Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors

Article abstract of DOI:10.1016/j.ejmech.2017.06.024

The α₄βδ subtype of the γ-aminobutyric acid (GABA) type A receptors (GABA_ARs) has been shown to be implicated in high-affinity binding of the neuromodulator γ-hydroxybutyric acid (GHB), but may not be the only GHB high-affinity bindi

Full text of DOI:10.1016/j.ejmech.2017.06.024

Accepted Manuscript
Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the
development of modulators for GHB high-affinity binding sites and α β δ GABA
receptors
1 2
A
Maria Damgaard, Anas Al-Khawaja, Mia Nittegaard-Nielsen, Rebekka F. Petersen,
Petrine Wellendorph, Bente Frølund
PII:
S0223-5234(17)30470-1
10.1016/j.ejmech.2017.06.024
EJMECH 9519
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 March 2017
Revised Date: 19 May 2017
Accepted Date: 14 June 2017
Please cite this article as: M. Damgaard, A. Al-Khawaja, M. Nittegaard-Nielsen, R.F. Petersen, P.
Wellendorph, B. Frølund, Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the
development of modulators for GHB high-affinity binding sites and α β δ GABA receptors, European
1 2
A
Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.06.024.
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ACCEPTED MANUSCRIPT
OH
O
GHB high-affinity
binding modulators
GABAA receptor
modulators
O
NH
N
S
OH
OH/H
H
Monastrol, 1
O
O
O
O
NH
NH
N
H
S
N
H
O
29
34,37

ACCEPTED MANUSCRIPT
Monastrol, a 3,4-dihydropyrimidin-2(1H)-thione, as structural scaffold for the development of
modulators for GHB high-affinity binding sites and α1β2δ GABAA receptors
Maria Damgaard, Anas Al-Khawaja, Mia Nittegaard-Nielsen, Rebekka F. Petersen, Petrine
Wellendorph* and Bente Frølund*
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University
of Copenhagen, Denmark
* Corresponding authors: Chemistry: Bente Frølund, phone: +45 35 33 64 95 e-mail:
bfr@sund.ku.dk. Pharmacology: Petrine Wellendorph, phone: +45 35 33 63 97, e-mail:
pw@sund.ku.dk.
1

ACCEPTED MANUSCRIPT
Abstract
The α4βδ subtype of the γ-aminobutyric acid (GABA) type A receptors (GABAARs) has been
shown to be implicated in high-affinity binding of the neuromodulator γ-hydroxybutyric acid
(GHB), but may not be the only GHB high-affinity binding sites. Monastrol has been identified as a
modulator of GHB high-affinity binding and is furthermore reported as an allosteric modulator
selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two
targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing
the Biginelli cyclocondensation and examined for modulation of GHB high-affinity binding using
the GHB-specific ligand [3H]NCS-382 [(E,RS)-6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-
ylidene)acetic acid] in rat brain homogenate. Only limited modifications were allowed on the
monastrol scaffold in order to maintain modulation of GHB high-affinity binding. However, three
analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [3H]NCS-382 to a higher
maximal level than seen for monastrol itself. Selected compounds were further characterized as
modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have δ-
specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB
high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and
24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a
GHB high-affinity binding preferring modulator. However, compounds 34 and 37 were shown to be
α1β2δ GABAAR selective modulators, without modulatory effects on GHB high-affinity binding.
Thus, our study shows that minor modifications in the structure of monastrol affects the selectivity
profile for the two targets under study enabling separation of the dual activity.
Keywords: max 6
GHB high-affinity binding sites, GABAA receptor, allosteric modulators, monastrol analogues,
Biginelli cyclocondensation
Highlights (3-5 bullet points (max 85 characters))
-
-
-
39 structural diverse monastrol analogues were synthesized
Structural determinants for modulatory activity and selectivity were identified
Modulators selective for α1β2δ GABAAR and high-affinity GHB binding were identified
2

