Synthesis and Antiproliferative Activity of Prenylated Chalcone Mannich Base Derivatives

Article abstract of DOI:10.1007/s10600-021-03380-4

Prenylated chalcones xanthohumol (1) and 2′-hydroxy-3,4,4′-trimethoxy-6′-O-prenyl chalcone (2) were synthesized through the Claisen–Schmidt condensation, O-prenylation, and Claisen rearrangement and deprotection respectively, using phloroglucinol and appropriate benzaldehydes as starting materials. Based on the Mannich reaction of prenylated chalcone 1 or 2 with various secondary amines and formaldehyde in acid alcohol solvent, 10 novel prenylated chalcone Mannich base derivatives 3a–3e and 4a–4e were synthesized. Furthermore, all synthetic compounds were evaluated for antiproliferative activities in vitro against four human cancer cell lines (Aspc-1, SUN-5, HepG-2, and HCT-116) by MTT assay. The results showed that most of them exhibit moderate to good antiproliferative activities against the four human cancer cells with IC₅₀ values of 2.52 to 47.67 μM.

Full text of DOI:10.1007/s10600-021-03380-4

DOI 10.1007/s10600-021-03380-4
Chemistry of Natural Compounds, Vol. 57, No. 3, May, 2021
SYNTHESIS AND ANTIPROLIFERATIVE ACTIVITY
OF PRENYLATED CHALCONE MANNICH BASE
DERIVATIVES
Liang Su, Ke-Xiong Liu, Pei-Pei Han,
and Qiu-An Wang*
Prenylated chalcones xanthohumol (1) and 2′-hydroxy-3,4,4′-trimethoxy-6′-O-prenyl chalcone (2) were
synthesized through the Claisen–Schmidt condensation, O-prenylation, and Claisen rearrangement and
deprotection respectively, using phloroglucinol and appropriate benzaldehydes as starting materials.
Based on the Mannich reaction of prenylated chalcone 1 or 2 with various secondary amines and formaldehyde
in acid alcohol solvent, 10 novel prenylated chalcone Mannich base derivatives 3a–3e and 4a–4e were
synthesized. Furthermore, all synthetic compounds were evaluated for antiproliferative activities in vitro
against four human cancer cell lines (Aspc-1, SUN-5, HepG-2, and HCT-116) by MTT assay. The results
showed that most of them exhibit moderate to good antiproliferative activities against the four human cancer
cells with IC values of 2.52 to 47.67 μM.
50
Keywords: prenylated chalcones, xanthohumol, Mannich bases, synthesis, antiproliferative activity.
Chalcones are precursors of flavonoids in plants. Chemically, they consist of open-chain flavonoids, in which the two
aromatic rings are joined by a three-carbon α,β-unsaturated carbonyl system. Prenylated chalcones are a unique class of
naturally occurring flavonoids characterized by the presence of a prenylated side chain in the chalcone skeleton [1, 2]. Owing
to their unique structure and extensive bioactivities, prenylated chalcones have attracted a great deal of attention. Various
prenylated chalcones have been investigated as antimalarial, antibacterial, and anticancer agents [3–6].
Xanthohumol is a representative prenylated chalcone extracted from the hop plant, Humulus lupulus, which has been
characterized as having a broad spectrum of anticancer research [7]. Besides its remarkable antiproliferative activity against
different cancer cell lines, xanthohumol also exhibited apoptotic activity and showed chemopreventive effects. Despite the
well-documented anticancer activity of xanthohumol, its efficacy is moderate and its bioavailability is low due to poor solubility
[8, 9]. Hence, it is of interest to improve its potency and increase its solubility in water.
The building block of Mannich base is an important model of drug biological activity. Studies demonstrated that
compounds bearing phenolic Mannich base moieties exhibited good biological activities. Aminomethylation of drugs can
increase the hydrophilic properties of drugs by introducing polar functional groups into the drug structure so as to improve the
solubility of the drug in the human body. These results make Mannich bases promising for anticancer drug design [10, 11].
Due to their structural uniqueness and potent bioactivity, the synthesiss of prenylated chalcone products has attracted
much attention in recent years. Based on our previous experience on flavonoid Mannich base derivatives as antitumor agents
[12–14], we have synthesized two series of 10 prenylated chalcone Mannich base derivatives 3a–3e and 4a–4e and evaluated
in vitro their antiproliferative activities against a panel of four human cancer cell lines including Aspc-1 (human pancreatic
cancer), SUN-5 (human gastric cancer), HepG-2 (human hepatocellular carcinoma), and HCT-116 (human colon cancer),
using the thiazolyl blue tetrazolium bromide (MTT) method with staurosporine as the positive control drug.
College of Chemistry and Chemical Engineering, Hunan University, 410082, Changsha, P. R. China, e-mail:
wangqa@hnu.edu.cn. Published in Khimiya Prirodnykh Soedinenii, No. 3, May–June, 2021, pp. 364–369. Original article
submitted March 18, 2020.
