Discovery of novel quinazoline-2,4(1H,3H)-dione derivatives as potent PARP-2 selective inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.05.052

The PARP-2 selective inhibitor is important for clarifying specific roles of PARP-2 in the pathophysiological process and developing desired drugs with reduced off-target side effects. In this work, a series of novel quinazoline-2,4(1H,3H)-dione derivatives was designed and synthesized to explore isoform selective PARP inhibitors. As a result, compound 11a (PARP-1 IC₅₀?=?467?nM, PARP-2 IC₅₀?=?11.5?nM, selectivity PARP-1/PARP-2?=?40.6) was disclosed as the most selective PARP-2 inhibitor with high potency to date. The binding features of compound 11a within PARP-1 and PARP-2 were investigated respectively to provide useful insights for the further construction of new isoform selective inhibitors of PARP-1 and PARP-2 by using CDOCKER program.

Full text of DOI:10.1016/j.bmc.2017.05.052

Accepted Manuscript
Discovery of novel quinazoline-2,4(1H,3H)-dione derivatives as potent PARP-2
selective inhibitors
Hailong Zhao, Ming Ji, Guonan Cui, Jie Zhou, Fangfang Lai, Xiaoguang Chen,
Bailing Xu
PII:
S0968-0896(17)30707-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.052
BMC 13770
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
2 April 2017
22 May 2017
24 May 2017
Please cite this article as: Zhao, H., Ji, M., Cui, G., Zhou, J., Lai, F., Chen, X., Xu, B., Discovery of novel
quinazoline-2,4(1H,3H)-dione derivatives as potent PARP-2 selective inhibitors, Bioorganic & Medicinal
Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.05.052
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Discovery of novel quinazoline-2,4(1H,3H)-dione derivatives as
potent PARP-2 selective inhibitors
Hailong Zhaoa,#, Ming Jib,#, Guonan Cuia, Jie Zhoua, Fangfang Laib, Xiaoguang Chenb,*, Bailing
Xua,*
aBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences＆Peking Union Medical College, Beijing,
100050, China
bState Key Laboratory of Bioactive Substances and Functions of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences＆Peking Union Medical
College, Beijing, 100050, China
Abstract: The PARP-2 selective inhibitor is important for clarifying specific roles of
PARP-2 in the pathophysiological process and developing desired drugs with reduced
off-target side effects. In this work, a series of novel quinazoline-2,4(1H,3H)-dione
derivatives was designed and synthesized to explore isoform selective PARP
inhibitors. As a result, compound 11a (PARP-1 IC50 = 467 nM, PARP-2 IC50 = 11.5
nM, selectivity PARP-1/PARP-2 = 40.6) was disclosed as the most selective PARP-2
inhibitor with high potency to date. The binding features of compound 11a within
PARP-1 and PARP-2 were investigated respectively to provide useful insights for the
further construction of new isoform selective inhibitors of PARP-1 and PARP-2 by
using CDOCKER program.
Keywords: Quinazoline-2,4(1H,3H)-dione; PARP-2 selective inhibitor; Anti-tumor agents.
1. Introduction
ADP-ribosyltransferases (ARTs) represent a large family of enzymes containing
17 members at least, and they can modify their target proteins by ADP-ribosylating.
These ARTs bind to nicotinamide adenine dinucleotide (NAD+) in their catalytic
domain, cleave the NAD+ and transfer ADP-ribose moiety onto specific amino acid
residues of acceptor proteins1-3. Six members (ARTs 1-6) of this family can function
as poly(ADP-ribose)polymerases2-4 to transfer multiple ADP-ribose moieties
consecutively and form the linear or branched ADP-ribose polymers on the target
proteins. PARP-1 was first identified in 1963 and its role in the DNA repair pathway
5
was well explored4,
Presently, as PARP-1 inhibitors, Olaparib (AZD-2281),
Rucaparib (AG014699) and Niraparib (MK4827) have been approved by FDA for the
#The first two authors contributed equally.
*Corresponding author. B. Xu, xubl@imm.ac.cn; X. Chen, chxg@imm.ac.cn

treatment of ovarian cancer in patients with or without BRCA mutations6-8. In the late
1990’s9, 10 , PARP-2 was confirmed as the second member of this family. In fact, the
detection of residual DNA-dependent PARP activity in PARP-1-/- deficient mouse
fibroblasts led to the discovery of PARP-2. PARP-1 and PARP-2 were the closest
homologs and they possessed 69% similarity in the catalytic domain10. Therefore,
many initially recognized PARP-1 inhibitors such as AZD-2281, ABT-888,
AG014699, MK-4827 and BMN673 also showed comparable PARP-2 inhibitory
activities, since these inhibitors were designed to compete with NAD+ in the catalytic
domain 6-8, 11-14
While both PARP-1 and PARP-2 were involved in the DNA breaks repair
.
1, 15-17
,
their specific roles in the repair pathway have not been well understood. The delayed
and persistent accumulation at UV laser-induced damaged cells15 demonstrated that
PARP-2 participated in later steps of the DNA repair process. Moreover, while
PARP-1 can bind with both single-strand breaks and double-strand breaks18, PARP-2
can bind with single-strand breaks with greater specificity. Meanwhile, the
biochemical evidence proved that PARP-2 took part in many other physiological
processes, such as spermatogenesis, adipogenesis, and T-cell development15, 19-21
.
Depletion of PARP-2 in mice led to chronic anemia which was not detected in mice
lacking PARP-122. Consequently, it was speculated that PARP-1 selective inhibitors
might work as anti-cancer drugs, bearing less off-target side effects. Interestingly,
PARP-2 has been proved very recently to possess pleiotropic influences on the
hallmarks of cancer, such as genomic instability, dysregulated cellular metabolism,
angiogenesis, inflammation, tumor invasion and immune evasion23. So, the selective
inhibition of PARP-2 might cause a multipronged attack on tumorigenesis. Obviously,
the development of PARP-1 selective inhibitors and PARP-2 selective inhibitors are
highly desired to clarify the biological functions of individual PARPs and to achieve
the improved therapeutic agents compared with the known non-selective inhibitors.
By now, several PARP-1 selective inhibitors have been reported24-27. In contrast,
only one inhibitor (compound I) was described as the most selective PARP-2 inhibitor
with a 9.3-fold selectivity and an IC50 value of 1.5 µM against PARP-228.
Figure 1.The chemical structures of known PARP-2 selective inhibitors (I, II), and the general
structure of designed compounds
In our previous work, we found that a quinazoline-2,4(1H,3H)-dione derivative
(compound II, Figure 1)29 was about 15-fold more potent against PARP-2 with the
IC50 value at nanomolar level. The predicated binding mode showed that the

quinazoline-2,4(1H,3H)-dione scaffold occupied the nicotinamide-ribose binding site
(NI site) and the N-Boc-pyrrolidin-3-yl fragment extended into the adenine-ribose
binding site (AD site)29. The benzyl group served as a spacer to direct those fragments
into the NI site and AD site, and interacted with the key amino acids in PARP-1, such
as Tyr889, Tyr896 and Gly894 and PARP-2, such as Tyr455, Tyr462 and Gly460. By
comparison with the highly conserved NI site, the AD site in PARP-1 and PARP-2
differs from each other to some extent. For example, the AD site in PARP-1 consists
of Glu763, Asp766 and Leu769 amino acid residues, whereas the corresponding
amino acid residues in PARP-2 were Gln332, Glu335 and Gly338. The differentiation
of amino acid residues in PARP-1 and PARP-2 could be useful for the design of
isoform selective inhibitors30, 31. Based on the above structural features of PARP-1
and PARP-2, we envisioned that modifications on the spacer and the
N-Boc-pyrrolidin-3-yl subunit of compound II could tune their interactions with the
key amino acids in the spacer site and AD site, and therefore might create novel
isoform selective inhibitors. Herein, we present the synthesis of the designed
compounds and their enzymatic inhibitory activities against PARP-1 and PARP-2. The
structure-activity relationships were investigated preliminarily and led to the
discovery of a highly potent and selective PARP-2 inhibitor (Compound 11a).
2. Chemistry

