G-protein-coupled receptor 84 (GPR84) is a proinflammatory orphan G-protein-coupled receptor implicated in several inflammatory and fibrotic diseases. Several agonist and antagonist ligands have been developed that target GPR84; however, a noncompetitive receptor blocker that was progressed to phase II clinical trials failed to demonstrate efficacy. New high-quality antagonists are required to investigate the pathophysiological role of GPR84 and to validate GPR84 as a therapeutic target. We previously reported the discovery of a novel triazine GPR84 competitive antagonist 1. Here, we describe an extensive structure-activity relationship (SAR) of antagonist 1 and also present in silico docking with supporting mutagenesis studies that reveals a potential binding pose for this type of orthosteric antagonist. Lead compound 42 is a potent GPR84 antagonist with a favorable pharmacokinetic (PK) profile suitable for further drug development.
G-protein-coupled receptor 84 (GPR84) is a proinflammatory orphan G-protein-coupled receptor implicated in several inflammatory and fibrotic diseases. Several agonist and antagonist ligands have been developed that target GPR84; however, a noncompetitive receptor blocker that was progressed to phase II clinical trials failed to demonstrate efficacy. New high-quality antagonists are required to investigate the pathophysiological role of GPR84 and to validate GPR84 as a therapeutic target. We previously reported the discovery of a novel triazine GPR84 competitive antagonist 1. Here, we describe an extensive structure-activity relationship (SAR) of antagonist 1 and also present in silico docking with supporting mutagenesis studies that reveals a potential binding pose for this type of orthosteric antagonist. Lead compound 42 is a potent GPR84 antagonist with a favorable pharmacokinetic (PK) profile suitable for further drug development.
GPR84 is a poorly characterized
rhodopsin-like G-protein-coupled
receptor expressed predominantly by immune cell types.[1,2] Upregulation of GPR84 occurs in proinflammatory conditions, and
thus it is considered a promising therapeutic target in inflammatory
conditions, including ulcerative colitis and fibrotic diseases.[3]GPR84 is activated by medium-chain free
fatty acids (MCFAs) with
chain lengths of C10–12. However, even decanoic acid I (Figure ) has only moderate potency as an agonist and, as such, GPR84 is
still classed as an orphan receptor.[4,5] Studies have
demonstrated that GPR84 is also activated by hydroxylated MCFAs such
as 3-hydroxydodecanoic acid II (Figure ).[6] The first
synthetic orthosteric GPR84 agonist to be reported was 6-octylaminouracil
(6-OAU) III (Figure ), discovered from a high-throughput screen (HTS) of
a small-molecule library using a GTPγS binding assay.[6,7] In a different HTS, the thiopyrimidine agonist 2-(hexylthio)pyrimidine-4,6
diol (2-HTP) IV (Figure ) was discovered to have a markedly higher potency
than the MCFAs.[8] Subsequent optimization
of 2-(hexylthio)pyrimidine-4,6 diol (2-HTP) led to the development
of the 2,4-dihydroxypyridine agonist LY-237 V (Figure ) with 3 orders of
magnitude improved potency over 2-(hexylthio)pyrimidine-4,6 diol (2-HTP).[9] Embelin VI (Figure ), a 2-hydroxy-1,4-benzoquinone natural product
isolated from the plant Embelia ribes (Myrsinaceae), also activates GPR84 with moderate potency.[10] A structure–activity relationship (SAR)
study of embelin led to an analogue VII (Figure ) with 45-fold higher potency
at human GPR84 over embelin.[11] Diindolylmethane
(DIM) VIII (Figure ) is another naturally occurring compound that activates
GPR84.[12] However, DIM acts as an ago-allosteric
agonist of GPR84, binding at a site distinct from MCFAs and the other
orthosteric ligands described above.[13,14] SAR studies
of DIM resulted in the development of the tetrafluoro analogue PSB-16671 IX (Figure ) as a more potent GPR84 activator.[15,16] Biased agonist
DL-175 X was discovered from a library screen against
a predictive quantitative structure–activity relationship (QSAR)
model and subsequent hit optimization.[17]
Figure 1
Chemical
structures of known GPR84 activators.
Chemical
structures of known GPR84 activators.Proinflammatory stimuli increase the expression
of GPR84 and, therefore
there is significant interest in assessing whether antagonism of the
receptor might provide therapeutic benefit in a range of inflammatory
conditions, either by restricting the movement of immune cells into
inflamed tissue or by enhancing disease resolution.[3,4,6] Liminal Biosciences (previously Prometic
Life Sciences, Inc.) has identified a low-affinity GPR84 antagonist,
2-(3-pentylphenyl)acetic acid sodium salt, PBI-4050 XI (Figure ) that also
binds to other targets, which reached phase III clinical trials for
Alström Syndrome.[18,19] Recently Galapagos
NV used a small-molecule HTS to identify GPR84 antagonists.[20] Subsequent optimization generated a series of
GPR84 antagonists including 9-cyclopropylethynyl-2-((S)-1-[1,4]dioxan-2-ylmethoxy)-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one, GLPG 1205 XII (Figure ) a potent and selective GPR84
antagonist.[21] This compound was shown to
reduce disease activity index score and neutrophil infiltration in
a mouse dextran sodium sulfate-induced chronic inflammatory bowel
disease model and, although failing to achieve predefined efficacy
endpoints in clinical studies of ulcerative colitis this ligand has
also been assessed for the treatment of idiopathic pulmonary fibrosis.
Recently, Chen et al. published a series of phosphodiester GPR84 antagonists
and showed the ability of an exemplar molecule (XIII)
to inhibit the chemotaxis of mouse neutrophils and macrophages upon
GPR84 activation.[22]
Figure 2
Chemical structures of
selected GPR84 antagonists.
Chemical structures of
selected GPR84 antagonists.Better understanding of the pathophysiological
roles of GPR84,
and its potential as a therapeutic target in a range of conditions
would be enhanced by the availability of a wider range of receptor
antagonists. Therefore, there is currently a need to identify new
ligands for GPR84 that can be used as chemical probes or lead compounds
for drug development.We recently reported the discovery of
3-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)methyl)-1H-indole 1 (Figure ) as the first, high-affinity and highly
selective competitive antagonist of human GPR84.[23] In this work, we report a substantial SAR of this compound
and novel highly potent analogues with improved druglike properties.
The SAR analysis together with a docking study provides a better understanding
of the potential antagonist mode of binding to GPR84 and build a foundation
for further compound optimization.
Figure 3
Chemical structure, calculated physicochemical
properties, and
bioactivity of hit 1,2,4-triazine compound 1.
Chemical structure, calculated physicochemical
properties, and
bioactivity of hit 1,2,4-triazine compound 1.
Results and Discussion
Chemistry
1,2,4-Triazines and their
derivatives are a class of azaheterocycles with a broad spectrum of
biological activities and are thus privileged scaffolds in medicinal
chemistry.[24,25]The predominant method
used for the synthesis of substituted 1,2,4-triazines is the condensation
reaction of a 1,2-dicarbonyl with the corresponding acid hydrazide.[26] This chemistry is most often used for preparing
1,2,4-triazines for which the 5- and 6-substituents are identical.
A limitation of this chemistry is that reaction of unsymmetrical 1,2-diketones
produces regioisomeric mixtures.[27]SAR optimization was initiated with the aim to improve the potency
and druglike properties of compound 1 (Figure ). The general synthetic strategy
used for the preparation of this series of compounds is summarized
in Scheme . The required
hydrazides were either sourced commercially or prepared by reported
procedures.[28−30] Hydrazides were prepared from the corresponding esters
by refluxing with hydrazine in ethanol. The hydrazides were then reacted
with symmetrical and unsymmetrical 1,2-diketones (Scheme ) in the presence of ammonium
acetate and acetic acid to afford the 3,5,6-trisubstituted 1,2,4-triazines 4–72. Regioisomers produced by reacting
unsymmetrical 1,2-diketones were separated by supercritical fluid
chromatography (SFC).
Scheme 1
Synthesis of 3,5,6-Trisubstituted 1,2,4-Triazines 4-72 via Condensation of 1,2-Diketones
Unsymmetrical 1,2-diketones react with hydrazides
to produce mixtures
of 5,6-diaryl triazine regioisomers. The accurate structure of this
series of molecules was identified by X-ray crystallography for compounds 37 and 52 (Figure ) and extrapolation through relative reversed-phase
high-performance liquid chromatography (RP-HPLC) retention times.
X-ray
crystal structures of (A) 37 and (B) 52 (carbon
= gray, hydrogen = white, oxygen = red, nitrogen = blue).After confirming the configuration of 52 with phenyl
in the 5-position and benzoic acid in the 6-position of the 1,2,4-triazines,
we exploited the carboxylic acid functionality to structurally diversify
and produce amides 73–81 (Scheme ). Similarly, the
other regioisomer 51 was functionalized leading to compounds 82–90.
Scheme 2
Late-Stage Structural Diversification
of (A) 52 to Yield 73–81 (B) 51 to Yield Amides 82–90 (R1 and R2 Defined in Table ).
In Silico Docking
A molecular docking
study was carried out to provide a structural hypothesis of an antagonist
binding mode. An experimentally determined structure of GPR84 is not
available. The AlphaFold structure of human GPR84 generated by the
AlphaFold deep learning algorithm[31] was
thus used for antagonist docking. In our previous work, this structure
was used effectively in docking of the agonist compounds 2-HTP, 6-OAU,
and DL-175 and to provide an explanation of their functional bias,[32] so it was applicable to the current study. A
key feature of both this model and our previously generated homology
model[13] is that Arg172, located within
extracellular loop 2 (ECL2), points inward toward the binding cavity
to coordinate the carboxylate of medium-chain fatty acids that are
the native activators of the receptor. This is very different from
other homology models[33,34] where the fatty acid was predicted
to have the opposite orientation, with the carboxylate located deep
with the transmembrane helix bundle and interacting with Asn104. The
preferred binding pose of compound 1 is shown in Figure . Similar to the
agonists, compound 1 is predicted to sit within the helical
bundle forming direct contacts with helices 2, 3, 6, and 7 and extracellular
loop 2 (ECL2). This is consistent with functional and pharmacological
studies that show compound 1 to act as a competitive
antagonist of the agonists noted above.[23] In particular, the indole functionality is predicted to project
from the 3-position of the triazine ring into the bottom of the extracellular
cavity. This indole is likely to form a H-bond with the backbone carbonyl
of Leu1003.32 (The GPCR Ballesteros–Weinstein numbering
is provided in the superscript.[35]) A π-stacking
interaction is also predicted to occur between Tyr692.53 and the indole ring.
