarbonylethyl group at 5th position of quinazolinone practically did not change the

lipophilicity (compounds 6a vs 4a and 6c vs 4c, Tables 2,3) while introduction of
carboxyphenyl group in this position had negative impact (compounds 7a vs 4a,
Tables 2,3).
Selected compounds were tested for the solubility 1b,c, 6a,b and 7a,b at
physiological pH (Table 4). These data indicate that removal of phenyl group from
tetrahydrofurane and the installation of ionazable group at the 5-position was
beneficial for improving solubility of inhibitors 6a,b and 7a,b compared to parent
compounds 1b,c, however the solubility was found to be still very low.
Table 4 Solubility of Inhibitors 1b,c, 6a,b and 7a,b in 0.05 M TRIS buffer at pH 7.4
Solubilitya
Entry
Compound
<10 g/Lb
<10 g/Lb
1b
1c
6a
100 g/L
7a
6b
7b
30 g/L
10 g/L
170 g/L
aSolubility was determined by HPLC as the residual compound concentration after its precipitation with
aqueous TRIS-buffer (pH=7.4) from DMSO stock solution; bdetection limit

2.2. Chemistry
The previously reported strategy was applied to prepare 2-aminoquinazolin-4(3H)-
ones 2-7 using 4,6-substituted 2-aminobenzamides 15a-e as key intermediates
(Scheme 1) [29]. Commercially available 4-bromo- or 4-chloro-2-nitrobenzoic acids
(8a,b) were coupled with 2-aminomethyltetrahydrofuran derivatives 9a (R3 = Ph) or
9b (R3 = H) to give amides 10a-c. Two of intermediates 10a,c bearing bromide were
subjected to Suzuki–Miyaura reaction with corresponding boronic acid 11 or
pinacolboranes 12a,b to give the coupling products 13a,d (R2 = Ph); 13b, (R2 = p-
(Ph(CH2)3)C6H4) and 13c, (R2 = p-(n-Pentyl)C6H4). This was followed by directed
ortho-C-H activation/iodination in the presence of catalytic amount of Pd(OAc)2
providing iodides 14a-d. Reduction of nitro group in compounds 14a-d provided the
desired 2-aminobenzamides 15a-d. Formation of thiourea intermediate from aniline
15a-d with BzSCN and subsequent cyclisation afforded protected 2-aminoquinazolin-
4(3H)-one building blocks 16a-d. Last steps involved Suzuki–Miyaura or Mizoroki-
Heck reactions to install R1 substituents. Benzoyl group cleavage was achieved with
hydrazine hydrate which served also as hydrogen transfer reagent for hydrogenation
of unsaturated bonds in the presence of Pd/C catalyst to give the final products 2a-g;
3a-b; 6a-b; 7a-b.
Intermediate 10b bearing chloride substituent was converted to 2-aminoquinazolin-
4(3H)-one building block 16e in three step sequence which involved directed ortho-
C-H activation/iodation of 10b to give amide 14e. The reduction of nitro group in this
compound provided 4,6-substituted 2-aminobenzamide 15e, which was cyclised to
give building block 16e. Selective Sonogashira reaction with chloride 16e was used to
install alkynyl group in R2 position and Mizoroki-Heck reaction with t-butyl acrylate
was used to introduce substituent in R1 position. Subsequent cleavage of t-butyl and
benzoyl groups followed by in situ saturation of unsaturated bonds provided product
6c.
Products 4a-b were synthesized from known building block 16f [29] using Suzuki–
Miyaura reaction with pinacolboranes 12a,b and subsequent benzoyl group cleavage
with hydrazine hydrate. Sonogashira reaction with building blocks 16f,g [29], benzoyl
group cleavage and in situ saturation of alkynyl group provided final products 4c, 5.

Scheme 1 Synthesis of 2-aminoquinazolin-4(3H)-ones. Reagents and conditions: A)
(COCl)2, cat. DMF, DCM, then Et3N, amine 9a,b, DCM; B) R2-B(OH)2 11 or R2-
B(Pin) 12a,b, Pd(PPh3)4, K2CO3, THF, H2O; C) PhI(OAc)2, I2, Pd(OAc)2, DMF; D)
Na2S.9H2O, THF, MeOH; E) BzSCN, Et2O, then EDCI*HCl, Et3N, DCM; F) boronic
acid 11b-g or R2-B(Pin) 12a,b, Pd(PPh3)4, K2CO3, DMF, then N2H4*H2O, EtOH; G)
1-octyne, Pd[(PPh3)Cl]2, Et3N, CuI, DMF, then N2H4*H2O, Pd/C, EtOH; H) t-butyl
acrylate, Pd(PPh3)4, Et3N, DMF, then TFA, DCM, then N2H4*H2O, Pd/C, EtOH; I) t-
butyl acrylate, Pd(dppf)Cl2*DCM, K2CO3, DMF, then 1-octyne, Pd(Amphos)Cl2,
Cs2CO3, DMF, then TFA, DCM, then N2H4*H2O, Pd/C, EtOH.
3. SUMMARY
In summary, we have shown that exploiting the solvent exposed area of enzyme
provides an option to install polar groups (R1) in the 5-position of 2-aminoquinazolin-
4(3H)-one inhibitors such as carboxylic acid or pyridine without sacrificing enzymatic
potency. Moreover, introduction of R1 substituents increased selectivity factors of

compounds in this series up to 100-fold for Plm II, IV vs CatD inhibition. It was
found that the introduction of flap pocket substituent (R2) at 7-postion of 2-
aminoquinazolin-4(3H)-one allows to remove the Ph group from THF ring without
notably impairing Plm inhibitory potency. Taken these findings together, inhibitors
6a,b and 7a,b were developed which show Plm II and IV inhibitory potency in low
nanomolar range and remarkable selectivity against Cathepsin D. These modifications
were also beneficial to decrease the lipophilicity and increase the aqueous solubility.
4. EXPERIMENTAL SECTION
4.1. General
Reagents and starting materials were purchased from commercial sources and used as
received. The solvents were purified and dried by standard procedures prior to use.
Flash chromatography was carried out using Kieselgel (35−70μm) silica gel. TLC was
performed on Kieselgel 60 F254 (Merck) and was visualized by staining with KMnO4
or UV. NMR spectra were recorded on 300 and 400 MHz spectrometers with
chemical shift values (δ) in ppm using the residual solvent signal as an internal
refercence. Exact molecular masses (HRMS) were determined on a hybrid quadrupole
time-of-flight mass spectrometer equipped with an electrospray ion source. The purity
for all compounds tested in bioassays was 95+%, determined by UPLC analysis on a
Waters Acquity column: Acquity UPLC BEH-C18 (2.1 mm×50 mm, 1.7μm,
(30.0±5.0)°C); gradient, 0.01% TFA in water/CH3CN 90%/10% to 10%/90%;flow
rate, 0.5 mL/min; run time, 8 min; detector, PDA (photodiode matrix), 220−320 nm.
4.2. General procedure A for the synthesis of compounds 10a-c
(COCl)2 (1.5 equiv.) was added dropwise to the solution of benzoic acid (5 mmol, 1
equiv.) and few drops of DMF in DCM (20 mL) at 0 oC. Reaction mixture was stirred
at r. t. for 4 h, then solvent evaporated. Obtained crude benzoyl chloride was
dissolved in DCM (20 mL). Et3N (3 equiv.) and amine (1.1 equiv.) was added and
mixture stirred at r. t. overnight. Then 5% aq. KHSO4 solution was added and product
extracted with DCM, dried over anh. Na2SO4. Purification by column
chromatography.