Scheme 1 Reagents and conditions: (a) urea, 160 °C, 76.1%; (b) HMDS, conc. H2SO4, toluene, reflux; (c)
NBS, AIBN, CCl4, 28.0%-41.5%; (d) substituted bromomethylbenzene, 130 °C; (e) MeOH, dioxane, 70 °C,
27.6%-97.0%; (f) Raney-Ni, H2, THF, MeOH, 53.6%-93.0%; (g) PPh3, hexachloroacetone, THF, 22.4%-70.0%;
(h)TFA, DCM, 71.4%-88.2%; (i) 10% Pd-C, H2, THF, MeOH, 62.0%; (j) NaBH3CN, NaOAc, DCM, MeOH or
Pd2dba3, Xantphos, Cs2CO3, Tol, 5.6%-62.2%.
The synthesis of quinazoline-2,4(1H,3H)-dione derivatives (9a-9i, 10a-10g
and11a-11i) was accomplished according to the synthetic route as outlined in Scheme
1. The condensation of 2-aminobenzoic acid with urea delivered compound 232 under
a neat reaction condition. Upon treatment with hexamethyldisilazane (HMDS) and
concentric sulfuric acid in toluene, compound 2 was converted into the silylated
compound 3; then, compound 3 reacted with compounds 5a-5c and 5e-5f, which were
prepared by treating 4a-4c and 4e-4f with N-bromobutanimide and
azodiisobutyronitrile in CCl4, to generate the N-1 substituted intermediates 6a-6c and
6e-6f. Removal of the silyl group in 6a-6c and 6e-6f in MeOH gave rise to
compounds 7a-7c, 7e-7f and 7h in 27%-97% yields. The catalytic hydrogenation of
compounds 7a-7c, 7e-7f and 7h provided the corresponding amines 8a-8c, 8e-8f and
8h in 53%-93% yields. In addition, compound 8d was prepared from 7b and
compound 8g was prepared from 6g. In the presence of PPh3 and hexachloroacetone,
the coupling reaction between 8a-8h with N-Boc-β-proline yielded compounds 9a-9h
in a yield of 22%-70%. Under the reaction conditions of Raney-Ni and H2 in THF,
compound 9g was transformed into compound 9i in 53% chemical yield. The
deprotection of Boc group of compounds 9a-9g with TFA produced the target
compounds 10a-10g in good yields. The alkylation reaction of 10a afforded 11a-11i in
the reasonable yields.
3. Biological results and discussion
The inhibitory activities of all target compounds (9a-9i, 10a-10g and 11a-11i)
were evaluated against PARP-1 and PARP-2. The clinical drug AZD2281 was
selected as a reference compound. The corresponding results were expressed as IC50
values and presented in Table 1 and Table 2.
Initially, modifications on the benzyl spacer were carried out by changing atom
X and substituent Y in order to investigate the impact of these variations on the
inhibition and selectivity toward PARP-1 and PARP-2. As shown in Table 1, the
variation of Y substituent on the pyridine spacer led to the formation of compounds

9a-9d. Compounds 9a and 9b with a fluorine or a chlorine atom on the pyridine ring
displayed a stronger inhibition toward PARP-2 over PARP-1 (9a, PARP-1 IC50 = 39.8
nM, PARP-2 IC50 = 8.6 nM, selectivity PARP-1/PARP-2 = 4.6; 9b, PARP-1 IC50 =
232 nM, PARP-2 IC50 = 12.2 nM, selectivity PARP-1/PARP-2 = 19.0), showing a
4.6-fold and 19-fold selectivity, respectively. In terms of potency and selectivity, these
two compounds are comparable with compound II. By comparison, using a hydrogen
atom or a bromine atom as the Y substituent (compounds 9c and 9d) led to a drastic
decrease in potency against both PARP-1 and PARP-2.
The use of benzyl group as a spacer along with the further variation of Y group
yielded compounds 9e-9i. Compound 9e with a chlorine atom exhibited the similar
inhibitory activities toward PARP-1 and PARP-2 with IC50 values in the single digit
nanomolar level. The introduction of a bromine atom as the Y group (compound 9f)
lowered the potency toward both PARP-1 and PARP-2, and favored the binding
toward PARP-2 with a 5.1-fold selectivity. The incorporation of other substituents
(compounds 9g-9i), such as the nitro, amine and hydroxyl groups into the benzyl ring
resulted in the loss of potency.
Taken together, the phenyl group or pyridine ring could be taken as the spacer,
which was supposed to interact with the tyrosine residues around it via π-π stacking
interactions. The incorporation of a fluorine or a chlorine atom into the spacer could
generate highly potent PARP-2 inhibitors (compounds 9a, 9b, 9e and compound II)
with IC50 values at the nanomolar level. In contrast, regarding the inhibition toward
PARP-1, only the fluorine substituted compound 9a and compound II had the
comparable potency with IC50 at the double digit nanomolar level. The installation of
many other substituents was not tolerated on the Y position.
As shown in Table 1, the removal of Boc group of compounds 9a-9g furnishing
compounds 10a-10g generally reduced the inhibitory activity against PARP-2
markedly and had little impact on the PARP-1 inhibition. These results suggested that
the incorporation of a variety of substituents on the nitrogen could be beneficial for
PARP-2 potency and consequently might produce PARP-2 selective inhibitors.
Surprisingly, the removal of Boc group on compound 9d increased the inhibition
toward PARP-1 and gave rise to a PARP-1 selective inhibitor (compound 10d).
Table 1
The chemical structures and inhibitory activities against PARP-1 and PARP-2 of compounds 9a–9i
and 10a-10g.a,b,c