0009-3130/21/5703-0425 ©2021 Springer Science+Business Media, LLC
425

MOMO
O
OH
O
OH
b
c
a
OMOM
MOMO
MOMO
OMOM
O
MOMO
OMOM
O
OMOM
5
7
6
3'
3
MOMO
HO
OMOM
OMOM
OH
OH
HO
OH
OH
f
e
α
1'
5
R R N CH
1
2
1
2
6'
β
R
O
O
O
H CO
3
H CO
3
3a-e
8, 9
1
d
8
9
O
OH
O
OH
g
b
H CO
3
HO
OCH
H CO
3
HO
OCH
3
3
10
11
R R N
2
1
CH
3'
2
H CO
3
H CO
3
OH
O
OCH
3
OH
4 OCH
3
3
6
f
α
1'
5'
3
OCH
1
OCH
3
β
O
O
O
4a-e
2
8: R = OH; 9: R = OCH
3
N
; 3c, 4c: NR R =
1 2
3a, 4a: NR R = N(CH CH CH ) ; 3b, 4b: NR R =
N
1
2
2
2
3 2
1
2
N
O
; 3e, 4e: NR R =
3d, 4d: NR R =
N
N
CH
3
1
2
1 2
a. 4-Methyloxymethylbenzaldehyde, KOH, EtOH; b. prenylbromide, K CO , acetone, 50°C; c. florisil, toluene, 100°C; d. (CH ) SO ,
2
3
3 2
4
K CO , acetone, r.t.; e. 3M HCl (aq.); f. HCHO (aq.), CH OH, amines, HCl, 80°C; g. 3,4-dimethoxybenzaldehyde, KOH, EtOH
2
3
3
Scheme 1
The synthesis of naturally occurring C-prenylated chalcone xanthohumol (1), O-prenylated chalcone analogs (2),
and their Mannich base derivatives 3a–3e and 4a–4e is outlined in Scheme 1. 2,4-Dimethoxymethoxy-6-hydroxyacetophenone
(5) was prepared from commercially available phloroglucinol via Friedel–Crafts acetylation and selective O-methoxymethylation
[13, 14]. Claisen–Schmidt aldol condensation of 5or 10 with MOM-protected 4-hydroxybenzaldehydes or 3,4-dimethoxybenzaldehydes
gave the corresponding chalcones 6 or 11, which were refluxed with prenylbromide in K CO –acetone for 24 h to obtain
2
3
O-prenylated chalcones 2 or 7 in high yield. Claisen rearrangement of O-prenylated chalcone 7 using Florisil as an effective
and environmentally benign heterogeneous catalyst in toluene gave the MOM-protected C-prenylated chalcone 8 [15]. The
1
para-rearranged product was obtained selectively in 85% yield and was the only product observed in the H NMR spectrum
of the product signals for the –CH -CH=C(CH ) in 1 at 3.16 ppm (2H, d). Methylation with dimethyl sulfate gave 9.
2
3 2
Deprotection with HCl (aq.) in MeOH (reflux) gave the prenylated chalcone xanthohumol (1). The spectroscopic data of 1
1
were identical to those reported in the literature [16]. E-Chalcone is characterized by a large J , H NMR coupling constant
α β
in the trans olefin bond region of 15–16 Hz.
426

TABLE 1. Half-Inhibitory Concentration of Compounds 1, 2, 3a–3e, and 4a–4e on Human Cancer Cell Lines (IC , μM)
50
Compound
Aspc-1
SUN-5
HepG-2
HCT-116
1
2
14.57 1.21
> 50
16.20 1.35
> 50
16.37 1.46
> 50
9.32 1.02
> 50
3a
3b
3c
28.5 1.16
11.23 0.96
9.29 1.06
> 50
9.29 0.68
> 50
16.82 1.45
13.37 1.22
> 50
6.05 1.08
10.71 0.98
> 50
3d
3e
4a
4b
4c
4d
4e
35.29 1.64
> 50
> 50
6.0 0.26
5.37 0.65
13.61 0.86
14.49 1.06
0.052 2.68
34.66 1.26
36.51 1.36
> 50
47.67 1.68
22.77 1.64
> 50
4.62 1.06
4.83 1.27
17.96 0.88
5.97 1.06
0.0575 1.95
15.06 1.20
19.81 1.52
6.98 1.24
2.52 1.16
2.54 1.04
8.47 0.86
4.72 0.96
0.02 1.46
> 50
10.49 1.02
12.96 0.68
15.28 0.55
0.0208 2.55
Staurosporinea
______
a
Staurosporine was employed as a positive control.
Further modifications of xanthohumol (1) and O-prenylated chalcone (2) were achieved through the introduction of
an amino methyl functional group by means of the Mannich reaction. Compounds 1 or 2 reacted with various secondary
amines and formaldehyde in acid alcohol solvent. Ten novel prenylated chalcone Mannich base derivatives 3a–3e and 4a–4e
were synthesized. The Mannich reaction can be applied to aromatic rings as long as the hydroxyl group is available in the
ortho-position of phenol. The synthesized compounds are classified as aminoalkylphenols. The exclusive formation of
ortho-aminoalkylated products is attributed to the directing effect of the hydrogen bond between nitrogen and the o-hydroxyl
group, which affords a stabilized six-membered ring. The electron density of the B ring is higher than that of the A ring due to
the existence of multiple electron-donating groups in the B ring; the C-5′ or C-3′ position on the B-ring of chalcone is most
nucleophilic and thus prone to electrophilic aromatic substitution. The difference in reactivity allows the regioselectivity for
the Mannich reaction.