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1. Introduction
γ-Hydroxybutyric acid (GHB) is a small endogenous compound proposed to be a neuromodulator in
the mammalian brain.1 GHB is both chemically and pharmacologically related to γ-aminobutyric
acid (GABA), the main inhibitory neurotransmitter in the brain. GHB was first synthesized as a
brain permeable GABA analogue in the 1960’s2 and simultaneously GHB was found to be a
naturally occurring metabolite.3 GHB has a complex pharmacology, comprising both high- and
low-affinity binding sites.1, 4 GABAB receptors are well-established low-affinity binding sites for
GHB at which GHB acts as a weak partial agonist.5, 6 Many of the effects observed with
exogenously administered GHB have been ascribed to the GABAB receptors, e.g. hypothermia,
dopamine release and sedation.4, 7, 8, 9, 10 The α4βδ GABAA receptors (GABAARs) have, however,
been shown to be implicated in GHB high-affinity binding11 and were shown to account for up to
40% of the GHB high-affinity binding, whereas the remaining 60% of GHB high-affinity binding
sites remain elusive.1
The GABAARs are pentameric ligand-gated chloride ion-channels. They can be assembled from 19
12
different GABAAR subunits α1-6, β1-3, γ1-3, δ, ε, θ, π, and ρ1-3 forming receptors showing distinct
function and distribution depending of the subunit composition.13 Synaptic GABAARs are
predominantly composed of α1, α2, and/or α3 in combination with β2/3 and γ2 subunits, the α1β2γ2
representing the major subtype, whereas δ-containing GABAAR have been shown to be located
extrasynaptically and mediate tonic inhibition.13-14 Specifically, α1/δ-containing GABAAR have
been shown to be present in hippocampal interneurons, possibly mediating high sensitivity towards
ethanol.15 α1β2δ receptors have been suggested to be silent receptors that upon modulation by
endogenous neurosteroids could exert profound inhibition.16
Selective ligands targeting the orthosteric GHB high-affinity binding sites are highly useful
pharmacological tools for studying these in isolation.1 NCS-382 is representing a structural class of
GHB analogues showing selectivity and high nanomolar affinity for the GHB high-affinity binding
sites.17 So far, the main focus has been on the development of GHB-like ligands for targeting the
orthosteric GHB high-affinity binding sites and only recently allosteric modulators have been
reported.18
Allosteric modulators have, on the other hand, been widely used to target and study the GABAARs
including benzodiazepines, barbiturates, steroids and naturally occurring compounds. Most
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GABAAR modulators are non-selective towards the GABAAR subtypes19 but a few compounds
have been shown to have some selectivity towards the δ-containing receptors. DS1, DS220,
AA2950421,
ethyl
4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (monastrol, 1) and the monastrol analogue JM-II-43A22 are such compounds. More
specifically, the synthetic compound monastrol is reported to selectively modulate the α1β2δ
GABAAR subtype.22 Recently, monastrol and catechin were identified as the first GHB high-
affinity allosteric modulators in a library screening of natural compounds and known GABAAR
modulators.18 The aim of this study was to dissect the structural determinants of monastrol for
modulating GHB high-affinity binding and α1β2δ GABAAR activity, respectively, assuming these
effects are mediated via different molecular targets.
O
HO
a.
OH
O
O
HO
H2N
OH
OH
GHB
GABA
NCS-382
S
NH2
H
N
N
O
N
HN
O
O
N
H
Cl
AA29504
DS2
OH
O
O
O
NH
O
NH
N
S
N
O
H
H
Monastrol, 1
JM-II-34, 37
b.
O
R1
R1 = aryl, Me
R2 = H, Me
R3,4 = alkyl, Ph
R5 = OH, OMe, OBn, NEt2, Me, Et
X = O,S
R2
X
R5
N
R4
N
R3
2–41
Figure 1. a Structures of GHB, GABA, NCS-382, and the δ selective GABAA receptors modulators
DS2, AA29504, monastrol (1) and JM-II-34 (37). B General structure of the target monastrol
analogues (2-41).
2. Results
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2.1. Chemistry
A series of monastrol analogues were synthesized to elucidate the structure-activity relationship
(SAR) for the modulatory effect of monastrol on GHB high-affinity binding and α1β2δ GABAAR
activity. The structural design of analogues was based on systematic investigation of different parts
of the 3,4-dihydropyrimidin-2(1H)-thione scaffold constituting the core scaffold of monastrol
(Figure 1): the aromatic ring system and substituents (R1), the urea moiety (X, R2, R3), the methyl
substituent (R4) and the acyl substituent (R5).
The synthetic procedures for the synthesis of compounds 1-41 are depicted in Scheme 1.
Compounds 1-11, 13, 14, 17, 19-21, 25, 28-30, 32-34 and 36-37 were synthesized according to
modified procedures previously described in the literature (please refer to Experimental Section for
further information). The Biginelli three component reaction involving cyclocondensation of a 1,3-
dicarbonyl, an aromatic aldehyde and urea or thiourea23, 24 was used to synthesize the target
compounds as racemic mixtures. Analogues (1-17, 24-39) differing in the R1, R4, R5 and X regions
were synthesized using a microwave-mediated Biginelli reaction employing ytterbium(III)
trifluoromethanesulfonate (Yb(OTf)3) to catalyze the reaction.25 The yields varied greatly
depending on the substituents. This procedure showed not to be applicable for N-substituted
thioureas. Instead, analogues 18-23 were synthesized by a modified Biginelli reaction using TMSCl
to catalyze the reaction of 3-hydroxybenzaldehyde, ethyl acetoacetate and N-substituted thioureas.26
The bicyclic product 41 was synthesized by TBDMS-protection of the phenol group of monastrol to
give 40, thereafter cyclization by the use of n-BuLi and 1,2-dibromoethane to obtain the cyclized
product, subsequent deprotection with MeONa gave the desired compound 41 as previously
described for similar compounds.27 The two enantiomers of monastrol were separated on a
Chiralpak IF semi-prep column (10x250 mm, 5µm), using a flow of 6 ml/min, injection size of app.
5 mg and heptane/2-propanol (90:10) as the mobile phase. Identification of the enantiomers was
confirmed by optical rotation, comparable to literature values.28
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Scheme 1. Synthesis of monastrol analogues 1-41a
aReagents and conditions: (a) Yb(OTf)3 cat, THF or MeCN, 120 °C (microwave), 1-97%, (b)
TMSCl, DMF, rt, 5-80%, (c) TBDMSCl, imidazole, DMF, 80 %, (d) 1. N-BuLi, THF, -10°C, 2.
BrCH2CH2Br, THF, -10°C, 8 %, (e) MeONa, MeOH, 28 %.
2.2. Pharmacology
2.2.1. Modulation of GHB high-affinity binding
The synthesized monastrol analogues were initially pharmacologically characterized using the well-
known [3H]NCS-382 binding assay on rat cortical membranes29 to investigate their modulatory
effect on the GHB high-affinity binding, similar to previously reported for monastrol.18 Table 1
gives an overview of the synthesized compounds and their ability to enhance [3H]NCS-382 binding.
In the majority of cases, structural modifications of the lead compound, monastrol (1), led to loss of
modulatory effect on the high-affinity GHB binding, i.e. no enhancement of [3H]NCS-382 binding
was observed. However, of the 39 synthesized analogues, six compounds (11, 12, 18, 24, 25, 29)
did show modulatory effects to the same or higher level than monastrol (1) itself (Table 1).
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Interestingly, in terms of the modulatory capacity, no apparently stereoselectivity was seen for
monastrol as both stereoisomers increased the cooperativity factor (
the racemate.
α value) to a similar extent as
Table 1. Modulatory effects of monastrol (1) and the synthesized analogues (2–41) on [3H]NCS-
382 binding (rat cortical membranes). The cooperativity factor (α) and the dissociation constant of
the modulator (Kb) were estimated by curve fitting (see Experimental section). Average data are
based on triplicate measurements from at least three independent experiments, except for the
stereoisomers of monastrol, which were tested only twice.
OH
OH
N
N N
N
N
R1
O
O
O
O
R5
R4
R2
X
N
O
NH
O
NH
O
NH
O
NH
N
N
H
S
N
H
S
N
S
N
S
R3
H
Monastrol, 1
2-39
15
16
41
Compound
Chemical
Kb (µM)
(pKb ± SEM) ± SEM (% of control)
α ± SEM
Maximal binding
α
modification
(log
2.00
(0.31 ± 0.05) (3.96 ± 0.16)
2.35 111
(0.37 ± 0.02) (3.95 ± 0.07)
1.87 329
Monastrol, 1a
(S)-monastrolb
(R)-monastrolb
110
185 ± 7.1
208 ± 12
171 ± 5.3
(0.27 ± 0.02) (3.48 ± 0.11)
R1 modification
R1 = Ph
NA
NA
NA
-
NA
NA
NA
-
2
R1 = 3-Me-C6H4
R1 = 4-Me-C6H4
R1 = 2-Me-C6H4
3
4
5c
7

