Development of coumarine derivatives as potent anti-filovirus entry\ninhibitors targeting viral glycoprotein
Filoviruses, including Ebola virus (EBOV), Marburgvirus (MARV) and Cuevavirus, cause hemorrhagic fevers in humans with up to 90% mortality rates. In the 2014-2016 West Africa Ebola epidemic, there are 15,261 laboratory confirmed cases and 11,325 total deaths. The lack of effective vaccines and medicines for the prevention and treatment of filovirus infection in humans stresses the urgency to develop antiviral therapeutics against filovirus-associated diseases. Our previous study identified a histamine receptor antagonist compound CP19 as an entry inhibitor against both EBOV and MARV. The preliminary structure-activity relationship (SAR) studies of CP19 showed that its piperidine, coumarin and linker were related with its antiviral activities. In this study, we performed detailed SAR studies on these groups with synthesized CP19 derivatives. We discovered that 1) the piperidine group could be optimized with heterocycles, 2) the substitution groups of C3 and C4 of coumarin should be relatively large hydrophobic groups and 3) the linker part should be least substituted. Based on the SAR analysis, we synthesized compound 32 as a potent entry inhibitor of EBOV and MARV (IC50=0.5 \xce\xbcM for EBOV and 1.5 \xce\xbcM for MARV). The mutation studies of Ebola glycoprotein and molecular docking studies showed that the coumarin and its substituted groups of compound 32 binds to the pocket of Ebola glycoprotein in a similar way to the published entry inhibitor compound 118a. However, the carboxamide group of compound 32 does not have strong interaction with N61 as compound 118a does. The coumarin skeleton structure and the binding model of compound 32 elucidated by this study could be utilized to guide further design and optimization of entry inhibitors targeting the filovirus glycoproteins.
Introduction<!>Results<!>The SAR study on the carboxamide group<!>The SAR study on the C-3 of coumarin<!>The SAR study on the linker<!>The SAR study on the C-4 of coumarin<!>The synergy of Rt, R2 and R3 groups<!>The binding model of compound 32 in GP protein.<!>Cell culture<!>Generation of Zaire Ebolavirus glycoprotein mutants.<!>Pseudovirion infection assay and cell viability assay<!>General Methods for Chemistry.<!>Synthesis of derivatives of CP19<!>1-(3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carbox\namide (1)<!>3-benzyl-7-(2-hydroxy-3-(prop-2-yn-1-ylamino)propoxy)-4-methyl-2H-chromen-2-one\n(2)<!>3-benzyl-7-(3-((cyclopropylmethyl)amino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2-one\n(3)<!>3-benzyl-7-(2-hydroxy-3-(phenylamino)propoxy)-4-methyl-2H-chromen-2-one\n(4)<!>3-benzyl-7-(2-hydroxy-3-((2-hydroxy-5-nitrophenyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(5)<!>3-benzyl-7-(3-((4-bromophenyl)amino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2-one\n(6)<!>3-benzyl-7-(3-(benzylamino)-2-hydroxypropoxy)-4-methyl-2H-chromen-2-one\n(7)<!>3-benzyl-7-(2-hydroxy-3-((3-hydroxypropyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(8)<!>3-benzyl-7-(2-hydroxy-3-((6-hydroxyhexyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(9)<!>3-benzyl-7-(2-hydroxy-3-((tetrahydro-2H-pyran-4-yl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(10)<!>3-benzyl-7-(2-hydroxy-3-((2-(pyridin-2-yl)ethyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(11)<!>3-benzyl-7-(2-hydroxy-3-((2-(pyridin-3-yl)ethyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(12)<!>3-benzyl-7-(2-hydroxy-3-((2-(pyridin-4-yl)ethyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(13)<!>3-benzyl-7-(2-hydroxy-3-((thiophen-2-ylmethyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(14)<!>3-benzyl-7-(2-hydroxy-3-(thiazol-2-ylamino)propoxy)-4-methyl-2H-chromen-2-one\n(15)<!>7-(3-((1H-1,2,3-triazol-1-yl)amino)-2-hydroxypropoxy)-3-benzyl-4-methyl-2H-chromen-2-one\n(16)<!>7-(3-((1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)-3-benzyl-4-methyl-2H-chromen-2-one\n(17)<!>3-benzyl-7-(2-hydroxy-3-((6-methylbenzo[d]thiazol-2-yl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(18)<!>1-(2-hydroxy-3-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(19)<!>1-(3-((3-(4-(tert-butyl)benzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(20)<!>1-(3-((3-(4-fluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(21)<!>1-(3-((3-(3-fluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine\n-4-carboxamide (22)<!>1-(3-((3-(4-chlorobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine\n-4-carboxamide (23)<!>1-(3-((3-(4-bromobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(24)<!>1-(3-((3-(4-fluoro-2-methylbenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(25)<!>1-(3-((3-(2,4-difluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperi\ndine-4-carboxamide (26)<!>1-(3-((3-(2-chloro-4-fluorobenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(27)<!>1-(3-((3-(2-bromo-4-methoxybenzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropy\nl)piperidine-4-carboxamide (28)<!>1-(2-hydroxy-3-((4-methyl-3-(3-nitrobenzyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(29)<!>1-(2-hydroxy-3-((4-methyl-2-oxo-3-(4-(trifluoromethyl)benzyl)-2H-chromen-7-yl)oxy)propyl)\npiperidine-4-carboxamide (30)<!>1-(2-hydroxy-3-((4-methyl-2-oxo-3-(2-(trifluoromethyl)benzyl)-2H-chromen-7-yl)oxy)propyl)\npiperidine-4-carboxamide (31)<!>1-(2-hydroxy-3-((4-methyl-2-oxo-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-7-yl)oxy)prop\nyl)piperidine-4-carboxamide (32)<!>Methyl-4-((7-(3-(4-carbamoylpiperidin-1-yl)-2-hydroxypropoxy)-4-methyl-2-oxo-2H-chrome\nn-3-yl)methyl)benzoate (33)<!>1-(2-hydroxy-3-((4-methyl-3-(naphthalen-1-ylmethyl)-2-oxo-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(34)<!>1-(3-((3-(4-(3,5-dimethylisoxazol-4-yl)benzyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(35)<!>1-(3-((3-([1,1\xe2\x80\xb2-biphenyl]-4-ylmethyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(36)<!>1-(3-((3-((4\xe2\x80\xb2-fluoro-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(37)<!>1-(3-((3-((4\xe2\x80\xb2-chloro-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(38)<!>1-(2-hydroxy-3-((3-((2\xe2\x80\xb2-methoxy-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(39)<!>1-(3-((3-((2\xe2\x80\xb2-cyano-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hyd\nroxypropyl)piperidine-4-carboxamide (40)<!>1-(2-hydroxy-3-((4-methyl-2-oxo-3-((4\xe2\x80\xb2-(trifluoromethyl)-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-2H-ch\nromen-7-yl)oxy)propyl)piperidine-4-carboxamide (41)<!>1-(3-((3-((3\xe2\x80\xb2-fluoro-4\xe2\x80\xb2-formyl-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)\noxy)-2-hydroxypropyl)piperidine-4-carboxamide (42)<!>1-(3-((3-((4\xe2\x80\xb2-cyano-3\xe2\x80\xb2-fluoro-[1,1\xe2\x80\xb2-biphenyl]-4-yl)methyl)-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(43)<!>(R)-1-(2-hydroxy-3-((4-methyl-2-oxo-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-7-yl)oxy)p\nropyl)piperidine-4-carboxamide (44)<!>(S)-1-(2-hydroxy-3-((4-methyl-2-oxo-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(45)<!>1-(3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-(2-fluorophenoxy)propyl)piperidine-4-carboxamide\n(46)<!>1-(3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-(4-fluorophenoxy)propyl)piperidine-4-carboxamide\n(47)<!>1-(3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-(4-methoxyphenoxy)propyl)piperidine-4-carboxamide\n(48)<!>1-(3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)-2-(4-nitrophenoxy)propyl)piperidine-4-carboxamide\n(49)<!>1-(2-(4-(2-aminothiazol-4-yl)phenoxy)-3-((3-benzyl-4-methyl-2-oxo-2H-chromen-7-yl)oxy)propyl)piperidine-4-carboxamide\n(50)<!>3-benzyl-7-(2-methoxy-3-((2-(pyridin-4-yl)ethyl)amino)propoxy)-4-methyl-2H-chromen-2-one\n(51)<!>3-benzyl-4-methyl-7-(3-(thiazol-2-ylamino)propoxy)-2H-chromen-2-one\n(52)<!>1-(3-((3-benzyl-4-cyclopropyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-ca\nrboxamide (53)<!>1-(3-((3-benzyl-4-cyclopropyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-ca\nrboxamide(54)<!>1-(3-((4-cyclopropyl-2-oxo-2H-chromen-7-yl)oxy)-2-hydroxypropyl)piperidine-4-carboxamide\n(55)<!>7-(2-hydroxy-3-(thiazol-2-ylamino)propoxy)-4-methyl-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-2-one\n(56)<!>7-(2-hydroxy-3-((2-(pyridin-4-yl)ethyl)amino)propoxy)-4-methyl-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-2-one\n(57)<!>3-([1,1\xe2\x80\xb2-biphenyl]-4-ylmethyl)-7-(2-hydroxy-3-(thiazol-2-ylamino)propoxy)-4-methyl-2H-chromen-2-one\n(58)<!>7-(2-hydroxy-3-(phenethylamino)propoxy)-4-methyl-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-2-one\n(59)<!>Molecular docking<!>Discussion<!>
<p>Filoviruses, consisting of Marburgvirus (MARV), Ebolavirus (EBOV), and
Ceuvavirus, are negative-sense RNA enveloped viruses with filamentous morphology.
The Marburgvirus was the first discovered filovirus as the infectious agent
responsible for a few small outbreaks in Marburg, Germany in 1967, which led to 7
deaths among the 31 reported cases. The Ebolavirus was discovered later in 1976 in
the Democratic Republic of Congo (formerly Zaire). Since then small Ebolavirus
outbreaks occurred sporadically in Africa, during which EBOV typically infected less
than 500 people with high mortality rate (up to 90%). The world witnessed the
biggest Ebolavirus outbreak in 2014-2016 West Africa Ebola epidemics which results
in 15,261 laboratory-confirmed infections and 11,325 reported deaths (74% mortality
rate). The 2018 Kivu Ebola outbreak started 1st August 2018 again in the
Democratic Republic of Congo. Although a few experimental treatment options like
rVSV-ZEBOV Ebola vaccine, Zmapp, GS-5734, REGN monoclonal antibody combination, and
mAb114 have been approved for use in the current outbreak 1–6, they haven't shown dramatically effect against
ebolavirus disease spread. The case figures are still rising and a total of 3444
cases (confirmed and probable) and 2264 deaths (66% mortality rate) have been
reported as of 8th March 2020 7. Novel treatment options, especially small molecule based
new drugs which are more affordable and easier to store and deliver in these
economically disadvantaged countries, are in urgent need to prevent and control
future outbreaks and potential bioterrorism attacks.</p><p>Viral entry has been proved to be a suitable target for antiviral development
since the approval of anti-HIV drug maraviroc and T-20 by FDA, which target HIV-1
entry receptor CCR5 and HIV-1 fusion with the target cell respectively 8. Filovirus entry is mediated by a
single viral glycoprotein (GP), which is composed of two subunits, GP1 and GP2
910. GP1 mediates the initial
attachment of virion to the host cell surface which triggers the viral
internalization via macropinocytosis and trafficking of the virion to late
endosomes/lysosomes where the mucin-like domain and the glycan cap of GP1 are
removed by cathepsin B/L to expose the receptor binding domain (RBD) 11–15. Then the interaction of RBD and Niemann-Pick C1 (NPC1)
protein anchors the virion and induces conformational change of GP2, leading to
viral-endosomal membrane fusion 16–19. The
critical role of viral GP in the indispensable entry process makes it a promising
target protein for developing small molecule entry inhibitors.</p><p>We previously identified compound CP19 as a potent entry
inhibitor against EBOV and MARV from a focused GPCR antagonist library 20. In this study, we performed SAR
studies with 58 derivatives of CP19 on both EBOV and MARV. From these
SAR studies, compound 32 was identified as a potent inhibitor of EBOV
and MARV (IC50=0.5 Î¼M for EBOV and 1.2
Î¼M for MARV). The molecular docking analysis revealed
that compound 32 binds in the same pocket on EBOV glycoprotein as a few
previous reported EBOV entry inhibitors (e.g. toremifene and compound
118a) do 21, 22. However, the mutation studies
showed that compound 32 did not form vital interactions with N61 or
D522, which have been proved to be important for the binding of compound
118a. The coumarin and its hydrophobic substituted groups may bind
to the pocket close to Y517 which is occupied by the hydroxyphenol ring of compound
118a. Our studies showed that the carboxamide group should interact with R164 on the
surface of Ebola glycoprotein and the coumarin skeleton of compound 32
could be further optimized for more potent anti-filovirus lead compounds with better
drug-like properties.</p><!><p>The previous preliminary SAR studies of compound CP19 mainly
focused on the substitutions of its piperidine ring (Figure 1.Cyan oval). The carboxamide group on the piperidine ring was
important for CP19's activity and replacement with alkyl
substitution groups resulted in dramatic antiviral activity loss. These results
indicated that the piperidine and carboxamide group may interact with amino acid
residues possibly via H-bond. Thus, groups with hetero atoms that can form H-bond
interactions would probably show better activity. Moreover, the benzyl substitution
at the C-3 of coumarin (Figure 1.Magentas oval)
was also a key group for the activity because the unsubstituted counterpart was
totally inactive. Based on these results, herein we performed SAR studies on the
carboxamide, C-3 and C-4 (Figure 1.Green oval)
positions of coumarin. We also studied the SAR on the secondary hydroxyl group
(Figure 1.Yellow circle) in the linker,
which has not been reported before.</p><p>We conducted the SAR studies with the general synthesis method (scheme 1).</p><!><p>We first tested if the hydrophobic alkyne (2), cyclopropane
(3), phenyl (4-6) and benzyl (7)
groups could increase the antiviral activity compared with CP19
(1). Only benzyl (7) group displayed comparable
anti-filovirus activities and SI values than CP19. This result
provided partial support for our postulation that the nitrogen atom of
carboxamide could form H-bond with residues in the active site. We then tested
if the oxygen atom in the alcohol (8, 9) and cyclic ether
substitution groups (10) could form the desired interactions as
carboxamide dose. However, these modifications did not significantly improve the
activity. Then compounds with hetero cycles (11-18) were
synthesized and most of them exhibited slightly better antiviral activities but
more cytotoxicity in A549 cell line when compared with CP19. The
compounds with ethyl-o-pyridine (13) showed the
best inhibitory activity on EBOV entry and best SI in this series of compounds.
However, other larger hetero cycles (17-18) did not increase the
activity, which indicated that the binding pocket for this part is relatively
small or has strict requirement for ligands.</p><p>
          
</p><!><p>Previous reported data suggested that the benzyl groups on the C-3 of
coumarin was vital for the activity. Consistent with this result, the coumarin
without any substitutions (19) lost most of the activity. Thereof,
the effects of large aromatic groups with different substitutions on this site
were evaluated. First, we synthesized compounds with substituted benzyl rings
(20-33). Basically, most the halogen substitutions
(21-27) afforded better antiviral activities than
CP19 and compound 21 and 23 showed
good SI. Moreover, most other larger phenyl groups with strong
electro-withdrawing substitutions (28-32) also displayed improved
activity. Compound 32 showed the strongest antiviral activity
against EBOV. This indicated that the binding pocket for this part could be
relatively large and thus large aromatic substitution groups would benefit the
binding. However, these modifications did not improve the cytotoxicity
significantly, indicating that the metabolism properties of halogen and strong
electro-withdrawing groups on the aromatic ring should be optimized.</p><p>To further exploit the possibly large binding pocket for this part, we
synthesized compounds with larger aromatic groups such as naphthalene
(34) and phenyl-heterocycle group (35). The
naphthalene group displayed similar IC50 and SI value as
CP19. We then increased the size of substitution by installing
bi-phenyl groups (36-43). Compound 36 showed better antiviral
activities for EBOV and MARV with better SI values than CP19. These
results showed that the binding pocket could accommodate ligands with groups as
large as bi-phenyl.</p><p>
          
</p><!><p>The SAR of the linker in CP19 has not been studied. Herein,
we focused on the substitutions of the secondary hydroxyl group of the linker.