Figure 5
Proposed binding mode of compound 1 at the
human GPR84
receptor Alphafold (AF-Q9NQS5-F1). The overall view of the ligand–receptor
complex and the zoomed ligand binding site are on the left and right.
The ligand and the amino acids are shown by orange and blue sticks,
respectively. Hydrogen bonds, π–π, and cation−π
interactions are in pink, cyan, and green dashed lines, respectively.
A section of helix 7 was hidden for a clear view in the zoomed image.
The 1,2,4-triazine ring is numbered.
Proposed binding mode of compound 1 at the
human GPR84
receptor Alphafold (AF-Q9NQS5-F1). The overall view of the ligand–receptor
complex and the zoomed ligand binding site are on the left and right.
The ligand and the amino acids are shown by orange and blue sticks,
respectively. Hydrogen bonds, π–π, and cation−π
interactions are in pink, cyan, and green dashed lines, respectively.
A section of helix 7 was hidden for a clear view in the zoomed image.
The 1,2,4-triazine ring is numbered.In the selected docking pose, the anisole substituent
in the 6-position
of the 1,2,4-triazine forms π–π interactions with
Phe1013.33 and Phe3356.51 and cation−π
interaction with Arg172 of ECL2. The 4-methoxy group then projects
up toward the extracellular opening and makes a H-bond interaction
with Ser169 of ECL2. The anisole substituent in the 5-position is
predicted to be buried in a pocket beneath the β-sheets of ECL2.
In this pocket, Trp3607.43 may form a T-shaped π–π
stacking interaction with the anisole ring. A H-bond between the side-chain
hydroxyl of Tyr812.65 and the methoxy group is also likely
to occur. However, there appears to be limited space for aryl substituents
in this pocket as two disulfide bridges appear to restrict the mobility
of ECL2.Initial mutagenesis studies provided support for this
docking pose
where an alteration to Ala of each of Phe1013.33, Phe3356.51, and Trp3607.43 resulted in substantial loss
of affinity of compound 1 to antagonize PSB-16671-promoted
binding of [35S]GTPγS (Figure ). The obtained binding pose of compound 1 was thus used to rationalize the SAR of analogues.
Figure 6
[35S]GTPγS binding was performed on membrane preparations
from Flp-In T-REx 293 cells expressing the indicated GPR84-Gαi2 fusion proteins. Conversion of each of Phe101, Phe335, and
Trp360 to Ala resulted in a substantial loss of affinity of compound 1 to block effects of the allosteric agonist PSB-16671. PSB-16671
showed pEC50 = 6.70 ± 0.05 at wild type, 6.97 ± 0.29 at
Phe101Ala, 5.88 ± 0.02 at Phe335Ala, and 7.17 ± 0.08 at
Trp360Ala.
[35S]GTPγS binding was performed on membrane preparations
from Flp-In T-REx 293 cells expressing the indicated GPR84-Gαi2 fusion proteins. Conversion of each of Phe101, Phe335, and
Trp360 to Ala resulted in a substantial loss of affinity of compound 1 to block effects of the allosteric agonist PSB-16671. PSB-16671
showed pEC50 = 6.70 ± 0.05 at wild type, 6.97 ± 0.29 at
Phe101Ala, 5.88 ± 0.02 at Phe335Ala, and 7.17 ± 0.08 at
Trp360Ala.
Structure–Activity Relationship
The structural requirements of symmetrical 3-methylindole-5,6-trisubstituted
1,2,4-triazines were investigated first (Table ). Hit compound 1 incorporates
anisole groups in the 5- and 6-positions of the triazine ring that
face ECL2 in the docking model. Substitution of these rings with halides
(compounds 5–7) decreased activity
in an atomic-size-dependent manner. The larger and more electronegative
pyridine analogue 8 also lost significant activity. A
decrease in activity is observed for compound 9, which
incorporates isopropyl groups with central sp3-hybridized
carbons, suggesting hindrance with the buried ECL2 in the binding
site. Indeed, compound 9 could be docked to the site
only when the van der Waals radii of receptor atoms were reduced.
The two predicted Cys11–Cys166 and Cys93–Cys168 disulfide
bridges between ECL2 and the N-terminus of helix 3 would restrain
the position of the loop and reduce its flexibility. Likewise, analogue 10 incorporating a large substituent on the phenyl ring loses
significant activity. Increasing the size of these aryl substituents
is clearly disfavored with a steady decrease in potency as the size
increases. Removing the aryl substituents altogether gave the best
results with diphenyl analogue 4, the most potent ligand
identified (pIC50 8.14 ± 0.14), although with a lower
than desired LLE value. Thiophene analogue 11, which
is smaller, although more polar than the phenyl analogue 4 resulted in a modest decrease in activity.
Table 1
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 4–11
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04.Attention then turned to the role of the indole ring
for activity.
Three analogues of 1 were produced in this series (Table ). Analogue 12 incorporates an electronegative fluorine atom at the 5-position
of the indole, which results in a decrease in activity. The indole
NH is predicted to form a H-bond with the backbone carbonyl of Leu1003.32. This could explain why N-methyl analogue 13 is not active. These data further support our proposed
binding hypothesis in which the indole is predicted to be bound in
a deep narrow pocket.
Table 2
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 12–14
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04; NA, not applicable.To further probe the steric and electronic requirements
of the
indole ring for binding, and taking into account the apparent limitations
of anisole groups in both the 5- and 6-positions of the triazine ring,
we switched to preparing analogues with phenyl groups in these positions
(Table ). Again, substitution
in either the 5- or 6-positions of the indole ring was not tolerated
(compounds 15 and 16). It is of note that
in these docked structures the diphenyl analogues move up the binding
pocket to try and fit these substituents into the indole pocket.
Table 3
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 15–20
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04; NA, not applicable.A series of azaindole analogues was prepared to investigate
the
effect of incorporating H-bond acceptor nitrogen heteroatoms into
the indole ring and to reduce the lipophilicity of the molecules (Table ). The three azaindole
analogues 17–19 retain potency yet,
again with a log unit less in calculated lipophilicity. Both the 5-
and 7-azaindoles have less activity than indole analogue 4. 6-Azaindole retains activity and has the most favorable LLE in
the series. Imidazopyridine 20 incorporates a nitrogen
atom connecting the two aromatic rings and as a result converts the
indole NH into a H-bond acceptor. This analogue was the only molecule
in this series to lose significant activity, supporting the proposed
role of the indole H-bond donor for binding.A series of molecules
with a diverse set of substituents in the
3-position of the triazine ring was explored next (Table ). However, none of these compounds
were more active than indole analogue 4 or 6-azaindole
analogue 18.
Table 4
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 21–25
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04; NA, not applicable.In our binding model, the 5- and 6-aryl substituents
of the 1,2,4-triazine
ring appear to bind in distinct pockets. A series of unsymmetrical
3-methylindole-5,6-trisubstituted 1,2,4-triazine analogues 26–58 (Table ) and 3-(methyl-6-azaindole)-5,6-trisubstituted 1,2,4-triazine 59–70 (Table ) were produced to interrogate the SAR of
the 5- and 6-aryl substituents. The regioselective synthesis of these
molecules is currently not possible, and so in each case, the two
regioisomers were synthesized and separated using chiral SFC where
necessary. The isomers were assigned based on retention time and in
comparison with structurally assigned analogues 37 and 52 (vide supra) using X-ray crystallography. The antagonist
activity of the analogues presented in Table is entirely consistent with our proposed
binding mode for this class of molecule. From compound docking, we
see that 6-aryl substituents are predicted to have access to the extracellular
opening providing a space to accommodate a larger substituent, whereas
the pocket accommodating 5-aryl substitutions is sterically restricted.
Of particular note are 4-benzyl acetate analogues 37 and 38. Compound 37 (pIC50 8.30 ±
0.35) is the most potent compound discovered during this SAR investigation.
Table 5
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 26–58
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P.
Only one isomer was isolated and
regiochemistry could not be assigned; GLPG 1205(XII), pIC50 = 7.27 ± 0.04; NA, not applicable.
Table 6
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 59–70
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P.Only one isomer was isolated and
regiochemistry could not be assigned; GLPG 1205(XII), pIC50 = 7.27 ± 0.04; NA, not applicable.Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04.In considering pharmacokinetic (PK) parameters suitable
to take
compounds forward, we evaluated the CLog P and LLE of these molecules. The 6-azaindole analogues are consistently
predicted to have better solubility, with CLog P range 1.06–3.58, compared to CLog P range 2.01–4.83 for the indole analogues.5-Azaindole
analogues 71 and 72 were
prepared for direct comparison with the corresponding 6-azaindole
analogues 67 and 68. As expected the physicochemical
properties are predicted to be the same; however, the biological activity
is one log unit higher for the 6-azaindole analogue vs the 5-azaindole
analogue (Table ).
Table 7
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 71 and 72
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) binding assay.
Data are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) binding assay.
Data are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency
(LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04.Next, we exploited the carboxylic acid of 51 and 52 to structurally diversify and produce a series
of amides 73–90 (Table ), each series with
defined regiochemistry (Scheme ). Again, this series of molecules was active in the [35S]GTPγS assay, with methyl amide 82 (pIC50 8.01 ± 0.06) being most active.
Table 8
Physicochemical Properties and Activity
Data of 3,5,6-Trisubstituted 1,2,4-Triazines 73–90c
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.
Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).
Logarithm of the partition coefficient
for n-octanol/water (CLog P) was calculated using ChemDraw2019.Negative log of the IC50 in molar (pIC50); guanosine 5′-O-[gammathio]triphosphate
([35S]GTPγS) assay. Data
are expressed as mean ± standard error of the mean (SEM).Ligand lipophilic efficiency (LLE)
= pIC50 – CLog P; GLPG 1205(XII),
pIC50 = 7.27 ± 0.04.
