Synthesis and Evaluation of Diphenyl Conjugated Imidazole Derivatives as Potential Glutaminyl Cyclase Inhibitors for Treatment of Alzheimer's Disease

Article abstract of DOI:10.1021/acs.jmedchem.7b00648

High expression of glutaminyl cyclase (QC) contributes to the initiation of Alzheimer's disease (AD) by catalyzing the generation of neurotoxic pyroglutamate (pE)-modified β-amyloid (Aβ) peptides. Preventing the generation of pE-Aβs by QC inhibition has been suggested as a novel approach to a disease-modifying therapy for AD. In this work, a series of diphenyl conjugated imidazole derivatives (DPCIs) was rationally designed and synthesized. Analogues with this scaffold exhibited potent inhibitory activity against human QC (hQC) and good in vitro blood-brain barrier (BBB) permeability. Further assessments corroborated that the selected hQC inhibitor 28 inhibits the activity of hQC, dramatically reduces the generation of pE-Aβs in cultured cells and in vivo, and improves the behavior of AD mice.

Full text of DOI:10.1021/acs.jmedchem.7b00648

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Article
Synthesis and Evaluation of Diphenyl Conjugated Imidazole Derivatives as
Potential Glutaminyl Cyclase Inhibitors for Treatment of Alzheimer’s Disease
Manman Li, Yao Dong, Xi Yu, Yue Li, Yongdong Zou, Yizhi Zheng,
Zhendan He, Zhigang Liu, Jun-Min Quan, Xianzhang Bu, and Haiqiang Wu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00648 • Publication Date (Web): 12 Jul 2017
Downloaded from http://pubs.acs.org on July 12, 2017
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Synthesis and Evaluation of Diphenyl Conjugated Imidazole Derivatives as Potential Glutaminyl
Cyclase Inhibitors for Treatment of Alzheimer’s Disease
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Manman Li,§,#,¦ Yao Dong,§,#,¦ Xi Yu,§,# Yue Li,§ Yongdong Zou,# Yizhi Zheng,# Zhendan He,§ Zhigang
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Liu,¶ Junmin Quan,† Xianzhang Bu,‡ Haiqiang Wu§,#,φ,
*
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§ Department of Pharmacy, School of Medicine, Shenzhen University, Shenzhen, 518060, China
# College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, 518060, China
¶ School of Medicine, Shenzhen University, Shenzhen, 518060, China
† Key Laboratory of Structural Biology, School of Chemical Biology & Biotechnology, Peking
University, Shenzhen Graduate School, Shenzhen 518055, China
‡ School of Pharmaceutical Science, Sun Yatꢀsen University, Guangzhou, 510006, China
φ Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles,
CA, 90095, USA
¦ Manman Li and Yao Dong contributed equally to this work.
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Abstract:
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High expression of glutaminyl cyclase (QC) contributes to the initiation of Alzheimer’s disease (AD)
by catalyzing the generation of neurotoxic pyroglutamate (pE)ꢀmodified βꢀamyloid (Aβ) peptides.
Preventing the generation of pEꢀAβs by QC inhibition has been suggested as a novel approach to a
diseaseꢀmodifying therapy for AD. In this work, a series of diphenyl conjugated imidazole derivatives
(DPCIs) was rationally designed and synthesized. Analogues with this scaffold exhibited potent
inhibitory activity against human QC (hQC), and good in vitro blood brain barrier (BBB) permeability.
Further assessments corroborated that the selected hQC inhibitor 28 inhibits the activity of hQC and
dramatically reduces the generation of pEꢀAβs in cultured cells and in vivo, and improves the behavior
of AD mice.
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INTRODUCTION
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Alzheimer’s disease manifests as a progressive dementia that currently affects over 40 million people
worldwide.1 The devastating impact of AD on public health, the global economy, and society in general
is widely recognized.2 The hallmarks of AD are the presence of extracellular amyloid plaques
composed of Aβ peptides (mainly Aβ42 and Aβ40) surrounded by activated glia and by intracellular
neurofibrillary tangles composed of hyperphosphorylated tau protein, which makes Aβs and tau
protein the prime drug targets for the development of antiꢀAD agents.3
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The pathogenesis of AD has been extensively investigated, and the Aβ pathology, in which the Aβ
peptides produced from proteolytic cleavage of the amyloid precursor protein (APP) by βꢀ and γꢀ
secretase, are believed to trigger AD pathogenesis via their aggregating forms, is well documented.4, 5
In this regard, targeting Aβ pathology was considered a promising approach for prevention of AD and
possibly other dementias. Unfortunately, though the behavior of AD can be improved in AD model
mice by inhibiting the production, aggregation or increasing clearance of Aβ peptides, many key
elements of the disease neurobiology remain controversial, and there is no reliable cure or diseaseꢀ
modifying therapy available for use before AD progresses to major memory loss and functional
decline.6 The consistent failure of drug candidates targeting lateꢀstage Aβ pathology in clinical trials
has led to a major shift towards exploring new causes involved in the very early phases of AD.7, 8
During the past decades a variety of Nꢀtruncated Aβs starting with amino residue alaꢀ2,
pyroglutamylated gluꢀ3, pheꢀ4, argꢀ5, pyroglutamylated gluꢀ11, and so on, have been identified in AD
brains.7 Aβs undergo Nꢀterminal truncation and cyclization of Nꢀterminal glutamate (E) to
pyroglutamate (pE), generating pEꢀAβ3ꢀ40/42 and pEꢀAβ11ꢀ40/42. These pEꢀAβs, especially pEꢀAβ3ꢀ42
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account for more than 50% of the total Aβ plaques in the AD brains.9, 10 These modified Aβs have
proteolytic resistance and increased hydrophobicity, they aggregate more rapidly than normal Aβs, seed
further Aβ aggregation, and are more neurotoxic.11ꢀ13 Importantly, pEꢀAβs may be key initiators of AD
pathogenesis in the earlyꢀstages of AD.9, 10 Reducing the generation of pEꢀAβs should promote more
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efficient Aβ proteolysis and prevent aggregation by clearance of a major nucleation factor, and thus
help prevent the initiation of the disease process.
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The pyroglutamylation of Nꢀtruncated, Nꢀternimal gluꢀAβs is catalyzed by glutaminyl cyclase
(QC, also known as QPCT, EC 2.5.2.3)13, 14 which is widely distributed in mammalian brain with
robust expression in the hippocampus and cortex.15, 16 Consistently increased expression of QC is
correlated with the generation of pEꢀAβ3ꢀ42 and other Aβs in AD brains. Moreover, higher levels of QC
mRNA are found in AD compared to ageꢀmatched normal brains, and a positive correlation between
QC expression and the severity of AD has been confirmed.17 It is revealed that QC inhibition attenuated
the generation of pEꢀAβ3ꢀ4 in an AD model mouse brain and diminished the formation of Aβ plaques,
and significantly improved the ADꢀlike pathology.18, 19 Thus, QC inhibitors may offer a new option for
the development of novel antiꢀAD agents.20 In view of this, a few QC inhibitors have been reported.21ꢀ
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Typically, these inhibitors contain an aromatic motif tethered to an imidazole moiety, where the
aromatic ring matches the hydrophobic space at the entrance of the active site, and the imidazole
moiety is used to bind the catalytic zinc ion at the bottom of the pocket. For instance, the representative
analogue 1 (PBD150, Figure 1A, upper), which was developed by Buchholz, et al.,21 was found to be
efficient in reducing the generation of pEꢀAβ in a transgenic Drosophila model and also reduced AD
pathology in several transgenic mouse models. Despite this proof of principle, however, these QC
inhibitors have poor BBB permeability.25 Therefore, the discovery of QC inhibitors with high activity
and appropriate BBB penetrability is urgently needed to bring these novel antiꢀAD drugs to the clinic.
Here, we describe the design and discovery of a series of DPCIs as potential QC inhibitors. These
DPCIs retained the general properties required for reported inhibitory mechanisms but with enhanced
inflexibility and BBB penetration, through conformational restriction and polarity tuning based on
rational design. Our optimal DPCI shows higher QC inhibitory potency and improved in vitro BBB
penetrability than 1, and its beneficial effects on the pEꢀAβ production and AD pathogenesis were
validated in cell culture and in B6C3ꢀTg (APPswe/PSEN1dE9) doubleꢀtransgenic mice.
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RESULTS AND DISCUSSION
Design and synthesis of DPCIs
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To search for efficient QC inhibitors, the interaction of reported analogue 1 with the human QC (hQC)
was first based on the crystal structure of hQC in complex with 1 (PDB: 3PBB).26 Of interest is the πꢀπ
stacking between the aromatic motif of 1 and phe325 at the hydrophobic entrance of the active site, the
orthogonallyꢀdirected thiocarbamide starting linker, and the imidazole ring at the other terminal that
runs deep into the narrow active site and binds with the catalytic zinc ion at the bottom of the pocket
(Figure 1B and C). Therefore, it was predicted that flexibility of the thiocarbamide starting linker may
limit the binding of 1 with QC; moreover, the presence of the thiocarbamide motif makes the linker
hydrophilic, which would be expected to contribute to poor BBB penetrability. After these
considerations, we replaced the thiocarbamide motif by an additional phenyl ring to generate a
diphenyl moiety, and introduced an imidazole functionalized linker at the orthoꢀposition of the new
phenyl ring, to generate the new DPCI analogues. These modifications are anticipated to result in a
more rigid conformation that will match the binding features by restricting the orthogonal conformation
between the aromatic motif with the imidazole attached linker (Fig. 1BꢀC), and increase the
hydrophobicity to favor BBB penetration. The basic structure of DPCIs is shown in Fig. 1A (bottom).
According to this concept, a series of DPCIs (2ꢀ41, Scheme 1) with various substitutions and
different linker lengths were designed and synthesized (Scheme 1). Briefly, bromoaniline derivatives
were coupled with phenylboronic acid derivatives to give the diphenyl intermediates i by Suzukiꢀ
Miyaura reactions under the catalysis of Pd(DPPF)2Cl2 and K2CO3, in 1,4ꢀdioxane. Then the
intermediates i reacted with 1,3ꢀdibromopropane or 1,2ꢀdibromoethane provided the intermediate ii in
the presence of K2CO3 in acetonitrile. Finally, the nucleophile substitution reaction between ii and
imidazole derivatives afforded the desired products in the presence of K2CO3 in acetonitrile.
