Discovery of Antimetastatic Chiral Ionone Alkaloid Derivatives Targeting HIF-1α/VEGF/VEGFR2 Pathway

Article abstract of DOI:10.1002/cmdc.202100072

Novel chiral ionone alkaloid derivatives were synthesized and their antimetastatic effects were evaluated in human breast cancer cells using chemotaxis assay. Compared with positive control LY294002, a PI3 K inhibitor, derivatives 10 a, 11 a, 11 c, 11 g,

Full text of DOI:10.1002/cmdc.202100072

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Discovery of Antimetastatic Chiral Ionone Alkaloid
Derivatives Targeting HIF-1α/VEGF/VEGFR2 Pathway
Jing-Jing Liu+,[a] Xin-Yao Liu+,[a] Jiang-Ping Nie,[a] Mei-Qi Jia,[a] Yang Yu,[a] Nan Qin,*[a] and
Hong-Quan Duan*[a, b]
Novel chiral ionone alkaloid derivatives were synthesized and
their antimetastatic effects were evaluated in human breast
cancer cells using chemotaxis assay. Compared with positive
control LY294002, a PI3 K inhibitor, derivatives 10a, 11a, 11c,
11g, 11j, 11k and 11w exhibited significant inhibitory effects
against cancer cell migration. Especially, the IC50 for compound
11g was as low as 0.035�0.004 μM. Further investigations on
compound 11g revealed that it could exert inhibitory effects on
the adhesion, migration and invasion of MDA-MB-231 cells. The
mechanisms for the antitumor metastatic effects of 11g might
be through the inhibition of HIF-1α/VEGF/VEGFR2/Akt pathway,
which suppressed the downstream signaling molecules, includ-
ing Akt1/mTOR/p70S6K and Akt2/PKCζ/integrin β1 pathways.
Taken together, chiral ionone alkaloid derivative 11g has the
potential to be developed into an antitumor metastatic agent
for breast cancer.
1. Introduction
Breast cancer is the leading cause of deaths from malignancies
in women. The major obstacle in the management of breast
cancer, especially at advanced stages, is metastasis.[1] Metastasis
is a complex, multistep biological process, involving a multitude
of genes and biomolecules.[2] Hypoxia has been shown to be a
hallmark of cancer progression and is associated with the
metastatic potential of multiple solid tumors, where tumor cells
proliferate rapidly and form large solid tumor masses, leading
Figure 1. The structure of chiral ionone alkaloid derivative
to obstruction and compression of the blood vessels surround- 2. Results and Discussion
ing these masses.[3] Activation of hypoxia-inducible factor-1α
(HIF-1α) transcription factor is the most recognized pathway
adopted by hypoxic cells.[4] Accumulating evidences have
shown a strong correlation between elevated HIF-1α expression
and tumor metastasis, angiogenesis as well as poor patient
prognosis. Therefore, HIF-1α has been recognized as a potential
target for cancer therapy.
We previously reported that, a novel chiral ionone alkaloid
derivative (1) was able to inhibit EGF-induced invasion of MDA-
MB-231 cells.[5] In this study, the structure optimization of 1 was
performed and their antimetastatic activity was screened (Fig-
ure 1). Moreover, the molecular mechanisms of active deriva-
tives were investigated.
2.1. Chemistry
As shown in Scheme 1, the 6R-α-ionone was an appropriate
starting material. Several attempts were made to get 6R-α-
ionone from α-ionone. Although 6R-α-ionone could be total
synthesized through multiple steps,[6] it was not performed due
to expensive and toxic metal reagents. The multiple resolving
crystallization method used in our previous study was also
discarded because of low yields. It was reported that 6S- and
6R-α-ionone could be obtained by stereoselective lipase-
mediated esterification of α-ionol.[7] According to this, we
obtained a large amount of 6R-α-ionone (2) from α-ionone. The
[α]20 D (c=0.8 CHCl3) value of 2 was +441, which was as
similar as the total synthesis 6R-α-ionone. To get acid 3, 6R-α-
[a] J.-J. Liu,+ X.-Y. Liu,+ Dr. J.-P. Nie, M.-Q. Jia, Dr. Y. Yu, Dr. N. Qin, Prof. H.-
Q. Duan
°
ionone reacted with NaClO in EtOH for 8 h at 0 C. Compound 3
Tianjin Key Laboratory on Technologies Enabling Development Clinical
Therapeutics and Diagnostics (Theragnostic)
Tianjin Medical University
Tianjin (China)
E-mail: qinnan@tmu.edu.cn
was reacted with 3-fluorobenzylamine in the presence of HOBt,
EDCI and triethylamine in CH2Cl2 for 12 h at room temperature.
The resulted amide 4 was transformed into the corresponding
amines 5 using the catalytic hydrogenation with Raney nickel.
Then 5 was reduced to 6 with LiAlH4 at room temperature for
8 h. After the amine was protected by the (Boc)2O, the allyl
position was oxidized to get 8. Before reductive amination with
the corresponding aldehyde, the protecting group was re-
moved. Target compounds 10a–10e were synthesized by
nucleophilic substitution reaction. Compounds 11a–11x were
[b] Prof. H.-Q. Duan
Research Center of Basic Medical Sciences
Tianjin Medical University
Tianjin (China)
E-mail: duanhq@tmu.edu.cn
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202100072
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Scheme 1. The synthesis of compound 10a-10e and 11a-11x
prepared using reductive amination. To get 11n–11x, inter- 3. Biological assessment
mediate 12a–12k were synthesized by Suzuki coupling reac-
tion.
3.1. Derivatives screening and structure-activity relationship
Tumor cell migration is one of the prominent features of cancer
metastasis. Therefore the antimigratory effect of all derivatives
was evaluated using in vitro transwell chemotaxis with MDA-
MB-231 cells. The purities of compounds were determined by
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Table 1. Inhibitory effects of derivatives on the migration of MB-MDA-231 cells.
No.
IC50[a] [μM]
No.
IC50[a] [μM]
10a
10b
10c
10d
10e
11a
11b
11c
11d
11e
11f
11 g
11 h
11i
11j
11k
0.618�0.012
18.57�0.219
14.28�0.203
>20
11 l
1.179�0.018
12.850�0.155
1.399�0.028
8.880�0.079
9.891�0.046
>20
7.488�0.025
3.395�0.026
3.435�0.028
5.835�0.042
1.096�0.029
0.840�0.015
2.150�0.037
1.010�0.035
0.127�0.006
11 m
11n
11o
11p
11q
11r
11 s
11 t
11u
11v
>20
0.686�0.027
5.103�0.072
0.534�0.017
1.181�0.033
7.921�0.102
2.713�0.063
0.035�0.004
1.300�0.038
7.416�0.143
0.943�0.009
0.670�0.011
11w
11x
LY294002
1
[a] The data are represented as mean � S.D. of three independent experiments.
HPLC method, and the non-cytotoxic concentration of each
compound, which was determined by MTT assay (see Support-
ing Information, Table S1 and S2), was used for the chemotaxis
assay. The IC50 of the tested derivatives were listed in Table 1. In
comparison with LY294002 (a PI3K inhibitor served as positive
control[8]), derivatives 10a, 11a, 11c, 11g, 11j, 11k and 11w
showed obvious inhibitory effects on the migration of MDA-
MB-231 cells. Among them, derivative 11g was of highest
potential with IC50 value of 0.035 μM.
is a key component for molecular recognition.[11] Therefore, 11g
was chosen for the further investigation on the antimetastatic
activity and molecular mechanism.
3.2. The antimetastatic effect of derivative 11g
Derivative 11g supressed cell invasion at concentrations of 0.1–
25 μM in transwell assay (Figure 2) and cell migration at
concentrations of 0.1–25 μM in wound healing assay (Figure 3),
both in a dose-dependent manner. Furthermore, 11g (0.1–
Our previous study showed that fluorobenzene fragment in
compound 1 had excellent antimetastatic effects. In this work,
N-methyl group was optimized to obtain the compounds with
better activity. At the first round of this structure optimization,
10a–10e were designed and synthesized. The results showed
that amide, ester and acid derivatives had lower activities. The
alkynyl derivative 10a exhibited good anti-invasion effect with
IC50 of 0.618 μM. In some instances, aromatic ring and ethyln
group were considered as bioisosteres.[9] Their replacement was
widely used in structure optimization of lead compound. Based
on this, N-benzylamine derivatives 11a–11k were synthesized
in the second-round optimization. The biological evaluation of
these compounds showed that electron-deficient aromatic ring
derivatives 11a, 11c, 11g exhibited better anti-invasion activity.
The IC50 value of benzonitrile derivative 11g was four-fold less
than the lead compound 1. However, the substituted benzoni-
trile derivatives 11j and 11k did not exhibited better activities
than 11g. At the third-round of optimization, we constructed
the biaromatic rings derivatives, because biaromatic rings
structures often existed in small molecule tumor immunity
inhibitors.[10] Unfortunately, 11l–11x exhibited no better activity
than that of 11g. It was suggested that the nitrile group played
a crucial role in the anti-invasive effects. Nitrile-containing drugs
with diverse structures are widely used in a variety of medical
treatments. Surveying the interactions of the nitrile group
within these pharmaceuticals and drug candidates revealed
that the biological function of the nitrile group varies consid-
erably. Under some circumstances, the nitrile merely polarizes
the adjacent electron density, whereas in other cases the nitrile
Figure 2. Anti-invasive effect of 11g on MDA-MB-231 cells by transwell
assay. MDA-MB-231 cells were cultured in the upper chamber with matrigel
matrix. The cells that actively migrated to the lower surface of the filter were
determined. The data are represented as mean � S.D. of three independent
experiments. (*p<0.05, ***p<0.001, compared with control).
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the hypoxic area of tumor tissue to meet the demand for
oxygen and nutrients. Studies have shown that the angiogenic
function of VEGF was mediated by binding to its cognate
receptor VEGF receptor 2 (VEGFR-2)[15], which further promotes
the migration and invasion of tumor cells. The effect of 11g on
VEGF secretion in MDA-MB-231 cells was determined using
ELISA kit, and the expression and phosphorylation of VEGFR2
were determined by western blot. Results showed that 11g
dose-dependently down regulated both secreted (extracellular)
and intracellular VEGF (Figure 5). 11g also inhibited the
phosphorylation of VEGFR-2 in a dose-dependent manner
(Figure 6).
3.4. 11g suppressed hypoxia-induced HIF-1α expression
VEGF expression is regulated by HIF-1α, which binds to hypoxia
response elements within the gene promoter region of VEGF.[16]
HIF-1α is the most critical nuclear transcriptional regulator in
the regulation of hypoxic response. Hypoxia is closely related to
tumor invasion, metastasis, angiogenesis, and poor prognosis.[17]
Under non-hypoxic conditions, HIF-1α is rapidly degraded
through proteasomal degradation to the hydroxylation of
pro402 and/or 564 by prolyl hydroxylase enzyme (PHD), which
utilizes O2 as substrate. However, under hypoxic conditions,
decreased PHD activity results in the stabilization of HIF-1α
protein, which binds to the hypoxia response elements (HREs)
of the target gene promoter and activates downstream gene
transcription involved in angiogenesis and metastasis.[18] There-
fore identifying novel HIF-1α inhibitor has become a popular
trend in antitumor drugs research and development. The effect
of 11g on HIF-1α expression was determined by western blot.
Figure 3. Antimigratory effect of 11g on MDA-MB-231 cells by wound
healing assay. MDA-MB-231 cells were seeded in plates and generate a clean
wound area. After 24 h, cells were monitored and photographed under
microscope. The data are represented as mean � S.D. of three independent
experiments. (*p<0.05, ***p<0.001, compared with control).
25 μM) significantly decreased the adhesive capability of MDA-
MB-231 cells in adhesion assay (Figure 4).
3.3. 11g inhibited VEGF secretion and the phosphorylation of
VEGFR2 in MDA-MB-231cells
It is reported that β-Ionone exerts antitumor effects through
the Akt pathway.[12] To further reveal the molecular mechanism
of ionone alkaloid, we examined its effect on the expression
and secretion of vascular endothelial growth factors (VEGFs).
VEGFs are key mediators of angiogenesis, and are also reported
to be important upstream signaling molecules of Akt[13] and
crucial for the development and metastasis of tumors.[14] Tumor
cells secrete VEGFs to promote the growth of blood vessels in
Figure 5. The protein level of VEGF was detected by ELISA. (A) The
supernatant of breast cancer cells cultured with the compound in normoxia
or hypoxia was collected, then the content of VEGF was measured. (B) The
cell lysate of breast cancer cells cultured with the compound in hypoxia was
collected, then the content of VEGF was measured. The data are represented
as mean � S.D. of three independent experiments. (*p<0.05, **p<0.01,
compared with control).
Figure 4. Antiadhesive effect of 11g on MDA-MB-231 cells. The data are
represented as mean � S.D. of three independent experiments. (*p<0.05,
**p<0.01, compared with control).
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different concentrations of 11g, and the cells were cultured in
anoxic culture boxes filled with nitrogen of high purity. The
expression of HIF-1α protein was time-dependent under
hypoxia and higher than that in normal oxygen (Figure 7A). The
results showed that 11g significantly inhibited HIF-1α protein
expression with dose-dependence (Figure 7B). In addition, we
measured the expression of HIF-1α mRNA under hypoxia as
well (Figure 8A). After cells were starved and incubated with
compound 11g for 12 h, they were transferred to hypoxic
condition for 1 hour before mRNA extraction. The results
showed that 11g inhibited HIF-1α mRNA expression with dose-
dependence (Figure 8B).
In order to determine whether the inhibition of HIF-1α was
critical for the antimetastatic role of 11g, an agonist of HIF-1α,
dimethyloxallyl glycine (DMOG), was adopted. Results showed
that, upon administration of DMOG, the antimigratory activity
of 11g was abolished (Figure 9). Further investigation showed
that the inhibitory effect of 11g on pAkt was also eliminated by
DMOG (Figure 10). These rusults revealed that 11g is a HIF-1α
inhibitor that suppressed metastatic behavior of MDA-MB-231
cells through HIF-1α/VEGF/VEGFR2/Akt pathway.
Figure 6. Effects of 11g on the protein expression and phosphorylation of
VEGFR2 in MDA-MB-231 cells. The relative band density was determined by
densitometry with Image J software. (*p<0.05, **p<0.01, compared with
control).
