Subtle modifications to a thieno[2,3-d]pyrimidine scaffold yield negative allosteric modulators and agonists of the dopamine D2 receptor

Article abstract of DOI:10.1016/j.ejmech.2019.01.061

We recently described a structurally novel series of negative allosteric modulators (NAMs) of the dopamine D₂ receptor (D₂R) based on thieno[2,3-d]pyrimidine 1, showing it can be structurally simplified to reveal low molecular weight, fragment-like NAMs that retain robust negative cooperativity, such as 3. Herein, we report the synthesis and functional profiling of analogues of 3, placing specific emphasis on examining secondary and tertiary amino substituents at the 4-position, combined with a range of substituents at the 5/6-positions (e.g. aromatic/aliphatic carbocycles). We identify analogues with diverse pharmacology at the D₂R including NAMs with sub-μM affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i).

Full text of DOI:10.1016/j.ejmech.2019.01.061

Accepted Manuscript
Subtle modifications to a thieno[2,3-d]pyrimidine scaffold yield negative allosteric
modulators and agonists of the dopamine D receptor
2
Tim J. Fyfe, Barrie Kellam, Shailesh N. Mistry, Peter J. Scammells, J. Robert Lane,
Ben Capuano
PII:
S0223-5234(19)30080-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.061
EJMECH 11068
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 November 2018
Revised Date: 24 January 2019
Accepted Date: 24 January 2019
Please cite this article as: T.J. Fyfe, B. Kellam, S.N. Mistry, P.J. Scammells, J.R. Lane, B. Capuano,
Subtle modifications to a thieno[2,3-d]pyrimidine scaffold yield negative allosteric modulators and
agonists of the dopamine D receptor, European Journal of Medicinal Chemistry (2019), doi: https://
2
doi.org/10.1016/j.ejmech.2019.01.061.
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ACCEPTED MANUSCRIPT
Subtle Modifications to a Thieno[2,3-d]pyrimidine Scaffold Yield Negative
Allosteric Modulators and Agonists of the Dopamine D2 Receptor
Tim J. Fyfe,†,§,∥Barrie Kellam,∥ Shailesh N. Mistry,∥ Peter J. Scammells,† J. Robert
,
,†
≠
Lane,* Ben Capuano*
†Medicinal Chemistry, and §Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash
University, Parkville, Victoria 3052, Australia
∥School of Pharmacy, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD,
U.K.
≠School of Life Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham NG7 2UH, U.K.
Abstract: We recently described a structurally novel series of negative allosteric modulators
(NAMs) of the dopamine D2 receptor (D2R) based on thieno[2,3-d]pyrimidine 1, showing it can be
structurally simplified to reveal low molecular weight, fragment-like NAMs that retain robust
negative cooperativity, such as 3. Herein, we report the synthesis and functional profiling of
analogues of 3, placing specific emphasis on examining secondary and tertiary amino substituents at
the 4-position, combined with a range of substituents at the 5/6-positions (e.g. aromatic/aliphatic
carbocycles). We identify analogues with diverse pharmacology at the D2R including NAMs (19fc)
with sub-µM affinity (9h) and, surprisingly, low efficacy partial agonists (9d and 9i).
Keywords:
negative allosteric modulators, dopamine D2 receptor, thieno[2,3-d]pyrimidines,
NAMs, agonists.
1. Introduction
The D2R is a class A G protein-coupled receptor (GPCR) implicated in the pathophysiology and
treatment of a number of central nervous system (CNS) disorders, including schizophrenia (SCZ).
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As such, drugs targeting this receptor have traditionally targeted the orthosteric site - where the
endogenous ligand DA binds. Clinically used antipsychotic drugs (APDs) targeting this site act as
D2R antagonists or low efficacy partial agonists, and are termed typical or atypical APDs based on
their propensity to cause extrapyramidal side effects. Unfortunately, the efficacy of these drugs is
largely limited to the positive symptoms of this disorder. Typical APDs are associated with extra-
pyramidal motor symptoms (EPS) and hyperprolactinaemia, which are mediated by blockade of
D2R signalling in the nigrostriatal and tuberoinfundibular DA pathways, respectively.1-6 Atypical
APDs show a reduced incidence of EPS, but display other off-target side-effects mediated through
the interaction with other aminergic receptors, including metabolic disorders and weight gain.7
Alternative approaches to target the D2R have emerged via the identification of homo- and
heterodimeric complexes.8 Such complexes in specific tissues may provide novel pharmacological
targets for compounds with distinctive functional profiles and improved therapeutic windows.9-11
Another proposition which entails targeting binding sites topographically distinct to the orthosteric
site of GPCRs might also be advantageous. Negative allosteric modulators (NAMs) of the D2R may
represent a safer therapeutic approach for the treatment of SCZ symptoms. A D2R NAM with
limited negative cooperativity with DA may display antipsychotic efficacy but avoid EPS through
partial blockade of the D2R akin to the action of atypical partial agonist APDs such as
aripiprazole.12 Furthermore, as a NAM will allow DA to bind, the spatiotemporal pattern of DA
signalling is more likely to be maintained. Finally, NAMs may display greater selectivity for the
D2R over other targets via the targeting of less evolutionarily conserved sites and consequently
reduced off target toxicities. Drug-like (NAMs) of DA receptors have recently been identified
including the scaffold that is the focus of the present study.13-19
Our previous study described the pharmacological validation of a virtual ligand screen hit
(1, Figure 1), confirming that it binds with low µM functional affinity and exerts its effect via
negative allosteric cooperativity, primarily by modulation of dopamine signalling efficacy.18 Based
on the scaffold of 1, we synthesised a small library of structural analogues to further understand key
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molecular features that were responsible for changes in affinity and negative allosteric
cooperativity. Of several promising analogues identified, 2 (Figure 1) maintained low µM affinity
(KB = 1.92 µM) and significantly attenuated DA signalling efficacy (β = 0.001). This compound was
devoid of the morpholinomethyl moiety present on 1, and also incorporated replacement of the ((3-
trifluoromethyl)phenyl)amino- substituent with an N,N-diethylamino motif. In addition to this, our
previous work focused on assessing various substituents at the 5/6-positions (e.g. phenyl,
cyclohexyl) in place of the fused cyclohexane system. Moreover, we discovered that the scaffold
could be structurally simplified, to reveal a low molecular weight fragment-like starting point that
maintained µM affinity and negative cooperativity (MW = 207, KB = 4.56 µM, β = 0.13) (3, Figure
1). Thus, we identified an appropriate starting point for further structural interrogation and
elaboration of the core scaffold using 2 and 3 as our lead compounds with the aim to identify higher
affinity NAMs.
Fig. 1. Promising structural analogues of 1 arising from a preliminary structure-activity
relationship (SAR) investigation. Compound 1 was identified from a virtual ligand screen and
formed the basis of our previous SAR study. Compound 2 significantly attenuates DA signalling
efficacy at a concentration of 10
allosteric cooperativity despite being a structurally simplified fragment-like analogue (MW: 207, KB
= 4.56 M, = 0.13) of 1.
