Elaboration of a proprietary thymidylate kinase inhibitor motif towards anti-tuberculosis agents

Article abstract of DOI:10.1016/j.bmc.2016.08.041

We report the design and synthesis of a series of non-nucleoside MtbTMPK inhibitors (1–14) based on the gram-positive bacterial TMPK inhibitor hit compound 1. A practical synthesis was developed to access these analogues. Several compounds show promising MtbTMPK inhibitory potency and allow the establishment of a structure–activity relationship, which is helpful for further optimization.

Full text of DOI:10.1016/j.bmc.2016.08.041

Accepted Manuscript
Elaboration of a Proprietary Thymidylate Kinase Inhibitor Motif Towards Anti-
Tuberculosis Agents
Lijun Song, Martijn D.P. Risseeuw, Matheus Froeyen, Izet Karalic, Jan
Goeman, Davie Cappoen, Johan Van der Eycken, Paul Cos, Hélène Munier-
Lehmann, Serge van Calenbergh
PII:
S0968-0896(16)30649-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.08.041
BMC 13224
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 April 2016
20 August 2016
22 August 2016
Please cite this article as: Song, L., Risseeuw, M.D.P., Froeyen, M., Karalic, I., Goeman, J., Cappoen, D., Eycken,
J.V.d., Cos, P., Munier-Lehmann, H., Calenbergh, S.v., Elaboration of a Proprietary Thymidylate Kinase Inhibitor
Motif Towards Anti-Tuberculosis Agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.08.041
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Elaboration of a Proprietary Thymidylate Kinase Inhibitor
Motif Towards Anti-Tuberculosis Agents
Lijun Song,a Martijn D.P. Risseeuw,a Matheus Froeyen,b Izet Karalic,a Jan Goeman,c Davie
Cappoen,d Johan Van der Eycken,c Paul Cos, d and Hélène Munier-Lehmann,e,f Serge van
Calenbergh*,a
a Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, Ghent University.
Ottergemsesteemweg 460, B-9000, Ghent Belgium.b Medicinal Chemistry (Rega Institute),
Department of Pharmaceutical and Pharmacological Sciences, KU LEUVEN.
Minderbroedersstraat 10 blok x - box 1030, 3000 Leuven Belgium.
c Laboratory for Organic and Bioorganic Synthesis, Department of Organic and Macromolecular
Chemistry, Ghent University. Krijgslaan 281, S4, B-9000, Ghent Belgium.
d Laboratory for Microbiology, Parasitology and Hygiene (LMPH), Department of
Pharmaceutical Sciences, University of Antwerp, Campus Drie Eiken, Universiteitsplein 1
e Institut Pasteur, Unit of Chemistry and Biocatalysis, Department of Structural Biology and
Chemistry, 28 Rue du Dr. Roux, 75724 Paris Cedex 15, France.
f CNRS UMR3523, Paris, France.
* Laboratory for Medicinal Chemistry, Faculty of Pharmaceutical Sciences, UGent,
Ottergemsesteenweg 460, B-9000 Gent, Belgium. Phone: +32 9 264 81 24. Fax: + 32 9 264 81
46. E-mail: serge.vancalenbergh@ugent.be
1

Keywords: thymine derivatives; Mycobacterium tuberculosis; thymidine monophosphate kinase;
inhibitors
Abbreviations: HIV, human immunodeficiency virus; LTBI, latent tuberculosis infection; MDR-
TB, multidrug-drug resistant tuberculosis; Mtb, Mycobacterium tuberculosis; TMPK, thymidine
monophosphate kinase; dTMP, thymidine 5’-monophosphate; dTDP, thymidine 5’-diphosphate;
dTTP, thymidine 5’-triphosphate; XDR-TB, extensively drug-resistant tuberculosis.
2

Graphical Abstract
3

Abstract
We report the design and synthesis of a series of non-nucleoside MtbTMPK inhibitors (1
- 14) based on the gram-positive bacterial TMPK inhibitor hit compound 1. A practical synthesis
was developed to access these analogues. Several compounds show promising MtbTMPK
inhibitory potency and allow the establishment of a structure–activity relationship, which is
helpful for further optimization.
1. Introduction
According to the global tuberculosis (TB) report in 2015, worldwide TB causes more deaths
than any other disease caused by a single infectious agent. As a result, TB is ranked as the most
lethal infectious disease on par with the human immunodeficiency virus HIV.1 It is estimated
that one-third of the world population is infected with asymptomatic latent TB (LTBI), with
around 10% lifetime risk of developing active TB. This is especially relevant for persons with a
compromised immune system2 (one-third HIV deaths were due to TB in 20151). For drug-
susceptible active TB, treatment requires a standard 6 month course of 4 frontline drugs, which
frequently results in poor treatment compliance. Moreover, due to the inappropriate treatment,
multidrug-drug resistant (MDR) TB is now widespread (MDR-TB is defined as a rifampicin-
resistant form of TB that has additional resistance to isoniazid3). Mycobacterium tuberculosis
strains of the Beijing lineage are responsible for the massive spread of MDR-TB in Eurasia.4
Furthermore extensively drug-resistant (XDR) TB has been reported by 105 countries by the end
of 2014 (XDR-TB is defined as a form of MDR-TB that does not respond to any fluoroquinolone
4

and at least one second-line injectable drug3). Therefore, there is an urgent need for effective and
new TB drugs to shorten the treatment regime and cure MDR-TB and XDR-TB.
Thymidylate kinase (TMPK, also called thymidine 5’-monophosphate kinase) phosphorylates
thymidine 5’-monophosphate (dTMP) to thymidine 5’-diphosphate (dTDP) utilizing ATP as its
preferred phosphoryl donor. The synthesis of dTDP is unique in that it requires TMPK, while
other deoxynucleoside diphosphates are directly produced by ribonucleotide reductase. Further
phosphorylation of dTDP gives thymidine triphosphate (dTTP), which is one of the building
blocks of DNA. TMPKs are subdivided into two types based on the position of basic residues
within the active site.5 Notably, M. tuberculosis TMPK (MtbTMPK) does not belong to type I
nor to the type II enzyme, revealing its unique catalytic mechanism.6 Additionally, it also has a
mere 22% sequence identity to human TMPK.5 Moreover, TMPK is the last specific enzyme for
the synthesis of dTTP. Therefore, MtbTMPK inhibitors have the potential for being effective and
selective anti-TB and anti-MDRTB drugs with low toxicity.
The current MtbTMPK inhibitors can be categorized into two types: thymine-like inhibitors
and non-thymine-like inhibitors. The thymine-like inhibitors can be further subdivided into
nucleoside and non-nucleoside inhibitors. The nucleoside family is mainly composed of
thymidine analogues with modifications at position 5 of the thymine base7 and at the 3’- and/or
5’-positions of the sugar ring,8 which give Ki values in the low micromolar range and poor anti-
bacterial activity.9 In the non-nucleoside family, most of the analogues still bear the thymine
heterocyclic head with either an acyclic tail10 or a substituted benzyl at the N-1 position of the
thymine ring.11 Some inhibitors of this family have submicromolar Ki values (Figure 1).12 Two
recently discovered 3-cyanopyridone and 1,6-naphthyridin-2-one MtbTMPK inhibitors that show
nanomolar inhibitory activity have a non-thymine-like structure.13
5

