Design, Synthesis, and Characterization of Ogerin-Based Positive Allosteric Modulators for G Protein-Coupled Receptor 68 (GPR68)

Article abstract of DOI:10.1021/acs.jmedchem.9b00869

G protein-coupled receptor 68 (GPR68) is an understudied orphan G protein-coupled receptor (GPCR). It is expressed most abundantly in the brain, potentially playing important roles in learning and memory. Pharmacological studies with GPR68 have been hinde

Full text of DOI:10.1021/acs.jmedchem.9b00869

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Article
Design, Synthesis and Characterization of Ogerin-Based Positive
Allosteric Modulators for G Protein-Coupled Receptor 68 (GPR68)
Xufen Yu, Xi-Ping Huang, Terry P. Kenakin, Samuel T.
Slocum, Xin Chen, Michael L. Martini, Jing Liu, and Jian Jin
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00869 • Publication Date (Web): 12 Jul 2019
Downloaded from pubs.acs.org on July 13, 2019
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Design, Synthesis and Characterization of Ogerin-Based Positive
Allosteric Modulators for G Protein-Coupled Receptor 68 (GPR68)
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†,#
‡,§,#,
‡
‡
†
Xufen Yu, Xi-Ping Huang,
* Terry P. Kenakin, Samuel T. Slocum, Xin Chen, Michael L.
†
†,
†,
Martini, Jing Liu, * and Jian Jin *
†
Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and
Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY
1
0029.
‡
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599
The National Institute of Mental Health Psychoactive Drug Screening Program (NIMH PDSP),
§
Department of Pharmacology, University of North Carolina, Chapel Hill, NC 27599
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ABSTRACT: GPR68 is an understudied orphan G protein-coupled receptor (GPCR). It is expressed
most abundantly in the brain, potentially playing important roles in learning and memory.
Pharmacological studies with GPR68 have been hindered by lack of chemical tools that can selectively
modulate its activity. We previously reported the first small-molecule positive allosteric modulator
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(PAM), ogerin (1), and showed that 1 can potentiate proton activity at the GPR68-G pathway. Here, we
report the first comprehensive structure-activity relationship (SAR) study on the scaffold of 1. Our lead
compound resulted from this study, MS48107 (71), displayed 33-fold increased allosteric activity
compared with 1. 71 demonstrated high selectivity over closely related proton GPCRs and 48 common
drug targets, and was bioavailable and brain penetrant in mice. Thus, our SAR study has resulted in an
improved GPR68 PAM for investigating the physiological and pathophysiological roles of GPR68 in
vitro and in vivo.
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INTRODUCTION
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GPR68 was originally discovered as a clinically related receptor in an ovarian cancer cell line as
1
“
OGR1” for ovarian cancer G protein-coupled receptor. While it is widely expressed in most cells and
tissues including spleen, testes, small intestine, peripheral blood leukocytes, heart, lung, placenta,
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kidney, and bone, it is most abundantly expressed in the brain, particularly in the hippocampus - the
brain region that is critical for learning and memory. Despite being known for over two decades, GPR68
4
remains an understudied orphan GPCR according to IUPHAR. Generally known as a proton-sensing
GPCR, it couples to multiple signaling pathways through various G proteins (G
s
, G
q i
, G , and G12/13) in
2
,5–7
different cells.
However, it remains to be established whether GPR68 couples to the β-arrestin
signaling pathway. Consistent with its capacity to couple to multiple signaling pathways and its wide
expression pattern, GPR68 has been implicated in many biological processes, including pH
2,8
6,7,9–11
12–14
9,15,16
homeostasis, tumor growth and metastasis,
inflammation,
osteoclast development,
1
7,18
19,20
insulin secretion,
and epithelial barrier function.
Interestingly, the receptor also functions as a
mechanotransducer that senses blood flow (shear stress) in arteries, thus modulating vascular
21
physiology. Thus, GPR68, a potentially important therapeutic target, is one of the National Institutes
2
2
of Health (NIH)’s Illuminating the Druggable Genome (IDG) GPCRs. Presently, the underlying
molecular mechanisms have not been determined between proton-sensing and these physiological or
pathological processes. Pharmacological studies of GPR68 have previously been hindered by the lack of
small-molecule ligands. It is clear that potent and selective GPR68 ligands that can be used to activate
or block its activity are urgently needed to elucidate GPR68 pharmacology and clarify its potential roles
2
3
in the aforementioned biological processes.
Previous studies have shown that receptor activity is reduced or completely lost if extracellular
histidine residues are mutated, presumably by affecting protonation states responding to extracellular
2
pH changes. Therefore, it was proposed that alkaline pH conditions stabilize GPR68 in an inactive
conformation by hydrogen bonding interactions involving extracellular histidine residues, while
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acidification and histidine protonation breaks hydrogen bond interactions, initiating receptor activation.
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The putative histidine binding sites for protons are scattered throughout different extracellular loops
rendering challenges to directly activate or block this receptor with orthosteric ligands. This, in turn,
2
4,25
makes allosteric binding site more attractive to target as with many other GPCRs.
Allosteric
modulators rely on endogenous protons to produce function and can preserve the spatial and temporal
nature of the proton-mediated activation of GPR68. Allosteric modulators bind to GPR68 at a site
different from that of protons and function by fine-tuning proton activity through increasing or reducing
proton affinity (α-cooperativity), efficacy (β-cooperativity), or both.
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For conventional GPCRs, such as metabotropic glutamate receptors (mGluRs), binding and
functional assays can be carried out in biological systems that the concentration of the orthosteric ligand
(i.e. glutamate) can be controlled. However, for GPR68, the orthosteric ligand is proton, which always
presents in the receptor compartment. Therefore, the allosteric binding affinity (K ), defined as the
B
dissociation equilibrium constant of allosteric modulator in the absence of orthosteric ligand (i.e.
proton), does not have corresponding biological meaning and cannot be measured in binding assays.
Instead, we can estimate the allosteric affinity cooperativity α, efficacy cooperativity β, and allosteric
2
6–28
binding affinity K
The observed binding affinity of allosteric modulators can be calculated by the equation K
1+[A]/K )/(1+ α[A]/K ), in which [A] is the proton concentration and K is the equilibrium
B
from functional assays according to the standard allosteric operational model.
B
=
(
A
A
A
dissociation constant of protons at GPR68 in the absence of modulators. In structure-activity
relationship (SAR) studies, we actually can optimize the potency of the modulators in proton-mediated
signaling by optimizing α, β and/or K
which rely on binding affinity (K ) for selectivity, allosteric modulators achieve high selectivity through
allosteric αβ cooperativity in addition to allosteric binding affinity (K The overall allosteric
B
. In contrast to traditional orthosteric agonists or antagonists
i
2
9,30
B
).
3
1
B
activity can be quantified with a characteristic allosteric index, Log(αβ/K ), which contains
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information about allosteric cooperativity and can be calculated and used as a guiding parameter for
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SAR studies and optimization.
Development of small-molecule ligands for GPR68 remains a challenging task. A small number
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of isoxazole compounds have been reported to possess GPR68 agonist activity. However, several
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reports suggested that the weak activity of isoxazoles could be non-specific.
Divalent metal ions,
2
+
2+
2,19
such as Cu and Zn , were initially used to inhibit GPR68 activity. Recently, these divalent ions, in
2
+
2+
35–37
addition to Co and Mn , were reported to be GPR68 agonists.
However, these recent reports did
not fully control pH, nor used a broad enough pH range to address potential allosteric mechanisms. In
2
+
38–40
addition, Zn is a well-known allosteric modulator for the orphan receptor GPR39.
Moreover, these
divalent ions are biologically-important metals or trace elements and play critical roles in many
4
1–43
biological processes.
Therefore, these divalent metals are unlikely GPR68 selective tools.
In 2015, we reported the discovery of ogerin (1) as the first positive allosteric modulator (PAM)
4
4
s
of GPR68. Compound 1 potentiates proton activity in GPR68-mediated G -cAMP production, but has
no activity at related proton-sensing GPCRs, including GPR4 and GPR65. This selective ligand led to
the discovery that GPR68 activation suppresses contextual- but not cue-dependent learning and
44
memory, and does so in wild-type but not GPR68 knockout mice. These data provide the first
evidence for a role of GPR68 in cognition. Specifically, sustained hippocampal GPR68 activation may
contribute to memory deficits. Compound 1, which is now commercially available, has been used by the
21,45
research community and its PAM activity has been confirmed by other groups.
Compound 1 was
identified from a ZINC sub-library of about 3.1 million compounds through a virtual screening
4
4,46
campaign targeting GPR68.
Thorough SAR studies on the scaffold of compound 1 have not been
reported to date. Here, we report a comprehensive SAR study at the following three regions of
compound 1: (1) the aminotriazine core, (2) the benzyl alcohol moiety, and (3) the benzylamine moiety
(Figure 1). We describe design, synthesis and biological characterization of various analogs. This study
has resulted in the identification of significantly improved GPR68 PAMs.
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RESULTS
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Biological Assay and Data Analysis. We used a GloSensor cAMP assay to characterize all of
the synthesized putative GPR68 PAMs. More specifically, HEK293 T cells were transiently transfected
s
with receptor and GloSensor cAMP plasmids for proton-mediated receptor activation of G -cAMP
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production. To determine allosteric modulator activity, proton concentration responses in the absence
and presence of increasing concentrations of putative PAMs were quantified and results were analyzed
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6–28
using a standard allosteric operational model
in GraphPad Prism. Several previously reported
4
4
compounds (1, 20, 30, 55, 61, 62 and 63) were included in this study for comparison.