Selectivity
We have previously shown
that compound 4 shows high selectivity for GPR84 against
a panel of 167 other human GPCRs.[23] However,
although the assessed GPCR set did include each of the long-chain
fatty acid receptors FFAR1 (aka GPR40) and FFAR4 (aka GPR120), it
did not include either FFAR2 or FFAR3 that respond instead to short-chain
fatty acids. Compounds 1, 4, 42, and 76 displayed no ability to block the actions of
the C3 fatty acid propionate at either FFAR2 or FFAR3 when tested
at 10 μM (Figure ). We have also shown that compound 1 and compound 4 are unable to antagonize mouse GPR84 and provided a molecular
basis for this lack of activity via comparison of homology models
of human and mouse GPR84 linked to mutagenesis studies.[23] To extend this, we tested the antagonist activity
of all of the compounds reported herein at mouse GPR84. At 10 μM,
very few of the compounds displayed any inhibitory activity (SI Figure 1). Compounds that did produce greater
than 50% inhibition at 10 μM were assessed further in concentration–response
curves. None produced pIC50 within 1000-fold of its activity
at human GPR84.
Figure 7
[35S]GTPγS binding was performed on membrane
preparations
from Flp-In T-REx 293 cells induced to express human FFAR2 (A), human
FFAR3 (B), or a human GPR84-Gαi2 fusion protein (C).
Compounds 1, 4, 42, and 76 (at 10 μM) did not block effects of C3 at either
FFAR2 or FFAR3.
[35S]GTPγS binding was performed on membrane
preparations
from Flp-In T-REx 293 cells induced to express human FFAR2 (A), human
FFAR3 (B), or a human GPR84-Gαi2 fusion protein (C).
Compounds 1, 4, 42, and 76 (at 10 μM) did not block effects of C3 at either
FFAR2 or FFAR3.
Drug Metabolism and Pharmacokinetics (DMPK)
A series of analogues were selected based on activity and predicted
physicochemical properties to be profiled in some preliminary in vitro
DMPK assays (Table ). Highly potent analogue 4 is rapidly metabolized in
both human liver microsomes and mouse hepatocytes, is highly protein
bound, and has poor kinetic solubility. Its azaindole analogue (compound 18) has improved LLE, resulting from lower Log D7.4, improved kinetic solubility, and a modest
improvement in metabolic stability. Similar improvements in LLE and
stability were achieved through substitution of the 6-phenyl ring
with the more polar 4-benzylmorpholine group (42) or
2-(hydroxyethyl)benzamide (76). Compound 68 incorporates both of these substitutions, namely, replacing the
indole with a 6-azaindole and also the 6-phenyl ring with more polar
functionality. As expected the combination of these two groups resulted
in lower Log D7.4 and plasma protein
binding (PPB) with a significant increase in kinetic solubility and
a modest improvement in metabolic stability in comparison to compound 4.
Table 9
Experimental Physicochemical and In
Vitro ADME Properties of Selected Compoundsac
analog
GTPγS
(pIC50)
Log D7.4
LLE
kinetic solubility
(μM)
mouse heps t1/2 (min)
human mics t1/2 (min)
mouse PPB
(%free)
4
8.14 ± 0.14
4.24
3.9
1.4
4.4
21.8
<1
18
8.07 ± 0.16
3.49
4.58
71
9.7
66.2
1.9
42
8.28 ± 0.11
3.84
4.44
14
16
12.8
<1
68
7.29 ± 0.05
2.71
4.58
146
12.5
39.9
3.8
76
7.46 ± 0.07
3.09
4.37
61
47.3
57.2
1.1
Guanosine 5′-O-[gammathio]triphosphate ([35S]GTPγS) assay, data
are expressed as mean ± standard error of the mean (SEM); Log D7.4 (distribution coefficient).
Guanosine 5′-O-[gammathio]triphosphate ([35S]GTPγS) assay, data
are expressed as mean ± standard error of the mean (SEM); Log D7.4 (distribution coefficient).Ligand lipophilic efficiency (LLE)
= pIC50 – Log D7.4; mouse hepatocyte (mouse heps); human microsomes (human mics); mouse
plasma protein binding (mouse PPB).Based on these data, compounds 42 and 76 were selected for in vivo PK profiling (Table ). Compound 42 has a good elimination
half-life (2.51 h), moderate rate of clearance, and bioavailability.
Analogue 76 has a slower rate of clearance, moderate
bioavailability, and shorter elimination half-life (1.22 h).
Table 10
Experimental in Vivo Mouse PK Properties
of Selected Compoundsa
IV (1 mg/kg)
PO (10 mg/kg)
analog
half-life
(h)
CL (mL/min/kg)
Vss (L/kg)
C0 (ng/mL)
AUCall (ng/mL·h)
Cmax (ng/mL)
Tmax (h)
AUCall (ng/mL·h)
bioavailability
(%)
42
2.51 (6.55)
38.9
(13.9)
7.24 (9.2)
394 (19.1)
420 (12.5)
402 (31.8)
1
1590 (18.7)
36.8
(18.7)
76
1.22 (13.5)
22.0 (11.3)
1.81 (15.2)
1170 (29.6)
757 (10.8)
1080 (16.1)
1
3160 (8.82)
41.5
(8.73)
Intravenous (IV); per os (PO),
percentage of relative standard deviation (CV %) provided in parenthesis,
calculated using formula CV % = SD/M × 100 (CL
= clearance level); volume of distribution at steady state (Vss); area under curve (AUCall); peak drug concentration
(Cmax); peak time (Tmax).
Intravenous (IV); per os (PO),
percentage of relative standard deviation (CV %) provided in parenthesis,
calculated using formula CV % = SD/M × 100 (CL
= clearance level); volume of distribution at steady state (Vss); area under curve (AUCall); peak drug concentration
(Cmax); peak time (Tmax).
Conclusions
The development of small-molecule
antagonists that target GPR84
represents a promising potential therapeutic strategy for the treatment
of inflammatory diseases. High-quality small-molecule antagonists
and methods to interrogate GPR84 structure and function are invaluable
to realize the potential of GPR84 as a therapeutic target. Here, we
present a potential binding mode and substantial SAR study of GPR84
antagonist 1 that was previously discovered by us in
an HTS. Molecular docking revealed that antagonist 1 sits
within the helical bundle forming several direct contacts with the
GPR84 receptor helices and ECL2. This binding mode was used to rationalize
the SAR of analogues. The structural requirements of the indole ring,
and 5- and 6-substituents of the triazine core scaffold of antagonist 1 were investigated. A series of four potent analogues were
progressed to in vitro DMPK assays. Compounds 42 and 76 were subsequently progressed to in vivo PK profiling. Of
these compounds, compound 42 displays potent antagonist
activity and a favorable PK profile. These data provide an understanding
of antagonist mode of binding and lead compound 42 is
a promising novel GPR84 antagonist for further drug development.Antagonist 4 was shown to be highly selective for
GPR84 when assessed against a panel of 167 other GPCRs[23] and although we demonstrate that compound 42 lacks activity at other members of the free fatty acid
group of GPCRs whether this compound will be equally selective across
the broader family of GPCRs remains to be assessed.
Experimental Section
General Information
Chemicals and
solvents were purchased from commonly used suppliers and used without
further purification. Celite was purchased from Acros Organics or
Fisher. Hydrophobic frits and SCX columns were purchased from Telos
or Biotage. Telos columns were purchased from Telos. Daicel OJ-H columns
and Daicel AD-H columns were purchased from Daicel. Reacti-Vials were
purchased from Thermo Fisher. Chromatographic purifications were performed
using prepacked Telos silica columns using a Biotage Isolera Purification
system (Uppsala, Sweden). Deuterated solvent was obtained from Cambridge
Isotopes. All NMR spectra were recorded using a Bruker Avance 400
MHz spectrometer. Chemical shifts (δ) are given in ppm relative
to the solvent peak, and coupling constants (J) are
reported in hertz. Reactions under microwave radiation were performed
on Biotage Initiator Eight or Sixty under standard operating conditions.
The purity of all final compounds is >95%, as determined by liquid
chromatography–mass spectrometry (LC–MS).
Method A: General Method for the Synthesis
of 1,2-Diketones
Synthesis of α-hydroxy ketones required
for the synthesis of 1,2-diketones was performed according to published
literature methods.[36,37] The desired ethanone (1 equiv)
dissolved in dimethyl sulfoxide (DMSO) (3.00 mL) was stirred at room
temperature, and N-bromosuccinimide (NBS) (1 equiv)
was added in one portion. The resultant solution was stirred at room
temperature overnight. Worked up by partitioning mixture between water
(30 mL) and ethyl acetate (3 × 10 mL). Combined extracts washed
with water, brine, dried over Na2SO4, filtered,
and concentrated to dryness under reduced pressure to afford symmetrical
1,2-diketones. Used without further purification.
Method B: General Method for the Synthesis
of Methyl Esters
To a solution of the desired acid (1 equiv)
in methanol (5 mL) was added H2SO4 (95.0%, 3
equiv), and the mixture was microwaved at 100 °C for 10 min,
worked up by a basifying mixture by the addition of sat. NaHCO3, and concentrated under reduced pressure to remove methanol.
The residue was partitioned between water and ethyl acetate (2 ×
10 mL), and the combined organics were washed with brine, dried over
Na2SO4, filtered, and concentrated to dryness
under reduced pressure to afford the corresponding methyl esters.
Method C: General Method for the Synthesis
of Hydrazides
A solution of methyl ester (1 equiv) was transferred
to a 5 mL microwave vial and dissolved in ethanol (2 mL). Hydrazine
hydrate (2 equiv) was added in one portion, the vial was capped, and
the mixture was heated at 90 °C for 5.5 h. The mixture was cooled
to room temperature, further hydrazine hydrate (2 equiv) was added,
the mixture was heated at 90 °C overnight, and worked up by concentrating
reaction mixture to dryness under reduced pressure to afford the subsequent
hydrazides.
Method D: General Method for the Synthesis
of 3,5,6-Trisubstituted 1,2,4-Triazines with Symmetrical 1,2-Diketones
A solution of desired acetohydrazide (1.0 equiv), symmetrical 1,2-diketones
(1.0 equiv), and ammonium acetate (10.0 equiv) was transferred to
a 5 mL microwave vial. Acetic acid (2.5 mL) was added, and the resultant
suspension was subjected to microwave radiation at 180 °C for
5 min. The reaction mixture was concentrated to dryness under reduced
pressure. Residue was partitioned between water (20 mL) and dichloromethane
(DCM) (2 × 10 mL). Combined extracts were washed with brine,
dried over Na2SO4, filtered, and concentrated
to dryness under reduced pressure. The residue was purified by flash,
preparative HPLC or SFC as indicated.
Method E: General Method for the Synthesis
of 3,5,6-Trisubstituted 1,2,4-Triazines with Unsymmetrical 1,2-Diketones
To a microwave vial were added acetohydrazide (1.0 equiv), unsymmetrical
1,2-diketones (1.0 equiv), and acetic acid (2 mL). Then, the vial
was sealed and subjected to microwave radiation for 5 min at 180 °C.