We first studied whether the stereochemistry of the secondary carbon could
affect the activity. The two isomers of compound 32 (44,
45) showed little difference in activity and cytotoxicity, which
indicated that this secondary hydroxyl group had not stereospecific interactions
in the binding site. So we did not consider stereochemistry in further SAR
studies on the linker. The phenyl ring with EWG and EDG (46-49)
caused obvious loss of activity compared with CP19, which indicated
that the aromatic substitution groups in this site may not be favored. To test
whether the hydroxyl group itself may have important El-bond at the active site,
we installed a group with multiple heteroatoms (50), but compound
50 did not afford better activity. Then we proposed that the
binding pocket for the linker was small and thus had no vital interaction with
the linker part. Compound 51 with smallest methyl group afforded
less anti-viral activity than compound 13. Then we removed the
hydroxyl group on the linker (52) and it showed lower antiviral
activity and SI compared with compound 15. These results indicated
that the binding site of the linker should be tunnel-like and so the linker
should not have large substitution groups.</p><p>
          
</p><!><p>The previous study showed that the substitutions on the C-4 of coumarin
could reduce the cytotoxicity but had little effect on the antiviral activity of
CP19. In this study, the cyclopropane (53) and
phenyl (54) groups on this site were evaluated but they did not
display better activity and cytotoxicity. Considering the steric hindrance
between the substitution groups on the sterically adjacent C-3 and C-4, we
removed the benzyl group on C-3 (55) but this compound lost most of
the antiviral activity. These results confirmed that R3 group had vital roles
for the activity its functions on the binding pocket and could not be replaced
by the adjacent R4 group.</p><p>
          
</p><!><p>To test whether the synergic effects could be acquired from the groups
of R1, R2 and R3, we accordingly synthesized
compounds with the substitution groups that had afforded best activity or had
reduced toxicity in our SAR studies (56-59). Among them, compound
(56) afforded strong activity and low cytotoxicity. The
synergic effect was not as high as expected (SI EBOV =66 for compound
56 and SIEBOV =29 for CP19).
Considering that the linker group took minor roles for the activity, we proposed
that the C3 on coumarin and the terminal carboxamide groups
influenced each other and cross talk between these groups should be
considered.</p><p>
          
</p><!><p>Compound CP19 has been shown to target a late step of Ebola
virus entry, very likely via binding to the GP to prevent GP-mediated fusion
event. Its analog compound 32 has a similar structure to compound
118a, which has been reported to be able to bind EBOV GP and
inhibit virus entry. Thus, based on the crystal structure of EBOV GP complexed
with compound 118a, we located a few key amino acid residues which
might be involved in the GP-compound 32 interaction. Then we
evaluated the inhibitory activities of compound 32 against a few
EBOV pseudovirions carrying mutated glycoprotein. Compound 32 did
not show obvious loss of activity against EBOV pseudovirions carrying
glycoprotein mutant N61A, N61V, N61D (Figure
2A) or D522A (Figure 2B). These
data suggest that the carboxamide piperidine group of compound 32
does not have key interaction with N61 or D522 of Ebola GP, which is
substantially different to the interaction profile of compound
118a, even though these two compounds have the same carboxamide
group. Moreover, compound 32 showed ~3 fold lower activity
against the infection of pseudovirion carrying Y517S mutant compared with the WT
pseudovirions (Figure 2C). It has been
shown that methylpyrazole group of compound 118a interacts with
Y517 via ring stacking and chlorophenyl ring of compound 118a binds
to a hydrophobic pocket formed by V106, A101, L515 and Y517. These data
indicates that the trifluomethoxybenzyl group of compound 32 binds
to the hydrophobic pocket close to Y517 via hydrophobic interaction or/and ring
stacking interaction like compound 118a.</p><p>To further elucidate the binding mechanism of compound 32,
we performed molecular docking simulations with the crystal structure of Ebola
glycoprotein complexed with compound 118a. The co-crystal structure
(PDB: 6HRO) was prepared by removing all ligands and unrelated water molecules
and the whole protein was used as binding pocket for compound 32.
The docking results showed that the model of compound 32 with the
highest binding affinity score bound to the same pocket as the reported molecule I of compound 118a22 (Figure 3A). Thus
this pocket was chosen for further docking studies. As shown in Figure 3B, except the carboxamide part, compound
32 has a similar binding pose to that of compound 118a: (1) The
linker moiety of compound 32 takes almost the same pose as the ethyl moiety of
118a. Although the linker of compound 32 is one carbon-carbon bond longer than
that of 118a and possesses a hydroxyl group that 118a does not have, neither the
linker chain nor the hydroxyl group produces key interactions with EBOV GP.
Moreover, the docking results of compound 32 showed that the hydroxyl groups is
in the "neck" of the tunnel-like binding pocket, which is
consistent with the SAR results that the substitutions on the hydroxyl group
reduced activity (Figure
S1). (2) The coumarin moiety of compound 32 occupies similar space as
the phenol group of 118a. Due to the lack of ring structure between coumarin and
benzyl group on C-3, compound 32 does not form ring stacking with Y517 as the
methylpyrazole group of compound 118a does. The oxygen atoms of the lactone
structure of compound 32 does not form hydrogen bond with GP protein, which is
comparable with the fact that the phenol group of 118a is not involved in key
interactions with GP protein. (3) The trifluomethoxybenzyl group of compound 32
also has a very similar pose as the o-chlorobenzene of 118a. However, because
lack of the σ-hole in the fluorine atom, compound 32 (Figure 3C) cannot form the critical halogen bond with
G67 which was an important contributor for the high efficacy of 118a (Figure 3D). In the SAR studies of this part,
we synthesized compounds (23, 24 27 and 28) which had halogen substitutions on
the benzene, but none of them increased antiviral activities. We also performed
docking studies of compound 23 and 24 with 4-chlorobenzyl and 4-bromobenzyl
groups respectively, but these two compounds did not form halogen bond with G67
in the docking results. These results are consistent with the halogen-bond
requirement that the carbonyl group of amino acid residues must strictly point
to the σ-hole in the halogen atom of the ligand to form the electrostatic
interactions. Moreover, the docking results show that the carboxamide group of
compound 32 forms H-bond with R164 but does not interact with N61 or R587 as
compound 118a does, probably due to that glycerol linker of compound 32 is
longer than the ethyl linker of compound 118a, which may afford more freedom for
carboxamide and cause more entropy loss in binding process. Finally, we compared
the binding mechanism of compound 32 in glycoprotein of Ebola virus (PDB: 6HRO)
and Marburg Virus (PDB: 6BP2) with docking studies. Compound 32 is shown to bind
in the same binding pocket in glycoprotein of Marburg virus as it does in Ebola
GP, but in an obviously different pose. The oxygen atom of coumarine ring may
form hydrogen bond with Gly547 and the nitrogen atom of piperidine ring may form
hydrogen bind with Glu45 of Marburg GP (Figure S2).</p><!><p>Human embryonic kidney cells (239T, ATCC# CRL-1573), human lung
epithelial cells (A549, ATCC#CCL185) were cultured in DMEM (Cellgro, Manassas,
VA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO,
USA), 100 Î¼g/mL of streptomycin and 100 units of penicillin (Invitrogen,
Carlsbad, CA, USA).</p><!><p>Plasmid containing the Zaire Ebolavirus glycoprotein gene was mutated
using the Agilent Technologies QuickChange Lightning Site-Directed Mutagenesis
Kit (Agilent, Santa Clara, CA, USA). Primer pairs were designed using
QuickChange Primer Design program and purchased through Sigma (Sigma, St. Louis,
MO, USA). Plasmids containing GP mutations were made following
manufacturer's instructions and were sequenced to ensure the correct
mutation.</p><!><p>The HIV/MARV and HIV/EBOV pseudovirions were generated by transient
co-transfection of replication-defective HIV vector (pNL4–3.Luc.R-E-) and
plasmid encoding MARV GP or EBOV GP into 293T cells, using a polyethylenimine
(PEI)-based transfection protocol. Six hours after transfection, cells were
washed with phosphate-buffered saline (PBS) and 20 mL of fresh media was added
to each plate (150 mm). Twenty-four hours post-transfection, the supernatants
were collected and filtered through 0.45 Î¼m pore size filter (Nalgene,
Rochester, NY, USA). The pseudovirion stocks were stored at 4 °C prior to
use.</p><p>Low-passage A549 cells were seeded at the density of 5000/well in
96-well plates 24 h before infection. The synthesized compounds were prepared as
3-fold serial dilutions and mixed with HIV/MARV or HIV/EBOV pseudotyped virus or
fresh media for IC50 and CC50 evaluation. The A549 cells
were incubated with 100 gL/well of above-mentioned mixtures for 48 h. The
luciferase activity of infected A549 cells was quantified using the neolite
Reporter Gene Assay System (PerkinElmer, Waltham, MA, USA). The cell viability
was measured with the CellTiter-Glo kit (Promega, Madison, WI, USA). Cells
incubated with virus or medium alone was used as the negative controls and the
signals from these cells were used for data normalization. IC50 and
CC50 values were determined by fitting the dose-response curves
with logistic regression in GraphPad.</p><!><p>All starting materials were commercially available and used without
further purification. All reactions were carried out with the use of
standard techniques under an inert atmosphere (Ar or N2). NMR spectra were
generated on a Bruker 500 or 400MHz instrument and obtained as CDCl3 or
DMSO-d6 solutions (reported in ppm), using CDCl3 as the reference standard
(7.26 ppm) or DMSO-d6 (2.50 ppm). Mass spectral data (ESI) were gathered on
Thermofisher LCQ or QE Mass spectrometry. HPLC (Agilent Prostar 218 or 1200)
was employed for purity determination, using the following method: Eclipse
XDB C18 column, 5 gm, 4.6 mm*150 mm, column temperature 40 °C;
solvent A: H2O or 0.1 formic acid in H2O; solvent B:
methanol or ACN; gradient of 40–70% B (0–10 min),
70–90% B (10–15 min), 90–40% B (15–20 min), with
minor adjustoin according to the retention time of the target compounds;
flow rate of 1.5 mL/min. Compound purity was determined by high pressure
liquid chromatography (HPLC) with a confirming purity of ⩾95% for all
of the final biologically tested compounds.</p><!><p>The syntheses of derivatives of CP 19 in this study were performed
according to the methods in Scheme 2 with
minor modifications as described in each compounds.</p><p>Reagents and conditions:(a) Ethyl acetoacetate. Benzyl bromide,
CH3ONa, Methonal, RT→68°C, 0.5h; (b) Resorcino,
PPA, 65°C, 4h, 67%; (c) Epichlorohydrin, K2CO3,
TBAB, 80°C 2.3h, 81%;(d)Organic amine, AcNMe2, 50 °C,
24h, 85%.</p><p>
          
</p><p>Reagents and conditions:(a) Ethyl acetoacetate,
4-(Trifluoromethoxy)benzyl bromide, CH3ONa, Methonab
RT→68°C, 0.5 h; (b) Resorcino, PPA, 65°C, 4 h, 67%; (c)
(R)-(−)-Epichlorohydrin or (S)-(+)-Epichlorohydrin,
K2CO3, TBAB, 80°C, reflux 2.3 h, 53%;(d)
Hexahydroisonicotinamide, AcNMe2, 50°C, 24 h, 89%.</p><!><p>A mixture of ethyl acetoacetate (5 mmol) and CH3ONa (1
eq) in MeOH (4 rnL) was stirred at room temperature for 10 minutes. Then,
benzyl bromide derivatives (1.1 eq) in 5 mL MeOH was added dropwise when the
reaction solution was slightly boiling. The reaction mixture was continued
to heat under reflux until it was almost neutral. After cooling, the
reaction solution was filtered off and concentrated by vacuum to give ethyl
acetoacetate derivatives.</p><p>To a stirred solution of the above mentioned products in PPA 3 g,
resorcino (1 eq) was added. The reaction mixture was stirred at 65 C for 4
h. The mixture solution had to rest overnight, then diluted with 100 mL
water. The precipitated solid was filtered off, washed with water to give
the crude product. The crude product was purified by silica gel
chromatography using cyclohexane-ethyl acetate as eluate to give
7-hydroxy-chromen-2-one derivatives (67 %).</p><p>A mixture of the above products, epichlorohydrin (1-3 mL), potassium
carbonate (2 eq) and TBAB (1 eq) was stirred at 80 C for 2.3 h. Then the
mixture was extracted with ethyl acetate, washed with water. The organic
phase was collected and die solvent was removed under vacuum. The product
was purified by silica gel chromatography using cyclohexane-ethyl acetate as
eluate to give 4-methyl-7-(oxiran-2-yhnethoxy)-chromen-2-one derivatives(81
%).</p><p>The above mentioned products was added to a stirred solution of
organic amine (2.5 eq) in 1-2 mL dimentylacetamide then the reaction mixture
was stirred at 50 C for 24 h. The reaction solution was allowed to cool to
room temperature and concentrated by vacuum. The residue was dissolved in
acetone, absorbed onto silica and purified by flash column chromategraphy
using dichloromethane-methanol as eluate to give compound NO.1
(85%).</p><p>1H NMR (500 MHz, DMSO) Î´ 7.71 (d,
J = 9.5 Hz, 1H), 7.25 (t, J = 7.4 Hz,
3H), 7.17 (dd, J = 17.2, 8.6 Hz, 4H), 6.99 – 6.90
(m, 2H), 4.11 – 4.01 (m, 1H), 3.97 – 3.94 (m, 2H), 3.93 (s,
2H), 2.90 (d, J = 11.2 Hz, 1H), 2.84 (d, J
= 11.2 Hz, 1H), 2.40 (d, J = 6.6 Hz, 3H), 2.33 (dd,
J = 12.6, 5.8 Hz, 1H), 2.08 – 1.86 (m, 4H), 1.61
(d, J = 2.4 Hz, 2H), 1.57 – 1.46 (m, 2H). ESI-MS
m/z: 451.07 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 178.42 (s), 163.11 (s),
155.18 (s), 150.19 (s), 141.16 (s), 130.25 (s), 129.86 (s), 128.42 (s),
127.89 (s), 122.82 (s), 115.34 (s), 114.31 (s), 111.37 (s), 102.89 (s),
73.61 (s), 68.27 (s), 62.97 (s), 55.50 (d, J = 9.7 Hz),
43.47 (s), 33.97 (s), 30.45 (s), 17.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.:H NMR (500 MHz, DMSO)
δ 7.71 (d, J = 9.6 Hz, 1H), 7.27 – 7.22 (m,
2H), 7.20 (d, J = 7.0 Hz, 2H), 7.16 (d, J
= 7.1 Hz, 1H), 6.98 – 6.92 (m, 2H), 4.07 (dd, J =
10.0, 4.1 Hz, 1H), 3.98 – 3.93 (m, 2H), 3.93 (s, 2H), 3.75 (dd,
J = 5.7, 2.4 Hz, 1H), 3.08 (s, 1H), 3.04 (t,
J = 2.4 Hz, 1H), 2.93 (s, 1H), 2.77 (s, 1H), 2.67
– 2.64 (m, 2H), 2.62 (s, 1H), 2.41 (s, 3H), 1.97 (s, 1H), 1.94 (s,
1H). ESI-MS m/z: 377.16 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.02 (s), 155.17 (s),
150.22 (s), 141.15 (s), 130.26 (s), 129.85 (s), 128.43 (s), 127.90 (s),
122.84 (s), 115.36 (s), 114.36 (s), 102.86 (s), 84.66 (s), 75.63 (s), 74.85
(s), 73.20 (s), 70.17 (s), 69.75 (s), 67.05 (s), 52.74 (s), 48.37 (s), 39.46
(s), 33.96 (s), 31.62 (s), 17.2 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
δ 7.28 – 7.14 (m, 6H), 6.95 (dd, J = 6.0, 2.4
Hz, 2H), 4.07 (dd, J = 10.0, 4.3 Hz, 1H), 3.97 (dd,
J = 10.0, 6.1 Hz, 1H), 3.93 (s, 2H), 3.90 (d,
J = 4.6 Hz, 1H), 2.70 (d, J = 4.9 Hz,
1H), 2.65 (d, J = 6.9 Hz, 1H), 2.44 (s, 1H), 2.41 (s, 3H),
2.33 (d, J = 12.6 Hz, 1H), 0.39 (ddd, J =
8.0, 5.6, 4.0 Hz, 2H), 0.09 (dt, J = 15.4, 7.6 Hz, 2H).
