Characterization and structure-activity relationship study of iminodipyridinopyrimidines as novel hepatitis C virus inhibitor

Article abstract of DOI:10.1016/j.ejmech.2017.09.010

Upon high-throughput screening of synthetic small molecule libraries with the infectious hepatitis C virus (HCV) cell culture system, we identified an iminodipyridinopyrimidine (IDPP) scaffold. IDPP did not inhibit HCV replication, but exhibited very potent inhibitory activity on early and late steps of HCV life cycle. Applying an intensive structure-activity relationship (SAR) study, a promising IDPP Lead compound (12c) with excellent potency (EC₅₀ = 10 nM), high safety margin (SI > 2000), and an acceptable stability in human and rat liver microsomes (t_1/2 >60 min) was identified. Overall, our results suggest that the IDPP scaffold could be used for the development of novel HCV interventions.

Full text of DOI:10.1016/j.ejmech.2017.09.010

Accepted Manuscript
Characterization and structure-activity relationship study of iminodipyridinopyrimidines
as novel hepatitis C virus inhibitor
Dong-Sik Park, Eunji Jo, Jihyun Choi, MyungEun Lee, Soohyun Kim, Hee-Young Kim,
Jiyon Nam, Sujin Ahn, Jong Yeon Hwang, Marc Peter Windisch
PII:
S0223-5234(17)30692-X
10.1016/j.ejmech.2017.09.010
EJMECH 9725
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 April 2017
Revised Date: 23 August 2017
Accepted Date: 5 September 2017
Please cite this article as: D.-S. Park, E. Jo, J. Choi, M. Lee, S. Kim, H.-Y. Kim, J. Nam, S.
Ahn, J.Y. Hwang, M.P. Windisch, Characterization and structure-activity relationship study of
iminodipyridinopyrimidines as novel hepatitis C virus inhibitor, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.09.010.
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ACCEPTED MANUSCRIPT
Graphical Abstract

Characterization and structure-activity relationship study of iminodipyridinopyrimidines as novel
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hepatitis C virus inhibitor
Dong-Sik Parka,#, Eunji Job,#, Jihyun Choia, MyungEun Leeb, Soohyun Kimb, Hee-Young Kimb, Jiyon Namc,
Sujin Ahnc, and Jong Yeon Hwanga,d,*, Marc Peter Windischb,*
aMedicinal Chemistry Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea;
bApplied Molecular Virology, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea;
cDMPK Group, Institut Pasteur Korea, Seongnam-si, Gyeonggi-do, Republic of Korea;
dBio&Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon
305-600, Republic of Korea
#These authors equally contributed to this work.
*Corresponding authors. Email: jyhwang@krict.re.kr and marc.windisch@ip-korea.org
Abstract
Upon high-throughput screening of synthetic small molecule libraries with the infectious hepatitis C virus
(HCV) cell culture system, we identified an iminodipyridinopyrimidine (IDPP) scaffold. IDPP did not
inhibit HCV replication, but exhibited very potent inhibitory activity on early and late steps of HCV life
cycle. Applying an intensive structure-activity relationship (SAR) study, a promising IDPP Lead compound
(12c) with excellent potency (EC50 = 10 nM), high safety margin (SI >2,000), and an acceptable stability in
human and rat liver microsomes (t1/2 >60 min) was identified. Overall, our results suggest that the IDPP
scaffold could be used for the development of novel HCV interventions.
1. Introduction

About 3% of the global population is chronically infected with the hepatitis C virus (HCV) which is a major
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cause of chronic liver diseases potentially leading to hepatocellular carcinoma (HCC) [1]. For decades the
standard-of-care (SOC) was a combination of pegylated interferon-alpha (PEG-IFNα) and ribavirin (RBV)
associated with an unsatisfactory sustained virologic response rate (SVR) of only 70-80 % accompanied
with partially severe side effects [2-5]. With the recent FDA approval of direct-acting antiviral agents
(DAAs) such as sofosbuvir, daclatasvir, etc. powerful drugs to tackle chronic hepatitis C (CHC) are now
available for therapy [6, 7]. However, all drugs exclusively interfere with viral replication whereas drugs
targeting other steps of the HCV life cycle have not been developed yet. Utilizing the infectious HCV cell
culture (HCVcc) system, which enabled to study the entire viral life cycle, led to the identification of small
molecules, such as, green-tea polyphenolepigallocatechin-3-gallate (EGCG), HCV II-1, flunarizine, etc.
shown to interfere with glycoprotein E1 and E2 mediated HCV entry[8-13]. Targeting entry tackles a viral
infection at its initial step prior to the multiplication of the virus genomic material which may reduce the
emergence of viral drug resistance. Furthermore, HCV entry inhibitors are predestinated of preventing
reinfection of transplanted livers and of inhibiting the vertical transmission of the virus.
In this context, we set up the infectious HCV genotype 2 cell culture system and screened small molecule
compound libraries to identify new antiviral agents with a novel mechanism of action (MoA) [14]. In order
to enrich for hits with a novel MoA, we excluded hits active in the HCV replicon system, thereby removing
replication inhibitors which are dominant drug candidates in clinical development. Furthermore, selected
hits were evaluated and characterized in a series of secondary assays and to identify the putative molecular
target. From this rational drug discovery approach, we identified an iminodipyridinopyrimidine (IDPP)
scaffold very potently inhibiting the HCV life cycle, which encouraged us to explore further the potential of
IDPPs as a novel anti-HCV agent by conducting a structure-activity relationship (SAR) study. Here we
report the identification, synthesis and SAR study of IDPPs.
2. Results and discussion
2.1. High throughput screening campaign and hit selection.