The reaction mixture was cooled, ammonium acetate (10 equiv) was added,
and the vial was resealed before subjecting it again to microwave
radiation for 5 min at 180 °C. The mixture was concentrated,
and the resulting residues were purified on preparative HPLC (basic
conditions). The desired fractions were concentrated to afford a solid
or oil. The mixture of regioisomers was separated by SFC using a Daicel
AD-H column (10 × 250 mm2, 20% methanol, 15 mL/min).
Method F: General Method for the Synthesis
of 3,5,6-Trisubstituted 1,2,4-Triazines with Unsymmetrical 1,2-Diketones
To a microwave vial were added acetohydrazide (1.0 equiv), unsymmetrical
1,2-diketones (1.0 equiv), and acetic acid (2 mL). Then, the vial
was sealed and subjected to microwave radiation for 5 min at 180 °C.
The reaction mixture was cooled, ammonium acetate (10 equiv) was added,
and the vial was resealed before subjecting it again to microwave
radiation for 5 min at 180 °C. The reaction mixture was concentrated
and passed through an SCX cartridge eluting the product with 2 M NH3/methanol. The resulting residue was purified by preparative
HPLC (basic conditions) to give a mixture of regioisomers. The regioisomers
were separated by SFC using a Daicel OJ-H (10 × 250 mm2, 30% methanol, 15 mL/min).
Method G: General Method for the Synthesis
of Amide Bond Formation
To a solution of desired acid (1
equiv) in DCM (1 mL) was added propylphosphonic anhydride (50% in
ethyl acetate,1.5 equiv), and the resultant solution was stirred at
room temperature for 10 min. A solution of desired amine (1.2 equiv)
and N,N-diisopropylethylamine (3
equiv) in DCM (0.5 mL) was added, and the resultant orange solution
was stirred at room for 3 h. The crude mix was partitioned between
water/brine (20 mL) and DCM (3 × 10 mL), and the organic layers
were separated using a hydrophobic frit, combined, and concentrated
to dryness under reduced pressure. The resulting residues were purified
by flash chromatography using 0–5% methanol in DCM. The relevant
fractions were combined and concentrated to dryness.
Method H: General Method for Boc Protection
of Amine
To a solution of desired amine (1 equiv) in DCM
(5.00 mL) were added di-tert-butyl dicarbonate (1.5
equiv) and triethylamine (3 equiv), and the mixture was gently heated
to 40 °C for 4 h. The mixture was further diluted in DCM and
washed with brine. The organic layers were passed through a phase
separator and concentrated to dryness.
Preparative HPLC Method
Preparative
HPLC was carried out on a Waters HPLC comprising a Waters 2767 Sample
Manager, a Waters 2545 Binary Gradient Module, a Waters Systems Fluidics
Organiser, a Waters 515 ACD pump, a Waters 2998 Photodiode Array Detector,
and a Waters XBridge Prep OBD C18, 5 μm, 19 mm × 50 mm
i.d. column and a flow rate of 20 mL/min. The general method that
may be used to purify compounds are: acidic reversed-phase HPLC (water/acetonitrile/0.1%
trifluoroacetic acid (TFA)) using a standard gradient of 5% acetonitrile/95%
water to 100% acetonitrile or basic reversed-phase HPLC (water/acetonitrile/0.01
M ammonia solution) using a standard gradient of 10% acetonitrile/90%
water to 100% acetonitrile. UV detection, e.g., 254 nM, is used for
the collection of fractions from HPLC.
Supercritical Fluid Chromatography (SFC)
SFC was carried out on a Waters Investigator SFC comprising a Waters
05962 fluid delivery module, a Waters 07419 autosampler, a Waters
2489 UV/vis detector, a Waters 08005 column oven, a Waters 279002192
heat exchanger, a Waters ABPR-20A back-pressure regulator, and a Waters
08127 fraction collection module. The general method used liquid CO2 and the appropriate modifier as stated in the experimental
as an isocratic mix unless otherwise stated. The preparative column
used was as stated in the experimental. UV detection, e.g., 254 nM,
was used for the collection of fractions from SFC.
Analytical Methods for the Pairs of Separate
Isomers
Method A
Phenomenex Luna C18
(2)-HST column (2.5 μm, 50 × 2.0 mm2). Mobile
phase A contained 0.1% formic acid in 18 MΩ water, and mobile
phase B contained 0.1% formic acid in HPLC-grade acetonitrile. A flow
rate of 1.00 mL/min was used over a 5.5 min gradient starting with
99% mobile phase A gradually increasing to 100% mobile phase B. The
samples were monitored at 254 nm.
Method B
Phenomenex Luna C18
(2)-HST column (2.5 μm, 50 × 2.0 mm2). Mobile
phase A contained 0.1% formic acid in 18 MΩ water, and mobile
phase B contained 0.1% formic acid in HPLC-grade acetonitrile. A flow
rate of 1.00 mL/min was used over a 5.5 min gradient starting with
90% mobile phase A gradually increasing to 100% mobile phase B. The
samples were monitored at 254 nm.
Method C
Phenomenex Kinetix
EVO C18 (2.6 μm, 50 × 2.1 mm2) column. Mobile
phase A contained 0.1% formic acid in 18 MΩ water, and mobile
phase B contained 0.1% formic acid in HPLC-grade acetonitrile. A flow
rate of 1.00 mL/min was used over a 5.5 min gradient starting with
90% mobile phase A gradually increasing to 100% mobile phase B. The
samples were monitored at 254 nm.
LCMS
Low-resolution mass spectrometry
data (m/z) were obtained from an
Agilent 6140 series quadrupole mass spectrometer with a multimode
source attached to an Agilent 1200 series HPLC.
Prepared according to
method D. The residue was purified by flash chromatography eluting
with 0–2.5% methanol in DCM and then re-purified by flash chromatography
using 0–100% ethyl acetate in heptane to give a pale orange
solid. The solid was sonicated with ether (10 mL), filtered off, washed
with ether, and dried under suction to give the title compound 1 (47 mg, 19%). 1H NMR (400 MHz, CDCl3) δ 8.07–8.21 (br s, 1 H), 7.90–8.01 (m, 1 H),
7.55–7.63 (m, 2 H), 7.47–7.54 (m, 2 H), 7.33–7.42
(m, 2 H), 7.12–7.26 (m, 2 H), 6.79–6.95 (m, 4 H), 4.70
(s, 2 H), 3.85 (s, 3 H), 3.84 (s, 3 H); m/z: calcd for C26H23N4O2 [M + H]+, 423.0; found, 423.0.
Prepared according to
method D. The residue was purified by flash chromatography eluting
with 0–30% ethyl acetate in heptane to give a pale orange solid.
The solid was sonicated with ether (2 mL), solid filtered off, washed
with ether, and dried under suction to give the title compound 4 (80 mg, 38%). 1H NMR (400 MHz, CDCl3) δ 8.16 (br s, 1H), 7.96 (m, 1H), 7.54 (m, 4H), 7.30–7.47
(m, 8H), 7.20 (m, 2H), 4.73 (s, 2H); m/z: calcd for C24H19N4 [M + H]+, 363.4; found, 363.4.
Prepared according to
method D. The residue was purified by flash chromatography eluting
with 0–30% ethyl acetate in heptane to give a pale orange solid.
The solid was sonicated with ether (2 mL), filtered off, washed with
ether, and dried under suction to give the title compound 5 (65 mg, 28%). 1H NMR (400 MHz, CDCl3) δ
8.08–8.21 (m, 1H), 7.89–7.98 (m, 1H), 7.47–7.61
(m, 4H), 7.32–7.41 (m, 2H), 7.14–7.26 (m, 2H), 7.00–7.12
(m, 4H), 4.72 (s, 2H); m/z: calcd
for C24H17F2N4 [M + H]+, 399.1; found, 399.2.
Prepared according to
method D. The residue was purified by flash chromatography eluting
with 0–50% ethyl acetate in heptane. The resulting solid was
further purified by preparative HPLC (basic conditions) to give the
title compound 6 (8 mg, 3%). 1H NMR (400 MHz,
CDCl3) δ 8.05–8.27 (br s, 1H), 7.80–7.93
(m, 1H), 7.08–7.48 (m, 12H), 4.78 (s, 2H); m/z: calcd for C24H17Cl2N4 [M + H]+, 431.1; found, 431.0.
Prepared according to
method D. The residue was purified by flash chromatography eluting
with 0–40% ethyl acetate in heptane. The resulting solid was
further purified by sonicating with ether (2 mL), and the resultant
yellow solid was filtered off, washed with further ether (5 mL), and
dried under suction to give the title compound 7 (192
mg, 32%). 1H NMR (400 MHz, CDCl3) δ 8.06–8.15
(br s, 1H), 7.89–7.96 (m, 1H), 7.51 (m, 4H), 7.35–7.45
(m, 5H), 7.31–7.35 (m, 1H), 7.20–7.26 (m, 1H), 7.13–7.19
(m, 1H), 4.71 (s, 2H); m/z: calcd
for C24H17Br2N4 [M + H]+, 519.0; found, 520.8.
Prepared according to method D. The resulting
residue was purified by flash chromatography eluting with a gradient
0–50% ethyl acetate in heptane. The resulting residues were
further purified by preparative HPLC (basic conditions) to afford
the title compound 10 (73 mg, 5%). 1H NMR
(400 MHz, CDCl3) δ 8.08–8.16 (br s, 1H), 7.89–8.08
(m, 5H), 7.58 (dd, J = 8.5, 2.8 Hz, 4H), 7.32–7.43
(m, 2H), 7.13–7.27 (m, 2H), 4.75 (s, 2H), 3.95 (s, 6H); m/z: calcd for C28H23N4O4 [M + H]+, 479.2; found 479.0.
Prepared according to
method D. The resulting residue was purified by flash chromatography
eluting with a gradient 0–5% methanol in DCM. The appropriate
fractions were combined, and the was solvent removed at reduced pressure
to afford the title compound 11 (30 mg, 15%). 1H NMR (400 MHz, DMSO-d6) δ 10.88–11.04
(m, 1H), 7.82–7.96 (m, 2H), 7.68–7.76 (m, 1H), 7.41–7.47
(m, 1H), 7.28–7.38 (m, 3H), 7.21 (dd, J =
3.76, 5.02 Hz, 1H), 6.93–7.16 (m, 3H), 4.49 (s, 2H); m/z: calcd for C20H15N4S2 [M + H]+, 375.1; found, 375.0.