PARP-1
IC50/nM
±SD
PARP-2
IC50/nM
±SD
PARP-1
IC50/nM
±SD
PARP-2
IC50/nM
±SD
Selectivity
PARP-1/2
Selectivity
PARP-1/2
Cpd.
X
Y
Cpd.
X
Y
N
N
N
N
C
C
C
C
C
F
Cl
4.6
19.0
--
N
N
N
N
C
C
C
F
Cl
0.18
4.4
--
9a
9b
9c
9d
9e
9f
39.8±11.6
8.6±1.7
10a
10b
10c
10d
10e
10f
13.7±11.3 80±20.4
232±80.4 12.2±1.8
174±13.4
>100
40±9.4
>100
Br
>100
>100
>100
>100
Br
H
H
--
0.04
--
9.9±0.9
>100
220±85
>100
Cl
0.7
5.1
--
Cl
6.2±0.1
346±45
>100
9.1±2.1
68±21.5
>100
Br
Br
NO2
>100
>100
--
NO2
OH
NH2
>100
>100
--
9g
9h
9i
10g
>100
>100
--
>100
>100
--
a Concentration for 50% inhibition in PARP-1 enzyme assay (IC50); IC50 for AZD-2281 was 8.1 nM.
b Concentration for 50% inhibition in PARP-2 enzyme assay (IC50); IC50 for AZD-2281 was 1.7 nM.
cThe IC50 value was the average of two or three independent experiments; SD, standard deviation.
Based on the results mentioned above, we chose compound 9a as a template to
probe the SAR of the N-substituents and to search for the isoform selective inhibitors
by changing the Boc group with other various subunits. As shown in Table 2,
substitution of Boc group with 2,2,2-trifluoroethyl moiety led to a significant drop in
potency toward PARP-1 and a little variation in potency toward PARP-2, thus
resulting in a highly selective PARP-2 inhibitor (compound 11a) with about 40-fold
selectivity. To the best of our knowledge, compound 11a showed the highest
selectivity toward PARP-2 over PARP-1 with high potency as compared with the
known PARP-2 selective inhibitors. Although grafting other alkyl hydrophobic groups
such as a trifluoropropyl, cyclopropylmethyl or cyclopropylethyl moiety onto the
nitrogen of the pyrrolidine ring could not prodcuce the selective inhibition between
PARP-1 and PARP-2 at all, these compounds (11b-11d) served as highly potent
inhibitors of PARP-1 and PARP-2 with IC50 values at low nanomolar level. The
placement of a 4,4-difluorocyclohexyl (11e) or cyclopropanecarbonyl substituent (11f)
on the nitrogen atom produced a moderate selectivity toward PARP-2 over PARP-1.
Also, we attempted the installation of aromatic groups on the nitrogen atom, and
found that compounds (11g-11i) showed remarkable inhibition toward PARP-2 with
IC50 values of 4.1 nM-5.8 nM and less potency against PARP-1 (IC50, 13.3 nM-92.6
nM). Among these compounds examined, compound 11i exhibited a strong potency
and favorable selectivity (PARP-1/PARP-2 = 22.8) toward PARP-2. The chemical
modifications on the benzene ring of compound 11i may further improve the isoform
selectivity.
Table 2
The chemical structures and inhibitory activities against PARP-1 and PARP-2 of compounds
11a-11i.a,b,c

PARP-1
IC50/nM
±SD
PARP-2
IC50/nM
±SD
PARP-1
IC50/nM
±SD
PARP-2
IC50/nM
±SD
Selectivity
PARP-2/1
Selectivity
PARP-2/1
Cpd.
R
Cpd.
R
11.5±
40.6
1.0
12.0
2.5
11a
467±33.4
3.1±0.1
11f
54±1.3
4.5±0.9
5.4±0.8
0.9
11b
11c
2.9±0.2
3.6±1.0
1.0±0.2
11g
11h
11i
13.3±2.5
1.6
10.7
22.8
5.7±0.7
62±10.0
5.8±1.3
1.8
6.8
11d
11e
1.4±0.5
81±14.6
92.6±12.0 4.1±0.5
11.9±
2.3
a Concentration for 50% inhibition in PARP-1 enzyme assay (IC50); IC50 for AZD-2281 was 8.1 nM.
b Concentration for 50% inhibition in PARP-2 enzyme assay (IC50); IC50 for AZD-2281 was 1.7 nM.
cThe IC50 value was the average of two or three independent experiments; SD, standard deviation.
With an aim to probe the binding features of the most selective PARP-2 inhibitor
(11a) in the binding site of PARP-1 and PARP-2, we performed the molecular docking
by using CDOCER protocol integrated in Accelrys Discovery Studio 2.533. As shown
in Figure 2(A-C), the quinazolinedione and the pyridine fragments bound to the NI
site of PARP-1 and PARP-2 in a very similar orientation. As anticipated, the
quinazolinedione scaffold formed the crucial interactions with PARP-1 through the
conserved amino acids Gly863, Ser904 and Tyr907 and with PARP-2 through Gly429,
Ser470 and Tyr473. The pyridine spacer resided at the subpocket lined with Tyr889
(Tyr445) and Tyr896 (Tyr462). Although the trifluoroethyl substituted pyrrolidine
ring extended into AD site, its orientation in PARP-1 and PARP-2 was noticeably
different (Figure 2, C-E). Due to the restriction of the amino acid residue Glu763 in
PARP-1 through the hydrogen bonds with neighboring residues, the α5 helix was not
easily shifted away by the induced fit effect of the trifluoroethyl group25. As a

consequence, the trifluoroethyl group stretched into a water-exposed surface and no
hydrogen bonding interactions with Arg878 was observed in PARP-1. However, as
far as PARP-2 is concerned, the Glu763 residue in PARP-1 was replaced with Gln332.
The side chain of Gln332 is more flexible and the α5 helix can be shifted away to
make a favorable accommodation for the trifluoroethyl group. Importantly, the
trifluoroethyl fragment in this binding orientation could form H-bonding interactions
with Arg444 in PARP-2 (Figure 2, F). We speculated that this type of distinct
binding feature of 11a within PARP-2 made significant contributions to its inhibition
toward PARP-2, which consequently resulted in a high selectivity toward PARP-2
over PARP-1.
Figure 2.CDOCKER-modeled binding mode of compound 11a within PARP-1(carbon atoms
colored gold) and PARP-2 (carbon atoms colored blue). (A) The binding interactions of compound
11a within the binding site of PARP-1; (B) The binding interactions of compound 11a within the
binding site of PARP-2; (C) Comparison of the binding poses of 11a in PARP-1 and PARP-2, the
α5 helix shifted away in PARP-2 in comparison with PARP-1; (D) Close-up view of the binding

orientation of the trifluoroethyl group in PARP-1; (E) Close-up view of the binding orientation of
the trifluoroethyl group in PARP-2; (E) The hydrogen bonding network was observed between the
trifluoroethyl moiety and Arg444 in PARP-2. Molecular image was generated with UCSF
Chimera34.
4. Conclusion
In summary, a series of structurally novel quinazoline-2,4(1H,3H)-dione
derivatives were designed and synthesized by varying the spacer and the substituents
on the pyrrolidine ring of our lead compound, which showed selectivity toward
PARP-2 over PARP-1 to some degree. Introducing the pyridine ring into the spacer
improved the structural novelty and conferred this series of inhibitors with distinct
physicochemical properties. Among all the target molecules, three compounds
(11b-11d) strongly inhibited PARP-1 and PARP-2 with IC50 in the single digit
nanomolar level, although no selectivity was observed. Compound 11i possessed the
very potent activity as well as a moderate PARP-2 selectivity. Remarkably, compound
11a was discovered as the most selective PARP-2 inhibitor with high potency for the
present. The molecular docking of 11a with PARP-1 and PARP-2 offered an insight
into its preference for PARP-2 binding. These results will deepen our understanding
on the distinct features of the AD site within PARP-1 and PARP-2, and facilitate to
develop more isoform selective PARP inhibitors.
5. Experimental section
5.1. General
Melting points were measured on a Yanaco micro melting point apparatus and
are uncorrected.1H NMR (300 MHz or 400 MHz) on a Varian Mercury 300 or 400
spectrometer was recorded in DMSO-d6, acetone-d6 or CDCl3. Chemical shifts are
reported in δ (ppm) units relative to the internal standard tetramethylsilane (TMS).
High resolution mass spectra (HRMS) were obtained on an Agilent Technologies
LC/MSD TOF spectrometer. All chemicals and solvents used were of reagent grade
without purified or dried before use. All the reactions were monitored by thin-layer
chromatography (TLC) on pre-coated silica gel G plates at 254 nm under a UV lamp.
Column chromatography separations were performed with silica gel (200–300 mesh).