4.2.1. rac-4-Bromo-2-nitro-N-((tetrahydrofuran-2-yl)methyl)benzamide (10a)
Prepared from benzoic acid 8a and amine 9b. Yield 1.3 g (79%), white amorphous
1
solid. H NMR (300 MHz, CDCl3) δ: 8.19 (d, J = 1.9 Hz, 1H), 7.79 (dd, J = 8.1, 1.9
Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 6.19 (s, 1H), 4.09 (qd, J = 7.2, 3.3 Hz, 1H), 3.89 –
3.73 (m, 3H), 3.33 (ddd, J = 13.8, 7.4, 4.8 Hz, 1H), 2.12 – 1.87 (m, 3H), 1.72 – 1.63
(m, 1H). 13C NMR (101 MHz, CDCl3) δ: 165.5, 146.9, 136.4, 131.6, 129.9, 127.4,
123.6, 77.3, 68.0, 43.6, 28.5, 25.8. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C12H14BrN2O4 329.0137; Found 329.0143.
4.2.2. rac-4-Chloro-2-nitro-N-((tetrahydrofuran-2-yl)methyl)benzamide (10b)
Prepared from acid 8b and amine 9b. Yield 4.59 g (88%), white amorphous solid. 1H
NMR (400 MHz, CDCl3) δ: 8.02 (d, J = 1.9 Hz, 1H), 7.62 (dd, J = 8.1, 1.9 Hz, 1H),
7.46 (d, J = 8.1 Hz, 1H), 6.36 (brs, 1H), 4.06 (qd, J = 7.2, 3.4 Hz, 1H), 3.86 – 3.78 (m,
1H), 3.78 – 3.67 (m, 2H), 3.35 – 3.25 (m, 1H), 2.03 (m, 1H), 1.92 (m, 2H), 1.71 –
1.59 (m, 1H). 13C NMR (101 MHz, CDCl3) δ: 165.7, 147.2, 136.5, 133.7, 131.4,
130.0, 125.0, 77.5, 68.3, 43.9, 28.7, 26.0. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd
for C12H14ClN2O4 285.0642; Found 285.0645.
4.2.3.
4-Bromo-2-nitro-N-(((2S*,5R*)-5-phenyltetrahydrofuran-2-
yl)methyl)benzamide (10c)
1
Prepared from acid 8a and amine 9a. Yield 2.85 g (59%), white amorphous solid. H
NMR (400 MHz, CDCl3) δ: 8.12 (d, J = 1.9 Hz, 1H), 7.70 (dd, J = 8.1, 1.9 Hz, 1H),
7.29 (d, J = 8.1 Hz, 1H), 7.28 – 7.24 (m, 4H), 7.23 – 7.17 (m, 1H), 6.35 (brs, 1H),
4.86 (dd, J = 7.7, 6.7 Hz, 1H), 4.31 – 4.09 (m, 1H), 3.87 (ddd, J = 13.7, 6.9, 3.4 Hz,
1H), 3.40 (ddd, J = 13.8, 7.5, 4.6 Hz, 1H), 2.38 – 2.24 (m, 1H), 2.19 – 2.05 (m, 1H),
1.94 – 1.73 (m, 2H). 13C NMR (101 MHz, CDCl3) δ: 165.8, 147.1, 142.2, 136.7,
131.7, 130.0, 128.6, 127.7, 126.0, 124.0, 81.7, 78.1, 44.3, 34.0, 29.0. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C18H18BrN2O4 405.0450; Found 405.0445.
4.3. General procedure B for the synthesis of compounds 13a-d
To a solution of bromobenzene (3 mmol, 1 equiv.) in THF (10 mL) and H2O (2 mL),
boronic acid (1.2 equiv.) or pinacolborane (1.2 equiv.), Pd(PPh3)4 (2.5 mol%), and
K2CO3 (3 equiv.) were added. The resulting mixture was heated at 80 °C. After the

reaction was completed (monitored by TLC), the solution was cooled to r. t. and then
partitioned between H2O and EtOAc. The organic layer was concentrated in vacuo
and product purified by column chromatography.
4.3.1. rac-3-Nitro-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-4-carboxamide
(13a)
Prepared from bromobenzene 10a and phenylboronic acid 11. Yield 0.91 g (92%),
1
white amorphous solid. H NMR (400 MHz, CDCl3) δ: 1.66-1.75 (m, 1H), 1.91-1.99
(m, 2H), 2.03-2.11 (m, 1H), 3.35-3.42 (m, 1H), 3.74-3.90 (m, 3H), 4.09-4.15 (m, 1H),
6.24 (brs, 1H), 7.43-7.53 (m, 3H), 7.58-7.62 (m, 3H), 7.86 (dd, J = 2.0 Hz, 7.8 Hz,
13
1H), 8.26 (d, J = 2.0 Hz, 1H). C NMR (101 MHz, CDCl3) δ: 166.6, 147.3, 144.1,
137.9, 131.9, 131.5, 129.4, 129.3, 129.1, 127.3, 123.1, 77.6, 68.3, 43.9, 28.8, 26.1.
HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C18H19N2O4 327.1345; Found
327.1353.
4.3.2.
rac-3-Nitro-4'-(3-phenylpropyl)-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-
biphenyl]-4-carboxamide (13b)
Prepared from bromide 10a pinacolborane 12a. Yield 1.3 g (86%), white amorphous
1
solid. H NMR (400 MHz, CDCl3) δ: 8.24 (d, J = 1.7 Hz, 1H), 7.84 (dd, J = 7.9, 1.8
Hz, 1H), 7.55 (dd, J = 16.4, 8.1 Hz, 4H), 7.35 – 7.25 (m, 3H), 7.21 (d, J = 0.7 Hz,
3H), 6.29 – 6.21 (m, 1H), 4.17 – 4.06 (m, 1H), 3.89 – 3.71 (m, 4H), 3.38 (ddd, J =
13.8, 7.3, 4.9 Hz, 1H), 2.70 (dt, J = 11.8, 7.7 Hz, 4H), 2.11 – 1.90 (m, 4H), 1.76 –
1.65 (m, 1H). 13C NMR (101 MHz, CDCl3) δ: 166.6, 147.3, 144.0, 143.6, 142.1,
135.4, 131.6, 131.2, 129.5, 129.2, 128.6, 128.5, 127.2, 126.0, 122.8, 77.6, 68.3, 43.8,
35.5, 35.2, 32.9, 28.7, 26.1. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C27H29N2O4 445.2127; Found 445.2124.
4.3.3.
rac-3-Nitro-4'-pentyl-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-4-
carboxamide (13c)
Prepared from bromide 10a pinacolborane 12b in DMF. Yield 1.5 g (75%), white
amorphous solid. 1H NMR (300 MHz, CDCl3) δ: 8.22 (d, J = 1.7 Hz, 1H), 7.83 (dd, J
= 7.9, 1.7 Hz, 1H), 7.54 (dd, J = 13.7, 8.0 Hz, 3H), 7.30 (d, J = 8.1 Hz, 2H), 6.37 (t, J
= 5.7 Hz, 1H), 4.14 – 4.04 (m, 1H), 3.88 – 3.71 (m, 3H), 3.37 (ddd, J = 13.7, 7.3, 4.9
Hz, 1H), 2.71 – 2.63 (m, 2H), 1.98 – 1.87 (m, 2H), 1.75 – 1.59 (m, 3H), 1.35 (h, J =

13
3.7, 3.2 Hz, 4H), 1.25 (td, J = 7.2, 0.6 Hz, 1H), 0.94 – 0.85 (m, 3H). C NMR (101
MHz, CDCl3) δ: 166.7, 147.3, 144.3, 144.0, 135.2, 131.6, 131.1, 129.5, 129.2, 127.1,
122.7, 77.6, 68.3, 43.8, 35.7, 31.6, 31.2, 28.7, 26.1, 22.7, 14.1. HRMS (ESI/TOF-Q)
m/z: [M + H]+ Calcd for C23H29N2O4 397.2127; Found 397.2135.
4.3.4. 3-Nitro-N-(((2S*,5R*)-5-phenyltetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-4-
carboxamide (13d)
Prepared from bromide 10c and boronic acid 11. Yield 1.7 g (85 %), white amorphous
1
solid. H NMR (400 MHz, CDCl3) δ: 8.16 (d, J = 1.8 Hz, 1H), 7.75 (dd, J = 7.9, 1.8
Hz, 1H), 7.56 – 7.49 (m, 2H), 7.50 – 7.35 (m, 4H), 7.30 – 7.20 (m, 4H), 7.22 – 7.13
(m, 1H), 6.28 (t, J = 5.9 Hz, 1H), 4.85 (t, J = 7.0 Hz, 1H), 4.23 (tdd, J = 7.2, 6.3, 3.4
Hz, 1H), 3.89 (ddd, J = 13.8, 6.9, 3.4 Hz, 1H), 3.43 (ddd, J = 13.8, 7.4, 4.7 Hz, 1H),
13
2.36 – 2.22 (m, 1H), 2.19 – 2.04 (m, 1H), 1.94 – 1.76 (m, 2H). C NMR (101 MHz,
CDCl3) δ: 166.6, 147.2, 144.1, 142.3, 137.9, 131.9, 131.4, 129.4, 129.2, 129.1, 128.6,
127.7, 127.3, 126.1, 123.1, 81.6, 78.3, 44.3, 34.1, 29.0. HRMS (ESI/TOF-Q) m/z: [M
+ H]+ Calcd for C24H23N2O4 403.1658; Found 403.1660.
4.4. General procedure C for the synthesis of compounds 14a-e
Mixture of benzamide (1 mmol, 1 equiv.), PhI(OAc)2 (1 equiv.), I2 (1 equiv.) and
o
Pd(OAc)2 (10 mol%) in DMF (5 mL) was heated at 100 C. After 24 h saturated aq.
NaHCO3 added, and mixture extracted with EtOAc. Organic layer washed with 10%
aq. Na2S2O3, brine and dried over anh. Na2SO4. Purification by column
chromatography.
4.4.1.
rac-3-Iodo-5-nitro-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-4-
carboxamide (14a)
1
Prepared from benzamide 13a. Yield 0.27 g (65%), white amorphous solid. H NMR
(300 MHz, CDCl3) δ: 1.72.-1.79 (m, 1H), 1.91-1.99 (m, 2H), 2.04-2.13 (m, 1H), 3.37-
3.43 (m, 1H), 3.75-3.80 (m, 1H), 3.85-3.91 (m, 2H), 4.16-4.22 (m, 1H), 6.14 (brs,
1H), 7.44-7.54 (m, 3H), 7.56-7.60 (m, 2H), 8.33 (d, J = 2.0 Hz, 1H), 8.34 (d, J = 1.6
Hz, 1H). 13C NMR (101 MHz, CDCl3) δ: 166.5, 146.9, 144.6, 143.2, 136.6, 136.3,
129.5, 127.3, 123.0, 96.0, 77.3, 68.3, 43.8, 28.9, 26.1. HRMS (ESI/TOF-Q) m/z: [M +
H]+ Calcd for C18H18IN2O4 453.0311; Found 453.0313.