µM (KB = 1.92 µM, β = 0.001). Compound 3 retains negative
µ
β
To this end, we sought to investigate the influence of varying the nature (both aliphatic and
aromatic) of the 4-amino moiety of 2 (series 1, Figure 2). In addition, a second series of compounds
was designed to further explore the effect of 5/6-thiophene substitution with respect to altering
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functionality at the 4-position (series 2, Figure 2). This would permit further refinement of the
structural determinants of D2R affinity and negative cooperativity. Through the parallel synthesis of
an additional thirty-seven structural analogues of 2, we have identified molecules that exert a range
of modulatory behaviour including, surprisingly, derivatives that display agonism. To confirm a
D2R-mediated mechanism of action, all compounds with agonist profiles together with selected
NAMs were assessed for their ability to modulate our functional readout in the absence of the D2R.
Fig. 2. Areas of structural modification conducted during SAR investigation of 2.
2. Chemistry
The synthesis of all compounds generally followed established methods for the synthesis of 1 and
other related compounds previously reported by our research group.18 Each compound detailed in
this study was easily accessed in four steps (schemes 1-3). Briefly, Scheme 1 depicts the synthesis
of selected analogues (9a-j), beginning with Gewald20 chemistry to facilitate the one-pot
construction of the ethyl 2-amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (5),18
followed by cyclisation with formamide (6) to afford the corresponding 5,6,7,8-
tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidinone (7).18 POCl3 was employed to convert pyrimidinone
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7 to the corresponding key intermediate chloropyrimidine 8,18 followed by nucleophilic aromatic
substitution (SNAr) with the desired amine, in the presence of Hünig’s base in propan-2-ol. This
methodology afforded compounds 9a-j in high purity after a simple work-up and/or
chromatography.
Scheme 1. Synthesis of analogues of 2 modified at the 4-positiona
aReagents and conditions: (i) S8, cyclohexanone, morpholine, rt, ~18 h, 81%; (ii) 170 °C, 12 h, 85%;
(iii) POCl3, DMF(cat.), toluene, 4 h, 68%; (iv) amine, DIPEA, i-PrOH, reflux, 1-3 h, 68-88%; (v) 9j,
TFA, DCM, rt, 4 h, 96%.
2.1. Analogues of 2 varied at the 4-(N,N-diethylamino) substituent
Compound 2 was originally synthesised as an analogue of 1 to observe the effect of simultaneously
removing the morpholinomethyl moiety whilst incorporating a 4-(N,N-diethylamino) group in in the
context of retaining the
tricyclic structure of the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine. As this molecule was found to be a potent NAM of DA efficacy at the D2R, an
additional series of compounds was synthesised to investigate further changes to the 4-position of
the pyrimidine ring (Scheme 1). Preliminary modifications included incorporating 6-membered
secondary aromatic and aliphatic amino substituents (e.g. anilino (9a) and cyclohexylamino (9b)
together with 6-membered cyclic aliphatic tertiary amine moieties, including piperidino (9c),
morpholino (9d) and piperazino (10), followed by the smaller pyrrolidino (9e) to assess the
influence of a five-membered cyclic aliphatic system. The piperazino analogue 10 was obtained via
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the SNAr reaction between commercially available N-Boc piperazine and 8 under microwave
conditions as outlined previously (Scheme 1) to afford 9j as a crystalline solid requiring no further
purification. Standard trifluoroacetic acid-mediated N-Boc de-protection followed by an alkaline
work-up furnished 10. This molecule is unique relative to its counterparts in that it will impart a
positive charge at physiological pH. Moreover, we investigated both monocyclic (cyclopropylamino
(9f), cyclopropylmethylamino (9g), cyclobutylamino (9h)) and acylic aliphatic secondary amine
substituent (tert-butylamino (9i)). The tert-butylamino substituent was installed to assess the effect
of an acylic aliphatic secondary amine as a comparison to N,N-diethylamino. These syntheses were
all achieved using chemistry as previously outlined (Scheme 1), and successfully afforded ten
analogues (9a-i, and 10) in yields varying from 68-88% following SNAr of 8 with the appropriate
amine.
2.2. Fragment analogues of 3
We previously reported a low molecular weight fragment (N,N-diethylthieno[2,3-d]pyrimidin-4-
amine, (3)) that retains low
µM functional binding affinity and negative allosteric cooperativity at
the D2R (KB = 4.56 M,
µ
β
= 0.13, Figure 1).18 This molecule was originally synthesised using a
deletion strategy to examine the effect of switching from a 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine to the corresponding thieno[2,3-d]pyrimidine scaffold, in conjunction with
incorporating a tertiary aliphatic amine at the 4-position. As such, we generated two additional
analogues of 3 bearing 4-cyclopropylamino and 4-cyclobutylamino substituents. Their synthesis is
depicted in Scheme 2 and begins with the construction of ethyl 2-aminothiophene-3-carboxylate
(12)18 using ethyl cyanoacetate (4) and 1,4-dithiane-2,5-diol (11) under modified Gewald
conditions. Refluxing 12 in neat formamide afforded the corresponding pyrimidinone (13)18 in good
yield. Subsequent chemistry as outlined previously included deoxychlorination to give 14,18
followed by SNAr with cyclopropylamine or cyclobutylamine to afford the corresponding analogues
15a and 15b, respectively.
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Scheme 2. Synthesis of Thieno[2,3-d]pyrimidine Analogues of 2a
aReagents and conditions: (i) Et3N, DMF, 45 °C, 0.5 h, 55%; (ii) formamide (neat), 170 °C, 12 h,
76%; (iii) POCl3, DMF(cat.), toluene, 100 °C 4 h, 77%; (iv) amine, DIPEA, i-PrOH, 100 °C, 1-3 h,
76% (15a), 79% (15b).
2.3. Single modifications to the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine moiety of 2
whilst varying the nature of the 4-substituent
Our previously reported SAR study of 1 focused on structural modifications to the fused
cyclohexane system, including switching from a 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine to the corresponding thieno[2,3-d]pyrimidine, 5- and 6-aryl substitution, 5- and 6-
cyclohexyl substitution, 5,6-dimethyl substitution, as well as incorporation of fused cyclopentane,
cycloheptane, and cyclooctane systems.18 These structural modifications were mostly employed in
the presence of a 3-(trifluoromethyl)anilino substituent at the 4-position, and a 2-morpholinomethyl
substituent. Only the fused cyclopentane and the 6-cyclohexyl modifications preserved the allosteric
pharmacology seen with this scaffold, while the other structural modifications rendered the
corresponding molecule functionally inactive. This study, however, had demonstrated the
encouraging effects of incorporating N,N-diethylamino, cyclopropylamino, or cyclobutylamino to
the 4-position of the pyrimidine core, increasing functional affinity as well as maintaining the
magnitude of negative allosteric cooperativity whilst bearing the fused cyclohexane system (Series
1, Figure 2). To this end, we wanted to evaluate combining the structural modifications to the
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thiophene, whilst concurrently incorporating the aforementioned 4-amino substituents (Series 2,
Figure 2). These compounds would further refine the SAR and provide insight into the design of
further high affinity NAMs. These modifications are depicted in Figure 2 and their chemical
synthesis is outlined in Scheme 3. All compounds were accessed using established chemistry as
detailed in our previous work, beginning with construction of the appropriate thiophene via
Gewald20 chemistry (16a-h),18 formation of the corresponding pyrmidinones (17a-h)18 and
chloropyrimidines (18a-h),18 followed by SNAr with the appropriate amine to furnish a further
twenty-four analogues (19aa-hc).