5.2. Synthesis of target compounds 9a-9i, 10a-10g, 11a-11i
5.2.1.
(S)-Tert-butyl
3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-yl)ca
rbamoyl)pyrrolidine-1-carboxylate (9a)
A mixture of (3S)-1-(tert-butoxycarbonyl)-3-pyrrolidinecarboxylic acid (70 mg,
0.32 mmol) and triphenylphosphine (170 mg, 0.63 mmol) in dried tetrahydrofuran
(4.0 mL) was stirred under argon and cooled to 0 °C. Hexachloroacetone (85 mg, 0.32
mmol) in dried tetrahydrofuran (2.0 mL) was added dropwise and the mixture was
stirred for 1h.The acyl chloride solution was then treated with a solution of 8a (60 mg,
0.21 mmol) in dried tetrahydrofuran (4.0 mL)dropwise followed by triethylamine (32
mg, 0.32 mmol) in dried tetrahydrofuran (1.0 mL). The reaction mixture was then
allowed to reach room temperature and stirred for 5 h and then the mixture was
evaporated. The crude product was obtained and purified with column
chromatography (petroleum ether/methylene chloride/ethyl acetate = 1:1:1 to
methylene chloride/methanol/tetrahydrofuran = 40:1:1) to give the corresponding
25
product 9a as a light yellow solid (50 mg, 49.5%); mp 212-214 °C; [α]D+22.00
1
(c=0.43, CHCl3:MeOH=10:1); HNMR (400 MHz, CDCl3)δ (ppm): 8.83 (d, J = 9.2
Hz, 1H), 8.63 (s, 1H), 8.23 (d, J = 7.6 Hz, 1H), 7.82 (brs, 1H), 7.63 (t, J = 8.0 Hz, 1H),
7.52 (brs, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 5.33 (s, 2H),
3.55-3.76 (m, 3H), 3.35-3.46 (m, 1H), 3.02-3.12 (m, 1H), 2.16-2.26 (m, 2H), 1.47 (s,
9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 161.72,153.45 (d, J = 234.6 Hz),
153.31, 150.65, 140.53, 135.28, 131.53, 130.79 (d, J = 4.2 Hz),127.70, 122.83, 121.46
(d, J = 27.5 Hz), 119.44, 115.99, 114.79, 78.31, 48.24 (48.14), 45.32 (45.15), 43.71
(42.84), 42.33, 29.12 (28.29), 28.13; HR-MS (ESI): m/z, calcd. for
C24H26N5O5FNa[M+Na]+506.1810, Found: 506.1791.
5.2.2.
(S)-Tert-butyl
3-((2-chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-yl)ca
rbamoyl)pyrrolidine-1-carboxylate (9b)
Following the preparation protocol of compound 9a, starting from 8b (87 mg,
0.29 mmol), the title compound 9b was obtained as a white solid (80 mg, 58.0%); mp
1
25
234-236 °C;[α]D+16.11 (c=0.42, CHCl3:MeOH=10:1); HNMR (400 MHz, DMSO-d6)
δ (ppm): 11.75 (s, 1H), 9.79 (s, 1H), 8.27 (s, 1H), 8.09 (s, 1H), 8.03 (d, J = 7.6 Hz,
1H), 7.67 (t, J = 8.0 Hz, 1H), 7.30 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H),5.35 (s,
2H), 3.46-3.54 (m, 1H), 3.32-3.41 (m, 2H), 3.22-3.29 (m, 2H), 1.96-2.14 (m, 2H),
1.40 (s, 9H); HR-MS (ESI): m/z, calcd. for C24H26N5O5ClNa[M+Na]+522.1515,
Found: 522.1504.

5.2.3.
(S)-Tert-butyl
3-((2-bromo-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-yl)ca
rbamoyl)pyrrolidine-1-carboxylate (9c)
Following the preparation protocol of compound 9a, starting from 8c (90 mg,
0.26 mmol), the title compound 9c was obtained as a white solid (70 mg, 70.0%); mp
1
25
239-241 °C;[α]D+15.13 (c=0.26, CHCl3:MeOH=10:1); HNMR (400 MHz, DMSO-d6)
δ (ppm): 11.75 (s, 1H), 9.76 (s, 1H), 8.27 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.92 (s,
1H), 7.67 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.8Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 5.33 (s,
2H), 3.45-3.54 (m, 1H), 3.31-3.42 (m, 2H), 3.24 (brs, 2H), 1.95-2.15 (m, 2H), 1.39 (s,
9H); HR-MS (ESI): m/z, calcd. for C24H26N5O5BrNa[M+Na]+566.1010, Found:
566.0991.
5.2.4.
(S)-Tert-butyl
3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-yl)carbamoyl)
pyrrolidine-1-carboxylate (9d)
Following the preparation protocol of compound 9a, starting from 8d (77 mg,
0.29 mmol), the title compound 9d was obtained as a white solid (30 mg, 22.4%); mp
163-165 °C; [α] +10.94 (c=0.79, CHCl3:MeOH=10:1); 1HNMR (400 MHz,
25
D
DMSO-d6) δ (ppm): 11.80 (s, 1H), 10.20 (s, 1H), 8.76 (s, 1H), 8.34 (s, 1H), 8.04 (d, J
= 7.6 Hz, 1H), 7.81 (s, 1H), 7.68 (t, J = 8.4 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.27 (t,
J = 7.6 Hz, 1H), 5.34 (s, 2H), 3.45-3.52 (m, 1H), 3.33-3.40 (m, 2H), 3.18-3.28 (m,
1H), 3.09 (brs, 1H), 1.91-2.13 (m, 2H), 1.39 (s, 9H); 13C NMR (100 MHz, DMSO-d6)
δ(ppm):161.74, 153.31, 150.62, 142.91, 140.59, 139.72, 135.76, 135.34, 132.18,
127.69, 123.71, 122.85, 119.44, 115.89, 114.88, 78.31, 48.15 (48.06), 45.33 (45.15),
44.18 (43.30), 42.81, 29.12 (28.28), 28.13; HR-MS (ESI): m/z, calcd. for
C24H28N5O5[M+H]+466.2085, Found: 466.2080.
5.2.5.
(S)-Tert-butyl
3-((2-chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbam
oyl)pyrrolidine-1-carboxylate (9e)
Following the preparation protocol of compound 9a, starting from 8e (100 mg,
0.33 mmol), the title compound 9e was obtained as a white solid (135 mg, 52.0%);
1
25
mp 158-160 °C;[α] D +7.12 (c=0.39, CHCl3:MeOH=10:1); HNMR (400 MHz,
DMSO-d6) δ (ppm): 11.75 (s, 1H), 9.64 (s, 1H), 8.02 (d, J = 7.6 Hz, 1H), 7.63-7.67 (m,
2H), 7.44 (d, J = 8.0 Hz, 1H), 7.23-7.27 (m, 2H), 7.13 (d, J = 8.0 Hz, 1H),5.29 (s, 2H),
3.47-3.54 (m, 1H), 3.33-3.43 (m, 2H), 3.19-3.29 (m, 2H), 1.95-2.15 (m, 2H), 1.40 (s,
9H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 171.34, 161.74, 153.33, 150.62,