Compared to the well-explored nucleoside family, non-nucleoside MtbTMPK inhibitors
warrant further investigation particularly since some of them offer promising antimycobacterial
activity, e.g. the sulfoxide- and sulfone-containing cyanopyridone derivatives.13
Figure 1. Representative MtbTMPK inhibitors
In 2012, gram-positive bacterial TMPK inhibitors with picomolar activity were reported and
further optimization of their physico-chemical properties and pharmacokinetics afforded a class
6

of promising antibacterial agents.14 Since no evaluation of these analogues on MtbTMPK was
reported, we set out to resynthesize compound 1, which featured as the parent inhibitor for lead
generation, and found it to be a fairly potent MtbTMPK inhibitor (Ki = 1.5 µM), comparable
with the most potent nucleoside MtbTMPK inhibitors.9
Encouraged by this result, we decided to further investigate its SAR (Figure 2). We first
changed the configuration of the lipophilic biaryl ether from the meta- to para-position of the
internal phenyl group (compound 2). In order to explore the chiral preference of the enzyme, the
pure enantiomers of 1 and compound 2 (compounds 3-6) were prepared. In two more analogues
(7 and 8) the piperidine ring was substituted for a pyrrolidine ring. To explore the effect of
additional distance and flexibility between the thymine and piperidinyl or pyrrolidinyl rings,
analogues having a methylene linkage inserted between these heterocycles (9-12) were prepared.
In two final analogues (13 and 14) the tertiary amine was replaced with a tertiary amide in order
to investigate the importance of the positively charged nitrogen on the piperidine ring. Based on
the established X-ray crystal structure of MtbTMPK, we performed molecular modeling studies
on these piperidinyl thymine analogues in order to better understand the observed SAR and to
shed light on further modifications.
7

Figure 2. Overview of non-nucleoside thymine derivatives synthesized in this study.
2. Results and Discussion
2.1.Chemistry
In the original synthesis of the hit compound 1,14 the key intermediate 15 is prepared
using the highly moisture-sensitive isocyanate species 17, which was obtained from methyl
methacrylate using a potentially hazardous multi-step synthesis.15 In order to access intermediate
15 by an easier and safer means, alternative synthetic routes were explored. According to
Rejman and coworkers,15 there are two alternative methods for the synthesis of the 1-(piperidin-
3-yl)thymine 15 in addition to the cyclization between substituted isocyanate 17 and primary
amine 16 mentioned above. The first method connects the piperidine and thymine rings using a
Mitsunobu reaction, the second one involves an alkylation reaction (Scheme 1).16
8

In the Mitsunobu reaction, thymine can react as an ambident nucleophile yielding a
mixture of N- and O-linked isomers. Therefore, the N3-position of thymine was protected with a
benzoyl- (Bz-, 19) or a benzyloxymethyl- (BOM-, 20) as to avoid formation of the N3-
regioisomer.17 Despite using the conditions for the Mitsunobu reaction as recommended by
Rejman and coworkers,16 no product was obtained. Further attempts to optimize the reaction
conditions proved fruitless.
Scheme 1. Three routes towards piperidinylthymine: isocyanate based pyrimidine synthesis,
Mitsunobu reaction and alkylation using a mesylate
The alkylation reaction between thymine and mesylate (21)18 is typically performed at high
temperature (e.g., 100 ℃ or 120 ℃) under basic condition (NaH or Cs2CO3) with the best
obtained yield being 15%.16 In an attempt to increase the yield of the alkylation, a first trial was
conducted using N3-Bz-thymine (1917) and cesium carbonate. Since compound 19 was
hydrolyzed back to thymine under the reaction conditions, we switched to N3-BOM-protected
9

thymine (20), which gave similar yields with cesium carbonate (21% and 16% at 100 ℃ and
130 ℃, respectively). A commonly observed feature of the alkylation reaction under these
conditions was the large amount of unreacted thymine in the reaction mixture. A possible reason
was the elimination of mesylate (21) into compounds 22 and 23 at high temperature (Scheme 2).
Based on these observations we could eventually raise the yield of product 25 substantially (to
51%) by lowering the reaction temperature to 80 ℃ with portionwise addition of the mesylate
(21) and base (K2CO3) over the course of the reaction (4 days).19
Scheme 2: Alkylation of N3-protected thymine using piperidinylmesylates
The alkylation of compound 20 with the azacyclic mesylates (21 and 26 - 31) provided the
Boc-protected intermediates, which could be deprotected to give the required secondary amine
intermediates (34 and 32-38). Next, reductive amination with aromatic aldehydes gave the N3-
BOM-protected products, which were directly deprotected with TFA at 72 ℃ to afford the final
products 1 - 12.20 The alkylation reaction was found to occur mostly by the SN2 nucleophilic
substitution reaction, which was demonstrated by chiral purity analysis (cf. experimental part and
supplementary information).
For the synthesis of piperidinylamide analogues, corresponding aromatic carboxylic acids
were condensed to 34, followed by hydrogenolysis to provide the final products 13 and 14
10

alongside their corresponding hemiaminals 13a and 14a, which could be further hydrolyzed to
the final products (Scheme 3).
n
n
n
HN
N
N
a
b
OMs
N
HN
BocN
R
O
O
N
O
N
O
N
O
O
m
m
H
1, m = 2, n = 0, R = R1, rac.
m
BOM
BOM
2, m = 2, n = 0, R = R2, rac.
3
20
,
m = 2, n = 0, R = R1, R
26, m = 1, n = 0, R
27, m = 1, n = 0, S
21
, m = 2, n = 0, rac.
28, m = 2, n = 0, R
29, m = 2, n = 0, S
30
, m = 1, n = 1, rac.
32, m = 1, n = 0, R
33, m = 1, n = 0, S
34
, m = 2, n = 0, rac.
35, m = 2, n = 0, R
36, m = 2, n = 0, S
37
, m = 1, n = 1, rac.
4, m = 2, n = 0, R = R2, R
5, m = 2, n = 0, R = R1, S
6
,
m = 2, n = 0, R = R2, S
7, m = 1, n = 0, R = R1, R
8, m = 1, n = 0, R = R1, S
9
,
m = 2, n = 1, R = R1, rac.
31, m = 2, n = 1, rac.
38, m = 2, n = 1, rac.
10, m = 2, n = 1, R = R2, rac.
11. m = 1, n = 1, R = R1, rac.
12
. m = 1, n = 1, R = R2, rac.
HO
BOM
H
N
O
O
O
O
N
O
c
d
R1 =
O
N
O
O
O
N
N
N
R
N
HN
R
N
O
R2 =
34
13a 14a
,
13,
R = R1
14, R = R2
Scheme 3. Synthesis of piperidinyl- and pyrrolidinylthymine MtbTMPK inhibitors. Reagents
and conditions: (a) i) K2CO3, dry DMF, 80 ℃, overnight (compound 37 and 38), 24 h
(compound 32 and 33), 4 days (compound 34 - 36); ii) 10% TFA/ CH2Cl2, 2 h - 4 h, rt; (b) i)
substituted aromatic aldehydes, NaBH(OAc)3, rt, overnight; ii) TFA, 72 ℃, 30 min – 1 h; (c) i)
EDC, 4-DMAP, dry CH2Cl2, overnight; ii) H2, Pd/C, EtOH, 6 h, rt. (d) THF/H2O, rt, 4 h.
2.2 Biological evaluation and structure-activity relationship (SAR)
The inhibitory potency of compounds (1 - 14) was evaluated on MtbTMPK catalytic activity
(determination of the inhibitory constant Ki) and the results are summarized in Table 1.
11