ESI-MS m/z: 394.23 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.02 (s), 155.18 (s),
150.19 (s), 141.16 (s), 130.25 (s), 129.86 (s), 128.43 (s), 127.89 (s),
122.85 (s), 115.36 (s), 114.35 (s), 102.86 (s), 73.22 (s), 69.60 (s), 55.88
(s), 53.65 (s), 33.97 (s), 17.02 (s), 12.64 (s), 5.08 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
δ 7.74 – 7.68 (m, 1H), 7.26 – 7.14 (m, 5H), 7.04 (dd,
J = 8.4, 7.4 Hz, 2H), 6.97 (dd, J =
6.9, 2.4 Hz, 2H), 6.60 (d, J = 7.7 Hz, 2H), 6.50 (t,
J = 7.2 Hz, 1H), 4.12 (dd, J = 9.8,
3.8 Hz, 1H), 4.06 – 3.97 (m, 2H), 3.93 (s, 2H), 3.24 – 3.18
(m, 1H), 3.13 – 3.04 (m, 1H), 2.41 (s, 3H), 1.40 – 1.18 (m,
2H). ESI-MS m/z: 415.97 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.04 (d,
J = 8.2 Hz), 155.17 (s), 150.60 (s), 150.19 (s), 141.15
(s), 130.72 (s), 130.26 (s), 129.86 (s), 128.44 (s), 127.90 (s), 122.87 (s),
117.57 (s), 115.39 (s), 114.36 (s), 113.96 (s), 102.87 (s), 72.88 (s), 69.22
(s), 47.91 (s), 33.97 (s), 17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 9.3 Hz, 1H), 7.44 (dd,
J = 8.6, 2.7 Hz, 1H), 7.31 (d, J = 2.7
Hz, 1H), 7.27 – 7.22 (m, 2H), 7.20 (d, J = 7.1 Hz,
2H), 7.16 (t, J = 7.1 Hz, 1H), 7.01 – 6.96 (m, 2H),
6.78 (d, J = 8.6 Hz, 1H), 4.14 – 4.10 (m, 1H), 4.09
– 4.05 (m, 2H), 3.93 (s, 2H), 3.15 (d, J = 5.0 Hz,
3H), 2.93 (s, 1H), 2.77 (s, 1H), 2.41 (s, 3H). ESI-MS m/z: 475.64 [M+H]
−.</p><p>13C NMR (101 MHz, DMSO) δ 163.35 – 163.11
(m), 162.96 (d, J = 20.6 Hz), 155.16 (s), 152.85 (s),
150.17 (s), 142.34 (s), 141.14 (s), 139.70 (s), 130.25 (s), 129.85 (s),
128.43 (s), 127.89 (s), 122.90 (s), 115.26 (d, J = 38.6
Hz), 114.16 (d, J = 31.8 Hz), 113.99 – 113.71 (m),
105.03 (s), 102.88 (s), 72.82 (s), 68.96 (s), 47.64 (s), 33.97 (s), 17.01
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.73 – 7.69 (m, 1H), 7.25 (t, J = 7.4 Hz,
2H), 7.20 (d, J = 7.1 Hz, 2H), 7.18 – 7.15 (m, 3H),
6.97 (dd, J = 5.7, 2.4 Hz, 2H), 6.57 (d, J
= 8.9 Hz, 2H), 4.09 (dd, J = 10.0, 4.1 Hz, 1H), 4.03 (dd,
J = 10.0, 5.9 Hz, 1H), 3.97 (dd, J =
10.3, 5.2 Hz, 1H), 3.93 (s, 2H), 3.20 (dd, J = 12.8, 6.4
Hz, 1H), 3.07 (dd, J = 12.5, 6.4 Hz, 1H), 2.41 (s, 3H).
ESI-MS m/z: 494.20 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.01 (d,
J = 12.6 Hz), 155.17 (s), 150.19 (s), 149.93 (s),
141.15 (s), 133.18 (s), 130.26 (s), 129.86 (s), 128.44 (s), 127.90 (s),
122.89 (s), 115.86 (s), 115.42 (s), 114.35 (s), 108.03 (s), 102.89 (s),
72.74 (s), 69.12 (s), 47.83 (s), 33.97 (s), 17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
δ 7.70 (d, J = 9.6 Hz, 1H), 7.34 – 7.17 (m,
10H), 6.94 (dd, J = 7.2, 2.4 Hz, 2H), 4.09 (dd,
J = 9.9, 4.1 Hz, 1H), 3.97 (dd, J =
9.9, 6.2 Hz, 2H), 3.93 (s, 2H), 3.71 (s, 2H), 2.61 (ddd, J
= 18.3, 11.9, 5.8 Hz, 2H), 2.41 (s, 3H), 2.32 (d, J = 14.1
Hz, 1H). ESI-MS m/z: 430.24 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.06 (s), 155.18 (s),
150.20 (s), 142.62 (s), 141.17 (s), 130.25 (s), 130.08 – 129.47 (m),
127.89 (s), 122.83 (s), 115.33 (s), 114.37 (s), 102.84 (s), 73.24 (s), 69.89
(s), 54.89 (s), 53.40 (s), 33.97 (s), 17.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.71 (s, 1H), 7.24 (d, J = 7.2 Hz, 3H), 7.21 (s,
3H), 7.17 (d, J = 7.1 Hz, 2H), 6.95 (d, J
= 2.5 Hz, 1H), 4.07 (dd, J = 9.6, 3.8 Hz, 2H), 4.00
– 3.96 (m, 2H), 3.93 (s, 3H), 3.45 (t, J = 6.2 Hz,
4H), 2.93 (s, 1H), 2.81 – 2.77 (m, 1H), 2.77 (s, 1H), 2.69 (dd,
J = 9.2, 4.7 Hz, 4H), 2.40 (s, 3H), 1.94 (s, 1H), 1.60
(d, J = 6.7 Hz, 2H), 1.22 (s, 2H). ESI-MS m/z: 397.90 [M+H]
+</p><p>13C NMR (101 MHz, DMSO) Î´ 162.99 (d,
J = 16.5 Hz), 155.16 (s), 150.19 (s), 141.15 (s),
130.26 (s), 129.86 (s), 128.44 (s), 127.90 (s), 122.89 (s), 115.41 (s),
114.34 (s), 102.88 (s), 72.99 (s), 68.88 (s), 60.91 (s), 53.38 (s), 48.27
(s), 33.97 (s), 33.30 (s), 17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 9.6 Hz, 1H), 7.24 (d,
J = 7.3 Hz, 2H), 7.20 (d, J = 7.0 Hz,
2H), 7.17 (d, J = 7.1 Hz, 1H), 6.95 (dd, J
= 6.1, 2.5 Hz, 2H), 4.06 (dd, J = 10.0, 4.2 Hz, 1H), 3.95
(dd, J = 10.1, 6.3 Hz, 1H), 3.93 (s, 2H), 3.88 –
3.82 (m, 1H), 3.37 (d, J = 6.5 Hz, 2H), 2.64 (d,
J =5.2 Hz, 1H), 2.62 (d, J = 5.0 Hz,
1H), 2.57 (s, 1H), 2.56 (s, 1H), 2.40 (d, J = 9.0 Hz, 3H),
2.22 (s, 1H), 1.39 – 1.36 (m, 5H), 1.26 – 1.24 (m, 4H). ESI-MS
m/z: 439.98 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.07 (s), 155.18 (s),
150.20 (s), 141.16 (s), 130.25 (s), 129.85 (s), 128.41 (s), 127.89 (s),
122.82 (s), 115.32 (s), 114.34 (s), 102.84 (s), 73.28 (s), 69.80 (s), 62.49
(d, J = 6.9 Hz), 54.12 (s), 51.30 (s), 34.68 – 33.89
(m), 31.47 (s), 28.58 (s), 27.34 (s), 27.10 (s), 17.2 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.71 (d, J = 9.2 Hz, 1H), 7.31 – 7.11 (m,
5H), 6.96 (d, J = 7.9 Hz, 2H), 4.7 (dd, J
= 10.0, 4.2 Hz, 1H), 3.97 (dd, J = 10.1, 6.1 Hz, 2H), 3.93
(s, 2H), 3.85 (d, J = 4.9 Hz, 1H), 3.81 – 3.74 (m,
2H), 3.27 – 3.17 (m, 2H), 2.70 (dd, J = 11.8, 5.1
Hz, 1H), 2.64 – 2.54 (m, 2H), 2.40 (d, J = 7.9 Hz,
3H), 1.73 (dd, J = 8.5, 4.5 Hz, 2H), 1.38 (s, 1H), 1.21
(dd, J = 18.3, 11.5 Hz, 2H). ESI-MS m/z: 424.21 [M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.05 (s), 155.18 (s),
150.18 (s), 141.16 (s), 130.25 (s), 129.85 (s), 128.41 (s), 127.89 (s),
122.83 (s), 115.33 (s), 114.36 (s), 102.84 (s), 73.24 (s), 70.08 (s), 67.64
(s), 55.23 (s), 50.58 (s), 35.06 (s), 33.97 (s), 17.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.47 (d, J = 4.8 Hz, 1H), 7.71 (t,
J = 7.1 Hz, 2H), 7.40 (d, J = 7.8 Hz,
1H), 7.27 (s, 1H), 7.24 (d, J = 7.3 Hz, 2H), 7.20 (d,
J = 7.0 Hz, 3H), 7.17 (d, J = 7.2 Hz,
1H), 6.95 (d, J = 7.9 Hz, 2H), 4.09 (dd, J
= 10.0, 4.2 Hz, 1H), 3.98 (dd, J = 9.9, 6.2 Hz, 1H), 3.93
(s, 3H), 3.81 (d, J = 2.2 Hz, 2H), 2.68 (dd,
J = 11.8, 5.1 Hz, 1H), 2.61 (dd, J =
11.9, 6.5 Hz, 1H), 2.41 (s, 3H), 1.38 (s, 1H). ESI-MS m/z: 430.92 [M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.07 (d,
J = 5.4 Hz), 162.02 (s), 155.18 (s), 150.60 (s), 150.20
(s), 141.16 (s), 138.28 (s), 130.26 (s), 129.86 (s), 128.42 (s), 127.89 (s),
123.67 (d, J = 7.3 Hz), 122.83 (s), 115.34 (s), 114.37 (s),
102.85 (s), 73.17 (s), 69.93 (s), 56.52 (s), 53.64 (s), 33.97 (s), 17.2
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.43 (d, J = 1.8 Hz, 1H), 8.37 (dd,
J = 4.7, 1.5 Hz, 1H), 7.73 – 7.68 (m, 1H), 7.63
(d, J = 7.8 Hz, 1H), 7.28 – 7.26 (m, 1H), 7.24 (d,
J = 7.3 Hz, 2H), 7.21 (d, J = 7.0 Hz,
2H), 7.16 (t, J = 7.1 Hz, 1H), 6.94 (dd, J
= 5.8, 2.4 Hz, 2H), 4.04 (dd, J = 10.0, 4.3 Hz, 1H), 3.95
(d, J = 6.2 Hz, 1H), 3.93 (s, 2H), 3.86 (d,
J = 5.2 Hz, 1H), 2.76 (d, J = 6.2 Hz,
1H), 2.70 (dd, J = 12.0, 5.7 Hz, 3H), 2.66 (s, 1H), 2.63
– 2.57 (m, 1H), 2.41 (s, 3H). ESI-MS m/z: 444.83 [M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.06 (d,
J = 5.7 Hz), 155.18 (s), 151.70 (s), 150.19 (s), 148.96
(s), 141.17 (s), 137.86 (d, J = 16.7 Hz), 130.26 (s),
129.86 (s), 128.41 (s), 127.89 (s), 125.13 (s), 122.84 (s), 115.34 (s),
114.33 (s), 102.84 (s), 73.22 (s), 69.86 (s), 53.86 (s), 52.49 (s), 34.76
(s), 33.97 (s), 17.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.45 (d, J = 5.9 Hz, 1H), 8.40 (d,
J = 5.9 Hz, 2H), 7.70 (d, J = 9.6 Hz,
1H), 7.27 (s, 1H), 7.25 (s, 1H), 7.23 (s, 2H), 7.22 (d, J =
4.9 Hz, 3H), 7.20 (s, 1H), 7.16 (t, J = 7.1 Hz, 1H), 6.95
(s, 2H), 6.93 (d, J = 2.5 Hz, 1H), 5.01 (d,
J = 4.9 Hz, 1H), 4.05 (d, J = 4.3 Hz,
1H), 4.3 (d, J = 4.3 Hz, 1H), 3.95 (d, J =
6.1 Hz, 1H), 3.93 (d, J = 4.6 Hz, 2H), 3.86 (d,
J = 5.3 Hz, 1H), 3.15 (d, J = 5.2 Hz,
1H), 2.78 (d, J = 6.6 Hz, 2H), 2.72 (d, J
= 6.9 Hz, 2H), 2.69 (s, 1H), 2.66 (s, 1H), 2.60 (dd, J =
11.9, 6.6 Hz, 1H), 2.41 (s, 3H). ESI-MS m/z: 444.99[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.68 (d, J = 7.9 Hz),
153.80 (s), 150.50 – 150.07 (m), 149.87 (d, J = 21.1 Hz), 148.83 (s),
139.79 (s), 128.88 (s), 128.48 (s), 127.05 (s), 126.52 (s), 124.70 (s),
121.47 (s), 113.97 (s), 112.97 (s), 101.47 (s), 71.82 (s), 68.47 (s), 52.44
(s), 50.33 (s), 35.54 (s), 32.60 (s), 15.66 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
Î´ 7.72 (d, J = 8.7 Hz, 1H), 7.35 (dd,
J = 4.7, 1.5 Hz, 1H), 7.29 (s, 1H), 7.27 (s, 1H), 7.25
(s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 7.17 (s, 1H), 7.15 (s, 1H), 6.96 (d,
J = 3.4 Hz, 2H), 6.94 (s, 2H), 4.09 (dd,
J = 9.8, 3.9 Hz, 1H), 3.98 (t, J = 4.9
Hz, 2H), 3.94 (s, 2H), 3.92 (s, 2H), 2.73 – 2.62 (m, 2H), 2.42 (s,
3H). ESI-MS m/z: 435.94 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.06 (s), 162.86 (s),
155.16 (s), 152.86 (s), 150.17 (s), 142.34 (s), 141.14 (s), 139.70 (s),
130.25 (s), 129.85 (s), 128.43 (s), 127.89 (s), 122.90 (s), 115.45 (s),
115.6 (s), 114.32 (s), 114.00 (s), 105.03 (s), 102.88 (s), 72.82 (s), 68.96