2-(5-Fluoro-1H-indol-3-yl)acetohydrazide
(12a)
Prepared according to method C. Worked
up by concentrating reaction mixture to dryness under reduced pressure
to give the title compound 12a (108 mg, 60%). 1H NMR (400 MHz, CDCl3) δ 7.21–7.34 (m, 3H),
6.88 (td, J = 9.1, 2.6 Hz, 1H), 3.58 (d, J = 2.5 Hz, 2H).
Prepared according to method C. Worked
up by concentrating reaction mixture to dryness under reduced pressure
to afford the title compound 13b, (98.4 mg, 65%). 1H NMR (400 MHz, CD3OD) δ 7.51–7.57
(m, 1H), 7.27–7.33 (m, 1H), 7.12–7.19, (m, 1H), 6.99–7.09
(m, 2H), 4.85 (s, 3H), 3.74 (s, 3H), 3.59 (s, 2H).
Prepared according to
method D. The resulting residue was purified by flash chromatography
with a gradient 0–50% ethyl acetate in heptane to give the
title compound 15 (109 mg, 30%). 1H NMR (400
MHz, DMSO-d6) δ 11.03 (br s, 1H),
7.71 (m, 1H), 7.31–7.51 (m, 11H), 7.08–7.16 (m, 1H),
6.87 (m, 1H), 4.56 (s, 2H); m/z:
calcd for C24H18FN4 [M + H]+, 381.1; found, 381.2.
Prepared according to
method D. The resulting residue was purified by flash chromatography
eluting with a gradient 0–50% of ethyl acetate in heptane.
The appropriate fractions were combined, and the solvent was removed
at reduced pressure to afford the title compound 16 (59
mg, 32%). 1H NMR (400 MHz, CDCl3) δ 8.10
(br s, 1H), 7.77–7.88 (m, 1H), 7.48–7.62 (m, 4H), 7.22–7.47
(m, 8H), 7.16 (s, 1H), 6.93–7.05 (m, 1H), 4.70 (s, 2H); m/z: calcd For C25H20N4 [M + H]+, 377.2; found, 377.0.
Prepared according to method D. The solution was filtered
and concentrated under reduced pressure. The resulting residue was
purified by flash chromatography eluting with a gradient 0–50%
of 9:1:0.1 DCM/methanol/NH4OH in DCM. The resulting residue
was loaded onto an SCX cartridge washed with methanol and then eluted
2 M NH3 in methanol. The basic fractions were combined
to give the title compound 17 (13 mg, 23%). 1H NMR (400 MHz, CDCl3) δ 9.24 (br s, 1H), 8.61 (s,
1H), 8.11 (m, 1H), 7.65 (m, 1H), 6.99–7.41 (m, 12H), 4.53 (s,
2H); m/z: calcd for C23H17N5 [M + H]+, 364.1; found, 364.2.
Prepared according to method D. The resulting residue
was purified by flash chromatography eluting with a gradient 0–50%
of 9:1:0.1 DCM/methanol/NH4OH in DCM. The resulting residue
was loaded onto an SCX cartridge, washed with methanol, and then eluted
2 M NH3 in methanol. The basic fractions were combined
to give the title compound 18 (37 mg, 33%). 1H NMR (400 MHz, DMSO-d6) δ 4.61
(s, 2H), 7.27–7.53 (m, 10H), 7.60–7.75 (m, 2H), 8.02–8.17
(m, 1H), 8.63–8.82 (m, 1H), 11.43–11.63 (m, 1H); m/z: calcd for C23H18N5 [M + H]+, 364.1; found, 364.0.
Prepared according to method D. Residue was purified by
flash chromatography on silica eluting with 0–3% methanol in
DCM. The resulting residues were washed with ether and dried to give
the title compound 19 (37 mg, 23%). 1H NMR
(400 MHz, DMSO-d6) δ 4.59 (s, 2H),
6.97–7.13 (m, 1H), 7.27–7.62 (m, 11H), 8.04–8.29
(m, 2H) 11.51 (br s, 1H); m/z: calcd
for C23H18N5 [M + H]+,
364.1; found, 364.0.
Prepared according
to method D. The material was purified by flash chromatography using
3–5% MeOH in DCM. The relevant fraction was concentrated to
give a yellow gum, which was further purified by preparative HPLC
(basic conditions), concentrated, further purified by preparative
HPLC (acidic conditions), and concentrated. The resulting residues
were taken up in methanol and passed through an SCX cartridge eluting
with methanol and then 2 M NH3 in methanol. The NH3/methanol fractions were combined and concentrated to give
the title compound 20 (56 mg, 29%). 1H NMR
(400 MHz, CDCl3) δ 4.86 (s, 2H), 6.85–6.90
(m, 1H), 7.19–7.24 (m, 1H), 7.31–7.47 (m, 6H), 7.49–7.55
(m, 4H), 7.66 (d, J = 9.3 Hz, 1H), 7.75–7.76
(m, 1H), 8.56 (d, J = 7.0 Hz, 1H); m/z: calcd for C23H19N5 [M + H]+, 364.1; found, 365.2.
Prepared according to method D. The reaction
mixture was evaporated to dryness and then partitioned between water
and DCM and passed through a hydrophobic frit. The organics were concentrated
and then purified by preparative HPLC (acidic conditions) to give
the title compound 21 (37 mg, 15%). 1H NMR
(400 MHz, CDCl3) δ 7.44–7.51 (m, 4H), 7.23–7.39
(m, 6H), 3.07–3.21 (m, 2H), 1.82–1.99 (m, 2H), 1.28–1.46
(m, 4H), 0.83–0.93 (m, 3H); m/z: calcd for C20H21N3 [M + H]+, 304.2; found, 304.2.
Preparation of 3-(3-Pyridyl)propanehydrazide
(22a)
Prepared according to method D. The reaction
mixture was evaporated to dryness and then partitioned between water
and DCM and passed through a hydrophobic frit. The resulting residues
were purified by preparative HPLC (acidic conditions). The relevant
fractions were passed through an SCX cartridge eluting the desired
product with 2 M NH3/methanol and concentrated to give
the title compound 22 (37 mg, 17%). 1H NMR
(400 MHz, CDCl3) δ 3.33–3.41 (m, 2H), 3.54–3.63
(m, 2H), 7.22–7.28 (m, 2H), 7.32–7.51 (m, 6H), 7.52–7.60
(m, 3H), 7.64–7.72 (m, 1H), 8.50 (m, 1H), 8.57–8.62
(m, 1H); m/z: calcd for C22H19N4 [M + H]+, 339.2; found, 339.0.
2-(5-Methyl-1H-pyrazol-3-yl)acetohydrazide
(23a)
Prepared according to method C. 1H NMR (400 MHz, DMSO-d6) δ 2.14
(s, 3H), 3.27 (s, 2H), 3.80–4.44 (m, 2H), 5.83 (s, 1H), 9.06
(s, 1H).
Prepared according to method D. The mixture
was evaporated to dryness, then partitioned between water and DCM,
passed through a hydrophobic frit, and evaporated to dryness. The
crude product was purified by prep HPLC (acidic conditions). Fractions
containing the desired product were concentrated. The resulting residues
were further purified by preparative HPLC (basic conditions). The
fractions containing the product were extracted with DCM and passed
through a hydrophobic frit, and the solvent was removed under reduced
pressure and dried to give the title compound 23 (32
mg, 15%). 1H NMR (400 MHz, CDCl3) δ 7.51–7.59
(m, 4H), 7.30–7.48 (m, 6H), 6.13 (s, 1H), 4.59 (s, 2H), 2.30
(s, 3H); m/z: calcd for C20H18N5 [M + H]+, 328.1; found, 328.0.
3-Bromo-5,6-diphenyl-1,2,4-triazine (75.0
mg, 0.240 mmol) and K2CO3 (0.0996 g, 0.721 mmol)
were combined in acetonitrile (0.500 mL). 3-Pyridylmethanamine (0.0312
g, 0.288 mmol) was added, and the reaction was heated to 150 °C
for 30 min under microwave radiation. The reaction was diluted with
DCM and water. The layers were separated. The organics were washed
with brine, dried over Na2SO4, filtered, and
the solvent was removed at reduced pressure. The resulting residue
was purified by flash chromatography eluting with a gradient 0–50%
methanol in DCM. The appropriate fractions were combined, and the
solvent was removed at reduced pressure to afford the title compound 24 (46 mg, 56%). 1H NMR (400 MHz, CDCl3) δ 8.73 (dd, J = 2.3, 0.8, 1H), 8.58 (dd, J = 4.8, 1.7, 1H), 7.81 (d, J = 8.0, 1H),
7.52–7.28 (m, 11H), 5.88 (br s, 1H), 4.88 (d, J = 6.2, 2H); m/z: calcd for C21H18N5 [M + H]+, 340.1; found,
340.2.
3-Bromo-5,6-diphenyl-1,2,4-triazine (75 mg, 0.240 mmol),
5-methyl-1H-pyrazol-3-amine (33 mg, 0.336 mmol),
Xantphos (17 mg, 0.0288 mmol), K2CO3 (166 mg,
1.20 mmol), and tris(dibenzylideneacetone)dipalladium(0) (14 mg, 0.0240
mmol) were combined in 1,4-dioxane (1.00 mL) in a sealed vial. The
vial was evacuated and argon-bubbled through the suspension for 10
min. The reaction was heated to 150 °C for 1 h. The reaction
was diluted with DCM and washed with water. The layers were separated
and the organics were dried over Na2SO4, filtered,
and the solvent was removed at reduced pressure. The resulting residue
was purified by flash chromatography eluting with a gradient 0–50%
of 90/10/1DCM/methanol/NH4OH in DCM. The appropriate fractions
were combined, and the solvent was removed at reduced pressure. The
resulting residues were further purified by preparative HPLC (basic
conditions). The appropriate fractions were combined, and the solvent
was removed at reduced pressure to afford the title compound 25 (7 mg, 9%). 1H NMR (400 MHz, DMSO-d6) δ 11.92–12.13 (m, 1H), 10.24–10.44
(m, 1H), 7.28–7.56 (m, 10H), 6.39–6.53 (m, 1H), 2.24
(s, 3H); m/z: calcd for C19H17N7 [M + H]+, 329.1; found, 329.2.