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6c
7
R1 = 3-Br-C6H4
-
-
R1 = 3-F-C6H4
NA
NA
NA
NA
210
NA
R1 = 3-Cl-C6H4
R1 = 3-NO2-C6H4
R1 = 4-OH-C6H4
R1 = 3,4-OH-C6H3
NA
8
NA
9
NA
10
11e
2.62
246 ± 21
(0.42 ± 0.03)* (3.68 ± 0.02)
12e
R1 = 3,5-OH-C6H3
3.06 134
247 ± 3.0
(0.49 ± 0.01)** (3.83 ± 0.06)
R1 = 3-pyridine
R1 = 3-OMe-C6H4
R1 = Het.cycle
R1 = Het.cycle
R1 = Me
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
13
14
15
16
17
R2 and R3 modifications
R3 = Me
1.97
87
177 ± 1.3
18
(0.29 ± 0.01) (4.06 ± 0.01)
R3 = Et
WA
NA
NA
NA
NA
WA
NA
NA
NA
NA
19
20
21
22
23
R3 = Bu
R3 = Ph
R3 = Bn
R2 = R3 = Me
R4 modification
R4 = Et
2.66
109
245 ± 13
24
(0.42 ± 0.04) (3.98 ± 0.08)
25d
26
27
28
R4 = Pr
R4 = iPr
R4 = tBu
R4 = Ph
-
-
198 ± 17
WA
WA
WA
NA
WA
NA
R5 modification
29e
R5 = CO2Me
R5 = CO2H
2.37
347
194 ± 3.7
NA
(0.38 ± 0.01)* (3.46 ± 0.06)
NA
30
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R5 = CO2Bn
R5 = CONEt2
R5 = COCH3
NA
NA
WA
NA
NA
WA
31
32
33
Mixed profile
X = O
NA
NA
NA
NA
NA
WA
WA
NA
34
35
36
37
38
39
41
R3 = Me, R5 = CO2Me
X = O, R1 = Ph
NA
NA
NA
WA
WA
NA
X = O, R1 = Ph, R5 = CO2Me
R4 = Et, R5 = COCH2CH3
R4 = CH2OCH3, R5 = CO2Me
R3, R4 =(CH2)3-
NA: no activity (defined as compounds that did not significantly increase the binding compared to
control; student’s t-test; data not shown); WA: weak activity (defined as compounds that only
enhanced the binding to max. 130% at 100 µM and consequently not tested further).
a Data from ref18
b In one of the experiment for (R)-monastrol, the plateau of the concentration-response curve was
not fully reached. Due to shortage of compounds, the pooled data were therefore used in a similar
manner to fit both (S) and (R)-stereoisomers of monastrol. Hence, no statistical analysis was
conducted on these values. Using this method did not change the values for (S)-monastrol.
c Compound insolubility precluded testing.
d Due to solubility problems, the plateau of the concentration-response curve was not reached.
Consequently the data could not be analyzed by nonlinear regression. Therefore, no value for α or
Kb was obtained for this compound.
e log
α means were compared and analyzed statistically by one-way ANOVA followed by Dunnett’s
multiple comparison test with significance levels indicated; * p<0.05 and **p<0.01.
The 3-hydroxyphenol part of monastrol was found to be crucial for modulation of [3H]NCS-382
binding. Any variation of the aromatic substitution pattern of the 4-aryl moiety (2–10, 13–17) was
shown to be detrimental for the enhancement of [3H]NCS-382 binding. However, the 3,4-, 3,5-
dihydroxy and C6-ethyl analogues, 11, 12 and 24 enhanced the maximal binding of [3H]NCS-382
to a higher maximal level than seen for monastrol (1) itself (Figure 2 and Table 1). The effect seen
for the 3-hydroxy containing compounds could not be achieved by substituting the 3-hydroxy group
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for a pyrazol or triazole moiety, 15 and 16, set up for accepting a hydrogen bond in the
corresponding position.
Figure 2. Concentration-dependent modulation of [3H]NCS-382 (16 nM) binding to rat cortical
membranes by monastrol (1) and selected analogues (12 and 32). Each point is mean ± S.D. of
triplicate measurements of a single representative experiment with at least two additional
experiments giving similar results. Average
α and Kb values are summarized in Table 1.
Introduction of a methyl group on the N1 nitrogen in the 3,4-dihydropyrimidin-2(1H)-thione
scaffold was allowed (18) whereas larger substituents such as an ethyl (19), a butyl (20) and a
phenyl (21) group abolished the modulatory effect. Neither introduction of an additional methyl
group in the N3 nitrogen (23), nor cyclisation of the allowed side chains in the 6- and N1-position
(41) retained modulatory effect. Substitution of the methyl group at the 6-position of the 3,4-
dihydropyrimidin-2(1H)-thione scaffold by ethyl (24) led to a compound that increased the
maximal binding of the radioligand to a higher level than monastrol (1). A propyl in this position
(25) also retained modulatory effect, but larger alkyl groups (26–27) or a phenyl group (28)
abolished activity.
Furthermore, only small modifications in the ester group was accepted, where the methyl ester (29)
did show modulatory effect, but could not be replaced by the corresponding benzylic ester (31),
carboxylic acid (30) or diethylamide (32). Interestingly, exchanging the thiocarbonyl group at the 2-
position in monastrol for an oxocarbonyl (34) was detrimental for activity.
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Overall, these results show that only minor modifications are allowed to retain the modulatory
effect at high-affinity GHB binding. A summary of the allowed modifications in the core scaffold
of monastrol is shown in Figure 3.
R1
R1 = 3-OH, 3,4-OH, 3,5-OH
Thiocarbonyl essentiel
O
R5 = OMe, OEt
R4 = Et, Pr
R5
NH
R4
N
S
R3 = Me
R3
Figure 3. Representative structure of monastrol and the allowed modifications that retain the
modulatory properties on GHB high-affinity binding.
2.2.2. GABA-potentiating effects of selected compounds
Monastrol (1) and compound 37 (JM-II-43A) have previously been shown to selectively modulate
the α1β2δ GABAAR.22 Therefore, monastrol (1), 37 and five other analogues (11, 18, 24, 29 and 34)
were further characterized on different GABAAR subtypes representing the synaptic (α1β2γ2s) and
extrasynaptic (α1β2δ) GABAARs and on α1β2 GABAARs for comparison, expressed in Xenopus
laevis oocytes, by use of the two-electrode voltage clamp technique (TEVC). Compounds 11, 18,
24 and 29 were chosen based on the observed modulatory effect on the GHB high-affinity binding,
whereas 34, the oxycarbonyl analog of monastrol, was selected based on previous report on
modulatory effect of 37 on α1β2δ GABAA receptors.22
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α1β2
A
α1β2δ
B
GABA
GABA
1
37
34
11
24
29
18
DS2
1
37
34
11
24
29
18
100 nA
60 s
200 nA
2 min
α1β2γ2S
α1β2δ
C
D
GABA
GABA
18 29 24 11 34 37
1
DS2
1
37
34
11
24
29
18
100 nA
60 s
200 nA
2min
Figure 4. Pharmacological characterization of monastrol (1) and analogues (11, 18, 24, 29, 34, and
37) at human α1β2δ (5:1:5), α1β2 (1:1) and α1β2γ2s (5:1:5) GABAA receptors expressed in
Xenopus laevis oocytes. Representative traces showing the effect of 1 mM of monastrol and
analogues on currents evoked by 1 mM GABA at α1β2δ (A), α1β2 (B) and α1β2γ2s (C) GABAA
receptors. Monastrol and the analogues showed no or only minimal agonistic effects in a
concentration of 1 mM at α1β2δ receptors (D). DS2 is a positive allosteric modulator of the δ
subunit-containing GABAA receptors and was applied to confirm the expression of the δ subunit.
Data are from 4 to 15 oocytes from at least two different oocyte batches.
Compounds 1, 11, 18, 24, 29, 34, and 37 were tested at a concentration of 1 mM for their effect on
the currents evoked by 1 mM GABA at the human α1β2δ, α1β2γ2s and α1β2 GABAAR. This
concentration was previously used for monastrol (1) as a modulator of α1β2δ GABAARs.22 At the
α1β2δ receptors, all compounds seemed to increase the amplitude of the GABA-mediated current.
The thiocarbonyl-containing compounds (1, 11, 18, 24, and 29) showed pronounced rapid rebound
currents upon termination of compound application (Figure 4A), the amplitudes of which were,
however, not considered feasible to permit quantification. The amplitudes of the initial responses
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are given in Table 2 together with the amplitudes for compounds 34 and 37, lacking the
thiocarbonyl group and showing no tail responses.
The GABA-potentiating effects were δ-specific, as the compounds did not potentiate the GABA
currents at the α1β2 (Figure 4B) as well as at the α1β2γ2s (Figure 4C) GABAARs. In fact, tendencies
for reduced GABA responses appeared especially for monastrol (1). Furthermore, all compounds
had no or only minimal agonistic effects on their own (Figure 4D).
Table 2. Effects of 1 mM monastrol (1) and 6 analogues (11, 18, 24, 29, 34, and 37) on the
response of 1 mM GABA at human α1β2δ (5:1:5) GABAA receptors expressed in X. laevis oocytes.
Compounds 11, 18, 24 and 29 gave rebound effects upon termination of compound application. The
initial response for all compounds is given as fold current increase compared to the response of 1
mM GABA (mean ± S.E.M., n=6).
Fold current increase
(IA/Icontrol ± S.E.M.)
Initial response
2.9 ± 0.27
2.7 ± 0.28
3.1 ± 0.54
1.4 ± 0.22
1.3 ± 0.22
6.2 ± 0.43
5.9 ± 0.41
Monastrol, 1
11
18
24
29
34
37
To further investigate the rebound current observed for some of the compounds, the concentration-
response relationship of monastrol (1), which showed the most pronounced rebound trace, was
examined (Figure 5).
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Figure 5. Representative traces showing the effects of varying concentrations of monastrol (1) on
currents evoked by 1 mM GABA at human α1β2δ (5:1:5) GABAA receptors expressed in Xenopus
laevis oocytes. Data are from 6 to 9 oocytes from two different oocyte batches.
At a concentration of 100 µM and below, no rebound current for monastrol (1) was observed, while
the rebound currents were present and increased in amplitude with higher concentrations,
suggesting distinct concentration-dependent receptor effects.
3. Discussion
The dihydropyrimidin-2(1H)-thione monastrol is a well-studied molecule, originally described as a
kinesin inhibitor arresting mammalian cell mitosis.30 Later, Lewis et al. showed that monastrol
potentiated GABA currents in recombinant α1β2δ GABAAR expressed in HEK293T cells to a
similar extent as phenobarbital.22 Furthermore, monastrol was found to significantly reduce
[3H]NCS-382 dissociation rates and induce conformational changes in the high-affinity GHB
binding site, demonstrating a positive allosteric modulation of radioligand binding.18
Considering the dual activity of monastrol as a δ-selective GABAAR allosteric modulator22 and a
modulator of GHB high-affinity binding18, monastrol comprises an interesting lead compound in an
effort to separate the structural determinants for these modulatory effects. Furthermore, the single
step Biginelli three-component reaction23 enables efficient synthesis of a considerable number of
compounds covering a systematic SAR study on the 3,4-dihydropyrimidin-2(1H)-thione scaffold of
monastrol.
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Based on the obtained SAR for the monastrol analogues of the present study in modulating GHB
high-affinity binding and α1β2δ GABAA receptor activity, the structural determinants for effect
differ for the two targets. 41 compounds were characterized for modulation of GHB high-affinity
binding at rat cortical membranes in a [3H]NCS-382 binding assay. For retaining modulatory effect
of GHB high-affinity binding only very limited modifications were allowed on the structural
scaffold of monastrol. Variation of the aromatic substitution pattern of the 4-aryl moiety in 4-aryl-
3,4-dihydropyrimidin-2(1H)-thione was not tolerated. However, a few compounds did enhance the
level of modulation of [3H]NCS-382 binding. The 3,4- and 3,5-dihydroxy analogues, 11 and 12, and
compound 24 with an ethyl C6 side chain enhanced the maximal binding of [3H]NCS-382 to a
higher maximal level than seen for monastrol (1) itself. Also, the thiourea substituent was crucial
for the ability to enhance [3H]NCS-382 maximal binding.
A limited number of compounds (1, 11, 18, 24, 29, 34, 37) were characterized in different
GABAAR subtypes expressed in Xenopus laevis oocytes, representing synaptic and extrasynaptic
α1β2-containing GABAARs. The tested compounds were shown to modulate the α1β2δ subtype
selectively over α1β2 and α1β2γ2s GABAARs. In contrast to what has been reported for monastrol at
α4β3δ receptors expressed in Xenopus laevis oocytes18, no direct agonist activity on α1β2δ receptors
in the absence GABA was observed for any of the tested compounds. At the α1β2δ GABAAR,
GABA showed a low efficacy compared to that at α1β2 and the α1β2γ2s receptors (as exemplified in
Figure 4), which is in agreement with a previous TEVC study.16
The α1β2δ selective GABAA modulation displayed by monastrol (1) and 37 is consistent with
previous reports,22 however, 37 seems to be a more potent modulator in our hands.
In addition to the increase in the overall current amplitude, a significant rebound current was seen
for monastrol (1) and the thiocarbonyl containing analogues 11, 18, 24 and 29 at the end of the
application period, which correlates with previous findings for monastrol.22 It is difficult to explain
the exact mechanism for these effects other than to suggest that the compound could bind with
different affinities stabilizing distinct conducting states of the receptor. Rebound currents at
GABAARs have previously been reported for the barbiturate, pentobarbital31, which blocks the
receptors in millimolar concentrations and potentiates the GABA response at lower concentrations.
Hence, the large tail observed upon termination of pentobarbital application has been suggested to
reflect recovery from receptor block when the compound is washed away, which is translated into a
repopulation of the conducting state of the channel.31 Assuming a similar mechanism for monastrol
15