More than 200,000 small molecules were screened at 6 µM concentration against the entire HCV life cycle.
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Hits were selected according >70% HCV inhibition and >70% cell viability criteria and confirmed by 10-
point dose-response curve (DRC) analysis in the high throughput screening (HTS) assay. Thereby, the half
maximal effective concentration against entirety inhibit the entire HCV life cycle (EC50), as well as the
cytotoxic concentration (CC50), was determined to calculate selectivity index (SI) values (CC50/EC50).
Confirmed active hits with an SI >10 were triaged in HCV pseudoparticle (HCVpp) and replicon (HCVrp)
systems to determine inhibitory effects on viral entry and viral RNA replication, respectively. Following this
approach, we identified a series of IDPPs with potent anti-HCV activity in the infectious cell culture system
(Figure 1). The representative IDPP 1 exhibited strong inhibitory activity against HCVcc without any
cytotoxicity (Figure 2A). The preliminary SAR analysis with IDPP analogs, screened with DRC analysis in
the primary HTS assay, revealed that hydrophilic or short alkyl substitution such as ethyl, propyl, isopropyl,
morpholine, or methoxyethyl on either R2 or R3 of IDPPs showed very weak or no inhibitory HCV activity
(data not shown). IDPP 1 was shown to be inactive in the HCV replicon system (EC50 >30 µM) (Figure 2B).
Interestingly, IDPP inhibited specifically E1/E2-mediated viral entry as shown by experiments with HCVpp,
whereas vesicular stomatitis virus pseudoparticle (VSVpp) were not inhibited (Figure 2C). Furthermore,
utilizing a virus supernatant transfer assay, it was demonstrated that IDPP interfered with the secretion of
infectious HCV particle (data not shown). Taken together, these results demonstrate that IDPP has a novel
MoA by interfering with the early and late steps of the HCV life cycle which encouraged us to conduct an
in-depth SAR study with the goal to move from hit towards Lead.
Figure 1. The representative structure of IDPP (left), active hit IDPP 1 (middle), and inactive IDPP (right).

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Figure 2. Biological activity of IDPP. (A) Evaluation of IDPP 1 in the HCVcc system. Huh-7.5 cells were
seeded in 384-well plate and exposed to compounds at indicated concentrations (0.01, 0.1 and 1 µM) for 2 h
followed by infection of HCVcc. Sofosbuvir, an HCV RNA polymerase inhibitor, was used as a positive
control. Anti-HCV activity was determined at day 3 post infection as described in material and methods. (B)
Evaluation of IDPP 1 in the HCVrp system. Replicon cells were seeded in 384-well plate and treated with
compounds and analyzed as described above. (C) Evaluation of IDPP 1 in the HCVpp system. Huh-7.5 cells
were seeded in 384-well plate and treated with compounds for 2 h followed by transduction with VSVpp or
HCVpp.
2.2. Chemistry.
The representative synthesis of IDPPs was depicted in Scheme 1. The key intermediate chloro-aldehyde 4
were prepared as described elsewhere [15]. Briefly, 2-hydroxypyridopyrimidinones 3 were prepared by
cyclization of 2-aminopyridines 2 and highly reactive bis (2,4,6-trichlorophenyl) malonate. Then 3 were
treated with POCl3 and DMF to afford chloro-aldehyde 4. Treatment of amines for R2 and subsequent 1 N
aqueous HCl hydrolysis gave aldehyde 5. Aldehydes 5 were subjected to Knoevenagel condensation with
ethylcyanoacetate to afford ester IDPPs 6 [16] that were hydrolyzed to carboxylic acids 7 by the treatment of
LiOH. Carboxylic acids 7 were converted to the desired IDPPs 8 by treatment with various amines using
BOP as a coupling agent. IDPPs 9 bearing cyano, carboxamide, and sulfonyl were prepared from
intermediate 5a by treatment of the corresponding nitriles such as malononitrile, cyanoacetamide,
sulfonylacetonitrile, respectively.

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Scheme 1. General synthetic method for IDPP analogs.
Scheme 1. Reagents and conditions: (a) bis(2,4,6-trichlorophenyl) malonate, acetone, rt, 18 h; (b) POCl3,
DMF, 0°C to rt, 2 h; (c) i) R2NH2, TEA, 70°C, 18 h; ii) 1 N aq. HCl/THF (1:3), 70°C, 1 h; (d) i) 2-
methoxyethylamine, MeOH, 50 °C, 18 h; ii) ethylcyanoacetate, CHCl3, 70°C, 18 h; (e) ethylcyanoacetate,
piperidine, molecular sieve, EtOH, rt, 1-2 days; (f) LiOH, H2O, THF, rt, 24 h; (g) R3NH2, BOP, NMM, DMF,
rt, 18 h; (h) nitriles, piperidine, EtOH, rt.
2.3. Structure-activity relationship study
Upon identification of IDPP 1 as an active hit, we set out to understand the key structural elements that were
responsible for anti-HCV inhibitory potency. First, the tolerability of amide in IDPPs by replacement with
several functional groups including ester, acid, nitrile, carboxamide, and sulfone was examined (Table 1).
IDPP 1 showed very potent anti-HCV activity with low cytotoxicity (EC50= 0.007 µM and CC50> 30 µM),

thus providing an excellent SI value (SI >4,285). The nitrile 9a, carboxamide 9b, and sulfone 9c showed at
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least 14-fold reduced potencies (EC50= 0.35, 0.16, 0.1 µM, respectively) compared to 1. Only ester 6a
displayed a slightly reduced activity and increased cytotoxicity (EC50= 0.015 µM and CC50= 17 µM) thus
showing lower SI value (SI= 1,121) compared to 1 (SI >4,286). Carboxylic acids 7a displayed poor anti-
HCV activity (EC50= 6.0 µM).
Table 1. Anti-HCV activity of IDPP 1, 6a, 7a, and 9a-9c.
SI
EC50 (µM)
CC50(µM)
No.
X
(CC50/EC50)
> 4,286
1,121
-CONHBn
-CO2Et
-CO2H
-CN
-CONH2
-SO2Ph
0.007
0.015 ± 0.001
6.0
0.35 ± 0.01
0.16
> 30
17.0 ± 2.12
17.2
1
6a
7a
9a
9b
9c
2.8
> 57
> 125
> 200
> 20
> 20
> 20
0.10 ± 0.03
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate
measurements.
Table 2. Anti-HCV activity of IDPP 10a-10p.
Metabolic stability
SI
(CC50/EC50)
R3R4NH
Human / Rat
(t1/2, min)
EC50 (µM)
CC50(µM)
No.
H2N
0.007
> 30
> 4,286
55/9
1