Screening of DPCIs as hQC inhibitors
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The hQCꢀglutamic acid dehydrogenase linked assay was used to evaluate the inhibitory potency of the
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resulting compounds. The percentages of inhibition at 100 µM (% at 100 µM) toward hQC were
recorded. As expected, compound 2 significantly inhibited the activity of hQC (85.34 %, Table 1). By
modifying substitutions on ring A, we observed that the methyl substitution at R3 (3), R4 (5), or
isopropyl substitution at R5 (6) sharply decreased the inhibitory activity, while replacing the methyl at
R3 (4) with smaller F recovered the activity to an even higher level, indicating hinderance occurred by
methyl substitution at the R3, R4, and R5 positions. In contrast, the methyl substitution at R6 (7)
moderately increased the inhibitory potency (96.14 %), and F substitution at R6 did not affect the
activity as compared 10 with 8. These observations are consistent with the predicted mode (Fig. 1B),
wherein A ring presents at the entrance of the deep pocket, therefore the methyl substitution at inner
positions R3, R4, or R5 may limit the interaction of the compounds with hQC, while the R6 points to the
outside and allows substitutions with appropriate volume. In addition, replacing the 3’,4’ꢀ dimethoxy at
B ring with 3’ꢀ or 4’ꢀ F substitution apparently did not affect the binding as compared with the
inhibitory activities of 2 with 8, 11, and 7 with 9, respectively.
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The 2nd batch of evaluation was focused on the analogues (12ꢀ20, Table 2) with a 2’ꢀmethyl
substituted imidazole ring (C ring), and similar substitution patterns in rings A and B. These analogues
generally showed low to moderate activities, and the 2’ꢀmethyl substitution at the C ring dramatically
decreased the activity as compared to the inhibitory potency of 12, 13, 14, 15, 16, 17 and 19 vs 3, 5, 6,
8, 9, 10 and 11, respectively. According to the proposed binding model, the zinc atom located at the
bottle of the active site of hQC could be chelated by the 3’ꢀN atom in the imidazole moiety. Therefore,
it could be concluded that the decrease of their activities may be attributed to the blockage of the
chelation by 2’ꢀmethyl substitution of the C ring.
Interestingly, unlike the 2’ꢀmethyl substitution, 4’ꢀmethyl substitution at the C ring moderately or
slightly increased the activity as compared with the inhibition rate of 21, 23, 27, 28, 30, 31 and 32 vs 2,
4, 7, 8, 9, 10 and 11, respectively (Table 3). More strikingly, compound 5, which contains a methyl
substitution in the R4 position, was the least potent, nevertheless an additional introduction of methyl in
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the 4’ꢀposition of imidazole motif led to compound 24 with sharply increased inhibitory potency
(94.48 %). We reasoned that the 4’ꢀmethyl substitution at the imidazole ring may shift the binding site
of 24 in hQC by the hydrophobic force between 4’ꢀmethyl and other residues in the native pocket. This
would contribute to the chelation of the 3’ꢀN atom in the imidazole motif with the zinc atom, and favor
the direct interaction between the molecule and enzyme. In the presence of an R3 methyl (3), or R5
isopropyl (6), however, the 4’ꢀmethyl substitution in imidazole cannot achieve the optimal binding
position as indicated by the dramatically decreased activity of 22 and 26, respectively.
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While designing the molecules, we noticed that a propyl linker is sufficient to deliver the imidazole
motif to the zinc ion located at the bottom of the active site. To explore the effect of linker length on the
inhibitory potency, analogues with a shorter ethyl linker instead of the propyl linker and similar
substitution partners on rings A and C were further designed, synthesized and evaluated. All these
compounds (33ꢀ41) showed poor potency on hQC inhibition as listed in Table 4, and the reduction of
the linker length dramatically decreased the activity as compared to 33, 35, 36, 37, 38, 39, 40, 41 with
2, 21, 8, 15, 28, 11, 19, 32, respectively. This observation indicated that the distance between the
imidazole and biphenyl motif is critical to achieve favorable chelation of imidazole with the zinc ion on
the bottom and the formation of πꢀπ stacking of the aromatic motif with phe325 at the entrance of the
hQC active site.
Based on the results mentioned above, the inhibitory IC50 values toward hQC of the analogues with
percentages of inhibition > 80 % at 100 µM (2, 4, 7, 8, 9, 10, 21, 23, 24, 25, 27, 28, 29, 30, 31, 32)
were determined using 1 as a positive control. All compounds except 32 demonstrated a subꢀ or low
micromolar IC50 (0.50ꢀ3.02 µM), which is lower than that of 1 (3.19 µM; Table 1 and 3). The structureꢀ
activity relationship (SAR) revealed by IC50 values is in accordance with rules indicated by the
screening, wherein the 4’ꢀmethyl substitution at the imidazole motif generally increases the potency of
hQC inhibition (2, 4, 8, 9 and 10 vs 21, 23, 28, 30 and 31 respectively), and methyl substitutions closer
to the linker are more harmful to the activity (24, 25, 27, 29 and 30). Moreover, F substitution (2 vs 4,
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21 vs 23) at R3, as well as F (8 vs 10, 28 vs 31) or methyl (2, 8, 21 and 28 vs 7, 9, 27 and 30,
respectively) substitution at R6 increases activity to a variable extent.
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To further understand the observed hQC inhibitory activity, molecular docking analysis of several
potent inhibitors (2, 23, and 28) and the inactive inhibitors (36 and 37) was performed. The top ranked
docked conformations for each compound were analyzed. The docked poses of the potent inhibitors (2,
23, and 28) resemble that of 1. Both the πꢀπ stacking between the terminal aromatic motif of the
inhibitors and phe325 at the hydrophobic entrance of the active site, and the chelation between the
imidazole ring and the catalytic zinc ion at the bottom of the pocket were conserved (Figure 2AꢀD).
The binding of these potential DPCIs with hQC in the catalytic active region is predicted to be stronger.
These compounds also exhibited increased potency of hQC inhibition compared with the positive
control. In contrast, the poses of 36 and 37 deviate markedly from that of the potent inhibitors, the πꢀπ
stacking between the terminal aromatic motif of the inhibitors and phe325 of hQC is disrupted due to
the shorter linker (Figure 2E and F). This also illuminated the SAR and hQC inhibitory potency of
these designed DPCIs.
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Compound 28 exhibits improved penetrability than 1 in PAMPAꢀBBB mode
One of the main concerns in the development of active chemicals targeting the central nervous system
(CNS) is the BBB permeation. In the current work, the new derivatives were expected to have
improved BBB permeability attributed to the introduction of the diphenyl moiety. This expectancy was
supported by the prediction with ADMETꢀblood brain barrier descriptors using professional drug
design software Discovery Studio. Two parameters, ADMET_BBB and ADMET_AlogP98, were used
to evaluate the selected analogues and 1, wherein ADMET_BBB means the base 10 logarithm of (brain
concentration)/(blood concentration) as predicted by a robust (leastꢀmedianꢀofꢀsquares) regression
derived from literature in vivo brain penetration data, and ADMET_AlogP98 is the octanol/water
partition coefficient which is another key molecular characteristic for bioactive chemicals. Here, 1
showed low scores with both ADMET_BBB and ADMET_AlogP98 (Table S1, see Supporting
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Information), which were in line with expectations. Among the evaluated DPCI analogues, 9, 10, 28,
29, 30, 31 and 32 exhibited higher scores than 2, 4, 7, 21, 23, 24, 25 and 27 with 3’,4’ꢀdimethoxy
substitutions in B ring, indicating that these analogues exhibited significantly enhanced lipophilicity
which may contribute to improved permeability of these DPCIs.
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To verify the prediction, we selected 28 as the representative compound for in vitro BBB penetration
examination after a combined consideration of inhibitory activity, predicted ADMET_BBB score, and
molecular weight, in which 28 and 30 were distinguished from others, but 30 is unfortunately less
soluble in the test buffer. The in vitro BBB penetration was evaluated using a parallel artificial
membrane permeation assay (PAMPAꢀBBB) following the generally reported protocols.27ꢀ29 The
UV/vis absorptions of 28 and 1 after permeating through a porcine polar brain lipid (PBL) membrane
were recorded from 290 to 310 nm and the effective permeabilities (Pe) were calculated. As described
in Figure 3, the Pe values of 28 in various wavelengths were significant higher than that of 1,
demonstrating a much stronger permeability of 28. This result is consistent with our prediction.
Compound 28 inhibits QC and reduces generation of pEꢀAβ3ꢀ42 in transfected HEK293T cells
Then, the hQC inhibitory potency of 28 was investigated in hQC and APP695 (NLQ) transfected
human embryonal kidney cells HEK293T which overꢀexpress recombinant hQC and Aβ3ꢀ42. It was
found that 28 had no effect on the expression of hQC in transfected HEK293T cells (data not shown),
but the activity of cellular hQC was effectively blocked by addition of 28 in the culture at a
concentration of 10 ꢁM. The percentage inhibition increased along with the culture time, and a
maximal percentage inhibition was obtained after 24 h incubation (Figure 4A). Noticeably, when the
culture time was extended to 48 h, a decreased percentage inhibition was observed, which could be
attributed to a change in the expression and activity of recombinant hQC protein in the cell cycle
different stages. The transfected HEK293T cells were therefore incubated with different concentrations
of 28 for 24 h (Figure 4B), and a significant doseꢀdependent inhibition was observed as expected,
which is associated with the doseꢀdependent decreased generation of pEꢀAβ3ꢀ42 as determined by
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ELISA (Figure 4C). These results indicated that 28 prevents the generation of pEꢀAβ3ꢀ42 by the
inhibition of hQC in cultured cells.
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Compound 28 inhibits QC, reduces generation of pEꢀAβ3ꢀ42 and improves the behavior of B6C3ꢀ
Tg mice
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Based on the above results of 28 on the hQC inhibition at both molecular and cellular levels, we
would expect it to exhibit beneficial effects on AD pathogenesis by inhibiting the activity of QC and
reducing the generation of toxic pEꢀAβ3ꢀ42 in vivo. Therefore, compound 28 was applied to 3ꢀmonthꢀold
female B6C3ꢀTg mice for 2 months by intraperitoneal injection with two doses, L and H (2 and 10 mg
per kg weight respectively). QC activity assay revealed a doseꢀdependent decrease of QC activity in the
hippocampus, cortex and total brain of the 28 treated mice, respectively (Figure 5A and B, P < 0.05),
which is associated with the doseꢀdependent decrease of pEꢀAβ3ꢀ42 as revealed by ELISA (Figure 5C)
and immunohistochemistry (Figure 5E and F). Interestingly, the accumulation of total soluble Aβs in
the AD brain increased doseꢀdependently after the treatment of 28 (Figure 5D), which is attributed to
the low conversion of normal Aβs to pEꢀAβs caused by QC inhibition. As pEꢀAβs are believed to serve
as seed and promote the formation of Aβ plaques, especially in the early stage, the significant doseꢀ
dependent reduction of Aβ plaques (Figure 5G and H) may also resulted from the inhibition of pEꢀAβs
generation by 28 treatment.
Subsequently, compound 28 treatment resulted in a significant behavioral improvement in B6C3ꢀtg
mice as measured by a Nestꢀbuilding test, reported as a useful index of behavioral deficits.30, 31
Compared with the mice in control group, compound 28ꢀtreated mice exhibited an obvious
improvement in nestꢀbuilding capacity (Figure 5I and S1, see Supporting Information). These findings
validated that the QC inhibitor 28 specifically blocks the generation of pEꢀAβs, reduces the Aβ plaques
in AD brain, and finally improves function in B6C3ꢀTg mice by inhibiting the activity of QC.