Modifications of the Aminotriazine Core. To investigate the SAR of the aminotriazine core,
we replaced the free amino group on the triazine core with a few small alkylamino groups. We also
switched the triazine core to a pyrimidine core (Scheme 1, Table 1). These designed compounds were
prepared using the synthetic routes outlined in Scheme 1. Intermediates 4 and 9 were prepared
4
4
according to the previously reported procedures from 2,4,6-trichloro-1,3,5-triazine (2) and 2,6-
dichlorpyrimidin-4-amine (8). Briefly, alkylamine substitution of one chloro group on trichlorotriazine 1
at low temperature provided alkylamino substituted dichlorotriazines 3. Benzylamine substitution of one
chloro group on dichlorotriazines 3 or dicholoropyrimidine 8 at an elevated temperature yielded
intermediates 4 or 9. Suzuki coupling between intermediates 4 or 9 and commercially available (2-
4
4
(hydroxymethyl)phenyl)boronic acid provided the designed compounds, 5–7 and 10 (Scheme 1).
As indicated in Table 1, a small mono-substituted amino group on the triazine core is well
tolerated. Either methylamino (5) or ethylamino (6) group has a minimal effect on allosteric parameters
or binding affinity. However, dimethyl amino group (7) completely eliminates the allosteric activity.
Replacing the triazine core with a pyrimidine (10) also greatly reduces allosteric activity to barely
detectable levels. These results suggest the amino substituted triazine core is critical for the PAM
activity. Therefore, the aminotriazine core is maintained in the following SAR studies.
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Modifications of the Benzyl Alcohol Moiety. For the benzyl alcohol moiety, we explored a
1
2
variety of substituents on the phenyl ring (Scheme 2, Table 2). The synthesis of these analogs started
with 2-amino-4,6-dichloro-1,3,5-triazine (11). Benzylamine substitution of one chloro group on 11
provided the common intermediate 12. Suzuki coupling between intermediate 12 and a set of
commercially available phenylboronic acids provided compounds 13–15, 21, 22-29 and 31-32 (Scheme
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A and 2D). Compound 16 was obtained by reducing acetophenone 15 (Scheme 2B). Compounds 18
and 19 were synthesized through reductive aminations of benzaldehyde 17 with different amines
Scheme 2C). Compounds 37 and 38 were prepared through Suzuki coupling between intermediate 12
(
and synthesized phenylboronic acids 36 bearing ethoxy and isopropoxy groups, respectively, followed
by formal group reduction (Scheme 2E).
Switching the hydroxymethyl group (1) to hydrogen (13), methyl (14), methyl ketone (15), and
1
-hydroxylethyl (16) groups is not tolerated and leads to either weak or inactive analogs. Replacing the
hydroxymethyl group with an aminomethyl group (18) retains affinity cooperativity and efficacy
cooperativity but with reduced allosteric binding affinity, rendering a less effective GPR68 PAM
compared with 1. The N-methyl substituted aminomethyl (19) group further reduces allosteric activities
and results in a weak PAM. Previously, we have demonstrated the importance of the position of the
hydroxymethyl group: the ortho-hydroxymethyl isomer was much more effective than the meta-
4
4
s
hydroxymethyl isomer to activate GPR68 G -cAMP pathway. We have confirmed this trend by
retesting 20 and by comparing the activities of 1 and 20. This trend has also been confirmed by
comparing the activities of 15 and 21 (two methyl ketone analogs). Based on these SAR results, we kept
the ortho-positioned hydroxymethyl group in subsequent SAR studies and explored additional
substituents, such as halides and alkoxy groups on the phenyl ring. First, we installed fluoro groups at 3-
(22), 4- (23), 5- (24), and 6- (25) positions on the phenyl ring. Compared with 1, the 3-fluoro analog 22
is a less effective GPR68 PAM due to the decreased efficacy cooperativity and allosteric binding
affinity. The 4-fluoro analog 23 shows slightly increased PAM activity compared with compound 1.
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Interestingly, the 6-fluoro analog 25 has significantly improved affinity cooperativity (Log (α) = 1.15 ±
.14). However, its binding affinity (-Log (K ) = 3.69 ± 0.12) is greatly reduced, resulting in an overall
slightly worse PAM (Log(αβ/K ) = 5.19 ± 0.03) than compound 1 (Log (αβ/K ) = 5.31 ± 0.05). The 5-
fluoro analog 24 is a slightly better PAM (Log (αβ/K ) = 5.69 ± 0.04) than 1 due to its slightly
improved affinity cooperativity (Log (α) = 0.91 ± 0.07) and allosteric binding affinity (-Log (K ) = 4.58
0.08). Next, we explored compounds with a chloro group at 4- (26), 5- (27), and 6- (28) positions on
the phenyl ring. Similar to 25, all of these chloro analogs show improved affinity cooperativities (Log
α) = 1.01 ± 0.08 (26); 1.20 ± 0.11 (27); and 1.25 ± 0.19 (28)). In addition, these compounds display
improved allosteric binding affinities (-Log (K ) = 4.76 ± 0.14 (26); 4.49 ± 0.19 (27); and 4.43 ± 0.20
28)) albeit reduced efficacy cooperativities, resulting in slightly better PAMs (2- to 4-fold) than 1. We
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also explored analogs bearing a methoxy group at different substitution positions (29–32). A methoxy
group in general leads to reduced allosteric index compared to 1. Among the four substitution positions,
the 4-position (30) is more tolerated while the 6-position (32) shows least PAM activity. Consequently,
we installed ethoxy (37) and isopropoxy (38) groups at the 4-postion to explore steric effects. The 4-
ethoxy group (37) slightly improves affinity cooperativity (Log (α) = 0.87 ± 0.11), maintains allosteric
binding affinities, and slightly decreases efficacy cooperativity, resulting in a comparable allosteric
index compared to 1. The 4-isopropoxy group (38) shows slightly improved allosteric activity
B B
(Log(αβ/K ) = 5.74 ± 0.07) compared to 1 due to the increased allosteric binding affinities (-Log (K ) =
4.91 ± 0.12). These SAR results indicate that additional substituents on the benzyl alcohol moiety are
tolerated, and appropriate substitutions on the phenyl ring could lead to enhanced allosteric activity.
Several substituents were identified to improve the modulators’ affinity cooperativities and/or allosteric
binding affinities. However, none of the substituents increased the modulators’ efficacy cooperativities.
Modifications of the Benzylamine Moiety. To evaluate the effects of the benzylamine moiety
on allosteric activity, we designed a variety of compounds with differentially substituted benzylamines.
In addition, we also explored different carbon linker length between the phenyl and amino groups
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(Table 3). These compounds were prepared using SNAr and Suzuki coupling reactions described in
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Scheme 3.
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Initially, a series of electron withdrawing groups (EWGs) such as fluoro (41 – 43), chloro (44 –
46), and trifluoromethyl (47 – 49) were introduced to the phenyl moiety to explore their effects at
different substitution positions. 2-, 3- and 4-fluoro groups lead to slightly improved allosteric indexes
mainly due to the increased affinity cooperativity with Log (α) values at 0.98 ± 0.15, 0.70 ± 0.13, and
1.09 ± 0.30, respectively. Compared with the fluoro substitutions, the chloro substitutions provide more
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B
significant allosteric activity improvement because of the higher allosteric binding affinities ((-Log (K )
=
5.07 ± 0.08 (44), 4.97 ± 0.19 (45), and 5.22 ± 0.07 (46)). Similarly, the trifluoromethyl analogs show
B
improved allosteric index due to improved allosteric binding affinities ((-Log (K ) = 5.05 ± 0.10 (47),
5
.11 ± 0.12 (48), and 5.03 ± 0.08 (49)). Thus, EWGs are able to improve the modulators’ allosteric
activities by increased affinity cooperativities or allosteric binding affinities. However, the substitution
positions only have moderate effects on the allosteric indexes. Next, we explored a couple of electron
donating groups (EDGs): methyl (50 – 52) and methoxy (52 – 55). The methyl analogs show slightly
improved allosteric indexes mainly due to slightly higher allosteric binding affinity (50) or affinity
cooperativity (51 and 52). Although the 2-, 3- and 4-methoxy groups improve the affinity
cooperativities (Log (α) = 0.98 ± 0.17 (53), 0.89 ± 0.18 (54), and 0.78 ± 0.11 (55)), they reduce the
allosteric binding affinities, resulting in analogs with reduced allosteric indexes. We subsequently
explored a few larger alkoxy groups, such as ethoxy (56), isopropoxy (57), cyclopropoxy (58), and
phenoxy (59), at the 4-postion. Interestingly, most of these larger ether substituents diminish the
efficacy cooperativities, leading to GPR68 α-PAMs, positive allosteric modulators with affinity
cooperativities (α) greater than 1 and efficacy cooperativities (β) equals or is close to 1. Compared with
4
B
-methoxy group (55), these larger alkoxy groups enhance allosteric binding affinities ((-Log (K ) =
4.77 ± 0.03 (56), 4.99 ± 0.09 (57), 5.07 ± 0.12 (58), and 5.19 ± 0.14 (59)), leading to improved allosteric
activities. In particular, the 4-phenoxy analog (59) is an α-PAM with the most improved allosteric
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activity compared to compound 1. Previously, we have shown that longer carbon linkers between the
1
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1
1
1
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1
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4
phenyl and amino groups resulted in analogs with higher PAM activity. We have now confirmed this
SAR trend by testing compounds 60 – 63. While a shorter linker (60) results in reduced an allosteric
B
index mainly due to the reduced allosteric binding affinity ((-Log (K ) = 3.92 ± 0.10 (60)), longer
linkers (61 – 63) enhance allosteric binding affinities ((-Log (K ) = 4.50 ± 0.21 (61), 5.22 ± 0.04 (62),
B
0
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and 5.52 ± 0.12 (63)), leading to improved allosteric indexes. We also extended the carbon linker length
of the 4-phenoxy substituted benzylamine analog 59, leading to three analogs 64 – 66. These analogs,
however, do not show enhanced allosteric activities compared to 59. Instead, longer linkers lead to
reduced allosteric activities. Interestingly, these analogs all retain high affinity cooperativity α values.