Substitution at the 5′-position for 1 and the 3′-position for 2 on the B-ring of chalcones was observed based upon the
H NMR. Compound 1 exhibited specific peaks at 6.10 ppm corresponding to 5′-positions, 2 exhibited specific peaks at 5.88 ppm
1
corresponding to 3′-positions. All of the Mannich products exhibited methylene (CH ) peaks around 3.60–3.85 ppm in the
2
1
13
H NMR, and a new sign appeared between 61.96–67.26 ppm in the C NMR. With the notable absence of any peaks
at 6.10 or 5.88 ppm, it is suggested that substitution occurred at the 5′-position for 1 and the 3′-position for 2 of the B-ring.
It should be noted that in all cases the electrophilic substitution occurred solely in the B-ring, even at the large excess of the
reagent and under more severe conditions.
The antiproliferative activity of all the synthesized compounds 1, 2, 3a–3e, and 4a–4e was investigated in vitro
against a panel of four human cancer cell lines, Aspc-1, SUN-5, HepG-2, and HCT-116, by MTT cell proliferation assays.
In these experiments, staurosporine, a known anticancer drug, was used as positive control. The antiproliferative activities of
the tested compounds are described by the half maximal inhibitory concentrations (IC ) value in Table 1. The results showed
50
that most of them exhibited moderate to good antiproliferative activities against the four human cancer cells with IC values
50
of 2.52 to 47.67 μM. As shown in Table 1, the C-prenylated chalcone xanthohumol 1 was more active than the O-prenylated
chalcone 2. The aminomethylene group substitution at the C-5′ position for 1 and the C-3′ position for 2 significantly
enhanced the antiproliferative activity. For the Aspc-1 cell line, most of the compounds showed antiproliferative activity
with IC from 5.37 to 35.29 μM. Compounds 4b and 4c showed the highest activity (IC 6.0 μM and 5.37 μM). For the
50
50
SUN-5 cell line, compounds 3b, 4c, 4d, and 4e exhibited higher activity (IC 9.29–15.28 μM) compared to xanthohumol
50
(IC 16.20 μM). For the HepG-2 cell line, most of the compounds showed antiproliferative activity with IC from 4.62 to
50
50
47.67 μM. Compounds 3b, 4b, 4c, and 4e exhibited higher activity (IC 4.62–13.37 μM) compared to xanthohumol
50
(IC 16.37 μM). For HCT-116, most of the compounds showed antiproliferative activity with IC from 2.52 to 19.81 μM.
50
50
Compounds 4b and 4c showed the highest activity (IC 2.52 and 2.54 μM, respectively). Compounds 3c against Aspc-1 cell
50
lines (IC 9.29 μM) and 4a against HCT-116 (IC 6.98 μM) cell lines showed selective antiproliferative activity, respectively.
50
50
Compounds 3b, 3d, 4c, and 4e showed antiproliferative activities with IC values ranging from 4.72 to 47.67 μM against all
50
427

cancer lines. Compound 4c, having the piperidinomethyl group at the 3′-position, proved to be the most active of the synthesized
prenylated chalcone Mannich base tested, with IC value ranging from 2.54 to 10.49 μM against all four cancer cell lines.
50
The presence of a Mannich base group in natural products may increase the biological potency due to the greater number of
molecular sites for electrophilic attack by cellular constituents, as well as due to the cascade effect of preferential
chemosensitization.
In summary, we have synthesized prenylated chalcones xanthohumol (1) and 2′-hydroxy-3,4,4′-trimethoxy-6′-O-prenyl
chalcone (2) and their 10 Mannich base derivatives. After antiproliferative activity screening of those compounds 4b, 4c, and 4e
against Aspc-1, HepG-2, and HCT-116 cells, and 3b against SUN-5 cells, these compounds were identified as the most active
with IC values ranging from 2.52 to 14.49 μM, and they showed more potency than the parent compounds prenylated
50
chalcones 1 and 2. Substitution of the aminomethylene group such as pyrrolidinomethyl, piperidinomethyl, and 4-methyl-
piperazinomethyl at the C-5′ position for 1 and the C-3′ position for 2 significantly enhanced the antiproliferative activity.
EXPERIMENTAL
1
13
General Information Melting points were determined via an XRC-1 apparatus. H NMR and C NMR spectra
were recorded on a Bruker-AV 400 spectrometer at 400 and 100 MHz, respectively, with TMS as internal standard in CDCl
3
or DMSO-d . Mass spectra (MS) and high-resolution mass spectra (HR-MS) were determined on a VG Autospec-3000 or
6
Mat 95 XP spectrometer by the EI or ESI method. Column chromatography was carried out on 200–300 mesh silica gel
(Qingdao Ocean Chemical Products, China). Commercially availableAR or chemically pure reagents were used, and anhydrous
solvents were dehydrated and redistilled using standard experimental procedures.