3.5. 11g suppressed the downstream molecules of AKT
pathway
The hypoxic cell model was established by pre-starvation of
cells for 12 hours using starvation medium (containing only 1%
FBS). Then, it was replaced with fresh starvation media with
As key signaling molecules during tumor development, Akt
promotes tumor metastasis by activating downstream mole-
Figure 7. Influences of hypoxia and 11g on the protein expression level of HIF-1α in MDA-MB-231 cells. (A) the protein expression of HIF-1α in hypoxia-
induced MDA-MB-231 cells were determined by western blot at different times ; (B) MDA-MB-231 cells was induced by hypoxia for 24 h in the presence of
different concentrations of 11g; thereafter, the protein levels of HIF-1α was examined by western blot. (C, D) was HIF-1α relative protein level. The relative
band density was determined by densitometry with Image J software. (*p<0.05, **p<0.01, ***p<0.001, compared with control).
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Figure 10. Effect of DMOG combined with 11g on the phosphorylation of
Akt in hypoxia. The relative band density was determined by densitometry
with Image J software. (**p<0.01, compared with control).
Akt2 is more involved in metastatic transmission.[20] Akt2 has
been found to be able to promote tumor metastasis through
regulating downstream PKCζ, integrinβ1 and FAK.[21] Our results
showed that 11g reduced the phosphorylations of Akt2 and
Akt1. 11g also reduced the phosphorylations of mTOR, p70S6K,
4EBP1, PKCζ, integrin β1 and FAK, respectively. The above data
were consistent with the previous phenotypic results (Fig-
ure 11).
Figure 8. Influences of hypoxia and 11g on the HIF-1α mRNA level in MDA-
MB-231 cells. (A) the mRNA expression of HIF-1α in hypoxia-induced MDA-
MB-231 cells was determined by qRT-PCR at different times ; (B) MDA-MB-
231 cells was induced by hypoxia for 1 h in the presence of different
concentrations of 11g; thereafter, the mRNA expression of HIF-1α was
determined by qRT-PCR. The data are represented as mean � S.D. of three
independent experiments. (*p<0.05, **p<0.01, compared with control).
Recent studies have shown that β-ionone has properties of
antiproliferation, antimetastasis and apoptosis in vitro and
in vivo.[22] However, the antitumor activity of α-ionone alkaloid
is rarely reported. In this paper, a series of α-ionone alkaloid
derivatives were synthesized and screened, and a compound
(11g) with significant antitumor metastatic activity was inves-
tigated.
The development of HIF-1α inhibitors has attracted increas-
ing attention in cancer research: These inhibitors have different
mechanisms. (1) Inhibit HIF-1α mRNA expression. (2) Inhibit HIF-
1α translation. (3) Inhibit HIF-1α stabilization (including Hsp90
inhibitors and Histone deacetylase (HDAC) inhibitors). (4) Inhibit
HIF-1 dimerization. After nuclear translocation of HIF-1α, it
forms a dimer with HIF-1β to bind to downstream hypoxia
response elements. Inhibition of dimerization can inhibit the
activity of HIF-1α. (5) Inhibit HIF-1/DNA binding. (6) Inhibit HIF-1
transcriptional activity. Interruption of HIF-1α and HIF-1β bind-
ing to the co-factor p300 to form an active transcription
complex is the final step towards HIF-1 transactivation.[23] In this
study, compound 11g dose-dependently down regulated the
mRNA expression of HIF-1α. That may be one of the mecha-
nisms of 11g.
These inhibitors suppress tumor growth and metastasis
through different signaling pathways. For example, SYP-5
inhibited the tumor angiogenesis and cell invasion by decreas-
ing HIF-1α and HIF-1β expression through down regulated
EGFR/PI3K/Akt and MAPK/ERK pathway;[24] benzophenone-1B
inhibits the lung and breast cancer cells migration and invasion
by down regulated HIF-1α nuclear translocation and inhibited
Figure 9. Antimigratory effect of DMOG and 11g combined on MDA-MB-
231 cell in hypoxia by wound healing assay. The data are represented as
mean � S.D. of three independent experiments. (***p<0.001, compared
with control).
cules including mTOR, p70S6K and 4EBP1.[19] Studies have
shown that Akt1 is critical for the growth of breast cancer, while
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Figure 11. Effects of 11g on the protein expressions and phosphorylations of Akt, mTOR, p70S6K, FAK, 4EBP1, PKCζ and integrin β1 in MDA-MB-231 cells.
(A,C) The protein expressions and phosphorylations in MDA-MB-231 cells were determined by western blot analysis; (B,D) were relative protein level. The
relative bands density was determined by densitometry with Image J software. (*p<0.05, **p<0.01, ***p<0.001, compared with control).
the expressions of MMP-2 and MMP-9;[25] ursolic acid derivative 4. Conclusion
inhibited Hep3B cancer cell growth by suppressing the HIF-1
and VEGF expression;[26] bishonokiol A inhibited the breast
cancer cell migration and invasion through suppressing the
accumulation of HIF-1α, which was regulated by PI3K/Akt
pathway;[27] the HIF-1α inhibitor echinomycin suppressed
leukaemia cell growth through NOTCH signalling pathway.[28] In
this article,we reported a novel chiral ionone alkaloid derivative
11g, which inhibited breast cancer cell adhesion, migration and
invasion through the downregulation of the HIF-1α/VEGF/
VEGFR2/Akt pathway, and suppression of the downstream
molecules, Akt1/mTOR/p70S6K and Akt2/PKCζ/integrin β1/FAK.
The potential mechanism of 11g was showed as Figure 12.
To summarize, in this study, a series of novel chiral ionone
alkaloid derivatives were synthesized and their antitumor
activity were screened. Compound 11g exhibited significant
inhibitory effect on breast cancer cell adhesion, migration, and
invasion through the inhibition of HIF-1α and VEGF/VEGFR2/Akt
pathway, which suppressed the downstream molecules, Akt1/
mTOR/p70S6K and Akt2/PKCζ/integrin β1/FAK. Therefore, chiral
ionone alkaloid derivative 11g has the potential to be
developed into an antitumor metastatic agent for breast cancer.
Experimental Section
Chemistry
General procedure for synthesis of 3
To a solution of (6R)-α-ionone (10 g) in EtOH (110 mL) was added
°
NaClO solution (230 mL, 13%) in portions at 0 C. The reaction
mixture was stirred at room temperature for 8 h. When the reaction
was completed, the mixture was acidified to pH=1 with concen-
trated HCl. The reaction mixture was extracted with CH2Cl2
(150 mL), washed with water (50 mL), then washed with brine
(50 mL) and dried over MgSO4. After removal of the solvent, the
residue 3 was immediately used in the next reaction without
purification.
General procedure for synthesis of 4
To a solution of 3 (4.1 g, 21.1 mmol, 1 eq.) and 3-fluorobenzylamine
°
(2.65 mL, 23.2 mmol, 1.1 eq.)in CH2Cl2 (40 ml) at 0 C were added
HOBt (3.14 g, 23.2 mmol, 1.1 eq.), EDCI (3.14 g, 23.2 mmol, 1.1 eq.)
Figure 12. Potential antitumor metastasis mechanism of 11g
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and triethylamine(7.3 mL, 52.8 mmol, 2.5 eq.). The reaction mixture
was stirred at room temperature for 12 h and then concentrated
under reduced pressure. The residue was diluted with CH2Cl2
(70 mL) and the solution was washed with aqueous HCl (0.06 M,
20 mL), saturated aqueous NaHCO3 (40 mL), and brine (20 mL), the
organic layer was dried over MgSO4. After removal of the solvent,
the residue was purified by flash column chromatography (PE/EA=
2/1) to give 4.
a pad of celite and evaporated to dryness. The residue was purified
by column chromatography over silica gel (PE/EA=10/1) to afford
the crude product 8 which could be used in the next reaction.
Tert-butyl (R)-(3-fluorobenzyl)
(3-(1,1,5-trimethyl-3-oxocyclohex-4-en-6-yl)propyl)carbamate
(8)
Yield 57.3%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.28 (m, 1H,
ArÀ H), 7.07 (m, 2H, ArÀ H), 6.94 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.79
(s, 2H, Ar-CH2), 2.63 (t, J=6.8 Hz, 2H, 9-H), 2.36 (d, J=17.2 Hz, 1H, 2-
H), 2.01 (d, J=17.2 Hz, 1H, 2-H), 1.97 (s, 3H, 13-H), 1.86 (t, J=5.2 Hz,
1H, 6-H), 1.74 (m, 1H, 7-H), 1.60 (m, 2H, 8-H), 1.47 (s, 9H, (CH3)3), 1.43
(m, 1H, 7-H), 1.05 (s, 3H, 11-H), 1.02 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.2 (3-C), 165.5 (5-C), 163.0 (d, J=244.4 Hz, ArÀ C), 156.5
(CO), 138.5 (d, J=6.9 Hz, ArÀ C), 129.9 (d, J=8.1 Hz, ArÀ C), 125.2 (4-
C), 123.7 (d, J=2.2 Hz, ArÀ C), 114.9 (d, J=21.1 Hz, ArÀ C), 113.9 (d,
J=21.0 Hz, ArÀ C), 79.8 (C), 65.3 (Ar-CH2), 51.0 (6-C), 47.2 (2-C), 47.1
(9-C), 36.3 (1-C), 30.6 (12-C), 28.7 (7-C), 28.4 ((CH3)3), 27.4 (8-C), 27.1
(11-C), 24.6 (13-C).
(R, E)-N-(3-fluorobenzyl)-3-(1,1,5-trimethylcyclohex-4-en-6-yl)
acrylamide (4)
1
Yield 73.5%, colorless oil. H NMR (400 MHz, CDCl3): δ 7.21 (m, 1H,
ArÀ H), 7.00 (m, 1H, ArÀ H), 6.90 (m, 2H, ArÀ H), 6.68 (dd, J=15.2 Hz,
9.6 Hz, 1H, 7-H), 5.86 (d, J=15.2 Hz, 1H, 8-H), 5.43 (s, 1H, 4-H), 4.38
(d, J=5.6 Hz, 2H, Ar-CH2), 2.20 (d, J=9.6 Hz, 1H, 6-H), 1.99 (br s, 2H,
3-H), 1.54 (s, 3H, 13-H), 1.43 (m, 1H, 2-H), 1.16 (m, 1H, 2-H), 0.89 (s,
3H, 12-H), 0.82 (s, 3H, 11-H). 13C NMR (100 MHz, CDCl3): δ 166.2 (9-
C), 162.9 (d, J=244.7 Hz, ArÀ C), 145.7 (7-C), 141.2 (d, J=6.9 Hz,
ArÀ C), 132.2 (5-C), 130.0 (d, J=8.2 Hz, ArÀ C), 124.5 (8-C), 123.1 (d,
J=2.8 Hz, ArÀ C), 122.3 (4-C), 114.4 (d, J=21.6 Hz, ArÀ C), 114.1 (d,
J=20.9 Hz, ArÀ C), 53.8 (6-C), 42.9 (Ar-CH2), 32.4 (2-C), 31.2 (1-C),
27.7 (12-C), 26.8 (11-C), 23.0 (3-C), 22.9 (13-C).
General procedure for synthesis of 9
Compound 8 (2.3 g, 5.7 mmol, 1.0 eq.) was dissolved in CH2Cl2
(40 mL), TFA (1.27 mL, 17.1 mmol, 3.0 eq.) was added. Then the
reaction was stirred at room temperature for 5 h. The reaction
mixture was treated with saturated Na2CO3 solution until a pH of 8–
9 and extracted by CH2Cl2 (50 mL), washed with brine (50 mL). The
organic layers were dried by MgSO4, and the solvent was removed
in vacuo to afford the residue to yield the desired product 9.
General procedure for synthesis of 5
Weighed 3 g of compound 4 and dissolved it in THF(40 mL), added
Raney nickel mixture(15 mL), stirred at room temperature under
hydrogen for 8 h. The reaction mixture was filtered through a pad
of celite, collected the filtrate and concentrated to obtain crude 5,
which can be used for the next step without purification.
(R)-6-(3-((3-fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (9)
General procedure for synthesis of 6
Yield 92.3%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.27 (m, 1H,
ArÀ H), 7.07 (m, 2H, ArÀ H), 6.94 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.79
(s, 2H, Ar-CH2), 2.63 (t, J=7.2 Hz, 2H, 9-H), 2.36 (d, J=17.2 Hz, 1H, 2-
H), 2.01 (d, J=17.2 Hz, 1H, 2-H), 1.97 (s, 3H, 13-H), 1.86 (t, J=5.2 Hz,
2H, 9-H), 1.74 (m, 2H, 7-H), 1.60 (m, 2H, 8-H), 1.44 (m, 2H, 7-H), 1.05
(s, 3H, 12-H), 1.01 (s, 3H, 11-H). 13C NMR (100 MHz, CDCl3): δ 199.3
(3-C), 165.6 (5-C), 163.0 (d, J=244.4 Hz, ArÀ C), 142.5 (d, J=6.9 Hz,
ArÀ C), 129.9 (d, J=8.2 Hz, ArÀ C), 125.1 (4-C), 123.7 (d, J=2.2 Hz,
ArÀ C), 114.9 (d, J=21.1 Hz, ArÀ C), 113.9 (d, J=21.2 Hz, ArÀ C), 53.3
(d, J=1.4 Hz, Ar-CH2), 51.1 (6-C), 49.5 (9-C), 47.1 (2-C), 36.3 (1-C),
29.7 (12-C), 28.7 (7-C), 27.8 (8-C), 27.1 (11-C), 24.7 (13-C).
To the solution of crude amide 5 (3.16 g) in anhydrous THF (40 mL),
°
LiAlH4 (593 mg, 15.62 mmol) was added in portions at 0 C under
nitrogen. The reaction was stirred at room temperature for 24 h,
and then quenched by careful addition of 4 N aqueous NaOH
°
(5 mL) and saturated potassium sodium tartrate (40 mL) at 0 C. The
mixture was filtered in vacuo and the filtrate was evaporated. The
mixture of 6 or the mixture of 6 thus obtained was immediately
used in the next reaction without purification.
General procedure for synthesis of 7
To the mixture of crude 6 (5.6 g) and KHCO3 (4.84 g, 29.03 mmol) in
CH2Cl2 (100 mL) was added a solution of Boc2O (10.5 g, 21.29 mmol)
in CH2Cl2 (5 mL) when the mixture was stirred in an ice bath. The
mixture was stirred at room temperature for 12 h, evaporated just
to dryness with a vacuum evaporator. The reaction mixture was
extracted with CH2Cl2 (50 mL), washed with water (50 mL), then
washed with brine (50 mL), and the organic layer was dried over
MgSO4. The solvent was evaporated under reduced pressure to give
the crude product, which was separated by column chromatog-
raphy (PE/EA=20/1) to obtain pure 7.
General procedure for synthesis of 12a–12k
3-Bromo-2-fluorobenzaldehyde (1.0 eq.) was dissolved in solvent
(1,4-dioxane/H2O=5/1), Substituted phenylboronic acid (1.8 eq.),
K2CO3 (5.0 eq.), Pd[P(C6H5)3]4 (0.07 eq.) were added. Then the
reaction was stirred at reflux temperature. The reaction mixture was
filtered through a pad of celite. The filtration was finally purified by
column chromatography in PE/EA as eluent to afford the target
compounds 12a–12k.