Thus, extending the carbon linker length between the phenyl group and amino group does not always
lead to better PAMs. Importantly, these SAR results suggest that, similar to the benzyl alcohol moiety,
modifications of the benzylamine moiety could improve the modulators’ affinity cooperativities and/or
allosteric binding affinities, but not their efficacy cooperativities.
Combined Modifications of Different Moieties. The SAR study described above indicates that
several functional groups, such as 6-fluoro (25) and 5-chloro (27) substituted hydroxymethylphenyl
moieties and 2-fluoro (41), 4-fluoro (43), 3-methyl (51), 2-methoxy (53), and 4-phenoxy (59)
substituted benzylamine, lead to high affinity cooperativities and/or improved allosteric binding
affinities. Using these preferred moieties, we designed a number of combination compounds, to further
improve the allosteric activity and obtain potent GPR68 α-PAMs. We also explored different linker
length between the phenyl group and amino group (n = 1-4). Synthesis of these compounds is
summarized in Scheme 3 and SAR results of these compounds are summarized in Table 4.
Compared to 25, 2-fluoro (67), 4-fluoro (68), 3-methyl (69), 2-methoxy (70), and 4-phenoxy
(71) substituted benzyl groups retain relatively high affinity cooperativity values and show low efficacy
cooperativity values. Most strikingly, the 4-phenoxy analog 71 displays greatly enhanced allosteric
binding affinity (-Log (K
B
) = 5.85 ± 0.12 versus 3.69 ± 0.12 for compound 25), resulting in a very
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Journal of Medicinal Chemistry
potent GPR68 α-PAM. Compared with 1, compound 71 has 33-fold better allosteric activity
Log(αβ/K ) = 6.83 ± 0.08 versus 5.31 ± 0.05 for compound 1). We have shown that the linker length
1
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(
B
between the phenyl and amino groups has different effects on the allosteric activities, depending on
5
different R groups (60 – 63 and 59, 64 – 65 (see above)). Extending the carbon linker length of 25 to 2
–
4 results in compounds 72 – 74. Compared with 25, all of these longer linker analogs maintain high
affinity cooperativity values and show low efficacy cooperativity values. For allosteric binding affinity,
the 2-carbon linker (72, -Log (K ) = 5.04 ± 0.09) is optimal among the 4 linker lengths explored.
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Extending the carbon linker length of 71 to 2 – 3 leads to α-PAMs 75 and 76 with retained high affinity
cooperativities, low efficacy cooperativities, but reduced allosteric binding affinities.
To improve the allosteric activity of 27, we also increased the linker length between the phenyl
group and the amino group. The longer linker analogs 77 – 79 maintain high affinity cooperativities and
low efficacy cooperativities, and enhance allosteric binding affinities (-Log (K
B
) = 5.14 ± 0.11 (77),
5
.35 ± 0.06 (78), and 4.98 ± 0.10 (79)) with the 3-carbon linker (78) being optimal. Lastly, we replaced
the benzylamine moiety of 27 with a 4-phenoxy substituted benzylamine moiety (80) or a 4-phenoxy
substituted homobenzylamine moiety (81). Compounds 80 and 81 preserve the α-PAM activity.
Compared with 27, both compounds show similar allosteric binding affinities (-Log (K
B
) = 4.70 ± 0.14
(80), and 4.59 ± 0.18 (81)) and similar overall allosteric activities (i.e., Log(αβ/K ) values).
B
Our SAR study has identified several GPR68 PAMs with better allosteric activities than
compound 1. For example, 46, an αβ-PAM (α and β > 1), has shown 6-fold higher allosteric activity
B B
(Log(αβ/K ) value) than 1. α-PAM 71 has the highest Log(αβ/K ) value, which is 33-fold higher than
that of 1. Compounds 77 and 78, α-PAMs, have approximately 10-fold better allosteric activity than 1.
We selected these four GPR68 PAMs for further characterization.
Selectivity Profiles of Selected GPR68 PAMs. To determine selectivity of 46, 71, 77, and 78
for GPR68 over other closely related proton GPCRs, such as GPR4 and GPR65, we tested these GPR68
PAMs against GPR4 and GPR65 using GloSensor cAMP assays. As shown in Figure 2A-2D, GPR4 and
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GPR65 have different response profiles to proton stimulation as compared to GPR68 in the absence of
modulators. The proton potency values (pEC50) were pH 8.04 (hill slope of 5.25) for GPR4, pH 7.47
(hill slope of 4.57) for GPR65, and pH 6.73 (hill slope of 3.38) for GPR68. In the presence of the lead
PAM 71 (Figure 2B) as well as 1 (Figure 2A), proton activity at GPR68 is potentiated in a PAM
concentration-dependent manner (proton concentration response curves were shifted leftward (affinity
cooperativity α>1) and upward (efficacy cooperativity β>1)), while compound 71 has no PAM activity
at GPR4 (Figure 2C) and weak potentiation of proton activity at GPR65 only at high concentrations
1
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(Figure 2D). These data indicate that our lead PAM 71 is selective for GPR68 over the functionally
related receptors GPR4 and GPR65. Similarly, as illustrated in Supplementary Figure 2, compounds 46,
77, and 78 show no effect or weak activity only at high concentrations at GPR4 and GPR65.
Compounds 46, 71, 77, and 78 were further screened in radioligand binding assays against a
panel of 48 common drug targets, including GPCRs, neurotransmitter transporters, and hERG ion
channels. As indicated in Figure 2E and Supplementary Figure 3, most of these compounds have
affinity values higher than 300 nM (pK
has moderate binding affinity (pK = 6.50 – 7.00) to 71, 77, and 78, with K
and 295 nM respectively. In addition, adenosine 2A (A2A) receptor has moderate binding affinity to 46
with K value of 300 nM. The compounds with binding affinities between 300 – 100 nM (pK = 6.50 –
.00) were further tested in functional assays to determine if they acted as agonists or antagonists. At 5-
HT2B receptors, 71, 77, and 78 show no agonist activity but display weak antagonist activity
(Supplementary Figure 4A and 4B) with functional affinity (K ) values of 310 nM, 678 nM, and 212
nM, respectively, which are comparable to their corresponding binding affinities. At A2A receptors
Supplementary Figure 4C), 46 displays no agonist activity, but shows weak inverse agonist activity
i
= 6.50) at all targets tested. Among the tested GPCRs, 5-HT2B
i
i
values of 219 nM, 209 nM,
i
i
7
i
(
with potency of 1,700 nM in the absence of reference agonist and antagonist activity with functional
affinity of 6,700 nM.
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Journal of Medicinal Chemistry
For lead PAMs with the potential to serve as molecular probes for chemical biology studies, it is
1
2
also desirable to determine their off-target agonist activities at other human GPCRs. Therefore, 46, 71,
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77, and 78 were tested against all human GPCRome in our PRESTO-Tango screening assays. As
illustrated in Figure 2F, across the whole human GPCRome, compound 71 has agonist activity only at
the MT
1 2
and MT receptors. Compound 46 shows no agonist activity while compounds 77 and 78
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display agonist activity at an atypical chemokine receptor 3 (ACKR3) (previously known as
4
8,49
CXCR7)
weak full agonist at the MT
receptor (EC50 = 540 nM; Emax = 24%) (Supplementary Figure 6). Consistently, 71 displays low binding
affinities to MT (5,900 nM) and MT (1,100 nM) receptors. Compounds 77 and 78 show full agonist
(Supplementary Figure 5). Subsequent concentration response assays indicate that 71 is a
1
receptor (EC50 = 320 nM) and a weak partial agonist activity at the MT
2
1
2
activities at ACKR3 with EC50 values of 1,700 nM and 690 nM, respectively (Supplementary Figure 6).
Characterization of Selected Lead Modulators at Mouse GPR68. To assess whether selected
human GPR68 PAMs also function as PAMs at mouse GPR68 receptor, we determined the PAM
s
activity at mouse GPR68 in the absence and presence of lead modulators for proton mediated G -cAMP
production. As illustrated in Figure 3 and Supplementary Figure 7, in the absence of modulators, mouse
GPR68 responds to proton stimulation in a very similar way as human GPR68 with a potency of pH
6.68 (hill slope of 2.95). As with human GPR68, the proton activity at mouse GPR68 is also potentiated
by the lead PAMs 71 (Figure 3), 46, 77 and 78 (Supplementary Figure 7) in a concentration dependent
manner – shifting proton response curves to the left with increased maxima. Extracted allosteric
parameters determined by fitting the curves to the operational allosteric model are listed in Table 5.
Compared with the allosteric activity of these PAMs at human GPCR68, the compound display 2 – 3-
fold less allosteric activity at mouse GPR68, suggesting a potential small species difference in
sensitivity to GPR68 PAMs.