Table 1. Inhibition of MtbTMPK with non-nucleoside thymine derivatives
Compound
m
n
R
Stereochemistry
Ki (µM)
1.5 0.4
2
2
2
2
2
2
1
1
2
2
1
1
2
2
0
0
0
0
0
0
0
0
1
1
1
1
0
0
R1
racemic
racemic
R
1
2
R2
R1
R2
R1
R2
R1
R1
R1
R2
R1
R2
R1
R2
22.3 0.6
8.8 0.3
3
R
138.5 9.2
0.8 0.1
4
S
5
S
21.5 2.4
8.2 0.8
6
R
7
S
7.8 1.4
8
racemic
racemic
racemic
racemic
racemic
racemic
186.3 24.3
NI at 0.2 mM
110.7 9.0
267.3 25.4
2.1 0.3
9
10
11
12
13
14
14.4 2.5
A clear trend emerges in that the potency of the analogues with the meta-biphenyl ether tail is
typically approximately 15-fold higher than that of their para-substituted congeners (comparing
pairs 1/2, 3/4, 5/6). For the piperidinyl thymine analogues (comparing pairs 3/5 and 4/6), the S-
enantiomers were about 10-fold more potent than the R-enantiomers, and offered the most
effective compound 5 (Ki = 0.8 µM) in this series. This trend was less pronounced in the
pyrrolidin-3-ylthymine analogues (compound 7 and 8). Additionally, these pyrrolidine analogues
12

are less potent than their piperidinyl counterparts (cf. couples 5/8 and 3/7). Introduction of the
methylene linker greatly impaired inhibitory potency (cf. pairs 1/9, 2/10), which indicates that
the saturated azacycle should be connected directly to the thymine ring. Although there is no
benefit from the substitution of the amide moiety (cf. pairs 1/13 and 2/14), bioisosteres of the
amide could be probed as a further modification. Compound 5 was found to show promising
activity against a Mtb H37Ra strain (MIC = 0.9 µM or 0.35 µg/mL) and a selectivity index of 35
versus MRC-5 cells.
To assess the binding mode of these thymine analogues, docking of the piperidinyl thymines 3,
5 and 6, the pyrrolidinyl thymine 8 and both enantiomers of amide 13 was performed. The
typical interactions found between the nucleobase of dTMP and MtbTMPK are likely to be
conserved with compound 55: (1) π-π stacking between the pyrimidine ring and Phe-70; (2) two
hydrogen bonds between the O4-thymine and Arg-74; (3) a hydrogen bond between Asn-100
and the N3-thymine ring. Moreover, one extra hydrogen bond is formed through the oxygen of
the meta-biphenyl ether and Arg-95 (Figure 3), which may contribute to the higher affinity for
the enzyme. The docking results indicate that none of the other compounds can form more polar
interactions with the enzyme (Figure 4). The thymine ring of compound 3 forms similar
interactions as compound 5 (with the Arg-74 and Asn-100 residues), but the meta-biphenyl ether
of 3 lacks a hydrogen bond interaction with the enzyme, which may explain the inferior
inhibitory potency of this R-enantiomer. Similar differences are also observed for compound 6
and 8, implying that the meta-biphenyl ether tail and piperidine ring are superior to the para-
substituted biphenyl ether tail and pyrroline ring, respectively. It is noteworthy that the aromatic
tail of 3 is surrounded by residues Ala-35, Phe-36, Pro-37, Tyr 39, Asp-94, Arg-95 and Arg-160,
which stabilize binding by hydrophobic interactions. Adding functional groups to this aromatic
13

tail may further increase interactions with those residues. Docking of the two enantiomers of
compound 13 into MtbTMPK, indicates that the R-enantiomer may occupy a similar pose as the
other inhibitors, while the S-enantiomer shows a completely different interaction with the
enzyme resulting in a lower docking score (Figure 4). Considering the promising inhibitory
activity of the racemate 13, it may be interesting to investigate the inhibitory potency of the pure
enantiomers in the future.
Figure 3. Compound 5 (green stick) docked in the active site of MtbTMPK. All residues
interacting with the inhibitors including hydrophobic contact (dark gray wire) and hydrogen-
bonding interaction (residues in yellow wire, hydrogen bonds indicated in pink) were calculated
using Ligplot21. Illustration was created using Chimera22.
14

Figure 4. Two-dimensional representation for the interacting mode of compound 3, 5, 6, 8
and 13 with MtbTMPK; it is created using the Ligplot21 program.
Conclusion
Inspired by the published Gram-positive bacterial TMPK inhibitor 1, we described the design
and synthesis of a new series of non-nucleoside MtbTMPK inhibitors. A convenient and less
15

hazardous synthetic method for 1-substituted azacyclo-thymine analogues has been developed.
By exploring different scaffolds, we have demonstrated that there are several spatial
requirements with respect to the connection between the thyminyl and piperidinyl ring. First,
analogues with S-stereochemistry were found to be superior inhibitors of MtbTMPK compared to
their enantiomers. Furthermore, piperidinyl analogues have been shown to have a higher potency
compared to their pyrrolidinyl counterparts. Additionally, the docking model suggests it is
favourable to introduce functional groups to the meta-biphenyl ether tail. For compounds with
amide moiety, more attention needs to be paid to the pure enantiomers. Finally we came to the
conclusion that the best scaffold of this series is the known S-enantiomer (compound 5)14a and
future efforts should be focused on piperidinyl thymine analogues with S-stereochemistry
combined with a meta-substituted biphenyl ether tail.
4. Experimental section
4.1. Spectrophotometric binding assay
Activity was determined as described in Blondin et al.23 Using a coupled spectrophotometric
assay at 334 nm in an Eppendorf ECOM 6122 photometer. The reaction medium (0.5 ml final
volume) contained 50 mM Tris-HCl pH 7.4, 50 mM KCl, 2 mM MgCl2, 0.2 mM NADH, 1 mM
phosphoenol pyruvate and 2 units each of lactate dehydrogenase, pyruvate kinase and nucleoside
diphosphate kinase. One unit of enzyme activity corresponds to 1 mole of the product formed in
1 min. at 30°C. The concentrations of ATP and dTMP were kept constant at 0.5 mM and 0.05
mM respectively, whereas the concentrations of analogues varied between 0.01 and 1 mM.
4.2 Biological Assays on Mycobacterium tuberculosis
16

4.2 Biological Assays on Mycobacterium tuberculosis
The Minimal Inhibitory Concentration (MIC) against mycobacteria was evaluated by serial
dilution method. The in vitro assay was based on a method in which a luminescent
Mycobacterium tuberculosis H37Ra strain Lehmann &Neumann (ATCC® 25177™)
transformed with pSMTB1 luciferase reporter plasmid is used. The tested compound was
solubilized in DMSO (Sigma-Aldrich) at stock concentration of 10 mM. Serial dilutions were
made in liquid 7H9 medium [Middlebrook 7H9 broth based (Difco)] with 10% oleic acid,
albumin, dextrose, catalase (OADC) enrichment. Volumes of 20 µL of the serial dilutions were
added in triplicate to 96 well, flat-bottomed micro well plates. The bacterial suspension was
made by thawing and dissolving a frozen Mycobacteria pellet in 7H9-10% OADC. The dissolved
pellet was passed through a 5.0 µM filter (Millipore) to eliminate clumps and left for 1 hour to
recover at 37 °C, 5% CO2. Next, the bacterial suspension was diluted in 7H9-10% OADC to
obtain 50,000 Relative Light Units (RLU)/mL and a volume of 180 µL of bacteria was added to
each well. A bacterial replication was analyzed by luminometry after 7 days of incubation. The
bacterial suspension from each well was collected and transferred to a black 96-well plate to
evade cross luminescence between wells. The luminescent signal was evoked by addition of the
substrate for the bacterial luciferase, 1% n-decanal in ethanol to each well by the Discover multi-
plate reader from Promega and the light emission in each well was measured.
4.3 Molecular modeling
All molecular modeling calculations were performed using the software packages AutoDock 4.2
on Windows Cygwin and AutodockTools-1.5.6.24 The previously reported X-ray structure of the
17