General procedure for synthesis of 8
2-fluoro-2’-methoxy-[1,1’-biphenyl]-3-carbaldehyde (12a)
1
To a suspension of CrO3 (11.7 g, 115.5 mmol, 10.0 eq.) in CH2Cl2
Yield 94.2%, white solid, H NMR (400 MHz, CDCl3): δ 10.42 (s, 1H,
°
(120 mL) at À 20 C, 3,5-dimethylpyrazole (11.25 g, 115.5 mmol,
CHO), 7.86 (m, 1H, ArÀ H), 7.60 (m, 1H, ArÀ H), 7.40 (m, 1H, ArÀ H),
7.28 (m, 2H, ArÀ H), 6.99 (m, 2H, ArÀ H), 3.81 (s, 3H, OCH3).13C NMR
(100 MHz, CDCl3): δ 187.7 (d, J=7.9 Hz, CHO), 162.2 (d, J=258.6 Hz,
ArÀ C), 156.9 (ArÀ C), 138.3 (d, J=5.1 Hz, C), 131.5 (ArÀ C), 131.2 (d,
J=1.0 Hz, ArÀ C), 130.1 (ArÀ C), 127.8 (d, J=9.7 Hz, C), 127.6 (d, J=
°
10.0 eq.) was added and the resulting mixture was stirred at À 20 C
for 10 min. A solution of 7 (4.5 g, 11.55 mmol, 1.0 eq.) in CH2Cl2
(8 mL) was then added dropwise and the mixture was stirred
continuously for 5 h. The reaction mixture was then filtered through
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1.8 Hz, C), 124.2 (d, J=4.1 Hz, ArÀ C), 123.5 (ArÀ C), 120.7 (ArÀ C),
111.1 (ArÀ C), 55.7 (d, J=2.3 Hz, OCH3).
7.22 (m, 3H, ArÀ H). 13C NMR (100 MHz, CDCl3): δ 187.0 (d, J=7.8 Hz,
CHO), 161.7 (d, J=259.8 Hz, ArÀ C), 150.9 (dd, J=247.5 Hz, 12.9 Hz,
ArÀ C), 148.1 (dd, J=249.6 Hz, 13.2 Hz, ArÀ C), 137.6 (dd, J=4.3 Hz,
1.2 Hz, ArÀ C), 128.9 (d, J=2.0 Hz, ArÀ C), 126.1 (dd, J=3.1 Hz, 1.1 Hz,
ArÀ C), 124.7 (d, J=4.4 Hz, ArÀ C), 124.5 (d, J=8.7 Hz, ArÀ C), 124.3
(dd, J=4.6 Hz, 2.3 Hz, ArÀ C), 124.2 (d, J=2.8 Hz, ArÀ C), 123.7 (dd,
J=15.2 Hz, 2.4 Hz, ArÀ C), 117.6 (d, J=17.1 Hz, ArÀ C).
2-fluoro-2’-methyl-[1,1’-biphenyl]-3-carbaldehyde (12b)
1
Yield 95.1%, white solid, H NMR (400 MHz, CDCl3): δ 10.42 (s, 1H,
CHO), 7.89 (m, 1H, ArÀ H), 7.52 (m, 1H, ArÀ H), 7.31 (m, 4H, ArÀ H),
7.21 (m, 1H, ArÀ H), 2.22 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3): δ
187.5 (d, J=7.7 Hz, CHO), 161.7 (d, J=256.6 Hz, ArÀ C), 137.9 (d, J=
5.1 Hz, ArÀ C), 136.7 (ArÀ C), 134.2 (ArÀ C), 130.6 (d, J=16.6 Hz, ArÀ C),
130.2 (ArÀ C), 130.0 (ArÀ C), 128.6 (ArÀ C), 127.8 (d, J=1.6 Hz, ArÀ C),
125.9 (ArÀ C), 124.5 (d, J=4.3 Hz, ArÀ C), 124.3 (d, J=8.9 Hz, ArÀ C),
20.0 (d, J=2.7 Hz, CH3).
2,2’-difluoro-3’-methyl-[1,1’-biphenyl]-3-carbaldehyde (12h)
1
Yield 93.6%, white solid, H NMR (400 MHz, CDCl3): δ 10.42 (s, 1H,
CHO), 7.90 (m, 1H, ArÀ H), 7.63 (m, 1H, ArÀ H), 7.33 (m, 1H, ArÀ H),
7.26 (m, 1H, ArÀ H), 7.19 (m, 1H, ArÀ H), 7.13 (m, 1H, ArÀ H), 2.34 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3): δ 187.3 (d, J=7.7 Hz, CHO),
161.9 (d, J=259.1 Hz, ArÀ C), 158.2 (d, J=245.4 Hz, ArÀ C), 137.9 (d,
J=4.3 Hz, ArÀ C), 137.8 (d, J=4.4 Hz, ArÀ C), 132.0 (d, J=5.3 Hz,
ArÀ C), 128.8 (d, J=1.9 Hz, ArÀ C), 128.1 (d, J=1.7 Hz, ArÀ C), 125.5
(d, J=17.2 Hz, ArÀ C), 125.2 (d, J=15.4 Hz, ArÀ C), 124.5 (d, J=
4.2 Hz, ArÀ C), 123.8 (d, J=4.3 Hz, ArÀ C), 121.7 (d, J=16.2 Hz, ArÀ C),
14.7 (d, J=4.4 Hz, ArÀ C).
2’-chloro-2-fluoro- [1,1’-biphenyl] À 3-carbaldehyde (12c)
1
Yield 91.5%, white solid, H NMR (400 MHz, CDCl3): δ 10.42 (s, 1H,
CHO), 7.93 (m, 1H, ArÀ H), 7.58 (m, 1H, ArÀ H), 7.52 (m, 1H, ArÀ H),
7.36 (m, 4H, ArÀ H). 13C NMR (100 MHz, CDCl3): δ 187.2 (d, J=7.4 Hz,
CHO), 161.7 (d, J=258.2 Hz, ArÀ C), 137.8 (d, J=4.3 Hz, ArÀ C), 133.5
(d, J=19.4 Hz, ArÀ C), 131.5 (ArÀ C), 129.9 (ArÀ C), 129.8 (ArÀ C), 128.5
(d, J=1.5 Hz, ArÀ C), 128.2 (d, J=15.6 Hz, ArÀ C), 126.9 (ArÀ C), 124.4
(d, J=4.5 Hz, ArÀ C), 124.2 (ArÀ C).
2’-chloro-2-fluoro-3’-methoxy-[1,1’-biphenyl]-3-carbaldehyde
(12i)
1
Yield 87.6%, white solid, H NMR (400 MHz, CDCl3): δ 10.43 (s, 1H,
3’,4’-dichloro-2-fluoro-[1,1’-biphenyl]-3-carbaldehyde (12d)
CHO), 7.93 (m, 1H, ArÀ H), 7.57 (m, 1H, ArÀ H), 7.34 (m, 1H, ArÀ H),
7.03 (m, 1H, ArÀ H), 6.95 (m, 1H, ArÀ H), 3.97 (s, 3H, OCH3). 13C NMR
(100 MHz, CDCl3): δ 187.3 (d, J=7.4 Hz, CHO), 161.7 (d, J=258.5 Hz,
ArÀ C), 155.4 (ArÀ C), 137.8 (d, J=3.9 Hz, ArÀ C), 135.0 (ArÀ C), 128.4
(d, J=1.3 Hz, ArÀ C), 127.7 (ArÀ C), 127.3 (ArÀ C), 124.3 (d, J=6.8 Hz,
ArÀ C), 124.1 (d, J=6.9 Hz, ArÀ C), 123.6 (ArÀ C), 122.2 (d, J=15.4 Hz,
ArÀ C), 111.9 (ArÀ C), 56.4 (CH3).
1
Yield 89.4%, white solid, H NMR (400 MHz, CDCl3): δ 10.44 (s, 1H,
CHO), 7.90 (m, 1H, ArÀ H), 7.66 (m, 2H, ArÀ H), 7.56 (d, J=8.0 Hz, 1H,
ArÀ H), 7.37 (m, 2H, ArÀ H). 13C NMR (100 MHz, CDCl3): δ 187.1 (d, J=
8.1 Hz, CHO), 161.6 (d, J=259.1 Hz, ArÀ C), 136.5 (d, J=4.2 Hz,
ArÀ C), 134.2 (ArÀ C), 132.8 (d, J=17.1 Hz, ArÀ C), 130.9 (d, J=2.9 Hz,
ArÀ C), 130.7 (ArÀ C), 128.6 (d, J=1.7 Hz, ArÀ C), 128.3 (d, J=3.2 Hz,
ArÀ C), 128.0 (d, J=13.0 Hz, ArÀ C), 125.0 (d, J=4.4 Hz, ArÀ C), 124.8
(d, J=8.8 Hz, ArÀ C).
2’-fluoro-3’-formyl-[1,1’-biphenyl]-2-carbonitrile (12j)
1
Yield 84.6%, white solid, H NMR (400 MHz, CDCl3): δ 10.45 (s, 1H,
2-fluoro-2’,3’-dimethyl-[1,1’-biphenyl]-3-carbaldehyde (12e)
CHO), 8.00 (m, 1H, ArÀ H), 7.83 (m, 1H, ArÀ H), 7.71 (m, 2H, ArÀ H),
7.56 (m, 2H, ArÀ H), 7.42 (m, 1H, ArÀ H).13C NMR (100 MHz, CDCl3): δ
186.8 (d, J=7.8 Hz, CHO), 161.4 (d, J=258.9 Hz, ArÀ C), 138.0 (ArÀ C),
137.4 (d, J=3.7 Hz, ArÀ C), 133.4 (ArÀ C), 132.8 (ArÀ C), 130.9 (d, J=
1.3 Hz, ArÀ C), 129.5 (d, J=2.0 Hz, ArÀ C), 128.9 (ArÀ C), 127.1 (d, J=
14.6 Hz, ArÀ C), 124.9 (d, J=4.5 Hz, ArÀ C), 124.7 (d, J=8.7 Hz, ArÀ C),
117.7 (CN), 112.9 (ArÀ C).
1
Yield 92.3%, white solid, H NMR (400 MHz, CDCl3): δ 10.41 (s, 1H,
CHO), 7.88 (m, 1H, ArÀ H), 7.51 (m, 1H, ArÀ H), 7.31 (m, 1H, ArÀ H),
7.20 (m, 2H, ArÀ H), 7.06 (m, 1H, ArÀ H), 3.25 (s, 3H, CH3), 2.11 (s, 3H,
CH3). 13C NMR (100 MHz, CDCl3): δ 187.5 (d, J=7.4 Hz, CHO), 161.6
(d, J=256.5 Hz, ArÀ C), 138.1 (d, J=4.8 Hz, ArÀ C), 137.3 (ArÀ C), 135.1
(ArÀ C), 134.3 (ArÀ C), 131.2 (d, J=16.5 Hz, ArÀ C), 130.1 (ArÀ C), 127.8
(ArÀ C), 127.6 (d, J=1.8 Hz, ArÀ C), 125.6 (ArÀ C), 124.5 (d, J=4.3 Hz,
ArÀ C), 124.2 (d, J=9.1 Hz, ArÀ C), 20.6 (CH3), 16.8 (d, J=2.1 Hz, CH3).
2-fluoro-2’-nitro-[1,1’-biphenyl]-3-carbaldehyde (12k)
1
Yield 81.2%, white solid, H NMR (400 MHz, CDCl3): δ 10.37 (s, 1H,
2,2’-difluoro-3’-methoxy-[1,1’-biphenyl]-3-carbaldehyde (12f)
CHO), 7.94 (m, 1H, ArÀ H), 7.71 (m, 2H, ArÀ H), 7.61 (m, 2H, ArÀ H),
7.45 (m, 1H, ArÀ H), 7.38 (m, 1H, ArÀ H).13C NMR (100 MHz, CDCl3): δ
186.3 (d, J=7.5 Hz, CHO), 161.6 (d, J=258.5 Hz, ArÀ C), 138.2 (ArÀ C),
137.3 (d, J=3.7 Hz, ArÀ C), 133.4 (ArÀ C), 132.8 (ArÀ C), 131.0 (d, J=
1.3 Hz, ArÀ C), 129.4 (d, J=2.0 Hz, ArÀ C), 128.9 (ArÀ C), 127.0 (d, J=
14.6 Hz, ArÀ C), 124.8 (d, J=4.5 Hz, ArÀ C), 124.8 (d, J=8.7 Hz, ArÀ C),
112.7 (ArÀ C).
1
Yield 90.3%, white solid, H NMR (400 MHz, CDCl3): δ 10.42 (s, 1H,
CHO), 7.91 (m, 1H, ArÀ H), 7.64 (m, 1H, ArÀ H), 7.35 (m, 1H, ArÀ H),
7.18 (m, 1H, ArÀ H), 7.06 (m, 1H, ArÀ H), 6.94 (m, 1H, ArÀ H), 3.93 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3): δ 187.3 (d, J=7.9 Hz, CHO),
161.8 (d, J=259.6 Hz, ArÀ C), 149.6 (d, J=247.4 Hz, ArÀ C), 148.1 (d,
J=10.8 Hz, ArÀ C), 137.8 (d, J=2.5 Hz, ArÀ C), 137.7 (d, J=2.3 Hz,
ArÀ C), 128.4 (d, J=1.9 Hz, ArÀ C), 124.7 (d, J=15.5 Hz, ArÀ C), 124.4
(d, J=4.4 Hz, ArÀ C), 124.1 (d, J=4.8 Hz, ArÀ C), 122.8 (d, J=13.0 Hz,
ArÀ C), 122.4 (d, J=2.7 Hz, ArÀ C), 113.5 (d, J=1.7 Hz, ArÀ C), 56.3
(OCH3).
General procedure for synthesis of 10a-10b
To the solution of compound 9 (1.0 eq.) in acetone, K2CO3 (3.0 eq.)
and corresponding bromides were added in portions.The reaction
°
was stirred at 50 C for 6 h, and then quenched by careful addition
2,2’,3’-trifluoro-[1,1’-biphenyl]-3-carbaldehyde (12g)
of 1 N aqueous NaOH. The residue was diluted with EA and the
solution was washed with brine, the organic layer was dried over
MgSO4. After removal of the solvent, the residue was purified by
flash column chromatography (PE/EA =10/1).