In Vivo Pharmacokinetic Properties of Compound 71. After obtaining the promising in vitro
potency and selectivity results for compound 71, we assessed in vivo pharmacokinetic properties of this
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lead compound in mice. Since GPR68 is most abundantly expressed in the brain and is associated with
learning and memory, it is necessary to test whether this compound can penetrate the blood-brain barrier
(BBB) in animals. A single intraperitoneal injection at the dose of 25 mg/kg led to high exposure levels
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(above 10 µM) in both plasma and brain at 0.5 h in mice (Figure 4). The high compound exposure levels
in both plasma and brain were maintained for 2 h. These results have demonstrated that compound 71 is
bioavailable and can readily cross the BBB in mice. Furthermore, there were no clinical signs observed
in test mice. Taken together, these results suggest that compound 71 is a valuable chemical tool for
investigating biological function of GPR68 in vivo.
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Characterization of Selected Inactive Analogs. Compounds 7, 20 (a known inactive analog of
4
4
1) and 21 do not show PAM activity at GPR68, GPR4, and/or GPR65 (Supplementary Figures 1, 2).
There are two possible mechanisms for the lack of apparent PAM activity by these compounds in
functional assays: they do not bind GPR68, or they bind but function as silent allosteric modulators
(SAMs, with α = 1 and β = 1). Such allosteric modulators have been described as molecules that bind to
5
0
51,52
the allosteric site without producing cooperative activity on the agonist for opioid receptors
and
53
mGLuR5 receptors . Functional assays can potentially differentiate the lack of binding from silent
allosteric modulation as the latter can be detected through interference with the observed PAM activity.
We therefore tested the effects of these apparently inactive analogs (at 10 µM) on the positive allosteric
activity of 71 (0.3 µM). As shown in Supplementary Figure 8, proton responses are left shifted by 71 as
expected and are minimally affected by the presence of the inactive analogs, suggesting that the inactive
analogs do not bind to the allosteric site of GPR68 to antagonize the PAM activity of 71. In addition,
inactive analogs 7 and 21 show minimal binding affinities to common drug targets (Supplementary
Figure 3). Moreover, 7 and 21 display no off-target agonist activity across the human GPCRome
(Supplementary Figure 5). Therefore, compounds 7, 20, and 21 are potentially useful inactive controls
for studying GPR68 pharmacology.
DISCUSSIONS AND CONCLUSIONS
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Journal of Medicinal Chemistry
It is worth to note the unique nature of GPR68 pharmacology as the co-binding ligand for the
1
2
allosteric effect, namely hydrogen ion, is always present. Therefore, GPR68 allosteric modulators will
likely have direct consequences to cellular signaling: PAMs will produce direct agonism while negative
allosteric modulators (NAMs) will produce inverse agonism. In addition, differences exist for PAMs
depending on whether the allosterism primarily affects affinity (α-effect) or efficacy (β-effect).
Specifically, α-PAMs (α > 1 and β ≈ 1) will produce greater agonism at neutral pH conditions while β-
PAMs (α ≈ 1 and β > 1) will produce relatively more agonism in acidic environments. The differences
in signaling produced by α- versus β-modulators are described in detail in the Supporting Information.
In the present study, we are able to achieve potent and selective GPR68 α-PAMs (e.g., compounds 71,
3
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7, and 78) and αβ-PAMs (e.g., compound 46), but not β-PAMs.
There are examples that precipitous changes in allosteric activity may come from apparently
small changes in molecular structure, known specifically as ‘activity switching’ in the PAMs for
5
4–56
mGluRs.
In this SAR study, with compound 1 as a scaffold, we also observed large changes in
allosteric activity (from active PAMs to inactive analogs) with small structural modifications to the
aminotriazine core and benzylalcohol group. Further SAR and mutational studies are needed to
elucidate underlying molecular mechanisms, which will aid in designing better modulators.
a
Depending on the pK values, the physiochemical properties of modulators, such as net charges
and H-bonding potential, could change under different pH conditions. Correspondingly, binding
interactions of modulators with GPR68 could be pH dependent, leading to different levels of receptor
activation in both a pH and concentration dependent manner. Therefore, our functional assay is designed
to use a wide pH range and is done in the absence and presence of increasing concentrations of
modulators to generate at least 6 sets of 16-point proton concentration response curves (each in
quadruplicate) in a single 384-well plate for proper comparison.
To achieve novel brain penetrant GPR68 PAMs, we tried to maintain low tPSA values by
utilizing lipophilic substituents on the triazine scaffold. While this strategy successfully led to BBB
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permeable compound 71, these lipophilic groups could also reduce the aqueous solubility of the
compounds. Indeed, we have observed bell-shaped proton concentration-response curves at high
compound concentrations (10 and/or 30 µM) for compounds 57 – 59, 63 – 66, 75 – 78, and 81. The
1
2
3
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1
1
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6
assay signals waned when modulators were tested at pH conditions below pH 7.40. This is likely due to
57,58
the reduced aqueous solubility of these compounds under acidic conditions or aggregation.
Addition
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of 1 mg/mL BSA in assay buffers may have improved solubility but did not affect the bell-shaped
curves. Under these circumstances, we may have slightly underestimated the allosteric parameters of
these compounds, especially with regard to the efficacy cooperativity β. In any case, this effect
prevented high concentrations of modulators from being included in the functional assays. This, in turn,
prevented observation of the characteristic saturation (termed the “ceiling effect”) of allosteric
modulation, further leading to increased levels of variation in estimated allosteric parameters.
In summary, we have performed a comprehensive SAR study by exploring three regions of the
scaffold represented by compound 1. While structural changes on the aminotriazine core did not
improve the allosteric activity, modifications to the benzylalcohol and benzylamino groups did lead to
slightly more active GPR68 PAMs with increased affinity cooperativities and allosteric binding
affinities. The subsequently designed combination compounds with the substituents showing best α and
K
B
values from each region ultimately resulted in much improved GPR68 α-PAMs including compound
7
1, which has 33-fold increased allosteric activity compared with 1. We also identified a significantly
improved αβ-PAM, compound 46. We further determined that compounds 71 and 46 and two additional
GPR68 PAMs were selective for GPR68 over GPR4 and GPR65, two closely related proton GPCRs. In
addition, these four compounds had no or low off-target agonist activity across the human GPCRome,
and had weak to moderate binding affinity to 48 common GPCRs, ion channels and transporters.
Moreover, we demonstrated that our lead compound, 71, is bioavailable in mice and has excellent brain
penetration. Last, we identified several potentially useful inactive controls. These novel GPR68 PAMs
and inactive controls are valuable chemical tools for studying GPR68 biology and pharmacology.
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Journal of Medicinal Chemistry
Furthermore, our study paved the way for developing the next generation of GPR68 PAMs and
allosteric modulators for other GPCRs.
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6
EXPERIMENTAL SECTION
Chemistry General Procedures. All chemical reagents were purchased from commercial vendors and
used without further purification. Dry solvents were purchased from commercial vendors with a verified
anhydrous standard. Microwave-heated reactions were performed with a Discover SP microwave
system with an Explorer 12 Hybrid Autosampler by CEM (Buckingham, UK). EMD Milipore 210-270
µm 60-F254 silica gel plates were used to perform analytical thin-layer chromatography (TLC). The
plates were examined under a UV Lamp. The flash column chromatography was conducted using a
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+
Teledyne ISCO CombiFlash Rf instrument. This instrument was also equipped with a variable
wavelength UV detector and a fraction collector. RediSep Rf normal phase silica columns were used for
purification. Preparative high-performance liquid chromatography (HPLC) was used to purify all final
compounds. HPLC was conducted on an Agilent Prep 1200 series with the UV detector set to 254 nm.
Samples were injected into a Phenomenex Luna 750 x 30 mm, 5 µm C18 column. Separation was
performed at room temperature with a flow rate of 40 mL/min. The linear gradient program was set as
2
follows: 10% of methanol (A) in H O containing 0.1% TFA (B) to 100% of methanol (A). All
compounds for biological evaluation were determined to have > 95% purity after the purification using
the aforementioned prep-HPLC methods. An Agilent 1200 series system with DAD detector and a 2.1
mm x 150 mm Zorbax 300SB-C18 5 µm column for chromatography were used to obtain HPLC spectra
for all compounds. Samples (0.5 µL) were injected onto a C18 column at room temperature. The
column flow rate was set to 0.4 mL/min. Chromatography was performed with water containing 0.1%
formic acid as Solvent A and acetonitrile containing 0.1% formic acid as solvent B. The linear gradient
was set as follows: 1% B (0-1 min), 1-99% B (1-4 min), and 99% B (4-8 min). An Agilent G1969A
API-TOF with an electrospray ionization (ESI) source was used to acquire high-resolution mass spectra
(HRMS) data in positive ion mode. All compounds were also characterized using a Bruker (Billerica,
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Journal of Medicinal Chemistry
Page 18 of 61
1
MA) DRX-600 Nuclear Magnetic Resonance (NMR) spectrometer (600 MHz, H-NMR). Chemical
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
shifts are reported in parts per million (ppm, δ) relative to residual solvent peaks. H-NMR data are
reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet), coupling constant, and integration.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedures for Preparation of Compounds 5-7.