0.030 ± 0.001
> 6.7
> 222
1.8/1.5
2.8/1.4
8.6/3.6
58/5.6
8a
8b
8c
8d
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0.140 ± 0.01
0.048 ± 0.007
0.105 ± 0.05
8.2 ± 1.9
10.1 ± 5.8
> 10
61
209
> 95
2.053 ± 0.21
0.040 ± 0.008
> 20
> 20
> 9.8
127.7/24
8e
> 445
7,000
8,847
n.d.
8f
8g
8h
0.001 ± 0.0003 7.00 ± 0.95
0.001 ± 0.0003 8.5 ± 0.95
3.0/1.5
4.3/2.0
0.034 ± 0.0009 15.6 ± 1.26
0.004 ± 0.0004 26.6 ± 6.27
459
6,650
45
40/18
15/6
8i
8j
0.190 ± 0.06
8.4 ± 0.33
17.1
1.4/1.3
8k
0.020 ± 0.0003
854
> 60/12.5
8l
0.005 ± 0.004
0.004 ± 0.0008
> 20
> 4,000
> 1,668
34.4/7.5
4.7/1.9
8m
8n
> 6.67
0.003 ± 0.001
0.003 ± 0.002
> 6.67
> 6.67
> 2,223
> 2,223
2.8/2.4
5.3/4.9
8o
8p
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate
measurements. Not determined (n.d.).
Second, the effect of R3R4 substituents in IDPP was examined and the data was summarized in Table 2.
Secondary amides such as N-methyl 8a, isoquinoline 8b, phenylpiperazine 8c, phenylpiperidine 8d, and
morpholine 8e generally reduced the anti-HCV activity (EC50= 0.030-2.1 µM ranges). Particularly,
hydrophilic morpholine substituted IDPP 8e exhibited a drastically reduced potency. The phenyl 8f
displayed weaker inhibitory activity (EC50= 0.040 µM) than benzyl 1. Elongation of benzyl to phenethyl 8g
and phenylpropyl 8h increased the potency (EC50= 0.001 µM), however these modifications also increased
cytotoxicity. Methyl substitution at immediately adjacent to nitrogen atom such as compound 8i and 8j
showed good inhibitory activity (EC50= 0.034 and 0.004 µM, respectively). Interestingly, S-isoform (8j)
with methyl was approx. 9-fold more potent than the R-isoform (8i). Compounds 8k and 8l containing

hydrophilic carbonyl group decreased anti-HCV activity (EC50= 0.19 and 0.020 µM, respectively) in
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comparison with 8 g and 8h. Hydrophobic aliphatic amines including n-butyl 8m, cylcohexylmethyl 8n,
adamantyl 8o, cyclohexyl 8p showed very good inhibitory activity (EC50= 0.004-0.005 µM ranges). In
addition, the metabolic stability of IDPPs was evaluated in human and rat liver microsomal to predict in vivo
pharmacokinetics. Compounds were incubated with liver microsomes and the half-life time (t1/2, min) was
determined. Surprisingly, the majority of compounds displayed poor microsomal stability against either
human or rat (Table 2).
Table 3. Anti-HCV activity of IDPP 10a-10c.
SI
EC50 (µM)
CC50(µM)
No.
R2
(CC50/EC50)
0.004 ±
0.0004
Bn
26.6 ± 6.27
6,650
8j
H
Ph
Inactive
0.42 ± 0.25
> 30
> 20
18.5
-
10a
10b
10c
> 47.6
4,593
CH2-cHexcyl 0.004 ± 0.001
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate
measurements.
Third, the effect of R2 in IDPP was investigated (Table 3). In the primary HTS data analysis hydrophilic
substituents containing oxygen or nitrogen in the R2 group and short aliphatic side chain such as Me, Et, etc.
were inactive. Given our early screening data, three compounds having H (10a), Ph (10b), and
cyclohexylmethyl (10c) were evaluated for their anti-HCV activity. IDPP 10a (R2= H) was inactive and 10b
(R2= Ph) had a significant lower anti-HCV activity (EC50= 0.42 µM). The cyclohexyl methyl compound
(10c) only showed equivalent potency (EC50= 0.004 µM) with 8j.
Table 4. Anti-HCV activity of IDPP 11a-11j.

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Metabolic
stability
Human / Rat
(t1/2, min)
15 / 6
10.7 / 3.6
9.5 / 4.9
7.0 / 4.3
8.47 / 4.4
n.d.
SI
R1
EC50 (µM)
CC50(µM)
No.
(CC50/EC50)
H
0.004 ± 0.0004 26.6 ± 6.27
6650
> 221,385
> 68,172
1,581
8j
8-Me
9-Me
10-Me
10-MeO
8-F
10-F
8,10-F2
8-Cl
10-Cl
9-CF3
0.00009
0.00013
0.004 ± 0.001
> 20
8.69
5.60 ± 0.6
11a
11b
11c
11d
11e
11f
11g
11h
11i
0.002 ± 0.0008 7.47 ± 0.74
3,628
0.002 ± 0.001
0.014 ± 0.015
0.003 ± 0.002
0.001 ± 0.001
0.06 ± 0.023
0.007± 0.0004
> 20
> 20
> 20
> 20
> 20
> 20
> 20,000
> 20,000
> 7,576
> 13,841
> 20,000
> 2,719
n.d.
n.d.
n.d.
n.d.
n.d.
11j
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate
measurements. Not determined (n.d.).
We next studied the substituent effect on R1 group of IDPPs and summarized the data in Table 4.
Interestingly, all of the compounds investigated in the series showed strong inhibitory activity irrespectively
with substituent R1. Particularly, 8-Me (11a) and 9-Me (11b) substituted IDPP exhibited excellent anti-HCV
activity with picomolar range (EC50= 90 pM and 130 pM, respectively). In addition, 11a displayed no
cytotoxicity in the maximum concentration of the assay, resulting in an outstanding SI value (SI >221,385).
However, all compounds substituents on the R1 group had low human and rat microsomal stability. To
overcome this issue, metabolite analysis of IDPP 1 in rat microsomal was performed, and the result
suggested that major metabolites were hydroxylated or debenzylated IDPP as shown in Figure 3.