140.68, 136.04, 135.21, 134.92, 129.68, 127.62, 126.15, 124.69, 122.76, 115.90,
114.97, 78.29, 48.23 (48.18), 45.33 (45.16), 44.54, 43.61(42.72), 29.11 (28.33), 28.14;
HR-MS (ESI): m/z, calcd. for C25H27N4O5ClNa[M+Na]+521.1562, Found: 521.1536.
5.2.6.
(S)-Tert-butyl
3-((2-bromo-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carba
moyl)pyrrolidine-1-carboxylate (9f)
Following the preparation protocol of compound 9a, starting from 8f (100 mg,
0.29 mmol), the title compound 9f was obtained as a white solid (90 mg, 57.0%); mp
1
25
163-165 °C;[α]D+7.84 (c=0.1, CHCl3:MeOH=10:1); HNMR (400 MHz, DMSO-d6) δ
(ppm): 11.75 (s, 1H), 9.61 (s, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.0 Hz, 1H),
7.60 (d, J = 8.0 Hz, 1H), 7.51 (brs, 1H), 7.23-7.27 (m, 2H), 7.08 (d, J = 8.0 Hz, 1H),
5.28 (s, 2H), 3.46-3.55 (m, 1H), 3.34-3.43 (m, 2H), 3.14-3.29 (m, 2H), 1.96-2.16 (m,
2H), 1.40 (s, 9H); HR-MS (ESI): m/z, calcd. for C25H27N4O5BrNa[M+Na]+565.1057,
Found: 565.1030.
5.2.7.
(S)-Tert-butyl
3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-nitrophenyl)carbamo
yl)pyrrolidine-1-carboxylate (9g)
Following the preparation protocol of compound 9a, starting from 8g (200 mg,
0.64 mmol), the title compound 9g was obtained as a gray solid (90 mg, 27.6%); mp
1
25
142-144 °C;[α]D+5.48 (c=0.15, CHCl3:MeOH=10:1); HNMR (400 MHz, DMSO-d6)
δ (ppm): 10.62 (s, 1H), 8.92 (s, 1H), 8.69 (s, 1H),8.24 (d, J = 8.0 Hz, 1H), 8.19 (d, J =
8.8 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 7.26-7.31 (m, 1H), 7.03 (d, J = 8.4 Hz, 2H),
5.40 (s, 2H), 3.54-3.83 (m, 3H), 3.45 (brs, 1H), 3.16 (brs, 1H), 2.21-2.30 (m, 2H),
1.48 (s, 9H); HR-MS (ESI): m/z, calcd. for C25H27N5O7Na[M+Na]+532.1803, Found:
532.1797.
5.2.8.
(S)-Tert-butyl3-((5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-hydrox
yphenyl)carbamoyl)pyrrolidine-1-carboxylate (9h)
Following the preparation protocol of compound 9a, starting from 8h (80 mg,
0.28 mmol), the title compound 9h was obtained as a gray solid (80 mg, 59.0%); mp
1
25
208-210 °C;[α]D+6.35 (c=0.15, CHCl3:MeOH=10:1); HNMR (400 MHz, DMSO-d6)
δ (ppm): 11.71 (s, 1H), 9.76 (s, 1H), 9.32 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.74 (s,
1H), 7.64 (t, J = 8.0 Hz, 1H), 7.20-7.30 (m, 2H), 6.88 (d, J = 8.0 Hz, 1H), 6.80 (d, J =
8.4 Hz, 1H),5.18 (s, 2H), 3.49 (t, J = 8.4 Hz, 1H), 3.16-3.41 (m, 4H), 1.90-2.12 (m,
2H),1.40 (s, 9H); HR-MS (ESI): m/z, calcd. for C25H28N4O6Na[M+Na]+503.1901,

Found: 503.1882.
5.2.9.
(S)-Tert-butyl
3-((2-amino-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)carbam
oyl)pyrrolidine-1-carboxylate (9i)
The mixture of compound 9g (60 mg, 0.12 mmol) and Raney nickel (40 mg) in
methanol (8.0 mL) and tetrahydrofuran (8.0 mL) was hydrogenated at room
temperature and 1 atm for 18 h. Tetrahydrofuran (30.0 mL) was added and the Raney
nickel was filtered over a pad of Celiteand, the solution was concentrated to afford the
crude compound. The crude product was purified with column chromatography
(DCM/MeOH = 50:1-30:1) to afford compound 9i as a white solid (30mg, 53.6%);
1
25
mp 149-151 °C;[α] D +8.40 (c=0.13, CHCl3:MeOH=10:1); HNMR (400 MHz,
DMSO-d6) δ (ppm):11.68 (s, 1H), 9.26 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.65 (t, J =
7.6 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.23 (t, J = 7.2 Hz, 1H), 7.15 (s, 1H), 6.89 (d, J
= 7.6 Hz, 1H), 6.68 (d, J = 8.0 Hz, 1H), 5.14 (brs, 2H), 4.82 (brs, 2H), 3.51 (t, J = 9.2
Hz, 1H),3.34-3.41 (m, 2H), 3.07-3.28 (m, 2H), 1.93-2.14 (m, 2H), 1.40 (s, 9H);
HR-MS (ESI): m/z, calcd. for C25H30N5O5[M+H]+480.2242, Found: 480.2245.
5.2.10.
(S)-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-yl
)pyrrolidine-3-carboxamide2,2,2-trifluoroacetate(10a)
To a stirred solution of 9a (45 mg, 0.09 mmol) in DCM (4.0 mL) was added TFA
(0.5 mL) dropwise, the reaction mixture was then allowed to stir at room temperature
overnight. DCM and excessive TFA were then removed under reduced pressure. The
crude product was purified by crystallization from a solvent mixture of methyl alcohol
and diethyl ether to afford compound 10a as a yellow solid (34 mg, 77.3%); mp
1
25
205-207 °C; [α]D+3.11 (c=0.26, MeOH); H NMR (400 MHz, DMSO-d6) δ (ppm):
11.76 (s, 1H), 10.23 (s, 1H), 8.84 (brs, 2H), 8.37 (d, J = 9.2 Hz, 1H), 8.09 (s, 1H),
8.03 (d, J = 7.6 Hz, 1H), 7.68 (t, J = 8.0 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.27 (t, J =
7.6 Hz, 1H), 5.34 (s, 2H), 3.34-3.45 (m, 3H), 3.14-3.24 (m, 2H), 2.16-2.28 (m, 1H),
1.97-2.08 (m, 1H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 171.64, 161.77, 158.30
(q, J = 31.4 Hz), 153.41 (d, J = 234.4 Hz), 150.66, 141.55, 140.54, 140.10 (d, J = 14.9
Hz), 135.37, 132.49 (q, J = 245.3 Hz), 131.22, 130.87 (d, J = 4.1 Hz), 127.73, 122.92,
121.36 (d, J = 27.7 Hz), 115.95, 114.82, 46.78, 44.90, 42.50, 42.31, 28.80; HR-MS
(ESI): m/z, calcd. for C19H19N5O3F [M+H]+384.1466, Found: 384.1456.