4.4.2. rac-3-Iodo-5-nitro-4'-(3-phenylpropyl)-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-
biphenyl]-4-carboxamide (14b)
1
Prepared from benzamide 13b. Yield 1.3 g (76%), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.32 (qd, J = 1.7, 0.6 Hz, 2H), 7.55 – 7.45 (m, 2H), 7.34 – 7.27
(m, 4H), 7.24 – 7.16 (m, 3H), 6.18 – 6.13 (m, 1H), 4.18 (qd, J = 7.1, 3.5 Hz, 1H), 3.92
– 3.83 (m, 2H), 3.77 (dt, J = 8.2, 6.8 Hz, 1H), 3.40 (ddd, J = 13.6, 7.3, 4.7 Hz, 1H),
2.70 (dt, J = 13.5, 7.7 Hz, 4H), 2.13 – 2.05 (m, 1H), 2.03 – 1.89 (m, 4H), 1.76 (ddt, J
13
= 12.1, 8.5, 7.1 Hz, 1H). C NMR (101 MHz, CDCl3) δ: 166.4, 146.7, 144.4, 144.0,
142.8, 141.9, 135.8, 133.9, 129.5, 128.42, 128.36, 127.1, 125.9, 122.6, 95.8, 77.2,
68.2, 43.7, 35.4, 35.0, 32.8, 28.7, 25.9. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C27H28IN2O4 571.1094; Found 571.1093.
4.4.3. rac-3-Iodo-5-nitro-4'-pentyl-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-
4-carboxamide (14c)
1
Prepared from benzamide 13c. Yield 1.1 g (57%), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.32 (d, J = 1.7 Hz, 1H), 8.31 (d, J = 1.7 Hz, 1H), 7.49 (d, J =
8.2 Hz, 2H), 7.31 (d, J = 8.2 Hz, 2H), 6.13 (t, J = 5.1 Hz, 1H), 4.19 (qd, J = 7.0, 3.3
Hz, 1H), 3.92 – 3.84 (m, 2H), 3.80 – 3.73 (m, 1H), 3.40 (ddd, J = 13.7, 7.3, 4.7 Hz,
1H), 2.67 (t, J = 7.5 Hz, 2H), 2.14 – 2.03 (m, 1H), 2.00 – 1.91 (m, 2H), 1.82 – 1.70
13
(m, 1H), 1.65 (m, 2H), 1.37 – 1.31 (m, 4H), 0.91 (t, J = 7.1 Hz, 3H). C NMR (101
MHz, CDCl3) δ: 166.4, 146.7, 144.7, 144.5, 142.8, 135.8, 133.8, 129.4, 127.0, 122.6,
95.8, 77.2, 68.2, 43.7, 35.6, 31.4, 31.0, 28.7, 25.9, 22.5, 14.0. HRMS (ESI/TOF-Q)
m/z: [M + H]+ Calcd for C23H28IN2O4 523.1094; Found 523.1091.
4.4.4.
3-Iodo-5-nitro-N-(((2S*,5R*)-5-phenyltetrahydrofuran-2-yl)methyl)-[1,1'-
biphenyl]-4-carboxamide (14d)
Prepared from benzamide 13d. Yield 0.15 g (56 %, 75 % brsm), white amorphous
1
solid. H NMR (300 MHz, CDCl3) δ: 8.25 (s, 2H), 7.54 – 7.36 (m, 5H), 7.31 – 7.07
(m, 5H), 6.15 (dd, J = 7.1, 4.3 Hz, 1H), 4.85 (t, J = 7.0 Hz, 1H), 4.30 (qd, J = 7.2, 3.4
Hz, 1H), 3.95 (ddd, J = 13.7, 7.1, 3.5 Hz, 1H), 3.44 (ddd, J = 13.7, 7.6, 4.3 Hz, 1H),
13
2.36 – 2.23 (m, 1H), 2.22 – 2.08 (m, 1H), 1.97 – 1.79 (m, 2H). C NMR (101 MHz,
CDCl3) δ: 166.6, 146.8, 144.6, 143.1, 142.3, 136.6, 136.2, 129.5, 128.6, 127.7, 127.3,

126.1, 123.0, 96.0, 81.7, 77.9, 44.4, 34.1, 29.2. HRMS (ESI/TOF-Q) m/z: [M + H]+
Calcd for C24H22IN2O4 529.0624; Found 529.0599.
4.4.5. rac-4-Chloro-2-iodo-6-nitro-N-((tetrahydrofuran-2-yl)methyl)benzamide (14e)
1
Prepared from benzamide 10b.Yield 1.8 g (31 %), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.13 (m, 2H), 6.09 (brs, 1H), 4.16 (qd, J = 7.2, 3.4 Hz, 1H),
3.92 – 3.81 (m, 2H), 3.81 – 3.67 (m, 1H), 3.35 (ddd, J = 13.7, 7.4, 4.6 Hz, 1H), 2.14 –
2.01 (m, 1H), 1.94 (m, 2H), 1.87 – 1.63 (m, 1H). 13C NMR (101 MHz, CDCl3) δ:
165.67, 146.65, 144.36, 136.54, 136.50, 124.87, 95.82, 77.36, 68.32, 43.90, 28.88,
26.06. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C12H13ClIN2O4 410.9609;
Found 410.9612.
4.5. General procedure D for the synthesis of compounds 15a-e
Mixture of nitrobenzene (1.5 mmol, 1 equiv.) and Na2S.9H2O (3 equiv.) in THF (15
mL) and MeOH (15 mL) was refluxed for 1 – 2 h then solvents evaporated in vacuo.
Residue partitioned between H2O and EtOAc. The organic layer was washed 2 times
with H2O, brine and dried over anh. Na2SO4. After evaporation of solvent crude
product washed with PE/EtOAc (4:1), filtered.
4.5.1.
rac-3-Amino-5-iodo-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-biphenyl]-4-
carboxamide (15a)
Prepared from nitrobenzene 14a. Yield 0.52 g (83%), light yellow amorphous solid.
1H NMR (400 MHz, CDCl3) δ: 1.65-1.74 (m, 1H), 1.86-2.09 (m, 3H), 3.37-3.44 (m,
1H), 3.71-3.79 (m, 2H), 3.86-3.91 (m, 1H), 4.09-4.16 (m, 1H), 4.39 (brs, 2H), 6.29
13
(brs, 1H), 6.83 (d, J = 1.6 Hz, 1H), 7.33-7.44 (m, 4H), 7.47-7.51 (m, 2H). C NMR
(101 MHz, CDCl3) δ: 169.1, 146.0, 144.8, 139.3, 128.9, 128.2, 128.1, 127.2, 126.2,
114.7, 93.90, 77.4, 68.2, 43.5, 29.0, 26.0. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd
for C18H20IN2O2 423.0569; Found 423.0563.
4.5.2.
rac-3-Amino-5-iodo-4'-(3-phenylpropyl)-N-((tetrahydrofuran-2-yl)methyl)-
[1,1'-biphenyl]-4-carboxamide (15b)
Prepared from nitrobenzene 14b. Yield 1.0 g (96%), light yellow amorphous solid.
1H NMR (400 MHz, CDCl3) δ: 7.41 (dd, J = 4.9, 3.4 Hz, 3H), 7.32 – 7.26 (m, 2H),