MtbTMPK (PDB entry 1G3U)5 was used in all docking experiments. The 2D chemical structures
and PDB files of the ligands were drawn and created using ChemBioDraw 13. The PDBQT file
of ligands and receptor were prepared by AutodockTools-1.5.6, which includes atomic partial
charges, atom types and the information of the ligand torsional degrees. For the docking, a
default grid spacing of 0.375 Å and 60 × 60 × 60 number of grid points were used, which
centered the box on the active site of MtbTMPK (e.g. the typical π-π stacking between Phe-70
and thymine ring of the ligand)9. The Genetic Algorithm-Local Search (GA-LS) method was
adopted using default settings. 50 possible conformations were generated by Autodock 4.2 for
each docking. A manual selection procedure combining visual inspection in Chimera guided by
the Ligplot analysis together with the predicted free energy found for each conformation was
used to validate the docked conformations.
4.2. Chemical synthesis
General: Solvents were purchased from standard commercial sources and of analytical grade.
Building blocks and reagents were used as received without any further purification. TLC
analysis was performed using precoated Alugram Silica Gel F254 plates (Machery-Nagel). Spots
were examined under ultraviolet light at 254 nm. Column chromatography was carried out on a
Reveleris X2 (Grace) automated flash unit using the corresponding disposable silica gel
1
cartridges. H and 13C NMR spectra were recorded in CDCl3 or DMSO-d6 on a Varian Mercury
300/75 MHz spectrometer. Chemical shifts are given in parts per million (ppm δ), δ relative to
1
13
residual solvent peak or TMS for H and C. Structural assignment was confirmed with COSY,
18

HSQC and HMBC. Exact mass measurements (HRMS) were performed on a Waters LCT
Premier XETM Time of flight (TOF) mass spectrometer equipped with a standard electrospray
ionization (ESI) and modular LockSpray TM interface. Samples were infused in a CH3CN/H2O
(1:1) mixture at 100 µL/min. Preparative reversed phase HPLC chromatography was carried out
using a Phenomenex Luna C-18 (21.2x250mm) column using an aq. NH4HCO3/MeCN gradient.
Chiral analysis was carried out with a Daicel Chiralpak-IA HPLC column or a Daicel Chiralcel-
ODH HPLC column using hexane/ethanol 80:20 as eluent. Both columns, dimensions
4.6x250mm, 5µm particle size, were used at 35°C.
4.2.1. 3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (34): A
suspension of 3-((benzyloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (20, 1.06 g, 4.3
mmol), tert-butyl 3-((methylsulfonyl)oxy)piperidine-1-carboxylate (21, 1.80 g, 6.45 mmol) and
potassium carbonate (1.19 g, 8.6 mmol) in dry DMF (10 mL) was stirred at 80 ℃ for 48 h under
argon. Additional 21 (1.20 g, 4.3 mmol) and potassium carbonate (0.60 g, 4.3 mmol) were added
to the reaction mixture and the mixture was stirred at 80 ℃ for 24 h. Additional 21 (1.20 g, 4.3
mmol) and potassium carbonate (0.60 g, 4.3 mmol) were added and the mixture was stirred at
80°C another 24 h. After cooling to room temperature, the reaction mixture was diluted with
CH2Cl2 (100 mL), following by washing with water (100 mL) and brine (100 mL). The organic
layer was dried over sodium sulfate. After evaporation, the residue was purified by column
chromatography (50% hexane/ethyl acetate) to give the intermediate which was dissolved with
CH2Cl2 (20 mL), and TFA (2 mL) was added to the solution. The reaction mixture was stirred at
room temperature for 3 h, followed by evaporation in vacuo. The residue was dissolved in sat.
NaHCO3 solution (30 mL) and extracted with CH2Cl2 (50 mL × 3). The combined organic layers
were washed with brine (50 mL × 2) and dried over sodium sulfate. The water layer can be re-
19

extracted again if the product is found by TLC. After evaporation, the residue was purified by
column chromatography (0.1% Et3N/4%MeOH/ CH2Cl2) to afford the desired product 34 as
1
colorless gel (0.60 g, 42.4%). H NMR (300 MHz, DMSO-d6) δ: 1.60 - 2.02 (m, 7H, 5-CH3,
piperidin-4-yl and piperidin-5-yl), 2.49 (br. s., 1H, NH), 2.55 - 2.66 (m, 1H, piperidin-6a-yl),
2.73 (dd, J = 12.01, 10.25 Hz, 1H, piperidin-2a-yl), 2.99 - 3.10 (m, 1H, piperidin-6b-yl), 3.19 (dd,
J = 11.86, 2.78 Hz, 1H, piperidin-2b-yl ), 4.50 (ddd, J = 10.47, 6.52, 4.10 Hz, 1H, piperidin-3-yl),
13
4.71 (s, 2H), 5.51 (s, 2H), 7.18 (d, J = 1.17 Hz, 1H, 3-CH3), 7.21 - 7.41 (m, 5H, Ph). C NMR
(75 MHz, CDCl3) δ: 14.21 (5-CH3), 27.00 ( piperidin-5-yl), 30.55 (piperidin-4-yl), 46.66
(piperidin-6-yl), 51.06 (piperidin-2-yl), 54.50 (piperidin-3-yl), 71.81 (CH2, benzyloxy), 73.23 (3-
methyl), 110.67 (C-3) 128.53 (Ph), 128.58 (2C, Ph), 129.20 (2C, Ph), 136.76 (C-6), 139.06 (Ph),
152.50 (C-2), 164.25 (C-4). HRMS (ESI): calculated for [C18H23N3O3 + H]+ , 330.1812; found,
330.1809. The R/S-enantiomers (compound 35 and 36) were synthesized as described above.
The yield of compound 35 and 36 is 38.9% and 41.3% respectively.
4.2.2 General procedure for synthesis of compound 32, 33, 37 and 38.
A suspension of 3-((benzyloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (20, 1 eq), N-
Boc-protected mesylates (1.1 eq - 2 eq), potassium carbonate (1.5 eq - 2 eq) was stirred at 80 ℃
overnight (compound 37 and 38) or 24 h (compound 32 and 33) under argon. The work-up
procedure as well as the second step were performed as has been described for the synthesis of
compound 34.
4.2.2.1 (R)-3-((benzyloxy)methyl)-5-methyl-1-(pyrrolidin-3-yl)pyrimidine-2,4(1H,3H)-dione (32):
3-((benzyloxy)methyl)-5-methylpyrimidine-2,4 (1H,3H)-dione (20, 0.72g, 2.92 mmol), tert-butyl
(S)-3-((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (26, 1.55 g, 5.84 mmol) and potassium
20