(s), 50.43 (s), 47.64 (s), 33.97 (s), 17.01 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.72 (d, J = 9.5 Hz, 1H), 7.20 (qd,
J = 14.6, 7.4 Hz, 6H), 6.98 – 6.92 (m, 2H), 6.73
(d, J = 4.9 Hz, 1H), 4.18 – 4.11 (m, 1H), 3.99 (dt,
J = 14.3, 5.1 Hz, 2H), 3.93 (s, 2H), 3.89 (d,
J = 4.2 Hz, 1H), 3.86 (s, 1H), 3.74 (dd,
J = 14.0, 7.1 Hz, 1H), 2.93 (s, 1H), 2.77 (s, 1H), 2.41
(s, 3H). ESI-MS m/z: 423.13732[M+H] +, delta (ppm)
<0.04.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.67 (s), 161.35 (s),
153.76 (s), 148.81 (s), 139.76 (s), 130.34 (s), 128.88 (s), 128.48 (s),
127.12 (s), 126.53 (s), 121.62 (s), 114.18 (s), 112.95 (s), 101.52 (s), 71.0
(s), 67.23 (s), 49.85 (s), 32.61 (s), 15.68 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.89 (s, 1H), 7.73 (d, J = 8.8 Hz, 1H), 7.27 (s,
1H), 7.24 (d, J = 7.3 Hz, 2H), 7.20 (d, J
= 7.0 Hz, 2H), 7.17 (d, J = 7.1 Hz, 1H), 6.99 (t,
J = 2.8 Hz, 2H), 6.97 (s, 1H), 5.92 (s, 2H), 5.65 (d,
J = 4.4 Hz, 1H), 4.08 (s, 1H), 4.05 (d,
J = 4.2 Hz, 1H), 4.02 (d, J = 4.1 Hz,
1H), 3.98 (dd, J = 6.3, 3.6 Hz, 1H), 3.96 – 3.95 (m,
1H), 3.93 (s, 2H), 3.85 (dd, J = 14.5, 7.9 Hz, 1H), 2.90
(dd, J = 14.5, 7.2 Hz, 1H), 2.42 (s, 3H). ESI-MS m/z:
407.12 [M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.05 (s), 162.66 (s),
155.14 (s), 150.18 (s), 142.56 (s), 141.12 (s), 130.26 (s), 129.85 (s),
128.49 (s), 127.90 (s), 123.02 (s), 115.60 (s), 114.35 (s), 102.92 (s),
72.11 (s), 68.88 (s), 50.42 (s), 47.46 (s), 33.97 (s), 17.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.73 (d, J = 9.5 Hz, 1H), 7.28 – 7.23 (m,
2H), 7.21 (d, J = 7.1 Hz, 2H), 7.16 (dd, J
= 7.3, 3.6 Hz, 2H), 7.11 (d, J = 7.7 Hz, 1H), 6.99 (d,
J = 2.4 Hz, 1H), 6.97 (s, 1H), 6.90 (t,
J = 7.1 Hz, 1H), 6.82 (t, J = 7.2 Hz,
1H), 6.23 (s, 2H), 5.60 (d, J = 5.3 Hz, 1H), 4.17 (d,
J = 8.2 Hz, 1H), 4.12 (d, J = 4.2 Hz,
1H), 4.08 (dd, J = 10.0, 3.9 Hz, 1H), 4.06 – 4.00
(m, 2H), 3.93 (s, 2H), 2.42 (s, 3H). ESI-MS m/z: 455.93 [M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.06 (s), 162.73 (s),
157.14 (s), 155.14 (s), 150.19 (s), 144.48 (s), 141.14 (s), 136.71 (s),
130.26 (s), 129.87 (s), 128.50 (s), 127.90 (s), 122.98 (s), 122.11 (s),
119.90 (s), 116.59 (s), 115.53 (s), 114.33 (s), 109.72 (s), 102.89 (s),
72.11 (s), 69.54 (s), 46.74 (s), 33.98 (s), 17.04 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.70 (d, J = 8.9 Hz, 1H), 7.25 (t,
J = 7.5 Hz, 2H), 7.21 – 7.13 (m, 4H), 7.1 (d,
J = 8.2 Hz, 1H), 6.97 – 6.91 (m, 2H), 6.90 (d,
J = 2.3 Hz, 1H), 4.26 – 4.21 (m, 1H), 4.07 (dd,
J = 10.5, 3.9 Hz, 2H), 4.02 – 3.99 (m, 1H), 3.92
(s, 2H), 2.40 (s, 3H), 2.21 (s, 3H). ESI-MS m/z: 487.15 [M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 163.05 (s), 162.79 (s),
161.76 (s), 155.11 (s), 150.19 (s), 141.14 (s), 140.52 (s), 132.16 (s),
130.25 (s), 129.85 (s), 128.40 (d, J = 8.3 Hz), 127.90 (s),
123.97 (s), 123.62 (s), 122.92 (s), 115.45 (s), 114.29 (s), 111.69 (s),
111.36 (s), 102.81 (s), 72.59 (s), 68.49 (s), 47.82 (s), 33.97 (s), 22.21
(s), 17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.68 (d, J = 9.2 Hz, 1H), 7.19 (s, 1H), 6.99 (d,
J = 2.4 Hz, 1H), 6.97 (s, 1H), 6.69 (s, 1H), 6.20 (d,
J = 1.1 Hz, 1H), 4.08 (t, J = 6.4 Hz,
1H), 4.02 – 3.93 (m, 2H), 2.92 (d, J = 11.1 Hz, 1H),
2.86 (d, J = 11.1 Hz, 1H), 2.45 – 2.41 (m, 1H), 2.40
(d, J = 0.9 Hz, 3H), 2.35 (dd, J = 12.7,
5.9 Hz, 1H), 2.06 – 1.88 (m, 3H), 1.68 – 1.59 (m, 2H), 1.54
(qd, J = 11.8, 3.0 Hz, 2H). ESI-MS m/z: 361.24 [M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 162.38 (s),
160.63 (s), 155.18 (s), 153.88 (s), 126.90 (s), 113.52 (s), 112.88 (s),
111.55 (s), 101.76 (s), 72.26 (s), 66.88 (s), 61.58 (s), 54.12 (d,
J = 14.4 Hz), 42.09 (s), 29.07 (s), 18.59 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.68 (s, 1H), 7.28 (d, J = 8.3 Hz, 3H), 7.23 (t,
J = 7.3 Hz, 3H), 7.19 (t, J = 6.9 Hz,
4H), 7.14 (t, J = 7.3 Hz, 3H), 7.00 (s, 1H), 6.69 (s, 1H),
4.82 (s, 2H), 4.08 (s, 1H), 4.02 (t, J = 3.7 Hz, 1H), 4.00
(d, J = 4.1 Hz, 1H), 3.98 (s, 1H), 3.96 (d,
J = 4.9 Hz, 3H), 3.89 (s, 3H), 2.85 (d,
J = 11.3 Hz, 1H), 2.74 (d, J = 11.0
Hz, 1H), 2.44 (s, 1H), 2.42 (s, 3H), 2.39 – 2.34 (m, 1H), 2.28 (dd,
J = 12.6, 6.3 Hz, 1H), 2.12 (s, 1H), 2.05 – 1.96
(m, 2H), 1.91 (dt, J = 11.5, 8.7 Hz, 3H), 1.60 (t,
J = 11.9 Hz, 3H), 1.51 (dq, J = 11.8,
8.1 Hz, 3H), 1.24 (s, 12H), 1.23 (s, 8H), 1.21 (s, 2H), 1.15 (s, 1H). ESI-MS
m/z: 653.43 [M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.10 (s), 161.80 (s),
159.40 (s), 152.61 (s), 149.30 – 148.43 (m), 148.43 – 148.17
(m), 138.08 (s), 136.70 (s), 128.59 (s), 128.15 (d, J = 7.2
Hz), 126.97 (d, J = 9.0 Hz), 125.60 (s), 125.30 (s), 121.55
(s), 113.36 (s), 99.81 (s), 71.81 (s), 66.89 (s), 61.45 (s), 54.07 (d,
J = 13.5 Hz), 42.13 (s), 35.17 (s), 34.48 (d,
J = 4.2 Hz), 32.08 (s), 31.61 (s), 29.05 (s), 15.66
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 9.5 Hz, 1H), 7.24 (dd,
J = 8.5, 5.7 Hz, 2H), 7.17 (s, 1H), 7.7 (t,
J = 8.9 Hz, 2H), 6.98 – 6.91 (m, 2H), 6.67 (s,
1H), 4.87 (s, 1H), 4.06 (t, J = 6.3 Hz, 1H), 3.97 –
3.92 (m, 2H), 3.90 (s, 2H), 2.90 (d, J = 11.2 Hz, 1H), 2.84
(d, J = 11.1 Hz, 1H), 2.41 (s, 3H), 2.39 (d,
J = 5.5 Hz, 1H), 2.34 (d, J = 5.7 Hz,
1H), 2.31 (d, J = 5.3 Hz, 1H), 2.05 – 1.91 (m, 3H),
1.61 (d, J = 2.3 Hz, 2H), 1.52 (dd, J =
23.2, 11.5 Hz, 2H). ESI-MS m/z: 469.15 [M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 161.72 (d,
J = 11.7 Hz), 153.81 (s), 148.90 (s), 135.90 (s),
130.28 (d, J = 7.9 Hz), 127.09 (s), 121.37 (s), 115.62 (s),
115.45 (s), 113.93 (s), 112.96 (s), 101.50 (s), 72.23 (s), 66.88 (s), 61.57
(s), 54.11 (d, J = 12.2 Hz), 42.07 (s), 31.81 (s), 29.05
(s), 15.62 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.75 – 7.67 (m, 1H), 7.30 (dd, J = 14.3,
7.9 Hz, 1H), 7.17 (s, 1H), 7.7 – 6.93 (m, 5H), 6.68 (s, 1H), 4.08 (q,
J = 6.2 Hz, 1H), 4.01 (q, J = 7.1 Hz,
1H), 3.98 – 3.93 (m, 4H), 2.88 (dd, J = 29.9, 11.2
Hz, 2H), 2.44 – 2.38 (m, 4H), 2.36 – 2.31 (m, 1H), 2.04
– 1.93 (m, 4H), 1.89 (s, 1H), 1.67 – 1.58 (m, 2H), 1.53 (dd,
J = 23.3, 11.6 Hz, 2H), 1.38 (s, 1H), 1.16 (t,
J = 7.1 Hz, 1H). ESI-MS m/z: 469.06 [M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.06 (s), 163.66 (s),
162.19 – 161.59 (m), 153.86 (s), 149.27 (s), 142.76 (d,
J = 7.2 Hz), 130.72 (d, J= 8.3 Hz), 127.14 (s), 124.56
(s), 120.80 (s), 115.15 (s), 113.92 (s), 113.35 (d, J =
20.9 Hz), 113.26 – 113.25 (m), 112.96 (s), 101.51 (s), 72.24 (s),
66.88 (s), 61.57 (s), 54.11 (d, J = 12.0 Hz), 42.08 (s),
32.36 (s), 29.06 (s), 26.80 (s), 15.65 (s).</p><!><p>1H NMR (500 MHz, DMSO) Î´ 7.71 (d, J = 9.6 Hz, 1H),
7.65 (s, 1H), 7.31 (s, 1H), 7.29 (s, 1H),7.27 – 7.25 (m, 1H), 7.24
(s, 1H), 7.22 (s, 1H), 7.20 (s, 1H), 6.99 (s, 1H), 6.97 (s, 1H), 6.95 (d, J
=2.0 Hz, 1H), 6.70 (s, 1H), 6.18 (s, 1H), 4.07 (d, J = 6.6 Hz, 1H), 3.96 (d,
J = 7.4 Hz, 2H), 3.91 (s, 2H), 3.15 (s, 1H), 2.91 (dd, J = 22.3, 6.0 Hz,
2H), 2.40 (s, 3H), 2.37 (s, 1H), 2.05 (d, J = 15.4 Hz, 3H), 1.62 (s, 2H),
1.59 – 1.48 (m, 2H), 1.21 (s, 1H), 0.89 – 0.79 (m, 1H). ESI-MS
m/z: 485.40 [M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.96 (s), 161.73 (d, J
= 14.0 Hz), 153.84 (s), 149.13 (s), 138.86 (s), 131.13 (s), 130.39 (s),
128.79 (s), 127.14 (s), 121.05 (s), 113.94 (s), 112.99 (s), 101.53 (s),
72.17 (s), 66.70 (s), 61.40 (s), 53.99 (s), 49.06 (s), 41.86 (s), 31.99 (s),
28.85 (s), 15.66 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 9.5 Hz, 1H), 7.43 (d,
J = 8.1 Hz, 2H), 7.17 (d, J = 7.7 Hz,
3H), 6.96 (d, J = 6.4 Hz, 2H), 6.68 (s, 1H), 4.07 (d,
J = 6.3 Hz, 1H), 3.95 (d, J = 6.3 Hz,
2H), 3.89 (s, 2H), 2.91 (d, J = 8.8 Hz, 1H), 2.85 (d,
J = 8.1 Hz, 1H), 2.40 (s, 3H), 2.35 (s, 1H), 2.00 (d,
J =11.4 Hz, 2H), 1.61 (s, 2H), 1.53 (d,
J = 11.8 Hz, 2H), 1.21 (s, 1H). ESI-MS m/z: 530.94
[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.77 (s), 161.67 (d,
J = 8.0 Hz), 153.82 (s), 149.13 (s), 139.27 (s), 131.70
(s), 130.77 (s), 127.13 (s), 121.00 (s), 119.55 (s), 113.96 (s), 112.99 (s),
101.53 (s), 72.5 (s), 66.40 (s), 61.08 (s), 53.80 (s), 41.52 (s), 32.05 (s),
29.48 (s), 28.49 (s), 15.65 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.75 (d, J = 9.6 Hz, 1H), 7.22 (s, 1H), 7.16 (dd,
J = 15.1, 6.9 Hz, 1H), 7.6 (dd, J =
10.0, 2.6 Hz, 1H), 6.99 (dd, J = 6.9, 2.4 Hz, 2H), 6.84
(td, J = 8.5, 2.6 Hz, 1H), 6.81 – 6.77 (m, 1H), 6.73
(s, 1H), 6.54 – 6.49 (m, 1H), 4.93 (s, 1H), 4.10 (d,
J = 6.5 Hz, 1H), 3.97 (dd, J = 12.2,
5.5 Hz, 2H), 3.81 (s, 2H), 2.94 (d, J = 10.7 Hz, 1H), 2.87
(d, J = 10.7 Hz, 1H), 2.45 (d, J = 5.7 Hz,
1H), 2.42 (d, J = 4.6 Hz, 1H), 2.38 (s, 3H), 2.34 (s, 3H),
2.31 (d, J = 4.8 Hz, 1H), 2.08 -I. 93 (m, 3H), 1.63 (s,
2H), 1.55 (dd, J = 23.3, 11.6 Hz, 2H), 1.23 (s, 1H). ESI-MS