Synthesis of 2′,4,4′-Trimethoxymethoxy-6′-hydroxychalcone (6). A 100 mL round-bottom flask was charged with
4-methoxymethoxybenzaldehyde (0.4 g, 2.4 mmol), 2,4-dimethoxymethoxy-6-hydroxyacetophenone (5, 0.5 g, 2.0 mmol),
KOH (1.0 g, 0.018 mmol), and 20 mL of ethanol. After stirring at room temperature for 24 h, the reaction liquid changed from
the original yellow to orange red. TLC analysis showed the complete consumption of the raw materials. The solvent was removed,
50 mL of water added, and the aqueous layers extracted with CH Cl (15 mL × 3) and then washed with saturated brine. The
2
2
organic layer was dried over Na SO and concentrated. The crude product was purified by silica gel column chromatography
2
4
1
eluted with petroleum ether–ethyl acetate (20:1) to give a yellow solid 0.7 g, yield 89%, mp 83–85°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 13.84 (1H, s, OH), 7.77 (1H, d, J = 15.5, H-β), 7.70 (1H, d, J = 15.5, H-α), 7.48 (2H, d, J = 8.1, H-2, 6), 7.00
3
(2H, d, J = 8.2, H-3, 5), 6.25 (1H, s, H-3′), 6.18 (1H, s, H-5′), 5.22 (2H, s, OCH ), 5.15 (2H, s, OCH ), 5.12 (2H, s, OCH ), 3.47
2
2
2
13
(3H, s, OCH ), 3.42 (6H, s, OCH ). C NMR (100 MHz, CDCl , δ, ppm): 191.83, 166.28, 162.31, 158.81, 157.95, 141.42,
3
3
3
+
128.96, 128.19, 124.42, 115.50, 106.53, 96.48, 94.12, 93.73, 93.10, 55.86, 55.45, 55.16. MS (EI, 70 eV) m/z 404.14 [M] .
Synthesis of 2′,4,4′-Trimethoxymethoxy-6′-O-prenylchalcone (7). A mixture of compound 6 (0.5 g, 1.2 mmol) and
K CO (1.0 g, 7.2 mmol) in acetone (30 mL) was stirred at room temperature for 0.5 h, then 3,3-dimethylallylbromide (0.3 mL,
2
3
2.5 mmol) was added. The reaction was carried out for 1 h after the temperature was raised to 50°C. The reaction solution
changed from orange red to pale yellow; TLC analysis showed the complete consumption of the raw materials after 5 h. The
mixture was filtered through a Celite pad, and the filtrate was concentrated to give the crude product. It was purified by neutral
alumina column chromatography eluted with petroleum ether–ethyl acetate (30:1) to give 0.3 g of yellow solid substance,
1
yield 62%, mp 76–78°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.32 (2H, d, J = 6.7, H-2, 6), 7.18 (1H, d, J = 16.2, 1H, H-β),
3
6.88 (2H, d, J = 6.6, H-3, 5), 6.74 (1H, d, J = 16.0, H-α), 6.38 (1H, s, H-3′), 6.27 (1H, s, H-5′), 5.20 (1H, t, J = 12.9, CH=), 5.03
(4H, s, OCH ), 4.96 (2H, s, OCH ), 4.36 (2H, d, J = 6.4, CH ), 3.34 (3H, s, OCH ), 3.29 (3H, s, OCH ), 3.23 (3H, s, OCH ),
2
2
2
3
3
3
13
1.54 (3H, s, CH ), 1.51 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 194.22, 159.58, 158.98, 157.67, 155.77, 144.31,
3
3
3
137.59, 131.61, 129.86, 128.54, 128.27, 127.39, 119.58, 116.37, 116.06, 114.28, 95.90, 95.22, 94.43, 94.03, 65.54, 56.24,
+
25.60, 18.11. MS (EI, 70 eV) m/z 472.20 [M] .
Synthesis of 2′,4,4′-Trimethoxymethoxy-6′-hydroxy-3′-prenylchalcone (8). A round-bottom flask was charged
with compound 7 (0.5 g, 1.1 mmol) and Florisil (2.0 g) in toluene (40 mL), and the reaction was carried out at 100°C for 48 h.
The TLC was tracked and the material disappeared. The reaction solution was filtered to remove Florisil, 80 mL of water was
added, and the mixture was extracted with CH Cl (15 mL × 3); then the organic layer was washed with saturated brine, dried
2
2
over Na SO , and concentrated. The crude product was purified by silica gel column chromatography eluted with
2
4
1
petroleum ether–ethyl acetate (30:1) to give 0.4 g of yellow solid substance, yield 85%, mp 103–105°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 12.01 (1H, s, OH), 7.22 (2H, d, J = 8.0, H-2, 6), 6.94 (2H, d, J = 8.0, H-3, 5), 6.13 (1H, d, J = 15.6, H-β),
3
428

5.18 (1H, d, J = 15.6, Hα), 5.05 (6H, s, OCH ), 3.34 (9H, s, OCH ), 3.17 (2H, d, J = 7.0, CH ), 2.95 (1H, s, H-5′), 2.61 (1H,
2
3
2
13
t, J = 8.0, CH=), 1.66 (3H, s, CH ), 1.54 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 196.28, 162.83, 161.04, 160.56,
3
3
3
157.60, 150.89, 142.95, 131.81, 131.40, 130.19, 127.64, 122.45, 116.46, 110.68, 103.44, 94.33, 93.84, 93.46, 78.88, 56.27,
+
55.96, 43.29, 29.59, 25.79, 21.30, 17.76. MS (EI, 70 eV) m/z 472.21 [M] .