5.2.11.
(S)-N-(2-Chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-
yl)pyrrolidine-3-carboxamide2,2,2-trifluoroacetate(10b)
Following the preparation protocol of compound 10a, starting from 9b (40 mg,
0.08 mmol), the title compound 10b was obtained as a white solid (30 mg, 77.0%);
1
25
mp 207-209 °C; [α]D +12.76 (c=0.19, MeOH); HNMR (400 MHz, DMSO-d6) δ
(ppm): 11.76 (s, 1H), 9.99 (s, 1H), 8.82 (brs, 2H), 8.35 (s, 1H), 8.01-8.06 (m, 2H),
7.67 (t, J = 8.0 Hz, 1H), 7.33 (d, J = 8.8 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H),5.36 (s, 2H),
3.32-3.44 (m, 3H),3.20 (t, J = 7.2 Hz, 2H), 2.21-2.29 (m, 1H), 2.01-2.09 (m, 1H);
HR-MS (ESI): m/z, calcd. for C19H19N5O3Cl [M+H]+400.1171, Found: 400.1169.
5.2.12.
(S)-N-(2-Bromo-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-y
l)pyrrolidine-3-carboxamide2,2,2-trifluoroacetate(10c)
Following the preparation protocol of compound 10a, starting from 9c (40 mg,
0.07 mmol), the title compound 10c was obtained as a white solid (33 mg, 84.6%);
1
25
mp 201-202 °C; [α]D +13.01 (c=0.33, MeOH); HNMR (400 MHz, DMSO-d6) δ
(ppm): 11.76 (s, 1H), 9.98-10.00 (m, 1H), 8.88 (brs, 2H), 8.35 (s, 1H), 8.03 (d, J = 7.6
Hz, 1H), 7.87 (s, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H),7.27 (t, J = 7.6
Hz, 1H), 5.34 (s, 2H), 3.37 (brs, 3H), 3.16-3.26 (m, 2H), 2.19-2.30 (m, 1H), 2.01-2.12
(m, 1H); HR-MS (ESI): m/z, calcd. for C19H19N5O3Br [M+H]+444.0666, Found:
444.0651.
5.2.13.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)pyridin-3-yl)pyrroli
dine-3-carboxamide2,2,2-trifluoroacetate(10d)
Following the preparation protocol of compound 10a, starting from 9d (15 mg,
0.03 mmol), the title compound 10d was obtained as a white solid (10 mg, 71.4%);
1
25
mp 143-145 °C; [α]D+5.59 (c=0.14, MeOH); HNMR (400 MHz, DMSO-d6) δ (ppm):
11.80 (s, 1H), 10.40 (s, 1H), 8.81 (brs, 2H), 8.74 (s, 1H), 8.42 (s, 1H), 8.04 (d, J = 8.0
Hz, 1H), 7.81 (s, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.27 (t, J =
7.6 Hz, 1H), 5.36 (s, 2H), 3.32-3.41 (m, 2H), 3.15-3.27 (m, 3H), 2.14-2.26 (m, 1H),
1.98-2.08 (m, 1H); HR-MS (ESI): m/z, calcd. for C19H20N5O3 [M+H]+366.1561,
Found: 366.1553.

5.2.14.
(S)-N-(2-Chloro-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyr
rolidine-3-carboxamide2,2,2-trifluoroacetate(10e)
Following the preparation protocol of compound 10a, starting from 9e (40 mg,
0.08 mmol), the title compound 10e was obtained as a white solid (32 mg, 82.0%);
1
25
mp 223-225 °C; [α]D+2.52 (c=0.32, MeOH); H NMR (400 MHz, DMSO-d6) δ (ppm):
11.76 (s, 1H), 9.86 (s, 1H), 8.93 (s, 2H), 8.03 (d, J = 7.6 Hz, 1H), 7.65 (t, J = 8.0 Hz,
1H), 7.57 (s, 1H), 7.48 (d, J = 8.0 Hz, 1H),7.18-7.30 (m, 3H), 5.30 (s, 2H), 3.34-3.43
(m, 3H), 3.15-3.27 (m, 2H), 2.20-2.31 (m, 1H), 1.99-2.11 (m, 1H); HR-MS (ESI): m/z,
calcd. for C20H20N4O3Cl[M+H]+ 399.1218, Found: 399.1206.
5.2.15.
(S)-N-(2-Bromo-5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)phenyl)pyr
rolidine-3-carboxamide2,2,2-trifluoroacetate(10f)
Following the preparation protocol of compound 10a, starting from 9f (35 mg,
0.06 mmol), the title compound 10f was obtained as a pink solid (30 mg, 88.2%); mp
1
25
145-147 °C; [α]D+3.24 (c=0.21, MeOH); HNMR (400 MHz, DMSO-d6) δ (ppm):
11.75 (s, 1H), 9.83 (s, 1H), 8.84 (brs, 2H), 8.03 (d, J = 8.0 Hz, 1H), 7.65 (t, J = 8.4 Hz,
2H), 7.45 (d, J = 2.0 Hz, 1H), 7.22-7.28 (m, 2H), 7.16 (d, J = 8.4 Hz, 1H), 5.28 (s,
2H), 3.33-3.41 (m, 3H), 3.15-3.25 (m, 2H), 2.21-2.31 (m, 1H), 2.02-2.14 (m, 1H);
HR-MS (ESI): m/z, calcd. for C20H20N4O3Br [M+H]+443.0713, Found: 443.0702.
5.2.16.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-nitrophenyl)pyrr
olidine-3-carboxamide2,2,2-trifluoroacetate(10g)
Following the preparation protocol of compound 10a, starting from 9g (40 mg,
0.08 mmol), the title compound 10g was obtained as a yellow solid (32 mg, 82.5%);
1
25
mp 147-149 °C; [α]D +21.57 (c=0.21, MeOH); HNMR (400 MHz, DMSO-d6) δ
(ppm): 11.82 (s, 1H), 10.60 (s, 1H), 8.86 (brs, 2H), 8.05 (d, J = 8.0 Hz, 1H), 7.95 (d, J
= 8.8 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.4 Hz, 1H), 7.20-7.34 (m, 3H),
5.41 (s, 2H), 3.11-3.41 (m, 5H), 2.16-2.26 (m, 1H), 1.97-2.05 (m, 1H); HR-MS (ESI):
m/z, calcd. for C20H20N5O5 [M+H]+410.1459, Found: 410.1440.

5.2.17.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-y
l)-1-(2,2,2-trifluoroethyl)pyrrolidine-3-carboxamide (11a)
To a stirred solution of 10a (80 mg, 0.16 mmol) in tetrahydrofuran (6.0 mL)
were added Et3N (98
mg, 1.01 mmol) and 2,2,2-trifluoroethyl
trifluoromethanesulfonate (198 mg, 0.81 mmol). The reaction mixture was heated at
36 °C for 12 h and then was evaporated. After concentration, the crude product was
obtained and purified with column chromatography (methylene chloride/methanol =
50:1-30:1) to give compound 11a as a white solid (36 mg, 46.8%); mp 260-262°C;
1
25
[α]D +2.27 (c=0.41,CHCl3:MeOH=10:1); H NMR (400 MHz, DMSO-d6) δ (ppm):
11.75 (s, 1H), 9.92 (s, 1H), 8.40 (dd, J1 = 9.6 Hz, J2 = 2.4 Hz, 1H), 8.03 (dd, J1 = 7.6
Hz, J2 = 1.6 Hz, 1H), 7.99 (brs, 1H), 7.65-7.71 (m, 1H), 7.36 (d, J = 8.4 Hz, 1H),
7.24-7.29 (m, 1H), 5.33 (s, 2H), 3.14-3.31 (m, 3H), 3.01 (t, J = 8.8 Hz, 1H), 2.72-2.84
(m, 2H), 2.64-2.71 (m, 1H), 1.89-2.05 (m, 2H); HR-MS (ESI): m/z, calcd. for
C21H20N5O3F4[M+H]+466.1497, Found: 466.1486.
5.2.18.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-y
l)-1-(3,3,3-trifluoropropyl)pyrrolidine-3-carboxamide (11b)
Following the preparation protocol of compound 11a, starting from 10a (90mg,
0.18mmol) and 3,3,3-trifluoropropyl trifluoromethanesulfonate (234mg, 0.94mmol),
the title compound 11b was obtained as a white solid (56mg, 62.2%); mp 236-238 °C;
[α] +1.78 (c=0.41,CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ
25
D
(ppm):11.77 (s, 1H), 9.92 (s, 1H), 8.41 (d, J = 9.2 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H),
7.99 (s, 1H), 7.65-7.72 (m, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.27 (t, J = 7.2 Hz, 1H),
5.34 (s, 2H), 3.14 (brs, 1H), 2.83 (t, J = 8.0 Hz,1H), 2.55-2.68 (m, 5H), 2.38-2.48 (m,
2H), 1.89-2.04 (m, 2H);13C NMR (150 MHz, DMSO-d6) δ(ppm): 174.24, 162.21,
153.77 (d, J = 233.85 Hz), 151.12, 141.01, 139.85 (d, J = 14.25 Hz), 135.78, 131.53,
131.26 (d, J = 3.6 Hz), 128.18, 127.50 (q, J = 274.95 Hz), 123.33, 122.20 (d, J =
27.45 Hz), 116.45, 115.28, 57.24, 53.65, 47.99 (d, J = 2.7 Hz), 43.46, 42.82, 32.60 (q,
J = 26.4 Hz), 28.24; HR-MS (ESI): m/z, calcd. for C22H22N5O3F4[M+H]+480.1653,
Found: 480.1650.
5.2.19.
(S)-1-(Cyclopropylmethyl)-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)meth
yl)-2-fluoropyridin-3-yl)pyrrolidine-3-carboxamide (11c)
To a suspension of 10a (100 mg,0.21 mmol) in dichloromethane (4.0 mL),