1
Yield 88.7%, white solid, H NMR (400 MHz, CDCl3): δ 10.44 (s, 1H,
CHO), 7.95 (m, 1H, ArÀ H), 7.66 (m, 1H, ArÀ H), 7.38 (m, 1H, ArÀ H),
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(R)-6-(3-((3-fluorobenzyl) (prop-2-yn-1-yl) amino) propyl)-1,1,5-
trimethylcyclohex-4-en-3-one (10a)
General procedure for synthesis of 10d
To the solution of compound 9 (490 mg, 1.61 mmol, 1.0 eq.) in
DMF, Cs2CO3 (2.63 g, 8.07 mmol, 5.0 eq.), NaI (245 mg, 1.61 mmol,
1.0 eq.) and tert-butyl (2-bromoethyl) carbamate (1.81 g,
8.07 mmol, 5.0 eq.) were added in portions. The reaction was stirred
Yield 82.7%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.26 (m, 1H,
ArÀ H), 7.08 (m, 2H, ArÀ H), 6.94 (m, 1H, ArÀ H), 5.81 (s, 1H, 4-H), 3.61
(s, 2H, Ar-CH2), 3.31 (d, J=2.0 Hz, 2H, N-CH2), 2.54 (t, J=6.8 Hz, 2H,
9-H), 2.37 (d, J=17.2 Hz, 1H, 2-H), 2.26 (t, J=2.0 Hz, 1H, CH), 2.02 (d,
J=17.2 Hz, 1H, 2-H), 1.97 (s, 3H, 13-H), 1.86 (t, J=5.4 Hz, 1H, 6-H),
1.75 (m, 1H, 7-H), 1.58 (m, 2H, 8-H), 1.44 (m, 1H, 7-H), 1.05 (s, 3H, 11-
H), 1.01 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.2 (3-C), 165.4
(5-C), 162.9 (d, J=244.2 Hz, ArÀ C), 141.4 (d, J=3.9 Hz, ArÀ C), 129.8
(d, J=8.2 Hz, ArÀ C), 125.0 (4-C), 124.5 (d, J=2.6 Hz, ArÀ C), 115.6 (d,
J=21.1 Hz, ArÀ C), 114.1 (d, J=21.0 Hz, ArÀ C), 78.0 (C), 73.5 (CH),
57.4 (Ar-CH2), 53.5 (9-C), 51.1 (6-C), 47.1 (2-C), 41.5 (N-CH2), 36.3 (1-
C), 28.7 (12-C), 27.8 (7-C), 27.2 (11-C and 8-C), 24.7 (13-C). HR-ESIMS
m/z 342.2219 [M+H]+, calcd for C22H29FNO, 342.2233.
°
at 100 C for 48 h under nitrogen, and then quenched by careful
addition of 1 N aqueous NaOH. The residue was diluted with EA
and the solution was washed with brine, the organic layer was
dried over MgSO4. After removal of the solvent, the residue was
purified by flash column chromatography (PE/EA =6/1) to give
10d.
tert-Butyl (R)-(2-((3-fluorobenzyl)
(3-(1,1,5-trimethyl-3-oxocyclohex-4-en-6-yl) propyl) amino)
ethyl) carbamate (10d)
Yield 54.8%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.27 (m, 1H,
ArÀ H), 7.04 (m, 2H, ArÀ H), 6.94 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.55
(s, 2H, Ar-CH2), 3.19 (m, 2H, CH2), 2.54 (t, J=6.0 Hz, 2H, 9-H), 2.44 (t,
J=6.6 Hz, 2H, N-CH2), 2.35 (d, J=17.2 Hz, 1H, 2-H), 2.01 (d, J=
17.2 Hz, 1H, 2-H), 1.95 (s, 3H, 13-H), 1.82 (t, J=5.2 Hz, 1H, 6-H), 1.68
(m, 1H, 7-H), 1.53 (m, 2H, 8-H), 1.44 (s, 9H, (CH3)3), 1.35 (m, 1H, 7-H),
1.04 (s, 3H, 11-H), 1.00 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ
199.2 (3-C), 165.5 (5-C), 162.9 (d, J=244.3 Hz, ArÀ C), 155.9 (CO),
141.2 (d, J=5.7 Hz, ArÀ C), 129.8 (d, J=8.1 Hz, ArÀ C), 125.0 (4-C),
124.2 (d, J=2.1 Hz, ArÀ C), 115.4 (d, J=21.2 Hz, ArÀ C), 114.0 (d, J=
21.0 Hz, ArÀ C), 79.1 (C), 58.2 (Ar-CH2), 54.3 (N-CH2), 53.5 (9-C), 51.0
(6-C), 47.1 (2-C), 38.0 (CH2), 36.3 (1-C), 28.7 (12-C), 28.4 ((CH3)3), 28.0
(7-C), 27.2 (11-C), 26.9 (8-C), 24.8 (13-C). HR-ESIMS m/z 447.3002 [M
+H]+, calcd for C26H40FN2O3, 447.3023.
Ethyl(R)-N-(3-fluorobenzyl)-N-(3-(1,1,5-trimethyl-3-oxocyclohex
À 4-en-6-yl) propyl) glycinate (10b)
Yield 84.3%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.27 (m, 1H,
ArÀ H), 7.09 (m, 2H, ArÀ H), 6.93 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 4.16
(q, J=7.0 Hz, 2H, OCH2), 3.77 (s, 2H, Ar-CH2), 3.31 (s, 2H, Ar-CH2),
2.63 (t, J=6.8 Hz, 2H, 9-H), 2.35 (d, J=17.4 Hz, 1H, 2-H), 2.01 (d, J=
17.4 Hz, 1H, 2-H), 1.95 (d, J=1.0 Hz, 3H, 13-H), 1.84 (t, J=5.2 Hz, 1H,
6-H), 1.72 (m, 1H, 7-H), 1.55 (m, 2H, 8-H), 1.43 (m, 1H, 7-H), 1.27 (t,
J=7.0 Hz, 3H, CH3), 1.04 (s, 3H, 11-H), 1.00 (s, 3H, 12-H). 13C NMR
(100 MHz, CDCl3): δ 199.1 (3-C), 177.1 (CO), 165.4 (5-C), 163.0 (d, J=
244.2 Hz, ArÀ C), 142.0 (d, J=6.8 Hz, ArÀ C), 129.8 (d, J=8.1 Hz,
ArÀ C), 125.1 (4-C), 124.3 (d, J=2.6 Hz, ArÀ C), 115.5 (d, J=21.2 Hz,
ArÀ C), 114.0 (d, J=21.1 Hz, ArÀ C), 60.3 (OCH2), 58.0 (Ar-CH2), 54.5
(N-CH2), 54.2 (9-C), 51.1 (6-C), 47.2 (2-C), 36.3 (1-C), 28.8 (12-C), 27.7
(7-C), 27.5 (8-C), 27.1 (11-C), 24.7 (13-C), 14.3 (CH3). HR-ESIMS m/z
390.2428 [M+H]+, calcd for C23H33FNO3, 390.2444.
General procedure for synthesis of 10e
Compound 10d (1.5 g, 3.36 mmol, 1.0 eq.) was dissolved in CH2Cl2
(40 mL), TFA (0.75 mL, 10.1 mmol, 3.0 eq.) was added. Then the
reaction was stirred at room temperature for 5 h. The reaction
mixture was treated with 1 N aqueous NaOH and stirred for 1 h,
then extracted by CH2Cl2 (50 mL), washed with brine (50 mL). The
organic layers were dried by MgSO4, and the solvent was removed
in vacuo to afford the residue to yield the desired product. The
crude product in CH2Cl2 was added pyridine (1.4 mL, 17.35 mmol,
5.0 eq.) and succinic anhydride (1.74 g, 17.35 mmol, 5.0 eq.) when
the mixture was stirred in an ice bath. The mixture was stirred at
room temperature for 12 h, evaporated just to dryness with a
vacuum evaporator. The solvent was evaporated under reduced
pressure to give the crude product, which was separated by column
chromatography (CH2Cl2/MeOH=20/1) to obtain pure 10e.
General procedure for synthesis of 10c
To the solution of compound 10b (350 mg, 0.9 mmol, 1.0 eq.) in
MeOH, KOH (66 mg, 1.17 mmol, 1.3 eq.) was added in portions. The
reaction was stirred at room temperature for 5 h, and then
quenched by addition of saturated citric acid solution until a pH of
5–6, extracted by EA (50 mL), washed with brine (50 mL), the
organic layer was dried over MgSO4. After removal of the solvent,
the residue was purified by flash column chromatography (CH2Cl2/
MeOH=30/1) to give 10c.
(R)-N-(3-fluorobenzyl)-N-(3-(1,1,5-trimeth-
yl-3-oxocyclohex-4-en-6-yl) propyl) glycine (10c)
1
Yield 86.2%, yellow oil, H NMR (400 MHz, DMSO-d6): δ 12.08 (s, 1H,
(R)-4-((2-((3-fluorobenzyl) (3-(1,1,5-trimethyl-3-oxocyclohex-
4-en-6-yl) propyl) amino) ethyl) amino)-4-oxobutanoic acid
(10e)
OH), 7.37 (m, 1H, ArÀ H), 7.18 (m, 2H, ArÀ H), 7.09 (m, 1H, ArÀ H), 5.71
(s, 1H, 4-H), 3.81 (s, 2H, Ar-CH2), 3.32 (s, 2H, Ar-CH2), 2.62 (t, J=
6.8 Hz, 2H, 9-H), 2.30 (d, J=17.2 Hz, 1H, 2-H), 1.90 (m, 4H, 13-H and
2-H), 1.82 (t, J=5.4 Hz, 1H, 6-H), 1.64 (m, 1H, 7-H), 1.49 (m, 2H, 8-H),
1.30 (m, 1H, 7-H), 0.96 (s, 3H, 11-H), 0.92 (s, 3H, 12-H). 13C NMR
(100 MHz, DMSO-d6): δ 197.8 (3-C), 171.9 (CO), 165.6 (5-C), 162.2 (d,
J=241.7 Hz, ArÀ C), 141.7 (d, J=5.9 Hz, ArÀ C), 130.1 (d, J=8.2 Hz,
ArÀ C), 124.7 (d, J=2.6 Hz, ArÀ C), 124.2 (4-C), 115.3 (d, J=21.2 Hz,
ArÀ C), 113.9 (d, J=20.9 Hz, ArÀ C), 57.2 (Ar-CH2), 54.2 (N-CH2), 53.6
(9-C), 50.0 (6-C), 46.8 (2-C), 35.9 (1-C), 28.3 (12-C), 26.9 (7-C), 26.5
(11-C), 24.0 (8-C), 21.0 (13-C). HR-ESIMS m/z 362.2114 [M+H]+,
calcd for C21H29FNO3, 362.2131.
1
Yield 73.5%, yellow oil, H NMR (400 MHz, DMSO-d6): δ 12.08 (s, 1H,
OH), 7.79 (s, 1H, NH), 7.34 (m, 1H, ArÀ H), 7.15 (m, 2H, ArÀ H), 7.05 (m,
1H, ArÀ H), 5.70 (s, 1H, 4-H), 3.59 (s, 2H, Ar-CH2), 3.15 (q, J=6.1 Hz,
2H, CH2), 2.42 (m, 6H, CH2), 2.31 (m, 3H, 2-H and CH2), 1.91 (s, 3H,
13-H), 1.86 (m, 2H, 2-H and 6-H), 1.64 (m, 1H, 7-H), 1.48 (m, 2H, 8-H),
1.29 (m, 1H, 7-H), 0.97 (s, 3H, 11-H), 0.92 (s, 3H, 12-H). 13C NMR
(100 MHz, DMSO-d6): δ 197.8 (3-C), 173.7 (CO), 170.8 (CO), 165.6 (5-
C), 162.2 (d, J=241.5 Hz, ArÀ C), 143.0 (d, J=6.3 Hz, ArÀ C), 129.9 (d,
J=8.2 Hz, ArÀ C), 124.4 (d, J=2.6 Hz, ArÀ C), 124.1 (4-C), 115.0 (d, J=
21.1 Hz, ArÀ C), 113.5 (d, J=20.7 Hz, ArÀ C), 57.5 (Ar-CH2), 53.8 (N-
CH2), 53.0 (9-C), 50.2 (6-C), 46.9 (2-C), 39.7 (CH2), 36.6 (CH2), 35.9 (1-
C), 30.0 (CH2), 29.2 (CH2), 28.3 (12-C), 27.2 (7-C), 26.5 (11-C), 26.4 (8-
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C), 24.0 (13-C). HR-ESIMS m/z 447.2638 [M+H]+, calcd for
C25H36FN2O4, 447.2659.
(d, J=21.1 Hz, ArÀ C), 58.1 (Ar-CH2), 57.7 (9-C), 54.0 (Ar-CH2), 51.0 (6-
C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.2 (8-C), 26.8 (11-
C), 24.8 (13-C). HR-ESIMS m/z 430.2341 [M+H]+, calcd for
C26H31F3NO, 430.2358.
General procedure for synthesis of 11a–11x
°
To a solution of amine 9 (1.0 eq.) in MeOH was added at 0 C
(R)-6-(3-((3-fluorobenzyl) (4-methoxybenzyl) amino)
propyl)-1,1,5- trimethylcyclohex-4-en-3-one (11d)
NaBH3CN (1.5 eq.) and ZnCl2 (0.5 eq.) which had been dissolved by
MeOH followed by substituted aldehyde (3.0 eq.) and 0.5 mL of
H2O. The reaction mixture was warmed to room temperature,
stirred 12 h, evaporated just to dryness with a vacuum evaporator.
The viscous suspension was transferred to a separatory funnel and
extracted with CH2Cl2. The combined organic extracts were dried
(MgSO4) and concentrated in vacuo to give a yellow oil that was
purified by column chromatography (PE/EA) to afford 11a–11x.
Yield 77.2%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.25 (m, 3H,
ArÀ H), 7.08 (m, 2H, ArÀ H), 6.91 (m, 2H, ArÀ H), 6.85 (d, J=8.4 Hz, 1H,
ArÀ H), 5.79 (s, 1H, 4-H), 3.79 (s, 3H, OCH3), 3.51 (s, 2H, Ar-CH2), 3.49
(s, 2H, Ar-CH2), 2.39 (t, J=6.8 Hz, 2H, 9-H), 2.32 (d, J=17.2 Hz, 1H, 2-
H), 2.00 (d, J=17.2 Hz, 1H, 2-H), 1.89 (s, 3H, 13-H), 1.77 (t, J=5.6 Hz,
1H, 6-H), 1.64 (m, 2H, 7-H), 1.54 (m, 2H, 8-H), 1.00 (s, 3H, 12-H), 0.98
(s, 3H, 11-H). 13C NMR (100 MHz, CDCl3): δ 199.3 (3-C), 165.6 (5-C),
163.0 (d, J=243.9 Hz, ArÀ C), 158.7 (ArÀ C), 142.8 (d, J=6.4 Hz, ArÀ C),
129.9 (ArÀ C), 129.6 (d, J=8.9 Hz, ArÀ C), 125.1 (4-C), 124.2 (d, J=
2.6 Hz, ArÀ C), 115.3 (d, J=21.3 Hz, ArÀ C), 113.7 (d, J=20.8 Hz,
ArÀ C), 113.7 (ArÀ C), 58.1 (Ar-CH2), 55.2 (OCH3), 53.9 (Ar-CH2), 51.1 (6-
C), 47.16 (2-C), 36.3 (1-C), 28.7 (12-C), 28.0 (7-C), 27.2 (8-C), 26.9 (11-
C), 24.8 (13-C). HR-ESIMS m/z 424.2630 [M+H]+, calcd for
C27H35FNO2, 424.2652.