59
(
2-(4-(Benzylamino)-6-(methylamino)-1,3,5-triazin-2-yl)phenyl)methanol (5) . Intermediate 3 was
prepared according to previously published procedures starting with 4,6-dichloro-N-methyl-1,3,5-
triazin-2-amine (2) and benzylamine. The white solid 3 (24.9 mg, 0.1 mmol) was suspended in dioxane
(0.8 mL) and water (0.2 mL) in a microwave tube. Then, tetrakis(triphenylphosphine)palladium (0) (5.8
mg, 5 mol %), potassium carbonate (27.6 mg, 0.2 mmol), and (2-(hydroxymethyl)phenyl)boronic acid
o
(30.4 mg, 0.2 mmol)) were added. The mixture was heated to 120 C for 20 min under microwave
irradiation. The resulting mixture was filtered through Celite with ethyl acetate as eluent solvent. The
filter was washed with water and extracted with ethyl acetate. The combined organic layers were
2 4
washed with brine, dried over anhydrous Na SO , filtered, and concentrated in vacuo. The resulting
residue was dissolved in 1.5 mL of methanol and purified by preparative HPLC to yield 7 as white solid
1
(
27 mg, 84%). H NMR (600 MHz, CD
3
OD) δ 7.81 (d, J = 7.8 Hz, 1H), 7.60 (dd, J = 20.0, 7.4 Hz, 3H),
7
.50 (t, J = 7.6 Hz, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.35 (t, J = 7.8 Hz, 2H), 7.31 – 7.24 (m, 1H), 4.81 (s,
+
+
2H), 4.72 (d, J = 5.1 Hz, 2H), 3.05 (d, J = 5.3 Hz, 3H). HRMS m/z [M+H] calculated for C18
20 5
H N O
3
22.1662, found 322.1678.
(2-(4-(Benzylamino)-6-(ethylamino)-1,3,5-triazin-2-yl)phenyl)methanol (6). White solid (15.2 mg,
1
9
1
=
8%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.8 Hz, 1H), 7.69 – 7.57 (m, 2H), 7.51 (t, J = 7.6 Hz,
H), 7.35 (ddt, J = 41.4, 34.0, 7.3 Hz, 5H), 4.81 (s, 2H), 4.70 (s, 2H), 3.54 (q, J = 7.2 Hz, 2H), 1.22 (t, J
+
+
22 5
7.2 Hz, 3H). HRMS m/z [M+H] calculated for C19H N O 336.1819, found 336.1873.
(2-(4-(Benzylamino)-6-(dimethylamino)-1,3,5-triazin-2-yl)phenyl)methanol (7). White solid (32 mg,
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Page 19 of 61
Journal of Medicinal Chemistry
1
4
2%). H NMR (600 MHz, CD
3
OD) δ 7.90 (d, J = 7.8 Hz, 1H), 7.62 (dd, J = 24.7, 7.5 Hz, 2H), 7.52 (t,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
J = 7.6 Hz, 1H), 7.42 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.4 Hz, 2H), 7.28 (t, J = 7.3 Hz, 1H), 4.84 (s, 2H),
+
+
4.70 (s, 2H), 3.32 (d, J = 24.3 Hz, 6H). HRMS m/z [M+H] calculated for C19
22 5
H N O 336.1819, found
3
36.1832.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2-(6-Amino-2-(benzylamino)pyrimidin-4-yl)phenyl)methanol (10). Compound 10 was synthesized
following the same procedure for preparing 5 starting with 2,6-dichloropyrimidin-4-amine 8 and
1
benzylamine. The title compound 10 was obtained as white solid (27.2 mg, 92%). H NMR (600 MHz,
CD
3
OD) δ 7.57 (d, J = 5.2 Hz, 2H), 7.53 – 7.43 (m, 2H), 7.39 (d, J = 7.6 Hz, 2H), 7.34 (t, J = 7.6 Hz,
+
2
H), 7.27 (t, J = 7.3 Hz, 1H), 6.20 (s, 1H), 4.66 (s, 2H), 4.62 (s, 2H). HRMS m/z [M+H] calculated for
+
C
18
H
19
N
4
O 307.1553, found 307.1542.
General Procedures for Preparation of Compounds 13-15.
4
4
Intermediate 12 was prepared according to previously published procedures .
2
N -benzyl-6-phenyl-1,3,5-triazine-2,4-diamine (13). To a dried microwave tube was added,
intermediate 12 (23.6 mg, 0.1 mmol), phenylboronic acid (24.4 mg, 0.2 mmol),
tetrakis(triphenylphosphine)palladium (5.8 mg, 5 mol %), potassium carbonate (27.6 mg, 0.2 mmol)
o
dioxane (0.8 mL) and water (0.2 mL). The mixture was heated to 120 C for 20 min under microwave
irradiation. The resulting mixture was filtered through Celite with ethyl acetate as eluent solvent. The
filter was washed with water and extracted with ethyl acetate. The combined organic layers were
2 4
washed with brine, dried over anhydrous Na SO , filtered, and concentrated in vacuo. The resulting
residue was dissolved in 1.5 mL of methanol and purified by preparative HPLC to yield 13 as white
1
solid (26 mg, 94%). H NMR (600 MHz, CD
3
OD) δ 8.15 (t, J = 6.3 Hz, 2H), 7.69 (q, J = 8.9, 8.2 Hz,
1H), 7.57 (dt, J = 11.2, 7.5 Hz, 2H), 7.42 – 7.30 (m, 4H), 7.27 (q, J = 6.9 Hz, 1H), 4.69 (d, J = 3.8 Hz,
+
+
2
H). HRMS m/z [M+H] calculated for C16
H
16
N
5
278.1400, found 278.1415.
2
1
N -benzyl-6-(o-tolyl)-1,3,5-triazine-2,4-diamine (14). White solid (29 mg, 98%). H NMR (600 MHz,
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Journal of Medicinal Chemistry
Page 20 of 61
CD
.7 Hz, 1H), 4.69 (s, 2H), 2.52 (s, 3H). HRMS m/z [M+H] calculated for C17
292.1539.
3
OD) δ 7.60 (dd, J = 12.1, 7.8 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.43 – 7.31 (m, 6H), 7.28 (q, J = 7.2,
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
+
+
5
18 5
H N 292.1557, found
1-(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)phenyl)ethan-1-one (15). White solid (48 mg,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
5
4%). H NMR (600 MHz, CD
3
OD) δ 8.06 (d, J = 7.7 Hz, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.80 (dd, J =
7
.7, 3.2 Hz, 1H), 7.73 (dt, J = 11.0, 7.6 Hz, 1H), 7.44 – 7.23 (m, 5H), 4.71 (s, 2H), 2.01 (d, J = 2.4 Hz,
+
+
18 5
3H). HRMS m/z [M+H] calculated for C18H N O 320.1506, found 320.1531.
1-(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)phenyl)ethan-1-ol (16). Compound 15 (15 mg,
o
0
.05 mmol) was dissolved in THF (mL). After the mixture was cooled to 0 C, sodium borohydride (4
mg, 0.1 mmol) was added slowly. The reaction was monitored by TLC till 15 disappeared. Methanol (1
mL) was added slowly to quench the reaction. The mixture was filtered, and concentrated in vacuo. The
resulting residue was dissolved in 1.5 mL of methanol and purified by preparative HPLC to yield 16 as
1
white solid (16 mg, 51%). H NMR (600 MHz, CD
3
OD) δ 7.76 – 7.59 (m, 3H), 7.52 – 7.44 (m, 2H),
7.43 – 7.31 (m, 4H), 7.28 (dd, J = 9.2, 5.7 Hz, 1H), 5.27 – 5.18 (m, 1H), 4.70 (s, 2H), 1.53 (d, J = 6.5
+
+
Hz, 3H). HRMS m/z [M+H] calculated for C18
H
20
N
5
O 322.1662, found 322.1669.
2
6
-(2-(Aminomethyl)phenyl)-N -benzyl-1,3,5-triazine-2,4-diamine (18). Intermediate 17 (123 mg,
8
6%) was prepared following the same procedures that were used to prepare 13 starting with 2-
formylphenyl boronic acid and intermediate 12. To a suspension of intermediate 17 (47 mg, 0.15
.
mmol), magnesium sulfate (200 mg) and NH
3
H
2
O (0.75 mmol, 107 µL) in THF (2 mL) was added
sodium borohydride (5.7 mg, 0.15 mmol) slowly. After stirring for 2 d at room temperature, methanol (1
mL) was added slowly to quench the reaction. The mixture was filtered, and concentrated in vacuo. The
resulting residue was dissolved in 1.5 mL of methanol and purified by preparative HPLC to yield 18 as
1
yellow oil (21 mg, 45%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.8 Hz, 1H), 7.64 (t, J = 7.5 Hz,
1H), 7.60 (d, J = 7.5 Hz, 1H), 7.51 (q, J = 7.0, 6.5 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H), 7.37 – 7.31 (m,
+
3
H), 7.29 (q, J = 7.9, 7.2 Hz, 1H), 4.82 (s, 2H), 4.69 (s, 2H). HRMS m/z [M+H] calculated for
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Journal of Medicinal Chemistry
+
17 19 6
C H N 307.1666, found 307.1676.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
N -benzyl-6-(2-((methylamino)methyl)phenyl)-1,3,5-triazine-2,4-diamine (19). Compound 19 was
prepared following the general procedure for preparing compound 18 from intermediate 17 and
1
methylamine (2 M in THF). Yellow oil (22 mg, 67%). H NMR (600 MHz, CD
3
OD) δ 7.69 (dd, J = 7.6,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
.7 Hz, 1H), 7.66 – 7.49 (m, 3H), 7.39 – 7.15 (m, 5H), 4.62 (s, 2H), 4.39 (s, 2H), 2.79 (s, 3H). HRMS
+
+
21 6
m/z [M+H] calculated for C18H N 321.1882, found 321.1877.
General Procedures for Preparation of Compounds 21-29 and 31-32.
Compounds 21-19 and 31-32 were prepared following the general procedure for preparing compound
1
3 from intermediate 12 and commercially available substituted phenylbronic acids.