7.25 – 7.16 (m, 5H), 6.83 (d, J = 1.5 Hz, 1H), 6.26 (t, J = 5.9 Hz, 1H), 4.33 (brs, 2H),
4.14 (qd, J = 7.1, 3.6 Hz, 1H), 3.89 (ddd, J = 8.4, 7.0, 6.3 Hz, 1H), 3.79 – 3.73 (m,
2H), 3.41 (ddd, J = 13.8, 7.3, 5.3 Hz, 1H), 2.72 – 2.63 (m, 4H), 2.10 – 1.87 (m, 5H),
1.74 – 1.65 (m, 1H). 13C NMR (101 MHz, CDCl3) δ: 168.9, 145.8, 144.5, 142.4,
142.1, 136.6, 128.9, 128.4, 128.3, 127.8, 126.9, 125.83, 125.76, 114.3, 93.7, 77.6,
68.1, 43.4, 35.4, 35.0, 32.8, 28.8, 25.8. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C27H30IN2O2 541.1352; Found 541.1371.
4.5.3.
rac-3-Amino-5-iodo-4'-pentyl-N-((tetrahydrofuran-2-yl)methyl)-[1,1'-
biphenyl]-4-carboxamide (15c)
Prepared from nitrobenzene 14c. Yield 0.79 g (84%), light yellow amorphous solid.
1H NMR (300 MHz, CDCl3) δ: 7.40 (dt, J = 7.5, 1.7 Hz, 3H), 7.25 – 7.16 (m, 2H),
6.83 (d, J = 1.6 Hz, 1H), 6.23 (t, J = 5.9 Hz, 1H), 4.36 (brs, 2H), 4.14 (qd, J = 7.1, 3.7
Hz, 1H), 3.89 (dt, J = 8.4, 6.6 Hz, 1H), 3.81 – 3.67 (m, 2H), 3.41 (ddd, J = 13.8, 7.3,
5.3 Hz, 1H), 2.63 (dd, J = 8.7, 6.7 Hz, 2H), 2.12 – 1.84 (m, 3H), 1.78 – 1.53 (m, 3H),
1.39 – 1.26 (m, 4H), 0.95 – 0.85 (m, 3H). 13C NMR (101 MHz, CDCl3) δ: 169.0,
145.8, 144.6, 143.1, 136.4, 128.9, 127.9, 126.8, 125.8, 114.3, 93.7, 77.3, 68.1, 43.4,
35.6, 31.5, 31.1, 28.8, 25.8, 22.5, 14.0. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C23H30IN2O2 493.1352; Found 493.1352.
4.5.4.
3-Amino-5-iodo-N-(((2S*,5R*)-5-phenyltetrahydrofuran-2-yl)methyl)-[1,1'-
biphenyl]-4-carboxamide (15d)
1
Prepared from nitrobenzene 14d. Yield 0.41 g (59%), yellow amorphous solid. H
NMR (300 MHz, CDCl3) δ: 7.52 – 7.20 (m, 11H), 6.82 (d, J = 1.6 Hz, 1H), 6.30 (t, J
= 6.0 Hz, 1H), 4.92 (t, J = 7.1 Hz, 1H), 4.42 – 4.24 (m, 3H), 3.91 (ddd, J = 13.7, 6.7,
3.7 Hz, 1H), 3.52 (ddd, J = 13.2, 7.7, 4.8 Hz, 1H), 2.42 – 2.28 (m, 1H), 2.25 – 2.09
13
(m, 1H), 1.99 – 1.80 (m, 2H). C NMR (101 MHz, CDCl3) δ: 169.1, 146.0, 144.8,
142.3, 139.3, 128.9, 128.6, 128.2, 128.1, 127.6, 127.2, 126.1, 126.0, 114.7, 93.9, 81.7,
77.9, 44.2, 34.3, 29.3. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C24H24IN2O2
499.0882; Found 499.0893.
4.5.5. rac-2-Amino-4-chloro-6-iodo-N-((tetrahydrofuran-2-yl)methyl)benzamide (15e)

1
Prepared from nitrobenzene 14e. Yield 1.6 g (96%), light yellow amorphous solid. H
NMR (300 MHz, CDCl3) δ: 7.17 (d, J = 1.8 Hz, 1H), 6.64 (d, J = 1.8 Hz, 1H), 6.16
(brs, 1H), 4.39 (brs, 2H), 4.11 (qd, J = 7.1, 3.5 Hz, 1H), 3.94 – 3.83 (m, 1H), 3.82 –
3.61 (m, 2H), 3.47 – 3.21 (m, 1H), 2.10 – 1.86 (m, 3H), 1.74 – 1.57 (m, 1H). 13C
NMR (101 MHz, CDCl3) δ: 168.4, 146.4, 136.4, 128.2, 125.8, 115.7, 93.4, 77.3, 68.2,
43.6, 29.0, 25.9. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C12H15ClIN2O2
380.9867; Found 380.9865.
4.6. General procedure E for the synthesis of compounds 16a-e
Benzoyl isothiocyanate (1 equiv.) was added dropwise to the solution of benzamide (2
mmol, 1 equiv.) in Et2O (10 mL), and the mixture was stirred at r. t. for 2 h. The solid
was collected, giving the corresponding thiourea intermediate that was dissolved in
dry DCM (20 mL). Et3N (3 equiv.) and EDCI*HCl (1.2 equiv.) added and the solution
was stirred at r. t. overnight. Then solvent evaporated and product washed with
MeOH, filtered.
4.6.1.
rac-N-(5-Iodo-4-oxo-7-phenyl-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-2-yl)benzamide (16a)
Prepared from benzamide 15a. Yield 0.45 g (44 %), white amorphous solid. 1H NMR
(300 MHz, CDCl3) δ: 1.79-2.17 (m, 4H), 3.71-3.79 (m, 1H), 3.93-4.01 (m, 1H), 4.28-
4.33 (m, 1H), 4.57-4.75 (m, 2H), 7.41-7.64 (m, 9H), 8.22-8.29 (m, 3H), 14.51 (brs,
13
1H). C NMR (101 MHz, DMSO-d6) δ: 168.9, 167.1, 151.4, 142.2, 138.3, 135.2,
134.5, 133.1, 132.1, 132.0, 129.1, 128.6, 128.3, 127.3, 126.9, 120.0, 94.5, 76.6, 67.2,
43.3, 28.9, 25.2. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C26H23IN3O3
552.0784; Found 552.0791.
4.6.2.
rac-N-(5-Iodo-4-oxo-7-(4-(3-phenylpropyl)phenyl)-3-((tetrahydrofuran-2-
yl)methyl)-3,4-dihydroquinazolin-2-yl)benzamide (16b)
1
Prepared from benzamide 15b. Yield 0.52 g (42%), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.27 (d, J = 7.3 Hz, 2H), 8.21 (s, 1H), 7.57 – 7.38 (m, 7H), 7.34
– 7.18 (m, 7H), 4.70 (dd, J = 12.6, 8.4 Hz, 1H), 4.65 – 4.57 (m, 1H), 4.30 (dd, J =
12.6, 4.6 Hz, 1H), 4.00 – 3.93 (m, 1H), 3.80 – 3.73 (m, 1H), 2.76 – 2.63 (m, 4H), 2.14