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Figure 3. Detected metabolites induced by rat microsomes.
On the basis of the evaluation of IDPP analogs toward anti-HCV activity, cytotoxicity, and microsomal
stability, we synthesized additional IDPP analogs to improve the balance between activity and stability.
Among the additional compounds, four IDPP derivatives (12a-d) showing good microsomal stabilities in
both human and rat were summarized in Table 5. As observed earlier in Table 4, compounds substituted with
an 8-methyl at R1 displayed very good anti-HCV activity without cytotoxicity. In addition, blocking of the
potential metabolic labile site with substituents such as methyl, halogen, trifluoromethoxy, and phenyl in R2
or R3 successfully increased microsomal stabilities, thereby providing a promising Lead compound.
Table 5. Anti-HCV activity of IDPP 12a-12d.
CC50
SI
Metabolic
stability
Human /
R2
R3
R1
No.
EC50 (µM)
(CC50/
(µM)

EC50)
Rat
(t1/2, min)
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0.017
8-Me
> 20 > 1,176 > 60 / > 60
12a
± 0.0001
0.030
8-Me
8-Me
> 20 > 2,041 > 60 / > 60
> 20 > 2,000 > 60 / > 60
12b
12c
± 0.48
F
F
0.010
O
F
± 0.0003
0.050
8-Me
> 20 > 2,198 > 60 / > 60
12d
± 0.008
EC50 and CC50 values were determined by 10-point DRC analysis in duplicates with quadruplicate
measurements.
3. Conclusion
In conclusion, a series of iminodipyridinopyrimidines (IDPPs) was identified by a phenotypic HTS assay
using the infectious HCV cell culture system. IDPP 1 showed very potent anti-HCV activity with a favorable
SI value. In addition, IDPPs were shown to be inactive in the HCV replicon system, indicating it has a
distinct MoA from current DAAs which was reported by Lee et al. [17]. These results encouraged us to
perform an SAR study to optimize IDPPs pharmacological properties. Through our intensive SAR study, we
generated a promising IDPP Lead compound (12c) that showed excellent potency (EC50= 10 nM), a good
safety margin (SI >2,000), and an acceptable stability in human and rat liver microsomes (t1/2 >60 min).
4. Experimental section
4.1. Chemistry
All materials were obtained from commercial suppliers and used without further purification. Solvents in
this study were dried using an aluminum oxide column. Thin-layer chromatography was performed on pre-

coated silica gel 60 F254 plates. Purification of intermediates was carried out by normal phase column
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chromatography (MPLC, Silica gel 230-400 mesh). NMR spectra were recorded on a Varian 400 MHz.
LC/MS data were obtained using a Waters 2695 LC and Micromass ZQ spectrometer. Identity of final
compounds was confirmed by proton NMR and mass spectrometry.
4.1.1. The representative procedure for the preparation of compound 5. To a stirred solution of 2-
chloro-4-oxo-4H-pyrido[1,2-a]pyrimidine-3-carbaldehyde (100 mg, 0.479 mmol) in THF (3 mL) was added
benzylamine (0.058 mL, 0.527 mmol) and TEA (0.1 mL, 0.719 mmol). The reaction mixture was stirred at
70°C for 3 h. After the mixture was concentrated, 1 N aqueous HCl solution in THF (1/3 ratio, 4 mL). The
reaction mixture was stirred at 70°C for 1 h. After reaction was completed, the mixture was evaporated and
neutralized to approximately pH 7 by adding 1 N NaOH. The pale solid was collected by filtration and
washed with H2O to give 5a (117 mg, 87%) as a brown solid. 1H NMR (400 MHz, DMSO) δ 10.11 (s, 1H),
9.87 (t, J = 5.6 Hz, 1H), 8.81 (d, J = 8.0 Hz, 1H), 7.98 – 7.94 (m, 1H), 7.39 – 7.32 (m, 5H), 7.29 – 7.25 (m,
1H), 7.18 – 7.14 (m, 1H), 4.80 (d, J = 6.0 Hz, 2H)
4.1.2. The representative procedure for the preparation of compound 6. To a stirred solution of 5a (86
mg, 0.310 mmol) in MeOH (1 mL) was added 2-methoxyethanamine (0.030 mL, 0.341 mmol). The mixture
was stirred at 50°C overnight. After reaction was completed, the mixture was evaporated. To the residue in
chloroform (2 mL) was added ethyl cyanoacetate (0.036 mL, 0.340 mmol) and stirred at 70°C overnight.
After reaction was completed, the reaction mixture was diluted with EtOAc (20 mL) and washed with water
(40 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The crude product
was purified by flash column chromatography to give 6a (97 mg, 84%) as a yellow solid. 1H NMR (400
MHz, DMSO-d6) δ 9.27 (s, 1H), 8.90 (d, J = 6.4 Hz, 1H), 8.47 (s, 1H), 8.05 – 8.00 (m, 1H), 7.58 (d, J = 8.8
Hz, 1H), 7.38 (d, J = 7.4 Hz, 1H), 7.34 – 7.29 (m, 2H), 7.26 (t, J = 7.4 Hz, 2H), 7.20 – 7.17 (m, 1H), 5.67 (s,
2H), 4.29 (q, J = 14.4, 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H); LCMS (electrospray) m/z (M+H)+ 375
4.1.3. The representative procedure for the preparation of compound 7. To a stirred solution of 6a (97
mg, 0.259 mmol) in THF (1 mL), MeOH (0.3 mL), and H2O (0.6 mL) was added lithium hydroxide (62 mg,
2.6 mmol). The mixture was stirred at room temperature overnight. After reaction was completed, the
mixture was evaporated and 1 N HCl (10.0 mL) was added to the residue to neutralize (pH 6). The pale solid