1
-(3-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)phenyl)ethan-1-one (21). White solid (28 mg,
1
8
7%). H NMR (600 MHz, CD
3
OD) δ 8.80 (s, 1H), 8.44 (d, J = 7.8 Hz, 1H), 8.32 – 8.20 (m, 1H), 7.78 –
+
7.63 (m, 1H), 7.47 – 7.19 (m, 5H), 4.69 (s, 2H), 2.67 (s, 3H). HRMS m/z [M+H] calculated for
+
C
18
H
18
N
5
O 320.1506, found 320.1522.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-6-fluorophenyl)methanol (22). White solid (29 mg,
1
8
9%). H NMR (600 MHz, CD
3
OD) δ 7.63 (d, J = 7.8 Hz, 1H), 7.53 (td, J = 8.0, 5.1 Hz, 1H), 7.44 –
+
7.32 (m, 5H), 7.28 (t, J = 7.5 Hz, 1H), 4.83 (s, 2H), 4.69 (s, 2H). HRMS m/z [M+H] calculated for
+
C
17
H
17FN
5
O 326.1412, found 326.1435.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-5-fluorophenyl)methanol (23). White solid (30 mg,
1
9
1%). H NMR (600 MHz, CD
3
OD) δ 7.86 (dt, J = 10.6, 5.3 Hz, 1H), 7.43 – 7.30 (m, 5H), 7.30 – 7.15
+
(m, 2H), 4.84 (d, J = 5.1 Hz, 2H), 4.67 (d, J = 5.0 Hz, 2H). HRMS m/z [M+H] calculated for
+
C
17
H
17FN
5
O 326.1412, found 326.1426.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-4-fluorophenyl)methanol (24). White solid (28 mg,
1
8
6%). H NMR (600 MHz, CD
3
OD) δ 7.66 – 7.57 (m, 2H), 7.42 – 7.31 (m, 5H), 7.30 – 7.24 (m, 1H),
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Journal of Medicinal Chemistry
Page 22 of 61
+
+
4
.78 (s, 2H), 4.69 (s, 2H). HRMS m/z [M+H] calculated for C17
H17FN
5
O 326.1412, found 326.1407.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(
2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (25). White solid (31 mg,
1
9
4%). H NMR (600 MHz, CD
3
OD) δ 7.63 – 7.54 (m, 2H), 7.43 – 7.31 (m, 5H), 7.28 – 7.23 (m, 1H),
+
+
5
4.76 (s, 2H), 4.69 (s, 2H). HRMS m/z [M+H] calculated for C17H17FN O 326.1412, found 326.1419.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-5-chlorophenyl)methanol (26). White solid (16 mg,
1
6
8
4%). H NMR (600 MHz, CD
3
OD) δ 7.81 (dd, J = 8.4, 2.0 Hz, 1H), 7.65 (s, 1H), 7.48 (dd, J = 21.4,
.4 Hz, 1H), 7.41 – 7.30 (m, 4H), 7.27 (t, J = 7.5 Hz, 1H), 4.81 (s, 2H), 4.68 (s, 2H). HRMS m/z
+
+
5
[M+H] calculated for C17H17ClN O 342.1116, found 342.1111.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-4-chlorophenyl)methanol (27). White solid (27 mg,
1
9
4
3%). H NMR (600 MHz, CD
3
OD) δ 7.86 (d, J = 2.3 Hz, 1H), 7.65 – 7.56 (m, 2H), 7.42 – 7.32 (m,
+
H), 7.28 (t, J = 7.3 Hz, 1H), 4.79 (s, 2H), 4.69 (s, 2H). HRMS m/z [M+H] calculated for
+
C
17
H
17ClN
5
O 342.1116, found 342.1126.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-3-chlorophenyl)methanol (28). White solid (6.8
1
mg, 78%). H NMR (600 MHz, CD
3
OD) δ 7.42 – 7.30 (m, 3H), 7.26 – 7.09 (m, 5H), 4.53 (s, 2H), 4.49
+
+
(s, 2H). HRMS m/z [M+H] calculated for C17
H
17ClN
5
O 342.1116, found 342.1123.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-6-methoxyphenyl)methanol (29). White solid (13
1
mg, 84%). H NMR (600 MHz, CD
3
OD) δ 7.48 (t, J = 8.1 Hz, 1H), 7.40 (d, J = 7.2 Hz, 1H), 7.37 – 7.31
+
(m, 4H), 7.29 (dd, J = 7.8, 3.5 Hz, 2H), 4.82 (s, 2H), 4.69 (s, 2H), 3.91 (s, 3H). HRMS m/z [M+H]
+
20 5 2
calculated for C18H N O 338.1612, found 338.1618.
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-4-methoxyphenyl)methanol (31). White solid (28
1
mg, 86%). H NMR (600 MHz, CD
3
OD) δ 7.47 (dd, J = 8.6, 5.9 Hz, 1H), 7.44 (d, J = 2.8 Hz, 1H), 7.40
(d, J = 7.2 Hz, 1H), 7.35 (t, J = 7.6 Hz, 3H), 7.31 – 7.24 (m, 1H), 7.17 (dd, J = 8.5, 2.7 Hz, 1H), 4.72 (d,
+
+
20 5 2
J = 2.8 Hz, 2H), 4.69 (d, J = 2.9 Hz, 2H), 3.86 (s, 3H). HRMS m/z [M+H] calculated for C18H N O
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Page 23 of 61
Journal of Medicinal Chemistry
3
38.1612, found 338.1614.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-3-methoxyphenyl)methanol (32). White solid (16
1
mg, 48%). H NMR (600 MHz, CD
3
OD) δ 7.54 (td, J = 8.2, 3.0 Hz, 1H), 7.42 – 7.32 (m, 4H), 7.29 (tt, J
= 8.5, 4.8 Hz, 1H), 7.13 (dd, J = 7.9, 5.6 Hz, 2H), 4.68 (s, 2H), 4.68 (s, 2H), 3.88 (d, J = 1.6 Hz, 3H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
20 5 2
HRMS m/z [M+H] calculated for C18H N O 338.1612, found 338.1625.
General Procedures for Preparation of Compounds 37-38. Intermediate 36 was prepared according
6
0
to previously published procedures .
(2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-5-ethoxyphenyl)methanol (37). To
a
dried
microwave tube was added, intermediate 12 (23.6 mg, 0.1 mmol), intermediate 36 (39.2 mg, 0.2 mmol),
tetrakis(triphenylphosphine)palladium (0) (5.8 mg, 5 mol %), and potassium carbonate (27.6 mg, 0.2
o
mmol %), dioxane (0.8 mL) and water (0.2 mL). The mixture was heated to 120 C for 20 min under
microwave. The resulting mixture was filtered through Celite with ethyl acetate as eluent solvent. The
filter was washed with water and extracted with ethyl acetate. The combined organic layers were
washed brine, dried over anhydrous Na
2
SO
4
, filtered, and concentrated in vacuo. The resulting residue
o
was dissolved in 5 mL of THF. After the mixture was cooled to 0 C, sodium borohydride (11 mg, 0.3
mmol) was added slowly. The reaction was warmed to room temperature and stirred for 10 min, before
methanol was added slowly to quench the reaction. The mixture was washed with water and extracted
2 4
with ethyl acetate. Combined organic layers were washed with water, brine, dried over Na SO , filtered,
and concentrated in vacuo. The resulting residue was dissolved in 1.5 mL of methanol and purified by
1
preparative HPLC to yield 37 as white solid (3.7 mg, 23%). H NMR (600 MHz, CD
3
OD) δ 7.68 (d, J =
7
–
3
.9 Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.35 – 7.30 (m, 3H), 6.29 (s, 1H), 4.10 (s, 2H), 3.65 (s, 2H), 2.65
+
+
22 5 2
2.51 (q, J = 7.2 Hz, 2H), 1.49 (t, J = 7.2 Hz, 3H). HRMS m/z [M+H] calculated for C19H N O
52.1768, found 352.1762.
(
2-(4-Amino-6-(benzylamino)-1,3,5-triazin-2-yl)-5-isopropoxyphenyl)methanol (38). White solid
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 61
1
(
3
32 mg, 88%). H NMR (600 MHz, CD OD) δ 7.86 (d, J = 8.7 Hz, 1H), 7.39 (d, J = 7.3 Hz, 1H), 7.37 –
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
7
.30 (m, 3H), 7.27 (t, J = 7.3 Hz, 1H), 7.14 (d, J = 2.6 Hz, 1H), 7.00 (dd, J = 8.8, 2.7 Hz, 1H), 4.81 (s,
+
2H), 4.78 – 4.72 (m, 1H), 4.68 (s, 2H), 1.35 (d, J = 6.0 Hz, 6H). HRMS m/z [M+H] calculated for
+
C
20
H
24
N
5
O
2
366.1925, found 366.1934.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedures for Preparation of Compounds 41-54, 56-60 and 64-81.
Intermediate 40 was prepared from intermediates 3, 11 and commercially available amines
4
4
(
intermediate 39) according to the previously reported procedures. Compounds 41-54, 56-60 and 64-
66 were prepared following the general procedure for preparing compound 13 from intermediate 40 and
2-(hydroxymethyl)phenyl)boronic acid. Compounds 67-81 were prepared following the general
(
procedure for preparing compound 13 from intermediate 40 and substituted (2-
(hydroxymethyl)phenyl)boronic acids.
(2-(4-Amino-6-((2-fluorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (41). White solid (27
1
mg, 82%). H NMR (600 MHz, CD
3
OD) δ 7.82 (d, J = 7.7 Hz, 1H), 7.65 – 7.54 (m, 2H), 7.47 (dt, J =
2
6.2, 7.6 Hz, 2H), 7.33 – 7.25 (m, 1H), 7.13 (t, J = 7.7 Hz, 1H), 7.07 (t, J = 9.3 Hz, 1H), 4.79 (d, J = 3.9
+
+
5
Hz, 2H), 4.74 (s, 2H). HRMS m/z [M+H] calculated for C17H17FN O 326.1412, found 326.1443.