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(1) and its analogues (11, 18, 24, 29), the amplitude of the tail response, which reflects recovery
from block, could then be used as a measure of the modulatory effect of the compounds. However,
there are at least two caveats for quantifying the magnitude of these currents: 1) Since the
conventional TEVC technique utilized in this study does not allow for the accurate recording of the
very rapid currents exemplified in Figure 4, the amplitudes of the rebound traces shown in the
figure could be underestimated. Therefore, a fast application and recording system would have been
necessary. 2) Because the rebound effect is seen immediately after switching from compound
application to washing buffer, the exact concentration of the compound giving rise to the
modulatory effect is not known. Therefore, a direct comparison between the different compounds
would not be possible. In general, the oxocarbonyl containing compounds, 34 and 37, were shown
to be more effective than the thiourea analogues, monastrol (1), 11 and 18, as modulators of α1β2δ
GABAAR activity.
In the present study, selective modulators for each of the studied targets as well as dual active
modulators were identified. The thiocarbonyl containing analogue 29 displaying a low degree of
GABAAR modulation was shown to be a GHB high-affinity binding preferring modulator.
Furthermore, compound 11 proved to modulate GHB high-affinity binding to a higher extent than
monastrol (1) and was shown to be a less potent modulator at α1β2δ GABAARs than monastrol (1).
This indicates that GHB high-affinity binding sites and at α1β2δ GABAARs are separate molecular
targets. Leaving room for further development of GHB selective modulators based on the 3,4-
dihydropyrimidin-2(1H)-thione scaffold. A pronounced preference for the oxocarbonyl containing
analogues for modulation of α1β2δ GABAA receptor activity was observed, making compounds 34
and 37 selective for this specific target. Furthermore, apart from monastrol (1), three dual active
compounds were identified, 11, 18, and 24 being modulators of both GHB high-affinity binding and
α1β2δ GABAAR activity.
In general, dihydropyrimidin-2(1H)-ones or -thiones represents a class of heterocylic compounds
with significant pharmacological efficiency. They exhibit a diverse pharmacological profile like
calcium channel modulation, α1a-adrenoreceptor antagonism, antibacterial, antifungal and other
related properties.32 In this study, the synthesized monastrol analogues were only examined for
modulation of high-affinity GHB binding and activity of specific GABAAR subtypes. However, the
structural determinants reported for activity for the abovementioned targets, especially the absolute
stereochemistry at the C4 stereocenter and the hetero substituent at the 2-position, seem to differ
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from the pattern found for the targets of the present study. For instance, the (S)-monastrol has been
established as the effective enantiomer of monastrol regarding kinesin inhibition.33 As demonstrated
here, both enantiomers of monastrol seem to be modulators of GHB high-affinity binding. This
leaves some room for selectivity for the (R)-enantiomer in this context. In addition, substituting
oxygen for sulfur at position 2 results in the loss of kinesin inhibition, which differ from the
structural preference for α1β2δ GABAAR activity.33
In conclusion, a series of structural diverse analogues of monastrol has been synthesized by
employing the Biginelli cyclocondensation. These analogues have been pharmacologically
evaluated as modulators at two distinct targets: GHB high-affinity binding sites and α1β2δ
GABAARs. This SAR study highlighted some of the functional groups contributing to the
modulatory effect and selectivity for each target. For instance, the nature of the heteroatom in 2-
position was determinant for selectivity for either of the targets as shown for compounds 34, 37 and
24. The dual effect shown for monastrol (1) to positively modulate both GHB high-affinity binding
and α1β2δ GABAAR activity was also shown for the compounds 11 and 18 in the present study.
Thus, our study provides structural information for further development of the 4-aryl-3,4-
dihydropyrimidin-2(1H)-thione scaffold into compounds with higher modulatory activity and
selectivity towards the targets of the present study and potentially enlightening some of the
unknown factors regarding binding site localization and function.
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4. Experimental section
4.1. Chemistry
4.1.1. General methods
Starting material, reagents and solvents were obtained from commercial suppliers and used without
further purification unless otherwise specified. Dry DMF were obtained by using a solvent
purification system. Reactions involving air- and moisture sensitive reagents were performed under
nitrogen atmosphere using syringe-septum cap techniques and oven dried glassware. Analytical
thin-layer chromatography (TLC) was carried out using Merck silica gel 60 F254 plates and
visualized using UV light (254 nm), KMnO4 or ninhydrin spraying reagents. LC-MS was performed
using a Agilent 1100 HPLC systems with a XBridge 3.5 µm C-18 column (100 x 4.60 mm) using
gradient elution from buffer A (H2O:CH3CN:HCOOH, 95:5:0.1) to buffer B (H2O:CH3CN:
HCOOH, 5:95:0.05) over 8 min, coupled to an Hewlett Packard 1100 series mass spectrometer with
an electrospray ionization source. Analytical HPLC (Anal. HPLC) was performed using a LaChrom
HPLC system by Merck (Darmstadt, Germany), consisting of an L-7100 pump (4.0 mL/min), an L-
7200 autosampler, and an L-7400 UV-detector (254 nm) using a Chromolith SpeedROD RP-18
column (50 ´ 4.6 mm); gradient elution, 0 to 100 % solvent B (MeCN-H2O-TFA 90:10:0.1) in
solvent A (H2O-TFA 100:0.1) over 3.5 min. Data were acquired and processed using the
EZChromElite Software by Hitachi. All microwave reactions were carried out in a glass reactor
using a Biotage Initiator instrument. Dry column vacuum chromatography (DCVC) was performed
according to Pedersen and Rosenbohm34 using Merck silica gel 60 (0.015-0.040 mm). Melting
points were determined by OptiMelt from Stanford Research Systems in open capillary tubes and
1
are uncorrected. H-NMR and 13C-NMR spectra were recorded on a 400 MHz Bruker Avance
spectrometer at room temperature, unless otherwise statet, using MeOD or DMSO as solvents.
Solvent residue peak or tetramethylsilane (TMS) were used as reference signals. Chemical shifts (δ)
are quoted in ppm and coupling constants in Hertz (Hz). Reactions are not optimized for yields.
Anal. HPLC purity is ≥ 95 % unless otherwise stated.
4.1.1. General procedure 1, adapted from ref25:
The appropriate β-keto ester (0.75-3.0 mmol, 1.5 equiv.), the appropriate aldehyde (0.5-2.0 mmol,
1 equiv.), the appropriate urea or thiourea (0.5-2.0 mmol, 1 equiv.) and Yb(OTf)3 (0.05-0.20 mmol,
0.10 equiv.) were placed in a microwave oven vial (0.5-2.0 mL) with a magnetic stirring bar and
dissolved in THF or MeCN (0.5-1.0 mL). The resulting mixture is heated to 120 °C for 30-60 min
18