carbonate (0.81g, 5.84 mmol) yielded compound 32 as a colorless gel (0.53 g, 57.2%). 1H NMR
(300 MHz, CDCl3) δ: 1.77 - 1.98 (m, 4H, 5-CH3, pyrrolidin-4a-yl), 2.26 – 2.43 (m, 1H,
pyrrolidin-4b-yl), 2.89 - 3.12 (m, 2H, pyrrolidin-2a-yl, pyrrolidin-5a-yl), 3.17 (br. s., 1H, NH),
3.23 - 3.38 (m, 2H, pyrrolidin-2b-yl, pyrrolidin-5b-yl), 4.70 (s, 2H, CH2, 3-methylene), 5.00 -
5.13 (m, 1H, pyrrolidin-3-yl), 5.50 (s, 2H, CH2, benzyl), 7.13 - 7.44 (m, 6H, Ph, H-6). 13C NMR
(75 MHz, CDCl3) δ: 13.14 (5-CH3), 31.51 (pyrrolidin-4-yl), 46.15 (pyrrolidin-5-yl), 51.21
(pyrrolidin-2-yl), 56.30 (pyrrolidin-3-yl), 70.74 (3-methylene), 72.25 (CH2, benzyl), 110.63 (C-
5), 127.60 (3C, Ph), 128.21 (2C, Ph), 136.74 (C-6), 137.96 (Ph), 151.54 (C-2), 163.33 (C-4).
HRMS (ESI): calculated for [C17H21N3O3 + H]+ , 316.1656; found, 316.1651.
4.2.2.2 (S)-3-((benzyloxy)methyl)-5-methyl-1-(pyrrolidin-3-yl)pyrimidine-2,4(1H,3H)-dione (33):
3-((benzyloxy)methyl)-5-methylpyrimidine-2,4 (1H,3H)-dione (20, 1.45g, 6 mmol), tert-butyl
(R)-3-((methylsulfonyl)oxy)pyrrolidine-1-carboxylate (27, 3.18 g, 12 mmol) and potassium
1
carbonate (1.66 g, 12 mmol) yielded compound 33 as a colorless gel (0.96 g, 50.8%). H NMR
(300 MHz, CDCl3) δ 1.73 -1.87 (m, 1H, pyrrolidin-4a-yl), 1.92 (d, J = 1.17 Hz, 3H, 5-CH3), 2.26
- 2.41 (m, 1H, pyrrolidin-4b-yl), 2.89 - 3.09 (m, 2H, pyrrolidin-2a-yl, pyrrolidin-5a-yl), 3.19 -
3.32 (m, 2H, pyrrolidin-2b-yl, pyrrolidin-5b-yl), 4.70 (s, 2H, CH2, 3-methylene), 5.01 - 5.11 (m,
1H, pyrrolidin-3-yl), 5.50 (s, 2H, CH2, benzyl), 7.20 - 7.40 (m, 6H, Ph, H-6). 13C NMR (75 MHz,
CDCl3) δ: 13.32 (5-CH3), 31.87 (pyrrolidin-4-yl), 46.37 (pyrrolidin-5-yl), 51.70 (pyrrolidin-2-yl),
56.40 (pyrrolidin-3-yl), 70.89 (3-methylene), 72.38 (CH2, benzyl), 110.64 (C-5), 127.75 (3C, Ph),
128.36 (2C, Ph), 136.89 (C-6), 138.16 (Ph), 151.70 (C-2), 163.54 (C-4). HRMS (ESI): calculated
for [C17H21N3O3 + H]+ , 316.1656; found, 316.1650.
4.2.2.3. 3-((benzyloxy)methyl)-5-methyl-1-(pyrrolidin-3-ylmethyl)pyrimidine-2,4(1H,3H)-dione
(37): 3-((benzyloxy)methyl)-5-methylpyrimidine-2,4 (1H,3H)-dione (20, 1.5 mmol, 0.52 g), tert-
21

butyl 3-(((methylsulfonyl)oxy)methyl)pyrrolidine-1-carboxylate (30, 0.63 g, 2.25 mol,) and
potassium carbonate (0.42 g, 3 mmol) yielded compound 37 as a colorless gel (0.29 g, 58.3%).
1H NMR (300 MHz, CDCl3) δ: 1.54 (dd, J = 13.91, 6.59 Hz, 1H, pyrrolidin-4a-yl), 1.88 - 1.99
(m, 4H, 5-CH3, pyrrolidin-4b-yl), 2.58 - 2.69 (m, 1H, pyrrolidin-3-yl), 2.77 (dd, J = 11.13, 5.86
Hz, 1H, pyrrolidin-2a-yl), 2.97 - 3.17 (m, 3H, pyrrolidin-2b-yl, pyrrolidin-5-yl), 3.26 (br. s., 1H,
NH), 3.71 (dd, J = 7.62, 2.93 Hz, 2H, CH2, 1-methylene), 4.71 (s, 2H, CH2, benzyl), 5.50 (s, 1H,
CH2, 3-methylene), 7.08 (d, J = 1.17 Hz, 1H, H-6), 7.21 - 7.40 (m, 5H, Ph). 13C NMR (75 MHz,
CDCl3) δ: 13.14 (5-CH3), 29.64 (pyrrolidin-4-yl), 38.74 (pyrrolidin-3-yl), 46.10 (pyrrolidin-5-yl),
50.00 (pyrrolidin-2-yl), 52.37 (1-methylene), 70.84 (3-methylene), 72.38 (CH2, benzyl), 110.12
(C-5), 127.76 (3C, Ph), 128.39 (2C, Ph), 138.16 (Ph), 139.41 (C-6), 151.85 (C-2), 163.82 (C-4).
HRMS (ESI): calculated for [C18H23N3O3 + H]+ , 330.1812; found, 330.1810.
4.2.2.4
3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-ylmethyl)pyrimidine-2,4(1H,3H)-dione
(38): 3-((benzyloxy)methyl)-5-methylpyrimidine-2,4(1H,3H)-dione (20, 0.52 g, 2.1 mmol), tert-
butyl 3-(((methylsulfonyl)oxy)methyl)piperidine-1-carboxylate (31, 0.66 g, 2.25 mmol) and
potassium carbonate (0.44 g, 3.15 mmol) yielded compound 38 as colorless gel (0.26 g, 36%). 1H
NMR (300 MHz, CDCl3) δ: 1.12 - 1.30 (m, 1H, piperidin-4a-yl), 1.53 - 1.84 (m, 3H, piperidin-
4b-yl, piperidin-5-yl), 1.87 - 1.95 (d, J = 1.17 Hz, 3H, 5-CH3), 2.11 (dtd, J = 13.73, 6.68, 6.68,
3.37 Hz, 1H, piperidin-3-yl), 2.49 (dd, J = 12.30, 9.96 Hz, 1H, piperidin-2a-yl), 2.67 (td, J =
11.64, 3.08 Hz, 1H, piperidin-6a-yl), 3.03 - 3.17 (m, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.49
(br. s., 1H, NH), 3.52 - 3.72 (m, 2H, 1-methylene), 4.69 (s, 2H, CH2, benzyl), 5.48 (s, 2H, CH2,
13
3-methylene), 6.97 (d, J = 1.17 Hz, 1H, H-6), 7.19 - 7.38 (m, 5H, Ph). C NMR (75 MHz,
CDCl3) δ: 13.12 (5-CH3), 24.01 (piperidin-5-yl), 27.88 (piperidin-4-yl), 35.49 (piperidin-3-yl),
45.90 (piperidin-6-yl), 48.75 (piperidin-2-yl), 52.19 (1-methylene), 70.90 (3-methylene), 72.41
22