Scheme 3. Synthesis of Functionalised Analogues of 2a
aReagents and conditions: (i) aldehyde or ketone, S8, morpholine, rt 12-24 h, 38-84%, 16a-h; (ii)
formamide, 170 °C, 12 h, 75-85%, 17a-h; (iii) POCl3, DMF(cat.), toluene, 100 °C, 3-5 h, 77-91%,
18a-h; (iv) amine, DIPEA, i-PrOH, MWI, 1-3 h, 71-93%, 19aa-hc.
3. Pharmacology
3.1. Functional and off-target analyses of analogues of 2 using a BRET biosensor for cAMP
To measure the effect of these compounds upon the functional response of the D2R when stimulated
by DA we made use of a BRET biosensor of cAMP. This assay measures inhibition of forskolin-
stimulated cAMP accumulation through activation of the long isoform of the human D2R (hD2LR)
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stably expressed in FlpIn CHO cells and provides a robust measurement of D2R Gαi/o activation to
allow estimates of functional binding affinities and quantify the magnitude of modulatory effects.18
Application of an operational model of allostery to the DA concentration-response data obtained in
the presence and absence of increasing concentrations of our test compounds yielded an estimate of
their affinity for the unoccupied receptor (KB), cooperativity with DA affinity (
α) and modulation of
DA efficacy (β), plus the intrinsic efficacy of the allosteric ligand (τB).21 Values of
α
or β < 1
signify negative modulatory effects with DA, and values of
α or β > 1 signify positive modulatory
effects. Logarithms of affinity and cooperativity values are normally distributed, whereas the
absolute values (antilogarithms) are not.22 Thus, all interpretation of the SAR described below
(Tables 1-3) refer to the logarithmic values. For ease of interpretation, however, the allosteric
parameter antilogarithms are also highlighted in the main text for selected key analogues. As
reported previously, the lead compound 2 acted as a NAM at the D2R, with low µM functional
affinity (KB = 1.92 µM, Table 1).18 The depression in the DA dose-response curve caused by
increasing concentrations of 2 is characteristic of the action of a NAM of agonist efficacy and β was
fixed to 0.001 when fitting these data to signify high negative cooperativity (Figure 3A, Table 1).
To our surprise, we identified molecules based on this NAM scaffold that now appear to
display agonism. It was important, then, to confirm that these effects are D2R mediated. Luciferase
assays may identify false positive ‘hits’ through a variety of mechanisms, for example, by inhibition
of the luciferase enzyme.23-25 To allow us to discriminate between D2R-mediated activities and any
non-specific effects (Supplementary Figure 1), all compounds observed to display D2R agonism in
our cAMP assay were selected for further analysis (9c-d, 9i, 15a-b, 19ac, 19ca, 19da, 19db, 19dc,
19ec, and 19ha). In addition, a number of other compounds were selected, namely the parent NAMs
1 and 2, together with NAMs 9h, 19bb, 19fb, and 19fc. We measured their ability to modulate the
BRET signal in FlpIn CHO cells transfected with the CAMYEL biosensor,26 but not expressing the
D2R. All compounds were measured up to a concentration of 100 µM in both the presence and
absence of forskolin (10 µM). DA (10 µM) was also used as a control. Two compounds (9c, 19ca)
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were found to display effects independent of any action at the D2R. Importantly, the original VLS
hit (1), representative NAMs (2, 9h, 19bb, 19fb, and 19fc), as well as all other agonists exert an
effect dependent on the presence of the D2R.
4. Results and discussion
4.1. Functional analysis of 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine analogues of 2
We first examined the effect of introducing various monocyclic/acyclic aliphatic amines to the 4-
position of the core scaffold whilst still bearing the fused cyclohexane system. This was based on
our finding that 2, bearing a 4-(N,N-diethylamino) substituent, displayed high negative
cooperativity. As outlined in Table 1, anilino (9a) and cyclohexylamino (9b) substituents rendered
the corresponding analogues functionally inactive. Substitution with the smaller cyclopropylamino
substituent (9f) resulted in a modest increase in functional affinity (KB = 0.88 µM) and maintenance
of a negative modulatory effect on DA efficacy (β = 0.03, Figure 3C). The cyclopropylmethylamino
analogue (9g) maintained a functional affinity (KB = 1.19 µM) similar to that of 2, but more modest
modulatory effects upon dopamine efficacy (β = 0.29). Introduction of a cyclobutylamino
substituent (9h) not only maintained this negative modulatory action, but acted to increase affinity
(KB = 0.57 µM, α = 0.19, β = 0.22, Figure 3D). Conversely, the tert-butylamino analogue (9i) lost
~50-fold functional affinity relative to 2 (KB = 92.5 µM), but now, surprisingly, exerted agonism (τB
= 0.91). It is interesting to note that introduction of the tert-butylamino moiety (9i) abolished
negative cooperativity, whereas the N,N-diethylamino substituent (2) confers a high degree of
negative allosteric modulatory effects upon dopamine efficacy. These data demonstrate that the ring
size and nature of the substituent at the 4-position are crucial for both affinity and negative
modulatory action. We found that larger 6-membered cyclic substituents bearing secondary amines,
both of aromatic and aliphatic nature, are not tolerated, whereas three- and four-membered cyclic
substituents (cyclopropylamino and cyclobutylamino, respectively) increase affinity and maintain
modulatory properties in conjunction with the fused cyclohexane system. We next examined the
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effect of substituents bearing tertiary amines by incorporating various cyclic amines. The piperidino
analogue (9c) decreased the functional affinity by 10-fold (KB = 59.3 µM), and was devoid of any
modulatory effect on DA but, surprisingly, appeared to exert an intrinsic response in its own right.
However, our assay using FlpIn CHO cells that do not express the D2R revealed that this is effect is
most likely an off target effect (Supplementary Figure 1). The morpholino analogue (9d) further
decreased the functional affinity, some ~65-fold (KB = 126 µM), but also displayed agonism (τB =
1.66) with negligible modulatory action on DA an effect that required the expression of the D2R
(Figure 3B). Interestingly, isosteric replacement of the morpholine O with NH to give the
piperazino analogue (10), significantly changed the pharmacology as concentrations of 10 caused
no decrease in DA maximal response, whilst acting to cause a limitless rightward shift in the DA
dose-response curve. Such a pattern could be consistent with either very high negative cooperativity
with DA affinity, or conversely, a competitive mode of action. Accordingly, these data could also
be fit with a model of competitive antagonism (pA2 = 4.67 ± 0.09, Schild slope: 1.06 ± 0.12, Figure
3E). If this molecule is indeed competitive with DA we speculate that the presence of the piperazino
ionisable nitrogen atom at physiological pH may potentially confer greater affinity for the
orthosteric binding pocket, consequently converting the pharmacology from allosteric to
competitive. Decreasing the ring size by one carbon relative to 9c to give pyrrolidine analogue (9e),
maintained functional affinity (KB = 4.23 µM), and displayed robust negative allosteric modulatory
effects upon DA efficacy (β = 0.17).