cyclopropanecarbaldehyde (49 mg, 0.63 mmol), sodium acetate (84 mg, 1.04 mmol),
andmethanol (1.0 mL) were added. The resulting solution was stirred at 37 °C for 10
h. Then sodium cyanoborohydride (42 mg, 0.63 mmol) was addedand the mixture was
stirred for 8 h. The dichloromethane (30 mL) and methanol (3 mL) were added and
washed with saturated aqueous sodium bicarbonate (10 mL×2)and brine (10 mL×2),
dried over anhydrous magnesium sulfate, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (methylene
chloride/methanol = 50:1-30:1) to give the title compound 11c (45 mg, 45.9%);
1
25
yellow-green solid; mp 215-217 °C; [α]D +3.31 (c=0.23,CHCl3:MeOH=10:1); H
NMR (400 MHz, DMSO-d6) δ (ppm): 11.76 (brs, 1H), 10.02 (s, 1H), 8.44 (d, J = 7.6
Hz, 1H), 8.03 (dd, J1= 7.6 Hz, J2= 1.2 Hz, 1H), 7.97 (s, 1H), 7.65-7.71 (m, 1H), 7.36
(d, J = 8.4 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 3.05-3.15 (m, 1H), 2.82 (t, J
= 8.4 Hz,1H), 2.53-2.65 (m, 3H), 2.22-2.33 (m, 2H), 1.87-2.04 (m, 2H), 0.79-0.89 (m,
1H), 0.40-0.48 (m, 2H), 0.06-0.12 (m, 2H);13C NMR (100 MHz, DMSO-d6) δ
(ppm):174.07, 161.73, 153.15 (d, J = 233.8 Hz), 150.64, 140.53, 139.15 (d, J = 15.2
Hz), 135.30, 130.78 (d, J = 4.2 Hz), 127.70, 122.84, 121.83 (d, J = 27.3 Hz), 115.96,
114.80, 59.68, 56.95, 53.20, 43.09, 42.33, 27.62, 9.71, 3.62, 3.52; HR-MS (ESI): m/z,
calcd. for C23H25N5O3F [M+H]+438.1936, Found: 438.1919.
5.2.20.
(S)-1-(2-Cyclopropylethyl)-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)meth
yl)-2-fluoropyridin-3-yl)pyrrolidine-3-carboxamide (11d)
Following the preparation protocol of compound 11a, starting from 10a (96 mg,
0.2 mmol) and 2-cyclopropylethyl trifluoromethanesulfonate (220mg, 1.0mmol), the
title compound 11d was obtained as a white solid (42mg, 46.7%); mp 197-199 °C;
[α] +4.73 (c=0.34,CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ
25
D
(ppm):11.76 (s, 1H), 10.04 (s, 1H), 8.40 (dd, J1 = 9.2 Hz, J2 = 2.0 Hz, 1H), 8.01 (dd,
J1 = 7.6 Hz, J2 = 1.6 Hz, 1H), 7.99 (s, 1H), 7.64-7.70 (m, 1H), 7.36 (d, J = 8.4 Hz,
1H), 7.26 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 3.28-3.47 (m, 1H), 3.17 (s, 1H), 2.52-2.94
(m, 5H), 1.88-2.11 (m, 2H), 1.32-1.42 (m, 2H), 0.62-0.73 (m, 1H), 0.32-0.42 (m, 2H),
0.04-0.06 (m, 2H); HR-MS (ESI): m/z, calcd. for C24H27N5O3F [M+H]+452.2092,
Found: 452.2091.
5.2.21.
(S)-1-(4,4-Difluorocyclohexyl)-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)
methyl)-2-fluoropyridin-3-yl)pyrrolidine-3-carboxamide (11e)
Following the preparation protocol of compound 11c, starting from 10a (100 mg,
0.21 mmol) and 4,4-difluorocyclohexanone(84 mg, 0.63 mmol), the title compound
25
11e was obtained as a white solid (46 mg, 44.2%); mp 189-191 °C; [α]D+2.20 (c=0.42,
CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.76 (s, 1H), 9.97 (s,

1H), 8.43 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.6 Hz, 1H), 7.98 (s, 1H), 7.68 (t, J = 7.6
Hz, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 3.10 (brs, 1H),
2.81 (brs, 1H), 2.55-2.69 (m, 3H), 2.23 (s, 1H), 1.72-2.09 (m, 8H), 1.57 (brs, 2H); 13C
NMR (100 MHz, DMSO-d6) δ(ppm):173.94, 161.73, 150.64, 140.54, 139.14 (d, J =
15.0 Hz),135.30, 130.80 (d, J = 4.4 Hz), 127.70, 122.85, 121.65 (d, J = 27.5 Hz),
119.45, 115.96, 114.82, 58.60, 54.43, 50.73, 43.06, 42.34, 30.56 (t, J = 24.2 Hz),
27.68, 27.05; HR-MS (ESI): m/z, calcd. for C25H27N5O3F3[M+H]+502.2060, Found:
502.2085.
5.2.22.
(S)-1-(Cyclopropanecarbonyl)-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)
methyl)-2-fluoropyridin-3-yl)pyrrolidine-3-carboxamide (11f)
To a stirred solution of 10a (90 mg, 0.19 mmol) in methylene chloride (15.0 mL),
Et3N (49 mg, 0.47 mmol) and cyclopropanecarbonyl chloride (23 mg, 0.21 mmol)
were added at 0°C. The reaction mixture was then allowed to stir at room temperature
overnight. The dichloromethane(30 mL) and methanol(3 mL) were added and washed
with water (10 mL×2)and brine (10 mL×2), dried over anhydrous magnesium sulfate,
and concentrated under reduced pressure. The residue was purified by silica gel
column chromatography (methylene chloride/methanol = 50:1-30:1) to give the title
25
compound 11f (49 mg, 57.69%); light yellow solid;mp 208-210 °C; [α]D +49.3
(c=0.5,CHCl3:MeOH=10:1);1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H),
10.12 (s, 0.5H), 10.09 (s, 0.5H),8.42 (d, J = 9.6 Hz, 1H), 8.01 (t, J = 7.6 Hz, 2H),
7.64-7.71 (m, 1H), 7.36 (d, J = 8.8 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H),
3.84-3.90 (m, 0.5H), 3.67-3.78 (m, 1H), 3.55-3.66 (m, 1H), 3.36-3.48 (m, 1.5H),
3.21-3.30 (m, 1H), 2.05-2.26 (m, 1.5H), 1.91-2.00 (m, 0.5H), 1.69-1.72 (m, 1H),
0.67-0.74 (m, 4H); HR-MS (ESI): m/z, calcd. for C23H23N5O4F [M+H]+452.1729,
Found: 452.1718.
5.2.23.
(S)-1-Benzyl-N-(5-((2,4-dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropy
ridin-3-yl)pyrrolidine-3-carboxamide (11g)
Following the preparation protocol of compound 11c, starting from 10a (100 mg,
0.21 mmol) and benzaldehyde(68 mg, 0.63 mmol), the title compound 11g was
25
obtained as a white solid (53 mg, 54.1%); mp 218-220 °C; [α]D+34.65 (c=0.18,
CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.76 (s, 1H), 9.88 (s,
1H), 8.41 (dd, J1= 9.6 Hz, J2= 2.4 Hz, 1H), 8.03 (dd, J1= 7.6 Hz, J2= 1.6 Hz, 1H),
7.97 (brs, 1H), 7.66-7.70 (m, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.21-7.34 (m, 6H), 5.33 (s,
2H), 3.50-3.65 (m, 2H), 3.07-3.18 (m, 1H), 2.78 (t, J = 8.4 Hz, 1H), 2.52-2.62 (m,
2H), 2.42-2.49 (m, 1H), 1.92-2.02 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ
(ppm):173.86, 161.78, 152.09,150.68, 140.56, 139.31 (d, J = 17.1 Hz), 135.34, 131.02,