(CH2, benzyl), 110.16 (C-5), 127.78 (Ph), 127.81 (2C, Ph), 128.40 (2C, Ph), 138.10 (Ph), 139.46
(C-6), 151.91 (C-2), 163.80 (C-4). HRMS (ESI): calculated for [C19H25N3O3 + H]+ , 344.1969;
found, 344.1973.
4.2.3 General procedure for the synthesis of final compounds 1 - 12.
A suspension of compound 32 - 38 (1 eq), substituted aromatic aldehyde (1.5 – 2 eq) and sodium
triacetoxyborohydride (1.5 – 3 eq) in 1,2-dichloroethane (~ 0.03 M) was stirred at room
temperature under argon overnight. The reaction mixture was evaporated and dried with oil
pump vacuum for 0.5 h. The residue was purified by silica chromatography (CH2Cl2→90%
CH2Cl2/MeOH in a linear gradient elution) to afford pure intermediate, which was immediately
dissolved with TFA under argon. The reaction mixture was stirred at 72 ℃ for 30 min to 1 h. The
reaction was monitored by HRMS. After cooling to room temperature, the reaction mixture was
concentrated and dried under oil pump vacuum. The residue was dissolved in an equivolumar
mixture of MeCN/t-BuOH/H2O (1 – 2 mL) and purified by preparative liquid chromatography
(Phenomenex Luna C-18 (21.2x250mm), flow rate 17.5 mL/min) using a linear gradient from 10%
MeCN-90% aq. 10 mM ammonium bicarbonate → 100% MeCN over 20 min. After
lyophilization, products 1 – 12 were obtained as a white powder.
4.2.3.1
5-methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione
(1):
Following the general procedure, the use of 3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (34, 85.64 mg, 0.26 mmol), 3-phenoxybenzaldehyde (77.31 mg,
0.39 mmol) and sodium triacetoxyborohydride (110.22 mg, 0.52 mmol) yielded compound 1
1
(28.9 mg, 28.4%). H NMR (300 MHz, DMSO-d6) δ: 1.45 - 1.78 (m, 7H, 5-CH3, piperidin-4-yl,
piperidin-5-yl), 1.97 - 2.24 (m, 2H, piperidin-2a-yl, piperidin-6a-yl), 2.64 - 2.80 (m, 2H,
23

piperidin-2b-yl, piperidin-6b-yl), 3.51 (s, 2H, CH2, 1-benzyl), 4.29 - 4.46 (m, 1H, piperidin-3-yl),
6.89 (d, J = 8.20 Hz, 1H, Ph), 6.94 - 7.17 (m, 5H, Ph), 7.29 - 7.43 (m, 3H, Ph), 7.70 (s, 1H, H-6)
13
11.20 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ: 12.04 (5-CH3), 23.87 (piperidin-5-yl),
27.90 (piperidin-4-yl), 50.98 (piperidin-3-yl), 52.13 (piperidin-6-yl), 56.16 (piperidin-2-yl),
61.39 (CH2, 1-benzyl), 108.61 (C-5), 117.25, 118.55, 118.67, 123.39, 123.84, 129.81, 130.03 (7C,
Ph), 137.96 (C-6), 140.34 (Ph), 150.80 (C-2), 156.62 (Ph), 163.62 (C-4). HRMS (ESI):
calculated for [C23H25N3O3 + H]+, 392.1969; found: 392.1972.
4.2.3.2
5-methyl-1-(1-(4-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione
(2):
Following the general procedure the use of 3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (34, 82.70 mg, 0.251 mmol), 4-phenoxybenzaldehyde (74.70
mg, 0.377 mmol) and sodium triacetoxyborohydride (106.4 mg, 0.501 mmol) yielded compound
1
2 (29.48 mg, 30.0%). H NMR (300 MHz, DMSO-d6) δ: 1.46 - 1.63 (m, 1H, piperidin-5a-yl),
1.63 - 1.80 (m, 6H, 5-CH3, piperidin-4-yl, piperidin-5b-yl), 1.96 - 2.09 (m, 1H, piperidin-2a-yl),
2.19 (t, J = 10.25 Hz, 1H, piperidin-6a-yl), 2.64 - 2.81 (m, 2H, piperidin-2b-yl, piperidin-6b-yl),
3.48 (s, 2H, CH2, 1-benzyl), 4.33 - 4.47 (m, 1H, piperidin-3-yl), 6.92 - 7.02 (m, 4H, Ph), 7.09 -
7.16 (m, 1H, Ph), 7.27 - 7.33 (m, 2H, Ph), 7.34 - 7.42 (m, 2H, Ph), 7.67 - 7.73 (d, J = 3.0 Hz, 1H,
H-6), 11.20 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ: 12.06 (5-CH3), 23.93 (piperidin-5-yl),
28.07 (piperidin-4-yl), 51.04 (piperidin-3-yl), 52.16 (piperidin-6-yl), 56.28 (piperidin-2-yl),
61.30 (CH2, 1-benzyl), 108.64 (C-5), 118.29 (2C, Ph), 118.62 (2C, Ph), 123.42 (Ph), 130.03 (2C,
Ph), 130.42 (2C, Ph), 133.00 (Ph), 138.02 (C-6), 150.83 (C-2), 155.67 (Ph), 156.68 (Ph), 163.65
(C-4). HRMS (ESI): calculated for [C23H25N3O3 + H]+, 392.1969; found: 392.1970.
4.2.3.3 (R)-5-methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (3):
Following the general procedure the use of (R)-3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
24

yl)pyrimidine-2,4(1H,3H)-dione (35, 139.01 mg, 0.422 mmol), 3-phenoxybenzaldehyde (167.30
mg, 0.844 mmol) and sodium triacetoxyborohydride (268.34 mg, 1.266 mmol) yielded
1
compound 3 (45.09 mg, 27.3%). H NMR (300 MHz, DMSO-d6) δ: 1.44 - 1.82 (m, 7H, 5-CH3,
piperidin-4-yl, piperidin-5-yl), 1.96 - 2.11 (m, 1H, piperidin-6a-yl), 2.17 (t, J = 9.81 Hz, 1H,
piperidin-2a-yl), 2.63 - 2.82 (m, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.51 (s, 2H, CH2, 1-
methylene), 4.38 (br. s., 1H, piperidin-3-yl), 6.89 (d, J = 7.91 Hz, 1H, Ph), 6.94 - 7.04 (m, 3H,
13
Ph), 7.04 - 7.17 (m, 2H, Ph), 7.29 - 7.43 (m, 3H, Ph), 7.70 (s, 1H, H-6), 11.20 (s, 1H, NH). C
NMR (75 MHz, DMSO-d6) δ: 12.04 (5-CH3), 23.89 (piperidin-5-yl), 27.92 (piperidin-4-yl),
50.98 (piperidin-3-yl), 52.13 (piperidin-6-yl), 56.20 (piperidin-2-yl), 61.41 (1-methylene),
108.61 (C-5), 117.22 (Ph), 118.55 (2C, Ph), 118.65 (Ph), 123.39 (Ph), 123.83 (Ph), 129.81 (Ph),
130.01 (2C, Ph), 137.96 (C-6), 140.36 (Ph), 150.80 (C-2), 156.62 (2C, Ph), 163.62 (C-4). HRMS
(ESI): calculated for [C23H25N3O3 + H]+, 392.1969; found: 392.1970. ee: 84.8% (Daicel
Chiralpak-IA HPLC column, hexane/ethanol 80/20 as eluent).
4.2.3.4 (R)-5-methyl-1-(1-(4-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (4):
Following the general procedure the use of (R)-3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (35, 139.01 mg, 0.422 mmol), 4-phenoxybenzaldehyde (167.30
mg, 0.844 mmol) and sodium triacetoxyborohydride (268.34 mg, 1.266 mmol) yielded
1
compound 3 (44.10 mg, 26.7%). H NMR (300 MHz, DMSO-d6) δ: 1.43 - 1.81 (m, 7H, 5-CH3,
piperidin-4-yl, piperidin-5-yl), 1.93 - 2.09 (m, 1H, piperidin-6a-yl), 2.17 (t, J = 10.84 Hz, 1H,
piperidin-2a-yl), 2.63 - 2.80 (m, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.47 (s, 2H, CH2, 1-
methylene), 4.38 (br. s., 1H, piperidin-3-yl), 6.88 - 7.03 (m, 4H, Ph), 7.08 - 7.16 (m, 1H, Ph),
7.24 - 7.42 (m, 4H, Ph) 7.69 (s, 1H, H-6), 11.18 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ:
12.06 (5-CH3), 23.92 (piperidin-5-yl), 28.06 (piperidin-4-yl), 51.04 (piperidin-3-yl), 52.16
25