(R)-6-(3-((3-fluorobenzyl) (pyridin-4-ylmethyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11a)
Yield 77.3%, yellow oil, 1H NMR (600 MHz, CDCl3): δ 8.55 (d, J=
5.4 Hz, 2H, ArÀ H), 7.29 (d, J=5.4 Hz, 2H, ArÀ H), 7.27 (m, 1H, ArÀ H),
7.09 (m, 2H, ArÀ H), 6.95 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.55 (s, 4H,
Ar-CH2), 2.42 (t, J=6.9 Hz, 2H, 9-H), 2.33 (d, J=17.4 Hz, 1H, 2-H),
2.02 (d, J=17.4 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H), 1.80 (t, J=5.4 Hz, 1H,
6-H), 1.66 (m, 1H, 7-H), 1.55 (m, 2H, 8-H), 1.35 (m, 1H, 7-H), 1.01 (s,
3H, 11-H), 0.99 (s, 3H, 12-H). 13C NMR (150 MHz, CDCl3): δ 199.3 (3-
C), 165.4 (5-C), 163.0 (d, J=244.4 Hz, ArÀ C), 149.8 (C), 148.9 (C),
141.8 (d, J=7.1 Hz, ArÀ C), 129.9 (d, J=8.1 Hz, ArÀ C), 125.2 (4-C),
124.1 (d, J=2.6 Hz, ArÀ C), 123.6 (C), 115.3 (d, J=21.2 Hz, ArÀ C),
114.1 (d, J=21.0 Hz, ArÀ C), 58.3 (Ar-CH2), 57.7 (9-C), 54.4 (N-CH2-Ar),
51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.2 (8-C),
26.9 (11-C), 24.8 (13-C). HR-ESIMS m/z 395.2479 [M+H]+, calcd for
C25H32FN2O, 395.2499.
(R)-6-(3-((4-ethoxybenzyl) (3-fluorobenzyl) amino) propyl)-1,1,5-
trimethylcyclohex-4-en-3-one (11e)
Yield 76.4%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.19 (m, 3H,
ArÀ H), 7.01 (m, 2H, ArÀ H), 6.84 (m, 1H, ArÀ H), 6.77 (d, J=8.4 Hz, 1H,
ArÀ H), 5.71 (s, 1H, 4-H), 3.95 (q, J=6.8 Hz, 2H, OCH2), 3.43 (s, 2H, Ar-
CH2), 3.41 (s, 2H, Ar-CH2), 2.31 (t, J=6.8 Hz, 2H, 9-H), 2.25 (d, J=
17.6 Hz, 1H, 2-H), 1.92 (d, J=17.6 Hz, 1H, 2-H), 1.82 (s, 3H, 13-H),
1.69 (t, J=5.4 Hz, 1H, 6-H), 1.56 (m, 2H, 7-H), 1.46 (m, 2H, 8-H), 1.33
(t, J=6.8 Hz, 3H, CH3), 0.93 (s, 3H, 12-H), 0.90 (s, 3H, 11-H). 13C NMR
(100 MHz, CDCl3): δ 199.3 (3-C), 165.6 (5-C), 163.0 (d, J=243.7 Hz,
ArÀ C),157.2 (ArÀ C), 143.2 (d, J=6.8 Hz, ArÀ C), 130.1 (ArÀ C), 129.5 (d,
J=8.1 Hz, ArÀ C), 127.8 (ArÀ C), 127.6 (ArÀ C), 125.0 (4-C), 124.1 (d,
J=2.0 Hz, ArÀ C), 120.3 (ArÀ C), 115.4 (d, J=21.2 Hz, ArÀ C), 113.6 (d,
J=21.1 Hz, ArÀ C), 111.4 (ArÀ C), 64.1 (OCH2), 62.4 (Ar-CH2), 57.0 (Ar-
CH2), 52.8 (Ar-CH2), 50.1 (6-C), 46.1 (2-C), 35.3 (1-C), 27.7 (12-C), 27.0
(7-C), 26.12 (8-C), 25.9 (11-C), 23.7 (13-C), 13.9 (CH3). HR-ESIMS m/z
438.2775 [M+H]+, calcd for C28H37FNO2, 438.2808.
(R)-6-(3-(bis(3-fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex- 4-en-3-one (11b)
Yield 81.6%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.27 (m, 2H,
ArÀ H), 7.09 (m, 4H, ArÀ H), 6.93 (m, 2H, ArÀ H), 5.79 (s, 1H, 4-H), 3.53
(s, 4H, Ar-CH2), 2.41 (t, J=6.8 Hz, 2H, 9-H), 2.33 (d, J=10.8 Hz, 3H,
13-H), 2.03 (d, J=10.8 Hz, 3H, 13-H), 1.90 (s, 3H, 13-H), 1.78 (t, J=
5.4 Hz, 1H, 6-H), 1.66 (m, 1H, 7-H), 1.54 (m, 2H, 8-H), 1.33 (m, 1H, 7-
H), 1.01 (s, 3H, 11-H), 0.98 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ
199.3 (3-C), 165.6 (5-C), 163.0 (d, J=244.2 Hz, ArÀ C), 142.3 (d, J=
6.9 Hz, ArÀ C), 129.8 (d, J=8.2 Hz, ArÀ C), 125.1 (4-C), 124.2 (d, J=
2.4 Hz, ArÀ C), 115.4 (d, J=21.2 Hz, ArÀ C), 113.9 (d, J=21.0 Hz,
ArÀ C), 58.2 (Ar-CH2), 54.1 (9-C), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7
(12-C), 28.0 (7-C), 27.2 (11-C), 26.9 (8-C), 24.8 (13-C). HR-ESIMS m/z
412.2437 [M+H]+, calcd for C26H32F2NO, 412.2452.
(R)-6-(3-((3-ethoxybenzyl) (3-fluorobenzyl) amino) propyl)-1,1,5-
trimethylcyclohex-4-en-3-one (11f)
Yield 78.3%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.23 (m, 2H,
ArÀ H), 7.09 (m, 2H, ArÀ H), 6.91 (m, 3H, ArÀ H), 6.85 (d, J=7.2 Hz, 1H,
ArÀ H), 5.79 (s, 1H, 4-H), 4.02 (q, J=7.2 Hz, 2H, OCH2), 3.53 (s, 2H, Ar-
CH2), 3.52 (s, 2H, Ar-CH2), 2.41 (t, J=6.8 Hz, 2H, 9-H), 2.32 (d, J=
17.2 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H), 1.77 (t, J=5.2 Hz, 1H, 6-H), 1.66
(m, 2H, 7-H), 1.53 (m, 2H, 8-H), 1.41 (t, J=7.2 Hz, 3H, CH3), 1.01 (s,
3H, 12-H), 0.98 (s, 3H, 11-H). 13C NMR (100 MHz, CDCl3): δ199.2 (3-C),
165.5 (5-C), 163.0 (d, J=244.0 Hz, ArÀ C),159.1 (ArÀ C), 142.7 (d, J=
6.9 Hz, ArÀ C), 141.1 (ArÀ C), 129.6 (d, J=8.1 Hz, ArÀ C), 129.0 (ArÀ C),
125.1 (4-C), 124.2 (d, J=2.4 Hz, ArÀ C), 120.9 (ArÀ C), 115.4 (d, J=
21.3 Hz, ArÀ C), 115.0 (ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 112.9
(ArÀ C), 63.3 (OCH2), 58.8 (Ar-CH2), 58.3 (Ar-CH2), 54.1 (Ar-CH2), 51.2
(6-C), 47.2 (2-C), 36.3 (1-C), 28.7 (12-C), 28.0 (7-C), 27.1 (8-C), 27.1
(11-C), 24.7 (13-C), 14.9 (CH3). HR-ESIMS m/z 438.2787 [M+H]+,
calcd for C28H37FNO2, 438.2808.
(R)-6-(3-((3,4-difluorobenzyl) (3-fluorobenzyl) amino)
propyl)-1,1,5- trimethylcyclohex-4-en-3-one (11c)
Yield 83.5%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.29 (m, 1H,
ArÀ H), 7.19 (m, 1H, ArÀ H), 7.11 (m, 4H, ArÀ H), 6.96 (m, 1H, ArÀ H),
5.82 (s, 1H, 4-H), 3.54 (s, 2H, Ar-CH2), 3.51 (s, 2H, Ar-CH2), 2.41 (t, J=
6.8 Hz, 2H, 9-H), 2.34 (d, J=17.4 Hz, 1H, 2-H), 2.03 (d, J=17.4 Hz,
1H, 2-H), 1.93 (s, 3H, 13-H), 1.81 (t, J=5.4 Hz, 1H, 6-H), 1.66 (m, 1H,
7-H), 1.56 (m, 2H, 8-H), 1.36 (m, 1H, 7-H), 1.03 (s, 3H, 11-H), 1.00 (s,
3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.4 (3-C), 165.6 (5-C),
163.0 (d, J=244.2 Hz, ArÀ C), 150.3 (dd, J1 =246.3 Hz, J2 =12.7 Hz,
ArÀ C), 149.4 (dd, J1 =245.4 Hz, J2 =12.8 Hz, ArÀ C), 142.1 (d, J=
4.0 Hz, ArÀ C), 136.6 (d, J=2.4 Hz, ArÀ C), 129.8 (d, J=8.1 Hz, ArÀ C),
125.1 (4-C), 124.3 (), 124.1 (d, J=2.6 Hz, ArÀ C), 117.3 (d, J=17.2 Hz,
ArÀ C), 117.0 (d, J=16.9 Hz, ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 114.2
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2-H), 1.93 (s, 3H, 13-H), 1.81 (t, J=5.2 Hz, 1H, 6-H), 1.64 (m, 1H, 7-H),
(R)-4-(((3-fluorobenzyl)
1.56 (m, 2H, 8-H), 1.34 (m, 1H, 7-H), 1.02 (s, 3H, 11-H), 1.00 (s, 3H,
12-H). 13C NMR (100 MHz, CDCl3): δ 199.3 (3-C), 165.4 (5-C), 163.0 (d,
J=244.7 Hz, ArÀ C), 162.2 (d, J=256.8 Hz, ArÀ C), 141.7 (d, J=6.6 Hz,
ArÀ C), 136.8 (d, J=14.0 Hz, ArÀ C), 135.1 (d, J=7.7 Hz, ArÀ C), 133.1
(ArÀ C), 129.9 (d, J=8.1 Hz, ArÀ C), 125.2 (4-C), 124.1 (d, J=4.3 Hz,
ArÀ C), 116.4 (d, J=19.4 Hz, ArÀ C), 115.3 (d, J=21.3 Hz, ArÀ C), 114.3
(d, J=21.1 Hz, ArÀ C), 114.1(CN), 101.2 (d, J=15.5 Hz, ArÀ C), 58.2
(Ar-CH2), 57.2 (Ar-CH2), 54.5 (9-C), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C),
28.7 (12-C), 27.9 (7-C), 27.2 (11-C), 26.8 (8-C), 24.8 (13-C). HR-ESIMS
m/z 437.2387 [M+H]+, calcd for C27H31F2N2O, 437.2404.
(3-(1,1,5-trimethyl-3-oxocyclohex-4-en-6- yl) propyl) amino)
methyl) benzonitrile (11g)
Yield 76.2%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.63 (d, J=
7.6 Hz, 2H, ArÀ H), 7.45 (d, J=7.6 Hz, 2H, ArÀ H), 7.29 (m, 1H, ArÀ H),
7.08 (m, 2H, ArÀ H), 6.96 (m, 1H, ArÀ H), 5.82 (s, 1H, 4-H), 3.60 (s, 2H,
Ar-CH2), 3.55 (s, 2H, Ar-CH2), 2.42 (t, J=6.4 Hz, 2H, 9-H), 2.34 (d, J=
17.4 Hz, 1H, 2-H), 2.04 (d, J=17.4 Hz, 1H, 2-H), 1.92 (s, 3H, 13-H),
1.81 (t, J=5.2 Hz, 1H, 6-H), 1.64 (m, 1H, 7-H), 1.55 (m, 2H, 8-H), 1.36
(m, 1H, 7-H), 1.02 (s, 3H, 11-H), 1.00 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.4 (3-C), 165.5 (5-C), 163.0 (d, J=244.5 Hz, ArÀ C), 145.4
(ArÀ C), 141.8 (d, J=6.6 Hz, ArÀ C), 132.2 (ArÀ C), 129.9 (d, J=8.0 Hz,
ArÀ C), 129.4 (ArÀ C), 125.2 (4-C), 124.1 (ArÀ C), 118.9 (CN), 115.3 (d,
J=21.0 Hz, ArÀ C), 114.1 (d, J=21.1 Hz, ArÀ C), 110.9 (ArÀ C), 58.4 (Ar-
CH2), 58.3 (9-C), 54.3 (Ar-CH2), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.8
(12-C), 27.9 (7-C), 27.1 (8-C), 26.8 (11-C), 24.8 (13-C). HR-ESIMS m/z
419.2477 [M+H]+, calcd for C27H32FN2O, 419.2499.
(R)-2-chloro-3-(((3-fluorobenzyl)
(3-(1,1,5-trimethyl-3-oxocyclohex- 4-en-6-yl) propyl) amino)
methyl) benzonitrile (11k)
Yield 77.4%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.81 (d, J=
7.6 Hz, 1H, ArÀ H), 7.58 (d, J=7.6 Hz, 1H, ArÀ H), 7.37 (m, 1H, ArÀ H),
7.27 (m, 1H, ArÀ H), 7.07 (m, 2H, ArÀ H), 6.94 (m, 1H, ArÀ H), 5.80 (s,
1H, 4-H), 3.71 (s, 2H, Ar-CH2), 3.60 (s, 2H, Ar-CH2), 2.45 (t, J=6.8 Hz,
2H, 9-H), 2.31 (t, J=17.2 Hz, 1H, 2-H), 2.01 (d, J=17.2 Hz, 1H, 2-H),
1.91 (s, 3H, 13-H), 1.79 (t, J=5.2 Hz, 1H, 6-H), 1.64 (m, 1H, 7-H), 1.55
(m, 2H, 8-H), 1.32 (m, 1H, 7-H), 1.00 (s, 3H, 11-H), 0.99 (s, 3H, 12-H).