m/z: 483.63[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.94 (s), 163.83 (s),
161.37 (s), 153.89 (s), 146.67 (s), 131.30 (d, J = 6.1 Hz),
127.17 (s), 122.48 (d, J = 12.5 Hz), 118.64 (s), 113.89
(s), 113.00 (s), 111.85 (s), 111.75 (d, J = 21.0 Hz),
104.10 (s), 101.53 (s), 72.20 (s), 66.76 (s), 61.45 (s), 54.02 (s), 41.95
(s), 29.47 (s), 28.93 (s), 25.64 (s), 15.53 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.73 (d, J = 9.5 Hz, 1H), 7.20 (d,
J = 2.5 Hz, 1H), 7.18 (s, 1H), 7.17 (d,
J = 1.6 Hz, 1H), 6.98 (d, J = 2.4 Hz,
1H), 6.96 (s, 2H), 6.94 (d, J = 2.2 Hz, 1H), 6.92 (d,
J =2.1 Hz, 1H), 6.68 (s, 1H), 4.07 (d,
J = 6.5 Hz, 1H), 3.96 (d, J = 6.2 Hz,
2H), 3.89 (s, 2H), 2.89 (d, J = 23.7 Hz, 2H), 2.40 (s, 3H),
2.35 (s, 1H), 2.01 (d, J = 9.4 Hz, 2H), 1.62 (s, 2H), 1.53
(d, J =II 0 Hz, 2H), 1.22 (s, 2H). ESI-MS m/z: 487.02[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.97 (s), 161.83 (s),
161.37 (s), 153.89 (s), 149.60 (s), 131.30 (d, J = 6.1 Hz),
127.17 (s), 122.48 (d, J = 12.5 Hz), 119.65 (s), 113.89
(s), 113.00 (s), 111.85 (s), III 75 (d, J = 21.0 Hz),
104.10 (s), 101.53 (s), 72.20 (s), 66.76 (s), 61.45 (s), 54.02 (s), 41.95
(s), 29.47 (s), 28.93 (s), 25.54 (s), 15.51 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.76 – 7.71 (m, 1H), 7.44 (dd, J = 8.7, 2.1
Hz, 1H), 7.20 (s, 1H), 7.07 – 7.03 (m, 2H), 7.00 – 6.96 (m,
2H), 6.70 (s, 1H), 4.08 (d, J = 6.8 Hz, 1H), 3.96 (d,
J = 7.7 Hz, 2H), 3.93 (s, 2H), 3.15 (s, 2H), 2.87 (dd,
J = 31.6, 11.1 Hz, 2H), 2.43 – 2.37 (m, 1H),
2.33 (s, 3H), 2.6 – 1.89 (m, 4H), 1.61 (s, 2H), 1.52 (dt,
J = 11.9, 8.9 Hz, 2H). ESI-MS m/z: 503.55[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.09 (s), 161.71 (d,
J = 62.2 Hz), 159.94 (s), 154.04 (s), 150.36 (s),
133.98 (d, J = 10.4 Hz), 133.00 (s), 130.46 (s), 127.20
(s), 119.37 (s), 116.84 (s), 114.90 (s), 113.87 (s), 113.04 (s), 101.56 (s),
72.27 (s), 66.88 (s), 61.59 (s), 54.14 (d, J = 17.1 Hz),
49.06 (s), 42.09 (s), 29.91 (s), 29.08 (s), 15.66 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.73 (d, J = 9.5 Hz, 1H), 7.53 (d,
J = 8.8 Hz, 1H), 7.19 (s, 1H), 6.99 (s, 1H), 6.98
– 6.94 (m, 1H), 6.80 – 6.73 (m, 1H), 6.70 (s, 1H), 6.51
– 6.47 (m, 1H), 6.43 (d, J = 2.9 Hz, 1H), 4.91 (s,
1H), 4.08 (d, J = 6.6 Hz, 1H), 3.96 (d, J
= 6.8 Hz, 2H), 3.89 (s, 2H), 3.68 (s, 1H), 3.61 (s, 3H), 3.15 (d,
J = 4.8 Hz, 1H), 2.91 (d, J = 10.8 Hz,
1H), 2.85 (d, J = 10.9 Hz, 1H), 2.41 (d, J
= 10.1 Hz, 2H), 2.34 (d, J = 2.2 Hz, 1H), 2.31 (s, 3H),
2.06 – 1.91 (m, 4H), 1.61 (s, 2H), 1.53 (t, J = 12.0
Hz, 2H), 1.23 (dd, J = 15.0, 10.3 Hz, 1H), 0.89 –
0.77 (m, 1H). ESI-MS m/z: 561.38[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.08 (s), 161.95 (s),
161.54 (s), 159.30 (s), 154.05 (s), 150.40 (s), 139.35 (s), 133.67 (s),
127.20 (s), 119.48 (s), 115.44 (s), 114.72 (s), 113.84 (s), 113.37 (s),
113.2 (s), 101.59 (s), 72.26 (s), 66.87 (s), 61.59 (s), 55.72 (s), 54.13 (d,
J = 14.1 Hz), 42.09 (s), 33.36 (s), 29.06 (s), 15.72
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.06 (dd, J = 18.6, 9.0 Hz, 2H), 7.74 (t,
J = 9.9 Hz, 1H), 7.69 (dd, J = 17.5,
8.4 Hz, 1H), 7.60 – 7.51 (m, 1H), 7.20 (s, 1H), 7.02 – 6.93
(m, 2H), 6.71 (s, 1H), 4.14 – 4.02 (m, 3H), 3.97 (s, 2H), 2.91 (d,
J = 27.7 Hz, 2H), 2.47 (s, 3H), 2.39 (d,
J = 9.4 Hz, 1H), 2.02 (s, 3H), 1.63 (s, 2H), 1.55 (s,
2H), 1.20 (d, J = 9.6 Hz, 1H). ESI-MS m/z: 596.20[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.96 (s), 161.90 (s),
161.68 (s), 153.90 (s), 149.68 (s), 148.30 (s), 142.19 (s), 135.35 (s),
130.37 (s), 127.25 (s), 123.13 (s), 121.69 (s), 120.40 (s), 113.86 (s),
113.5 (s), 101.55 (s), 72.20 (s), 66.74 (s), 61.43 (s), 54.01 (s), 41.92
(s), 32.29 (s), 28.91 (s), 15.72 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.78 – 7.69 (m, 1H), 7.61 (d, J = 8.2 Hz,
2H), 7.44 (d, J = 8.1 Hz, 2H), 7.18 (s, 1H), 6.98 (s, 1H),
6.97 (d, J = 2.4 Hz, 1H), 4.07 (d, J = 6.6
Hz, 1H), 4.03 (s, 2H), 3.99 –3.92 (m, 2H), 2.91 (d,
J = 11.0 Hz, 1H), 2.85 (d, J = 10.9
Hz, 1H), 2.42 (s, 3H), 2.40 – 2.37 (m, 1H), 2.37 – 2.29 (m,
1H), 2.06 – 1.92 (m, 3H), 1.61 (d, J = 2.4 Hz, 2H),
1.52 (dd, J = 23.6, 11.7 Hz, 2H). ESI-MS m/z: 519.10[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.03 (s), 161.90 (s),
161.76 (d, J = 29.5 Hz), 153.89 (s), 149.47 (s), 144.81 (s), 129.30 (s),
127.44 (s), 127.18 (s), 125.71 (d, J = 3.2 Hz), 123.75 (s), 120.58 (s),
113.87 (s), 113.01 (s), 101.53 (s), 72.26 (s), 66.89 (s), 61.58 (s), 54.13
(d, J = 13.8 Hz), 42.09 (s), 32.54 (s), 29.07 (s), 15.70 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.76 (t, J = 7.4 Hz, 2H), 7.51 (t,
J = 7.5 Hz, 1H), 7.42 (t, J = 7.6 Hz,
1H), 7.21 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H), 7.02 (dd,
J = 4.7, 2.4 Hz, 1H), 7.00 (d, J = 2.5
Hz, 1H), 6.73 (s, 1H), 4.93 (s, 1H), 4.12 (s, 1H), 4.10 (s, 2H), 4.02
– 3.94 (m, 2H), 3.35 (s, 2H), 2.93 (d, J =11.0 Hz,
1H), 2.87 (d, J = 11.1 Hz, 1H), 2.44 (d, J
= 6.1 Hz, 1H), 2.42 (d, J = 5.5 Hz, 1H), 2.37 (d,
J = 5.8 Hz, 1H), 2.32 (s, 3H), 2.04 (ddd,
J = 15.5, 7.6, 3.8 Hz, 1H), 2.01 – 1.94 (m, 2H),
1.63 (d, J = 2.3 Hz, 2H), 1.60 – 1.49 (m, 2H).
ESI-MS m/z: 519.43[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 161.72 (d,
J = 11.7 Hz), 153.81 (s), 148.90 (s), 135.90 (s),
130.28 (d, J = 7.9 Hz), 127.09 (s), 121.37 (s), 115.62 (s),
115.45 (s), 113.93 (s), 112.96 (s), 101.50 (s), 72.23 (s), 66.88 (s), 61.57
(s), 54.11 (d, J = 12.2 Hz), 42.07 (s), 31.81 (s), 29.05
(s), 15.62 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.72 (d, J = 9.4 Hz, 1H), 7.33 (d,
J = 8.7 Hz, 2H), 7.24 (d, J = 8.1 Hz,
2H), 7.17 (s, 1H), 6.97 (d, J = 2.5 Hz, 1H), 6.95 (s, 1H),
6.67 (s, 1H), 4.06 (t, J = 6.4 Hz, 1H), 3.97 (s, 1H), 3.95
(s, 2H), 3.94 (s, 1H), 2.90 (d, J = 11.3 Hz, 1H), 2.84 (d,
J = 11.1 Hz, 1H), 2.42 (s, 3H), 2.39 (d,
J = 5.3 Hz, 1H), 2.34 (d, J = 5.8 Hz,
1H), 2.31 (d, J = 5.5 Hz, 1H), 2.06 – 1.87 (m, 3H),
1.66 – 1.58 (m, 2H), 1.53 (t, J = 11.7 Hz, 2H).
ESI-MS m/z: 535.2052 [M+H] +, Delta <0.5 ppm.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 162.11
– 161.90 (m), 161.73 (d, J = 22.5 Hz), 153.85 (s),
149.19 (s), 147.13 (s), 139.33 (s), 130.28 (s), 127.15 (s), 121.48 (s),
120.97 (s), 113.90 (s), 112.98 (s), 101.51 (s), 72.24 (s), 66.89 (s), 61.58
(s), 54.12 (d, J = 13.3 Hz), 42.09 (s), 40.48 (t,
J =10.5 Hz), 31.99 (s), 29.07 (s), 15.66 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
Î´ 7.87 (d, J = 8.2 Hz, 2H), 7.74 (d,
J = 9.6 Hz, 1H), 7.68 (d, J = 9.2 Hz,
1H), 7.37 (d, J = 8.2 Hz, 2H), 7.17 (s, 1H), 7.01 –
6.96 (m, 2H), 4.10 (d, J = 6.7 Hz, 1H), 4.03 (s, 2H), 4.00
– 3.93 (m, 2H), 3.83 (s, 3H), 2.89 (dd, J = 22.7,
11.2 Hz, 2H), 2.43 (s, 3H), 2.40 (s, 1H), 2.37 (d, J = 5.8
Hz, 1H), 2.33 (s, 1H), 2.07 – 1.95 (m, 3H), 1.64 (d,
J = 12.9 Hz, 2H), 1.59 – 1.48 (m, 2H). ESI-MS
m/z: 509.30[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.06 (s), 162.38 (s),
160.62 (s), 155.18 (s), 153.87 (s), 126.90 (s), 113.52 (s), 112.88 (s),
111.55 (s), 101.76 (s), 72.26 (s), 66.89 (s), 61.58 (s), 54.12 (d,
J = 14.5 Hz), 42.10 (s), 29.07 (s), 18.59 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.27 (d,J= 8.4 Hz, 1H), 8.23
(d,J= 4.9 Hz, 1H), 8.21 (s, 1H), 7.95 (t,
J= 7.3 Hz, 1H), 7.93 – 7.92 (m, 1H), 7.82 -
111 (m, 2H), 7.76 (s, 1H), 7.70 – 7.67 (m, 1H),
7.65 – 7.61 (m, 1H), 7.61 – 7.59 (m, 1H), 7.59
(t,J= 2.2 Hz, 1H), 7.58 – 7.56 (m, 1H), 7.55
– 7.53 (m, 1H), 7.52 (s, 1H), 7.44 – 7.39 (m, 1H), 7.38
– 7.34 (m, 1H), 7.32 (d, J= 7.9 Hz, 1H), 7.21 (s,
2H), 7.16 (d,J= 8.2 Hz, 1H), 7.03 (dd, .7=5.9, 2.4 Hz, 1H),
7.01 (d, .7=2.3 Hz, 1H), 6.99 (d,.7=3.7 Hz, 1H), 6.98 (d, J =
2.2 Hz, 1H), 6.96 (s, 1H), 6.71 (s, 1H), 6.55 – 6.49 (m,
1H), 5.54 (dd, J = 10.9, 5.0 Hz, 1H), 4.93 (s, 1H), 4.83
(s, 1H), 4.39 (s, 2H), 4.12 (t, .7= 6.7 Hz, 1H), 4.02 (dd,
J = 11.7, 5.7 Hz, 3H), 2.92 (dd, .7=29.2, 10.2 Hz, 3H),
2.35 (s, 3H), 2.18 (s, 1H), 2.04 (dd,T= 11.5, 4. Hz, 4H), 1.65 (s, 2H), 1.59
– 1.53 (m, 3H). ESI-MS m/z: 501.30[M+H] +.</p><p>13C NMR (126 MHz, DMSO) δ 177.03 (s), 162.24
– 161.99 (m), 161.79 (d, J= 20.7 Hz), 154.05 (s),
150.25 (s), 134.88 (s), 133.87 (s), 132.05 (s), 129.04 (s), 127.07 (s),
126.67 (s), 126.19 (d,T = 23.1Hz), 123.87 (d, .7 = 8.9 Hz), 120.26 (s),
114.04 (s), 113.01 (s), 101.71 (d,J= 19.2 Hz), 72.27 (s),
66.88 (s), 61.55 (s), 54.08 (s), 42.05 (s), 29.54 (s), 29.03 (s), 15.66
(s).</p><!><p>A solution of compound NO.24 (0.227 mmol),
phenylboronic acid or benzene boric acid ester derivatives (2 eq) in diox(4
mL) and saturated sodium bicarbonate solution (0.7 mL) was replaced the air
with argon for ten minutes then Pd(PPh3)4 (0.05 eq)
was added and the reaction mixture was stirred at 95°C overnight.