was collected by filtration and washed with H2O to give 7a (63 mg, 70%) as a brown solid. 1H NMR (400
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MHz, DMSO-d6) δ 9.30 (brs, 1H), 9.04 (d, J = 6.4 Hz, 1H), 8.99 (s, 1H), 8.14 – 8.10 (m, 1H), 7.71 (d, J =
8.8 Hz, 1H), 7.45 (t, J = 6.8 Hz, 1H), 7.40 – 7.27 (m, 6H), 5.86 (s, 2H) ); LCMS (electrospray) m/z (M+H)+
347.
4.1.4. The representative procedure for the preparation of compound 1, 8, 10, 11, and 12. To a stirred
solution of 7a (29 mg, 0.56 mmol) in DMF (1 mL) was added N-methylmorpholine (0.014 mL, 0.126 mmol),
benzylamine (0.011 mL, 0.101 mmol) and benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluorophosphate (45 mg, 0.101 mmol). The reaction mixture was stirred at room temperature overnight.
After reaction was completed, the reaction mixture was diluted with EtOAc (10 mL) and washed with
saturated brine (20 mL). The organic layer was dried over anhydrous MgSO4 and concentrated in vacuo. The
1
crude product was purified by flash column chromatography to give 1 (7.0 mg, 20%) as a yellow solid. H
NMR (400 MHz, DMSO-d6) δ 11.53 (t, J = 4.8 Hz, 1H), 9.25 (m, 1H), 9.16 (s, 1H), 8.97 (d, J = 7.2 Hz, 1H),
8.90 (d, J = 6.4 Hz, 1H), 8.10 (s, 1H), 8.03 – 7.96 (m, 2H), 7.77 (s, 1H), 7.61 – 7.55 (m, 1H), 7.39 – 7.17 (m,
7H), 5.65 (s, 2H), 4.46 (m, 2H); LCMS (electrospray) m/z (M+H) + 436
N,1-dibenzyl-2-imino-N-methyl-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8a). Yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 8.88 (d, J = 6.0 Hz, 1H), 7.97 (t, J = 8.0
Hz, 1H), 7.58 (s, 1H), 7.56 (s, 1H), 7.37 – 7.20 (m, 12H), 5.68 (s, 2H), 4.59 (brs, 2H), 2.87 (s, 3H); LCMS
(electrospray) m/z (M+H) + 450
1-benzyl-2-imino-3-(1,2,3,4-tetrahydroisoquinoline-2-carbonyl)-1H-dipyrido[1,2-a:2',3'-d]pyrimidin-
5(2H)-one (8b). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.98 (d, J = 7.0 Hz, 1H), 7.92 (s, 1H), 7.78 (t,
J = 8.8 Hz, 1H), 7.55 (d, J = 8.5 Hz, 1H), 7.44 (d, J = 7.4 Hz, 2H), 7.31 (t, J = 6.8 Hz, 3H), 7.21 - 7.11 (m,
6H), 5.84 (s, 2H), 4.78 (brs, 2H), 3.86 – 3.77 (m, 2H), 2.95 (s, 2H); LCMS (electrospray) m/z (M+H) + 462
1-benzyl-2-imino-3-(4-phenylpiperazine-1-carbonyl)-1H-dipyrido[1,2-a:2',3'-d]pyrimidin-5(2H)-one
(8c). Yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 7.0 Hz, 1H), 7.97 (t, J = 8.4 Hz, 1H), 7.61
– 7.57 (m, 2H), 7.39 (d, J = 7.5 Hz, 2H), 7.32 – 7.20 (m, 7H), 6.95 (d, J = 8.4 Hz, 2H), 6.81 (t, J = 7.2 Hz,
1H), 5.68 (s, 2H), 3.64 (brs, 4H), 3.14 (brs, 4H); LCMS (electrospray) m/z (M+H) + 491

1-benzyl-2-imino-3-(4-phenylpiperidine-1-carbonyl)-1H-dipyrido[1,2-a:2',3'-d]pyrimidin-5(2H)-one
ACCEPTED MANUSCRIPT
(8d). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.95 (d, J = 6.4 Hz, 1H), 7.76 (s, 1H), 7.63 (ddd, J = 8.8,
6.8, 1.6 Hz, 1H), 7.51 - 7.45 (m, 3H), 7.34 – 7.27 (m, 4H), 7.25 – 7.18 (m, 5H), 7.07 (t, J = 7.2 Hz, 1H),
5.79 (s, 2H), 3.21 – 2.91 (brs, 2H), 2.81 – 2.75 (m, 1H), 1.99 – 1.87 (brs, 2H), 1.78 – 1.65 (m, 4H); LCMS
(electrospray) m/z (M+H) + 490
1-benzyl-2-imino-3-(morpholine-4-carbonyl)-1H-dipyrido[1,2-a:2',3'-d]pyrimidin-5(2H)-one
(8e).
1
Yellow solid; H NMR (400 MHz, DMSO-d6) δ 8.90 (d, J = 7.6 Hz, 1H), 7.99 – 7.95 (m, 1H), 7.58 (d, J =
8.8 Hz, 1H), 7.55 (s, 1H), 7.46 (brs, 1H), 7.39 – 7.37 (m, 2H), 7.31 – 7.25 (m, 3H), 7.20 (d, J = 7.6 Hz, 1H),
5.66 (s, 2H), 3.63 – 3.56 (m, 4H), 3.55 -3.42 (m, 4H); LCMS (electrospray) m/z (M+H) + 416
1-benzyl-2-imino-5-oxo-N-phenyl-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxamide
(8f). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H), 9.04 (d, J = 6.8 Hz, 1H), 7.79 – 7.75 (m, 1H),
7.70 (d, J = 8.4 Hz, 2H), 7.55 – 7.48 (m, 2H), 7.39 – 7.27 (m, 8H), 7.16 – 7.12 (m, 1H), 7.06 (dd, J = 14.0,
7.2 Hz, 1H), 5.70 (s, 2H); LCMS (electrospray) m/z (M+H) + 422
1-benzyl-2-imino-5-oxo-N-phenethyl-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8g). Yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 9.66 (brs, 1H), 8.97 (d, J = 6.8 Hz, 1H),
8.48 (brs, 1H), 8.05 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.39 – 7.20 (m, 12H), 5.74 (s, 2H), 3.48 (q,
J = 14.0, 7.2 Hz, 2H), 2.84 (t, J = 6.8 Hz, 2H); LCMS (electrospray) m/z (M+H) + 450
1-benzyl-2-imino-5-oxo-N-(3-phenylpropyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8h). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.05 (d, J = 7.2 Hz, 1H), 8.95 (s, 1H), 7.84
(t, J = 8.0 H, 1H), 7.54 (d, J = 8.8, 1H), 7.39 – 7.30 (m, 4H), 7.27 - 7.24 (m, 4H), 7.20 - 7.12 (m, 5H), 5.75
(s, 2H), 3.44 (dd, J = 12.8, 6.7 Hz, 2H), 2.70 (t, J = 7.6 Hz, 2H), 1.94 (dt, J = 14.5, 7.3 Hz, 2H) ; LCMS
(electrospray) m/z (M+H) + 464
(R)-1-benzyl-2-imino-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8i). Yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 11.66 (s, 1H), 9.23 (brs, 2H), 8.96 (d, J =
6.9 Hz, 1H), 8.73 (brs, 1H), 8.37 (brs, 1H), 8.02 (t, J = 8.0 Hz, 1H), 7.72 (brs, 1H), 7.60 (d, J = 8.8 Hz, 1H),
7.36 - 7.23 (m, 8H), 5.70 (s, 2H), 5.15 – 5.06 (m, 1H), 1.45 (s, 3H); LCMS (electrospray) m/z (M+H) + 450