130.80 (d, J = 4.1 Hz),128.56, 128.17, 127.73, 126.88, 122.88, 121.77(d, J = 27.3 Hz),
115.99, 114.84, 59.08, 56.88, 53.31, 43.06, 42.35, 27.56; HR-MS (ESI): m/z, calcd.
for C26H25N5O3F [M+H]+474.1936, Found: 474.1916.
5.2.24.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-y
l)-1-(pyridin-4-ylmethyl)pyrrolidine-3-carboxamide (11h)
Following the preparation protocol of compound 11c, starting from 10a (100 mg,
0.21 mmol) and iso-nicotinaldehyde (72 mg, 0.63 mmol), the title compound 11h was
25
obtained as a light yellow solid (52 mg, 52.5%); mp 167-169 °C; [α]D+36.20
(c=0.44,CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.77 (s, 1H),
9.88 (s, 1H), 8.50 (d, J = 5.6 Hz, 2H), 8.41 (d, J = 8.0 Hz, 1H), 8.03 (d, J = 6.8 Hz,
1H), 7.99 (s, 1H), 7.68 (t, J = 7.2 Hz, 1H), 7.37 (d, J = 8.8 Hz, 1H), 7.33 (d, J = 5.6
Hz, 2H), 7.27 (t, J = 7.6 Hz, 1H), 5.33 (s, 2H), 3.56-3.70 (m, 2H), 3.18 (p, J = 7.6 Hz,
1H), 2.81 (t, J = 8.4 Hz, 1H), 2.54-2.63 (m, 3H), 1.94-2.06 (m, 2H); 13C NMR (100
MHz, DMSO-d6) δ(ppm): 173.68,168.51, 161.73, 159.75, 150.64, 149.49, 140.53,
139.39 (d, J = 14.1 Hz), 135.30, 131.19, 130.79, 127.70, 123.48, 122.85, 121.69 (d, J
= 27.2 Hz),115.96, 114.80, 57.76, 56.82, 53.39, 43.02, 42.31, 27.67; HR-MS (ESI):
m/z, calcd. for C25H24N6O3F [M+H]+475.1888, Found: 475.1885.
5.2.25.
(S)-N-(5-((2,4-Dioxo-3,4-dihydroquinazolin-1(2H)-yl)methyl)-2-fluoropyridin-3-y
l)-1-phenylpyrrolidine-3-carboxamide (11i)
To a stirred solution of 10a (200 mg, 0.52 mmol) in anhydrous toluene (3.0 mL)
were added Pd2dba3 (72 mg, 0.08mmol), Xantphos (132 mg, 0.24 mmol),
bromobenzene (250 mg, 1.56 mmol) and Cs2CO3 (676 mg, 2.08mmol). The reaction
mixture was heated by MW (100 W, 300 min, 120 °C, 150 psi) and then was
evaporated. After concentration, the crude product was obtained and purified with
column chromatography (methylene chloride/methanol/tetrahydrofuran= 50:1:1) to
25
give compound 11i as a light yellow solid (10 mg, 5.6%); mp 252-254 °C; [α]D+17.34
(c=0.44, CHCl3:MeOH=10:1); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.76 (s, 1H),
10.10 (s, 1H), 8.44 (dd, J1= 9.6 Hz, J2= 2.0 Hz, 2H),7.97-8.04 (m, 2H), 7.64-7.71 (m,
1H), 7.37 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.12-7.19 (m, 2H), 6.60 (t, J =
7.2 Hz, 1H), 6.54 (d, J = 8.0 Hz, 2H), 5.33 (s, 2H), 3.21-3.53 (m, 5H), 2.11-2.30 (m,
2H); HR-MS (ESI): m/z, calcd. for C25H23N5O3F [M+H]+460.1779, Found: 460.1772.
5.3. The assay for PARP-1 and PARP-2 inhibition
Plasmid pET32a-PARP1 was a gift from Prof. Satoh (Canada). Human recombinant

PARP1/2 were expressed and purified as described35. The ability of compounds to
inhibit PARP1/2 enzyme activity were tested using ELISA method and the chemical
quantitation of NAD+ method as described35,36. IC50 values were calculated using
GraphPad Prism 5 software.
5.4. Computational studies
All molecular computation studies were performed using CDOCER protocol
integrated in Accelrys Discovery Studio Client 2017 (Accelrys Software Inc., San
Diego, CA). The co-crystal structures of NMS118-PARP-1 complex (PDB ID: 5A00)
and NMS118-PARP-2 complex (PDB ID: 4ZZY) were chosen for molecular
modeling. The docking protocol of compound 11a with PARP-1 was chosen as a
representative. Using Prepare Protein tool of DS, the water molecules in protein were
removed and the protein were added hydrogens, corrected the incomplete residues and
refined with CHARMm force field. To define the binding site more rationally, we
superimpose 5WRQ (the co-crystal structure of 7TX-PARP1 complex) onto 5A00 and
then choose 7TX as the center to construct the binding site within 13 Å. Compound
11awas minimized using Prepare Ligands tool of DS and refined with CHARMm
force field. Then it was docked into the prepared PARP-1 and PARP-2 protein with
CDOCKER using the default parameters, respectively. The 40 final docked
conformations were ranked according to their binding free energy. The docking mode
was chosen on the basis of binding rationality.
Acknowledgment
This work is supported by National Natural Science Foundation of China (No. 81673300), the
PUMC Graduate Innovation Fund (No. 2015-1007-10) and PUMC Innovation Fund (No.
2016-12M-3-008).
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Graphical Abstract:
Discovery of novel quinazoline-2,4(1H,3H)-dione derivatives as potent PARP-2
selective inhibitors
Hailong Zhao, Ming Ji, Guonan Cui , Jie Zhou, Fangfang Lai, Xiaoguang Chen , Bailing Xu
A
series of novel quinazoline-2,4(1H,3H)-dione
derivatives was prepared and their inhibitory
activities were evaluated against both PARP-1 and
PARP-2. Compound 11a was identified as the highly
potent and selective PARP-2 inhibitor.