CONCLUSIONS
QC is a new target for the development of novel drugs for the prevention and treatment of AD because
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of the positive correlation between the expression of QC in the brain and the course of AD. Based on
rational design, a new series of DPCIs were prepared. These chemicals with enhanced inflexibility and
hydrophobicity exhibited powerful hQC inhibitory potency in vitro as expected. Fifteen compounds
were more potent than the positive control, and the improved permeability of several was also
confirmed by in silico and in vitro assays. In particular, the selected compound 28 showed remarkable
inhibitory activities in a doseꢀ and timeꢀ dependent manner in APP transfected HEK293T model cells.
Treatment with 28 significantly decreased the generation of pEꢀAβ3ꢀ42 in living cells and inhibited the
activity of hQC without interfering with its expression. Inhibitory efficacy was further validated in
animal studies by intraperitoneal injections of 28 in B6C3ꢀTg AD mice. The inhibitory effect of 28 in
vivo improved nestꢀbuilding behavior, an index of functional deficits in these AD model mice. We
believe these compounds represented a new class of hQC inhibitors that can contribute to the
development of novel antiꢀAD agents. Further efforts toward revealing in the depth mechanism and
activity of these inhibitors are now the focus of our ongoing investigations.
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EXPERIMENTAL SECTION
The 1HꢀNMR (400 MHz) data and 13CꢀNMR (100 MHz) was recorded on a Bruker Avance III, using
CDCl3 as solvent. The highꢀresolution positive ion electrospray mass spectra were obtained from a
LCMSꢀITꢀTOF (SHIMADZU). Thinꢀlayer chromatography (TLC) was performed on Merck silica gel
60F254 plates, and visualized with UV. Flash column chromatography was performed using silica gel
(200ꢀ300 mesh, Qingdao Haiyang Chemical Co. Ltd, China). Purities of the final compounds used for
biological evaluation were determined as above 95 % by HPLC/UV criteria (Agilent 1200). The
porcine polar brain lipid (PBL) was purchased from Avanti Polar Lipids, Inc., USA. SensoLyte® green
glutaminyl cyclase activity assay kit was purchased from AnaSpec, Inc. Amyloidꢀbeta (N3pEꢀ42)
ELISA kit was purchased from IBL International, Germany. Monoclonal antiꢀβꢀamyloid protein (mouse
IgG1 isotype) was purchased from Sigma, USA. Monoclonal mouse antibody against AβꢀpE3 was
purchased from Synaptic Systems GmbH, Germany. Other materials were purchased from Alfa Aesar
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Co. All other reagents were of analytical grade.
General procedure for the preparation of compounds 2ꢀ41.
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(1) To a solution of bromaniline or derivatives (1 equiv) and phenylboronic acid or derivatives (1.2
equiv) in 1,4ꢀdioxane (10 mL), Pd(DPPF)2Cl2 (0.06 equiv) and K2CO3 solution (2 mol/L, 10 mL) were
added. Under an atmosphere of Ar the mixture was warmed to 100 °C. After 3h the reaction was
quenched with a saturated NaCl solution (1 mL). The reaction mixture was extracted using ethyl
acetate (20 mL × 3). The combined organic phase was washed with saturated NaCl solution, dried with
anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified using
flash column chromatography (silica gel, ethyl acetate/hexane, gradient elution) to provide the
intermediate i.
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(2) To intermediate i (1 equiv) in anhydrous acetonitrile (6 mL), 1,3ꢀdibromopropane or 1,2ꢀ
dibromoethane (7 equiv) and anhydrous K2CO3 (2 equiv) was added; the mixture was warmed and
refluxed overnight. When cooled to room temperature, the acetonitrile was removed under reduced
pressure, water (20 mL) was added, and the water phase was extracted using ethyl acetate (20 mL × 3).
The combined organic phase was washed with saturated NaCl solution, dried with anhydrous Na2SO4,
filtered, and concentrated under reduced pressure. The residue was purified using flash column
chromatography (silica gel, ethyl acetate/hexane, gradient elution) to provide the intermediate ii.
(3) The nucleophile substitution reaction of ii (1 equiv) and imidazoles (1 equiv) provided the designed
inhibitors according to the procedure in (2).
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3',4'ꢀdimethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (2). Yield: 26%; H NMR
(400 MHz, CDCl3): δ 1.97ꢀ2.06 (m, 2H, CH2), 3.08ꢀ3.12 (m, 2H, CH2), 3.88 (s, 3H, OCH3), 3.93 (s,
3H, OCH3), 3.97ꢀ4.01 (m, 2H, CH2), 6.62ꢀ6.64 (d, J = 8.113 Hz, 1H, ArH), 6.76ꢀ6.81 (m, 1H, ArH),
6.86 (s, 1H, ArH), 6.91 (s, 1H, ArH), 6.95 (m, 2H, ArH), 7.06 (s, 1H, ArH), 7.09ꢀ7.12 (m, 1H, ArH),
7.20ꢀ7.23 (m, 1H, ArH), 7.26 (s, solv), 7.42 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 30.71, 40.83,
44.48, 56.02, 110.51, 111.83, 112.96, 117.48, 118.74, 121.45, 127.96, 129.60, 130.30, 131.86, 137.08,
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144.77, 148.54, 149.44. HRMS (EI): calculated for C20H23N3O2, 337.1790; found, C20H24N3O2,
338.1860 [M+H]+.
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3',4'ꢀdimethoxyꢀ3ꢀmethylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (3). Yield: 13%;
1H NMR (400 MHz, CDCl3): δ 1.79ꢀ1.85 (m, 2H, CH2), 2.31 (s, 3H, CH3), 2.81ꢀ2.85 (t, J = 13.663 Hz,
2H, CH2), 3.81ꢀ3.85 (t, J = 14.033 Hz, 2H, CH2), 3.88 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.76 (s, 1H,
ArH), 6.93ꢀ6.97 (m, 4H, ArH), 7.03ꢀ7.07 (m, 2H, ArH), 7.13ꢀ7.15 (d, J = 7.123 Hz, 1H, ArH), 7.28 (s,
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solv), 7.33 (s, 1H, ArH). C NMR (100 MHz, CDCl3): δ 18.86, 32.10, 44.50, 45.22, 55.97, 111.36,
112.43, 112.59, 118.66, 121.11, 121.46, 128.70, 128.93, 129.38, 130.56, 133.19, 133.61, 144.51,
148.25, 148.96. HRMS (EI): calcd for C21H25N3O2, 351.1947; found, C21H26N3O2, 352.2025 [M+H]+.
Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3ꢀfluoroꢀ3',4'ꢀdimethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (4). Yield: 33%;
1H NMR (400 MHz, CDCl3): δ 1.85ꢀ1.91 (m, 2H, CH2), 3.04ꢀ3.07 (t, J = 13.368 Hz, 2H, CH2), 3.87ꢀ
3.91 (m, 5H, CH2, OCH3), 3.95 (s, 3H, OCH3), 6.79 (s, 1H, ArH), 6.83ꢀ6.87 (m, 1H, ArH), 6.92 (s, 1H,
ArH), 6.94ꢀ6.96 (m, 2H, ArH), 6.98ꢀ6.99 (m, 1H, ArH), 7.01ꢀ7.04 (m, 2H, ArH), 7.28 (s, solv), 7.33 (s,
1H, ArH). 13C NMR (100 MHz, CDCl3): δ 32.00, 44.00, 44.24, 56.00, 111.51, 112.24, 115.04, 115.25,
118.64, 119.94, 121.11, 126.17, 128.52, 131.30, 133.27, 133.79, 136.99, 148.65, 149.20. HRMS (EI):
calcd for C20H22FN3O2, 355.1696; found, C20H23FN3O2, 356.1769 [M+H]+.
Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3',4'ꢀdimethoxyꢀ4ꢀmethylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (5). Yield:
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14.6%; H NMR (400 MHz, CDCl3): δ 2.01ꢀ2.07 (m, 2H, CH2), 2.36 (s, 3H, CH3), 3.11ꢀ3.14 (m, 2H,
CH2), 3.89 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 4.00ꢀ4.03 (m, 2H, CH2), 6.47 (s, 1H, ArH), 6.63ꢀ6.65
(d, J = 7.916 Hz, 1H, ArH), 6.89 (s, 1H, ArH), 6.92 (s, 1H, ArH), 6.95ꢀ6.96 (m, 2H, ArH), 7.01ꢀ7.03
(d, J = 7.916 Hz, 1H, ArH), 7.08 (s, 1H, ArH), 7.28 (s, solv), 7.46 (s, 1H, ArH). 13C NMR (100 MHz,
CDCl3): δ 21.67, 30.69, 40.72, 44.45, 55.96, 111.25, 111.58, 112.83, 118.19, 118.77, 121.45, 125.20,
129.62, 130.17, 131.76, 137.12, 138.52, 144.60, 148.27, 149.25. HRMS (EI): calcd for C21H25N3O2,
351.1947; found, C21H26N3O2, 352.2020 [M+H]+.
Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ5ꢀisopropylꢀ3',4'ꢀdimethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (6). Yield:
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15%; 1H NMR (400 MHz, CDCl3): δ 1.25 (s, 3H, CH3), 1.27 (s, 3H, CH3), 2.01ꢀ2.05 (t, J = 13.620 Hz,
2H, CH2), 2.83ꢀ2.90 (m, 1H, CH), 3.10ꢀ3.13 (t, J = 13.439 Hz, 2H, CH2), 3.91 (s, 3H, OCH3), 3.96 (s,
3H, OCH3), 4.00ꢀ4.03 (t, J = 13.894 Hz, 2H, CH2), 6.60ꢀ6.62 (d, J = 8.350 Hz, 1H, ArH), 6.89 (s, 1H,
ArH), 6.95 (s, 1H, ArH), 6.98ꢀ7.11 (m, 3H, ArH), 7.11 (s, 1H, ArH), 7.11ꢀ7.13 (d, J = 10.66 Hz, 1H,
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ArH), 7.28 (s, solv), 7.45 (s, 1H, ArH). C NMR (100 MHz, CDCl3): δ 24.23, 30.76, 33.16, 41.03,
44.56, 56.00, 110.61, 111.57, 112.76, 118.84, 121.45, 126.29, 127.86, 128.50, 129.24, 132.09, 137.02,
138.01, 142.76, 148.33, 149.21. HRMS (EI): calcd for C23H29N3O2, 379.2260; found, C23H30N3O2,
380.2334 [M+H]+.
Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3',4'ꢀdimethoxyꢀ6ꢀmethylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (7). Yield: 14%;
1H NMR (400 MHz, CDCl3): δ 1.96ꢀ1.99 (t, J = 13.494 Hz, 2H, CH2), 2.03 (s, 3H, CH3), 3.06ꢀ3.09 (t, J
= 13.465 Hz, 2H, CH2), 3.87 (s, 3H, OCH3), 3.93ꢀ3.96 (m, 5H, CH2, OCH3), 6.50ꢀ6.52 (d, J = 8.295
Hz, 1H, ArH), 6.69ꢀ6.71 (d, J = 7.585 Hz, 1H, ArH), 6.75ꢀ6.80 (m, 2H, ArH), 6.85 (s, 1H, ArH), 7.00ꢀ
7.02 (d, J = 8.014 Hz, 1H, ArH), 7.06 (s, 1H, ArH), 7.14ꢀ7.18 (t, J = 15.472 Hz, 1H, ArH), 7.28 (s,
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solv), 7.40 (s,1H, ArH). C NMR (100 MHz, CDCl3): δ 20.65, 30.56, 40.65, 44.33, 55.99, 107.78,
111.94, 113.08, 118.74, 119.09, 122.07, 127.55, 128.23, 129.62, 130.21, 137.09, 137.15, 145.37,
148.28, 149.66. HRMS (EI): calcd for C21H25N3O2, 351.1947; found, C21H26N3O2, 352.2024 [M+H]+.
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ4'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (8). Yield: 22%; H NMR (400
MHz, CDCl3): δ 1.99ꢀ2.06 (m, 2H, CH2), 3.09ꢀ3.12 (t, J = 13.178 Hz, 2H, CH2), 3.71 (s, 1H, NH),
3.98ꢀ4.01 (t, J = 13.868 Hz, 2H, CH2), 6.63ꢀ6.65 (d, J = 8.241 Hz, 1H, ArH), 6.77ꢀ6.81 (m, 1H, ArH),
6.86 (s, 1H, ArH), 7.05ꢀ7.08 (m, 2H, ArH), 7.12ꢀ7.17 (t, J = 17.530 Hz, 2H, ArH), 7.22ꢀ7.24 (m, 1H,
ArH), 7.26 (s, solv), 7.34ꢀ7.38 (m, 2H, ArH), 7.41 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 30.61,
40.72, 44.42, 110.53, 115.84, 116.05, 117.58, 118.71, 126.97, 128.97, 129.78, 130.43, 130.97, 135.09,
137.11, 144.59, 160.93, 163.38. HRMS (EI): calcd for C18H18FN3, 295.1485; found, C18H19FN3,
296.1556 [M+H]+.
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ4'ꢀfluoroꢀ6ꢀmethylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (9). Yield: 13%; H
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NMR (400 MHz, CDCl3): δ 1.96ꢀ2.00 (m, 5H, CH2, CH3), 3.06ꢀ3.09 (t, J = 13.147 Hz, 2H, CH2), 3.93ꢀ
3.97 (t, J = 13.878 Hz, 2H, CH2), 6.50ꢀ6.52 (d, J = 7.950 Hz, 1H, ArH), 6.69ꢀ6.71 (d, J = 7.666 Hz, 1H,
ArH), 6.85 (s, 1H, ArH), 7.06 (s, 1H, ArH), 7.15ꢀ7.21 (m, 5H, ArH), 7.28 (s, solv), 7.41 (s, 1H, ArH).
13C NMR (100 MHz, CDCl3): δ 20.70, 30.48, 40.58, 44.34, 107.85, 116.31, 116.52, 118.74, 119.23,
126.60, 128.48, 129.62, 131.77, 131.84, 133.67, 137.01, 145.12, 160.96, 163.41. HRMS (EI): calcd for
C19H20FN3, 309.1641; found, C19H21FN3, 310.1714 [M+H]+.
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ4',6ꢀdifluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (10). Yield: 15%; H NMR
(400 MHz, CDCl3): δ 1.98ꢀ2.04 (m, 2H, CH2), 3.08ꢀ3.11 (t, J = 13.455 Hz, 2H, CH2), 3.60 (s, 1H, NH),
3.97ꢀ4.00 (t, J = 13.491 Hz, 2H, CH2), 6.39ꢀ6.41 (d, J = 8.197 Hz, 1H, ArH), 6.51ꢀ6.55 (t, J = 17.335
Hz, 1H, ArH), 6.86 (s, 1H, ArH), 7.07 (s, 1H, ArH), 7.15ꢀ7.20 (m, 3H, ArH), 7.27 (s, solv), 7.29ꢀ7.32
(m, 2H, ArH), 7.50 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 30.46, 40.79, 44.48, 104.44, 104.63,
105.89, 116.16, 116.33, 118.75, 127.87, 129.36, 129.63, 129.71, 132.18, 132.24, 146.63, 161.54,
163.51. HRMS (EI): calcd for C18H17F2N3, 313.1391; found, C18H18F2N3, 314.1464 [M+H]+.
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Nꢀ(3ꢀ(1HꢀImidazolꢀ1ꢀyl)propyl)ꢀ3'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (11). Yield: 17%; H NMR (400
MHz, CDCl3): δ 2.01ꢀ2.05 (m, 2H, CH2), 3.09ꢀ3.11 (t, J = 13.490 Hz, 2H, CH2), 3.73 (s, 1H, NH),
3.98ꢀ4.00 (t, J = 14.156 Hz, 2H, CH2), 6.63ꢀ6.64 (d, J = 8.150 Hz, 1H, ArH), 6.77ꢀ6.80 (t, J = 15.640
Hz, 1H, ArH), 6.86 (s, 1H, ArH), 7.04ꢀ7.08 (m, 3H, ArH), 7.10ꢀ7.12 (d, J = 8.440 Hz, 1H, ArH), 7.17ꢀ
7.18 (d, J = 7.599 Hz, 1H, ArH), 7.22ꢀ7.25 (t, J = 17.061 Hz, 1H, ArH), 7.27 (s, solv), 7.29ꢀ7.43 (m,
2H, ArH). 13C NMR (100 MHz, CDCl3): δ 30.53, 40.64, 44.52, 110.62, 114.24, 114.45, 116.20, 116.41,
117.60, 124.99, 126.64, 129.24, 130.27, 130.51, 141.55, 144.36, 161.86, 164.32. HRMS (EI): calcd for
C18H18FN3, 295.1485; found, C18H19FN3, 296.1559 [M+H]+.
3',4'ꢀDimethoxyꢀ3ꢀmethylꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (12).
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Yield: 14%; H NMR (400 MHz, CDCl3): δ 1.92ꢀ1.96 (m, 2H, CH2), 2.03 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 3.05ꢀ3.09 (m, 2H, CH2), 3.84ꢀ3.89 (m, 5H, CH2, OCH3), 3.96 (s, 3H, OCH3), 6.50ꢀ6.55 (m, 2H,
ArH), 6.68ꢀ6.70 (d, J = 7.306 Hz, 1H, ArH), 6.74ꢀ6.75 (d, J = 1.965 Hz, 1H, ArH), 6.77ꢀ6.79 (m, 1H,
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ArH), 6.99ꢀ7.02 (m, 1H, ArH), 7.14ꢀ7.18 (t, J = 15.688 Hz, 1H, ), 7.28 (s, solv), 7.31 (s, 1H, ArH). 13C
NMR (100 MHz, CDCl3): δ 12.80, 18.89, 31.66, 43.63, 45.31, 55.96, 111.27, 112.35, 118.82, 121.08,
121.38, 126.88, 128.75, 128.77, 130.54, 133.21, 133.41, 144.22, 144.55, 148.19, 148.90. HRMS (EI):
calcd for C22H27N3O2, 365.2103; found, C22H28N3O2, 366.2177 [M+H]+.
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3',4'ꢀDimethoxyꢀ4ꢀmethylꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (13).
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Yield: 17%; H NMR (400 MHz, CDCl3): δ 1.77ꢀ1.80 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.31 (s, 3H,
CH3), 2.81ꢀ2.84 (m, 2H, CH2), 3.73ꢀ3.76 (m, 2H, CH2), 3.88 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.46
(s, 1H, ArH), 6.91ꢀ6.96 (m, 4H, ArH), 7.05ꢀ7.06 (d, J = 7.487 Hz, 1H, ArH), 7.12ꢀ7.14 (d, J = 7.113
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Hz, 1H, ArH), 7.21 (s, 1H, ArH), 7.28(s, solv). C NMR (100 MHz, CDCl3): δ 13.57, 18.88, 32.06,
44.40, 45.25, 55.95, 111.34, 112.42, 112.43, 115.12, 121.12, 121.39, 128.69, 128.76, 128.89, 130.55,
133.22, 133.53, 144.55, 148.24, 148.95. HRMS m/z: calcd for C22H27N3O2, 365.2103; found,
C22H28N3O2, 366.2177 [M+H]+.
5ꢀIsopropylꢀ3',4'ꢀdimethoxyꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine
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(14). Yield: 20%; H NMR (400 MHz, CDCl3): δ 1.25 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.96ꢀ2.03 (m,
2H, CH2), 2.22 (s, 3H, CH3), 2.83ꢀ2.90 (m, 1H, CH), 3.09ꢀ3.12 (t, J = 13.434 Hz, 2H, CH2), 3.88ꢀ3.93
(m, 5H, CH2, OCH3), 3.96 (s, 3H, OCH3), 6.58ꢀ6.63 (m, 2H, ArH), 6.96 (s, 1H, ArH), 6.98 (s, 2H,
ArH), 7.00ꢀ7.01 (d, J = 2.226 Hz, 1H, ArH), 7.11ꢀ7.14 (m, 1H, ArH), 7.28 (s, solv), 7.31 (s, 1H, ArH).
13C NMR (100 MHz, CDCl3): δ 12.64, 23.22, 29.72, 32.15, 40.04, 43.30, 54.98, 109.60, 110.55,
111.75, 114.22, 120.45, 125.27, 126.80, 127.46, 131.11, 135.20, 136.91, 137.63, 141.81, 147.31,
148.20. HRMS (EI): calcd for C24H31N3O2, 393.2416; found, C24H32N3O2, 394.2486 [M+H]+.
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4'ꢀFluoroꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (15). Yield: 32%; H
NMR (400 MHz, CDCl3): δ 1.94ꢀ2.00 (m, 2H, CH2), 2.31 (s, 3H, CH3), 3.11 (s, 2H, CH2), 3.69 (s, 1H,
NH), 3.87ꢀ3.90 (t, J = 13.833 Hz, 2H, CH2), 6.62ꢀ6.64 (d, J = 7.880 Hz, 1H, ArH), 6.75ꢀ6.76 (d, J =
1.360 Hz, 1H, ArH), 6.78ꢀ6.81 (t, J = 15.721 Hz, 1H, ArH), 6.90 (s, 1H, ArH), 7.06ꢀ7.08 (m, 1H, ArH),
7.12ꢀ7.16 (t, J = 17.136 Hz, 2H, ArH), 7.22ꢀ7.24 (m, 1H, ArH), 7.26 (s, solv), 7.34ꢀ7.38 (m, 2H, ArH).
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13C NMR (100 MHz, CDCl3): δ 12.92, 30.29, 40.68, 43.42, 110.50, 115.82, 116.03, 117.59, 118.89,
125.97, 127.44, 128.97, 130.40, 130.97, 131.05, 135.11, 144.60, 160.93, 163.38. HRMS (EI): calcd for
C19H20FN3, 309.1641; found, C19H21FN3, 310.1715 [M+H]+.