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Table 1 Functional parameters derived from cAMP BRET assay for analogues of 1 modified with
various amines at the 4-position
#
2
R1
pKB (KB, µM)a
LogτB (τB)b
Log α (α)c
Log β (β)d
5.72 ± 0.13 (1.92)
= -3.0
-
= -3.0
nd
nd
9a
9b
9c
9d
4.23 ± 0.13 (59.3)
3.90 ± 0.27 (126)
NSE
= 0
= 0
= 0
= 0
-0.22 ± 0.15
(1.66)
-0.76 ± 0.06
(0.17)
5.37 ± 0.13 (4.23)
6.05 ± 0.09 (0.88)
5.92 ± 0.19 (1.19)
6.25 ± 0.12 (0.57)
4.03 ± 0.31 (92.5)
= 0
= 0
9e
9f
-1.48 ± 0.08
(0.03)
-0.54 ± 0.08
(0.29)
-0.65 ± 0.07
(0.22)
-0.06 ± 0.14
(0.86)
-0.72 ± 0.12
(0.19)
9g
9h
9i
-0.04 ± 0.15
(0.91)
= 0
= 0
4.70 ± 0.09 (20.0)
= -3.0
= 0
10
aEstimate of the negative logarithm of the equilibrium dissociation constant as determined in an cAMP functional assay.
bEstimate of the logarithm of the net cooperativity factor between the modulator and dopamine as determined in an
c
cAMP functional assay. Estimate of the intrinsic efficacy of the modulator. nd = inactive at concentrations up to 100
µM. NSE, non-specific effect, compound determined to inhibit forskolin-stimulated cAMP accumulation in the absence
of the D2R. Values represent the mean ± S.E.M. from at least three independent experiments performed in duplicate.
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Fig. 3. Modification to the thieno[2,3-d]pyrimidine scaffold 4-substituent results in analogues
with distinct functional effects at the hD2LR. (A) N,N-Diethylamino substitution confers negative
allosteric cooperativity on DA signalling efficacy (2). (B) Introduction of the cyclic tertiary
aliphatic morpholino moiety confers allosteric agonism (9d). (C, D) Introduction of
cyclopropylamino or cyclobutylamino substituents to the 4-position in conjunction with the fused
cyclohexane system results in analogues with sub-µM affinities that maintain negative allosteric
cooperativity (9f, 9h). (E) Introduction of the piperazino moiety (10) converts allosteric to
competitive pharmacology. (F) Removal of the fused cyclohexane in conjunction with a
cyclobutylamino substituent converts allosteric pharmacology to agonism (15b). All data used in
these graphs are detailed in Tables 1 and 2 and are presented as mean ± SEM from three
independent experiments performed in duplicate.
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4.2. Functional analysis of thieno[2,3-d]pyrimidine fragment analogues of 2
We recently reported that incorporating an N,N-diethylamino substituent to the 4-position of the
thieno[2,3-d]pyrimidine scaffold devoid of both the 2-morpholinomethyl moiety and fused
cyclohexane systems (3), resulted in a low molecular weight fragment-like NAM of the D2R
(Figure 1).18 As 4-cyclopropylamino and 4-cyclobutylamino substituents on the thieno[2,3-
d]pyrimidine moiety conferred sub-µM analogues with robust negative cooperativity, we further
examined the effect of introducing these substituents to the 4-position of 3 in place of the N,N-
diethylamino functionality (Table 2). We found that introducing the cyclopropylamino substituent
(15a) did not affect functional affinity, however caused a limitless rightward shift in the DA dose-
response curve in addition to stimulating a modest agonist response in its own right. If cooperativity
values of
α and β were constrained to -3.0 and 0, respectively, we could derive a value of affinity
and efficacy (KB = 21.4 µM, τB = 0.22, Table 2). However, this pattern of ligand action is also
consistent with that of a competitive low efficacy partial agonist. Furthermore, introduction of a
cyclobutylamino substituent (15b) maintained affinity but abolished modulatory effects and
engendered agonism (KB = 7.38 µM, τB = 0.48, Table 2, Figure 3F). Thus, relatively subtle
structural modifications significantly change pharmacology, from compounds which negatively
modulate the behaviour of DA from a secondary binding site, to molecules that activate the receptor
with no cooperativity. Of note, the D2R orthosteric agonists pramipexole and sumanirole are both
low molecular weight heterocyclic compounds bearing acyclic secondary amines.
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Table 2 Functional parameters derived from cAMP BRET assay for fragment analogues of 2 with
modifications to the 4-position
#
3
R1
pKB (KB, µM)a
LogτB (τB)b
Log α (α)c
Log β (β)d
5.31 ± 0.07 (4.6)
= 0
-0.89 ± 0.04 (0.13)
4.67 ± 0.08 (21.4) -0.65 ± 0.07 (0.22)
= -3.0
= 0
15a
15b
5.13 ± 0.19 (7.38) -0.32 ± 0.06 (0.48)
= 0
= 0
aEstimate of the negative logarithm of the equilibrium dissociation constant determined in an cAMP functional assay.
bEstimate of the logarithm of the net cooperativity factor between the modulator and dopamine determined in an cAMP
functional assay. c Estimate of the intrinsic efficacy of the modulator. nd = inactive at concentrations up to 100 µM.
Values represent the mean ± S.E.M. from at least three independent experiments performed in duplicate.
4.3. Functional analysis (cAMP) of fused cyclohexane modified analogues of 2
Our previous study revealed that incorporating various substituents (fused cyclopentane,
cycloheptane, cyclooctane, 5- and 6-cyclohexyl, 5- and 6-phenyl, 5,6-dimethyl) to the 5-/6-positions
of the thieno[2,3-d]pyrimidine generally rendered the corresponding analogues functionally
inactive. However, these substituents were all previously examined on molecules also bearing 2-
morpholinomethyl and 4-(3-(trifluoromethyl)anilino) substituents. As such, we further extended our
study to monitor the effect of incorporating favourable amino substituents (N,N-diethylamino,
cyclopropylamino, or cyclobutylamino) onto the 4-position of the thieno[2,3-d]pyrimidine whilst
being devoid of the 2-morpholinomethyl moiety present in 1, and bearing variation in the nature of
substituents at the 5- and/or 6-positions (Table 3).