(2-(4-Amino-6-((3-fluorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (42). White solid (27
1
mg, 83%). H NMR (600 MHz, CD
3
OD) δ 7.82 (d, J = 7.8 Hz, 1H), 7.65 – 7.56 (m, 2H), 7.49 (t, J = 7.9
Hz, 1H), 7.32 (td, J = 8.0, 5.7 Hz, 1H), 7.21 – 7.10 (m, 2H), 6.98 (td, J = 8.5, 2.6 Hz, 1H), 4.81 (s, 2H),
+
+
5
4.68 (s, 2H). HRMS m/z [M+H] calculated for C17H17FN O 326.1412, found 326.1435.
(2-(4-Amino-6-((4-fluorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (43). White solid (26
1
mg, 80%). H NMR (600 MHz, CD
3
OD) δ 7.81 (dt, J = 7.9, 4.7 Hz, 1H), 7.72 – 7.53 (m, 3H), 7.49 (dd,
J = 7.9, 4.8 Hz, 1H), 7.39 (dt, J = 9.7, 5.0 Hz, 1H), 7.03 (tt, J = 8.7, 4.5 Hz, 2H), 4.80 (t, J = 4.9 Hz,
+
+
2H), 4.64 (d, J = 4.9 Hz, 2H). HRMS m/z [M+H] calculated for C17
5
H17FN O 326.1412, found
3
26.1438.
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Page 25 of 61
Journal of Medicinal Chemistry
(
2-(4-Amino-6-((2-chlorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (44). White solid (32
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mg, 94%). H NMR (600 MHz, CD
3
OD) δ 7.84 (d, J = 7.7 Hz, 1H), 7.68 – 7.58 (m, 2H), 7.54 – 7.47
(m, 2H), 7.41 (dd, J = 7.2, 2.0 Hz, 1H), 7.29 (ddt, J = 9.4, 6.9, 4.0 Hz, 2H), 4.83 (s, 2H), 4.79 (s, 2H).
+
+
5
HRMS m/z [M+H] calculated for C17H17ClN O 342.1116, found 342.1113.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2-(4-Amino-6-((3-chlorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (45). White solid (26
1
mg, 78%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.4 Hz, 1H), 7.70 – 7.57 (m, 2H), 7.51 (td, J =
7.5, 1.5 Hz, 1H), 7.43 (d, J = 2.1 Hz, 1H), 7.36 – 7.19 (m, 3H), 4.83 (s, 2H), 4.69 (s, 2H). HRMS m/z
+
+
5
[M+H] calculated for C17H17ClN O 342.1116, found 342.1102.
(2-(4-Amino-6-((4-chlorobenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (46). White solid (18
1
mg, 52%). H NMR (600 MHz, CD
3
OD) δ 7.83 (dd, J = 7.9, 1.2 Hz, 1H), 7.67 – 7.58 (m, 2H), 7.51 (td,
J = 7.5, 1.5 Hz, 1H), 7.38 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 9.0 Hz, 2H), 4.82 (s, 2H), 4.67 (s, 2H).
+
+
5
HRMS m/z [M+H] calculated for C17H17ClN O 342.1116, found 342.1108.
(2-(4-Amino-6-((2-(trifluoromethyl)benzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (47). White
1
solid (35 mg, 93%). H NMR (600 MHz, CD
3
OD) δ 7.86 (d, J = 7.7 Hz, 1H), 7.72 (t, J = 6.7 Hz, 1H),
7.67 – 7.55 (m, 4H), 7.53 (td, J = 7.5, 1.5 Hz, 1H), 7.47 (td, J = 7.4, 2.2 Hz, 1H), 4.93 (s, 2H), 4.85 (s,
+
+
2
17 3 5
H). HRMS m/z [M+H] calculated for C18H F N O 376.1380, found 376.1388.
(2-(4-Amino-6-((3-(trifluoromethyl)benzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (48). White
1
solid (35 mg, 94%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.6 Hz, 1H), 7.73 – 7.46 (m, 7H), 4.83
+
+
(s, 2H), 4.78 (s, 2H). HRMS m/z [M+H] calculated for C18
H
17
F
3
N
5
O 376.1380, found 376.1367.
(2-(4-Amino-6-((4-(trifluoromethyl)benzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (49). White
1
solid (36 mg, 96%). H NMR (600 MHz, CD
3
OD) δ 7.83 (dd, J = 7.8, 1.3 Hz, 1H), 7.67 – 7.55 (m, 3H),
+
+
-6.76 (m, 1H), 4.82 (s, 2H), 4.66 (s, 2H), 3.78 (s, 3H). HRMS m/z [M+H] calculated for C18
17 3 5
H F N O
376.1380, found 376.1374.
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Journal of Medicinal Chemistry
Page 26 of 61
(
2-(4-Amino-6-((2-methylbenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (50). White solid (28
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
mg, 88%). H NMR (600 MHz, CD
3
OD) δ 7.82 (d, J = 7.7 Hz, 1H), 7.64 – 7.54 (m, 2H), 7.54 – 7.44
(m, 1H), 7.35 – 7.22 (m, 1H), 7.19 – 7.12 (m, 3H), 4.80 (s, 2H), 4.68 (s, 2H), 2.35 (s, 3H). HRMS m/z
+
+
20 5
[M+H] calculated for C18H N O 322.1662, found 322.1654.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(2-(4-Amino-6-((3-methylbenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (51). White solid (29
1
mg, 92%). H NMR (600 MHz, CD
3
OD) δ 7.81 (d, J = 7.7 Hz, 1H), 7.68 – 7.55 (m, 2H), 7.50 (td, J =
7.5, 1.6 Hz, 1H), 7.25 – 7.13 (m, 3H), 7.08 (d, J = 7.3 Hz, 1H), 4.81 (s, 2H), 4.65 (s, 2H), 2.31 (s, 3H).
+
+
20 5
HRMS m/z [M+H] calculated for C18H N O 322.1662, found 322.1657.
(2-(4-Amino-6-((4-methylbenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (52). White solid (22
1
mg, 69%). H NMR (600 MHz, CD
3
OD) δ 7.81 (d, J = 7.7 Hz, 1H), 7.70 – 7.57 (m, 2H), 7.55 – 7.47
(m, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.15 (d, J = 7.7 Hz, 2H), 4.81 (s, 2H), 4.64 (s, 2H), 2.29 (s, 3H).
+
+
20 5
HRMS m/z [M+H] calculated for C18H N O 322.1662, found 322.1672.
(2-(4-Amino-6-((2-methoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (53). White solid (27
1
mg, 79%). H NMR (600 MHz, CD
3
OD) δ 7.80 (d, J = 7.8 Hz, 1H), 7.66 – 7.58 (m, 2H), 7.49 (t, J = 7.6
Hz, 1H), 7.38 – 7.18 (m, 2H), 7.04 – 6.74 (m, 2H), 4.80 (s, 2H), 4.67 (s, 2H), 3.85 (s, 3H). HRMS m/z
+
+
20 5 2
[M+H] calculated for C18H N O 338.1612, found 338.1623.
(2-(4-Amino-6-((3-methoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (54). White solid (29
1
mg, 87%). H NMR (600 MHz, CD
3
OD) δ 7.83 (dd, J = 7.8, 1.3 Hz, 1H), 7.67 – 7.55 (m, 3H), -6.76 (m,
+
+
20 5 2
1H), 4.82 (s, 2H), 4.66 (s, 2H), 3.78 (s, 3H). HRMS m/z [M+H] calculated for C18H N O 338.1612,
found 338.1618.
(2-(4-Amino-6-((4-ethoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (56). White solid (32
1
mg, 93%). H NMR (600 MHz, CD
3
OD) δ 7.81 (t, J = 6.4 Hz, 1H), 7.69 – 7.55 (m, 2H), 7.55 – 7.46 (m,
1H), 7.31 (td, J = 7.8, 7.0, 3.6 Hz, 1H), 6.95 – 6.79 (m, 3H), 4.81 (s, 2H), 4.60 (s, 2H), 4.00 (q, J = 6.7
+
+
Hz, 2H), 1.36 (t, J = 6.7 Hz, 3H). HRMS m/z [M+H] calculated for C19
H
22
N
5
O
2
352.1768, found
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Page 27 of 61
Journal of Medicinal Chemistry
3
52.1765.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(2-(4-Amino-6-((4-isopropoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (57). White solid
1
(3.2 mg, 52%). H NMR (600 MHz, CD
3
OD) δ 7.83 (dd, J = 7.8, 1.3 Hz, 1H), 7.70 – 7.55 (m, 2H), 7.51
(td, J = 7.6, 1.5 Hz, 1H), 7.36 – 7.16 (m, 1H), 6.98 – 6.73 (m, 3H), 4.81 (s, 2H), 4.61 (s, 2H), 4.60 –
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
4
.53 (m, 1H), 1.29 (d, J = 6.0 Hz, 6H). HRMS m/z [M+H] calculated for C20
H
24
N
5
O
2
366.1925, found
3
66.1928.
(2-(4-Amino-6-((4-cyclopropoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (58). White solid
1
(30 mg, 84%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.8 Hz, 1H), 7.62 (dt, J = 21.8, 7.4 Hz, 2H),
7
.51 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 8.2 Hz, 2H), 4.81 (s, 2H), 4.62 (s, 2H),
+
3
.80 – 3.71 (m, 1H), 0.77 (t, J = 6.3 Hz, 2H), 0.66 (t, J = 2.9 Hz, 2H). HRMS m/z [M+H] calculated for
+
C
20
H
22
N
5
O
2
364.1768, found 364.1776.