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4'ꢀFluoroꢀ6ꢀmethylꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (16). Yield:
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13%; H NMR (400 MHz, CDCl3): δ 1.89ꢀ1.96 (m, 2H, CH2), 1.99 (s, 3H, CH3), 2.30 (s, 3H, CH3),
3.06ꢀ3.10 (t, J = 13.097 Hz, 2H, CH2), 3.82ꢀ3.86 (t, J = 13.692 Hz, 2H, CH2), 6.50ꢀ6.52 (d, J = 8.169
Hz, 1H, ArH), 6.69ꢀ6.71 (d, J = 7.495 Hz, 1H, ArH), 6.73ꢀ6.74 (d, J = 1.361 Hz, 1H, ArH), 6.91 (s, 1H,
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ArH), 7.15ꢀ7.21 (m, 5H, ArH), 7.28 (s, solv). C NMR (100 MHz, CDCl3): δ 12.87, 20.70, 30.21,
40.54, 43.32, 107.80, 116.30, 116.51, 118.90, 119.24, 126.59, 127.28, 128.49, 131.77, 131.85, 133.65,
136.98, 145.15, 160.95, 166.28. HRMS (EI): calcd for C20H22FN3, 323.1798; found, C20H23FN3,
324.1873 [M+H]+.
4',6ꢀDifluoroꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (17). Yield: 20%;
1H NMR (400 MHz, CDCl3): δ 1.94ꢀ2.00 (m, 2H, CH2), 2.37 (s, 3H, CH3), 3.12 (s, 2H, CH2), 3.61 (s,
1H, NH), 3.87ꢀ3.90 (t, J = 14.039 Hz, 2H, CH2), 6.40ꢀ6.42 (d, J = 8.199 Hz, 1H, ArH), 6.52ꢀ6.57 (t, J =
17.440 Hz, 1H, ArH), 6.77 (s, 1H, ArH), 6.95 (s, 1H, ArH), 7.17ꢀ7.22 (m, 3H, ArH), 7.28 (s, solv),
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7.30ꢀ7.33 (m, 2H, ArH). C NMR (100 MHz, CDCl3): δ 12.66, 30.07, 40.70, 43.53, 104.52, 104.70,
105.86, 116.17, 116.34, 118.94, 127.84, 129.66, 129.75, 132.17, 132.24, 146.59, 159.47, 161.54,
163.51. HRMS (EI): calcd for C19H19F2N3, 327.1547; found, C19H20F2N3, 328.1624 [M+H]+.
4'ꢀFluoroꢀ4ꢀmethylꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (18). Yield:
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15%; H NMR (400 MHz, CDCl3): δ 2.01ꢀ2.04 (m, 2H, CH2), 2.19 (s, 3H, CH3), 2.35 (s, 3H, CH3),
3.11ꢀ3.13 (t, J = 13.519 Hz, 2H, CH2), 3.95ꢀ3.98 (t, J = 13.846 Hz, 2H, CH2), 6.45 (s, 1H, ArH), 6.62ꢀ
6.64 (d, J = 7.860 Hz, 2H, ArH), 6.96ꢀ6.97 (d, J = 7.482 Hz, 1H, ArH), 7.12ꢀ7.15 (t, J = 17.449 Hz,
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3H, ArH), 7.27 (s, solv), 7.33ꢀ7.35 (m, 2H, ArH). C NMR (100 MHz, CDCl3): δ 12.87, 20.70, 30.21,
40.54, 43.32, 107.80, 116.30, 116.51, 118.90, 119.24, 125.59, 127.28, 128.49, 131.77, 131.85, 133.65,
133.68, 136.98, 145.15, 160.95. HRMS (EI): calcd for C20H22FN3, 323.1798; found, C20H23FN3,
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324.1872 [M+H]+.
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3'ꢀFluoroꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (19). Yield: 19%; H
NMR (400 MHz, CDCl3): δ 1.98ꢀ2.02 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.08ꢀ3.11 (t, J = 13.664 Hz,
2H, CH2), 3.90ꢀ3.93 (t, J = 13.674 Hz, 2H, CH2), 6.57 (s, 1H, ArH), 6.64ꢀ6.65 (d, J = 2.014 Hz, 1H,
ArH), 6.76ꢀ6.81 (m, 1H, ArH), 7.05ꢀ7.09 (m, 2H, ArH), 7.11ꢀ7.13 (d, J = 5.998 Hz, 1H, ArH), 7.17ꢀ
7.19 (d, J = 6.014 Hz, 1H, ArH), 7.23ꢀ7.25 (d, J = 5.875 Hz, 1H, ArH), 7.26 (s, solv), 7.31 (s, 1H,
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ArH), 7.39ꢀ7.44 (m, 1H, ArH). C NMR (100 MHz, CDCl3): δ 12.75, 30.21, 40.58, 43.51, 110.60,
114.25, 114.46, 116.21, 116.42, 117.67, 118.95, 125.00, 126.87, 129.25, 130.26, 130.51, 144.34,
161.85, 164.31. HRMS (EI): calcd for C19H20FN3, 309.1641; found, C19H21FN3, 310.1709 [M+H]+.
3'ꢀFluoroꢀ5ꢀisopropylꢀNꢀ(3ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine
(20).
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Yield: 19%; H NMR (400 MHz, CDCl3): δ 1.22 (s, 3H, CH3), 1.24 (s, 3H, CH3), 1.93ꢀ2.01 (m, 2H,
CH2), 2.35 (s, 3H, CH3), 2.80ꢀ2.89 (m, 1H, CH), 3.08ꢀ3.13 (t, J = 13.007 Hz, 2H, CH2), 3.88ꢀ3.93 (t, J
= 13.996 Hz, 2H, CH2), 6.58ꢀ6.61 (d, J = 8.203 Hz, 1H, ArH), 6.78 (s, 1H, ArH), 6.93 (s, 1H, ), 6.96ꢀ
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6.97 (d, J = 2.190 Hz, 1H, ArH), 7.03ꢀ7.21 (m, 4H, ArH), 7.26 (s, solv), 7.38ꢀ7.45 (m, 1H, ArH). C
NMR (100 MHz, CDCl3): δ 12.61, 24.14, 30.35, 33.13, 40.95, 43.59, 108.51, 110.89, 114.11, 116.25,
116.41, 118.94, 125.01, 125.95, 128.39, 130.45, 138.34, 142.37, 162.11, 164.07. HRMS (EI): calcd for
C22H26FN3, 351.2111; found, C22H27FN3, 352.2184 [M+H]+.
3',4'ꢀDimethoxyꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (21). Yield:
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19%; H NMR (400 MHz, CDCl3): δ 1.94ꢀ2.03 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.07ꢀ3.12 (m, 2H,
CH2), 3.83ꢀ3.91 (m, 5H, CH2, OCH3), 3.93 (s, 3H, OCH3), 6.56 (s, 1H, ArH), 6.62ꢀ6.65 (d, J = 7.996
Hz, 1H, ArH), 6.76ꢀ6.81 (m, 1H, ArH), 6.91 (s, 1H, ArH), 6.95 (s, 2H, ArH), 7.09ꢀ7.12 (dd, J = 9.012
Hz, 1H, ArH), 7.20ꢀ7.22 (m, 1H, ArH), 7.26 (s, solv), 7.31 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3):
δ 12.84, 30.32, 40.69, 43.44, 55.98, 110.41, 111.57, 112.72, 117.45, 118.92, 121.39, 127.20, 127.88,
128.64, 130.27, 130.56, 131.72, 144.74, 148.39, 149.29. HRMS (EI): calcd for C21H25N3O2, 351.1947;
found, C21H26N3O2, 352.2023 [M+H]+.
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3',4'ꢀDimethoxyꢀ3ꢀmethylꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (22).
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Yield: 14%; H NMR (400 MHz, CDCl3): δ 1.76ꢀ1.80 (m, 2H, CH2), 2.20 (s, 3H, CH3), 2.31 (s, 3H,
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CH3), 2.81ꢀ2.84 (t, J = 14.081 Hz, 2H, CH2), 3.73ꢀ3.76 (t, J = 13.765 Hz, 2H, CH2), 3.88 (s, 3H,
OCH3), 3.95 (s, 3H, OCH3), 6.46 (s, 1H, ArH), 6.93ꢀ6.95 (m, 4H, ArH), 7.05ꢀ7.06 (d, J = 7.373 Hz,
1H, ArH), 7.12ꢀ7.14 (d, J = 7.145 Hz, 1H, ArH), 7.21 (s, 1H, ArH), 7.28 (s, solv). 13C NMR (100 MHz,
CDCl3): δ 13.57, 18.88, 32.06, 44.40, 45.25, 55.97, 111.34, 112.43, 115.12, 121.12, 121.39, 128.69,
128.75, 128.89, 130.53, 130.55, 133.22, 133.53, 144.55, 148.24, 148.95. HRMS (EI): calcd for
C22H27N3O2, 365.2103; found, C22H28N3O2, 366.2179 [M+H]+.
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3ꢀFluoroꢀ3',4'ꢀdimethoxyꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (23).
Yield: 22%; 1H NMR (400 MHz, CDCl3): δ 1.80ꢀ1.88 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.03ꢀ3.06 (t, J
= 13.102 Hz, 2H, CH2), 3.79ꢀ3.82 (t, J = 14.252 Hz, 2H, CH2), 3.89 (s, 3H, OCH3), 3.95 (s, 3H,
OCH3), 6.48 (s, 1H, ArH), 6.82ꢀ6.87 (m, 1H, ArH), 6.92ꢀ6.96 (m, 4H, ArH), 6.98ꢀ7.04 (m, 1H, ArH),
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7.20 (s, 1H, ArH), 7.28 (s, solv). C NMR (100 MHz, CDCl3): δ 13.63, 31.92, 44.01, 44.11, 55.97,
111.48, 112.25, 115.08, 115.24, 119.81, 119.89, 121.12, 126.15, 131.35, 133.19, 133.75, 136.07,
138.48, 148.64, 149.20. HRMS (EI): calcd for C21H24FN3O2, 369.1853; found, C21H25FN3O2, 370.1922
[M+H]+.
3',4'ꢀDimethoxyꢀ4ꢀmethylꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (24).
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Yield: 15%; H NMR (400 MHz, CDCl3): δ 1.92ꢀ1.96 (m, 2H, CH2), 2.03 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 3.05ꢀ3.09 (t, J = 13.564 Hz, 2H, CH2), 3.84ꢀ3.89 (m, 5H, CH2, OCH3), 3.96 (s, 3H, OCH3), 6.50ꢀ
6.55 (m, 2H, ArH), 6.68ꢀ6.70 (d, J = 7.402 Hz, 1H, ArH), 6.74ꢀ6.75 (d, J = 1.866 Hz, 1H, ArH), 6.77ꢀ
6.79 (m, 1H, ArH), 6.99ꢀ7.02 (dd, J = 10.143 Hz, 1H, ArH), 7.14ꢀ7.18 (t, J = 15.855 Hz, 1H, ArH),
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7.28 (s, solv), 7.31 (s, 1H, ArH). C NMR (100 MHz, CDCl3): δ 13.56, 20.64, 30.47, 40.62, 44.23,
55.97, 107.76, 111.91, 113.07, 115.24, 119.00, 122.06, 127.49, 128.20, 130.20, 136.12, 137.10, 138.45,
145.38, 148.25, 149.62. HRMS (EI) m/z: calcd for C22H27N3O2, 365.2103; found, C22H28N3O2,
366.2179 [M+H]+.