13C NMR (100 MHz, CDCl3): δ 199.3 (3-C), 165.4 (5-C), 162.9 (d, J=
244.5 Hz, ArÀ C), 141.7 (d, J=6.8 Hz, ArÀ C), 139.2 (ArÀ C), 136.0
(ArÀ C), 134.4 (ArÀ C), 132.6 (ArÀ C), 129.9 (d, J=8.1 Hz, ArÀ C), 127.1
(ArÀ C), 125.2 (4-C), 124.1 (d, J=4.3 Hz, ArÀ C), 116.2 (ArÀ C), 115.3 (d,
J=21.4 Hz, ArÀ C), 114.2 (d, J=21.0 Hz, ArÀ C), 114.0 (CN), 58.6 (Ar-
CH2), 55.4 (Ar-CH2), 54.7 (9-C), 51.0 (6-C), 47.0 (2-C), 36.3 (1-C), 28.7
(12-C), 28.0 (7-C), 27.2 (11-C), 26.9 (8-C), 24.8 (13-C). HR-ESIMS m/z
453.1994 [M+H]+, calcd for C27H31ClFN2O, 453.2019.
(R)-6-(3-((4-(dimethylamino) benzyl) (3-fluorobenzyl) amino)
propyl)- 1,1,5-trimethylcyclohex-4-en-3-one (11h)
Yield 73.2%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.26 (m, 1H,
ArÀ H), 7.20 (d, J=7.6 Hz, 2H, ArÀ H), 7.11 (d, J=7.6 Hz, 2H, ArÀ H),
6.93 (m, 1H, ArÀ H), 6.73 (d, J=7.4 Hz, 2H, ArÀ H), 5.81 (s, 1H, 4-H),
3.52 (s, 2H, Ar-CH2), 3.49 (s, 2H, Ar-CH2), 2.96 (s, 6H, N(CH3)2), 2.41 (t,
J=6.4 Hz, 2H, 9-H), 2.34 (d, J=17.2 Hz, 1H, 2-H), 2.02 (d, J=17.2 Hz,
1H, 2-H), 1.93 (s, 3H, 13-H), 1.80 (t, J=5.2 Hz, 1H, 6-H), 1.68 (m, 1H,
7-H), 1.55 (m, 2H, 8-H), 1.36 (m, 1H, 7-H), 1.03 (s, 3H, 11-H), 1.00 (s,
3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.6 (3-C), 166.0 (5-C),
162.9 (d, J=244.0 Hz, ArÀ C), 149.8 (ArÀ C), 143.0 (d, J=7.3 Hz, ArÀ C),
129.7 (ArÀ C), 129.5 (d, J=8.3 Hz, ArÀ C), 126.9 (ArÀ C), 125.0 (4-C),
124.1 (d, J=2.1 Hz, ArÀ C), 115.3 (d, J=21.1 Hz, ArÀ C), 113.6 (d, J=
21.0 Hz, ArÀ C), 112.5 (ArÀ C), 58.0 (Ar-CH2), 57.9 (9-C), 53.7 (Ar-CH2),
51.1 (6-C), 47.1 (2-C), 40.7 (N(CH3)2), 36.3 (1-C), 28.7 (12-C), 27.9 (7-
C), 27.2 (8-C), 26.9 (11-C), 24.8 (13-C). HR-ESIMS m/z 437.2948 [M+
H]+, calcd for C28H38FN2O, 437.2968.
methyl(R)-4’-(((3-fluorobenzyl)
(3-(1,1,5-trimethyl-3-oxocyclohex- 4-en-6-yl) propyl) amino)
methyl)-[1,1’-biphenyl]-3-carboxylate (11l)
Yield 76.5%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 8.28 (m, 1H,
ArÀ H), 8.00 (m, 1H, ArÀ H), 7.78 (m, 1H, ArÀ H), 7.59 (d, J=8.0 Hz, 2H,
ArÀ H), 7.50 (m, 1H, ArÀ H), 7.43 (d, J=8.0 Hz, 2H, ArÀ H), 7.27 (m, 1H,
ArÀ H), 7.11 (m, 2H, ArÀ H), 6.93 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.94
(s, 3H, OCH3), 3.60 (s, 2H, Ar-CH2), 3.56 (s, 2H, Ar-CH2), 2.44 (t, J=
6.8 Hz, 2H, 9-H), 2.34 (d, J=17.2 Hz, 1H, 2-H), 2.01 (d, J=17.2 Hz,
1H, 2-H), 1.90 (s, 3H, 13-H), 1.79 (t, J=5.4 Hz, 1H, 6-H), 1.67 (m, 1H,
7-H), 1.56 (m, 2H, 8-H), 1.36 (m, 1H, 7-H), 1.02 (s, 3H, 11-H), 0.98 (s,
3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.4 (3-C), 167.0 (CO), 165.7
(5-C), 162.9 (d, J=244.1 Hz, ArÀ C), 142.5 (d, J=6.8 Hz, ArÀ C), 141.1
(ArÀ C), 139.1 (ArÀ C), 138.8 (ArÀ C), 131.4 (ArÀ C), 130.6 (ArÀ C), 129.6
(d, J=8.2 Hz, ArÀ C), 129.2 (ArÀ C), 128.9 (ArÀ C), 128.3 (ArÀ C), 128.1
(ArÀ C), 127.0 (ArÀ C), 125.0 (4-C), 124.1 (d, J=2.4 Hz, ArÀ C), 115.3 (d,
J=21.1 Hz, ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 58.2
(Ar-CH2), 54.0 (9-C), 52.2 (OCH3), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C),
28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 26.9 (8-C), 24.8 (13-C). HR-ESIMS
m/z 528.2883 [M+H]+, calcd for C34H39FNO3, 528.2914.
(R)-6-(3-((4-(diethylamino) benzyl) (3-fluorobenzyl) amino)
propyl)- 1,1,5-trimethylcyclohex-4-en-3-one (11i)
Yield 72.6%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.26 (m, 1H,
ArÀ H), 7.17 (d, J=7.6 Hz, 2H, ArÀ H), 7.11 (d, J=7.6 Hz, 2H, ArÀ H),
6.93 (m, 1H, ArÀ H), 6.66 (d, J=7.4 Hz, 2H, ArÀ H), 5.82 (s, 1H, 4-H),
3.53 (s, 2H, Ar-CH2), 3.47 (s, 2H, Ar-CH2), 3.36 (q, J=6.8 Hz, 4H,
(CH2)2), 2.39 (m, 3H, 9-H and 2-H), 2.02 (d, J=17.2 Hz, 1H, 2-H), 1.93
(s, 3H, 13-H), 1.80 (t, J=5.2 Hz, 1H, 6-H), 1.69 (m, 1H, 7-H), 1.56 (m,
2H, 8-H), 1.36 (m, 1H, 7-H), 1.17(t, J=6.8 Hz, 6H, (CH3)2), 1.03 (s, 3H,
11-H), 1.00 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.6 (3-C),
166.1 (5-C), 162.9 (d, J=243.6 Hz, ArÀ C), 146.9 (ArÀ C), 143.1 (d, J=
6.7 Hz, ArÀ C), 129.9 (ArÀ C), 129.5 (d, J=8.2 Hz, ArÀ C), 125.5 (ArÀ C),
124.9 (4-C), 124.2 (d, J=2.1 Hz, ArÀ C), 115.3 (d, J=21.1 Hz, ArÀ C),
113.6 (d, J=21.1 Hz, ArÀ C), 111.6 (ArÀ C), 58.0 (Ar-CH2), 57.7 (9-C),
53.7 (Ar-CH2), 51.1 (6-C), 47.1 (2-C), 44.4 ((CH2)2), 36.3 (1-C), 28.7 (12-
C), 27.9 (7-C), 27.2 (8-C), 26.9 (11-C), 24.8 (13-C), 12.6 ((CH3)2). HR-
ESIMS m/z 465.3265 [M+H]+, calcd for C30H42FN2O, 465.3281.
(R)-6-(3-((3-fluorobenzyl) ((1-methyl-3-phenyl-1H-pyrazol-5- yl)
methyl) amino) propyl)-1,1,5-trimethylcyclohex-4-en-3-one
(11m)
(R)-2-fluoro-5-(((3-fluorobenzyl)
Yield 76.8%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.76 (d, J=
7.2 Hz, 2H, ArÀ H), 7.37 (m, 2H, ArÀ H), 7.28 (m, 2H, ArÀ H), 7.04 (m,
2H, ArÀ H), 6.95 (m, 1H, ArÀ H), 6.45 (s, 1H), 5.79 (s, 1H, 4-H), 3.84 (s,
3H, N-CH3), 3.56 (s, 4H, Ar-CH2), 2.44 (t, J=6.8 Hz, 2H, 9-H), 2.31 (d,
J=17.2 Hz, 1H, 2-H), 2.00 (d, J=17.2 Hz, 1H, 2-H), 1.89 (s, 3H, 13-H),
1.77 (t, J=5.2 Hz, 1H, 6-H), 1.60 (m, 1H, 7-H), 1.53 (m, 2H, 8-H), 1.31
(m, 1H, 7-H), 0.99 (s, 3H, 11-H), 0.97 (s, 3H, 12-H). 13C NMR (100 MHz,
(3-(1,1,5-trimethyl-3-oxocyclohex- 4-en-6-yl) propyl) amino)
methyl) benzonitrile (11j)
Yield 76.5%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.58 (m, 2H,
ArÀ H), 7.29 (m, 1H, ArÀ H), 7.18 (m, 1H, ArÀ H), 7.06 (m, 2H, ArÀ H),
6.95 (m, 1H, ArÀ H), 5.81 (s, 1H, 4-H), 3.54 (s, 4H, Ar-CH2), 2.41 (t, J=
6.8 Hz, 2H, 9-H), 2.32 (t, J=17.2 Hz, 1H, 2-H), 2.02 (d, J=17.2 Hz, 1H,
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CDCl3): δ 199.3 (3-C), 165.5 (5-C), 162.8 (d, J=244.6 Hz, ArÀ C), 149.8
(C), 141.6 (d, J=6.7 Hz, ArÀ C), 140.6 (ArÀ C), 133.3 (C), 129.8 (d, J=
8.3 Hz, ArÀ C), 128.6 (ArÀ C), 127.5 (ArÀ C), 125.3 (ArÀ C), 125.1 (4-C),
124.4 (d, J=2.5 Hz, ArÀ C), 115.5 (d, J=21.1 Hz, ArÀ C), 114.1 (ArÀ C),
104.3 (CH), 58.4 (Ar-CH2), 54.2 (9-C), 50.9 (Ar-CH2), 49.5 (6-C), 47.0 (2-
C), 36.8 (N-CH3), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 26.6
(8-C), 24.8 (13-C). HR-ESIMS m/z 474.2895 [M+H]+, calcd for
C30H37FN3O, 474.2921.
(d, J=246.4 Hz, ArÀ C), 142.5 (d, J=7.0 Hz, ArÀ C), 135.0 (ArÀ C), 133.6
(ArÀ C), 131.5 (ArÀ C), 131.0 (d, J=4.6 Hz, ArÀ C), 130.2 (d, J=3.2 Hz,
ArÀ C), 129.6 (d, J=8.1 Hz, ArÀ C), 129.5 (ArÀ C), 126.9 (d, J=16.9 Hz,
ArÀ C), 126.6 (ArÀ C), 126.1 (d, J=15.1 Hz, ArÀ C), 125.0 (4-C), 124.1
(d, J=2.4 Hz, ArÀ C), 123.5 (d, J=4.1 Hz, ArÀ C), 115.3 (d, J=21.2 Hz,
ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 54.0 (9-C), 51.2 (Ar-
CH2), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.2
(11-C), 27.0 (8-C), 24.8 (13-C). HR-ESIMS m/z 522.2346 [M+H]+,
calcd for C32H35ClF2NO, 522.2375.
(R)-6-(3-(((2-fluoro-2’-methoxy-[1,1’-biphenyl]-3-yl) methyl) (3-
fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11n)
(R)-6-(3-(((3’,4’-dichloro-2-fluoro-[1,1’-biphenyl]-3-yl) methyl) (3-
fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11q)
Yield 90.2%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.38 (m, 2H,
ArÀ H), 7.25 (m, 3H, ArÀ H), 7.14 (m, 3H, ArÀ H), 7.01 (m, 2H, ArÀ H),
6.91 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H), 3.78 (s, 3H, OCH3), 3.69 (s, 2H,
Ar-CH2), 3.59 (s, 2H, Ar-CH2), 2.45 (t, J=6.8 Hz, 2H, 9-H), 2.33 (d, J=
17.2 Hz, 1H, 2-H), 2.00 (d, J=17.2 Hz, 1H, 2-H), 1.89 (s, 3H, 13-H),
1.78 (t, J=5.2 Hz, 1H, 9-H), 1.67 (m, 1H, 7-H), 1.58 (m, 2H, 8-H), 1.35
(m, 1H, 7-H), 1.00 (s, 3H, 11-H), 0.97 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.5 (3-C), 165.9 (5-C), 162.9 (d, J=243.9 Hz, ArÀ C), 158.5
(d, J=246.6 Hz, ArÀ C), 156.8 (ArÀ C), 142.7 (d, J=6.8 Hz, ArÀ C), 131.2
(ArÀ C), 130.6 (d, J=3.5 Hz, ArÀ C), 130.1 (d, J=4.5 Hz, ArÀ C), 129.6
(d, J=8.2 Hz, ArÀ C), 129.3 (ArÀ C), 126.2 (d, J=17.1 Hz, ArÀ C), 125.7
(d, J=15.5 Hz, ArÀ C), 125.1 (4-C), 124.9 (ArÀ C), 124.1 (d, J=2.1 Hz,
ArÀ C), 123.4 (d, J=3.9 Hz, ArÀ C), 120.5 (ArÀ C), 115.3 (d, J=21.2 Hz,
ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 111.0 (ArÀ C), 58.2 (Ar-CH2), 55.6
(OCH3), 54.0 (9-C), 51.4 (d, J=1.8 Hz, Ar-CH2), 51.0 (6-C), 47.1 (2-C),
36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 27.0 (8-C), 24.7 (13-C).
HR-ESIMS m/z 518.2841 [M+H]+, calcd for C33H38F2NO2, 518.2871.