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using microwave irradiation. The resulting mixture is poured onto an ice-water mixture and left for
precipitation. The resulting crude solids were purified by recrystallization, DCVC or flash
chromatography.
4.1.2. General method 2, adapted from ref26:
Ethyl acetoacetate (4 mmol, 1 equiv.), the appropriate aldehyde (4 mmol, 1 equiv.), and the
appropriate thiourea (4 mmol, 1 equiv.) were dissolved in DMF (10 mL) in a reaction tube. TMSCl
(3.0 mL, 24 mmol, 6 equiv.) was added drop wise to the solution. The tube was thoroughly sealed
and stirred at room temperature for 16-72 h. The reaction mixture was poured into H2O (50 mL).
The mixture was extracted with Et2O (3x100 mL). The combined organic phase was washed with
brine (50 mL) and H2O (2x50 mL) and dried with MgSO4. The dry organic phase was evaporated
to dryness to give the crude product. The crude product was purified by flash chromatography.
4.1.2.1.
Ethyl
4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (Monastrol, 1). Synthesized according to general method 1 using 3-
hydroxybenzaldehyde (1.22 g, 10 mmol), ethyl acetoacetate (1.9 mL, 15 mmol), thiourea (0.761 g,
10 mmol), Yb(OTf)3 (0.620 g, 1 mmol), MeCN (5 mL) and 1h of heating. A white solid was
isolated in 75% yield (2.19 g). Purity by anal. HPLC (254 nm) > 97%, mp: 184-185 °C, LC-
MS(ESP) : m/z = 293 [M+H+]. The spectroscopic data is in agreement with data previously
reported in literature25.
4.1.2.2. Ethyl 6-methyl-4-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (2).
Synthesized according to general method 1 using benzaldehyde (102 µL, 1.0 mmol), ethyl
acetoacetate (191 µL, 1.5 mmol), thiourea (76 mg, 1.0 mmol), Yb(OTf)3 (62 mg, 0.1 mmol),
MeCN (1.0 mL) and 45 min of heating. A white solid was isolated in 73% yield (197 mg). Purity
by anal. HPLC (254 nm) > 97%, mp: 204-206 °C, LC-MS(ESP) : m/z = 277 [M+H+]. The
spectroscopic data is in agreement with data previously reported in literature35.
4.1.2.3. Ethyl 6-methyl-2-thioxo-4-(m-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (3).
Synthesized according to general method 1 using 3-methylbenzaldehyde (60 mg, 0.5 mmol), ethyl
acetoacetate (96 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol), Yb(OTf)3 (31 mg, 0.05 mmol),
MeCN (0.5 mL) and 30 min of heating. A white solid was isolated in 49% yield (71 mg). Purity by
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anal. HPLC (254 nm) > 99%, mp: 186-187 °C, LC-MS(ESP): m/z = 291 [M+H+]. The
spectroscopic data is in agreement with data previously reported in literature36.
4.1.2.4. Ethyl 6-methyl-2-thioxo-4-(p-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carb4xylate (4).
Synthesized according to general method 1 using 4-methylbenzaldehyde (120 µL, 1.0 mmol), ethyl
acetoacetate (191 µL, 1.5 mmol), thiourea (76 mg, 1.0 mmol), Yb(OTf)3 (62 mg, 0.1 mmol),
MeCN (1.0 mL) and 35 min of heating. A yellow solid was isolated in 93% yield (285 mg). Purity
by anal. HPLC (254 nm) > 98%, mp: 187-189 °C, LC-MS(ESP): m/z = 291 [M+H+]. The
spectroscopic data is in agreement with data previously reported in literature36
4.1.2.5. Ethyl 6-methyl-2-thioxo-4-(o-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (5).
Synthesized according to general method 1 using 2-methylbenzaldehyde (130 µL, 1.05 mmol),
ethyl acetoacetate (201 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3 (65 mg, 0.1 mmol),
THF (0.5 mL) and 1h of heating. Recrystallized from EtOH to obtain a white solid in 73% yield
(223 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 149-150 °C, LC-MS(ESP): m/z = 291
1
[M+H+]. H NMR (DMSO) δ 10.24 (s, 1H), 9.54 (br. s., 1H), 7.13 - 7.23 (m, 4H), 5.41 (s, 1H),
3.93 (q, J = 7.03 Hz, 2H), 2.46 (s, 3H), 2.34 (s, 3H), 1.02 (t, J = 7.15 Hz, 3H). 13C NMR (DMSO) δ
173.4, 165.0, 144.8, 142.2, 135.0, 130.1, 127.6, 127.1, 126.6, 100.9, 59.4, 50.6, 18.6, 17.0, 13.9.
4.1.2.6. Ethyl 6-methyl-2-thioxo-4-(o-tolyl)-1,2,3,4-tetrahydropyrimidine-5-carboxylate (6).
Synthesized according to general method 1 using 3-bromobenzaldehyde (123 µL, 1.05 mmol),
ethyl acetoacetate (201 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3 (65 mg, 0.1 mmol),
THF (1.0 mL) and 1h of heating. The crude product was recrystallized from EtOH to obtain a white
solid in 64% yield (237 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 190-191 °C, LC-
MS(ESP): m/z = 355. The spectroscopic data is in agreement with data previously reported in
literature37.
4.1.2.7.
Ethyl
4-(3-fluorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (7). Synthesized according to general method 1 using 3-flurobenzaldehyde (113 µL,
1.05 mmol), ethyl acetoacetate (201 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3 (65
mg, 0.1 mmol), THF (1.0 mL) and 1h of heating. The crude product was recrystallized from EtOH
to obtain a white solid in 67% yield (207 mg). Purity by anal. HPLC (254 nm) > 96%, mp: 221-223
1
°C, LC-MS(ESP) : m/z = 295 [M+H+]. H NMR (DMSO) δ 10.39 (s, 1H), 9.68 (br. s., 1H), 7.41
20