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3',4'ꢀDimethoxyꢀ5ꢀmethylꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (25).
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Yield: 23%; H NMR (400 MHz, CDCl3): δ 1.95ꢀ2.02 (m, 2H, CH2), 2.21 (s, 3H, CH3), 2.29 (s, 3H,
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CH3), 3.07ꢀ3.11 (t, J = 13.342 Hz, 2H, CH2), 3.55ꢀ3.59 (t, J = 12.350 Hz, 2H, CH2), 3.90 (s, 3H,
OCH3), 3.94 (s, 3H, OCH3), 6.57ꢀ6.59 (d, J = 7.916 Hz, 2H, ArH), 6.92ꢀ6.96 (m, 4H, ArH), 7.04ꢀ7.06
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(d, J = 8.041 Hz, 1H, ArH), 7.28(s, solv), 7.32(s, 1H, ArH). C NMR (100 MHz, CDCl3): δ 13.52,
20.31, 31.08, 34.88, 41.08, 55.97, 110.85, 111.54, 112.71, 115.30, 121.35, 125.63, 125.69, 127.97,
128.01, 128.96, 131.01, 131.90, 142.49, 148.32, 149.24. HRMS (EI): calcd for C22H27N3O2, 365.2103;
found, C22H28N3O2, 366.2170 [M+H]+.
5ꢀIsopropylꢀ3',4'ꢀdimethoxyꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine
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(26). Yield: 17%; H NMR (400 MHz, CDCl3): δ 1.25 (s, 3H, CH3), 1.27 (s, 3H, CH3), 1.96ꢀ2.03 (m,
2H, CH2), 2.22 (s, 3H, CH3), 2.83ꢀ2.90 (m, 1H, CH), 3.09ꢀ3.12 (t, J = 13.614 Hz, 2H, CH2), 3.88ꢀ3.93
(m, 5H, CH2, OCH3), 3.96 (s, 3H, OCH3), 6.58ꢀ6.63 (m, 2H, ArH), 6.96 (s, 1H, ArH), 6.98 (m, 2H,
ArH), 7.00ꢀ7.01 (d, J = 2.339 Hz, 1H, ArH), 7.11ꢀ7.14 (m, 1H, ArH), 7.28 (s, solv), 7.31 (s, 1H, ArH).
13C NMR (100 MHz, CDCl3): δ 12.64, 23.22, 29.72, 32.15, 40.04, 43.30, 54.98, 109.60, 110.55,
111.75, 114.22, 120.45, 125.27, 126.80, 127.46, 131.11, 135.20, 136.91, 137.63, 141.81, 147.31,
148.20. HRMS (EI): calcd for C24H31N3O2, 393.2416; found, C24H32N3O2, 394.2485 [M+H]+.
3',4'ꢀDimethoxyꢀ6ꢀmethylꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (27).
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Yield: 15%; H NMR (400 MHz, CDCl3): δ 1.91ꢀ1.98 (m, 2H, CH2), 2.03 (s, 3H, CH3), 2.21 (s, 3H,
CH3), 3.06ꢀ3.09 (t, J = 13.551 Hz, 2H, CH2), 3.84ꢀ3.88 (m, 5H, CH2, CH3), 3.96 (s, 3H, OCH3), 6.50ꢀ
6.55 (m, 2H, ArH), 6.68ꢀ6.70 (d, J = 7.335 Hz, 1H, ArH), 6.74ꢀ6.79 (m, 2H, ArH), 7.00ꢀ7.02 (d, J =
8.067 Hz, 1H, ArH), 7.14ꢀ7.18 (t, J = 15.845 Hz, 1H, ArH), 7.28 (s, solv), 7.31 (s, 1H, ArH). 13C NMR
(100 MHz, CDCl3): δ 13.56, 20.64, 30.47, 40.62, 44.23, 55.90, 107.76, 111.91, 113.07, 115.24, 119.00,
122.06, 127.49, 128.20, 130.20, 135.12, 137.10, 138.45, 145.38, 148.25, 149.62. HRMS (EI): calcd for
C22H27N3O2, 365.2103; found, C22H28N3O2, 366.2176 [M+H]+.
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NMR (400 MHz, CDCl3): δ 1.98ꢀ2.02 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.08ꢀ3.11 (t, J = 13.381 Hz,
2H, CH2), 3.90ꢀ3.93 (t, J = 14.287 Hz, 2H, CH2), 6.57 (s, 1H, ArH), 6.63ꢀ6.65 (d, J = 8.270 Hz, 1H,
ArH), 6.77ꢀ6.80 (t, J = 15.947 Hz, 2H, ArH), 7.05ꢀ7.07 (d, J = 8.703 Hz, 1H, ArH), 7.13ꢀ7.16 (t, J =
166.900 Hz, 2H, ArH), 7.22ꢀ7.25 (m, 1H, ArH), 7.26 (s, solv), 7.34ꢀ7.37 (m, 2H, ArH). 13C NMR (100
MHz, CDCl3): δ 13.54, 30.48, 40.72, 44.40, 110.54, 115.33, 115.77, 115.98, 117.45, 126.88, 128.91,
130.35, 130.93, 131.01, 136.13, 138.31, 144.61, 160.86, 163.31. HRMS (EI): calcd for C19H20FN3,
309.1641; found, C19H21FN3, 310.1716 [M+H]+.
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15%; H NMR (400 MHz, CDCl3): δ 1.98ꢀ2.04 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.34 (s, 3H, CH3),
3.10ꢀ3.13 (t, J = 13.678 Hz, 2H, CH2), 3.94ꢀ3.97 (t, J = 13.640 Hz, 2H, CH2), 6.46 (s, 1H, ArH), 6.61ꢀ
6.63 (d, J = 7.442 Hz, 2H, ArH), 6.95ꢀ6.97 (d, J = 7.555 Hz, 1H, ArH), 7.11ꢀ7.15 (t, J = 17.322 Hz,
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3H, ArH), 7.26 (s, solv), 7.31ꢀ7.35 (m, 2H, ArH). C NMR (100 MHz, CDCl3): δ 12.80, 21.64, 30.43,
40.80, 45.24, 111.42, 115.79, 115.96, 118.49, 118.57, 124.44, 130.31, 131.00, 131.07, 135.14, 137.47,
138.85, 144.39, 161.09, 163.06. HRMS (EI): calcd for C20H22FN3, 323.1798; found, C20H23FN3,
324.1870 [M+H]+.
4'ꢀFluoroꢀ6ꢀmethylꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (30). Yield:
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15%; H NMR (400 MHz, CDCl3): δ 1.95ꢀ2.02 (m, 2H, CH2), 2.19 (s, 3H, CH3), 2.27 (s, 3H, CH3),
3.07ꢀ3.10 (t, J = 13.755 Hz, 2H, CH2), 3.90ꢀ3.94 (t, J = 13.556 Hz, 2H, CH2), 6.56ꢀ6.58 (d, J = 7.982
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Hz, 2H, ArH), 6.90 (s, 1H, ArH), 7.04ꢀ7.16 (m, 4H, ArH), 7.26 (s, solv), 7.33ꢀ7.37 (m, 2H, ArH). C
NMR (100 MHz, CDCl3): δ 12.87, 20.70, 30.21, 40.54, 43.32, 107.80, 116.30, 116.51, 118.90, 119.24,
126.59, 127.28, 128.49, 131.77, 131.85, 133.65, 136.98, 145.15, 160.95, 166.28. HRMS (EI): calcd for
C20H22FN3, 323.1798; found, C20H23FN3, 324.1865 [M+H]+.
4',6ꢀDifluoroꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (31). Yield: 20%;
1H NMR (400 MHz, CDCl3): δ 1.94ꢀ2.00 (m, 2H, CH2), 2.20 (s, 3H, CH3), 3.08ꢀ3.11 (t, J = 14.105 Hz,
2H, CH2), 3.60 (s, 1H, NH), 3.88ꢀ3.91 (t, J = 13.410 Hz, 2H, CH2), 6.40ꢀ6.41 (d, J = 8.202 Hz, 1H,
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ArH), 6.51ꢀ6.56 (m, 2H, ArH), 6.79 (s, 1H, ArH), 7.16ꢀ7.20 (m, 3H, ArH), 7.27 (s, solv), 7.29ꢀ7.32 (m,
2H, ArH). 13C NMR (100 MHz, CDCl3): δ 12.66, 30.07, 40.70, 43.53, 104.52, 104.70, 105.86, 116.17,
116.34, 118.94, 127.84, 129.66, 129.75, 132.17, 132.24, 146.59, 159.47, 161.54, 163.51. HRMS (EI):
calcd for C19H19F2N3, 327.1547; found, C19H20F2N3, 328.1622 [M+H]+.
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3'ꢀFluoroꢀNꢀ(3ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)propyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (32). Yield: 22%; H
NMR (400 MHz, CDCl3): δ 1.93ꢀ2.02 (m, 2H, CH2), 2.31 (s, 3H, CH3), 3.10ꢀ3.14 (t, J = 12.914 Hz,
2H, CH2), 3.80 (s, 1H, NH), 3.87ꢀ3.92 (t, J = 14.199 Hz, 2H, CH2), 6.63ꢀ6.66 (d, J = 7.987 Hz, 1H,
ArH), 6.77ꢀ6.78 (t, J = 3.616 Hz, 1H, ArH), 6.80ꢀ6.82 (d, J = 7.992 Hz, 1H, ArH), 6.90 (s, 1H, ArH),
7.04ꢀ7.10 (m, 2H, ArH), 7.13ꢀ7.19 (m, 2H, ArH), 7.22ꢀ7.25 (m, 1H, ArH), 7.26 (s, solv), 7.38ꢀ7.45 (m,
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1H, ArH). C NMR (100 MHz, CDCl3): δ 13.56, 30.47, 40.73, 44.35, 110.55, 115.31, 115.75, 115.96,
117.44, 126.68, 128.91, 130.35, 130.94, 136.11, 136.58, 138.36, 144.64, 160.84, 163.29. HRMS (EI):
calcd for C19H20FN3, 309.1641; found, C19H21FN3, 310.1714 [M+H]+.