4-(3-((1H-Indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzyl
acetate (37) (102 mg, 0.235 mmol) was suspended/dissolved
in methanol (10 mL), K2CO3 (95 mg, 0.690 mmol)
was added, and the reaction mixture was stirred at room temperature
for 4 h. The reaction was concentrated to dryness and partitioned
between DCM and 0.5 M HCl. The phases were mixed, separated, and the
aq. layer was back-extracted with DCM (2×). Combined organics
were washed with sat. NaCl solution, dried over sodium sulfate, filtered,
and concentrated to dryness. The resulting residues were purified
by flash chromatography using 0–3.5% methanol in DCM. The appropriate
fractions were collected and concentrated to dryness to give (4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)phenyl)methanol
(39) (92 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 8.13–8.09 (m, 1H), 7.97–7.95 (m, 1H),
7.56–7.51 (m, 4H), 7.44–7.32 (m, 7H), 7.25–7.15
(m, 2H), 4.76–4.71 (m, 4H), 1.75 (t, J = 5.9
Hz, 1H). m/z: calcd for C25H21N4O [M + H]+, 393.2; found, 393.0.
4-(3-((1H-Indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzyl
acetate (38) (100 mg, 0.230 mmol) was suspended/dissolved
in methanol (10 mL), K2CO3 (93 mg, 0.670 mmol)
added, and the reaction mixture stirred at room temperature for 4
h. The reaction was concentrated to dryness and partitioned between
DCM and 0.5 M HCl. The phases were mixed and separated, and the aq.
layer was back-extracted with DCM (2×). Combined organics were
washed with sat. NaCl solution, dried over sodium sulfate, filtered,
and concentrated to dryness. The resulting residue was purified by
flash column chromatography using 0–3.5% methanol in DCM. The
appropriate fractions were collected to give (4-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)phenyl)methanol (40) (90 mg, 90%). 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.97–7.95 (m, 1H), 7.56–7.52
(m, 4H), 7.46–7.32 (m, 7H), 7.25–7.15 (m, 2H), 4.76–4.72
(m, 4H), 1.77–1.72 (m, 1H).
Prepared according to method E. 6 mg, 5%. Analytical RP-HPLC
method A, retention time 1.88 min. 1H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.97–7.94 (m, 1H), 7.55–7.53
(m, 2H), 7.49–7.46 (m, 2H), 7.45–7.29 (m, 7H), 7.25–7.14
(m, 2H), 4.72 (s, 2H), 3.45 (s, 2H), 2.26 (s, 6H); m/z: calcd for C27H25N5 [M + H]+, 420.2; found, 420.2.
2-[[4-[3-(1H-Indol-3-ylmethyl)-5-phenyl-1,2,4-triazin-6-yl]phenyl]methyl]isoindoline-1,3-dione
and 2-(4-(3-((1H-Indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzyl)isoindoline-1,3-dione
(45a and 46a)
Prepared according
to method D. The solvents were removed under reduced pressure, and
the mixture was purified by flash chromatography using 0–50%
ethyl acetate in heptane. The appropriate fractions were collected
and concentrated to dryness to give 2-[[4-[3-(1H-indol-3-ylmethyl)-5-phenyl-1,2,4-triazin-6-yl]phenyl]methyl]isoindoline-1,3-dione
and 2-(4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzyl)isoindoline-1,3-dione,
155 mg of which was used in the next step without further purification.
tert-Butyl 3-((6-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-Butyl 3-((5-(4-((1,3-Dioxoisoindolin-2-yl)methyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (45b and 46b)
Prepared according to method H. The resulting residues
were purified by flash chromatography using 0–50% ethyl acetate
in heptane to give tert-butyl 3-((6-(4-((1,3-dioxoisoindolin-2-yl)methyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-((1,3-dioxoisoindolin-2-yl)methyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate, 97 mg of which was used without
further purification.
tert-Butyl 3-((6-(4-(Aminomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-Butyl 3-((5-(4-(Aminomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (45c and 46c)
To a solution of tert-butyl 3-((6-(4-((1,3-dioxoisoindolin-2-yl)methyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-((1,3-dioxoisoindolin-2-yl)methyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (97.0 mg, 0.186 mmol) in ethanol
(4 mL) was added hydrazine hydrate (0.060 mL), and the mixture esd
stirred at 40 °C for 18 h, and then concentrated to dryness.
The resulting residues were purified by flash chromatography using
0–10% methanol in DCM to give tert-butyl 3-((6-(4-(aminomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-(aminomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate, 65 mg of which was used without
further purification.
tert-Butyl 3-((6-(4-(Acetamidomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-Butyl 3-((5-(4-(Acetamidomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (45d and 46d)
To a solution of tert-butyl 3-((6-(4-(aminomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-(aminomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (36.0 mg, 0.0732 mmol) in DCM (5.00
mL) were added triethylamine (0.102 mL, 0.732 mmol) and acetyl chloride
(0.0631 mL, 0.884 mmol). The mixture was stirred at 40 °C for
3 h, after which the reaction mixture was concentrated to dryness.
The resulting residues were purified by flash column chromatography
using 0–10% methanol in DCM. The appropriate fractions were
collected and concentrated to dryness to give tert-butyl 3-((6-(4-(acetamidomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-(acetamidomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate, 16 mg of which was used without
further purification.
Preparation of N-(4-(3-((1H-Indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzyl)acetamide
(45) and N-(4-(3-((1H-Indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzyl)acetamide
(46)
A mixture of tert-butyl
3-((6-(4-(acetamidomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-(acetamidomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (16 mg, 0.03 mmol) was stirred at
40 °C in DCM (5.00 mL) and trifluoroacetic acid was added (0.06
mL). The reaction was stirred for 16 h and then concentrated to dryness.
The resulting residues were purified by preparative HPLC (basic conditions),
and the crude regioisomers were isolated to give 45 and 46.
tert-Butyl 3-[[5-[4-(Methanesulfonamidomethyl)phenyl]-6-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate and tert-Butyl 3-[[5-[4-(Methanesulfonamidomethyl)phenyl]-6-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate (47a and 47b)
To a solution of tert-butyl 3-((6-(4-(aminomethyl)phenyl)-5-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate and tert-butyl 3-((5-(4-(aminomethyl)phenyl)-6-phenyl-1,2,4-triazin-3-yl)methyl)-1H-indole-1-carboxylate (20 mg, 0.0407 mmol) at 0 °C
in DCM (5 mL) were added triethylamine (0.0567 mL, 0.407 mmol) and
methanesulfonyl chloride (0.00472 mL, 0.0610 mmol), and the reaction
was stirred at room temperature for 20 h. After this time, a further
portion of methanesulfonyl chloride (0.00472 mL, 0.0610 mmol) was
added and the mixture was stirred for 24 h. The reaction mixture was
concentrated, and the resulting residues were purified by flash chromatography
using 0–10% methanol in DCM to give tert-butyl
3-[[5-[4-(methanesulfonamidomethyl)phenyl]-6-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate and tert-butyl 3-[[5-[4-(methanesulfonamidomethyl)phenyl]-6-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate, 22 mg of which was used without
further purification.
Preparation of N-(4-(3-((1H-Indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzyl)methanesulfonamide
(47) and N-(4-(3-((1H-Indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzyl)methanesulfonamide
(48)
A mixture of regioisomers tert-butyl 3-[[5-[4-(methanesulfonamidomethyl)phenyl]-6-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate (8.00 mg, 0.0140 mmol) and tert-butyl 3-[[6-[4-(methanesulfonamidomethyl)phenyl]-5-phenyl-1,2,4-triazin-3-yl]methyl]-1H-indole-1-carboxylate (8.00 mg, 0.0140 mmol) was stirred
at 40 °C in DCM (5.00 mL) and TFA (0.06 mL) was added. The mixture
was stirred for 24 h and then concentrated to dryness. The resulting
residues were purified by preparative HPLC (basic conditions) to give 47 and 48.
Methyl 4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate
(49) (10 mg, 0.24 mmol) was suspended in methanol (0.500
mL) and stirred at room temperature, while NaOH (2.00 M, 35.7 μL,
0.0713 mmol) was added in one portion. The suspension was heated at
reflux for 1 h during which time the solid dissolved to give an orange
solution. Worked up by concentrating reaction mixture to dryness,
dissolving the residue in water (2 mL), and acidifying by addition
of 1 M HCl. The resulting suspension was filtered off and purified
by preparative HPLC (acidic conditions). The relevant fractions were
combined and concentrated to dryness to give 4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoic acid (51) (3 mg, 31%). Analytical RP-HPLC method A, retention time
2.50 min. 1H NMR (400 MHz, DMSO-d6) δ 13.03–13.20 (m, 1H), 10.90–11.03 (m,
1H), 7.93 (d, J = 8.3, 2H), 7.69–7.75 (m,
1H), 7.56–7.63 (m, 2H), 7.47 (d, J = 8.0,
3H), 7.34 (s, 4H), 7.04–7.11 (m, 1H), 6.96–7.03 (m,
1H), 4.54–4.64 (m, 2H); m/z: calcd for C25H19N4O2 [M + H]+, 407.1; found, 407.0.
Methyl 4-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
(50) (10 mg, 0.24) was suspended in methanol (0.5 mL)
and stirred at room temperature, while NaOH (2.00 M, 17.8 μL,
0.0357 mmol) was added in one portion. The suspension was heated at
reflux for 3 h. After this time, a further portion of NaOH (2.00 M,
17.8 μL, 0.0357 mmol) was added and the suspension was stirred
at reflux for a further 1 h, resulting in dissolution to give a brown
solution. Worked up by concentrating reaction mixture to dryness,
dissolving the residue in water (2 mL), and acidifying by the addition
of 1 M HCl. The resulting suspension was filtered and purified by
preparative HPLC (acidic conditions). The relevant fractions were
combined and concentrated to dryness to give 4-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoic acid (52) (1.2 mg, 13%). Analytical RP-HPLC method A, retention
time 2.45 min. 1H NMR (400 MHz, DMSO-d6) δ 13.12–13.24 (m, 1H), 10.92–11.00
(m, 1H), 7.86–7.92 (m, 2H), 7.70–7.74 (m, 1H), 7.54–7.60
(m, 2H), 7.33–7.49 (m, 7H), 7.05–7.10 (m, 1H), 6.97–7.03
(m, 1H), 4.56–4.62 (m, 2H); m/z: calcd for C25H19N4O2 [M + H]+, 407.0; found, 407.0.