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(dt, J = 6.15, 7.97 Hz, 1H), 6.90 - 7.21 (m, 3H), 5.20 (d, J = 3.51 Hz, 1H), 4.03 (ttd, J = 3.76, 7.20,
13
10.70 Hz, 2H), 2.30 (s, 3H), 1.11 (t, J = 7.03 Hz, 3H). C NMR (DMSO) δ 174.3, 164.9, 130.7,
19
130.6, 122.3, 114.5, 113.1, 112.9, 100.1, 59.6, 57.6, 17.1, 13.9. F NMR (DMSO) δ 108.08 (s, 1
F).
4.1.2.8.
Ethyl
4-(3-chlorophenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (8). Synthesized according to general method 1 using 3-chlorobenzaldehyde (70, 0.5
mmol), ethyl acetoacetate (96 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol), Yb(OTf)3 (31 mg, 0.05
mmol), MeCN (0.5 mL) and 30 min of heating. A white solid was isolated in 51% yield (81 mg).
Purity by anal. HPLC (254 nm) > 99%, mp: 202-204 °C, LC-MS(ESP): m/z = 311 [M+H+]. The
spectroscopic data is in agreement with data previously reported in literature38.
4.1.2.9. Ethyl 6-methyl-4-(3-nitrophenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(9). Synthesized according to general method 1 using 3-nitrobenzaldehyde (76 mg, 0.5 mmol), ethyl
acetoacetate (96 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol), Yb(OTf)3 (31 mg, 0.05 mmol),
MeCN (0.5 mL) and 30 min of heating. The product was isolated as a white solid in 50% yield (81
mg). Purity by anal. HPLC (254 nm) > 97%, mp: 209-210 °C, LC-MS(ESP): m/z = 322 [M+H]+.
The spectroscopic data is in agreement with data previously reported in literature39.
4.1.2.10.
Ethyl
4-(4-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (10). Synthesized according to general method 1 using 4-hydroxybenzaldehyde (128
mg, 1.0 mmol), ethyl acetoacetate (191 µL, 1.5 mmol), thiourea (76 mg, 1.0 mmol), Yb(OTf)3 (62
mg, 0.1 mmol), THF (1.0 mL) and 45 min of heating. A white solid was isolated in 94% yield (214
mg). Purity by anal. HPLC (254 nm) > 97%, mp: 196-198 °C, LC-MS(ESP): m/z = 293 [M+H+].
The spectroscopic data is in agreement with data previously reported in literature40.
4.1.2.11. Ethyl 4-(3,4-dihydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (11). Synthesized according to general method 1 using 3,4-dihydroxybenzaldehyde
(552 mg, 4 mmol), ethyl acetoacetate (764 µL, 6 mmol), thiourea (304 mg, 4 mmol), Yb(OTf)3
(248 mg, 0.4 mmol), MeCN (2.0 mL) and 30 min of heating. The crude product was purified by
DCVC (heptane:EtOAc) and the product was isolated in 12% yield (144 mg) as colourless crystals.
21

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Purity by anal. HPLC (254 nm) > 99%, mp: 212-213 °C, LC-MS(ESP): m/z = 309 [M+H]+. The
spectroscopic data is in agreement with data previously reported in literature41.
4.1.2.12. Ethyl 4-(3,5-dihydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (12). Synthesized according to general method 1 using 3,5-dihydroxybenzaldehyde
(552 mg, 4.0 mmol), ethyl acetoacetate (765 µL, 6.0 mmol), thiourea (304 mg, 4.0 mmol),
Yb(OTf)3 (248 mg, 0.4 mmol), MeCN (2.0 mL) and 1h of heating. Purified by flash
chromatography (heptane:EtOAc, 2:1). The product was obtained as white crystals though
recrystelization of the purified compound yielding 12 mg (1%) of pure product. Purity by anal.
HPLC (254 nm) > 99%, mp: 144-145 °C, LC-MS(ESP): m/z = 309 [M+H]+.1H NMR (MeOD): δ
6.25 (d, J = 2.2 Hz, 2H), 6.17 (t, J = 2.2 Hz, 1H), 5.17 (s, 1H), 4.11 (q, J = 7.1 Hz, 2H), 2.33 (s,
13
3H), 1.20 (t, J = 7.1 Hz, 3H). C NMR (DMSO): δ 174.7, 166.0, 158.4, 145.4, 144.1, 104.8,
101.80, 101.66, 59.9, 55.0, 16.2, 13.1
4.1.2.13. Ethyl 6-methyl-4-(pyridin-3-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate
(13). Synthesized according to general method 1 using nicotinaldehyde (214 mg, 2.0 mmol), ethyl
acetoacetate (382 µL, 3.0 mmol), thiourea (152 mg, 2.0 mmol), Yb(OTf)3 (124 mg, 0.2 mmol),
MeCN (1.0 mL) and 30 min of heating. Purified by flash chromatography (heptane:EtOAc, 2:1) to
obtain the pure product as a yellow solid in 15% yield (36 mg). Purity by anal. HPLC (254 nm) >
95%, mp: 204-205 °C, LC-MS(ESP): m/z = 278 [M+H]+. The spectroscopic data is in agreement
with data previously reported in literature40.
4.1.2.14.
Ethyl
4-(3-methoxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (14). Synthesized according to general method 1 using 3-methoxybenzaldehyde (100
µL, 0.8 mmol), ethyl acetoacetate (153 µL, 1.2 mmol), thiourea (62 mg, 0.8 mmol), Yb(OTf)3 (48
mg, 0.08 mmol), MeCN (1.0 mL) and 45 min of heating. The crude product was recrystallized
from EtOH and H2O to obtain a yellow solid in 83% yield (209 mg). Purity by anal. HPLC (254
nm) > 99%, mp: 143-145 °C, LC-MS(ESP) : m/z = 307 [M+H+]. The spectroscopic data is in
agreement with data previously reported in literature42.
4.1.2.15. Ethyl 6-methyl-4-(1-methyl-1H-pyrazol-5-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (15). Synthesized according to general method 1 using 1-methyl-1H-pyrazole-5-
carbaldehyde (55 mg, 0.5 mmol), ethyl acetoacetate (96 µL, 0.75 mmol), thiourea (38 mg, 0.5
22

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mmol), Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. The product was
isolated as a white solid in 29% yield (41 mg). Purity by anal. HPLC (254 nm) > 98%, mp: 185-187
°C, LC-MS(ESP): m/z = 281 [M+H]+. 1H NMR (DMSO): δ 10.42 (s, 1H), 9.66 (s, 1H), 7.28 (d, J =
1.7 Hz, 1H), 6.00 (d, J = 1.9 Hz, 1H), 5.34 (d, J = 3.3 Hz, 1H), 3.99 (q, J = 7.1 Hz, 2H), 3.87 (s,
3H), 2.31 (s, 3H), 1.07 (t, J = 7.1 Hz, 3H). 13C-NMR (DMSO): δ 174.6, 165.2, 145.8, 144.3, 138.0,
104.5, 99.9, 60.1, 45.9, 36.8, 17.5, 14.5.
4.1.2.16.
Ethyl
6-methyl-4-(1-phenyl-1H-1,2,3-triazol-4-yl)-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (16). Synthesized according to general method 1 using 1-
phenyl-1H-1,2,3-triazole-4-carbaldehyde (87 mg, 0.5 mmol), ethyl acetoacetate (96 µL, 0.75
mmol), thiourea (38 mg, 0.5 mmol), Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of
heating. The product was isolated as a white solid in 65% yield (112 mg). Purity by anal. HPLC
1
(254 nm) > 99%, mp: 223-224 °C, LC-MS(ESP): m/z = 344 [M+H]+. H NMR (DMSO): δ 10.39
(d, J = 1.5 Hz, 1H), 9.65 (dd, J = 3.8, 1.8 Hz, 1H), 8.60 (s, 1H), 7.90 (q, J = 1.5 Hz, 1H), 7.88 (dt, J
= 1.5, 0.9 Hz, 1H), 7.61-7.56 (m, 2H), 7.50-7.46 (m, 1H), 5.42 (d, J = 3.9 Hz, 1H), 4.04 (qd, J =
13
7.1, 0.9 Hz, 2H), 2.32 (s, 3H), 1.12 (t, J = 7.1 Hz, 3H). C-NMR (DMSO): δ 175.3, 165.3, 150.3,
146.6, 137.0, 130.3, 129.1, 120.6, 120.5, 99.4, 60.0, 47.0, 17.7, 14.6.
4.1.2.17. Ethyl 4,6-dimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylate (17).
Synthesized according to general method 1 using acetoaldehyde (59 µL, 1.05 mmol), ethyl
acetoacetate (201 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3 (65 mg, 0.1 mmol), THF
(0.5 mL) and 1h of heating. A yellow solid was isolated in 97% yield (219 mg). Purity by anal.
HPLC (254 nm) > 97%, mp: 189-191 °C, LC-MS(ESP): m/z = 215 [M+H+]. The spectroscopic
data is in agreement with data previously reported in literature43.
4.1.2.18. Ethyl 4-(3-hydroxyphenyl)-1,6-dimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (18). Synthesized according to general method 2 using 3-hydroxybenzaldehyde (488
mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1-methylthiourea (361 mg, 4 mmol), TMSCl (3
mL, 24 mmol), DMF (10 mL) and 16h of stirring. Purified by flash chromatography
(heptane:EtOAc, 2:1) to obtain the pure product as a white solid in 75% yield (916 mg). Purity by
anal. HPLC (254 nm) > 95%, mp: 135-136 °C, LC-MS(ESP): m/z = 307. 1H NMR (DMSO): δ 9.78
(d, J = 4.7 Hz, 1H), 9.43 (s, 1H), 7.11 (t, J = 8.0 Hz, 1H), 6.66-6.61 (m, 3H), 5.13 (d, J = 4.7 Hz,
23