Yield 89.4%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.62 (s, 1H,
ArÀ H), 7.50 (d, J=8.4 Hz, 1H, ArÀ H), 7.45 (m, 1H, ArÀ H), 7.36 (m, 1H,
ArÀ H), 7.27 (m, 2H, ArÀ H), 7.19 (m, 1H, ArÀ H), 7.10 (m, 1H, ArÀ H),
6.92 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.69 (s, 2H, Ar-CH2), 3.59 (s, 2H,
Ar-CH2), 2.45 (t, J=6.8 Hz, 2H, 9-H), 2.33 (d, J=17.2 Hz, 1H, 2-H),
2.00 (d, J=17.2 Hz, 1H, 2-H), 1.91 (s, 3H, 13-H), 1.79 (t, J=5.4 Hz, 1H,
6-H), 1.67 (m, 1H, 7-H), 1.58 (m, 2H, 8-H), 1.35 (m, 1H, 7-H), 1.01 (s,
3H, 11-H), 0.97 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.5 (3-
C), 165.9 (5-C), 162.9 (d, J=243.8 Hz, ArÀ C), 158.1 (d, J=246.4 Hz,
ArÀ C), 142.5 (d, J=7.0 Hz, ArÀ C), 135.0 (ArÀ C), 133.6 (ArÀ C), 131.5
(ArÀ C), 131.0 (d, J=4.6 Hz, ArÀ C), 130.2 (d, J=3.2 Hz, ArÀ C), 129.6
(d, J=8.1 Hz, ArÀ C), 129.5 (ArÀ C), 126.9 (d, J=16.9 Hz, ArÀ C), 126.6
(ArÀ C), 126.1 (d, J=15.1 Hz, ArÀ C), 125.0 (4-C), 124.1 (d, J=2.4 Hz,
ArÀ C), 123.5 (d, J=4.1 Hz, ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8
(d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 54.0 (9-C), 51.2 (Ar-CH2), 51.0 (6-
C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.2 (11-C), 27.0 (8-
C), 24.8 (13-C). HR-ESIMS m/z 556.1963 [M+H]+, calcd for
C32H34Cl2F2NO, 556.1986.
(R)-6-(3-(((2-fluoro-2’-methyl-[1,1’-biphenyl]-3-yl) methyl) (3-
fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11o)
(R)-6-(3-(((2-fluoro-2’,3’-dimethyl-[1,1’-biphenyl]-3-yl) methyl)
(3- fluorobenzyl) amino)
Yield 89.2%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.41 (m, 1H,
ArÀ H), 7.26 (m, 4H, ArÀ H), 7.16 (m, 5H, ArÀ H), 6.92 (m, 1H, ArÀ H),
6.91 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H), 3.68 (s, 2H, Ar-CH2), 3.59 (s, 2H,
Ar-CH2), 2.45 (t, J=6.8 Hz, 2H, 9-H), 2.33 (d, J=17.6 Hz, 1H, 2-H),
2.19 (s, 3H, CH3), 2.00 (d, J=17.6 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H), 1.78
(t, J=5.2 Hz, 1H, 9-H), 1.66 (m, 1H, 7-H), 1.57 (m, 2H, 8-H), 1.34 (m,
1H, 7-H), 1.00 (s, 3H, 11-H), 0.98 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.5 (3-C), 165.8 (5-C), 162.9 (d, J=243.8 Hz, ArÀ C), 158.1
(d, J=246.6 Hz, ArÀ C), 142.6 (d, J=7.0 Hz, ArÀ C), 136.5 (ArÀ C), 135.8
(ArÀ C), 130.3 (d, J=8.9 Hz, ArÀ C), 130.3 (ArÀ C), 129.9 (d, J=9.6 Hz,
ArÀ C), 129.6 (d, J=8.2 Hz, ArÀ C), 129.2 (d, J=7.8 Hz, ArÀ C),127.9
(ArÀ C), 125.9 (d, J=15.4 Hz, ArÀ C), 125.6 (4-C), 125.0 (ArÀ C), 124.1
(d, J=2.2 Hz, ArÀ C), 123.6 (d, J=4.0 Hz, ArÀ C), 115.3 (d, J=21.2 Hz,
ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 54.1 (9-C), 51.4 (Ar-
CH2), 51.1 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1
(11-C), 27.0 (8-C), 24.8 (13-C), 20.0 (d, J=2.6 Hz, CH3). HR-ESIMS m/z
502.2903 [M+H]+, calcd for C33H38F2NO, 502.2921.
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11r)
Yield 88.6%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.40 (m, 1H,
ArÀ H), 7.25 (m, 1H, ArÀ H), 7.15 (m, 6H, ArÀ H), 7.05 (m, 1H, ArÀ H),
6.92 (m, 1H, ArÀ H), 5.80 (s, 1H, 4-H), 3.58 (m, 4H, Ar-CH2), 2.45 (t, J=
6.4 Hz, 2H, 9-H), 2.34 (s, 3H, Ar-CH3), 2.33 (d, J=17.2 Hz, 1H, 2-H),
2.07 (s, 3H, Ar-CH3), 2.00 (d, J=17.2 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H),
1.78 (t, J=5.2 Hz, 1H, 6-H), 1.67 (m, 1H, 7-H), 1.57 (m, 2H, 8-H), 1.34
(m, 1H, 7-H), 1.00 (s, 3H, 11-H), 0.98 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.4 (3-C), 165.8 (5-C), 162.9 (d, J=243.8 Hz, ArÀ C), 158.1
(d, J=244.5 Hz, ArÀ C), 142.6 (d, J=6.9 Hz, ArÀ C), 136.9 (ArÀ C), 135.9
(ArÀ C), 135.0 (ArÀ C), 130.4 (d, J=3.2 Hz, ArÀ C), 130.1 (d, J=4.4 Hz,
ArÀ C), 129.7 (d, J=17.5 Hz, ArÀ C), 129.5 (d, J=10.5 Hz, ArÀ C), 127.8
(ArÀ C), 125.8 (d, J=15.5 Hz, ArÀ C), 125.2 (ArÀ C), 125.0 (4-C), 124.1
(d, J=2.3 Hz, ArÀ C), 123.5 (d, J=4.1 Hz, ArÀ C), 115.3 (d, J=21.2 Hz,
ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 54.1 (9-C), 51.4 (Ar-
CH2), 51.1 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.2
(11-C), 27.0 (8-C), 24.8 (13-C), 20.0 (CH3), 16.7 (CH3). HR-ESIMS m/z
516.3050 [M+H]+, calcd for C34H40F2NO, 516.3078.
(R)-6-(3-(((2’-chloro-2-fluoro-[1,1’-biphenyl]-3-yl) methyl) (3-
fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11p)
(R)-6-(3-(((2,2’-difluoro-3’-methoxy-[1,1’-biphenyl]-3-yl) methyl)
(3- fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11s)
Yield 86.3%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.44 (m, 1H,
ArÀ H), 7.26 (m, 2H, ArÀ H), 7.15 (m, 4H, ArÀ H), 7.00 (m, 1H, ArÀ H),
6.92 (m, 2H, ArÀ H), 5.79 (s, 1H, 4-H), 3.92 (s, 3H, OCH3), 3.69 (m, 2H,
Ar-CH2), 3.60 (s, 2H, Ar-CH2), 2.45 (t, J=6.6 Hz, 2H, 9-H), 2.34 (d, J=
17.2 Hz, 1H, 2-H), 2.07 (s, 3H, Ar-CH3), 2.00 (d, J=17.2 Hz, 1H, 2-H),
1.90 (s, 3H, 13-H), 1.78 (t, J=5.2 Hz, 1H, 6-H), 1.67 (m, 1H, 7-H), 1.57
Yield 87.9%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.46 (m, 2H,
ArÀ H), 7.32 (m, 3H, ArÀ H), 7.25 (m, 1H, ArÀ H), 7.19 (m, 2H, ArÀ H),
7.11 (m, 2H, ArÀ H), 6.92 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H), 3.69 (s, 2H,
Ar-CH2), 3.60 (s, 2H, Ar-CH2), 2.46 (t, J=6.8 Hz, 2H, 9-H), 2.33 (d, J=
17.6 Hz, 1H, 2-H), 2.00 (d, J=17.6 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H),
1.78 (t, J=5.2 Hz, 1H, 6-H), 1.67 (m, 1H, 7-H), 1.57 (m, 2H, 8-H), 1.34
(m, 1H, 7-H), 1.00 (s, 3H, 11-H), 0.97 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.5 (3-C), 165.9 (5-C), 162.9 (d, J=243.8 Hz, ArÀ C), 158.1
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(m, 2H, 8-H), 1.34 (m, 1H, 7-H), 1.00 (s, 3H, 11-H), 0.97 (s, 3H, 12-H).
13C NMR (100 MHz, CDCl3): δ 199.6 (3-C), 166.0 (5-C), 162.9 (d, J=
244.0 Hz, ArÀ C), 158.2 (d, J=247.6 Hz, ArÀ C), 150.9 (ArÀ C), 148.4
(ArÀ C), 141.9 (d, J=11.1 Hz, ArÀ C), 142.5 (d, J=7.0 Hz, ArÀ C), 130.9
(d, J=4.6 Hz, ArÀ C), 130.3 (ArÀ C), 129.6 (d, J=3.2 Hz, ArÀ C), 126.2
(d, J=15.2 Hz, ArÀ C), 124.9 (4-C), 124.4 (d, J=13.2 Hz, ArÀ C), 124.1
(d, J=2.2 Hz, ArÀ C), 123.7 (ArÀ C), 123.3 (d, J=16.8 Hz, ArÀ C), 122.7
(ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 112.9
(ArÀ C), 58.3 (Ar-CH2), 56.3 (OCH3), 54.0 (9-C), 51.3 (d, J=1.8 Hz, Ar-
CH2), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1
(11-C), 27.0 (8-C), 24.7 (13-C). HR-ESIMS m/z 536.2744 [M+H]+,
calcd for C33H37F3NO2, 536.2776.
(R)-6-(3-(((2’-chloro-2-fluoro-3’-methoxy-[1,1’-biphenyl]-3- yl)
methyl) (3-fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11v)
Yield 84.9%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.44 (m, 1H,
ArÀ H), 7.27 (m, 2H, ArÀ H), 7.18 (m, 2H, ArÀ H), 7.11 (m, 2H, ArÀ H),
6.98 (m, 1H, ArÀ H), 6.92 (m, 2H, ArÀ H), 5.79 (s, 1H, 4-H), 3.94 (s, 3H,
OCH3), 3.60 (s, 2H, Ar-CH2), 3.60 (s, 2H, Ar-CH2), 2.46 (t, J=6.6 Hz, 2H,
9-H), 2.33 (d, J=17.2 Hz, 1H, 2-H), 2.33 (s, 3H, Ar-CH3), 1.99 (d, J=
17.2 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H), 1.78 (t, J=5.2 Hz, 1H, 6-H), 1.66
(m, 1H, 7-H), 1.57 (m, 2H, 8-H), 1.34 (m, 1H, 7-H), 1.00 (s, 3H, 11-H),
0.97 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.6 (3-C), 166.0 (5-
C), 163.0 (d, J=243.8 Hz, ArÀ C), 158.0 (d, J=246.3 Hz, ArÀ C), 155.3
(ArÀ C), 142.6 (d, J=6.9 Hz, ArÀ C), 136.7 (ArÀ C), 131.0 (d, J=4.5 Hz,
ArÀ C), 130.1 (d, J=2.8 Hz, ArÀ C), 129.7 (d, J=8.1 Hz, ArÀ C), 127.1
(d, J=17.0 Hz, ArÀ C), 127.0 (ArÀ C), 126.1 (d, J=15.1 Hz, ArÀ C), 125.0
(4-C), 124.2 (d, J=2.4 Hz, ArÀ C), 123.5 (d, J=4.0 Hz, ArÀ C), 123.3
(ArÀ C), 122.2 (ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8 (d, J=
21.1 Hz, ArÀ C), 111.4 (ArÀ C), 58.2 (Ar-CH2), 56.3 (OCH3), 54.0 (9-C),
51.3 (Ar-CH2), 51.0 (6-C), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C),
27.1 (11-C), 27.0 (8-C), 24.8 (13-C). HR-ESIMS m/z 552.2465 [M+H]+,
calcd for C33H37ClF2NO2, 552.2481.
(R)-6-(3-((3-fluorobenzyl) ((2,2’,3’-trifluoro-[1,1’-biphenyl]-3- yl)
methyl) amino) propyl)-1,1,5-trimethylcyclohex-4-en-3-one
(11t)
Yield 87.8%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.48 (m, 1H,
ArÀ H), 7.26 (m, 2H, ArÀ H), 7.20 (m, 2H, ArÀ H), 7.13 (m, 4H, ArÀ H),
6.92 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H), 3.70 (s, 2H, Ar-CH2), 3.60 (s, 2H,
Ar-CH2), 2.46 (t, J=6.7 Hz, 2H, 9-H), 2.34 (d, J=17.2 Hz, 1H, 2-H),
2.00 (d, J=17.2 Hz, 1H, 2-H), 1.90 (s, 3H, 13-H), 1.79 (t, J=5.2 Hz, 1H,
6-H), 1.66 (m, 1H, 7-H), 1.58 (m, 2H, 8-H), 1.34 (m, 1H, 7-H), 1.00 (s,
3H, 11-H), 0.98 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.5 (3-
C), 165.8 (5-C), 163.0 (d, J=243.9 Hz, ArÀ C), 158.2 (d, J=248.0 Hz,
ArÀ C), 150.8 (dd, J1 =246.5 Hz, J2 =13.1 Hz, ArÀ C), 148.0 (dd, J1 =
248.7 Hz, J2 =12.9 Hz, ArÀ C), 142.4 (d, J=6.7 Hz, ArÀ C), 131.4 (d, J=
4.6 Hz, ArÀ C), 130.1 (ArÀ C), 129.6 (d, J=8.1 Hz, ArÀ C), 126.5 (d, J=
15.1 Hz, ArÀ C), 126.2 (d, J=1.3 Hz, ArÀ C), 125.9 (d, J=12.4 Hz,
ArÀ C), 125.0 (4-C), 124.1 (d, J=2.0 Hz, ArÀ C), 123.9 (dd, J1 =4.7 Hz,
J2 =6.9 Hz, ArÀ C), 122.3 (dd, J1 =2.5 Hz, J2 =16.6 Hz, ArÀ C), 116.8 (d,
J=17.1 Hz, ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8 (d, J=21.1 Hz,
ArÀ C), 58.3 (Ar-CH2), 54.0 (9-C), 51.3 (d, J=1.9 Hz, Ar-CH2), 51.0 (6-C),
47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 27.0 (8-C),
24.7 (13-C). HR-ESIMS m/z 524.2547 [M+H]+, calcd for C32H34F4NO,
524.2577.