4.3.1. Fused cyclopentane analogues. Our previous work revealed converting the fused
cyclohexane system of 1 to the homologous cyclopentane system had no adverse impact on
functional affinity, nor the degree of negative cooperativity. Molecular docking studies conducted
using the D2R crystal structure as a template suggest that such fused hydrophobic rings interact with
a hydrophobic subpocket located between helices V and VI formed by the residues I184ECL2
,
16

ACCEPTED MANUSCRIPT
V1905.39, H3946.55 (where superscript numbers refer to Ballesteros-Weinstein numbering
system27).18,28 However, these interactions were predicted for compounds bearing a fused
hydrophobic ring in conjunction with additional substituents located at the 2-position
(morpholinomethyl) and 4-position (3-(trifluoromethyl)anilino). As these groups were now altered,
we wanted to re-examine the importance of this ring system for such compounds. The N,N-
diethylamino analogue (19aa) displayed an increase in functional affinity (KB = 0.74 µM) relative to
1 and acted to negatively modulate DA signalling efficacy (β = 0.14, Figure 4A). The
cyclopropylamino analogue (19ab) maintained low µM affinity (KB = 6.01 µM) and similarly acted
as a NAM of DA efficacy (β = 0.28). Conversely, the cyclobutylamino analogue (19ac) displayed a
>15-fold decrease in functional affinity (KB = 102 µM) and acted as an agonist (τB = 0.57). These
data were surprising as 9h, the corresponding fused cyclohexane variant of 19ac, displayed sub-µM
affinity at the D2R and acted as a NAM of DA efficacy.
4.3.2. Larger homologous fused rings (cycloheptane and cyclooctane). Increasing the hydrophobic
ring size by one carbon relative to 1 was previously shown to abolish D2R activity, potentially due
to the hydrophobic allosteric pocket failing to accommodate such extended ring sizes.18 Likewise,
further expansion of the hydrophobic ring by one additional carbon in the presence of the 3-
(trifluoromethyl)anilino substituent also previously rendered the corresponding analogue inactive.18
In the presence of a fused cycloheptane system devoid of the 2-morpholinomethyl substituent,
incorporation of the 4-(N,N-diethylamino) group (19ba) similarly abolished activity. However, the
cyclopropylamino analogue 19bb (KB = 2.96 µM,
β = 0.05, Figure 4B) and cyclobutylamino
analogue 19bc (KB = 0.91 µM, = 0.06, Figure 4C) remained NAMs of DA efficacy at the D2R,
β
with µM and sub-µM affinities, respectively. Interestingly, all cyclooctane analogues in this study
maintained activity at the D2R. The N,N-diethylamino analogue 19ca maintained low µM affinity
(KB = 14.5 µM) and negatively modulated DA signalling efficacy whilst exerting a slight degree of
agonism unlike the inactive structural analogue 19ba bearing the fused cycloheptane system.
However, this agonism proved to be a non-specific effect (Supplementary Figure 1). To our surprise
17

ACCEPTED MANUSCRIPT
the cyclopropylamino analogue 19cb displayed the highest improvement in affinity seen for any
analogue of 1 to date, some 10-fold compared to 2, coupled with robust negative modulation of DA
signalling efficacy (KB = 0.53 µM,
substituent (19cc) had no effects on affinity compared to 2 and this compound still acted as a NAM
of DA efficacy (KB = 3.98 µM, = 0.26).
β = 0.10, Figure 4D). Introduction of a cyclobutylamino
β
4.3.3. 5-Phenyl substituted analogues. Installing a phenyl substituent at the 5-position of 1 in the
presence of the 4-(3-(trifluoromethyl)anilino) substituent had previously been shown to abolish
activity.18 Surprisingly, the N,N-diethylamino analogue (19da) not only lost >10-fold affinity, but
caused a limitless rightward shift of the DA concentration response-curve, instead displaying weak
agonism. From this limited set of data, however, its mode of action cannot be conclusively
determined (i.e. allosteric agonist versus low efficacy competitive partial agonist). In order to derive
a value of affinity and efficacy, cooperativity values of
α and β were constrained to -3.0 and 0,
respectively (KB = 60.0 µM, τB = 0.27). Additionally, both the cyclopropylamino analogue (19db)
(KB = 1.93 µM, τB = 0.32) and cyclobutylamino analogue (19dc) (KB = 137 µM, τB = 0.39) lost their
modulatory effect upon DA and instead behaved as agonists.
4.3.4. 6-Phenyl substituted analogues. Converting the fused cyclohexane system of 1 to the
corresponding 6-phenyl-substituted thiophene in the presence of 3-(trifluoromethyl)anilino was
previously not tolerated.18 However, in the presence of 4-(N,N-diethylamino), 19ea showed
improved functional affinity (KB = 0.90 µM) relative to 1 and was a NAM of DA efficacy. The
cyclopropylamino analogue 19eb also maintained affinity and negative allosteric cooperativity (KB
= 6.35 µM,
α = 0.60, β = 0.43). Conversely, the cyclobutylamino analogue 19ec no longer
negatively modulated the action of DA but instead also acted as a weak agonist (KB = 4.50 µM, τB
=
0.28). These data further demonstrate that the nature of the amine at the 4-position can dramatically
affect the type of functional behaviour exerted by this scaffold at the D2R.
4.3.5. 5-Cyclohexyl substituted analogues. Integration of a 5-cyclohexyl substituent into the
scaffold of 1 was previously shown to abolish activity.18 In line with these data, the N,N-
18

ACCEPTED MANUSCRIPT
diethylamino analogue (19fa) was also inactive. However, despite this, analogues 19fb (KB = 11.6
µM, = 0.09) and 19fc (KB = 2.92 µM, = 0.03, Figure 4E) were both NAMs of DA efficacy at the
β
β
D2R. These data indicate that incorporation of a 5-cyclohexyl substituent may be favourable
depending on the nature of substituents at the 2- and 4-positions.
4.3.6. 6-Cyclohexyl substituted analogues. Modifying the thiophene with a cyclohexyl substituent at
the 6-position of 1 maintained low µM affinity and negative allosteric cooperativity.18 This
substituent changed observed pharmacology from negative modulation of DA efficacy (β) to
negative modulation of DA affinity (α). However, we found that incorporating the cyclohexyl
substituent into analogues devoid of the 2-morpholinomethyl moiety (19ga-c) abolished activity
regardless of the nature of the 4-substituent.
4.3.7. 5,6-Dimethyl substituted analogues. This structural modification in conjunction with the 4-(3-
(trifluoromethyl)anilino) group was previously shown to abolish activity at the D2R.18 In the
presence of an N,N-diethylamino in the 4-position, however, 19ha maintained low µM affinity (KB
= 19.5 µM) but failed to have any effects on DA binding and function, instead displaying agonism
(
τB = 0.24). Conversely, the cyclopropylamino analogue (19hb) was inactive in our functional
assay, while the cyclobutylamino analogue (19hc) maintained low µM affinity and was a NAM of
DA efficacy (KB = 2.88 µM, = 0.13, Figure 4F).