– 1.79 (m, 6H). 13C NMR (101 MHz, CDCl3) δ: 179.2, 159.0, 153.8, 148.0, 144.2,
142.1, 138.7, 138.1, 137.2, 134.5, 132.5, 129.5, 128.6, 128.5, 128.3, 127.3, 126.0,
114.9, 114.3, 94.9, 75.7, 68.1, 45.8, 35.5, 35.2, 32.9, 29.5, 25.6. HRMS (ESI/TOF-Q)
m/z: [M + H]+ Calcd for C35H33IN3O3 670.1567; Found 670.1557.
4.6.3. rac-N-(5-Iodo-4-oxo-7-(4-pentylphenyl)-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-2-yl)benzamide (16c)
1
Prepared from benzamide 15c. Yield 0.39 g (41%), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.25 (d, J = 7.6 Hz, 2H), 8.21 (d, J = 1.6 Hz, 1H), 7.53 (d, J =
7.9 Hz, 3H), 7.49 – 7.43 (m, 3H), 7.39 (brs, 1H), 7.32 – 7.26 (m, 2H), 4.76 – 4.53 (m,
2H), 4.30 (d, J = 12.6 Hz, 1H), 4.00 – 3.90 (m, 1H), 3.75 (d, J = 7.2 Hz, 1H), 2.71 –
2.61 (m, 2H), 2.14 – 2.03 (m, 2H), 1.98 – 1.75 (m, 2H), 1.65 (p, J = 7.5 Hz, 2H), 1.38
13
– 1.32 (m, 4H), 0.94 – 0.86 (m, 3H). C NMR (101 MHz, CDCl3) δ: 178.9, 158.9,
153.5, 147.8, 144.8, 138.7, 138.0, 137.0, 134.2, 132.4, 129.4, 128.2, 127.1, 114.8,
114.3, 94.7, 75.7, 68.1, 45.8, 35.7, 31.6, 31.1, 29.5, 25.5, 22.6, 14.1. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C31H33IN3O3 622.1567; Found 622.1570.
4.6.4. N-(5-Iodo-4-oxo-7-phenyl-3-(((2S*,5R*)-5-phenyltetrahydrofuran-2-yl)methyl)-
3,4-dihydroquinazolin-2-yl)benzamide (16d)
1
Prepared from benzamide 15d. Yield 0.21 g (20%), white amorphous solid. H NMR
(300 MHz, CDCl3) δ: 14.52 (brs, 1H), 8.29 (d, J = 7.1 Hz, 2H), 8.21 (d, J = 1.7 Hz,
1H), 7.64 – 7.35 (m, 11H), 7.23 – 7.14 (m, 3H), 5.00 – 4.85 (m, 2H), 4.80 – 4.70 (m,
13
1H), 4.52 (dd, J = 12.7, 5.1 Hz, 1H), 2.40 – 2.11 (m, 2H), 2.07 – 1.84 (m, 2H). C
NMR (101 MHz, CDCl3) δ: 179.3, 159.1, 153.8, 148.2, 142.7, 138.8, 138.3, 137.1,
132.5, 129.7, 129.6, 129.4, 128.4, 128.3, 127.4, 127.3, 126.0, 115.3, 114.6, 94.9, 81.8,
76.2, 46.5, 34.8, 29.9. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C32H27IN3O3
628.1097; Found 628.1073.
4.6.5.
rac-N-(7-Chloro-5-iodo-4-oxo-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-2-yl)benzamide (16e)
1
Prepared from benzamide 15e Yield 0.20 g (30%), white amorphous solid. H NMR
(400 MHz, CDCl3) δ: 8.24 (d, J = 7.1 Hz, 2H), 7.96 (d, J = 1.9 Hz, 1H), 7.61 – 7.42
(m, 3H), 7.24 (d, J = 1.9 Hz, 1H), 4.68 (dd, J = 12.7, 8.6 Hz, 1H), 4.62 – 4.52 (m,
1H), 4.25 (dd, J = 12.7, 4.4 Hz, 1H), 4.00 – 3.90 (m, 1H), 3.79 – 3.70 (m, 1H), 2.08

13
(m, 2H), 2.00 – 1.86 (m, 1H), 1.85 – 1.75 (m, 1H). C NMR (101 MHz, CDCl3) δ:
179.3, 158.4, 153.6, 141.0, 138.9, 138.7, 136.8, 132.6, 129.4, 128.2, 116.8, 114.4,
95.0, 75.4, 68.0, 45.8, 29.4, 25.4. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C20H18ClIN3O3 510.0081; Found 510.0095.
4.7. General procedure F for the synthesis of compounds 2a,c-g; 3a; 4a-b; 7a-b
To a solution of iodobenzene or bromobenzene (0.15 mmol, 1 equiv.) in DMF (1.5
mL), benzoic acid or pinacolborane (1.3 equiv.), Pd(PPh3)4 (5 mol%), and K2CO3 (3
equiv.) were added. The resulting mixture was heated at 80 °C. After the reaction was
completed (TLC), the solution was cooled to r. t., concentrated in vacuo and filtered
through a pad of silica gel with EtOAc:DCM = 1:1 to afford protected intermediate
that was used in the next step without additional purification. To a suspension of
protected intermediate (1 equiv.) in EtOH (5 mL), hydrazine hydrate (0.1 mL) was
added and the mixture was refluxed for 4 h. Solvent was evaporated, water was
added, product filtered, washed with wet MeOH, filtered or purified by column
chromatography.
4.7.1.
rac-4-(2-Amino-4-oxo-7-phenyl-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-5-yl)benzoic acid (2a)
Prepared from iodobenzene 16a and 4-boronobenzoic acid. Yield 33 mg (52%), white
amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ: 1.49-1.58 (m, 1H), 1.72-1.95 (m,
3H), 3.60-3.65 (m, 1H), 3.76-3.80 (m, 1H), 3.95-4.12 (m, 3H), 6.91 (brs, 2H), 7.09 (d,
J =1.6 Hz, 1H), 7.42-7.50 (m, 6H), 7.75 (d, J = 7.4 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H),
12.9 (brs, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 24.9, 28.5, 45.0, 67.3, 76.3, 112.2,
121.4, 123.0, 127.0, 128.1, 128.3, 128.7, 128.8, 129.0, 138.8, 142.7, 144.4, 147.0,
151.4, 152.8, 160.7, 167.3. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C26H24N3O4
442.1767 ; Found 442.1771.
4.7.2.
rac-2-Amino-7-phenyl-5-(pyridin-4-yl)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (2c)
Prepared from iodobenzene 16a and pyridin-4-ylboronic acid. Yield 14 mg (39%),
1
white solid. Tdecomp. = 180 oC. H NMR (400 MHz, CDCl3) δ: 1.51-1.61 (m, 1H),
1.87-1.95 (m, 2H), 2.07-2.14 (m, 1H), 3.78 (q, J = 7.0 Hz, 1H), 3.90-3.96 (m, 2H),

4.13 (q, J = 7.0 Hz, 1H), 4.45 (d, J = 14.9 Hz, 1H), 5.89 (brs, 2H), 7.16 (d, J = 2.0 Hz,
1H), 7.28 (d, J = 5.9 Hz, 2H), 7.38-7.48 (m, 3H), 7.61 (d, J = 1.6 Hz, 1H), 7.67 (d, J
13
= 7.4 Hz, 2H), 8.64 (d, J = 5.9 Hz, 2H). C NMR (101 MHz, CDCl3) δ: 25.6, 28.9,
46.7, 68.5, 79.4, 112.9, 123.0, 123.7, 124.8, 127.3, 128.5, 129.0, 139.3, 141.4, 146.0,
148.8, 150.8, 150.9, 153.7, 161.7. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C24H23N4O2 399.1821; Found 399.1821.
4.7.3.
rac-2-Amino-7-phenyl-5-(pyridin-3-yl)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (2d)
Prepared from iodobenzene 16a and pyridin-3-ylboronic acid. Yield 13 mg (36%),
1
white solid. Tdecomp. = 192 oC. H NMR (400 MHz, CDCl3) δ: 1.51-1.60 (m, 1H),
1.87-1.94 (m, 2H), 2.05-2.14 (m, 1H), 3.78 (dd, J =7.0 Hz, 14.9 Hz, 1H), 3.89-3.97
(m, 2H), 4.13 (q, J = 7.4 Hz, 1H), 4.43 (d, J =14.9 Hz, 1H), 5.89 (brs, 2H), 7.22 (d, J
=1.2 Hz, 1H), 7.32-7.41 (m, 2H), 7.44-7.48 (m, 2H), 7.61 (d, J =1.6 Hz, 1H), 7.69 (m,
3H), 8.61 (s, 2H). 13C NMR (101 MHz, CDCl3) δ: 25.6, 28.9, 46.6, 68.4, 79.4, 113.3,
122.2, 122.9, 125.6, 127.3, 128.4, 128.9, 135.9, 138.1, 139.3, 140.4, 145.9, 148.0,
149.2, 150.9, 153.7, 161.9. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C24H23N4O2
399.1821; Found 399.1825.
4.7.4.
rac-2-Amino-5-(3-aminophenyl)-7-phenyl-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (2e)
Prepared from iodobenzene 16a and (3-aminophenyl)boronic acid. Yield 29 mg
1
(39%), white amorphous solid. H NMR (400 MHz, CDCl3) δ: 1.51-1.61 (m, 1H),
1.82-1.95 (m, 2H), 1.99-2.13 (m, 1H), 3.74-3.80 (m, 1H), 3.86-3.96 (m, 2H), 4.08-
4.16 (m, 1H), 4.46-4.49 (m, 1H), 5.85 (brs, 2H), 6.67-6.77 (m, 3H), 7.15-7.23 (m,
13
2H), 7.35-7.46 (m, 5H), 7.53-7.57 (m, 1H), 7.64-7.71 (m, 2H). C NMR (101 MHz,
CDCl3) δ: 25.6, 28.9, 46.6, 68.5, 79.5, 113.4, 113.8, 115.4, 119.4, 121.7, 125.3, 127.2,
127.3, 128.2, 128.8, 128.9, 139.6, 143.8, 144.6, 145.5, 145.6, 153.5, 161.5. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C25H25N4O2 413.1978; Found 413.1971.
4.7.5.
rac-4-(2-Amino-4-oxo-7-phenyl-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-5-yl)benzamide (2f)
Prepared from iodobenzene 16a and (4-carbamoylphenyl)boronic acid. Yield 12 mg
1
(19%), light yellow amorphous solid. H NMR (400 MHz, DMSO-d6) δ: 1.52-1.61