Methyl 3-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
(53) (20 mg, 0.048 mmol) was stirred in tetrahydrofuran
(THF). Lithium hydroxide hydrate (24.0 mg, 0.571 mmol) and water (1
mL) were added, and the reaction was heated to 50 °C overnight
and then refluxed for 8 h. The solvent was removed at reduced pressure.
The residue was taken up in ethyl acetate and acidified with 1 M HCl.
The layers were separated. The organics were washed with water and
dried over Na2SO4, filtered, and concentrated
under reduced pressure. The resulting residue was purified using flash
chromatography eluting with a gradient 0–95/5/0.5 DCM/methanol/AcOH
in DCM. The appropriate fractions were collected and concentrated
to dryness. The resulting material was further purified by preparative
HPLC (acidic conditions) to give 3-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoic
acid (55) (16 mg, 86%). Analytical RP-HPLC method B,
retention time 2.35 min. 1H NMR (400 MHz, CDCl3) δ 8.31–8.26 (m, 1H), 8.10 (m, 2H), 7.93 (d, J = 7.8 Hz, 1H), 7.73–7.65 (m, 1H), 7.54–7.47
(m, 2H), 7.47–7.39 (m, 2H), 7.39–7.30 (m, 4H), 7.24–7.12
(m, 2H), 4.72 (d, J = 0.9, 2H); m/z: calcd for C25H19N4O2 [M + H]+, 407.1; found, 407.0.
Methyl 3-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate
(54) (33 mg, 0.0761 mmol) was stirred in THF. Lithium
hydroxide hydrate (24.0 mg, 0.571 mmol) and water (1 mL) were added,
and the reaction was heated to reflux overnight. The solvent was removed
at reduced pressure. The residue was taken up in ethyl acetate and
acidified with 1 M HCl. The layers were separated. The organics were
washed with water and dried over Na2SO4, and
the solvent was removed at reduced pressure. The resulting material
was further purified by preparative HPLC (acidic conditions) to give
3-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoic
acid (56) (13 mg, 57%). 1H NMR (400 MHz, CDCl3) δ 8.37–8.45 (m, 1H), 8.09–8.21 (m, 2H),
7.92–8.03 (m, 1H), 7.68 (m, 1H), 7.34–7.54 (m, 8H),
7.15–7.27 (m, 2H), 4.77 (s, 2H); m/z: calcd for C25H19N4O2 [M + H]+, 407.1; found, 407.0.
Methyl 4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate
(65) (14 mg, 0.033 mmol) was transferred to a microwave
vial in 0.5 mL of methanol. To this was added 2 mL of 40% methylamine
in water. The reaction mixture was then microwaved at 100 °C
for 1 h. The reaction mixture was extracted twice with DCM, and the
combined organics were concentrated to dryness. The resulting residues
were purified by preparative HPLC (basic conditions) to give 4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)-N-methylbenzamide (67) (9 mg, 64%). Analytical
RP-HPLC method C, retention time 1.33 min. 1H NMR (400
MHz, CD3OD) δ 8.62–8.69 (m, 1H), 8.02–8.10
(m, 1H), 7.72–7.82 (m, 3H), 7.61–7.65 (m, 1H), 7.59
(s, 2H), 7.32–7.51 (m, 5H), 4.69 (s, 2H), 2.90 (s, 3H); m/z: calcd for C25H21N6O [M + H]+, 421.2; found, 421.2.
Methyl 4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
(66) (22 mg, 0.052 mmol) was transferred to a microwave
vial in 0.5 mL of methanol. To this was added 2 mL of 40% methylamine
in water. The reaction mixture was then heated under microwave radiation
at 100 °C for 1 h. The reaction mixture was extracted twice with
DCM, and the combined organics were concentrated to dryness. The resulting
residues were purified by preparative HPLC (basic conditions) to give
4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)-N-methylbenzamide (68) (11 mg, 50%). Analytical
RP-HPLC method C, retention time 1.35 min. 1H NMR (400
MHz, CD3OD) δ 8.65 (s, 1H), 8.06 (d, J = 5.8, 1H), 7.76–7.84 (m, 3H), 7.54–7.65 (m, 3H),
7.48–7.53 (m, 2H), 7.41–7.47 (m, 1H), 7.28–7.38
(m, 2H), 4.69 (s, 2H), 2.91 (s, 3H); m/z: calcd for C25H21N6O [M + H]+, 421.2; found, 421.2.
Methyl
4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate
(65) (10 mg, 0.024 mmol) was transferred to a 2 mL microwave
vial, suspended in 2-aminoethanol (100 μL, 1.66 mmol), the vial
was capped, and the mixture was heated at 100 °C for 20 min resulting
in a brown solution. Worked up by cooling to room temperature and
diluting with methanol (0.2 mL) and DMSO (0.2 mL) and purifying directly
by preparative HPLC (basic conditions) to give 4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)-N-(2-hydroxyethyl)benzamide (69) (5 mg, 50%).
Analytical RP-HPLC method B, retention time 1.26 min. 1H NMR (400 MHz, CD3OD) δ 8.62–8.68 (m, 1H),
8.03–8.09 (m, 1H), 7.78 (d, J = 8.3, 3H),
7.61 (d, J = 12.3, 3H), 7.47 (d, J = 1.5, 3H), 7.38 (d, J = 7.5, 2H), 4.69 (s, 2H),
3.65–3.73 (m, 2H), 3.48 (s, 2H); m/z: calcd for C26H23N6O2 [M + H]+, 451.2; found, 451.2.
Methyl
4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
(66) (5 mg, 0.012 mmol) was transferred to a 2 mL microwave
vial, suspended in 2-aminoethanol (50.0 μL, 0.828 mmol), the
vial was capped, and the mixture was heated at 100 °C for 20
min. Worked up by cooling reaction mixture to room temperature diluting
with methanol (0.2 mL) and DMSO (0.2 mL) and purifying directly by
preparative HPLC (basic conditions) to give 4-(3-((1H-pyrrolo[2,3-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)-N-(2-hydroxyethyl)benzamide (70) (5 mg, 99%).
Analytical RP-HPLC method B, retention time 1.29 min. 1H NMR (400 MHz, CD3OD) δ 8.64–8.67 (m, 1H),
8.04–8.08 (m, 1H), 7.81–7.86 (m, 2H), 7.77–7.81
(m, 1H), 7.62 (s, 1H), 7.59 (s, 2H), 7.48–7.52 (m, 2H), 7.40–7.47
(m, 1H), 7.30–7.36 (m, 2H), 4.67–4.71 (m, 2H), 3.67–3.72
(m, 2H), 3.47–3.52 (m, 2H); m/z: calcd for C26H23N6O2 [M + H]+, 451.2; found, 451.2.
Methyl 4-(3-((1H-Pyrrolo[3,2-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
and Methyl 4-(3-((1H-Pyrrolo[3,2-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate (71a and 72b)
Prepared according to method
D. The resulting residues were purified by preparative HPLC (basic
conditions) to give methyl 4-(3-((1H-pyrrolo[3,2-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
and methyl 4-(3-((1H-pyrrolo[3,2-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate, 14 mg
of which was used directly in the next step.
Preparation of 4-(3-((1H-Pyrrolo[3,2-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)-N-methylbenzamide (71) and 4-(3-((1H-Pyrrolo[3,2-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)-N-methylbenzamide (72)
Methyl 4-(3-((1H-pyrrolo[3,2-c]pyridin-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)benzoate
and methyl 4-(3-((1H-pyrrolo[3,2-c]pyridin-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)benzoate (14 mg,
0.033 mmol) were transferred to a microwave vial in 0.5 mL of methanol.
To this was added 2 mL of 40% methylamine in water. The reaction mixture
was then microwaved at 100 °C for 1 h. The reaction mixture was
concentrated to dryness. The regioisomers were separated by SFC using
a Daicel OD-H (10 × 250 mm2, 30% methanol + 0.25%
diethylamine, 15 mL/min) to give 71 and 72.
Prepared according to method G. The resulting
residues were purified by flash chromatography eluting with 70–100%
ethyl acetate in heptane. The relevant fractions were combined to
afford a solid. The solid was further purified by preparative HPLC
(basic conditions) to give (4-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)phenyl)(morpholino)methanone
(74) (5 mg, 50%). Analytical RP-HPLC method A, retention
time 2.27 min. 1H NMR (400 MHz, CDCl3) δ
8.07–8.16 (m, 1H), 7.88–7.95 (m, 1H), 7.59 (d, J = 8.3, 2H), 7.47–7.54 (m, 2H), 7.31–7.45
(m, 7H), 7.16 (s, 2H), 4.72 (s, 2H), 3.55–3.87 (m, 6H), 3.29–3.51
(m, 2H); m/z: calcd for C29H26N5O2 [M + H]+, 476.2;
found, 476.0.
Prepared according to method G. The resulting
residues were purified by flash chromatography eluting with 0–10%
methanol in DCM. The relevant fractions were combined to afford a
yellow solid. The solid was further purified by preparative HPLC (basic
conditions) to give (4-(3-((1H-indol-3-yl)methyl)-5-phenyl-1,2,4-triazin-6-yl)phenyl)(4-methylpiperazin-1-yl)methanone
(77) (4.1 mg, 10%). Analytical RP-HPLC method B, retention
time 1.57 min. 1H NMR (400 MHz, CDCl3) δ
8.08–8.16 (m, 1H), 7.89–7.95 (m, 1H), 7.55–7.61
(m, 2H), 7.48–7.53 (m, 2H), 7.31–7.44 (m, 7H), 7.12–7.25
(m, 2H), 4.72 (s, 2H), 3.69–3.89 (m, 2H), 3.25–3.49
(m, 2H), 2.40–2.54 (m, 2H), 2.32 (s, 5H); m/z: calcd for C30H29N6O [M + H]+, 489.2; found, 489.0.
Prepared according
to method G. The resulting residues were purified by flash chromatography
using 0–5% methanol in DCM. The relevant fractions were combined
and concentrated to dryness. The resulting residues were further purified
by flash chromatography and eluting with 50–100% ethyl acetate
in heptane. The relevant fractions were combined to give 4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)-N-methylbenzamide (82) (10 mg, 34%). Analytical
RP-HPLC method A, retention time 2.34 min. 1H NMR (400
MHz, DMSO-d6) δ 10.94–11.01
(m, 1H), 8.44–8.55 (m, 1H), 7.77 (s, 2H), 7.70–7.75
(m, 1H), 7.50–7.56 (m, 2H), 7.32–7.49 (m, 7H), 7.05–7.11
(m, 1H), 6.97–7.03 (m, 1H), 4.54–4.63 (m, 2H), 2.72–2.80
(m, 3H); m/z: calcd for C26H22N5O [M + H]+, 420.2; found, 420.0.