Then, the mixture was extracted with ethyl acetate, washed with saline. The
organic phase was collected and the solvent was removed under vacuum. The
product was purified by silica gel chromatography using DCM-methaonl as
eluate to give compound NO. 35. Yield: 72 %. 1H NMR
(500 MHz, DMSO) δ 7.76 – 7.67 (m, 2H), 7.43 (d,
J = 8.4 Hz, 2H), 7.29 (dd, J = 20.6,
8.2 Hz, 3H), 7.17 (d,J= 8.2 Hz, 4H), 6.98 – 6.94 (m,
3H), 4.07 (d,J= 6.4 Hz, 2H), 3.99 –3.93 (m, 5H),
3.89 (s, 2H), 2.90 (d, 7= 11.1 Hz, 2H), 2.84 (d,J= 11.2 Hz,
2H), 2.40 (s, 4H), 2.35 (s, 3H), 2.31 (d,T=5.6Hz, 1H), 2.18 (s, 2H), 1.99
(ddd,T = 28.7, 15.9, 8.2 Hz, 5H), 1.61 (s,3H), 1.53 (t,J=
11.7 Hz, 3H). ESI-MS m/z: 546.10[M+H]+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 165.35 (s),
161.81 (s), 158.57 (s), 153.82 (s), 149.66 – 149.19 (m), 149.04 (d,
J = 20.9 Hz), 139.56 – 139.33 (m), 139.17 (d,
J = 27.9 Hz), 131.69 (s), 130.77 (s), 129.35 (s),
128.97 (s), 128.09 (s), 127.10 (s), 121.26 (s), 121.09 (d, J= 34.8 Hz),
119.55 (s), 116.12 (s), 113.92 (d, J = 5.8 Hz), 112.97 (s),
101.51 (s), 72.24 (s), 66.90 (s), 61.60 (s), 54.13 (d, J =
13.3 Hz), 42.10 (s), 32.21 (d, J = 39.3 Hz), 32.01 –
31.59 (m), 29.08 (s), 15.64 (s), 11.77 (s), 10.95 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
δ 7.74 – 7.70 (m, 1H), 7.59 (d, J = 7.3 Hz,
2H), 7.55 (d, J =8.2 Hz, 2H), 7.42 (t, J =
7.7 Hz, 2H), 7.31 (dd, J = 12.0, 7.8 Hz, 3H), 7.17 (s, 1H),
6.97 (dd, J = 4.6, 2.3 Hz, 2H), 4.07 (t, J
= 6.2 Hz, 1H), 3.97 (s, 2H), 3.96 (d, J = 6.9 Hz, 2H), 2.88
(dd, J = 29.8, 10.3 Hz, 2H), 2.45 (s, 3H), 2.41 (s, 1H),
2.35 (s, 1H), 2.06 – 1.92 (m, 3H), 1.61 (s, 2H), 1.54 (t,
J = 12.0 Hz, 2H). ESI-MS m/z: 527.37[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.02 (s), 161.74 (s),
153.82 (s), 148.89 (s), 140.43 (s), 139.08 (s), 138.51 (s), 129.37 (s),
129.21 (d, J = 33.1 Hz), 127.68 (s), 127.42 – 126.77
(m), 121.38 (s), 113.98 (s), 112.96 (s), 101.52 (s), 72.22 (s), 66.84 (s),
61.53 (s), 54.08 (d, J = 8.7 Hz), 42.03 (s), 32.27 (s),
29.01 (s), 15.70 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
δ 7.74 – 7.70 (m, 1H), 7.66 – 7.61 (m, 2H), 7.52 (d,
J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz,
2H), 7.24 (t, J = 8.9 Hz, 2H), 7.17 (s, 1H), 6.97 (td,
J = 4.8, 2.5 Hz, 2H), 4.07 (q, J = 6.0
Hz, 1H), 3.96 (d, J = 7.9 Hz, 4H), 2.88 (dd,
J = 29.8, 10.1 Hz, 2H), 2.44 (s, 3H), 2.39 (d,
J = 14.0 Hz, 1H), 2.35 (s, 1H), 2.01 (dd,
J = 13.5, 9.9 Hz, 3H), 1.61 (s, 2H), 1.53 (dd,
J = 24.0, 12.0 Hz, 2H). ESI-MS m/z: 545.10[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.02 (s), 163.16 (s),
161.74 (s), 161.22 (s), 153.82 (s), 148.90 (s), 139.07 (s), 137.47 (s),
136.91 (s), 129.10 (s), 128.90 (d, J = 8.0 Hz), 127.17 (s),
121.35 (s), 116.23 (s), 116.11 (d, J = 21.3 Hz), 113.97
(s), 112.96 (s), 101.52 (s), 72.21 (s), 66.83 (s), 61.53 (s), 54.08 (d,
J = 9.0 Hz), 42.02 (s), 32.25 (s), 29.00 (s), 15.69
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
δ 7.76 – 7.70 (m, 1H), 7.64 (d, J = 8.6 Hz,
2H), 7.57 (d, J =8.2 Hz, 2H), 7.48 (d, J =
8.6 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 7.20 (s, 1H), 6.98
(dd, J = 5.8, 2.4 Hz, 2H), 6.70 (s, 1H), 4.09 (d,
J = 6.4 Hz, 1H), 3.98 (s, 3H), 3.97 (s, 1H), 2.93 (d,
J = 11.2 Hz, 1H), 2.87 (d, J = 11.1
Hz, 1H), 2.46 (s, 3H), 2.43 (d, J = 5.0 Hz, 1H), 2.37 (d,
J = 5.6 Hz, 1H), 2.35 (d, J = 5.6 Hz,
1H), 2.09 – 1.94 (m, 3H), 1.64 (d, J = 12.8 Hz, 2H),
1.55 (td, J = 12.0, 2.9 Hz, 2H), 1.23 (s, 1H). ESI-MS m/z:
561.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.02 (s), 161.74 (d,
J = 4.6 Hz), 153.82 (s), 148.94 (s), 139.53 (s), 139.22
(s), 137.13 (s), 132.55 (s), 129.23 (d, J = 15.4 Hz),
128.70 (s), 127.13 (d, J = 9.5 Hz), 121.29 (s), 113.96 (s),
112.96 (s), 101.52 (s), 72.22 (s), 66.84 (s), 61.54 (s), 54.09 (d,
J = 9.9 Hz), 42.4 (s), 32.28 (s), 29.02 (s), 15.70
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
δ 7.76 – 7.69 (m, 1H), 7.35 (d, J = 8.2 Hz,
2H), 7.31 (d, J =1.7 Hz, 1H), 7.29 (s, 1H), 7.28 (d,
J = 1.7 Hz, 1H), 7.25 – 7.20 (m, 3H), 7.17 (s,
1H), 7.06 (d, J = 8.1 Hz, 1H), 6.99 (s, 1H), 6.98 (d,
J = 2.1 Hz, 1H), 6.97 (d, J = 2.3 Hz,
2H), 4.07 (t, J = 6.3 Hz, 1H), 3.96 (t, J
= 6.3 Hz, 4H), 3.72 (s, 3H), 2.91 (d, J = 11.2 Hz, 1H),
2.85 (d, J = 10.9 Hz, 1H), 2.46 (s, 3H), 2.40 (s, 1H), 2.34
(s, 1H), 1.99 (ddd, J = 22.5, 15.5, 8.5 Hz, 3H), 1.61 (s,
2H), 1.53 (dd, J = 23.7, 11.8 Hz, 2H). ESI-MS m/z:
557.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.04 (s), 161.73 (s),
156.53 (s), 153.81 (s), 148.82 (s), 138.30 (s), 136.43 (s), 130.71 (s),
129.76 (s), 129.15 (s), 128.12 (s), 127.10 (s), 121.18 (s), 112.11 (s),
101.52 (s), 72.23 (s), 66.87 (s), 61.57 (s), 55.85 (s), 54.11 (d, J = 10.3
Hz), 42.07 (s), 32.35 (s), 29.05 (s), 15.73 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
Î´ 7.91 (d, J = 7.7 Hz, 1H), 7.77 (d,
J = 1.2 Hz, 1H), 7.76 – 7.72 (m, 2H), 7.59
– 7.52 (m, 3H), 7.48 (d, J = 8.2 Hz, 2H), 7.37 (d,
J = 8.2 Hz, 2H), 7.17 (s, 1H), 6.98 (dd,
J = 5.7, 2.4 Hz, 2H), 6.68 (s, 1H), 4.87 (s, 1H), 4.08
(d, J = 6.6 Hz, 1H), 4.02 (s, 2H), 3.96 (d,
J = 7.0 Hz, 2H), 2.91 (d, J = 11.0 Hz,
1H), 2.85 (d, J = 10.4 Hz, 1H), 2.47 (s, 3H), 2.39 (d,
J = 6.2 Hz, 1H), 2.34 (d, J = 1.7 Hz,
1H), 2.03 – 1.93 (m, 4H), 1.61 (s, 2H), 1.53 (d, J =
11.9 Hz, 2H). ESI-MS m/z: 552.26[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.04 (s), 161.75 (d,
J = 10.3 Hz), 153.86 (s), 149.12 (s), 144.78 (s),
140.53 (s), 136.10 (s), 134.31 (s), 133.98 (s), 130.51 (s), 129.24 (s),
128.86 (s), 128.51 (s), 127.15 (s), 121.09 (s), 119.09 (s), 113.96 (s),
112.98 (s), 110.48 (s), 101.53 (s), 72.25 (s), 66.87 (s), 61.58 (s), 54.11
(d, J = 11.0 Hz), 42.08 (s), 32.41 (s), 29.05 (s), 15.76
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
Î´ 7.83 (d, J = 8.3 Hz, 2H), 7.77 (d,
J = 8.4 Hz, 2H), 7.74 – 7.70 (m, 1H), 7.63 (d,
J = 8.2 Hz, 2H), 7.35 (d, J = 8.2 Hz,
2H), 7.18 (s, 1H), 6.97 (dd, J = 4.6, 2.3 Hz, 2H), 4.07 (d,
J = 6.3 Hz, 1H), 3.99 (s, 2H), 3.96 (d,
J = 6.4 Hz, 2H), 2.89 (d, J = 20.6 Hz,
2H), 2.45 (s, 3H), 2.35 (d, J = 1.8 Hz, 1H), 2.09 –
1.85 (m, 3H), 1.62 (s, 2H), 1.53 (d, J = 12.0 Hz, 2H).
ESI-MS m/z: 557.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.99 (s), 161.75 (s),
153.83 (s), 149.03 (s), 144.41 (s), 140.29 (s), 136.89 (s), 129.27 (s),
127.65 (d, J = 16.7 Hz), 127.12 (s), 126.21 (s), 121.22
(s), 113.97 (s), 112.98 (s), 101.53 (s), 72.18 (s), 66.76 (s), 61.44 (s),
54.02 (s), 32.32 (s), 28.91 (s), 15.72 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
Î´ 10.23 (s, 1H), 7.89 (t, J = 7.9 Hz, 1H), 7.75 (s,
1H), 7.72 (d, J = 8.6 Hz, 2H), 7.69 (d, J
= 6.7 Hz, 2H), 7.37 (d, J = 8.2 Hz, 2H), 7.21 (s, 1H), 7.01
– 6.95 (m, 2H), 6.71 (s, 1H), 4.12 – 4.06 (m, 1H), 4.01 (s,
2H), 3.98 (d, J = 6.3 Hz, 2H), 2.96 (d, J
= 10.7 Hz, 1H), 2.90 (d, J = 10.7 Hz, 1H), 2.46 (s, 3H),
2.42 (d, J = 8.2 Hz, 1H), 2.11 – 2.00 (m, 3H), 1.91
(s, 1H), 1.64 (s, 2H), 1.61 – 1.51 (m, 2H), 1.34 (s, 1H), 1.23 (s,
1H), 0.85 (t, J = 6.7 Hz, 1H), 0.07 (s, 1H), 0.03 -
−0.14 (m, 1H). ESI-MS m/z: 573.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) δ 187.89 (s), 176.95 (s),
165.25 (s), 163.21 (s), 161.72 (s), 153.83 (s), 149.10 (s), 141.24 (s),
135.77 (s), 130.41 (s), 129.33 (s), 127.75 (s), 127.13 (s), 123.37 (s),
122.75 (s), 121.12 (s), 114.68 (d, J = 21.4 Hz), 114.59
– 114.40 (m), 113.96 (s), 112.99 (s), 101.53 (s), 72.17 (s), 66.71
(s), 61.39 (s), 53.99 (s), 41.86 (s), 32.37 (s), 28.85 (s), 15.72 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.35.1H NMR (500 MHz, DMSO)
δ 8.00 – 7.94 (m, 1H), 7.84 (dd, J = 11.1,
1.3 Hz, 1H), 7.75 (s, 1H), 7.73 (s, 1H), 7.72 – 7.69 (m, 2H), 7.37
(d, J = 8.3 Hz, 2H), 7.21 (s, 1H), 7.01 – 6.95 (m,
2H), 6.72 (s, 1H), 4.09 (d, J = 6.6 Hz, 1H), 4.01 (s, 2H),
4.00 – 3.92 (m, 2H), 2.96 (s, 1H), 2.90 (s, 1H), 2.46 (s, 3H), 2.42
(d, J = 3.1 Hz, 1H), 2.39 (d, J = 19.7 Hz,
1H), 2.06 (s, 3H), 1.91 (s, 1H), 1.65 (s, 2H), 1.57 (d, J =
11.5 Hz, 2H), 1.22 (s, 1H). ESI-MS m/z: 570.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 176.93 (s), 164.40 (s),
162.37 (s), 161.73 (d, J = 6.4 Hz), 153.83 (s), 149.11 (s),
148.03 (s), 141.45 (s), 135.33 (s), 134.70 (s), 129.35 (s), 127.81 (s),
127.13 (s), 123.82 (s), 121.08 (s), 114.64 (s), 113.96 (s), 112.99 (s),
101.53 (s), 98.82 (d, J = 15.3 Hz), 72.15 (s), 66.66 (s),
61.38 (s), 53.97 (s), 41.82 (s), 32.37 (s), 28.81 (s), 15.72 (s).</p><!><p>Step 1: A mixture of ethyl acetoacetate (1.3 mL, 10 mmol) and
CH3ONa (540 mg, 10 mmol, 1 eq) in MeOH (6 mL) was stirred at
room temperature for 10 minutes. Then, 4-(Trifluoromethoxy) benzyl bromide
(2805 mg, 11 mmol, 1.1 eq) in 5 mL MeOH was added dropwise when the reaction
solution was slightly boiling. The reaction mixture was continued to heat
under reflux condition until it was almost neutral. After cooling, the
reaction solution was filtered off and concentrated by vacuum to give ethyl
acetoacetate derivatives which was used in subsequent steps without further
purification.</p><p>Step 2: To a stirred solution of the above mentioned products in
PPA 5 g, resorcino (1102 mg, 10 mmol, 1 eq) was added. The reaction mixture
was stirred at 65°C for 4 h. The mixture solution had to rest
overnight, then diluted with 300 mL water. The precipitated solid was
filtered off, washed with water to give the crude product. The crude product
was purified by silica gel chromatography using 16% ethyl
acetate-cyclohexane as eluate to give the intermediate as a white solid
7-hydroxy-4-methyl-3-(4-(trifluoromethoxy)benzyl)-2H-chromen-2-one (1.4 g,
45 %).</p><p>Step 3: A mixture of the above products (700 mg, 2 mmol),
(R)-(−)-Epichlorohydrin (2 mL), potassium carbonate (553 mg, 4 mmol,
2 eq) and TBAB (645 mg, 2 mmol, 1 eq) was stirred at 80 for 2.3 h. Then the
mixture was extracted with ethyl acetate, washed with water. The organic
phase was collected and the solvent was removed under vacuum. The product
was purified by silica gel chromatography using 14% ethyl
acetate-cyclohexane as eluate to give the intermediate as a white solid
(R)-3-benzyl-4-methyl-7-(oxiran-2-yhnethoxy)-2H-chromen-2-one (410 mg, 51
%).</p><p>Step 4: The above mentioned products(390 mg, 0.96 mmol) was added
to a stirred solution of Hexahydroisonicotinamide (246 mg, 2 mmol, 2 eq) in
2 mL dimentylacetamide then the reaction mixture was stirred at 50□
for 24 h. The reaction solution was allowed to cool to room temperature and
concentrated by vacuum. The residue was dissolved in acetone, absorbed onto
silica and purified by flash column chromategraphy using 4%
methanol-dichloromethane as eluate to give white compound NO.44
(467 mg, 85%).1H NMR (500 MHz, DMSO) Î´ 7.71 (d,
J = 9.5 Hz, 1H), 7.33 (d, J= 8.7 Hz,
2H), 7.25 (s, 1H), 7.17 (s, 1H), 6.99 – 6.92 (m, 2H), 6.68 (s, 1H),
4.87 (s, 1H), 4.07 (d,J = 6.6 Hz, 1H), 3.96
(d,J= 8.3 Hz, 4H), 2.90 (d,J = 11.1
Hz, 1H), 2.84 (d,J= 11.2 Hz, 1H), 2.42 (s, 3H), 2.39 (d,
.7=5.4 Hz, 1H), 2.34 (d,T=5.8Hz, 1H), 2.32 (d,T=5.6Hz, 1H), 2.23
(t,J = 6.7 Hz, 1H), 2.05 – 1.92 (m, 3H),
1.77–1.71 (m, 1H), 1.62 (dd, J= 6.9, 3.8 Hz, 2H),
1.52 (dd, J = 23.3, 11.6 Hz, 2H). ESI-MS m/z: 535.20[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.04 (s), 161.84 (s),
161.65 (s), 153.87 (s), 149.19 (s), 147.14 (s), 139.35 (s), 130.29 (s),
127.16 (s), 121.49 (s), 120.98 (s), 113.91 (s), 112.99 (s), 101.52 (s),
72.26 (s), 66.90 (s), 61.59 (s), 54.13 (d, J = 13.4 Hz),
42.10 (s), 41.79 (s), 32.00 (s), 29.08 (s), 26.91 (s), 15.67 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.44 except using the
(S)-(+)-Epichlorohydrin to replace the
(R)-(−)-Epichlorohydrin.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 9.5 Hz, 1H), 7.33 (d,
J = 8.6 Hz, 2H), 7.24 (d, J = 8.3 Hz,
2H), 7.17 (s, 1H), 7.02 – 6.91 (m, 2H), 6.68 (s, 1H), 4.87 (s, 1H),
4.06 (t, J = 6.4 Hz, 1H), 3.95 (s, 4H), 2.90 (d,
J = 11.1 Hz, 1H), 2.84 (d, J = 11.1
Hz, 1H), 2.42 (s, 3H), 2.39 (d, J = 5.3 Hz, 1H), 2.34 (d,
J =5.7 Hz, 1H), 2.32 (d, J = 5.6 Hz,
1H), 2.23 (t, J = 6.7 Hz, 1H), 2.05 – 1.92 (m, 3H),
1.74 (dt, J = 12.6, 6.2 Hz, 1H), 1.66 – 1.58 (m,
2H), 1.52 (dd, J = 23.3, 11.6 Hz, 2H). ESI-MS m/z:
535.20[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.04 (s), 161.84 (s),
161.65 (s), 153.87 (s), 149.19 (s), 147.14 (s), 139.35 (s), 130.30 (s),
127.16 (s), 121.49 (s), 120.98 (s), 113.92 (s), 112.99 (s), 101.53 (s),
72.26 (s), 66.89 (s), 61.59 (s), 54.13 (d, J = 13.3 Hz),
42.10 (s), 41.79 (s), 32.00 (s), 29.08 (s), 15.67 (s).</p><!><p>A solution of compound NO.1 (0.267 mmol), phenol
derivative (2 eq), and PPh3(1.2 eq) was stirred in dry THF (4mL)
and DMF(0.5mL) at 0D under a nitrogen atmosphere. To this mixture was added
dropwise DIAD (1.2 eq) over a period of 5 min, and the reaction was
monitored by TLC. After 4.5h, the solvent was evaporated under reduced
pressure and the resulting oil purified by flash column chromatography,
eluting with 0-12% methanol in dichloromethane to give compound
NO.46.Yield: 68 %.1H NMR (500 MHz, DMSO)
Î´ 7.71 (d, J = 8.9 Hz, 1H), 7.62 (d,
J = 1.2 Hz, 2H), 7.61 (d, J = 1.2 Hz,
4H), 7.60 (s, 3H), 7.58 (s, 2H), 7.55 (d, J = 3.1 Hz, 3H),
7.54 (d, J = 2.9 Hz, 3H), 7.52 (s, 1H), 4.25 (ddd,
J = 18.5, 12.2, 5.6 Hz, 3H), 3.93 (s, 2H), 2.87 (s,
3H), 2.71 (s, 3H), 2.41 (s, 3H), 1.16 (d, J = 6.2 Hz, 11H).