Synthesis of 2′,4,4′-Trimethoxymethoxy-6′-methoxy-3′-prenylchalcone (9).Amixture of compound 8 (0.2 g, 0.4 mmol)
and K CO (0.2 g, 1.4 mmol) in acetone (20 mL) was stirred at room temperature for 20 min; then dimethyl sulfate (1 mL) was
2
3
added. The TLC was traced at 2 h, and the starting material point disappeared. The reaction solution was filtered, and the
filtrate was concentrated to give the crude product. It was purified by silica gel column chromatography eluted with
1
petroleum ether–ethyl acetate (20:1) to give 0.1 g of yellow solid material. Yield 52%, mp 108–110°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.40 (2H, d, J = 8.4, H-2, 6), 7.28 (1H, d, J = 16.0, H-β), 6.95 (2H, d, J = 8.4, H-3, 5), 6.85 (1H, d, J = 16.0,
3
H-α), 6.48 (1H, s, H-5′), 5.16 (2H, s, OCH ), 5.12 (4H, s, OCH ), 5.11 (1H, t, J = 8.0, CH=), 3.68 (3H, s, OCH ), 3.62 (3H, s,
2
2
3
13
OCH ), 3.43 (3H, s, OCH ), 3.39 (3H, s, OCH ), 3.24 (2H, d, J = 6.6, CH ), 1.69 (3H, s, CH ), 1.60 (3H, s, CH ). C NMR
3
3
3
2
3
3
(100 MHz, CDCl , δ, ppm): 194.60, 159.06, 157.54, 157.03, 156.26, 144.49, 131.14, 130.11, 128.64, 127.10, 123.28, 117.74,
3
+
116.86, 116.42, 94.56, 94.19, 62.84, 56.13, 56.03, 30.21, 29.70, 25.75, 22.68, 17.82. MS (EI, 70 eV) m/z 486.22 [M] .
Synthesis of Xanthohumol (1). Hydrochloric acid (3 mol/L, 3 drops) was added to a solution of compound 9 (0.1 g,
0.2 mmol) in anhydrous methanol (20 mL). The reaction was carried out at 40°C for 1 h. The solvent was evaporated, and
water (80 mL) was added. The mixture was extracted with ethyl acetate (15 mL × 3); then the organic layer was washed with
saturated brine, dried over Na SO , filtered, and the filtrate evaporated. The crude product was purified by silica gel column
2
4
chromatography eluted with petroleum ether–ethyl acetate (8:1) to give 40 mg of a yellow solid, yield 57%, mp 158–160°C.
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 14.65 (1H, s, OH), 10.57 (1H, s, OH), 10.08 (1H, s, OH), 7.78 (1H, d, J = 15.5,
3
H-β), 7.69 (1H, d, J = 15.5, H-α), 7.59 (2H, d, J = 8.0, H-2, 6), 6.86 (2H, d, J = 8.0, H-3, 5), 6.10 (1H, s, H-5′), 5.16 (1H, t,
13
J = 8.0, CH=), 3.88 (3H, s, OCH ), 3.16 (2H, d, J = 6.7, CH ), 1.71 (3H, s, CH ), 1.62 (3H, s, CH ). C NMR (100 MHz,
3
2
3
3
CDCl , δ, ppm): 192.15, 165.10, 162.87, 161.00, 160.39, 143.00, 130.97, 130.39, 126.54, 124.26, 123.50, 116.46, 107.81,
3
+
105.06, 91.42, 56.22, 25.95, 21.52, 18.14. MS (EI, 70 eV) m/z 354.9 [M] .
Synthesis of Xanthohumol Mannich Base Derivatives 3a–3e. A round bottom flask was charged with secondary
amines (3 mmol), methanol (10 mL), concentrated hydrochloric acid (3 drops), and 37% HCHO aqueous solution (1.0 mL). After
stirring at room temperature for 20 min, compound 1 (100 mg, 0.28 mmol) and methanol (20 mL) were added to the reaction
flask, and the whole heated to 80°C and reacted for 2 h. When TLC analysis showed the complete consumption of compound 1,
the mixture was concentrated, and then water (80 mL) was added. The mixture was extracted with dichloromethane (15 mL × 3),
then the organic layer was washed with saturated brine, dried over Na SO , filtered, and the filtrate evaporated. The crude
2
4
product was purified by silica gel column chromatography eluted with petroleum ether–ethyl acetate (3:1) to give 3a–3e.
1
5′-Dipropylaminomethylxanthohumol (3a). Light yellow solid 100 mg, yield 77%, mp 242–244°C. H NMR
(400 MHz, CDCl , δ, ppm, J/Hz): 7.73 (1H, d, J = 15.6, H-β), 7.65 (1H, d, J = 15.6, H-α), 7.40 (2H, d, J = 8.0, H-2, 6), 6.78
3
(2H, d, J = 7.8, H-3, 5), 5.19 (1H, t, J = 6.8, CH=), 3.83 (2H, s, CH N), 3.52 (3H, s, OCH ), 3.26 (2H, d, J = 6.7, CH ), 2.66
2
3
2
13
(4H, q, J = 6.6, NCH ), 1.71 (3H, s, CH ), 1.59 (3H, s, CH ), 1.09 (6H, t, J = 7.0, CH ). C NMR (100 MHz, CDCl , δ, ppm):
2
3
3
3
3
192.43, 163.86, 161.32, 158.89, 142.86, 131.80, 130.31, 128.74, 123.94, 122.66, 116.08, 115.49, 112.81, 106.22, 62.86,
+
50.03, 46.15, 31.43, 30.21, 29.71, 25.81, 21.70, 17.93, 10.68. MS (EI, 70 eV) m/z 467.1 [M] .