β
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Table 3 Functional parameters derived from cAMP BRET assay for analogues of 2 with
modifications to the 5/6-fused system and 4-position
#
2
n
R1
R2
R3
pKB (KB, µM)a LogτB (τB)b
Log α (α)c
Log β (β)d
5.72 ± 0.13
= -3.0
2
= 0
= -3.0
(1.92)
6.13 ± 0.14
(0.74)
-0.85 ± 0.07
(0.14)
1
1
1
3
3
3
4
4
4
-
= 0
19aa
19ab
19ac
19ba
19bb
19bc
19ca
19cb
19cc
19da
19db
19dc
19ea
5.22 ± 0.14
(6.01)
-0.13 ± 0.11 -0.55 ± 0.06
(0.75)
(0.28)
3.99 ± 0.50
(102)
-0.25 ± 0.29
(0.57)
= 0
= 0
nd
5.53 ± 0.07
(2.96)
-1.30 ± 0.07
(0.05)
= 0
= 0
6.04 ± 0.13
(0.91)
-1.22 ± 0.09
(0.06)
4.84 ± 0.13
(14.5)
NSE
= 0
-3.0
6.27 ± 0.18
(0.53)
-1.00 ± 0.11
(0.10)
= 0
5.40 ± 0.26
(3.98)
-0.58 ± 0.09
(0.26)
= 0
4.22 ± 0.33
(60.0)
-0.57 ± 0.14
(0.27)
H
H
H
= -3.0
= -3.0
= -3.0
= 0
= 0
= 0
5.72 ± 0.22
(1.93)
-0.47 ± 0.09
(0.32)
-
3.89 ± 0.52
(137)
-0.41 ± 0.31
(0.39)
-
= 0
6.04 ± 0.23
(0.90)
-
H
= -3.0
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5.92 ± 0.17
(6.35)
-0.22 ± 0.10 -0.37 ± 0.05
-
-
-
-
-
-
H
H
19eb
19ec
19fa
19fb
19fc
19ga
(0.60)
(0.43)
5.35 ± 0.24
(4.50)
0.55 ± 0.10
(0.28)
= 0
= 0
H
H
H
nd
4.93 ± 0.16
(11.6)
-1.04 ± 0.19
(0.09)
= 0
5.53 ± 0.13
(2.92)
-1.55 ± 0.19
(0.03)
= 0
H
nd
-
-
-
H
H
nd
nd
19gb
19gc
19ha
4.71 ± 0.30
(19.5)
-0.61 ± 0.11
(0.24)
CH3
CH3
CH3
CH3
= 0
= 0
-
CH3
nd
19hb
5.54 ± 0.16
(2.88)
-0.90 ± 0.08
(0.13)
-
CH3
19hc
aEstimate of the negative logarithm of the equilibrium dissociation constant determined in an cAMP functional assay.
bEstimate of the logarithm of the net cooperativity factor between the modulator and dopamine determined in an cAMP
c
functional assay. Estimate of the intrinsic efficacy of the modulator determined in an cAMP functional assay. nd =
inactive at concentrations up to 100 µM. NSE – non-specific effect, compound determined to inhibit forskolin-
stimulated cAMP accumulation in the absence of the D2R. Values represent the mean ± S.E.M. from at least three
independent experiments performed in duplicate.
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Fig. 4. Various aliphatic amino substituents at the 4-position can recover allosteric
pharmacology. Most modifications to the 5- and 6-positions of the thienopyrimidine were
previously found to be detrimental to the activity of the target compound. However, these activities
can be recovered and even enhanced when using N,N-diethylamino, cyclopropylamino or
cyclobutylamino substituents at the 4-position. (A) Fused cyclopentane with 4-(N,N-diethylamino)
substituent (19aa, KB = 0.74 µM, β = 0.14). (B) Fused cycloheptane with 4-(cyclopropylamino)
substituent (19bb, KB = 2.96 µM, β = 0.05). (C) Fused cycloheptane with 4-(cyclobutylamino)
substituent (19bc, KB = 0.91 µM, β = 0.06). (D). Fused cyclooctane with 4-(cycloproylamino)
substituent (19cb, KB = 0.53 µM, β = 0.10). (E) 5-Cyclohexyl with 4-(cyclobutylamino) substituent
(19fc, KB = 2.92 µM, β = 0.03). (F) 5,6-Dimethyl with 4-(cyclobutylamino) substituent (19hc, KB =
2.88 µM, β = 0.13). All data used in these graphs are detailed in Table 3 and are presented as mean
± SEM from three independent experiments performed in duplicate.
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Our data reveal that relatively subtle structural changes can cause a change in pharmacology
from that of a NAM to that of a weak D2R agonist. With two exceptions, the actions of the ligands
described within this study are mediated through interaction with the D2R. The phenomenon
whereby subtle modifications to a small molecule allosteric scaffold act to modulate modes of
pharmacology, presumably via a change in receptor conformation, have been coined as “molecular
switches”.29 This phenomenon has been documented for allosteric ligands targeting multiple GPCR
and non-GPCR targets, including muscarinic acetylcholine receptors (mAChRs),30-32 as well as
kinase33 and phospholipase34,35 allosteric ligands. Molecular switches have been reported to
encompass a number of subtle structural changes, for example, stereochemistry, ring size and
simple aryl substitution (i.e. fluoro vs methyl) to afford compounds with diverse pharmacology (e.g.
positive and negative alloseric modulators, partial antagonists, and agonists). Indeed it is not
surprising that such changes to allosteric ligands can cause dramatic changes in pharmacology
given that similarly subtle changes to orthosteric ligands can convert agonists to antagonists.
However, for those compounds that displayed agonism rather than the NAM activity of 1, the
nature of this agonism i.e. whether it is non-competitive (allosteric) or competitive (orthosteric),
cannot be conclusively confirmed due to their low D2R affinity. Thus it is not clear whether such
compounds bind to the same allosteric site as 1, and are examples of molecular switching, or
whether the relatively subtle structural changes investigated within this study confer the ability to
engage the orthosteric site.18 Indeed, our recent paper proposed that the allosteric binding site of 1
was in close proximity to the orthosteric site.
In summary, it is clear that the nature of substituents at the thieno[2,3-d]pyrimidine 4-, 5- and 6-
positions play a crucial role in both binding affinity and functional activity (e.g. anilino (9a) and
cyclohexylamino (9b) at the 4-position are not tolerated). However, other 6-membered substituents
(morpholino (9d) and piperazino (10)) convert NAMs to agonists and competitive antagonists,
respectively. Moreover, smaller acyclic and cyclic at the 4-position (N,N-diethylamino (2),
cyclopropylamino (9f), cyclopropylmethylamino (9g) and cyclobutylamino (9h)) are tolerated, with
23

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all compounds retaining modulatory activity as well as 9h showing a 10-fold increase in affinity.
Decreasing the fused ring size whilst bearing the cyclobutylamino substituent (19ac) abolishes any
negative modulatory effects, and instead confers agonism. However the N,N-diethylamino (19aa)
and cyclopropylamino (19ab) counterparts retain negative modulatory action. Interestingly,
increasing the ring size (cycloheptane (19bc), cyclooctane (19cc)) while bearing the
cyclobutylamino substituent maintains negative modulatory action. However, introduction of the
N,N-diethylamino substituent to the cycloheptane analogue (19ba) completely abolishes activity.