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Nꢀ(2ꢀ(1HꢀImidazolꢀ1ꢀyl)ethyl)ꢀ3',4'ꢀdimethoxyꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (33). Yield: 17%; H NMR
(400 MHz, CDCl3): δ 3.5 (m, 2H, CH2), 3.84 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.05 (s, 1H, NH),
4.11ꢀ4.14 (m, 2H, CH2), 6.66ꢀ6.68 (d, J = 7.953 Hz, 1H, ArH), 6.79ꢀ6.81 (m, 3H, ArH), 6.84 (s, 1H,
ArH), 6.89ꢀ6.91 (d, J = 8.444 Hz, 1H, ArH), 7.02 (s, 1H, ArH), 7.10ꢀ7.12 (d, J = 7.953 Hz, 1H, ArH),
7.23ꢀ7.25 (d, J = 8.12 Hz, 1H, ArH), 7.26 (s, solv), 7.40 (s, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ
44.55, 45.69, 55.98, 109.91, 111.58, 112.37, 117.78, 118.89, 121.23, 128.63, 129.68, 130.60, 131.26,
137.23, 143.71, 148.35, 149.23. HRMS (EI): calcd for C19H21N3O2, 323.1634; found, C19H22N3O2,
324.1705 [M+H]+.
3',4'ꢀDimethoxyꢀNꢀ(2ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (34). Yield: 14%;
1H NMR (400 MHz, CDCl3): δ 2.28 (s, 3H, CH3), 3.45ꢀ3.48 (m, 2H, CH2), 3.83 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 4.01ꢀ4.04 (m, 2H, CH2), 6.65ꢀ6.67 (d, J = 8.218 Hz, 1H, ArH), 6.74 (s, 1H, ArH), 6.76ꢀ
6.79 (m, 2H, ArH), 6.81ꢀ6.83 (d, J = 1.424 Hz, 1H, ArH), 6.88ꢀ6.92 (m, 2H, ArH), 7.10ꢀ7.12 (dd, J=
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6.504 Hz, 1H, ArH), 7.23ꢀ7.25 (m, 1H, ArH), 7.27 (s, solv). C NMR (100 MHz, CDCl3): δ 12.62,
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43.95, 53.50, 55.98, 109.83, 111.65, 112.39, 117.83, 119.19, 121.29, 126.65, 128.16, 128.64, 129.90,
130.62, 131.22, 143.74, 148.38, 149.24. HRMS (EI): calcd for C20H23N3O2, 337.1790; found,
C20H24N3O2, 338.1867 [M+H]+.
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3',4'ꢀDimethoxyꢀNꢀ(2ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (35). Yield: 14%;
1H NMR (400 MHz, CDCl3): δ 2.28 (s, 3H, CH3), 3.45ꢀ3.48 (m, 2H, CH2), 3.83 (s, 3H, OCH3), 3.91 (s,
3H, OCH3), 4.01ꢀ4.04 (m, 2H, CH2), 6.65ꢀ6.67 (d, J = 8.128 Hz, 1H, ArH), 6.74 (s, 1H, ArH), 6.79 (s,
1H, ArH), 6.81ꢀ6.82 (d, J = 6.750 Hz, 1H, ArH), 6.88 (s, 1H, ArH), 6.90 (s, 1H, ArH), 6.92 (s, 1H,
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ArH), 7.10ꢀ7.12 (dd, J = 9.043 Hz, 1H, ArH), 7.25 (s, 1H, ArH), 7.27 (s, solv). C NMR (100 MHz,
CDCl3): δ 12.57, 43.96, 44.76, 55.97, 109.83, 111.62, 112.37, 117.84, 119.22, 121.26, 126.28, 128.16,
128.63, 128.83, 130.61, 131.20, 143.71, 148.36, 149.22. HRMS (EI): calcd for C20H23N3O2, 337.1790;
found, C20H24N3O2, 338.1862 [M+H]+.
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Nꢀ(2ꢀ(1HꢀImidazolꢀ1ꢀyl)ethyl)ꢀ4'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (36). Yield: 16%; H NMR (400
MHz, CDCl3): δ 3.49 (m, 2H, CH2), 3.93 (s, 1H, NH), 4.03ꢀ4.06 (t, J = 12.127 Hz, 2H, CH2), 6.69ꢀ6.71
(d, J = 8.284 Hz, 1H, ArH), 6.74 (s, 1H, ArH), 6.82ꢀ6.86 (t, J = 14.763 Hz, 1H, ArH), 6.90 (s, 1H,
ArH), 7.02ꢀ7.07 (m, 3H, ArH), 7.10ꢀ7.12 (d, J = 7.815 Hz, 1H, ArH), 7.27ꢀ7.29 (t, J = 13.907 Hz, 2H,
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ArH), 7.36ꢀ7.42 (m, 1H, ArH). C NMR (100 MHz, CDCl3): δ 44.42, 45.69, 110.17, 115.88, 116.10,
117.98, 118.84, 127.31, 128.95, 129.70, 130.67, 130.86, 130.94, 137.21, 143.57, 160.92, 163.38.
HRMS (EI): calcd for C17H16FN3, 281.1328; found, C17H17FN3, 282.1404 [M+H]+.
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4'ꢀFluoroꢀNꢀ(2ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (37). Yield: 20%; H
NMR (400 MHz, CDCl3): δ 2.25 (s, 3H, CH3), 3.43ꢀ3.48 (m, 2H, CH2), 3.84 (s, 1H, NH), 4.00ꢀ4.04 (t,
J = 11.422 Hz, 2H, CH2), 6.66ꢀ6.70 (m, 2H, ArH), 6.78ꢀ6.84 (m, 1H, ArH), 6.87 (d, J = 1.324 Hz, 1H,
ArH), 7.05ꢀ7.06 (m, 1H, ArH), 7.08ꢀ7.09 (m, 1H, ArH), 7.12 (s, 1H, ArH), 7.18 (s, 1H, ArH), 7.20ꢀ
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7.21 (d, J = 2.336 Hz, 1H, ArH), 7.22ꢀ7.24 (t, J = 4.583 Hz, 1H, ArH), 7.26 (s, solv). C NMR (100
MHz, CDCl3): δ 12.79, 43.88, 44.60, 110.09, 115.87, 116.08, 117.96, 118.98, 127.13, 127.30, 128.95,
130.68, 130.86, 130.94, 134.60, 143.63, 160.92, 163.37. HRMS (EI): calcd for C18H18FN3, 295.1485;
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found, C18H19FN3, 296.1557 [M+H]+.
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NMR (400 MHz, CDCl3): δ 2.17 (s, 3H, CH3), 3.45ꢀ3.48 (t, J = 11.899 Hz, 2H, CH2), 3.84 (s, 1H, NH),
4.03ꢀ4.05 (t, J = 10.748 Hz, 2H, CH2), 6.50 (s, 1H, ArH), 6.65ꢀ6.69 (t, J = 15.111 Hz, 1H, ArH), 6.80ꢀ
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6.83 (t, J = 14.753 Hz, 1H, ArH), 7.06ꢀ7.12 (m, 5H, ArH), 7.21ꢀ7.24 (m, 2H, ArH), 7.26 (s, solv). C
NMR (100 MHz, CDCl3): δ 12.79, 43.88, 44.60, 110.09, 115.87, 116.08, 117.96, 118.98, 127.13,
127.30, 128.95, 130.68, 130.86, 130.94, 134.60, 143.63, 160.92, 163.37. HRMS (EI): calcd for
C18H18FN3, 295.1485; found, C18H19FN3, 296.1554 [M+H]+.
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Nꢀ(2ꢀ(1HꢀImidazolꢀ1ꢀyl)ethyl)ꢀ3'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (39). Yield: 19%; H NMR (400
MHz, CDCl3): δ 3.49 (m, 2H, CH2), 3.93 (s, 1H, NH), 4.03ꢀ4.06 (t, J = 11.939 Hz, 2H, CH2), 6.69ꢀ6.71
(d, J = 7.586 Hz, 1H, ArH), 6.74 (s, 1H, ArH), 6.82ꢀ6.86 (t, J = 14.977 Hz, 1H, ArH), 6.90 (s, 1H,
ArH), 7.02ꢀ7.07 (m, 3H, ArH), 7.10ꢀ7.12 (d, J = 8.241 Hz, 1H, ArH), 7.27ꢀ7.31 (t, J = 15.197 Hz, 2H,
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ArH), 7.36ꢀ7.42 (m, 1H, ArH). C NMR (100 MHz, CDCl3): δ 44.48, 45.76, 110.29, 114.31, 114.51,
116.15, 116.35, 118.01, 118.85, 124.81, 129.21, 129.69, 130.48, 137.13, 143.39, 161.84, 164.30.
HRMS (EI): calcd for C17H16FN3, 281.1328; found, C17H17FN3, 282.1403 [M+H]+.
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3'ꢀFluoroꢀNꢀ(2ꢀ(2ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (40). Yield: 33%; H
NMR (400 MHz, CDCl3): δ 2.29 (s, 3H, CH3), 3.49 (m, 2H, CH2), 3.93 (s, 1H, NH), 4.03ꢀ4.06 (t, J =
11.922 Hz, 2H, CH2), 6.69ꢀ6.71 (d, J = 8.184 Hz, 1H, ArH), 6.74 (s, 1H, ArH), 6.82ꢀ6.86 (t, J = 13.919
Hz, 1H, ArH), 6.90 (s, 1H, ArH), 7.07ꢀ7.12 (m, 3H, ArH), 7.27ꢀ7.31 (t, J = 14.475 Hz, 2H, ArH), 7.36ꢀ
7.42 (m, 1H, ArH). 13C NMR (100 MHz, CDCl3): δ 12.74, 43.91, 44.70, 110.24, 114.30, 114.51,
116.16, 116.37, 118.02, 118.98, 124.81, 127.21, 129.22, 130.49, 130.65, 143.49, 161.85, 164.31.
HRMS (EI): calcd for C18H18FN3, 295.1485; found, C18H19FN3, 296.1556 [M+H]+.
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3'ꢀFluoroꢀNꢀ(2ꢀ(4ꢀmethylꢀ1Hꢀimidazolꢀ1ꢀyl)ethyl)ꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀamine (41). Yield: 22%; H
NMR (400 MHz, CDCl3): δ 2.20 (s, 3H, CH3), 3.08ꢀ3.11 (t, J = 13.145 Hz, 2H, CH2), 3.90ꢀ3.93 (t, J =
12.877 Hz, 2H, CH2), 6.57 (s, 1H, ArH), 6.64ꢀ6.65 (d, J = 8.145 Hz, 1H, ArH), 6.76ꢀ6.81 (m, 1H,
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ArH), 7.05ꢀ7.09 (m, 2H, ArH), 7.11ꢀ7.13 (d, J = 7.869 Hz, 1H, ArH), 7.17ꢀ7.19 (d, J = 8.091 Hz, 1H,
ArH), 7.23ꢀ7.25 (d, J = 7.695 Hz, 1H, ArH), 7.26 (s, solv), 7.31 (s, 1H, ArH), 7.39ꢀ7.44 (m, 1H, ArH).