1
5′-Pyrrolidin-1-ylmethylxanthohumol (3b). Yellow solid 80 mg, yield 67%, mp 220–222°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.76 (1H, d, J = 16.2, H-β), 7.70 (1H, d, J = 15.8, H-α), 7.57 (2H, d, J = 7.9, H-2, 6), 6.86 (2H, d, J = 8.0,
3
H-3, 5), 5.18 (1H, t, J = 6.5, CH=), 4.01 (3H, s, OCH ), 3.60 (2H, s, CH N), 3.18 (2H, d, J = 6.9, CH ), 2.83–2.85 (4H, m,
3
2
2
13
NCH ), 1.88–1.85 (4H, m, CH ), 1.72 (3H, s, CH ), 1.62 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 193.38, 164.30,
2
2
3
3
3
158.58, 143.56, 131.00, 128.71, 124.69, 122.97, 116.59, 113.31, 106.16, 67.26, 63.64, 55.08, 53.37, 30.32, 26.44, 22.30,
+
18.52. MS (EI, 70 eV) m/z 455.93 [M + H O] .
2
1
5′-Piperidin-1-ylmethylxanthohumol (3c). Yellow solid 90 mg, yield 71%, mp 248–250°C. H NMR (400 MHz,
CDCl , δ, ppm, J/Hz): 7.69 (1H, d, J = 15.5, H-β), 7.64 (1H, d, J = 15.5, H-α), 7.40 (2H, d, J = 7.4, H-2, 6), 6.81 (2H, d, J = 7.4,
3
H-3, 5), 5.18 (1H, t, J = 6.5, CH=), 3.80 (3H, s, OCH ), 3.71 (2H, s, CH N), 3.54–3.52 (4H, m, CH ), 3.28 (2H, d, J = 5.3,
3
2
2
13
CH ), 1.72 (3H, s, CH ), 1.64 (3H, s, CH ), 1.62–1.60 (4H, m, CH ), 1.21–1.20 (2H, m, CH ). C NMR (100 MHz, CDCl ,
2
3
3
2
2
3
δ, ppm): 191.54, 163.07, 158.27, 157.88, 142.38, 131.44, 129.37, 126.39, 122.47, 121.26, 115.11, 111.59, 106.80, 104.34,
+
61.99, 54.12, 53.07, 52.48, 28.14, 24.79, 23.81, 22.26, 20.74, 16.90. ESI-MS m/z 452.07 [M + H] .
1
5′-Morpholine-1-ylmethylxanthohumol (3d). Pale yellow solid 90 mg, yield 71%, mp 230–232°C. H NMR
(400 MHz, CDCl , δ, ppm, J/Hz): 7.76 (1H, d, J = 16.2, H-β), 7.67 (1H, d, J = 15.8, H-α), 7.46 (2H, d, J = 7.1, H-2, 6), 6.79
3
429

(2H, d, J = 6.8, H-3, 5), 5.19 (1H, t, J = 6.5, CH=), 3.85 (2H, s, CH N), 3.76–3.73 (4H, m, OCH ), 3.54 (3H, s, OCH ), 3.27
2
2
3
13
(2H, d, J = 5.5, CH ), 2.58–2.55 (4H, m, NCH ), 1.73 (3H, s, CH ), 1.62 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm):
2
2
3
3
3
191.74, 162.66, 156.93, 141.92, 129.36, 127.06, 123.05, 121.33, 114.95, 111.67, 104.51, 65.62, 62.00, 53.44, 51.73, 29.19,
+
28.99, 28.68, 24.80, 20.65, 16.87. ESI-MS m/z 453.9 [M + H] .
5′-(4-Methyl)piperazin-1-ylmethylxanthohumol (3e). Light yellow solid 100 mg, yield 77%, mp 250–252°C.
1
H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.74 (1H, d, J = 15.6, H-β), 7.67 (1H, d, J = 15.5, H-α), 7.45 (2H, d, J = 7.9, H-2, 6),
3
6.78 (2H, d, J = 7.8, H-3, 5), 5.18 (1H, t, J = 6.8, CH=), 3.74 (2H, s, CH N), 3.51 (3H, s, OCH ), 3.25 (2H, d, J = 6.5, CH ),
2
3
2
13
2.88 (4H, t, J = 12.1, NCH ), 2.60 (4H, t, J = 19.0, CH ), 2.35 (3H, s, NCH ), 1.73 (3H, s, CH ), 1.62 (3H, s, CH ). C NMR
2
2
3
3
3
(100 MHz, CDCl , δ, ppm): 191.74, 162.60, 162.26, 158.11, 157.82, 142.21, 130.86, 129.44, 126.33, 122.60, 121.41, 115.21,
3
+
111.57, 107.15, 104.72, 61.96, 53.45, 52.85, 50.55, 44.34, 24.80, 20.64, 16.88. ESI-MS m/z 467.13 [M + H] .