ESI-MS m/z: 545.00[M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 178.40 (s), 164.14 (s),
163.06 (s), 158.00 (s), 150.17 (s), 141.14 (s), 135.08 (s), 133.97 (d,
J = 18.1 Hz), 133.32 (d, J = 9.7 Hz),
130.59 (d, J = 11.8 Hz), 130.25 (s),129.85 (s), 128.47 (s),
127.90 (s), 126.63 (s), 122.95 (s), 117.15 (s), 114.42 (s), 103.00 (s),
69.66 (s), 63.53 (s), 37.61 (s), 33.98 (s), 32.61 (s), 31.08 (s), 23.75 (s),
17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.46.1H NMR (500 MHz, DMSO)
Î´ 7.70 (d, J = 8.9 Hz, 1H), 7.25 (t,
J = 7.5 Hz, 2H), 7.20 (d, J = 7.1 Hz,
2H), 7.16 (t, J = 7.1 Hz, 2H), 7.08 (t, J
= 8.8 Hz, 1H), 7.01 (d, J = 2.4 Hz, 1H), 6.99 –6.93
(m, 2H), 6.66 (s, 1H), 4.26 (dd, J = 18.9, 5.9 Hz, 1H),
4.13 (dd, J = 19.2, 5.7 Hz, 1H), 4.01 (dd,
J = 14.2, 7.1 Hz, 1H), 3.93 (s, 2H), 2.40 (s, 3H), 1.97
(s, 1H), 1.38 (s, 4H), 1.18 – 1.12 (m, 5H). ESI-MS m/z: 545.33[M+H]
+.</p><p>13C NMR (101 MHz, DMSO) Î´ 178.39 (s), 163.05 (s),
162.69 (s), 156.57 (s), 155.18 (s), 150.16 (s), 141.14 (s), 130.25 (s),
129.85 (s), 128.17 (d, J = 55.6 Hz), 127.85 – 127.17
(m), 122.94 (s), 117.75 (s), 117.52 (s), 114.99 (d, J =
105.3 Hz), 102.98 (s), 68.30 (s), 63.58 (s), 61.59 (s), 51.48 (s), 43.82
(s), 33.97 (s), 31.07 (s), 28.18 (s), 23.63 (s), 17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.46.1H NMR (500 MHz, MeOD)
Î´ 8.52 (d, J = 8.8 Hz, 1H), 8.05 (d,
J = 7.3 Hz, 2H), 8.01 (d, J = 7.2 Hz,
3H), 7.97 (d, J = 7.3 Hz, 2H), 7.82 (d, J
= 2.3 Hz, 1H), 7.80 – 7.73 (m, 2H), 7.69 (d, J =9.1
Hz, 2H), 7.64 (s, 2H), 5.10 (d, J = 10.0 Hz, 1H), 4.92 (d,
J = 5.5 Hz, 1H), 4.82 (dd, J =
14.3,7.2 Hz, 2H), 4.74 (s, 2H), 4.48 (s, 3H), 3.22 (s, 4H), 2.78 (s, 2H),
2.70 (s, 1H), 2.45 (d, J = 11.8 Hz, 2H), 2.29 (d,
J = 10.9 Hz, 2H), 1.97 (t, J = 7.1 Hz,
5H). ESI-MS m/z: 557.17[M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 178.40 (s), 163.06 (s),
155.18 (s), 154.23 (s), 150.18 (s), 141.15 (s), 130.25 (s), 129.85 (s),
128.45 (s), 127.90 (s), 122.92 (s), 117.40 (s), 116.42 (s), 114.98 (d,
J =104.0 Hz), 114.38 – 114.16 (m), 102.98 (s),
63.65 (s), 57.18 (s), 51.38 (s), 43.83 (s), 33.97 (s), 31.7 (s), 23.67 (s),
17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.46.1H NMR (500 MHz, DMSO)
Î´ 8.18 (dd, J = 9.2, 4.8 Hz, 2H), 7.70 (d,
J = 8.9 Hz, 1H), 7.24 (d, J = 7.3 Hz,
3H), 7.20 (s, 1H), 7.18 (s, 2H), 7.16 (d, J = 4.7 Hz, 2H),
7.02 (s, 1H), 6.97 (dd, J = 8.9, 2.5 Hz, 1H), 6.67 (s, 1H),
4.31 (ddd, J = 23.7, 14.3, 4.9 Hz, 4H), 4.01 (dd,
J = 14.2, 7.1 Hz, 1H), 3.92 (s, 2H), 2.97 (d,
J = 11.0 Hz, 2H), 2.40 (s, 3H), 2.37 (s, 1H), 2.02 (d,
J = 11.9 Hz, 1H), 1.97 (s, 1H), 1.89 (s, 1H), 1.64 (d,
J = 11.0 Hz, 3H), 1.48 (d, J = 11.6
Hz, 2H), 1.38 (s, 1H), 1.16(t, J = 7.1 Hz, 1H). ESI-MS m/z:
572.27[M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 178.36 (s), 165.55 (s),
163.04 (s), 162.61 (s), 155.16 (s), 150.15 (s), 142.73 (s), 141.13 (s),
130.25 (s), 129.85 (s), 128.44 (s), 127.79 (d, J = 20.8
Hz), 122.96 (s), 117.90 (s), 117.02 (s), 115.54 (s), 114.47 (s), 111.36 (s),
103.01 (s), 68.67 (s), 68.28 (s), 63.37 (s), 51.38 (d, J =
20.8 Hz), 51.11 – 50.90 (m), 43.78 (s), 33.97 (s), 31.35 –
31.15 (m), 30.74 (d, J = 63.4 Hz), 28.17 (s), 19.96 (s),
17.03 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.46.1H NMR (500 MHz, DMSO)
δ 7.72 (dd, J = 8.8, 4.8 Hz, 3H), 7.28 – 7.25
(m, 2H), 7.21 (d, J = 7.1 Hz, 3H), 7.18 (d,
J = 7.2 Hz, 1H), 7.05 (d, J = 2.2 Hz,
1H), 7.01 (d, J = 2.3 Hz, 1H), 6.99 (d, J
= 2.5 Hz, 1H), 6.98 (s, 2H), 6.96 (s, 1H), 4.47 (s, 3H), 4.35 (d,
J = 16.5 Hz, 2H), 4.25 (d, J =16.1 Hz,
2H), 4.03 (q, J = 7.1 Hz, 2H), 3.94 (s, 2H), 2.42 (s, 3H),
1.98 (s, 2H), 1.91 (s, 1H), 1.17 (t, J = 7.1 Hz, 2H).
ESI-MS m/z: 625.30[M+H] +.</p><p>13C NMR (101 MHz, DMSO) Î´ 169.91 (s), 163.04 (s),
155.16 (s), 151.40 (s), 150.17 (s), 141.13 (s), 130.26 (s), 129.85 (s),
128.58 (d, J = 16.9 Hz), 127.91 (s), 123.04 (s), 116.41
(s), 114.48 (s), 103.09 (s), 101.35 (s), 64.63 (s), 63.78 (s), 51.60 (s),
33.98 (s), 30.59 (s), 17.04 (s).</p><!><p>To a solution of compound NO.13 (0.112 mmol) in 5
mLTHF was added MeI(1.1 eq) slowly and the mixture was stirred vigorously
for 15 min at room temperature. To the resulting solution, Ag2O
(1.6 eq) was added slowly. The reaction mixture was stirred at room
temperature and the progress of the reaction was monitored by TLC. When
complete, the reaction mixture was extracted with ethyl acetate. The organic
layer was washed with brine two times and dried over anhydrous
Na2SO4. After filtration, organic layer is
concentrated under reduced pressure. The reaction mixture was concentrated
under reduced pressure. Then, the crude product was purified by column
chromatography to give compound NO.51. Reaction time: 24h.
Yield: 40.4%.1H NMR (500 MHz, DMSO) Î´ 8.39 (dd,
J = 4.4, 1.5 Hz, 2H), 7.75 – 7.71 (m, 1H), 7.68
(dd, J = 5.9, 3.2 Hz, 1H), 7.30 – 7.26 (m, 2H), 7.25
– 7.21 (m, 4H), 7.18 (t, J = 7.1 Hz, 1H), 6.94 (d,
J = 2.5 Hz, 1H), 6.92 (d, J = 2.4 Hz,
1H), 4.89 (s, 1H), 4.45 (s, 1H), 4.14 (t, J = 5.3 Hz, 1H),
3.99 (t, J = 6.4 Hz, 1H), 3.95 (d, J =
10.0 Hz, 2H), 3.93 – 3.85 (m, 2H), 3.45 – 3.38 (m, 2H), 2.72
(t, J = 8.3 Hz, 2H), 2.70 – 2.62 (m, 2H), 2.57 (dd,
J = 12.7, 6.3 Hz, 1H), 2.44 (s, 3H), 2.29 (s, 2H), 1.63
(dd, J = 12.0, 6.0 Hz, 1H), 1.40 – 1.33 (m, 1H),
1.30 (d, J = 8.5 Hz, 3H), 0.93 – 0.82 (m, 3H).
ESI-MS m/z: 459.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.71 (s), 153.80 (s),
150.02 (s), 149.70 (s), 148.84 (s), 139.79 (s), 132.06 (s), 129.12 (s),
128.89 (s), 128.48 (s), 127.04 (s), 126.52 (s), 124.70 (s), 121.45 (s),
113.95 (s), 112.92 (s), 101.42 (s), 71.94 (s), 67.88 (s), 67.31 (s), 63.26
(s), 60.16 (s), 58.60 (s), 43.11 (s), 38.55 (s), 32.53 (d,
J = 18.4 Hz), 30.27 (s), 28.83 (s), 23.72 (s), 22.86
(s), 15.67 (s), 14.36 (s), 11.27 (s).</p><!><p>To a solution of thiazol-2-amine (10 mmol) in 2 mL DMF,
K2CO3 (1.6 eq) was added slowly and the mixture
was stirred vigorously for 15 min at room temperature. To the resulting
solution, 1, 3-dibromopropane (3 equiv, 6057 mg,30 mmol) in 2 mL of DMF was
added dropwise. The reaction mixture was heated at 60 °C, and the
progress of the reaction was monitored by TLC. The reaction mixture was
quenched by addition of water and extracted with ethyl acetate. The organic
layer was washed with brine two times and dried over anhydrous
Na2SO4. After filtration, organic layer is
concentrated under reduced pressure. Then, the crude product was purified by
column chromatography using ethyl acetate-hexane. Reaction time: 24 h,
Yield: 11.5%.</p><p>To a solution of 3-benzyl-7-hydroxy-4-methyl-2H-chromen-2-one
(0.338 mmol) in DMF (2 mL) was added TBAI (2.2 eq) and CsCO3 (2
eq). To the resulting solution, the above-mentioned products (2 eq) was
added dropwise. The mixture was stirred for 31 h at 100 °C, followed
by addition of 2 mL water. The mixture was extracted with 2×10 mL
ethyl acetate, and the organic phase was dried over anhydrous Na2SO4 and
concentrated. The residue was purified by column chromatography on silica
gel (hexane-EtOAc ) to give compound NO.52. Yield: 21 %.</p><p>1H NMR (600 MHz, DMSO) Î´ 7.73 (d,
J = 8.5 Hz, 1H), 7.61 (s, 1H), 7.27 (d,
J = 7.5, Hz, 2H), 7.23 (d, J = 7.4 Hz,
2H), 7.18 (t, J = 7.2 Hz, 1H),7.01 (d, J =
3.6 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.61(d,
J = 3.6 Hz, 1H), 4.16 (t, J = 6.2 Hz,
2H), 3.99 (t, J = 6.4 Hz, 1H), 3.95 (d, J
= 10.0 Hz, 2H), 3.93 – 3.85 (m, 2H), 3.45 – 3.38 (m, 2H), 2.72
(t, J = 8.3 Hz, 2H), 2.70 – 2.62 (m, 2H), 2.57 (dd,
J = 12.7, 6.3 Hz, 1H), 2.44 (s, 3H), 2.29 (s, 2H), 1.63
(dd, J = 12.0, 6.0 Hz, 1H), 1.40 – 1.33 (m, 1H),
1.30 (d, J = 8.5 Hz, 3H), 0.93 – 0.82 (m, 3H).