(
2-(4-Amino-6-((4-phenoxybenzyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (59). White solid
1
(13.4 mg, 84%). H NMR (600 MHz, CD
3
OD) δ 7.82 (dd, J = 7.8, 1.2 Hz, 1H), 7.65 – 7.56 (m, 3H),
7
.56 – 7.46 (m, 2H), 7.40 (d, J = 8.6 Hz, 1H), 7.32 (dd, J = 8.6, 7.3 Hz, 2H), 7.09 (t, J = 7.4 Hz, 1H),
+
+
6.95 (d, J = 8.5 Hz, 3H), 4.81 (s, 2H), 4.65 (s, 2H). HRMS m/z [M+H] calculated for C23
22 5 2
H N O
4
00.1768, found 400.1743.
(2-(4-Amino-6-(phenylamino)-1,3,5-triazin-2-yl)phenyl)methanol (60). White solid (28 mg, 96%).
1
3
H NMR (600 MHz, CD OD) δ 7.88 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 7.9 Hz, 2H), 7.64 (dt, J = 17.2, 7.7
Hz, 2H), 7.53 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7.9 Hz, 2H), 7.21 (t, J = 7.4 Hz, 1H), 4.87 (s, 2H). HRMS
+
+
16 5
m/z [M+H] calculated for C16H N O 294.1349, found 294.1341.
(2-(4-Amino-6-((4-phenoxyphenethyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (64). White solid
1
3
(32 mg, 77%). H NMR (600 MHz, CD OD) δ 7.82 (dd, J = 7.7, 1.3 Hz, 1H), 7.67 – 7.57 (m, 3H), 7.51
(td, J = 7.6, 1.4 Hz, 1H), 7.35 – 7.19 (m, 4H), 7.08 (t, J = 7.3 Hz, 1H), 6.93 (dd, J = 8.6, 6.8 Hz, 3H),
+
4
.79 (s, 2H), 3.73 (t, J = 7.3 Hz, 2H), 2.94 (t, J = 7.4 Hz, 2H). HRMS m/z [M+H] calculated for
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 61
+
24 24 5 2
C H N O 414.1925, found 414.1945.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
(2-(4-Amino-6-((3-(4-phenoxyphenyl)propyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol
(65).
1
White solid (18 mg, 87%). H NMR (600 MHz, CD
3
OD) δ 7.83 (d, J = 7.8 Hz, 1H), 7.70 – 7.56 (m,
3H), 7.51 (t, J = 7.3 Hz, 1H), 7.31 (t, J = 7.7 Hz, 1H), 7.21 (d, J = 8.2 Hz, 2H), 7.07 (t, J = 7.4 Hz, 1H),
.97 – 6.78 (m, 4H), 4.81 (s, 2H), 3.52 (t, J = 7.0 Hz, 2H), 2.71 (t, J = 6.1Hz, 2H), 2.08 – 1.86 (m, 2H).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
+
+
26 5 2
HRMS m/z [M+H] calculated for C25H N O 428.2081, found 428.2076.
(2-(4-Amino-6-((4-(4-phenoxyphenyl)butyl)amino)-1,3,5-triazin-2-yl)phenyl)methanol (66). White
1
solid (17 mg, 73%). H NMR (600 MHz, CD
3
OD) δ 7.82 (d, J = 7.8 Hz, 1H), 7.63 (dt, J = 22.8, 7.7 Hz,
3
H), 7.52 (t, J = 7.5 Hz, 1H), 7.30 (q, J = 6.2, 4.7 Hz, 1H), 7.19 (d, J = 8.2 Hz, 2H), 7.06 (t, J = 7.4 Hz,
H), 6.91 (dd, J = 23.1, 8.2 Hz, 4H), 4.81 (s, 2H), 3.52 (t, J = 6.4 Hz, 2H), 2.66 (q, J = 6.4 Hz, 2H), 1.70
1
+
+
28 5 2
(dt, J = 7.9, 3.9 Hz, 4H). HRMS m/z [M+H] calculated for C26H N O 442.2238, found 442.2234.
(2-(4-Amino-6-((2-fluorobenzyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (67). White
1
solid (33 mg, 96%). H NMR (600 MHz, CD
3
OD) δ 7.54 (td, J = 8.0, 5.5 Hz, 1H), 7.45 (td, J = 7.6, 1.7
Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.34 – 7.26 (m, 1H), 7.23 – 7.13 (m, 2H), 7.13 – 7.04 (m, 1H), 4.71
+
+
16 2 5
(s, 2H), 4.68 (s, 2H). HRMS m/z [M+H] calculated for C17H F N O 344.1317, found 344.1321.
(2-(4-Amino-6-((4-fluorobenzyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (68). White
1
solid (32 mg, 92%). H NMR (600 MHz, CD
3
OD) δ 7.58 (td, J = 8.0, 5.5 Hz, 1H), 7.44 – 7.31 (m, 3H),
+
7
.25 – 7.19 (m, 1H), 7.08 (t, J = 8.6 Hz, 2H), 4.72 (s, 2H), 4.65 (s, 2H). HRMS m/z [M+H] calculated
+
16 2 5
for C17H F N O 344.1317, found 344.1323.
(2-(4-Amino-6-((3-methylbenzyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (69). White
1
solid (32 mg, 94%). H NMR (600 MHz, CD
3
OD) δ 7.61 – 7.53 (m, 1H), 7.39 (d, J = 7.7 Hz, 1H), 7.26
+
–
7.15 (m, 4H), 7.14 – 7.05 (m, 1H), 4.73 (s, 2H), 4.64 (s, 2H), 2.34 (s, 3H). HRMS m/z [M+H]
+
5
calculated for C18H19FN O 340.1568, found 340.1562.
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Page 29 of 61
Journal of Medicinal Chemistry
(
2-(4-Amino-6-((2-methoxybenzyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (70). White
1
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9
1
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1
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6
1
solid (32 mg, 91%). H NMR (600 MHz, CD
3
OD) δ 7.62 – 7.54 (m, 1H), 7.42 – 7.37 (m, 1H), 7.33 (dd,
J = 7.4, 2.2 Hz, 1H), 7.31 – 7.26 (m, 1H), 7.26 – 7.20 (m, 1H), 6.98 (dd, J = 8.4, 2.1 Hz, 1H), 6.96 –
+
+
6
.88 (m, 1H), 4.73 (s, 2H), 4.66 (s, 2H), 3.86 (s, 3H). HRMS m/z [M+H] calculated for C18
H19FN
5
O
2
3
56.1517, found 356.1523.
0
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0
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0
(2-(4-Amino-6-((4-phenoxybenzyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (71). White
1
solid (16 mg, 75%). H NMR (500 MHz, CD
3
OD) δ 7.59 (td, J = 8.1, 5.6 Hz, 1H), 7.43 – 7.37 (m, 3H),
7
.36 – 7.30 (m, 2H), 7.27 – 7.21 (m, 1H), 7.13 – 7.07 (m, 1H), 6.99 – 6.95 (m, 4H), 4.73 (s, 2H), 4.65
1
3
(s, 2H); C NMR (126 MHz, CD
3
OD) δ 163.70, 163.17, 162.58, 160.59, 158.72, 158.44, 144.72,
134.40, 133.79, 131.03, 130.77, 125.63, 124.64, 120.03, 120.01, 116.34, 116.17, 62.66, 45.33. HRMS
+
+
m/z [M+H] calculated for C23
5 2
H21FN O 418.1674, found 418.1665.
(2-(4-Amino-6-(phenethylamino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (72). White solid (30
1
mg, 89%). H NMR (600 MHz, CD
3
OD) δ 7.60 (td, J = 8.2, 5.5 Hz, 1H), 7.41 (t, J = 8.8 Hz, 1H), 7.35 –
+
7.14 (m, 6H), 4.73 (d, J = 2.9 Hz, 2H), 3.71 (t, 2H), 2.95 (t, J = 7.5 Hz, 2H). HRMS m/z [M+H]
+
calculated for C18
H19FN
5
O 340.1568, found 340.1577.
(2-(4-Amino-6-((3-phenylpropyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (73). White
1
solid (25 mg, 71%). H NMR (600 MHz, CD
3
OD) δ 7.60 (td, J = 8.1, 5.6 Hz, 1H), 7.40 (d, J = 7.8 Hz,
1H), 7.29 – 7.23 (m, 2H), 7.23 – 7.10 (m, 4H), 4.73 (s, 2H), 3.50 (t, J = 7.2 Hz, 2H), 2.71 (t, J = 7.2 Hz,
+
+
2
5
H), 2.04 – 1.89 (m, 2H). HRMS m/z [M+H] calculated for C19H21FN O 354.1725, found 354.1735.
(2-(4-Amino-6-((4-phenylbutyl)amino)-1,3,5-triazin-2-yl)-3-fluorophenyl)methanol (74). White
1
solid (23 mg, 64%). H NMR (600 MHz, CD
3
OD) δ 7.65 – 7.56 (m, 1H), 7.40 (t, J = 6.6 Hz, 1H), 7.24
(ddt, J = 8.9, 5.1, 2.6 Hz, 3H), 7.21 – 7.17 (m, 2H), 7.14 (tt, J = 7.2, 1.5 Hz, 1H), 4.74 (s, 2H), 3.50 (t,
+
+
2
5
H), 2.67 (t, J = 7.0 Hz, 2H), 1.81 – 1.53 (m, 4H). HRMS m/z [M+H] calculated for C20H23FN O
3
68.1881, found 368.1883.
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