(m, 1H), 1.73-1.98 (m, 3H), 3.62-3.67 (m, 1H), 3.74-3.80 (m, 1H), 3.99-4.11 (m, 3H),
7.25 (d, J =1.6 Hz, 1H), 7.37 (brs, 1H), 7.40-7.56 (m, 6H), 7.77 -7.88 (m, 6H), 8.01
(brs, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 25.0, 28.5, 45.1, 67.3, 75.9, 111.5,
123.4, 124.5, 126.4, 127.0, 128.4, 128.7, 129.1, 132.3, 132.6, 138.2, 143.3, 144.3,
145.1, 152.2, 159.5, 167.7. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C26H25N4O3
441.1927; Found 441.1923.
4.7.6.
rac-3-(2-Amino-4-oxo-7-phenyl-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-5-yl)benzoic acid (2g)
Prepared from iodobenzene 16a and 3-boronobenzoic acid. Yield 60 mg (75%), white
amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ: 1.50-1.61 (m, 1H), 1.72-1.98 (m,
3H), 3.59-3.66 (m, 1H), 3.72-3.82 (m, 1H), 3.98-4.13 (m, 3H), 7.27 (brs, 2H), 7.43-
7.62 (m, 6H), 7.76-7.79 (m, 2H), 7.87-7.94 (m, 3H). 13C NMR (101 MHz, DMSO-d6)
δ: 25.5, 29.0, 45.8, 67.9, 75.6, 111.0, 114.1, 126.8, 127.6, 127.7, 128.3, 128.6, 129.7,
129.8, 130.5, 133.4, 137.8, 139.6, 141.0, 144.1, 146.7, 152.0, 158.5, 167.7. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C26H24N3O4 442.1767; Found 442.1771.
4.7.7.
4-(2-Amino-4-oxo-7-phenyl-3-(((2S*,5R*)-5-phenyltetrahydrofuran-2-
yl)methyl)-3,4-dihydroquinazolin-5-yl)benzoic acid (3a)
Prepared from iodobenzene 16d and 4-boronobenzoic acid. Yield 18 mg (77%), white
amorphous solid. 1H NMR (400 MHz, DMSO-d6) δ: 1.65-1.77 (m, 2H), 2.01-2.09 (m,
1H), 2.19-2.28 (m, 1H), 4.12-4.27 (m, 3H), 4.77 (t, J = 7.4 Hz, 1H), 7.15 (d, J = 1.6
Hz, 1H), 7.24-7.36 (m, 5H), 7.41-7.52 (m, 5H), 7.59-7.64 (m, 3H), 7.74-7.76 (m, 2H),
13
7.93 (d, J = 8.2 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ: 28.6, 33.7, 45.2, 76.3,
80.8, 112.0, 125.8, 127.0, 128.1, 128.1, 128.6, 128.7, 128.8, 129.0, 131.3, 131.4,
132.0, 133.1, 138.5, 142.3, 142.9, 144.7, 146.7, 152.5, 160.3, 167.3. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C32H28N3O4 518.2080; Found 518.2079.
4.7.8. rac-2-Amino-7-(4-pentylphenyl)-3-((tetrahydrofuran-2-yl)methyl)quinazolin-
4(3H)-one (4a)
Prepared from bromobenzene 16f [29] and pinacolborane 12b. Yield 19 mg (69%),
white amorphous solid. 1H NMR (400 MHz, CDCl3) δ: 0.89 (t, J = 7.0 Hz, 3H), 1.29-
1.39 (m, 4H), 1.59-1.75(m, 3H), 1.80-1.98 (m, 2H), 2.08-2.26 (m, 1H), 2.64 (dd, J =
6.5 Hz, 14.1 Hz, 2H), 3.73-3.81 (m, 1H), 3.90 (dt, J = 7.1, 14.2 Hz, 1H), 4.10-4.28

(m, 2H), 4.48 (d, J = 14.5 Hz, 1H), 5.85 (brs, 2H), 7.18-7.34 (m, 2H), 7.42 (dd, J =
1.7, 8.3 Hz, 1H), 7.52 (d, J = 1.7 Hz, 1H), 7.58 (d, J = 8.2 Hz, 2H), 8.13 (d, J = 8.3
Hz, 1H). 13C NMR (101 MHz, CDCl3) δ: 14.0, 22.5, 25.7, 28.7, 31.1, 31.5, 35.6, 46.4,
68.4, 79.5, 115.7, 122.0, 122.1, 127.1, 127.5, 128.9, 137.4, 143.2, 147.2, 149.3, 153.4,
163.0. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C24H30N3O2 392.2338; Found
392.2336.
4.7.9.
rac-2-Amino-7-(4-(3-phenylpropyl)phenyl)-3-((tetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (4b)
Prepared from bromobenzene 16f [29] and pinacolborane 12a. Yield 18 mg (75%),
1
white amorphous solid. H NMR (400 MHz, CDCl3) δ: 1.59-1.71 (m, 1H), 1.87-2.04
(m, 4H), 2.09-2.23 (m, 1H), 2.68 (dd, J = 6.6, 15.0 Hz, 4H), 3.72-3.82 (m, 1H), 3.85-
3.96 (m, 1H), 4.08-4.32 (m, 2H), 4.48 (d, J = 14.5 Hz, 1H), 5.83 (brs, 2H), 7.14-7.21
(m, 3H), 7.24-7.33 (m, 4H), 7.43 (dd, J = 1.8, 8.3 Hz, 1H), 7.52 (dd, J = 0.5 Hz, 1.7
Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 8.14 (dd, J = 0.5, 8.3 Hz, 1H). 13C NMR (101 MHz,
CDCl3) δ: 25.7, 28.8, 32.8, 35.1, 35.4, 46.4, 68.4, 79.5, 115.8, 122.0, 122.1, 125.7,
127.2, 127.6, 128.3, 128.4, 129.0, 137.6, 142.1, 142.5, 147.1, 149.2, 153.4, 162.9.
HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C28H30N3O2 440.2338; Found
440.2337.
4.7.10. rac-4-(2-Amino-4-oxo-7-(4-pentylphenyl)-3-((tetrahydrofuran-2-yl)methyl)-
3,4-dihydroquinazolin-5-yl)benzoic acid (7a)
Prepared from iodobenzene 16c and 4-boronobenzoic acid. Yield 20 mg (24%), white
amorphous solid.1H NMR (400 MHz, DMSO-d6) δ: 12.82 (brs, 1H), 7.93 – 7.89 (m,
2H), 7.66 (d, J = 8.3 Hz, 2H), 7.44 – 7.40 (m, 3H), 7.29 (d, J = 8.3 Hz, 2H), 7.08 (d, J
= 1.9 Hz, 1H), 6.88 (brs, 2H), 4.11 – 4.04 (m, 1H), 3.99 – 3.97 (m, 2H), 3.77 (dt, J =
8.3, 6.9 Hz, 1H), 3.63 (td, J = 7.8, 5.6 Hz, 1H), 2.61 (t, J = 7.6 Hz, 2H), 1.96 – 1.73
13
(m, 3H), 1.63 – 1.49 (m, 3H), 1.36 – 1.21 (m, 4H), 0.91 – 0.83 (m, 3H). C NMR
(101 MHz, DMSO-d6) δ: 167.4, 160.8, 152.9, 151.5, 147.2, 144.4, 142.8, 142.7,
136.2, 129.0, 128.9, 128.7, 128.2, 126.9, 123.0, 121.1, 112.1, 76.4, 67.3, 45.0, 34.7,
30.9, 30.5, 28.6, 25.0, 22.0, 13.9. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C31H34N3O4 512.2549; Found 512.2548.