(R)-2’-fluoro-3’-(((3-fluorobenzyl)
(3-(1,1,5-trimethyl-3-oxocyclohex-4-en-6-yl) propyl) amino)
methyl)-[1,1’-biphenyl]-2-carbonitrile (11w)
Yield 83.6%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.77 (d, J=
8.4 Hz, 1H, ArÀ H), 7.66 (m, 1H, ArÀ H), 7.50 (m, 3H, ArÀ H), 7.26 (m,
3H, ArÀ H), 7.11 (m, 2H, ArÀ H), 6.92 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H),
3.71 (s, 2H, Ar-CH2), 3.62 (s, 2H, Ar-CH2), 2.48 (t, J=6.8 Hz, 2H, 9-H),
2.34 (d, J=17.2 Hz, 1H, 2-H), 1.99 (d, J=17.2 Hz, 1H, 2-H), 1.91 (s,
3H, 13-H), 1.80 (t, J=5.2 Hz, 1H, 6-H), 1.67 (m, 1H, 7-H), 1.58 (m, 2H,
8-H), 1.35 (m, 1H, 7-H), 1.01 (s, 3H, 11-H), 0.98 (s, 3H, 12-H). 13C NMR
(100 MHz, CDCl3): δ 199.4 (3-C), 165.7 (5-C), 163.0 (d, J=244.0 Hz,
ArÀ C), 157.9 (d, J=247.1 Hz, ArÀ C), 142.4 (d, J=7.8 Hz, ArÀ C), 139.7
(ArÀ C), 133.1 (ArÀ C), 132.6 (ArÀ C), 132.0 (d, J=4.8 Hz, ArÀ C), 130.9
(ArÀ C), 130.0 (d, J=2.3 Hz, ArÀ C), 129.6 (d, J=8.1 Hz, ArÀ C), 128.2
(ArÀ C), 126.7 (d, J=15.1 Hz, ArÀ C), 125.9 (d, J=16.1 Hz, ArÀ C), 125.0
(4-C), 124.2 (d, J=2.5 Hz, ArÀ C), 124.0 (d, J=6.2 Hz, ArÀ C), 118.0
(CN), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8 (d, J=21.1 Hz, ArÀ C), 113.0
(ArÀ C), 58.3 (Ar-CH2), 54.1 (9-C), 51.2 (Ar-CH2), 51.0 (6-C), 47.2 (2-C),
36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 27.0 (8-C), 24.8 (13-C).
HR-ESIMS m/z 513.2695[M+H]+, calcd for C33H35F2N2O, 513.2717.
(R)-6-(3-(((2,2’-difluoro-3’-methyl-[1,1’-biphenyl]-3-yl) methyl)
(3- fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11u)
Yield 89.5%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 7.43 (m, 1H,
ArÀ H), 7.25 (m, 3H, ArÀ H), 7.17 (m, 3H, ArÀ H), 7.10 (m, 3H, ArÀ H),
6.92 (m, 1H, ArÀ H), 5.79 (s, 1H, 4-H), 3.69 (s, 2H, Ar-CH2), 3.60 (s, 2H,
Ar-CH2), 2.46 (t, J=6.7 Hz, 2H, 9-H), 2.34 (d, J=17.2 Hz, 1H, 2-H),
2.33 (s, 3H, CH3), 1.99 (d, J=17.2 Hz, 1H, 2-H), 1.89 (s, 3H, 13-H), 1.78
(t, J=5.2 Hz, 1H, 6-H), 1.67 (m, 1H, 7-H), 1.57 (m, 2H, 8-H), 1.34 (m,
1H, 7-H), 1.00 (s, 3H, 11-H), 0.97 (s, 3H, 12-H). 13C NMR (100 MHz,
CDCl3): δ 199.5 (3-C), 166.0 (5-C), 163.0 (d, J=244.0 Hz, ArÀ C), 158.4
(d, J=247.0 Hz, ArÀ C), 158.3 (d, J=245.5 Hz, ArÀ C), 142.6 (d, J=
6.9 Hz, ArÀ C), 131.3 (d, J=5.1 Hz, ArÀ C), 130.8 (d, J=4.6 Hz, ArÀ C),
130.4 (d, J=2.2 Hz, ArÀ C), 129.7 (d, J=8.1 Hz, ArÀ C), 128.9 (d, J=
2.0 Hz, ArÀ C), 126.2 (d, J=15.2 Hz, ArÀ C), 152.2 (d, J=17.7 Hz,
ArÀ C), 125.0 (4-C), 124.2 125.0 (d, J=23.8 Hz, ArÀ C), 123.9 (d, J=
23.0 Hz, ArÀ C), 123.7 (d, J=4.0 Hz, ArÀ C), 123.6 (d, J=4.3 Hz, ArÀ C),
123.3 (d, J=16.6 Hz, ArÀ C), 115.4 (d, J=21.2 Hz, ArÀ C), 113.9 (d, J=
21.1 Hz, ArÀ C), 58.4 (Ar-CH2), 54.0 (9-C), 51.4 (d, J=1.7 Hz, Ar-CH2),
51.1 (6-C), 47.1 (2-C), 36.4 (1-C), 28.7 (12-C), 28.0 (7-C), 27.2 (11-C),
27.0 (8-C), 24.8 (13-C), 14.8 (d, J=4.3 Hz, CH3). HR-ESIMS m/z
520.2814 [M+H]+, calcd for C33H37F3NO, 520.2827.
(R)-6-(3-(((2-fluoro-2’-nitro-[1,1’-biphenyl]-3-yl) methyl) (3-
fluorobenzyl) amino)
propyl)-1,1,5-trimethylcyclohex-4-en-3-one (11x)
Yield 80.7%, yellow oil, 1H NMR (400 MHz, CDCl3): δ 8.05 (m, 1H,
ArÀ H), 7.68 (m, 1H, ArÀ H), 7.55 (m, 1H, ArÀ H), 7.45 (m, 2H, ArÀ H),
7.25 (m, 3H, ArÀ H), 7.10 (m, 2H, ArÀ H), 6.92 (m, 1H, ArÀ H), 5.78 (s,
1H, 4-H), 3.64 (s, 2H, Ar-CH2), 3.58 (s, 2H, Ar-CH2), 3.60 (s, 2H, N-CH2),
2.43 (t, J=6.8 Hz, 2H, 9-H), 2.34 (d, J=17.2 Hz, 1H, 2-H), 1.99 (d, J=
17.2 Hz, 1H, 2-H), 1.91 (s, 3H, 13-H), 1.80 (t, J=5.4 Hz, 1H, 6-H), 1.67
(m, 1H, 7-H), 1.56 (m, 2H, 8-H), 1.33 (m, 1H, 7-H), 1.00 (s, 3H, 11-H),
0.98 (s, 3H, 12-H). 13C NMR (100 MHz, CDCl3): δ 199.5 (3-C), 165.9 (5-
C), 162.9 (d, J=243.8 Hz, ArÀ C), 157.8 (d, J=245.2 Hz, ArÀ C), 149.1
(ArÀ C), 142.4 (d, J=6.9 Hz, ArÀ C), 132.9 (ArÀ C), 132.5 (ArÀ C), 131.5
(d, J=4.5 Hz, ArÀ C), 130.8 (ArÀ C), 129.6 (d, J=8.2 Hz, ArÀ C), 128.9
(ArÀ C), 128.8 (d, J=2.7 Hz, ArÀ C), 126.0 (d, J=15.0 Hz, ArÀ C), 125.6
(d, J=16.9 Hz, ArÀ C), 125.0 (4-C), 124.5 (ArÀ C), 124.3 (d, J=4.0 Hz,
ArÀ C), 124.1 (d, J=2.5 Hz, ArÀ C), 115.3 (d, J=21.2 Hz, ArÀ C), 113.8
(d, J=21.1 Hz, ArÀ C), 58.3 (Ar-CH2), 54.0 (9-C), 51.1 (6-C), 51.0 (Ar-
CH2), 47.1 (2-C), 36.3 (1-C), 28.7 (12-C), 27.9 (7-C), 27.1 (11-C), 27.0
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ChemMedChem
(8-C), 24.7 (13-C). HR-ESIMS m/z 533.2485 [M+H]+, calcd for
C32H35F2N2O3, 533.2616.
at 570 nm. Relative adhesion rate(%)=OD value in sample/ OD
value cells in control ×100%.
Biological evaluation
Construction of the hypoxic cell model
To construct the hypoxic cell model, cells were incubated for 24 h
in culture dishes for adherence and pre-starved for 12 h with L15
starvation medium (containing only 1% FBS), which was replaced
with fresh starvation medium containing different concentrations
of 11g. Thereafter, the cells were placed into an anoxic culture
cassette, which was filled with high-purity nitrogen (purity of
nitrogen, >99.999%), sealed, and cultured in an incubator for
different durations.[32]
Chemotaxis assay
The chemotaxis assay was performed using nontoxic concentra-
tions of each compound. MDA-MB-231 cells were pretreated with
the compounds at the indicated concentrations for 24 h at 37 C in
six-well cell culture plates. Old medium and 10%FBS were loaded
into the lower chemotaxis chamber. Control (cells only) and
pretreated cells were resuspended in serum-free medium at a
density of 5×104 cells/mL and placed into the upper chamber
(50 μL/well). The transwell were inserted between the upper and
°
Western blotting
°
lower chambers. The cells were incubated at 37 C in 5% CO2 for
6 h; then the filter membrane was rinsed, fixed, and stained. The
number of migrating cells in three separate fields was counted
using light microscopy. The inhibitory ratio (IR) was calculated as
follows: IR%=(1-number of migrated cells in sample/number of
migrated cells in control) ×100%. The potencies of the compounds
were expressed as the median inhibitory concentration (IC50) values.
LY294002 (Camarillo, CA, USA) was used as a positive control
substance for this assay.[29]
MDA-MB-231 cells were cultured in 6-well plates and lysed on ice in
200 μL of RIPA buffer (containing 1% (v/v) PMSF) (Solarbio, Beijing,
China). The samples were electrophoresed on 10% SDS-polyacryla-
mide gels, transferred to polyvinylidene fluoride membranes,
blocked for 1 h in 2% (w/v) skim milk, and then incubated with
°
primary antibodies overnight at 4 C, followed by incubation with
appropriate secondary antibodies for 1 h at room temperature. The
bands were detected using chemiluminescent reagent and auto-
radiographic film.
Invasion assay
Real-time PCR (RT-PCR) analysis
Invasion of cells was measured in Matrigel (BD, Franklin Lakes, NJ,
USA) -coated transwell inserts (Costar, Manassas, VA, USA) contain-
ing polycarbonate filters with 8 μM pores. The inserts were coated
with 50 μL of Matrigel matrix according to the manufacturer’s
recommendations. 5×104 cells in 100 μL of cell suspension with
various concentrations of 11g were plated in the upper chamber,
whereas 750 μL of medium with 10% fetal bovine serum were
added to lower well. After incubating for 24 h, cells that migrated
to the lower surface of the membrane were fixed and stained. For
each membrane, three random fields were counted at 10 magnifi-
cation. Relative invasion rate (%)=number of migrated cells in
sample/number of migrated cells in control ×100%.
Total RNA from MDA-MB-231 cells was extracted using TRIzol
(Tiangen Biotechnology, Beijing, China). And the same amount of
RNA was taken for reverse transcription and RT-PCR using One Step
TB Green PrimeScript RT-PCR kit (Takara, Beijing, China) and 7500
Real Time PCR System (ThermoFisher Scientific, Waltham, MA, USA).
The human-specific primers were: HIF-1α (sense primer: 5’-
CTGGATGCTGGTGATTTGGA-3’; antisense primer: 5’-TGTCACCAT-
CATCTGTGAG-3’); GAPDH (sense primer: 5’-GTCATTGAGAGCAATGC-
CAG-3’; antisense primer: 5’-GTGTTCCTACCCCCAATGTG-3’). All
primers were purchased from GENEWIZ (Tianjin, China). GAPDH was
employed as the internal control.
Wound healing assay
Enzyme-linked immunosorbent assay (ELISA)
The assay was carried out as previously described.[30] MDA-MB-231
cells were seeded in 6-well plates and grown to full confluence. The
cell monolayer was wounded with a sterile micropipette tip to
generate a clean wound area across the center of the well.
Subsequently, cellular debris was rinsed away with PBS, and then
incubated in medium with or without compound for 24 h. Cell
migrated into the wound was monitored and photographed under
microscope. Relative migration rate (%)=migration distance in
sample/ migration distance in control ×100%.
The supernatant of breast cancer cells cultured with the compound
was collected and centrifuged, and the human VEGF ELISA kit (RD
Systems, USA) was used to detect the concentration of VEGF in
accordance with the protocol. The optical density was measured at
450 nm using an enzyme marker (BioTek, Winooski, USA). The
results are expressed as pg/mL. All the experiments were done
three times.
Statistical analysis
The results were expressed as means � SD from at least three
Adhesion assay
independent experiments. Statistical analyses were conducted by
Cell adhesion assay was performed as described[31] with modifica-
tions. The 96-well plates (BD Biosciences, Bedford, MA, USA) were
coated with fibronectin (20 μg/mL) at 4 C for overnight and then
GraphPad Prism.
significant.
P value <0.05 was considered statistically
°
blocked in BSA (1%) for 1 h. MDA-MB-231 cells were exposed to
11g (0.1, 1, 10 or 25 μM) for 24 h before seeding. Target cells were
suspended in medium. Cells (5×104 mLÀ 1
)
were seeded to
Acknowledgements
°
fibronectin coated plates and then incubated for 1 h at 37 C. Non-
adherent cells were removed by gentle washing with PBS. Then,
crystal violet staining assay was employed to analyze the adhesion
ability of cells. The OD value was measured using microplate reader
The authors gratefully acknowledge financial support from the
National Natural Science Foundation of China (NSFC) (81673310,
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Conflict of Interest
The authors declare no conflict of interest.
Keywords: Chiral ionone alkaloid derivatives
effect · HIF-1α · VEGFR2 · Akt
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FULL PAPERS
Novel chiral ionone alkaloid deriva-
tives were synthesized, and
J.-J. Liu, X.-Y. Liu, Dr. J.-P. Nie, M.-Q.
Jia, Dr. Y. Yu, Dr. N. Qin*, Prof. H.-Q.
Duan*
compound 11g with higher antiche-
motactic migration activity than the
lead compound was screened.
Compound 11g exerted inhibitory
effects on the adhesion, migration
and invasion of MDA-MB-231 cells.
The mechanisms for the antitumor
metastatic effects of 11g might be
that it inhibited HIF-1α expression to
down regulate the secretion of VEGF
and the phosphorylation of VEGFR2,
and then suppressed the down-
stream pathways, including Akt1/
mTOR/p70S6K and Akt2/PKCζ/
integrin β1.
1 – 17
Discovery of Antimetastatic Chiral
Ionone Alkaloid Derivatives
Targeting HIF-1α/VEGF/VEGFR2
Pathway