Additionally, introduction of a phenyl ring to the 5-position in the presence of any amine converts
all analogues (19da-c) to agonists. In most cases, a cyclohexyl substituent at this position appears
favourable (19fb-c) as these compounds retain affinity and cooperativity, whereas an N,N-
diethylamino substituent (19fa) abolishes activity. A phenyl substituent at the 6-posititon, however,
is tolerated in the presence of N,N-diethylamino (19ea) and cycloproylamino (19eb), yet, with
cyclobutylamino (19ec), this yields an agonist. Installing a cyclohexyl moiety at the 6-position
abolishes activity for 19ga-c, whereas the activity profile of analogues bearing a 5,6-dimethyl
substituent are highly dependent on the amine present at the 4-position (N,N-diethylamino (19ha) =
agonist, cyclopropylamino (19hb) = inactive, and cyclobutylamino (19hc) = NAM). All NAMs
identified were modulators of agonist efficacy, whereby structural modifications did not confer
modulation of agonist affinity.
5. Conclusions
In this study, we report the functional characterisation of a small library of structural analogues
based on a thieno[2,3-d]pyrimidine scaffold that we have previously shown to act as D2R NAMs.
The impact of structural modification was initially assessed with respect to the type of amine
substituent at the 4-position in the presence of the 5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-
d]pyrimidine moiety (series 1). Having identified favourable amines from series 1, we further
extended our study to the analysis of various substituents in place of the 5/6-fused cyclohexane
24

ACCEPTED MANUSCRIPT
system in conjunction with one of three selected amines (series 2) at the 4-position. While many of
these derivatives maintained NAM activity, some, surprisingly acted as D2R agonists. It is not clear
from the present data if this change in activity relates to a switch from an allosteric to an orthosteric
mode of engagement or whether these compounds bind to the same allosteric site yet display
distinct pharmacological effects. Future studies are needed to address this but such efforts are
complicated by the relatively low affinity of these ligands. In the presence of a fused cyclohexane
system, small cyclic aliphatic amines (cyclopropylamino, cyclobutylamino) were found to be vital
for negative modulatory action. Two of the highest affinity NAMs to arise from this scaffold were
identified, 9h (KB = 0.57 µM, fused cyclohexane moiety) and 19cb (KB = 0.53 µM, fused
cyclopentane moiety), that contain cyclobutylamino and cyclopropylamino substituents,
respectively. Conversely, larger heterocyclic substituents (morpholino) and acyclic amines (tert-
butylamino) conferred agonism and abolished modulatory activity (9d and 9i, respectively). In
combination with a range of different substituents at the 5/6-positions, however, small
cyclic/acyclic aliphatic amines gave a range of analogues with differential functional pharmacology,
ranging from NAMs of DA efficacy, to agonists and compounds with no apparent functional
activity. Taken together, this study demonstrates that the pharmacology of analogues functionalised
at the 4-, 5-, and 6-positions of the thieno[2,3-d]pyrimidine core is difficult to predict, resulting in
shallow SAR. Such observations are consistent with SAR studies of allosteric modulators of other
GPCR targets, highlighting the challenges associated with GPCR allosteric ligand design.
6. Experimental section
6.1. General methods for chemistry
Chemicals and solvents were purchased from standard suppliers and used without further
purification. Davisil silica gel (40−63 µm) for flash column chromatography was supplied by Grace
Davison Discovery Sciences (Victoria, Australia), and deuterated solvents were purchased from
Cambridge Isotope Laboratories, Inc. (USA, distributed by Novachem PTY. Ltd., Victoria,
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ACCEPTED MANUSCRIPT
Australia). Reactions were monitored by thin layer chromatography on commercially available
precoated aluminum-backed plates (Merck Kieselgel 60 F254). Visualization was by examination
under UV light (254 and 366 nm). A solution of ninhydrin (in ethanol) was used to visualize
primary and secondary amines. All organic extracts collected after aqueous workup procedures
were dried over anhydrous Na2SO4 before gravity/vacuum filtering and evaporation to dryness.
1
13
Organic solvents were evaporated in vacuo at ≤40 °C (water bath temperature). H NMR and C
NMR spectra were recorded on a Bruker Avance Nanobay III 400 MHz Ultrashield Plus
spectrometer at 400.13 and 100.62 MHz, respectively. Chemical shifts (δ) are recorded in parts per
million (ppm) with reference to the chemical shift of the deuterated solvent. Coupling constants (J)
are recorded in Hz, and the significant multiplicities described by singlet (s), doublet (d), triplet (t),
quadruplet (q), broad (br), multiplet (m), doublet of doublets (dd), and doublet of triplets (dt).
Spectra were assigned using appropriate COSY, distortionless enhanced polarization transfer
(DEPT), HSQC, and HMBC sequences. All NMR experiments were performed in CDCl3 to permit
comparison of the spectra of the various analogues. Experiments were performed in acetone-d6,
DMSO-d6, or MeOH-d4 where selected analogues lacked solubility in CDCl3. LCMS experiments
were run using one of two systems to verify reaction outcome and purity. System A was the default
unless otherwise stated. System A consisted of the following: an Agilent 6100 series single quad
coupled to an Agilent 1200 series HPLC instrument using the following buffers: buffer A, 0.1%
formic acid in H2O; buffer B, 0.1% formic acid in MeCN. The following gradient was used with a
Phenomenex Luna 3 µm C8(2) 15 mm × 4.6 mm column and a flow rate of 0.5 mL/min and total
run time of 12 min: 0−4 min 95% buffer A and 5% buffer B, 4−7 min 0% buffer A and 100% buffer
B, 7−12 min 95% buffer A and 5% buffer B. Mass spectra were acquired in positive and negative
ion modes with a scan range of 0−1000 m/z at 5 V. UV detection was carried out at 254 nm. System
B consisted of the following: an Agilent 6120 series single quad coupled to an Agilent 1260 series
HPLC instrument. The following buffers were used; buffer A, 0.1% formic acid in H2O; buffer B,
0.1% formic acid in MeCN. The following gradient was used with a Poroshell 120 EC-C18 50 mm
26

ACCEPTED MANUSCRIPT
× 3.0 mm, 2.7 µm column and a flow rate of 0.5 mL/min and total run time of 5 min: 0−1 min 95%
buffer A and 5% buffer B, from 1 to 2.5 min up to 0% buffer A and 100% buffer B, held at this
composition until 3.8 min, 3.8−4 min 95% buffer A and 5% buffer B, held until 5 min at this
composition. Mass spectra were acquired in positive and negative ion modes with a scan range of
100−1000 m/z. UV detection was carried out at 214 and 254 nm. All retention times (tR) are quoted
in minutes. System C: Analytical reverse-phase HPLC was performed on a Waters HPLC system
coupled directly to a photodiode array detector and fitted with a Phenomenex Luna C8 (2) 100 Å
column (150 mm × 4.6 mm, 5 µm) using a binary solvent system: solvent A, 0.1% TFA/H2O;
solvent B, 0.1% TFA/80% CH3CN/H2O. Gradient elution was achieved using 100% solvent A to
100% solvent B over 20 min at a flow rate of 1 mL/min. All compounds subjected to biological
testing were found to be >95% pure by HPLC at two wavelengths (λ of 254 and 214 nm).