(S)-1-benzyl-2-imino-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
ACCEPTED MANUSCRIPT
carboxamide (8j). Yellow solid; 1H NMR (400 MHz, DMSO-d6) δ 11.67 (s, 1H), 9.20 (brs, 2H), 8.95 (d, J
= 6.9 Hz, 1H), 8.72 (brs, 1H), 8.30 (brs, 1H), 8.02 (t, J = 7.2 Hz, 1H), 7.72 (brs, 1H), 7.60 (d, J = 8.8 Hz,
1H), 7.36 - 7.23 (m, 8H), 5.69 (s, 2H), 5.13 – 5.07 (m, 1H), 1.45 (s, 3H); LCMS (electrospray) m/z (M+H)+
450
methyl 2-(1-benzyl-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxamido)-
3-phenylpropanoate (8k). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.02 (d, J = 7.2 Hz, 1H), 8.97 (brs,
1H), 7.76 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 8.9 Hz, 1H), 7.37 – 7.29 (m, 4H), 7.24 - 7.12 (m, 9H), 5.62 (s, 2H),
5.00 (q, J = 13.2, 6.8 Hz, 1H), 3.67 (s, 3H), 3.21 – 3.11 (m, 2H); LCMS (electrospray) m/z (M+H) + 508
1-benzyl-N-butyl-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxamide
(8m). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.04 (d, J = 6.5 Hz, 1H), 7.78 (t, J = 6.8 Hz, 1H), 7.52 -
7.49 (m, 1H), 7.38 – 7.29 (m, 7H), 7.14 (t, J = 6.0 Hz, 2H), 5.68 (s, 2H), 3.41 (dd, J = 12.7, 6.9 Hz, 2H),
1.61 - 1.57 (m, 2H), 1.39 (dd, J = 15.2, 7.5 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H); LCMS (electrospray) m/z
(M+H)+ 402
1-benzyl-N-(cyclohexylmethyl)-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8n). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.04 (d, J = 6.8 Hz, 1H), 8.96 (s, 1H), 7.82
(t, J = 8.0 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.39 – 7.26 (m, 7H), 7.19 (t, J = 7.2 Hz, 1H), 5.75 (s, 2H), 3.26
(t, J = 6.4 Hz, 2H), 1.79 – 1.64 (m, 5H), 1.25 – 1.13 (m, 4H), 1.02 – 0.96 (m, 2H); LCMS (electrospray) m/z
(M+H) + 442
N-((1R,2S,5S)-adamantan-2-yl)-1-benzyl-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
1
d]pyrimidine-3-carboxamide (8o). Yellow solid; H NMR (400 MHz, DMSO-d6) δ 11.66 (d, J = 8.4 Hz,
1H), 8.97 (d, J = 7.2 Hz, 1H), 8.76 (s, 1H), 8.03 – 7.99 (m, 1H), 7.76 (s, 1H), 7.60 (d, J = 8.8 Hz, 1H), 7.37
– 7.25 (m, 6H), 5.72 (s, 2H), 4.08 – 4.05 (m, 1H), 1.85 – 1.72 (m, 10H), 1.70 (s, 2H), 1.56 (m, 2H); LCMS
(electrospray) m/z (M+H) + 480
1-benzyl-N-cyclohexyl-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (8p). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 9.07 (d, J = 7.6 Hz, 1H), 8.90 (s, 1H), 7.97

– 7.89 (m, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.40 – 7.26 (m, 8H), 5.87 (s, 2H), 3.91 (brs, 1H), 2.05 – 1.96 (m,
ACCEPTED MANUSCRIPT
2H), 1.80 – 1.71 (m, 2H), 1.44 – 1.32 (m, 5H), 1.30 – 1.19 (m, 1H); LCMS (electrospray) m/z (M+H) + 428
1-benzyl-2-imino-5-oxo-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-carbonitrile (9a). Yellow
solid; 1H NMR (400 MHz, CDCl3) δ 8.95 (d, J = 7.1 Hz, 1H), 8.20 (s, 1H), 7.85 – 7.81 (m, 1H), 7.54 (d, J =
8.0 Hz, 1H), 7.46 (d, J = 7.3 Hz, 2H), 7.31 - 7.28 (m, 2H), 7.24 – 7.21 (m, 1H), 7.14 (t, J = 6.4 Hz, 1H), 5.74
(s, 2H); LCMS (electrospray) m/z (M+H) + 328
1-benzyl-2-imino-3-(phenylsulfonyl)-1H-dipyrido[1,2-a:2',3'-d]pyrimidin-5(2H)-one (9c). Yellow solid;
1H NMR (400 MHz, CDCl3) δ 8.96 (d, J = 6.8 Hz, 1H), 8.79 (s, 1H), 8.56 (s, 1H), 7.96 (d, J = 7.6 Hz, 2H),
7.79 (ddd, J = 8.6, 6.7, 1.6 Hz, 1H), 7.61 (t, J = 7.4 Hz, 1H), 7.55 – 7.46 (m, 3H), 7.32 (d, J = 7.1 Hz, 2H),
7.23 – 7.17 (m, 3H), 7.10 (d, J = 6.8 Hz, 1H), 5.66 (s, 2H); LCMS (electrospray) m/z (M+H) + 443
(S)-2-imino-5-oxo-1-phenyl-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-
carboxamide (10b). Yellow solid; mp = 263°C; 1H NMR (400 MHz, CDCl3) δ 11.42 (s, 1H), 9.13 (s, 1H),
8.99 (d, J = 6.8 Hz, 1H), 7.68 – 7.54 (m, 3H), 7.38 (d, J = 7.6 Hz, 2H), 7.34 – 7.27 (m, 3H), 7.26 – 7.19 (m,
4H), 7.06 (t, J = 6.4 Hz, 1H), 6.67 (s, 1H), 5.33 – 5.28 (m, 1H), 1.54 (d, J = 6.8 Hz, 3H); LCMS
(electrospray) m/z (M+H) + 436
(S)-1-(cyclohexylmethyl)-2-imino-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (10c). Yellow solid; mp = 145°C; 1H NMR (400 MHz, CDCl3) δ 8.99 (d, J =
6.8 Hz, 1H), 8.91 (s, 1H), 7.90 (brs, 1H), 7.62 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5
Hz, 2H), 7.25 – 7.18 (m, 3H), 5.28 – 5.22 (m, 1H), 4.43 (brs, 2H), 1.94 (s, 1H), 1.77 - 1.61 (m, 5H), 1.26 -
1.11 (m, 5H) ; LCMS (electrospray) m/z (M+H) + 456
(S)-1-benzyl-2-imino-8-methyl-5-oxo-N-(1-phenylethyl)-1,5-dihydro-2H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11a). Yellow solid; mp = 215°C; 1H NMR (400 MHz, CDCl3); δ 8.90(s, 1H),
8.87(s, 1H), 7.89(d, J = 8.0Hz, 1H), 7.64(d, J = 8.8Hz, 1H), 7.28-7.34(m, 9H), 7.19-7.23(m, 2H), 5.98(q, J =
8.0Hz, 2H), 5.13(q, J = 8.8Hz, 1H), 2.50(s, 3H), 1.58(s, 3H); LC/MS (electrospray) m/z (M+H) +464.
(S)-1-benzyl-2-imino-9-methyl-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11b). Orange solid; mp = 144°C; 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H),
8.88 (d, J = 7.2 Hz, 1H), 7.36-7.26 (m, 10H), 7.16 (t, J = 7.2 Hz, 1H), 7.06 (d, J = 6.4 Hz, 1H), 5.95-5.70 (m,