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13
1H), 4.12 (q, J = 7.1 Hz, 2H), 3.48 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H). C NMR (DMSO): δ 178.5,
165.8, 157.9, 148.0, 144.1, 130.0, 116.9, 115.0, 113.4, 106.1, 60.6, 52.6, 36.7, 16.7, 14.5.
4.1.2.19. Ethyl 1-ethyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (19). Synthesized according to general method 2 using 3-hydroxybenzaldehyde (488
mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1-ethylthiourea (417 mg, 4 mmol), TMSCl (3
mL, 24 mmol), DMF (10 mL) and 16h of stirring. Purified by flash chromatography
(heptane:EtOAc, 2:1) to obtain the pure product as a white solid in 74% yield (949 mg). Purity by
anal. HPLC (254 nm) > 98%, mp: 149-150 °C, LC-MS(ESP): m/z = 321 [M+H]+. The
spectroscopic data is in agreement with data previously reported in literature44.
4.1.2.20. Ethyl 1-butyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-
5-carboxylate (20). Synthesized according to general method 2 using 3-hydroxybenzaldehyde (488
mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1-butylthiourea (529 mg, 4 mmol), TMSCl (3
mL, 24 mmol), DMF (10 mL) and 72h of stirring. Purified by flash chromatography
(heptane:EtOAc, 2:1) to obtain the pure product as a white solid in 69% yield (955 mg). Purity by
anal. HPLC (254 nm) > 98%, mp: 141-142 °C, LC-MS(ESP): m/z = 349 [M+H]+. The
spectroscopic data is in agreement with data previously reported in literature44.
4.1.2.21.
Ethyl
4-(3-hydroxyphenyl)-6-methyl-1-phenyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (21). Synthesized according to general method 2 using 3-
hydroxybenzaldehyde (488 mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1-phenylthiourea
(609 mg, 4 mmol), TMSCl (3 mL, 24 mmol), DMF (10 mL) and 16h of stirring. Purified by flash
chromatography (heptane:EtOAc, 3:1) to obtain the pure product as a white solid in 74% yield
(1097 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 66-68 °C, LC-MS(ESP): m/z = 369
[M+H]+. The spectroscopic data is in agreement with data previously reported in literature44.
4.1.2.22.
Ethyl
1-benzyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-
tetrahydropyrimidine-5-carboxylate (22). Synthesized according to general method 2 using 3-
hydroxybenzaldehyde (488 mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1-benzylthiourea
(665 mg, 4 mmol), TMSCl (3 mL, 24 mmol), DMF (10 mL) and 16h of stirring. Purified by flash
chromatography (heptane:EtOAc, 2:1) to obtain the pure product as a white solid in 70% yield
(1072 mg). Purity by anal. HPLC (254 nm) > 97%, mp: 159-160 °C, LC-MS(ESP): m/z = 383
24

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1
[M+H]+. H NMR (DMSO): δ 10.03 (d, J = 4.6 Hz, 1H), 9.48 (s, 1H), 7.20 (t, J = 3.2 Hz, 3H),
7.15-7.11 (m, 1H), 6.99-6.97 (m, 2H), 6.70 (ddd, J = 8.0, 2.2, 1.1 Hz, 1H), 6.66-6.63 (m, 2H), 6.07
(s, 1H), 5.20 (d, J = 4.5 Hz, 1H), 5.15 (s, 1H), 4.09 (q, J = 6.7 Hz, 2H), 2.35 (s, 3H), 1.15 (t, J = 7.1
Hz, 3H). 13C NMR (DMSO): δ 178.9, 165.8, 157.9, 146.9, 144.2, 138.2, 129.9, 128.8, 127.3, 126.6,
117.0, 115.1, 113.6, 107.5, 60.7, 58.2, 52.4, 51.0, 16.7, 14.5
4.1.2.23. Ethyl 4-(3-hydroxyphenyl)-1,3,6-trimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (23). Synthesized according to general method 2 using 3-hydroxybenzaldehyde (488
mg, 4 mmol), ethyl acetoacetate (524 µL, 4 mmol), 1,3-dimethylthiourea (417 mg, 4 mmol),
TMSCl (3 mL, 24 mmol), DMF (10 mL) and 16h of stirring. Purified by flash chromatography
(heptane:EtOAc, 2:1) and recrystallization to obtain the pure product as a white crystals in 5%
yield (63 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 127-131 °C, LC-MS(ESP): m/z = 321
[M+H]+. 1H NMR (DMSO): δ 9.46 (s, 1H), 7.13-7.09 (m, 1H), 6.66 (ddd, J = 8.1, 2.4, 1.1 Hz, 1H),
6.58-6.56 (m, 2H), 5.49 (s, 1H), 4.19-4.10 (m, 2H), 3.50 (s, 3H), 3.34 (s, 3H), 2.47 (d, J = 0.4 Hz,
3H), 1.22 (t, J = 7.1 Hz, 3H). 13C NMR (DMSO): δ 178.7, 165.0, 157.6, 147.2, 141.1, 129.7, 116.4,
114.9, 112.8, 105.0, 60.4, 60.1, 42.4, 37.7, 16.3, 14.1.
4.1.2.24.
Ethyl
6-ethyl-4-(3-hydroxyphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (24). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), ethyl 3-oxopentanoate (107 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol),
Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. A white solid was isolated
in 37% yield (57 mg). Purity by anal. HPLC (254 nm) > 98%, mp: 160-161 °C, LC-MS(ESP): m/z
= 307 [M+H]+. 1H NMR (MeOD): δ 7.15 (t, J = 7.8 Hz, 1H), 6.80-6.70 (m, 3H), 5.27 (s, 1H), 4.12
(q, J = 6.3 Hz, 2H), 2.85-2.70 (m, 2H), 1.24 (t, J = 5.7 Hz, 3H), 1.21 (t, J = 5.4 Hz, 3H).
13-C NMR (DMSO): δ 174.4, 164.8, 157.4, 150.3, 144.8, 129.4, 116.9, 114.6, 113.2, 99.9, 59.6,
53.8, 23.4, 13.9, 13.0
4.1.2.25.
Ethyl
4-(3-hydroxyphenyl)-6-propyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (25). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), ethyl 3-oxohexanoate (120 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol), Yb(OTf)3
(31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. A white solid was isolated in 69%
yield (111 mg). Purity by anal. HPLC (254 nm) > 96%, mp: 149-151 °C, LC-MS(ESP): m/z = 321
[M+H]+. The spectroscopic data is in agreement with data previously reported in literature44.
25