Prepared according to method G. The resulting residues were purified
by preparative HPLC (basic conditions) to give 4-(3-((1H-indol-3-yl)methyl)-6-phenyl-1,2,4-triazin-5-yl)-N-(2-amino-2-oxoethyl)benzamide (90) (3 mg, 25%). Analytical
RP-HPLC method B, retention time 2.02 min. 1H NMR (400
MHz, CDCl3) δ 8.08–8.15 (m, 1H), 7.91–7.97
(m, 1H), 7.77–7.83 (m, 2H), 7.58–7.64 (m, 2H), 7.47–7.54
(m, 2H), 7.41–7.46 (m, 1H), 7.30–7.40 (m, 4H), 7.12–7.24
(m, 2H), 6.91–6.98 (m, 1H), 5.89–6.00 (m, 1H), 5.42–5.53
(m, 1H), 4.72 (s, 2H), 4.17 (d, J = 5.0 Hz, 2H); m/z: calcd for C27H22N6O2 [M + H]+, 463.2; found, 463.0.
Assay
[35S]GTPγS Incorporation
Assay
The GTPγS functional assay was performed according
to the published procedures.[23] A brief
description of the binding assay is given below. Flp-ln TREx 293 cells
(lnvitrogen) were maintained in Dulbecco’s modified Eagle’s
medium without sodium pyruvate (lnvitrogen), supplemented with 10%
(w/v) fetal calf serum, 1% penicillin/streptomycin mixture, and 10
μg/mL blasticidin at 37 °C in a 5% CO2 humidified
atmosphere. To generate Flp-ln TREx 293 cells capable of inducibly
expressing the GPR84 receptor constructs, the cells were transfected
with a mixture containing the desired cDNA in pcDNA5/FRT/TO vector
and pOG44 vector (1:9) using 1 mg/mL polyethylenimine (PEI) (molecular
weight (MW)—25 000). Cells were grown until 60–80%
confluent then transfected with 8 μg of required plasmid DNA
and PEI (ratio 1:6 DNA/PEl) and diluted in 150 mM NaCl, pH 7.4. After
incubation at room temperature for 10 min, the mixture was added to
cells. After 48 h, the medium was changed to a medium supplemented
with 200 μg/mL hygromycin B to initiate the selection of stably
transfect cells. After isolation of resistant cells, expression of
the appropriate construct from the Flp-ln TREx locus was induced by
treatment with up to 100 ng/mL doxycycline for 24 h. Membrane proteins
were generated from Flp-In T-REx HEK293 cells treated with 100 ng/mL
doxycycline to induce expression of the receptor construct of interest.
The cells were washed with ice-cold phosphate-buffered saline, removed
from dishes by scraping, and centrifuged at 3000 rpm for 5 min at
4 °C. Pellets were resuspended in TE buffer (10 mM Tris–HCl,
0.1 mM ethylenediaminetetraacetic acid (EDTA); pH 7.5) containing
a protease inhibitor mixture (Roche Applied Science, West Sussex,
U.K.) and homogenized with a 5 mL hand-held homogenizer. This material
was centrifuged at 1500 rpm for 5 min at 4 °C, and the supernatant
was further centrifuged at 50 000 rpm for 45 min at 4 °C.
The resulting pellet was resuspended in TE buffer, and the protein
content was assessed using a bicinchoninic acid (BCA) protein assay
kit (Pierce, Fisher Scientific, Loughborough, U.K.). The following
assay can be used for the determination of GPR84 activation. The guanosine
5′-O-[γ-thio]triphosphate ([35S]GTPγS) functional assay measures the level of G protein activation
following agonist occupation of a GPCR, by determining the binding
of the poorly hydrolyzable analogue [35S]GTPγS to
Gα subunits. Initially, 5 μg of generated membrane protein
was preincubated for 15 min at 25 °C in assay buffer (20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic
acid (HEPES), 5 mM MgCl2; 160 mM NaCl; 1 μM GDP;
0.05% fatty acid-free bovine serum albumin; pH 7.5) containing the
indicated ligand agonist concentrations. To assess inhibition of agonist
stimulation, membrane preparations were preincubated with antagonist
compound for 15 min at room temperature prior to the addition of agonist.
The reaction was then initiated with the addition of [35S]GTPγS (50 nCi per tube), and the reaction was terminated
after 45 min incubation at 30 °C by rapid filtration through
GF/C glass filters using a 24-well Brandel cell harvester (Alpha Biotech,
Glasgow, U.K.). Unbound radioligand was removed from filters by three
washes with ice-cold phosphate-buffered saline (pH 7.4), and filters
were dried for 2–3 h at room temperature. The dried filters
were added to 3 mL of Ultima GoldTM XR (PerkinElmer Life Sciences,
Beaconsfield, U.K.), and [35S]GTPγS binding was determined
by liquid scintillation spectrometry
Molecular Docking
The obtained antagonists
were docked into the AlphaFold model of the human GPR84[31,32] using a standard precision docking protocol available in the Glide
module of Schrodinger software (2020-1).[38,39] The Alphafold structure of the receptor was obtained from the AlphaFold
protein structure database (https://alphafold.ebi.ac.uk/). The protein structures were
prepared with the protein preparation module, and the structure of
antagonists was assessed with the ligand preparation module of Schrodinger
software. Residues involving Tyr69, Phe101,
Arg172, Phe335, and Trp360 were selected
to center the docking box. Receptor docking grids with the receptor
van der Waals radius scaling of 1.0, 0.9, and 0.8 were generated to
probe the binding of bulky compound analogues. Docking poses were
evaluated with the Glide docking score. The most frequent docking
mode among compound analogues was selected as the most probable pose.
Short minimization of docking complexes was carried out with the MacroModel
module of Schrodinger software. A default protocol of minimization
in implicit solvent was used to obtain the final complexes. The OPLS_2005
force field was used in all calculations. The three-dimensional (3D)
images were created in Maestro 2020-1.
Method for In Vivo Pharmacokinetic Studies
Mouse pharmacokinetic studies were carried out by Xenogesis Ltd.
(Nottingham, U.K.). All life phases were conducted in accordance with
the local Ethics Review process.
IV Dosing
Male C57BL/6J mice (n = 3, Charles River, U.K.) were intravenously administered
with a dose of 1 mg/kg of the appropriate compound (formulation 5%
v/v DMSO in 20% w/v HPCD in water). Blood was taken at 0.033, 0.1,
0.17, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after dosing. Plasma
concentrations were determined by LC/MS/MS. LC/MS/MS analysis was
performed using a Thermo TSQ Quantiva with Thermo Vanquish UPLC system
on a Luna Omega C18 column. The plasma concentrations were simulated
by nonparametric analysis from the plasma concentrations obtained
in the PK study using Phoenix WinNonlin.
PO Dosing
Male C57BL/6J mice (n = 3, Charles River, U.K.) were orally administered with
a dose of 10 mg/kg of the appropriate compound (formulation 0.5% w/v
methylcellulose in water). Blood was taken at pre-dose, 0.25, 0.5,
1, 2, 4, 6, 8, 12, and 24 h after dosing. Plasma concentrations were
determined by LC/MS/MS. LC/MS/MS analysis was performed using a Thermo
TSQ Quantiva with Thermo Vanquish UPLC system on a Luna Omega C18
column. The plasma concentrations were simulated by nonparametric
analysis from the plasma concentrations obtained in the PK study using
Phoenix WinNonlin.
Authors: Frédéric Labéguère; Sonia Dupont; Luke Alvey; Florilène Soulas; Gregory Newsome; Amynata Tirera; Vanessa Quenehen; Thi Thu Trang Mai; Pierre Deprez; Roland Blanqué; Line Oste; Sandrine Le Tallec; Steve De Vos; Annick Hagers; Ann Vandevelde; Luc Nelles; Nele Vandervoort; Katja Conrath; Thierry Christophe; Ellen van der Aar; Emanuelle Wakselman; Didier Merciris; Céline Cottereaux; Cécile da Costa; Laurent Saniere; Philippe Clement-Lacroix; Laura Jenkins; Graeme Milligan; Stephen Fletcher; Reginald Brys; Romain Gosmini Journal: J Med Chem Date: 2020-09-23 Impact factor: 7.446
Authors: Matteo Gianella-Borradori; Ivy Christou; Carole J R Bataille; Rebecca L Cross; Graham M Wynne; David R Greaves; Angela J Russell Journal: Bioorg Med Chem Date: 2014-11-08 Impact factor: 3.641
Authors: Zobaer Al Mahmud; Laura Jenkins; Trond Ulven; Frédéric Labéguère; Romain Gosmini; Steve De Vos; Brian D Hudson; Irina G Tikhonova; Graeme Milligan Journal: Sci Rep Date: 2017-12-20 Impact factor: 4.379
Authors: Laura Jenkins; Sara Marsango; Sarah Mancini; Zobaer Al Mahmud; Angus Morrison; Stuart P McElroy; Kirstie A Bennett; Matt Barnes; Andrew B Tobin; Irina G Tikhonova; Graeme Milligan Journal: ACS Pharmacol Transl Sci Date: 2021-09-07
Authors: John Jumper; Richard Evans; Alexander Pritzel; Tim Green; Michael Figurnov; Olaf Ronneberger; Kathryn Tunyasuvunakool; Russ Bates; Augustin Žídek; Anna Potapenko; Alex Bridgland; Clemens Meyer; Simon A A Kohl; Andrew J Ballard; Andrew Cowie; Bernardino Romera-Paredes; Stanislav Nikolov; Rishub Jain; Demis Hassabis; Jonas Adler; Trevor Back; Stig Petersen; David Reiman; Ellen Clancy; Michal Zielinski; Martin Steinegger; Michalina Pacholska; Tamas Berghammer; Sebastian Bodenstein; David Silver; Oriol Vinyals; Andrew W Senior; Koray Kavukcuoglu; Pushmeet Kohli Journal: Nature Date: 2021-07-15 Impact factor: 49.962
Figure 1
Figure 2
Figure 3
Scheme 1
Figure 4
Scheme 2
Figure 5
Figure 6
Figure 7