2H), 5.22-5.12 (m, 1H), 2.50 (s, 3H), 1.56 (d, J = 6.4 Hz, 3H);LCMS (electrospray) m/z (M+H) + 464
ACCEPTED MANUSCRIPT
(S)-1-benzyl-2-imino-10-methyl-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11c). Brown solid; 1H NMR (400 MHz, CDCl3) δ 11.18 (br s, 1 H), 8.99 (s,
1H), 8.90 (d, J = 6.8 Hz, 1H), 7.64 (d, J = 6.8 Hz, 1H), 7.39-7.29 (m, 9 H), 7.21 (t, J = 7.2 Hz, 1H), 7.04 (t, J
= 6.8 Hz, 1H), 5.90-5.50 (m, 2H), 5.28 (quintet, J = 7.2 Hz, 1H), 2.44 (s, 3H), 1.52 (d, J = 6.8 Hz, 3H);
LCMS (electrospray) m/z (M+H) + 464.
(S)-1-benzyl-2-imino-10-methoxy-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11d). Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H), 8.66 (d, J =
6.7 Hz, 1H), 7.39 – 7.28 (m, 10H), 7.24 – 7.20 (m, 2H), 7.10 – 7.03 (m, 2H), 5.81 - 5.66 (m, 2H), 5.30 –
5.23 (m, 1H), 3.96 (s, 3H), 1.53 (s, 3H); LCMS (electrospray) m/z (M+H) + 480
(S)-1-benzyl-8-fluoro-2-imino-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
1
d]pyrimidine-3-carboxamide (11e). Brown solid; mp = 120°C; H NMR (400 MHz, CD3OD) δ 9.19 (s,
1H), 9.11 (t, J = 3.2 Hz, 1H), 8.20-8.15 (m, 1H), 7.83 (dd, J = 9.6, 4.8 Hz, 1H), 7.43 (d, J = 7.2 Hz, 2H),
7.37-7.24 (m, 8H), 6.03-5.92 (m, 2H), 5.26 (q, J = 6.8 Hz, 1H), 1.61 (d, J = 7.2 Hz, 3H); LCMS
(electrospray) m/z (M+H) + 468
(S)-1-benzyl-10-fluoro-2-imino-5-oxo-N-(1-phenylethyl)-1,5-dihydro-2H-dipyrido[1,2-a:2',3'-
1
d]pyrimidine-3-carboxamide (Compound 11f). Brown solid; mg = 235°C; H NMR (400 MHz, CDCl3);
δ 8.87(s, 1H), 8.83(s, 1H), 8.25(d, J = 7.2Hz, 1H), 7.87(d, J = 8.0Hz, 1H), 7.54(d, J = 7.2Hz, 1H), 7.25-
7.30(m, 9H), 7.15-7.20(m, 2H), 5.95(q, J = 8.0Hz, 2H), 5.11(q, J = 7.2Hz, 1H), 1.58(s, 3H); LC/MS
(electrospray) m/z (M+H)+ 468.
(S)-1-benzyl-8-chloro-2-imino-5-oxo-N-(1-phenylethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11h). Yellow solid; mp = 163°C; 1H NMR (400 MHz, CDCl3) δ 9.04 (d, J =
1.6 Hz, 1H), 8.98 (br s, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.45 (d, J = 9.6 Hz, 1H), 7.38-7.24 (m, 10H), 5.80-
5.50 (m, 2H), 5.27 (quintet, J = 7.2 Hz, 1H), 1.54 (d, J = 6.8 Hz, 3H); LCMS (electrospray) m/z (M+H)+484,
468 (Cl- isotope pattern).
(S)-1-benzyl-10-chloro-2-imino-5-oxo-N-(1-phenylethyl)-1,5-dihydro-2H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11i). Brown solid; mp = 237°C; 1H NMR (400 MHz, CDCl3); δ 8.90(s, 1H),