4.7.11. rac-4-(2-Amino-4-oxo-7-(4-(3-phenylpropyl)phenyl)-3-((tetrahydrofuran-2-
yl)methyl)-3,4-dihydroquinazolin-5-yl)benzoic acid (7b)
Prepared from iodobenzene 16b and 4-boronobenzoic acid using Pd(dppf)Cl2*CH2Cl2
1
as a catalyst. Yield 10 mg (46%), white amorphous solid. H NMR (400 MHz,
DMSO-d6) δ: 7.92 – 7.82 (m, 2H), 7.69 – 7.60 (m, 2H), 7.39 (d, J = 1.9 Hz, 1H), 7.36
– 7.31 (m, 2H), 7.29 – 7.22 (m, 4H), 7.20 – 7.11 (m, 3H), 7.04 (d, J = 1.9 Hz, 1H),
6.83 (brs, 2H), 4.07 – 4.00 (m, 1H), 3.98 – 3.91 (m, 2H), 3.77 – 3.70 (m, 1H), 3.62 –
3.55 (m, 1H), 2.65 – 2.55 (m, 4H), 1.95 – 1.71 (m, 5H), 1.58 – 1.45 (m, 1H). 13C
NMR (101 MHz, DMSO-d6) δ: 160.8, 152.9, 151.6, 144.3, 143.1, 142.4, 141.9, 136.4,
135.2, 134.3, 129.0, 128.6, 128.3, 128.2, 127.0, 125.7, 123.0, 121.1, 112.2, 76.4, 67.4,
45.1, 34.8, 34.4, 32.5, 28.6, 25.1. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for
C35H34N3O4 560.2549; Found 560.2535.
4.8. General procedure G for the synthesis of compounds 4c, 5
To a solution of bromobenzene (0.15 mmol, 1 equiv.) in DMF (0.5 mL), 1-octyne (1.1
equiv.), Pd[(PPh3)Cl]2 (10 mol%), Et3N (3 equiv.) and CuI (40 mol%) were added.
The resulting mixture was stirred at r. t. After the reaction was completed (TLC), the
solution was concentrated in vacuo and filtered through a pad of silica gel with
EtOAc/PE = 1:4 to afford alkyne intermediate that was used in the next step without
additional purification. To a suspension of protected intermediate (1 equiv.) in EtOH
(5 mL), hydrazine hydrate (0.1 mL) and 10% Pd/C (5 mg) was added and the mixture
was refluxed overnight. Solvent was evaporated and product purified by column
chromatography.
4.8.1.
2-Amino-7-octyl-3-(((2S*,5R*)-5-phenyltetrahydrofuran-2-
yl)methyl)quinazolin-4(3H)-one (5)
1
Prepared from bromobenzene 16g [29]. Yield 32 mg (66%), colorless oil. H NMR
(400 MHz, CDCl3) δ: 0.87 (t, J = 7.0 Hz, 3H), 1.63-1.39 (m, 10H), 1.60-1.70 (m, 2H),
1.84-1.96 (m, 2H), 2.19-2.41 (m, 2H), 2.66 (t, J = 7.0 Hz, 2H), 4.18 (dd, J = 7.1, 14.7
Hz, 1H), 4.33-4.42 (m, 1H), 4.65 (dd, J = 1.6, 14.8 Hz, 1H), 4.87-4.91 (m, 1H), 5.58
(brs, 2H), 7.04 (dd, J = 1.6, 8.1 Hz, 1H), 7.12 (d, J = 1.0 Hz, 1H), 7.22-7.38 (m, 5H),
8.03 (d, J = 8.1 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ: 10.1, 22.6, 28.8, 29.2, 29.4,
30.9, 31.9, 33.3, 36.2, 46.4, 79.8, 82.0, 115.0, 123.5, 124.2, 126.0, 126.9, 127.9,

128.6, 141.4, 149.0, 150.4, 153.0, 163.0. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd
for C27H36N3O2 434.2808; Found 434.2805.
4.8.2.
rac-2-Amino-7-octyl-3-((tetrahydrofuran-2-yl)methyl)quinazolin-4(3H)-one
(4c)
Prepared from bromobenzene 16f [29]. Yield 15 mg (25%), white amorphous solid.
1H NMR (400 MHz, CDCl3) δ: 8.01 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 1.5 Hz, 1H), 7.03
(dd, J = 8.2, 1.6 Hz, 1H), 5.71 (brs, 2H), 4.47 (d, J = 14.4 Hz, 1H), 4.27 – 4.10 (m,
2H), 3.98 – 3.84 (m, 1H), 3.84 – 3.71 (m, 1H), 2.67 (t, J = 7.5 Hz, 2H), 2.22 – 2.07
(m, 1H), 2.04 – 1.82 (m, 2H), 1.75 – 1.49 (m, 3H), 1.35 – 1.19 (m, 10H), 0.87 (t, J =
7.2 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ: 163.1, 153.3, 150.6, 149.1, 127.1, 124.3,
123.6, 115.1, 79.7, 68.6, 46.5, 36.4, 32.0, 31.0, 29.6, 29.4, 28.9, 25. 9, 22.8, 14.3.
HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C21H32N3O2 358.2495; Found
358.2493.
4.9. General procedure H for the synthesis of compounds 2b; 3b; 6a-b
To a solution of iodobenzene (0.15 mmol, 1 equiv.) in DMF (1 mL), Et3N (3 equiv.),
t-butyl acrylate (1.3 equiv.) and Pd(PPh3)4 (5 mol%) were added. The resulting
mixture was heated at 80 °C. After the reaction was completed (TLC), the solution
was cooled to r. t., and then partitioned between H2O and EtOAc. The organic layer
was concentrated in vacuo and product washed with MeOH to afford protected
intermediate that was used in the next step. To a suspension of protected intermediate
(1 equiv.) in DCM (5 mL), TFA (0.1 mL) added and mixture stirred at r. t. for 16 h.
Then reaction mixture concentrated in vacuo, EtOH (10 mL) and hydrazine hydrate
(0.1 mL), 10 % Pd/C (5 mg) was added and the mixture was refluxed for 6 h. Solvent
was evaporated, water was added resulting product filtered and washed with MeOH
or purified by column chromatography.
4.9.1.
rac-3-(2-Amino-4-oxo-7-phenyl-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-5-yl)propanoic acid (2b)
1
Prepared from iodobenzene 16a. Yield 16 mg (36%), white amorphous solid. H
NMR (400 MHz, DMSO-d6) δ: 1.55-1.64 (m, 1H), 1.73-1.82 (m, 1H), 1.83-2.01 (m,
2H), 2.52 (t, J = 7.6 Hz, 2H), 3.37 (t, J = 7.6 Hz, 2H), 3.59-3.64 (m, 1H), 3.76-3.81