ESI-MS m/z: 407.00[M+H] +.</p><p>13C NMR (151 MHz, DMSO) Î´ 169.65 (s), 161.71 (s),
161.49 (s), 153.84 (s), 148.84 (s), 139.79 (s), 139.23 (s), 128.89 (s),
128.49 (s), 127.08 (s), 126.53 (s), 121.49 (s), 114.01 (s), 112.97 (s),
106.45 (s), 101.43 (s), 66.42 (s), 41.69 (s), 32.60 (s), 28.67 (s), 15.66
(s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
δ 7.95 (s, 1H), 7.90 (s, 1H), 7.29 – 7.19 (m, 4H), 7.14 (s,
2H), 6.99 (s, 1H), 6.76 (s, 1H), 6.64 (s, 1H), 5.88 (d, J =
6.4 Hz, 1H), 5.84 (s, 1H), 4.15 – 3.88 (m, 5H), 3.65 (d,
J = 13.0 Hz, 1H), 3.04 – 2.96 (m, 1H), 2.83 (s,
1H), 2.76 (s, 1H), 2.67 – 2.57 (m, 1H), 2.32 (dd, J
= 7.4, 2.9 Hz, 1H), 2.29 – 2.23 (m, 1H), 2.14 (d, J
= 7.3 Hz, 1H), 1.96 (s, 1H), 1.91 (s, 1H), 1.87 (s, 1H), 1.72 (t,
J = 15.3 Hz, 1H), 1.60 (s, 2H), 1.55 – 1.46 (m,
2H), 1.41 (d, J = 4.5 Hz, 1H), 1.38 (d, J
= 4.7 Hz, 1H), 1.34 (s, 1H), 1.31 (d, J = 5.8 Hz, 1H), 1.28
(d, J = 3.5 Hz, 1H), 1.22 (s, 2H), 1.07 (dd,
J = 8.1, 2.3 Hz, 1H), 0.90 – 0.80 (m, 2H).
ESI-MS m/z: 477.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.06 (s), 176.19 (s),
161.16 (s), 160.04 (s), 141.03 (s), 129.00 (s), 128.83 (s), 128.65 (s),
126.30 (s), 105.63 (s), 66.87 (s), 61.54 (s), 54.11 (s), 44.92 (s), 42.09
(s), 38.86 (s), 29.28 (d, J = 56.3 Hz), 28.24 (s), 14.42
(s), 11.84 (s), 9.56 (s), 9.07 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.56 (d, J = 6.4 Hz, 1H), 7.53 (d,
J = 2.3 Hz, 1H), 7.52 (s, 1H), 7.50 (d,
J = 6.7 Hz, 1H), 7.33 – 7.29 (m, 1H), 7.26 (dd,
J = 7.6, 1.7 Hz, 1H), 7.21 – 7.16 (m, 4H), 7.15
– 7.09 (m, 2H), 7.06 (d, J = 2.5 Hz, 1H), 7.04 (s,
1H), 7.00 (t, J = 7.2 Hz, 2H), 6.89 (d, J
= 2.4 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.83 (s, 1H), 6.82
(s, 1H), 6.69 (d, J = 7.8 Hz, 1H), 4.89 (d,
J = 27.9 Hz, 1H), 4.14 (d, J = 5.2 Hz,
1H), 4.08 (d, J = 6.4 Hz, 1H), 4.04 – 4.00 (m, 1H),
3.96 (d, J = 6.5 Hz, 1H), 3.83 (d, J = 5.4
Hz, 1H), 3.61 (d, J = 4.8 Hz, 2H), 3.17 (d,
J = 4.9 Hz, 1H), 2.91 (d, J =11.0 Hz,
1H), 2.84 (t, J = 11.1 Hz, 1H), 2.74 (d, J
= 11.1 Hz, 1H), 2.42 (dd, J = 12.7, 5.4 Hz, 1H), 2.38
– 2.31 (m, 1H), 2.31 – 2.24 (m, 1H), 2.07 – 1.97 (m,
2H), 1.91 (dd, J = 21.2, 10.3 Hz, 1H), 1.61 (s, 2H), 1.58
– 1.46 (m, 2H). ESI-MS m/z: 513.10[M+H] +.</p><p>13C NMR (126 MHz, DMSO) δ 177.11 (s), 161.92 (s),
161.51 (s), 159.64 (s), 154.32 (s), 153.24 (s), 152.30 (s), 140.52 (s),
139.54 (d, J = 8.9 Hz), 134.70 (d, J = 9.2
Hz), 129.25 (d, J = 11.2 Hz), 128.93 – 128.85 (m),
128.69 (t, J = 13.4 Hz), 128.37 (s), 126.85 (s), 126.35 (d,
J = 22.3 Hz), 121.41 (s), 114.06 (s), 113.38 (s),
113.22 (s), 101.66 (s), 100.07 (s), 72.27 (s), 72.08 (s), 66.85 (s), 61.51
(s), 54.09 (d, J = 11.9 Hz), 49.07 (s), 42.06 (s), 35.42
(s), 33.75 (s), 29.02 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (400 MHz, DMSO)
δ 7.29 – 7.19 (m, 1H), 7.15 (t, J = 8.0 Hz,
2H), 7.03 – 6.92 (m, 1H), 6.66 (s, 1H), 6.51 (dd, J
= 8.2, 2.6 Hz, 2H), 4.05 – 3.86 (m, 5H), 3.85 – 3.78 (m, 1H),
3.73 (dd, J = 11.1, 4.6 Hz, 1H), 3.67 – 3.61 (m,
1H), 2.86 (s, 1H), 2.07 (s, 2H), 1.97 (s, 2H), 1.68 – 1.47 (m, 3H),
1.22 (s, 1H), 1.16 (t, J = 7.1 Hz, 2H), 1.08 (d,
J = 7.9 Hz, 1H), 0.89 – 0.81 (m, 1H). ESI-MS
m/z: 387.00[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 177.05 (s), 160.43 (s),
160.04 (s), 130.44 (s), 129.80 – 129.04 (m), 128.83 (d,
J = 42.9 Hz), 107.61 (s), 107.14 (s), 101.76 (s), 71.57
(s), 69.42 (s), 69.06 (s), 66.92 (s), 61.70 (s), 60.22 (s), 54.05 (s), 47.18
(s), 42.04 (s), 31.16 (s), 29.00 (s), 21.23 (s), 14.55 (s), 11.84 (s), 9.56
(s), 9.22 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.73 (d, J = 9.6 Hz, 1H), 7.34 (d,
J = 8.6 Hz, 2H), 7.24 (d, J = 8.2 Hz,
2H), 7.01 – 6.94 (m, 2H), 6.84 (d, J = 4.5 Hz, 1H),
6.15 (s, 1H), 4.16 (d, J = 6.9 Hz, 1H), 4.01 (ddd,
J = 16.1, 10.4, 5.2 Hz, 3H), 3.96 (s, 2H), 3.92 (d,
J = 3.9 Hz, 1H), 3.81 (dd, J = 13.9,
7.3 Hz, 1H), 2.43 (s, 3H), 1.21 (d, J = 4.8 Hz, 4H), 0.84
(t, J = 6.8 Hz, 1H). ESI-MS m/z: 507.11981[M+H]
+, delta <0.5 ppm.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.61 (s), 161.44 (s),
153.80 (s), 149.18 (s), 147.14 (s), 139.31 (s), 130.29 (s), 127.22 (s),
121.49 (s), 121.13 (s), 114.11 (s), 113.00 (s), 101.52 (s), 71.02 (s), 67.25
(s), 49.75 (s), 32.00 (s), 29.47 (s), 15.69 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 8.44 (dd, J = 15.6, 4.5 Hz, 6H), 7.73 (d, J = 9.4 Hz, 2H), 7.35 (d,
J =8.3 Hz, 3H), 7.29 (d, J = 6.6 Hz, 2H), 7.28 – 7.20 (m, 8H), 6.96
(s, 2H), 5.75 (s, 1H), 5.43 (s, 1H), 5.02 (d, J = 34.1 Hz, 1H), 4.06 (dd, J
= 9.8, 4.0 Hz, 1H), 3.97 (s, 2H), 3.95 – 3.92 (m, 2H), 3.89 (d, J =
6.9 Hz, 1H), 3.83 (s, 1H), 3.61 (dd, J = 10.9, 4.4 Hz, 2H), 3.53 (dd, J =
11.0, 5.3 Hz, 2H), 3.25 (d, J = 6.3 Hz, 2H), 2.81 (s, 2H), 2.76 –
2.71 (m, 5H), 2.69 (d, J = 4.7 Hz, 1H), 2.64 (d, J = 6.2 Hz, 1H), 2.44 (s,
3H), 2.07 (d, J = 10.3 Hz, 1H), 1.98 (d, J = 10.5 Hz, 1H), 1.76 (d, J = 16.2
Hz, 1H), 1.19 (dd, J = 21.0, 13.9 Hz, 1H), 1.07 (d, J = 30.0 Hz, 1H). ESI-MS
m/z: 529.40 [M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.69 (d, J = 9.6 Hz),
156.43 (s), 153.84 (s), 149.82 (d, J = 11.4 Hz), 148.69 (s), 147.14 (s),
139.33 (s), 130.28 (s), 127.15 (s), 124.70 (s), 121.48 (s), 120.99 (s),
113.91 (s), 113.01 (s), 101.47 (s), 71.82 (s), 68.84 (s), 68.43 (s), 65.29
(s), 52.40 (s), 50.29 (s), 47.8 (s), 41.08 (s), 35.48 (s), 34.91 (s), 31.99
(s), 15.67 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
δ 7.74 (d, J = 9.3 Hz, 1H), 7.61 – 7.55 (m,
7H), 7.54 (s, 1H), 7.51 (dd, J = 8.1, 3.2 Hz, 4H), 7.44
– 7.38 (m, 6H), 7.34 – 7.27 (m, 7H), 7.25 (d,
J = 8.2 Hz, 2H), 7.10 – 7.06 (m, 1H), 7.03
– 6.90 (m, 4H), 4.12 (s, 4H), 4.03 – 3.96 (m, 10H), 3.93 (dd,
J = 5.6, 4.1 Hz, 4H), 3.88 (dd, J =
15.0, 5.7 Hz, 9H), 3.76 – 3.71 (m, 3H), 3.64 (ddd, J
= 11.2, 7.0, 4.9 Hz, 3H), 2.45 (s, 2H), 1.97 (s, 3H), 1.89 (s, 2H), 1.21 (d,
J = 3.7 Hz, 5H), 1.15 (t, J = 7.1 Hz,
3H), 1.12 (s, 1H). ESI-MS m/z: 499.29[M+H] +.</p><p>13C NMR (126 MHz, DMSO) Î´ 172.51 (s), 161.69 (s),
160.05 (s), 140.58 (s), 140.42 (s), 131.16 (s), 130.67 (d,
J = 39.0 Hz), 130.27 – 129.98 (m), 129.98
– 129.27 (m), 129.09 (s), 127.58 (s), 127.21 (s), 126.93 (s), 121.57
(s), 112.98 (s), 107.54 (s), 102.71 – 101.92 (m), 101.68 (d,
J = 34.5 Hz), 100.22 (s), 70.90 (s), 70.29 (s), 69.44
(s), 69.05 (s), 67.27 (s), 67.09 (s), 60.22 (s), 50.47 (s), 49.6 (s), 47.16
(s), 34.96 (s), 32.28 (s), 29.47 (s), 21.56 (s), 21.23 (s), 14.55 (s).</p><!><p>The title compound was synthesized according to the procedure of
preparing Compound NO.1.1H NMR (500 MHz, DMSO)
Î´ 7.75 (d, J = 8.6 Hz, 1H), 7.34 (t,
J = 7.0 Hz, 3H), 7.30 (d, J = 7.4 Hz,
1H), 7.25 (d, J = 4.4 Hz, 2H), 7.24 – 7.19 (m, 3H),
6.99 (s, 1H), 6.97 (d, J = 2.5 Hz, 1H), 4.15 – 4.09
(m, 1H), 4.09 – 4.02 (m, 2H), 3.96 (s, 2H), 3.11 – 3.01 (m,
3H), 2.98 – 2.85 (m, 3H), 2.44 (s, 3H), 2.24 (t, J =
6.7 Hz, 1H), 1.90 (s, 1H), 1.77 – 1.70 (m, 1H), 1.67 – 1.59
(m, 1H), 0.88 – 0.81 (m, 1H). ESI-MS m/z: 528.00[M+H]
+.</p><p>13C NMR (126 MHz, DMSO) Î´ 161.62 (s), 161.37 (s),
153.84 (s), 149.18 (s), 147.17 (s), 139.32 (s), 138.59 (s), 130.31 (s),
129.05 (d, J = 11.5 Hz), 127.12 (d, J =
34.2 Hz), 121.51 (s), 121.20 (s), 114.16 (s), 113.05 (s), 101.58 (s), 71.14
(s), 66.11 (s), 50.53 (s), 49.47 (s), 41.80 (s), 33.15 (s), 32.01 (s), 26.91
(s), 15.70 (s).</p><!><p>The docking studies were prepared with Ligandscout (4.1) with MMFF 94
energy minimization. The HBV capsid protein (PDB: 6HRO) were prepared with
ligandscout (4.1) by removing the ligand and unnecessary water molecules. Then
the whole protein was selected for docking and the prepared molecules were
inserted. The docking was carried out by AutoDock Vina 1.1(Exhaustiveness =20,
Max. Energy difference=3). The halogen bond analysis was performed with
Flare.</p><!><p>We previously identified compound CP19 as an entry inhibitor
against Ebola virus infection. The preliminary SAR analysis of ten CP19 derivatives
indicated that the hydrophobic or aromatic substitutions on the C3 or C4 of coumarin
were vital for its antiviral activity and the carboxamide moiety was important for
its activity. However, the detailed SAR of these sites and the roles of the linker
between the coumarin and the piperidine were still elusive. In this research, we
performed a detailed SAR studies based on CP19 and elucidate the
different weights of these parts on their antivirus efficacy. Our analysis
discovered that the substitutions of large aromatic groups on the C3 and C4 of
coumarin could afford potent antiviral activities and the most important part of
CP19 derivatives was the substitution groups on C3 of coumarin. In
addition, the piperidine group could be optimized with heterocycles, but not with
small hydrophilic groups, which indicates that this moiety should have hydrogen bond
interactions with the GP protein. However, the substitutions on the linker would
decrease its activity, and the stereo structure had little influence on the
activity, which suggests that the linker structure did not take important role in
the interactions with the binding pocket.</p><p>Compound 32 was similar to compound 118a in its
carboxamide and piperidine groups, but compound 118a has more potent
activity to inhibit Ebolavirus entry. The crystal structure of Ebola GP complexed
with compound 118a and our studies strongly indicate that: (1) the
coumarin of compound 32 occupies almost the same pocket as the phenol
ring of compound 118a. However, the lactone of coumarin does not form
H-bond or strong electrostatic interactions with Ebola glycoprotein, and thus this
structure could be replaced by groups with smaller volume or better chemical
accessibility. (2) The trifluomethoxybenzyl group of compound 32
occupied more hydrophobic space in the binding pocket than the o-chlorobenzene group
of compound 118a, but the docking studies show that the halogen bond
between the chlorine atom of compound 118a and G67 of GP protein
contributes significantly to afford high binding score. Thereof, new derivatives of
compound 32 that has halogen bond with G67 probably could afford higher
activity.</p><!><p>Figure S1. The analysis of the binding mechanism of
hydroxyl group of compound 32 with docking studies. The
hydroxyl group (magenta arrow) was in the "neck" of the
tunnel-like binding pocket (PDB: 6HRO, Cyan) for the glycerol moiety of
compound 32 (Green) and the ethyl moiety of compound
118a (Yellow).</p><p>Figure S2. The studies binding mechanism of compound
32 in Marburg virus GP with Docking. (A). The
structure of Marburg virus GP (PDB: 6BP2, Green) is structurally similar
with Ebola virus GP (PDB: 6HRO, Salmon). The ligand of compound
118a (PDB: 6HRO, Yellow) was used to show the binding
pocket. (B) The binding model of compound 32
(Magenta) in the binding pocket of Marburg virus GP (PDB: 6BP2, Green).
Compound 32 binds in the same pocket as compound
118a (Yellow) but it showed a different pose. The coumarine
of compound 32 may form hydrogen bond with Gly547 and the
piperidine may form hydrogen bond with Glu45.</p>