Synthesis of 2′-Hydroxy-3,4,4′-trimethoxy-6-O-prenylchalcone (2). A mixture of compound 11 (0.5 g, 1.5 mmol)
and K CO (1.0 g, 7.2 mmol) in acetone (30 mL) was stirred at 50°C for 1 h; then 3,3-dimethylallyl bromide (0.3 mL,
2
3
2.5 mmol) was added. The reaction was allowed to continue for 4 h. TLC analysis showed the complete consumption of
compound 11, and the reaction liquid changed from the original orange red to pale yellow. The mixture was filtered and the
filtrate was concentrated. The crude product was chromatographed on a silica gel column eluted with petroleum ether–ethyl acetate
1
(15:1) to give the a yellow solid 0.4 g, yield 67%, mp 82–84°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 14.45 (1H, s, OH),
3
7.83 (1H, d, J = 14.6, H-β), 7.69 (1H, d, J = 14.2, H-α), 7.12 (1H, d, J = 8.2, H-6), 7.01 (1H, s, H-2), 6.78 (1H, d, J = 8.3,
H-5), 6.01 (1H, s, H-5′), 5.88 (1H, s, H-3′), 5.50 (1H, t, J = 5.7, CH=), 4.48 (2H, d, J = 6.6, CH ), 3.87 (3H, s, OCH ), 3.84
2
3
13
(3H, s, OCH ), 3.74 (3H, s, OCH ), 1.72 (3H, s, CH ), 1.66 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 191.59,
3
3
3
3
3
167.48, 164.98, 160.81, 149.94, 148.07, 141.36, 138.19, 127.71, 124.74, 121.20, 117.69, 110.12, 105.39, 92.73, 91.06, 86.68,
+
64.78, 57.54, 54.95, 54.52, 24.80, 17.30. ESI-MS m/z 439.9 [M + H O + Na] .
2
Synthesis of 2′-hydroxyl-3,4,4′-trimethoxy-6′-O-prenylchalcone Mannich base Derivatives 4a–4e. A solution of
37% HCHO aqueous solution (1 mL), amines (3.5 mmol), and concentrated hydrochloric acid (3 drops) in methanol (30 mL) was
cooled to room temperature and reacted for 20 min; compound 2 (300 mg, 0.7 mmol) was added to the reaction flask, the whole
warmed to 80°C, and the reaction continued for 3 h. When TLC analysis showed the complete consumption of compound 2, the
mixture was concentrated, and then water (80 mL) was added. The mixture was extracted with dichloromethane (15 mL× 3); then
the organic layer was washed with saturated brine, dried over Na SO , filtered, and the filtrate evaporated. The crude product was
2
4
purified by silica gel column chromatography eluted with petroleum ether–ethyl acetate (3:1) to give 4a–4e.
2′-Hydroxy-3′-dipropylaminomethyl-3,4,4′-trimethoxy-6′-O-prenylchalcone (4a). Yellow solid 300 mg,
1
yield 78%, mp 148–150°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 14.98 (1H, s, OH), 7.82 (1H, d, J = 15.6, H-β), 7.69
3
(1H, d, J = 15.3, H-α), 7.15 (1H, d, J = 8.0, H-6), 7.03 (1H, s, H-2), 6.81 (1H, d, J = 8.2, H-5), 6.00 (1H, s, H-5′), 5.52 (1H, t,
J = 6.5, CH=), 4.60 (2H, d, J = 4.8, CH ), 4.16 (2H, s, CH N), 4.01 (3H, s, OCH ), 3.87 (6H, s, OCH ), 3.06–2.91 (4H, m,
2
2
3
3
13
NCH ), 1.89–1.91 (4H, m, CH ), 1.74 (3H, s, CH ), 1.70 (3H, s, CH ), 1.62–1.60 (6H, m, CH ). C NMR (100 MHz, CDCl ,
2
2
3
3
3
3
δ, ppm): 192.56, 150.42, 149.02, 142.07, 129.88, 127.77, 127.48, 121.56, 118.27, 111.34, 109.77, 86.76, 67.11, 64.96, 55.94,
+
54.32, 37.71, 29.34, 28.67, 27.90, 24.77, 22.72, 21.96, 21.66, 17.81, 17.31, 13.04, 10.79, 9.94. ESI-MS m/z 511.9 [M + H] .
2′-Hydroxy-3′-pyrrolidin-1-ylmethyl-3,4,4′-trimethoxy-6′-O-prenylchalcone (4b). Yellow solid 310 mg,
1
yield 83%, mp 118–120°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.63 (1H, d, J = 15.6, H-β), 7.55 (1H, d, J = 15.6,
3
H-α), 7.19 (1H, d, J = 8.2, H-6), 7.12 (1H, s, H-2), 6.88 (1H, d, J = 8.0, H-5), 6.04 (1H, s, H-5′), 5.53 (1H, t, J = 6.8, CH=), 4.61
(2H, d, J = 6.4, CH ), 3.94 (6H, s, OCH ), 3.89 (3H, s, OCH ), 3.79 (2H, s, CH N), 2.65–2.62 (4H, m, NCH ), 1.79 (3H, s,
2
3
3
2
2
13
CH ), 1.74 (3H, s, CH ), 1.78–1.76 (4H, m, CH ). C NMR (100 MHz, CDCl , δ, ppm): 193.45, 162.96, 162.19, 160.06,
3
3
2
3
150.87, 149.08, 142.57, 138.45, 128.66, 126.65, 122.40, 119.28, 111.08, 110.65, 108.59, 105.54, 87.65, 65.94, 55.95, 55.58,
53.77, 47.91, 25.80, 23.55, 18.34, 1.03. ESI-MS m/z 482.1 [M + H] .