8.87(s, 1H), 8.27(d, J = 7.2Hz, 1H), 7.85(d, J = 5.2Hz, 1H), 7.34(d, J = 7.8Hz, 1H), 7.21-7.33(m, 9H), 7.20-
ACCEPTED MANUSCRIPT
7.22(m, 2H), 6.02(q, J = 8.0Hz, 2H), 5.13(q, J = 7.2Hz, 1H), 1.55(s, 3H); LC/MS (electrospray) m/z
(M+H)+ 484.
(S)-1-benzyl-2-imino-5-oxo-N-(1-phenylethyl)-9-(trifluoromethyl)-2,5-dihydro-1H-dipyrido[1,2-a:2',3'-
d]pyrimidine-3-carboxamide (11j). Yellow solid; mp = 165°C; 1H NMR (400 MHz, CDCl3) δ 11.39 (br s,
1H), 9.10 (d, J = 7.2 Hz, 1H), 9.03 (br s, 1H), 7.73 (s, 1H), 7.36-7.15 (m, 11H), 5.80-5.49 (m, 2H), 5.27
(quintet, J = 6.8 Hz, 1H), 1.54 (d, J = 6.4 Hz, 3H); LCMS (electrospray) m/z (M+H) + 518.
4-(trifluoromethyl)benzyl
1-(1-(3,4-dichlorophenyl)ethyl)-2-imino-8-methyl-5-oxo-1,5-dihydro-2H-
dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxylate (12a). Yellow solid; mp = 119°C; 1H NMR (400 MHz,
CD3OD ) δ 8.77 (s, 1H), 8.56 (brs, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.55 (d, J = 10.8
Hz, 3H), 7.47 – 7.40 (m, 2H), 7.31 (d, J = 8.0 Hz, 1H), 4.60 (s, 2H), 2.41 (s, 3H), 2.00(d, J = 6.8 Hz, 3H),;
LCMS (electrospray) m/z (M+H) + 600, 602 (Cl- isotope pattern).
1-(4-fluorophenyl)cyclopropyl
2-imino-8-methyl-5-oxo-1-(4-(trifluoromethoxy)benzyl)-1,5-dihydro-
1
2H-dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxylate (12b). Yellow solid; H NMR (400 MHz, CD3OD);
δ 8.91 (br s, 2H), 7.98 (d, J = 7.2 Hz, 1H), 7.63 (d, J = 9.2 Hz, 1H), 7.46-7.37 (m, 4H), 7.24 (d, J = 8.4 Hz,
2H), 7.02 (t, J = 8.8 Hz, 2H), 5.74 (br s, 2H), 2.48 (s, 3H), 1.36-1.27 (m, 4H); LCMS (electrospray) m/z
(M+H)+ 578.
4-(trifluoromethyl)benzyl
2-imino-8-methyl-5-oxo-1-(4-(trifluoromethoxy)benzyl)-1,5-dihydro-2H-
dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxylate (12c). Yellow solid. 1H NMR (400 MHz, CDCl3); δ 9.13
(s, 1H), 8.81 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.53-7.44 (m, 5H), 7.34 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.0
Hz, 2H), 5.82 (br s, 2H), 4.61 (d, J = 5.2 Hz, 2H), 2.43 (s, 3H); LCMS (electrospray) m/z (M+H)+ 602.
[1,1'-biphenyl]-4-ylmethyl
2-imino-8-methyl-5-oxo-1-(4-(trifluoromethoxy)benzyl)-1,5-dihydro-2H-
1
dipyrido[1,2-a:2',3'-d]pyrimidine-3-carboxylate (12d). Yellow solid; H NMR (400 MHz, CD3OD);
δ 9.23 (s, 1H), 8.98 (s, 1H), 8.08 (dd, J = 9.2, 2.0 Hz, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.63-7.56 (m, 4H), 7.48
(d, J = 8.0 Hz, 2H), 7.45-7.40 (m, 4H), 7.35-7.27 (m, 3H), 6.01 (br s, 2H), 4.65 (s, 2H), 2.51 (s, 3H); LCMS
(electrospray) m/z (M+H)+ 610.

ACCEPTED MANUSCRIPT
4.2. Biological evaluation
4.2.1. HCVcc assay
Cell culture derived infectious HCV (HCVcc) was produced by transfection of in vitro transcribed HCV
RNA genome as described previously [18]. Huh-7.5 cells were seeded in 384-well plates, incubated with
serially diluted compounds and inoculated with cell culture adapted HCVcc, expressing an NS5A-GFP
fusion protein, 2 h after compound treatment. At 72 h post-infection, HCV infection rates and cytotoxicity
were determined as previously reported [19].
4.2.2. HCVpp assay
HCV E1/E2-pseudotyped (or VSV-G pseudotyped as a control) lentiviral particles expressing a Firefly
luciferase reporter gene were used in HCV pseudoparticle (HCVpp) production by co-transfection of
plasmids expressing vesicular stomatitis virus (VSV) envelope and human immunodeficiency virus Gag-Pol
as described previously [20]. Comparable amounts of HCVpp and VSVGpp were used in every experiment.
Briefly, Huh-7 target cells were plated in 384-well plates, treated with compounds for 2 h followed by
transduction with HCVpp or VSVGpp. At 12 h post transduction, the cells were washed with PBS 3-times
and cells further incubated in fresh media until 72 h post transduction. Transduction rate and inhibition of
HCVpp infection were evaluated by DRC analysis measuring luciferase activity using Bright-Glo (Promega).
4.2.3. HCV replication assay
HCV subgenomic replicon cells expressing an NS5A-GFP fusion protein were seeded in 384-well plates and
incubated at 37°C with serially diluted compounds for 3 days [21]. HCV replication was assessed by
determining the percentage of GFP-positive cells. The EC50 and CC50 values were calculated by non-linear
regression using Prism 5.0v software (Graph Pad Software) [19].
4.3. Microsomal metabolic stability assay
Compounds (2 µM final concentration in 0.02 % DMSO) were incubated with 0.5 mg/mL human liver (pool
of 200, mixed gender, Xenotech) and rat male (BD Gentest) microsomes in potassium phosphate buffer. The

reaction was initiated by the addition of NADPH and stopped either immediately or at 10, 20, 30 and 60 min
ACCEPTED MANUSCRIPT
for a precise estimation of clearance. A triple quadrupole Quattro Premier™ mass spectrometer (Waters,
Milford, MA) with electrospray ionization (ESI) was employed for sample analysis. Samples were passed
through trapping cartridges (Acquity BEH RP18 50 mm×2.1 mm, 1.7 µm, Waters, Milford, MA) followed
by an analytical column. The mobile phases were (A) water with 0.1% of formic acid and (B) acetonitrile
with 0.1% of formic acid at a flow rate of 0.4 mL/min. The LC conditions were 5% B at 0min, a linear
gradient from 5 to 50% B over 0.5 min, held at 50% for 0.25 min, then ramped from 50 to 95% over 0.25
min, followed by 95% B for 0.75 min and back to 5% B over 0.25 min, then held at 5% B for the remaining
0.75 min. The percentage of the remaining compound was calculated by comparing with the initial quantity
at 0 min. Half-life was then calculated based on first-order reaction kinetics.
Acknowledgments
This work was supported by the National Research Foundation of Korea (NRF-No.
2014K1A4A7A01074644) grant funded by the Korean government (MSIP), Gyeonggi-do, and KISTI. We
are grateful to Drs. Charles M. Rice (Rockefeller University, New York, USA) and Ralf Bartenschlager
(University of Heidelberg, Germany) for kindly providing reagents.
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Highlights:
An iminodipyridinopyrimidine (IDPP) scaffold was identified by phenotypic HTS using
infectious HCV.
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IDPP exhibited anti-HCV activity on early and late steps of the viral cycle, but not on HCV
RNA replication.
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One IDPP derivative has a selectivity index >220,000 which is predestinated to conveniently
study the HCV life cycle.
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Furthermore, a drugable IDPP lead compound was identified which could be developed to a
therapeutic HCV intervention.
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