13C NMR (100 MHz, CDCl3): δ 12.79, 43.88, 44.60, 110.09, 115.87, 116.08, 117.96, 118.98, 127.13,
127.30, 128.95, 130.68, 130.86, 130.94, 134.60, 143.63, 160.92, 163.37. HRMS (EI): calcd for
C18H18FN3, 295.1485; found, C18H19FN3, 296.1556 [M+H]+.
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Design and in silico analysis
A molecular operating environment (MOE, 2011.10, Chemical Computing Group, Canada) was used.
The protein preparation steps involved 3D protonation, energy minimization, and active site
identification. The docking template structure of hQC was derived from the crystal structure of hQC
bound to the inhibitor 1 (PDB: 3PBB), in which 1 was extracted from the crystal structure of the
complex, and all crystal waters were removed. Protein was energy minimized and 3D protonated using
the structure preparation module of MOE. Ligand files for the molecular docking studies were prepared
in MOE and were followed by energy optimization at a standard MMFF94 force field level, with a
0.0001 kcal/mol energy gradient convergence criterion. The optimized geometries were saved in a
molecular data base file for further studies.
The optimized ligands were flexibly docked with the rigid hQC using the MOEꢀDock program. Thirty
independent docking runs were performed using the MOE docking simulation program. The docked
poses were analyzed and the best scored pose for each compound was chosen for further analysis. The
illustrated structures were made by PyMOL (Delano Scientific LLC, USA).
The ADMET_BBB and ADMET_AlogP98 of selected compounds were predicted by Discovery
Studio (Accelrys Co., Ltd., USA).
Inhibitive activities in vitro
Preparation of hQC. Cloning, expression and large scale preparation of hQC were performed
according to previous reports.32ꢀ34 The gene for hQC was inserted via the BamHI and XhoI restriction
sites into Escherichia coli expression vector pET32a with additional introduction of an Nꢀterminal
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His6ꢀtag (primer pair Cs/Cas). Primers, 5ꢀ3: ACCTGGATCCGCTTCTGCTTGGCCGG, BamHI; 5ꢀ3:
TATCCTCGAGTTACAGGTGCAGGTATTC, XhoI (Takara, China). E.coli strain DH5α was used for
all cloning procedures. The cDNA was verified by sequencing (Samgon, China). Plasmid DNA was
amplified, purified, and linearized. hQC was heterologously expressed in E.coli BL21(DE3) using
Fernbach flasks at room temperature overnight, and expression induced by addition of 0.2 mM IPTG
(isopropyl βꢀDꢀ1ꢀthiogalactopyranoside). Cells were disrupted with 1 mg/mL lysozyme and a freezeꢀ
thaw cycle. The purification of hQC protein followed two chromatographic steps: Ni2+ꢀIMAC
(immobilized metal affinity chromatography), and molecular sieve chromatography. QCꢀcontaining
fractions were pooled and purity was analyzed by SDSꢀPAGE (15%, sodium dodecyl sulfate
polyacrylamide gel electrophoresis) and Coomassie blue staining. The purified hQC enzyme was stored
at ꢀ80 °C without glycerol.
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hQC and inhibitive activities studies. The hQC activity was assayed as described elsewhere.35 Briefly,
for spectrophotometric assessment, the assay reactions (200 ꢁL) consisted of varying concentrations (0ꢀ
4 mM) of freshly prepared HꢀglnꢀglnꢀH, 30 units/ml glutamic acid dehydrogenase, 0.5 mM NADH/H+,
and 15mM αꢀketoglutaric acid in 0.05 M TrisꢀHCl, pH 8.0. Reactions were started by the addition of
hQC. Activity was monitored by recording the decrease in absorbance at 340 nm for 15 min.
For the inhibitor testing, the assay reaction composition was the same as described above, except for
the addition of different concentrations of the synthesized DPCIs and 1. The percentage of inhibition at
100 mM was calculated according to the formula: % inhibition = (VC ꢀ VS)/VC, where VC is the
reaction velocity of control, and VS is the reaction velocity of samples. All experiments were performed
in triplicate. The IC50 values were determined graphically from log concentration vs. % inhibition
curves.
BBB permeation assay
PAMPAꢀBBB assay in vitro was carried out according to previous reports.27ꢀ29 Simply, test
compounds were dissolved in DMSO (2 mg/mL) and the stock solutions were diluted in PBS at pH 8.0
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to make secondary stock solutions (100 ꢁg/mL, 5% DMSO). The Millipore MultiScreen® assay system
(Millipore, Bedford, MA) was used for the artificial membrane support and as the receiver plate. After
the required pretreatment, the filter membrane (hydrophobic PVDF) on the 96ꢀwell filtration plate was
coated with 6 ꢁL PBL solution in dodecane (20 mg/mL) in each well. The donor well was filled with
250 ꢁL of the secondary stock solution. Then the donor plate was securely placed on the top of an
acceptor plate which was preꢀfilled with equal volumes of blank receiving solution to form a
‘sandwich’. After 4 h at 25 °C, the absorbance of solutions in the accepter wells was determined by a
multiꢀwavelength UV plate reader at 290ꢀ310 nm. Pe was calculated according to the formula: Pe = ꢀ
VdVa/[(Vd + Va)St]ln(1ꢀAa/Ae), where Vd and Va are the mean volumes of the donor and accepter
solutions, S means the surface area of the artificial membrane, t the incubation time, Aa and Ae the UV
absorbance of the accepter well and the theoretical equilibrium absorbance, respectively. Each test
compound was analyzed in three wells, and at least in three runs, and the results are given as the mean
of the whole data set ± standard deviation.
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Inhibitory activities in cells
Vector, cell culture and transfect. The APP695ꢀA673V and the PCMV3ꢀQPCT (human glutaminyl
cyclase) plasmids were constructed. Human embryonal kidney cells HEK293T were purchased from
the Shanghai Cell Bank (Shanghai, China) and cultured in a humidified atmosphere of 5% CO2 at
37 °C. According to the manufacturer's manual, HEK293T cells were transfected with the APP695ꢀ
A673V and PCMV3ꢀQPCT plasmids using Lipofectamin2000 (Invitrogen, USA). Transiently
transfected cells were grown with serumꢀfree OptiꢀMEM for 6 h and afterwards incubated overnight
under normal DMEM conditions. Then, transfected HEK293T cells were cultured either in the presence
of 28 at a concentration of 10 ꢁM for 0, 6, 12, 24 and 48 h or in concentrations of 0, 1, 10, 50 and 100
ꢁM for 24 h. After the culture, cells and culture media were immediately collected for assay.
QC activities assay. Cells were lysed using tissue grinding tube on ice. The hQC activities in the
supernatants were determined using the same method mentioned in hQC and inhibitive activities
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pEꢀAβ3ꢀ42 concentrations assay. pEꢀAβ3ꢀ42 concentrations in the cell lysate supernatants and culture
media were determined using amyloidꢀbeta (N3pEꢀ42) ELISA kit. Briefly, 100 ꢁl of the sample or
100ꢁl of standard were added to 96ꢀwell microtiterꢀplates coated with antiꢀhuman Aβ38ꢀ42 rabbit IgG
affinity purify followed by incubation over night at 4 °C. The next day, plates were washed and
incubated with horseradish peroxidaseꢀconjugated antibodies for detecting pEꢀAβ3ꢀ42. After a final
washing step, bound enzyme activity was measured using a TMB peroxidase substrate in a colorimetric
reaction. The absorbance at 450 nm was determined using a Sunrise plate reader (Tecan, Switzerland).
The antiꢀAD effects in vivo
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Animals. All animal experiments were approved by the Animal Ethic Committee at Shenzhen
University and were carried out in accordance with the approved guidelines. The B6C3ꢀTg
(APPswe/PSEN1dE9) doubleꢀtransgenic mice (12 weeks, female) were selected and purchased from
Guangdong Medical Laboratory Animal Center (GDMLAC) (Foshan, China), and housed at a 12ꢀh
day/12ꢀh night cycle with free access to water and food pellets. Animals were treated with contrast
solution (control, C), 2 mg (low dose, L) or 10 mg compound 28 per kg weight (high dose, H) by
intraperitoneal injection for 8 weeks respectively.
Behavioral testing. Nesting behavior was used to measure changes in social behavior. Here, the nestꢀ
building task was administered with slight modifications as previously reported.30, 31At the end of the
treatment period, mice were individually housed. Eight pieces of cotton paper were introduced inside
the home cage to allow nesting behavior and 24h later the quality of the nest was determined.
QC activities, pEꢀAβ3ꢀ42 and soluble Aβs concentrations assay. One day after the behavioral testing,
mice were sacrificed by CO2 inhalation, the brains were removed, flushed with cold saline and placed
on filter paper. Separated hippocampus, cortex and one halfꢀbrain were homogenized in PBS (pH 7.6)
containing a protease inhibitor cocktail using tissue grinding tube on ice respectively. After
centrifugation, these supernatants were used for the followed assay or stored at ꢀ80 °C. QC activities
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were determined using SensoLyte® green glutaminyl cyclase activity assay kit and the
spectrophotometric method mentioned in hQC and inhibitive activities studies respectively. pEꢀAβ3ꢀ42
and total soluble Aβs concentrations in these supernatants were determined by amyloidꢀbeta (N3pEꢀ42)
ELISA kit and ELISA assay using monoclonal antiꢀβꢀamyloid protein (mouse IgG1 isotype) as the first
antibody, respectively.
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pEꢀAβ3ꢀ42 and total Aβs plaques assay. Mice were anaesthetized with pentobarbital and sequentially
perfused transcardially with PBS (pH 7.4) and 4% paraformaldehyde in PBS. The brains were removed
and placed in the same 4% paraformaldehyde solution for 24 h at 4 °C, followed by sequential
immersion in 10 %, 20 % and 30 % sucrose at 4 °C, at the end of which they had sunk to the bottom of
the container. When embedded in OCT, frozen 8 ꢁm sections were prepared with a freezing microtome
at ꢀ20 ꢀ ꢀ30 °C, and used for the immunohistochemical analysis. Briefly, after antigenꢀretrieval (5 min),
sections were stained with monoclonal mouse antibody against AβꢀpE3 and monoclonal antiꢀβꢀamyloid
protein (mouse IgG1 isotype) as first antibody for the detection of pEꢀAβ3ꢀ42 and total Aβs plaques,
respectively, over night at 4 °C. Primary antibodies were visualized with secondary horseradish
peroxidaseꢀconjugated antibodies and the diaminobenzidine reaction in the presence of H2O2. Brain
sections were washed extensively, mounted on slides and cover slipped. Plaques were observed and
photographed under white light or fluorescent microscopy (CX51, Olympus Corporation, Japan). The
photographs were further analyzed by ImageꢀPro Plus 6.0 and Graphpad Prism 5.
Statistical analysis
The results were expressed as the mean ± SD, or mean of means ± SE. The data of the studies in vivo
were also evaluated by oneꢀway analysis of variance (ANOVA) followed by a post hoc test, or tꢀtest. p
< 0.05 was considered to be significant.
Supporting Information
Evaluation of the house building capacity of the AD mice by Nesting test and the predicted
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