+
2′-Hydroxy-3′-piperidin-1-ylmethyl-3,4,4′-trimethoxy-6′-O-prenylchalcone (4c). Yellow solid 300 mg, yield 81%,
1
mp 108–110°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.45 (1H, d, J = 15.7, H-β), 7.19 (H, d, J = 15.7, H-1α), 7.07 (1H,
3
d, J = 8.1, H-6), 7.02 (1H, s, H-2), 6.78 (1H, d, J = 8.1, H-5), 5.95 (1H, s, H-5′), 5.39 (1H, t, J = 6.8, CH=), 4.49 (2H, d, J = 6.4,
CH ), 3.84 (6H, s, OCH ), 3.77 (3H, s, OCH ), 3.60 (2H, s, CH N), 2.45–2.42 (4H, m, CH ), 1.66 (3H, s, CH ), 1.63 (3H, s,
2
3
3
2
2
3
13
CH ), 1.53–1.52 (4H, m, CH ), 1.37–1.36 (2H, m, CH ). C NMR (100 MHz, CDCl , δ, ppm): 180.44, 179.49, 166.62,
3
2
2
3
161.59, 149.82, 148.04, 141.94, 127.53, 125.93, 121.55, 109.98, 109.36, 103.53, 98.93, 86.82, 64.93, 54.94, 54.55, 52.93,
+
25.89, 24.75, 21.60, 17.28, 9.73. ESI-MS m/z 496.1 [M + H] .
2′-Hydroxy-3′-morpholin-1-ylmethyl-3,4,4′-trimethoxy-6′-O-prenylchalcone (4d). Pale yellow solid 330 mg,
1
yield 82%, mp 144–146°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.65 (1H, d, J = 15.4, H-β), 7.59 (1H, d, J = 15.7,
3
H-α), 7.12 (1H, d, J = 8.1, H-6), 7.02 (1H, s, H-2), 6.80 (1H, d, J = 8.0, H-5), 5.95 (1H, s, H-5′), 5.48 (1H, t, J = 6.8, CH=), 4.54
430

(2H, d, J = 6.4, CH ), 3.85 (6H, s, OCH ), 3.82 (3H, s, OCH ), 3.65–3.62 (4H, m, CH O), 3.61 (2H, s, CH N), 2.52–2.50 (4H,
2
3
3
2
2
13
m, CH N), 1.71 (3H, s, CH ), 1.67 (6H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 192.19, 163.63, 162.62, 160.21,
2
3
3
3
149.95, 148.08, 141.58, 138.05, 127.61, 125.18, 121.29, 117.86, 110.10, 109.87, 106.39, 86.53, 65.82, 64.91, 57.25, 54.95,
+
54.62, 52.15, 49.06, 24.81, 17.34. ESI-MS m/z 497.9 [M + H] .
2′-Hydroxy-3′-(4-methyl)piperazin-1-ylmethyl-3,4,4′-trimethoxy-6′-O-prenylchalcone (4e). Light yellow solid
1
300 mg, yield 79%, mp 136–138°C. H NMR (400 MHz, CDCl , δ, ppm, J/Hz): 7.54 (1H, d, J = 15.6, H-β), 7.48 (1H, d, J = 15.8,
3
H-α), 7.10 (1H, d, J = 8.1, H-6), 7.02 (1H, s, H-2), 6.79 (1H, d, J = 8.1, H-5), 5.94 (1H, s, H-5′), 5.44 (1H, t, J = 6.8, CH=), 4.51
(2H, d, J = 6.4, CH ), 3.85 (6H, s, OCH ), 3.79 (3H, s, OCH ), 3.64 (2H, s, CH N), 2.55–2.52 (4H, m, NCH ), 2.42–2.40 (4H,
2
3
3
2
2
13
m, CH ), 2.20 (3H, s, NCH ), 1.69 (3H, s, CH ), 1.65 (3H, s, CH ). C NMR (100 MHz, CDCl , δ, ppm): 192.36, 149.89,
2
3
3
3
3
148.07, 141.66, 137.63, 127.59, 125.48, 121.39, 118.13, 110.05, 109.66, 107.33, 86.67, 64.91, 54.93, 54.55, 53.85, 51.50,
+
49.33, 44.90, 33.64, 26.34, 25.89, 24.78, 24.25, 17.32. ESI-MS m/z 511.1 [M + H] .
Antiproliferative Activity Screening. Using MTT assay, all the cells were cultured in an RPMI 1640 medium
containing 10%FBS and incubated at 37°C in a 5% CO humidified incubator to keep the cells growing in the exponential
2
phase. Briefly, MDA-MB-231 cells and K562 cells in a 100 μL culture medium were plated into a 96-well plate at
4000–5000 cells per well and subsequently cultured in the RPMI1640 medium containing 10% FBS, incubated at 37°C for
24 h prior to drug exposure. The selected compounds were weighed and dissolved in DMSO and then diluted with medium to
the needed concentrations. Cells were treated to a final concentration of 100, 50, 25, 12.5, 6.25, and 3.125 μM of the tested
compounds and incubated for 48 h; then 20 μL of 0.5% MTT solution was added to each well simultaneously and the whole
incubated at 37°C for 4 h. The formed formazan crystals were dissolved by adding 150 μL of DMSO. The optical density at
570 nm was determined on a microliter-plate reader. According to the inhibition ratios, the IC value was obtained.
50
ACKNOWLEDGMENT
We thank the National Natural Science Foundation of China (No. J1210040) and the Hunan Provincial Natural
Science Foundation of China (No. 2018JJ2034) for financial support.
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