6.2. General synthetic procedures.
6.3. General procedure A for the Synthesis of 9a-j, 15a-b, 19aa-hc
In a suitable microwave reaction vessel the required chloropyrimidine (1 equiv.) was taken up in i-
PrOH. To this was added the required amine (1.1 equiv.) and the mixture irradiated under stirring at
120 ˚C for 1-2 h. Upon completion of the reaction, the mixture was directly purified using FCC to
afford the compound. Similarly, any precipitate could be collected under vacuum and washed
several time with cold i-PrOH to afford the desired compound.
6.3.1. N-Phenyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (9a)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 105 mg of a white
amorphous solid (84%). LCMS (m/z): 281.9 [M+H]+. HPLC: tR 6.244 min, >95% purity (214 &
254 nm). HRMS (m/z): C16H15N3S requires 282.1071 [M+H]+; found 282.1059. 1H NMR (CDCl3) δ
8.48 (s, 1H), 7.64 (dt, J = 8.8, 1.7 Hz, 2H), 7.41 – 7.34 (m, 2H), 7.17 – 7.10 (m, 2H), 3.06 (dd, J =
27

ACCEPTED MANUSCRIPT
13
8.1, 3.9 Hz, 2H), 2.85 (dd, J = 8.0, 3.9 Hz, 2H), 2.03 – 1.89 (m, 4H). C NMR (CDCl3) δ 166.3,
155.0, 152.6, 138.5, 134.7, 129.1, 124.8, 124.0, 121.3, 116.6, 26.5, 25.5, 22.5, 22.4.
6.3.2. N-Cyclohexyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (9b)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 115 mg of a beige
amorphous solid (82%). LCMS (m/z): 288.2 [M+H]+. HPLC: tR 6.383 min, >95% purity (214 &
254 nm). HRMS (m/z): C16H21N3S requires 288.1533 [M+H]+; found 288.1529. 1H NMR (CDCl3) δ
8.35 (s, 1H), 4.23 – 4.12 (m, 1H), 2.91 (dd, J = 6.8, 5.1 Hz, 2H), 2.79 (dd, J = 8.1, 3.7 Hz, 2H), 2.12
– 2.03 (m, 2H), 1.96 – 1.85 (m, 4H), 1.75 (ddd, J = 13.3, 8.8, 5.0 Hz, 2H), 1.69 – 1.61 (m, 1H), 1.55
– 1.42 (m, 2H), 1.32 – 1.21 (m, 3H). 13C NMR (CDCl3) δ 165.3, 156.9, 153.3, 132.9, 125.5, 116.0,
48.9, 33.3, 26.5, 25.8, 25.5, 24.8, 22.7, 22.6.
6.5.3. 4-(Piperidin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (9c)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 95.2 mg of a white
amorphous solid (68%). LCMS (m/z): 274.1 [M+H]+. HPLC: tR 6.478 min, >95% purity (214 &
254 nm). HRMS (m/z): C15H19N3S requires 224.1374 [M+H]+; found 274.1372. 1H NMR (CDCl3) δ
8.51 (s, 1H), 3.36 – 3.28 (m, 4H), 2.96 – 2.91 (m, 2H), 2.90 – 2.85 (m, J = 6.3, 1.6 Hz, 2H), 1.97 –
13
1.90 (m, 2H), 1.81 (ddd, J = 12.1, 5.9, 2.6 Hz, 2H), 1.77 – 1.71 (m, 4H), 1.69 – 1.62 (m, 2H). C
NMR (CDCl3) δ 168.1, 162.9, 151.6, 134.4, 127.7, 121.5, 51.8, 26.7, 25.8, 25.7, 24.4, 23.1, 22.9.
6.5.4. 4-(5,6,7,8-Tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-yl)morpholine (9d)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 161 mg of a white
amorphous solid (88%). LCMS (m/z): 275.9 [M+H]+. HPLC: tR 5.179 min, >95% purity (214 &
254 nm). HRMS (m/z): C14H17N3OS requires 275.1065 [M+H]+; found 275.1087.1H NMR (400
MHz, CDCl3) δ 8.46 (s, 1H), 3.82 – 3.77 (m, 4H), 3.36 – 3.31 (m, 4H), 2.87 – 2.77 (m, 4H), 1.91 –
28

ACCEPTED MANUSCRIPT
1.84 (m, 2H), 1.79 – 1.71 (m, 2H). 13C NMR (CDCl3) δ 168.39, 162.08, 151.46, 135.46, 126.9,
121.4, 66.6, 51.1, 26.7, 25.8, 22.9, 22.8.
6.5.5. 4-(Pyrrolidin-1-yl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidine (9e)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 109 mg of a white
amorphous solid (82%). LCMS (m/z): 260.0 [M+H]+. HPLC: tR 5.270 min, >95% purity (214 &
254 nm). HRMS (m/z): C14H17N3S requires 259.1112 [M+H]+; found 259.1138. 1H NMR (CDCl3) δ
8.33 (s, 1H), 3.72 – 3.65 (m, 4H), 2.90 – 2.81 (m, 4H), 1.95 – 1.86 (m, 6H), 1.78 – 1.71 (m, 2H).
13C NMR (CDCl3) δ 167.7, 158.8, 150.9, 132.2, 127.5, 117.9, 50.9, 29.2, 25.8, 25.5, 23.3, 22.8.
6.5.6. N-Cyclopropyl-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (9f)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 105 mg of a white
amorphous solid (87%). LCMS (m/z): 246.0 [M+H]+. HPLC: tR 4.864 min, >95% purity (214 &
254 nm). HRMS (m/z): C13H15N3S requires 245.0954 [M+H]+; found 245.0981. 1H NMR (CDCl3) δ
8.47 (s, 1H), 5.51 (s, 1H), 2.96 – 2.89 (m, 1H), 2.83 (td, J = 5.9, 1.9 Hz, 2H), 2.78 (td, J = 6.0, 1.9
Hz, 2H), 1.95 – 1.83 (m, 4H), 0.95 – 0.90 (m, 2H), 0.61 – 0.57 (m, 2H). 13C NMR (CDCl3) δ 165.2,
158.3, 153.1, 133.4, 125.3, 116.2, 26.4, 25.4, 23.9, 22.5, 22.4, 7.6.
6.5.7. N-(Cyclopropylmethyl)-5,6,7,8-tetrahydrobenzo[4,5]thieno[2,3-d]pyrimidin-4-amine (9g)
General procedure A. Purification by FCC (eluent, 1:1 EtOAc/PE) afforded 88 mg of a light yellow
amorphous solid (85%). LCMS (m/z): 260.0 [M+H]+. HPLC: tR 5.519 min, >95% purity (214 &
254 nm). HRMS (m/z): C14H17N3S requires 260.1215 [M+H]+; found 260.1216. 1H NMR (CDCl3) δ
8.36 (s, 1H), 5.40 (s, 1H), 2.97 – 2.91 (m, J = 8.0, 4.1, 1.7 Hz, 1H), 2.82 – 2.78 (m, 2H), 1.98 – 1.85
(m, 4H), 1.20 – 1.08 (m, 1H), 0.62 – 0.55 (m, 2H), 0.33 – 0.28 (m, 2H). 13C NMR (CDCl3) δ
165.27, 157.40, 153.14, 133.1, 125.5, 116.1, 46.1, 26.4, 25.5, 22.7, 22.6, 10.8, 3.5.
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