(piperidin-6-yl), 56.27 (piperidin-2-yl), 61.29 (1-methylene), 108.64 (C-5), 118.29 (2C, Ph),
118.64 (2C, Ph), 123.43 (Ph), 130.04 (2C, Ph), 130.44 (2C, Ph), 132.99 (Ph), 138.01 (C-6),
150.83 (C-2), 155.69 (Ph), 156.66 (Ph), 163.65 (C-4). HRMS (ESI): calculated for [C23H25N3O3
+ H]+, 392.1969; found, 392.1972. ee: 90.4% (Daicel Chiralpak-IA HPLC column,
hexane/ethanol 80/20 as eluent).
4.2.3.5 (S)-5-methyl-1-(1-(3-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (5)14a:
Following the general procedure the use of (S)-3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (36, 139.01 mg, 0.422 mmol), 3-phenoxybenzaldehyde (167.30
mg, 0.844 mmol) and sodium triacetoxyborohydride (268.34 mg, 1.266 mmol) yielded
1
compound 5 (45.40 mg, 27.5%). H NMR (300 MHz, DMSO-d6) δ: 1.46 - 1.78 (m, 7H, 5-CH3,
piperidin-4-yl, piperidin-5-yl), 1.98 - 2.10 (m, 1H, piperidin-6a-yl), 2.17 (t, J = 10.25 Hz, 1H,
piperidin-2a-yl), 2.64 - 2.79 (m, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.50 (s, 2H, CH2, 1-
methylene), 4.30 - 4.45 (m, 1H, piperidin-3-yl), 6.89 (ddd, J = 8.13, 2.56, 1.03 Hz, 1H, Ph), 6.94
- 7.04 (m, 3H, Ph), 7.04 - 7.09 (m, 1H, Ph), 7.10 - 7.17 (m, 1H, Ph), 7.30 - 7.43 (m, 3H, Ph), 7.70
(d, J = 1.00 Hz, 1H, H-6), 11.20 (s, 1H, NH). 13C NMR (75 MHz, DMSO-d6) δ: 12.04 (5-CH3),
23.90 (piperidin-5-yl), 27.93 (piperidin-4-yl), 51.00 (piperidin-3-yl), 52.14 (piperidin-6-yl),
56.19 (piperidin-2-yl), 61.43 (1-methylene), 108.59 (C-5), 117.20 (Ph), 118.55 (2C, Ph), 118.64
(Ph), 123.37 (Ph), 123.81 (Ph), 129.80 (Ph), 130.01 (2C, Ph), 137.99 (C-6), 140.36 (Ph), 150.82
(C-2), 156.60 (Ph), 156.63 Ph), 163.62 (C-4). HRMS (ESI): calculated for [C23H25N3O3 + H]+,
392.1969; found: 392.1973. ee: 84.2% (Daicel Chiralpak-IA HPLC column, hexane/ethanol
80/20 as eluent).
4.2.3.6 (S)-5-methyl-1-(1-(4-phenoxybenzyl)piperidin-3-yl)pyrimidine-2,4(1H,3H)-dione (6):
Following the general procedure, (S)-3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
26

yl)pyrimidine-2,4(1H,3H)-dione (36, 130.11 mg, 0.395 mmol), 4-phenoxybenzaldehyde (156.59
mg, 0.79 mmol) and sodium triacetoxyborohydride (251.17 mg, 1.185 mmol) yielded compound
6 (41.78 mg, 27.2%). 1H NMR (300 MHz, DMSO-d6) δ: 1.45 - 1.78 (m, 7H, 5-CH3, piperidin-4-
yl, piperidin-5-yl), 1.96 - 2.08 (m, 1H, piperidin-6a-yl), 2.17 (t, J = 10.40 Hz, 1H, piperidin-2a-
yl), 2.62 - 2.80 (m, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.47 (s, 2H, CH2, 1-methylene), 4.31 -
4.45 (m, 1H, piperidin-3-yl), 6.89 - 7.01 (m, 4H, Ph), 7.08 - 7.15 (m, 1H, Ph), 7.24 - 7.41 (m, 4H,
13
Ph), 7.65 - 7.71 (m, 1H, H-6), 11.18 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ: 12.04 (5-
CH3), 23.92 (piperidin-5-yl), 28.04 (piperidin-4-yl), 51.03 (piperidin-3-yl), 52.14 (piperidin-6-yl),
56.27 (piperidin-2-yl), 61.29 (1-methylene), 108.61 (C-5), 118.27 (2C, Ph), 118.61 (2C, Ph),
123.40 (Ph), 130.03 (2C, Ph), 130.41 (2C, Ph), 132.99 (Ph), 138.01 (C-6), 150.82 (C-2), 155.66
(Ph), 156.65 (Ph), 163.64 (C-4). HRMS (ESI): calculated for [C23H25N3O3 + H]+, 392.1969;
found: 392.1971. ee: 86.4% (Daicel Chiralpak-IA HPLC column, hexane/ethanol 80/20 as
eluent).
4.2.3.7 (R)-5-methyl-1-(1-(3-phenoxybenzyl)pyrrolidin-3-yl)pyrimidine-2,4(1H,3H)-dione (7):
Following the general procedure, (R)-3-((benzyloxy)methyl)-5-methyl-1-(pyrrolidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (32, 157.69 mg, 0.50 mmol), 3-phenoxybenzaldehyde (198.22
mg, 1.00 mmol) and sodium triacetoxyborohydride (211.96 mg, 1.00 mmol) yielded compound 8
1
(53.40 mg, 28.3%). H NMR (300 MHz, DMSO-d6) δ: 1.60-1.80 (m, 4H, 5-CH3, pyrrolidin-4a-
yl), 2.14 - 2.37 (m, 2H, pyrrolidin-4b-yl, pyrrolidin-2a-yl), 2.48 – 2.56 (m, 1H, pyrrolidin-5a-yl),
2.60 - 2.75 (m, 1H, pyrrolidin-5b-yl), 2.89 - 3.03 (m, 1H, pyrrolidin-2b-yl), 3.48 - 3.70 (m, 2H,
CH2, benzyl), 4.87 - 5.01 (m, 1H, pyrrolidin-3-yl), 6.88 (dd, J = 7.91, 1.76 Hz, 1H, Ph), 6.93 -
7.01 (m, 3H, Ph), 7.05 - 7.15 (m, 2H, Ph), 7.28 - 7.40 (m, 3H, Ph), 7.62 (d, J = 1.17 Hz, 1H, H-6),
13
11.15 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ: 12.29 (5-CH3), 30.90 (pyrrolidin-4-yl),
27