(m, 1H), 4.06-4.17 (m, 3H), 7.27 (m, 3H), 7.32-7.49 (m, 4H), 7.69 (d, J = 7.0 Hz,
2H), 11.99 (brs, 1H). 13C NMR (101 MHz, DMSO-d6) δ: 25.5, 29.0, 30.9, 35.6, 45.5,
67.8, 76.6, 113.1, 119.0, 123.9, 127.4, 128.9, 129.5, 139.3, 144.3, 145.5, 152.6, 152.6,
161.5, 174.4. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C22H24N3O4 394.1767;
Found 394.1770.
4.9.2.
3-(2-Amino-4-oxo-7-phenyl-3-(((2S*,5R*)-5-phenyltetrahydrofuran-2-
yl)methyl)-3,4-dihydroquinazolin-5-yl)propanoic acid (3b)
Prepared from iodobenzene 16d. Yield 29 mg (97%), white solid. Tm = 124 – 126 oC.
1H NMR (400 MHz, DMSO-d6) δ: 1.67-1.82 (m, 2H), 2.01-2.13 (m, 1H), 2.20-2.30
(m, 1H), 2.44 (m, 2H), 3.37 (t, J = 7.43 Hz, 2H), 4.20-4.35 (m, 3H), 4.74-4.78 (m,
1H), 6.80 (brs, 2H), 7.17 (d, J =1.6 Hz, 1H), 7.20-7.23 (m, 2H), 7.29 (t, J = 7.0 Hz,
2H), 7.35-7.38 (m, 3H), 7.44 (t, J = 7.0 Hz, 2H), 7.66 (d, J = 7.4 Hz, 2H), 8.28 (brs,
1H). 13C NMR (101 MHz, DMSO-d6) δ: 28.7, 30.6, 33.8, 35.4, 45.1, 76.5, 80.9,
113.1, 120.1, 122.6, 125.8, 126.8, 127.1, 128.1, 128.9, 139.2, 142.3, 143.6, 144.5,
151.7, 152.3, 161.7, 174.1. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C28H28N3O4
470.2080; Found 470.2094.
4.9.3. rac-3-(2-Amino-4-oxo-7-(4-pentylphenyl)-3-((tetrahydrofuran-2-yl)methyl)-3,4-
dihydroquinazolin-5-yl)propanoic acid (6a)
1
Prepared from iodobenzene 16c. Yield 28 mg (46%), white amorphous solid. H
NMR (400 MHz, DMSO-d6) δ: 11.98 (brs, 1H), 7.62 (d, J = 7.7 Hz, 2H), 7.34 – 7.12
(m, 4H), 6.77 (brs, 2H), 4.22 – 4.04 (m, 3H), 3.89 – 3.77 (m, 1H), 3.70 – 3.60 (m,
1H), 3.44 – 3.35 (m, 2H), 2.67 – 2.59 (m, 2H), 2.03 – 1.75 (m, 4H), 1.67 – 1.55 (m,
13
4H), 1.39 – 1.24 (m, 4H), 0.92 – 0.82 (m, 3H). C NMR (101 MHz, DMSO-d6) δ:
174.1, 161.8, 152.4, 151.8, 144.6, 143.4, 142.6, 136.6, 128.9, 126.8, 122.5, 119.9,
112.9, 76.5, 67.4, 45.0, 35.4, 34.7, 30.9, 30.57, 30.56, 28.6, 25.1, 22.0, 13.9. HRMS
(ESI/TOF-Q) m/z: [M + H]+ Calcd for C27H34N3O4 464.2549; Found 464.2540.
4.9.4.
rac-3-(2-Amino-4-oxo-7-(4-(3-phenylpropyl)phenyl)-3-((tetrahydrofuran-2-
yl)methyl)-3,4-dihydroquinazolin-5-yl)propanoic acid (6b)
1
Prepared from iodobenzene 16b. Yield 24 mg (48%), white amorphous solid. H
NMR (400 MHz, DMSO-d6) δ: 11.78 (brs, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.31 – 7.22
(m, 5H), 7.21 – 7.15 (m, 4H), 6.74 (brs, 2H), 4.15 – 4.03 (m, 3H), 3.79 (q, J = 7.2 Hz,

1H), 3.64 – 3.55 (m, 1H), 3.36 (t, J = 7.6 Hz, 2H), 2.61 (m, 4H), 2.54 – 2.48 (m, 2H),
13
1.96 – 1.78 (m, 4H), 1.62 –1.53 (m, 2H). C NMR (101 MHz, DMSO-d6) δ: 174.1,
161.8, 152.4, 151.8, 144.5, 143.4, 142.2, 141.9, 136.7, 129.0, 128.3, 126.8, 125.7,
122.5, 119.9, 112.9, 112.6, 76.5, 67.4, 45.0, 35.4, 34.8, 34.4, 32.5, 30.6, 28.6, 25.1.
HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C31H34N3O4 512.2549; Found
512.2549.
4.10. Procedure
I
for the synthesis rac-3-(2-Amino-7-octyl-4-oxo-3-
((tetrahydrofuran-2-yl)methyl)-3,4-dihydroquinazolin-5-yl)propanoic acid (6c)
Procedure I: To a solution of iodobenzene 16e (323 mg, 1 equiv.) in DMF (10 mL),
K2CO3 (262 mg, 3 equiv.), t-butyl acrylate (0.111 mL, 1.2 equiv.) and
Pd(dppf)Cl2*DCM (50 mg, 10 mol%) were added. The resulting mixture was heated
at 100°C. After the reaction was completed, the solution was cooled to r. t., and
partitioned between 5% aq. KHSO4 and DCM. The organic layer was concentrated in
vacuo and alkene intermediate isolated by column chromatography (EtOAc/DCM/PE
= 1/1/4). To a solution of alkene intermediate (130 mg, 1 equiv.) in DMF (0.5 mL), 1-
octyne (0.045 mL, 1.2 equiv.), Pd(Amphos)Cl2 (3.6 mg, 2 mol%) and Cs2CO3 (249
o
mg, 3 equiv.) were added. The resulting mixture was stirred at 100 C for 2 days.
Then solution was concentrated in vacuo and mixture separated by column
chromatography EtOAc/PE = 1/8 to afford protected alkyne intermediate that was
dissolved in DCM (10 mL), TFA (0.1 mL) added and mixture stirred at r. t. for 48 h.
Then reaction mixture concentrated in vacuo, EtOH (5 mL), hydrazine hydrate (0.5
mL) and 10% Pd/C (5 mg) was added and the mixture was refluxed for 3 days.
Solvent was evaporated and product 6c washed with wet MeOH, filtered. Yield 19 mg
1
(44%), white amorphous solid. H NMR (400 MHz, CDCl3) δ: 9.12 (brs, 1H), 7.39
(brs, 2H), 7.02 (s, 1H), 6.90 (s, 1H), 4.47 (d, J = 14.5 Hz, 1H), 4.22 – 4.14 (m, 1H),
4.03 – 3.97 (m, 1H), 3.95 – 3.87 (m, 1H), 3.82 – 3.74 (m, 1H), 3.51 (t, J = 7.4 Hz,
2H), 2.70 (t, J = 7.8 Hz, 2H), 2.59 (t, J = 7.8 Hz, 2H), 2.23 – 2.09 (m, 1H), 1.92 (p, J
= 6.9 Hz, 2H), 1.67 – 1.53 (m, 3H), 1.35 – 1.15 (m, 10H), 0.85 (t, J = 6.8 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ: 178.6, 161.8, 153.9, 150.2, 147.9, 143.7, 126.8, 119.9,
112.0, 79.2, 68.5, 46.6, 36.5, 36.0, 31.8, 31.2, 30.8, 29.4, 29.3, 29.2, 28.9, 25.7, 22.6,
14.1. HRMS (ESI/TOF-Q) m/z: [M + H]+ Calcd for C24H36N3O4 430.2706; Found
430.2702.

4.11. Biological assays
4.11.1. Bioassay
A fluorescence resonance energy transfer (FRET) assay was performed to
evaluate ability of compounds to inhibit PlmI,II,IV, and CatD. Km of the substrate was
determined for each enzyme: PlmII = 2 ± 0.2 μM; PlmI = 2.7 ± 0.3μM; PlmIV = 2.8 ±
0.2μM; CatD = 1.8 ± 0.2 μM. A solution of compounds for testing (concentration
0.01−100 μM) on 96-well plate was added to the enzyme (PlmI,II,IV, or CatD) in
buffer (0.1 M NaOAc, pH = 4.5, 10% glycerol). The mixture was incubated for 30
min at 37 °C. Substrate (DABCYL-Glu-Arg-Nle-Phe-Leu-Ser-Phe-Pro-EDANS,
AnaSpec Inc.) was then added to reach a final concentration of 5 μM. Hydrolysis of
the substrate was detected as an increase in fluorescence (Em 490 nm, Ex 336 nm) at
37 °C. The data points were collected every 1 min within 8−15 min. For the rate
calculation, only the linear interval was used, which was slightly different for each
enzyme. Compounds were tested in triplicate experiments. IC50 values were
calculated using software Graph Pad Prism 5.0. Pepstatin A (IC50 = 0.42 ±0.02 nM
(Plm II); IC50 = 0.9 ±0.02 nM (Plm I); IC50 = 0.3 ±0.04 nM (Plm IV)) and resveratrol
(IC50 = 138 μM) were used as positive controls.
4.12. Computational studies
4.12.1. Docking
The crystal structure of Plm II and 1a complex (PDB ID 4z22) was prepared for
docking using the protein preparation wizard tool in Maestro 11.3.016 [30]. Hydrogen
atoms were added, and bond orders were assigned. Hydrogen bond networks were
optimized by choosing optimal orientations of hydroxyl groups, water molecules, and
amide groups of Asn and Gln and by selecting appropriate states and orientations of
histidine imidazoles. Inhibitors were prepared for docking using the standard protocol
implemented in LigPrep. The stereochemistry of the THF moiety’s 2- and 5-carbon
atoms was R and S, respectively [29]. The ligand and protein protonation states were
adjusted to pH 4.6, and Asp214 was [29, 31] protonated as this generally provided
better docking poses.
Docking was performed using the standard precision protocol in Glide [32]. Docked
complexes were further refined with Prime MM-GBSA (Molecular
Mechanics/Generalized Born Surface Area) method [33] using VSGB solvation
model [34] and OPLS3 force field [35]. For MM-GBSA calculations, a 7-Å active