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4.1.2.26. Ethyl 4-(3-hydroxyphenyl)-6-isopropyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (26). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), ethyl 4-methyl-3-oxopentanoate (121 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol),
Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. The crude product was
recrystallized from MeOH and H2O to obtain the product as white crystals in 43% yield (68 mg).
1
Purity by anal. HPLC (254 nm) > 99%, mp: 117-119 °C, LC-MS(ESP): m/z = 321 [M+H]+. H
NMR (DMSO): δ 9.72 (s, 1H), 9.57 (d, J = 2.4 Hz, 1H), 9.44 (s, 1H), 7.12 (t, J = 7.8 Hz, 1H),
6.66-6.63 (m, 3H), 5.08 (d, J = 3.9 Hz, 1H), 4.09 (m, 1H), 4.01 (q, J = 7.0 Hz, 2H), 1.20 (d, J = 7.1
13
Hz, 3H), 1.13 (d, J = 7.1 Hz, 3H), 1.10 (t, J = 7.1 Hz, 3H). C-NMR (DMSO): δ 175.2, 165.6,
157.9, 153.1, 145.0, 130.0, 117.4, 115.1, 113.7, 100.4, 60.2, 54.4, 27.2, 19.5, 19.0, 14.4.
4.1.2.27. Ethyl 6-(tert-butyl)-4-(3-hydroxyphenyl)-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (27). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), ethyl 4,4-dimethyl-3-oxopentanoate (130 µL, 0.75 mmol), thiourea (38 mg, 0.5
mmol), Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. A yellow solid was
isolated in 11% yield (19 mg). Purity by anal. HPLC (254 nm) > 97%, mp: 155-157 °C, LC-
1
MS(ESP): m/z = 335 [M+H]+. H NMR (CDCl3): δ 9.42 (s, 1H), 9.31 (s, 1H), 8.48 (s, 1H), 7.12
(t, J = 7.7 Hz, 1H), 6.72-6.61 (m, 3H), 4.90 (d, J = 3.2 Hz, 1H), 4.08-3.99 (m, 2H), 1.28 (s, 9H),
1.12 (t, J = 6.9 Hz, 3H). 13C NMR (DMSO): δ 174.5, 167.4, 157.4, 145.5, 143.3, 129.4, 117.1,
114.8, 113.4, 103.7, 60.4, 55.9, 35.2, 28.0, 13.7.
4.1.2.28.
Ethyl
4-(3-hydroxyphenyl)-6-phenyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (28). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), ethyl 3-oxo-3-phenylpropanoate (130 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol),
Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. A white solid was isolated
in 56% yield (99 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 196-197 °C, LC-MS(ESP): m/z
= 355 [M+H]+. The spectroscopic data is in agreement with data previously reported in literature35.
4.1.2.29. Methyl 4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (29). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (128
mg, 1.05 mmol), methyl acetoacetate (170 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3
26

ACCEPTED MANUSCRIPT
(65 mg, 0.1 mmol), THF (1.0 mL) and 30 min of heating. A white solid was isolated in 70% yield
(203 mg). Purity by anal. HPLC (254 nm) > 98%, mp: 217-218 °C, LC-MS(ESP) : m/z = 279
[M+H+]. The spectroscopic data is in agreement with data previously reported in literature45.
4.1.2.30. 4-(3-Hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-carboxylic
acid (30). KOH (0.10 g, 1.8 mmol) was dissolved in H2O (5 mL). Monastrol (180 mg, 0.62 mmol)
was added and the mixture was stirred for 72 h at room temperature. The mixture was acidified
with 1M HCl. The water was extracted with EtOAc. The organic phase was dried over MgSO4,
filtered and evaporated to dryness. The crude white product was washed with Et2O to obtain the
product in 22% yield (35 mg). Purity by anal. HPLC (254 nm) > 97%, mp: 163-165 °C, LC-
MS(ESP) : m/z = 264. The spectroscopic data is in agreement with data previously reported in
literature41.
4.1.2.31. Benzyl 4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (31). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (128
mg, 1.05 mmol), benzyl acetoacetate (273 µL, 1.6 mmol), thiourea (80 mg, 1.05 mmol), Yb(OTf)3
(65 mg, 0.1 mmol), MeCN (1.0 mL) and 30 min of heating. The oily crude product was purified by
DCVC (EtOAc:Heptane). Re-crystallised from EtOH to obtain pure white crystals in 28% yield
(191 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 168-169 °C, LC-MS(ESP) : m/z = 355
[M+H+]. 1H NMR (DMSO) δ 10.41 (s, 1H), 9.69 (d, J = 2.01 Hz, 1H), 9.50 (s, 1H), 7.34 - 7.39 (m,
3H), 7.24 (dd, J = 2.64, 6.90 Hz, 2H), 7.18 (t, J = 7.65 Hz, 1H), 6.69 - 6.76 (m, 3H), 5.20 (d, J =
13
3.76 Hz, 1H), 5.09 - 5.19 (m, 2H), 2.38 (s, 3H). C NMR (DMSO) δ 174.0, 164.9, 157.5, 145.6,
144.6, 136.3, 129.5, 128.3, 127.7, 127.5, 117.1, 114.7, 113.3, 100.2, 65.1, 53.9, 17.2.
4.1.2.32. N,N-diethyl-4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxamide (32). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), N,N-diethyl-3-oxobutanamide (118 µL, 0.75 mmol), thiourea (38 mg, 0.5 mmol),
Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. The product was isolated as
a white solid in 38% yield (60 mg). Purity by anal. HPLC (254 nm) > 98%, mp: decomp. > 250 °C,
LC-MS(ESP): m/z = 320 [M+H]+. The spectroscopic data is in agreement with data previously
reported in literature46.
27

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4.1.2.33.
1-(4-(3-hydroxyphenyl)-6-methyl-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-
yl)ethanone (33). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (122
mg, 1.0 mmol), pentane-2,4-dione (103 µL, 1.5 mmol), thiourea (76 mg, 1.0 mmol), Yb(OTf)3 (62
mg, 0.1 mmol), MeCN (0.5 mL) and 30 min of heating. The product was isolated as a white solid
in 48% yield (127 mg). Purity by anal. HPLC (254 nm) > 95%, decomp > 250°C, LC-MS(ESP):
m/z = 263 [M+H]+. The spectroscopic data is in agreement with data previously reported in
literature47.
4.1.2.34.
Ethyl
4-(3-hydroxyphenyl)-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (34). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
µL, 0.5 mmol), ethyl acetoacetate (96 µL, 0.75 mmol), urea (30 mg, 0.5 mmol), Yb(OTf)3 (31 mg,
0.05 mmol), THF (1.0 mL) and 40 min of heating. A white solid was isolated in 78% yield (107
mg). Purity by anal. HPLC (254 nm) > 99%, mp: 184-185 °C, LC-MS(ESP) : m/z = 277 [M+H+].
The spectroscopic data is in agreement with data previously reported in literature47.
4.1.2.35. Methyl 4-(3-hydroxyphenyl)-1,6-dimethyl-2-thioxo-1,2,3,4-tetrahydropyrimidine-5-
carboxylate (35). Synthesized according to general method 1 using 3-hydroxybenzaldehyde (61
mg, 0.5 mmol), methyl 3-oxobutanoate (91 µL, 0.75 mmol), 1-methylthiourea (45 mg, 0.5 mmol),
Yb(OTf)3 (31 mg, 0.05 mmol), MeCN (0.5 mL) and 30 min of heating. The crude product was
recrystallized from MeOH and H2O to obtain the product as a yellow solid in 15% yield (22 mg).
1
Purity by anal. HPLC (254 nm) > 97%, mp: 213-214 °C, LC-MS(ESP): m/z = 293 [M+H]+. H
NMR (DMSO): δ 9.80 (d, J = 4.7 Hz, 1H), 9.42 (s, 1H), 7.10 (t, J = 8.0 Hz, 1H), 6.66-6.63 (m, 1H),
6.62-6.60 (m, 2H), 5.13 (d, J = 4.8 Hz, 1H), 3.65 (s, 3H), 3.47 (s, 3H). 13C NMR (DMSO): δ 178.0,
165.8, 157.5, 147.9, 143.4, 129.5, 116.4, 114.6, 112.8, 105.2, 52.0, 51.5, 36.2, 16.3.
4.1.2.36. Ethyl 6-methyl-2-oxo-4-phenyl-1,2,3,4-tetrahydropyrimidine-5-carboxylate (36).
Synthesized according to general method 1 using benzaldehyde (107 µL, 1.05 mmol), methyl
acetoacetate (170 µL, 1.6 mmol), urea (63 mg, 1.05 mmol), Yb(OTf)3 (65 mg, 0.1 mmol), THF (0.5
mL) and 45 min of heating. The crude product was recrystallized from EtOH and H2O to obtain a
yellow solid in 78% yield (214 mg). Purity by anal. HPLC (254 nm) > 99%, mp: 205-206 °C, LC-
1
MS(ESP): m/z = 261 [M+H]+. H NMR (DMSO) δ 9.17 (s, 6H), 7.71 (br. s., 6H), 7.29 - 7.35 (m,
12H), 7.21 - 7.26 (m, 18H), 5.15 (d, J = 3.39 Hz, 1H), 3.98 (q, J = 7.11 Hz, 2H), 2.25 (s, 3H), 1.09
13
(t, J = 7.09 Hz, 3H). C NMR (DMSO) δ 165.3, 152.1, 148.3, 144.8, 128.4, 127.2, 126.2, 99.2,
28