52.45 (pyrrolidin-2-yl), 52.74 (pyrrolidin-3-yl), 58.27 (CH2, benzyl), 58.40 (pyrrolidin-5-yl),
109.16 (C-5), 117.28 (Ph), 118.33 (Ph), 118.44 (2C, Ph), 123.35 (Ph), 123.42 (Ph), 129.86 (Ph),
129.98 (2C, Ph), 137.72 (C-6), 141.23 (Ph), 150.77 (C-2), 156.66 (Ph), 163.67 (C-4). HRMS
(ESI): calculated for [C22H23N3O3 + H]+, 378.1812; found, 378.1806. ee: 98.8% (Daicel
Chiralcel ODH HPLC column, hexane/ethanol 80/20 as eluent).
4.2.3.8 (S)-5-methyl-1-(1-(3-phenoxybenzyl)pyrrolidin-3-yl)pyrimidine-2,4(1H,3H)-dione (8):
Following the general procedure, (S)-3-((benzyloxy)methyl)-5-methyl-1-(pyrrolidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (33, 126.15 mg, 0.40 mmol), 3-phenoxybenzaldehyde (158.58
mg, 0.80 mmol) and sodium triacetoxyborohydride (169.57 mg, 0.80 mmol) yielded compound 8
(42.30 mg, 28.0%). 1H NMR (300 MHz, DMSO-d6) δ: 1.64 - 1.83 (m, 4H, 5-CH3, pyrrolidin-4a-
yl), 2.17 - 2.38 (m, 2H, pyrrolidin-4b-yl, pyrrolidin-2a-yl), 2.48- 2.56 (m, 1H, pyrrolidin-5a-yl),
2.64 - 2.77 (m, 1H, pyrrolidin-5b-yl), 2.91 - 3.03 (m, 1H, pyrrolidin-2b-yl), 3.52 - 3.72 (m, 2H,
CH2, benzyl), 4.89 - 5.03 (m, 1H, pyrrolidin-3-yl), 6.90 (dd, J = 8.20, 1.76 Hz, 1H, Ph), 6.95 -
7.03 (m, 3H, Ph), 7.07 - 7.16 (m, 2H, Ph), 7.31 - 7.42 (m, 3H, Ph), 7.64 (d, J = 1.17 Hz, 1H, H-6),
13
11.17 (s, 1H, NH). C NMR (75 MHz, DMSO-d6) δ: 12.30 (5-CH3), 30.89 (pyrrolidin-4-yl),
52.44 (pyrrolidin-2-yl), 52.73 (pyrrolidin-3-yl), 58.26 (CH2, benzyl), 58.40 (pyrrolidin-5-yl),
109.15 (C-5), 117.26 (Ph), 118.31 (Ph), 118.43 (2C, Ph), 123.35 (Ph), 123.39 (Ph), 129.85 (Ph),
129.97 (2C, Ph), 137.71 (C-6), 141.24 (Ph), 150.76 (C-2), 156.66 (Ph), 163.66 (C-4). HRMS
(ESI): calculated for [C22H23N3O3 + H]+, 378.1812; found, 378.1806. ee: 99.2% (Daicel
Chiralcel ODH HPLC column, hexane/ethanol 80/20 as eluent).
28

4.2.3.9 5-methyl-1-((1-(3-phenoxybenzyl)piperidin-3-yl)methyl)pyrimidine-2,4(1H,3H)-dione (9):
Following the general procedure, 3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-yl)pyrimidine-
2,4(1H,3H)-dione (38, 102.00 mg, 0.297 mmol), 3-phenoxybenzaldehyde (88.41 mg, 0.446
mmol) and sodium triacetoxyborohydride (125.9 mg, 0.594 mmol) yielded compound 9 (37.58
mg, 31.2%).1H NMR (300 MHz, DMSO-d6) δ: 0.88 - 1.07 (m, 1H, piperidin-4a-yl), 1.28 - 1.46
(m, 1H, piperidin-5a-yl), 1.48 - 1.68 (m, 2H, piperidin-4b-yl, piperidin-5b-yl), 1.68 - 2.05 (m, 6H,
5-CH3, piperidin-2a-yl, piperidin-6a-yl), 2.58 (d, J = 9.67 Hz, 2H, piperidin-2b-yl, piperidin-6b-
yl), 3.34 - 3.60 (m, 4H, 2CH2, 1-methyl, phenoxybenzyl-), 6.83 - 7.07 (m, 5H, Ph), 7.09 - 7.17 (m,
13
1H, Ph), 7.27 - 7.42 (m, 3H, Ph), 7.44 (s, 1H, H-6), 11.19 (s, 1H, NH). C NMR (75 MHz,
DMSO-d6) δ: 11.91 (5-CH3), 23.93 (piperidin-5-yl), 27.22 (piperidin-4-yl), 35.35 (piperidin-3-
yl), 50.19 (CH2, 1-methylene), 53.38 (piperidin-6-yl), 56.51 (piperidin-2-yl), 61.99 (CH2,
phenoxybenzyl-), 108.24 (C-5), 117.00 (Ph), 118.47(Ph), 118.61 (2C, Ph), 123.40 (Ph), 123.72
(Ph), 129.67 (Ph), 130.03 (2C, Ph) 140.92 (Ph), 141.61 (C-6), 151.05 (C-2), 156.62 (Ph), 156.65
(Ph), 164.17 (C-4). HRMS (ESI): calculated for [C24H27N3O3 + H]+, 406.2125; 406.2142.
4.2.3.10
5-methyl-1-((1-(4-phenoxybenzyl)piperidin-3-yl)methyl)pyrimidine-2,4(1H,3H)-dione
(10): Following the general procedure, 3-((benzyloxy)methyl)-5-methyl-1-(piperidin-3-
yl)pyrimidine-2,4(1H,3H)-dione (38, 118.48 mg, 0.345 mmol), 4-phenoxybenzaldehyde (136.77
mg, 0.69 mmol), sodium triacetoxyborohydride (219.38 mg, 1.035 mmol) yielded compound 10
(44.10 mg, 32.7%). 1H NMR (300 MHz, DMSO-d6) δ: 0.89 - 1.06 (m, 1H, piperidin-4a-yl), 1.29
- 1.46 (m, 1H, piperidin-5a-yl), 1.48 - 1.67 (m, 2H, piperidin-4b-yl, piperidin-5b-yl), 1.67 - 1.82
(m, 4H, 5-CH3, piperidin-2a-yl), 1.83 - 2.02 (m, 2H, piperidin-3-yl, piperidin-6a-yl), 2.57 (d, J =
8.20 Hz, 2H, piperidin-2b-yl, piperidin-6b-yl), 3.34 - 3.54 (m, 4H, 2CH2, 1-methyl,
phenoxybenzyl-), 6.88 - 6.99 (m, 4H, Ph), 7.07 - 7.14 (m, 1H, Ph), 7.22 - 7.28 (m, 2H, Ph), 7.33
29