Total synthesis of novel skeleton flavan-alkaloids

Article abstract of DOI:10.3390/molecules25194491

The first total synthesis of novel skeleton natural compounds kinkeloids A and B, a group of newly discovered flavan alkaloids isolated fromthe African plant Combretum micranthum, are described in this study. The key and final step are achieved by Mannich reaction, through which the piperidine moiety couples to the flavan moiety. The identities of synthesized kinkeloids were further confirmed through a comparison with the ones in the plant leaves extract using LC/MS.

Full text of DOI:10.3390/molecules25194491

molecules
Article
Total Synthesis of Novel Skeleton Flavan-Alkaloids
Jing Zhen 1,2, James E. Simon 1,2,* and Qingli Wu 1,2,
*
1
Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers University,
160 Frelinghuysen Road, Piscataway, NJ 08854, USA; zhenjing89@gmail.com
The New Use Agriculture and Natural Plant Products Program, Department of Plant Biology,
Rutgers University, 59 Dudley Road, New Brunswick, NJ 08901, USA
2
*
Correspondence: jimsimon@rutgers.edu (J.E.S.); qlwu@sebs.rutgers.edu (Q.W.); Tel.: +1-848-932-6238 (J.E.S.);
+1-848-932-6239 (Q.W.)
Academic Editor: Jacek Skarzewski
Received: 11 September 2020; Accepted: 28 September 2020; Published: 30 September 2020
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The first total synthesis of novel skeleton natural compounds kinkeloids A and B, a group of
newly discovered flavan alkaloids isolated from the African plant Combretum micranthum, are described
in this study. The key and final step are achieved by Mannich reaction, through which the piperidine
moiety couples to the flavan moiety. The identities of synthesized kinkeloids were further confirmed
through a comparison with the ones in the plant leaves extract using LC/MS.
Keywords: Combretum micranthum; kinkeloids A; kinkeloids B
1. Introduction
Kinkeloids are a series of newly discovered natural compounds with a novel skeleton from the
leaves of the shrub plant Combretum micranthum, which is indigenous to Senegal and West Africa
and commonly called kinkeliba. The leaves of kinkeliba are widely consumed as an herbal tea in
West Africa and for the treatment of gastrointestinal problems, colic and vomiting [
brewed from the dried leaves is generally used as a panacea for possible weight loss and has shown
antihyperglycemic activity in vivo ]. Kinkeloids belong to the family of flavonoid alkaloids, which are
1,2]. The beverage
[3
chemically interesting as the skeletons can serve as a platform for targeted drug design due to their
amphoteric structure with phenolic acids and basic nitrogenous moieties. The flavonoid alkaloids
family has been shown to exhibit a wide range of biological activities including antiviral, anticancer,
and anti-inflammatory activities [
Buchenavia capitata have shown HIV-inhibitory activity with an EC50 of 0.26
4
]. For example, flavone alkaloids O-demethylbuchenavianine from
M [ ]. Flavopiridol
µ
5
derived from natural chrome alkaloid rohitukine that was extracted from Dysoxylum binectariferum
can inhibit the activity of cyclin-dependent kinases (CDKs), and is being developed in clinical trial for
cancer treatment [6] (Figure 1).
Kinkeloids were first discovered by our group [1]. Their structures are mainly made up of a flavan
moiety with a piperidine ring attached at either the 6 or 8 position. Kinkeloids A, B, C, and D differ
from each other only by the number of hydroxyl groups attached, resulting in consecutive mass unit
difference in mass spectra (Figure 1). Since kinkeloids have been identified as the major component
contained in C. micranthum, which has traditionally been used in herbal tea, it was of interest to explore
some of their biological activities. Due to the close physiochemical properties between theses kinkeloids
in the plant extract, direct separation and accumulation using chromatography is very challenging and
time-consuming, especially for large-scale production during pharmacological research.
Molecules 2020, 25, 4491; doi:10.3390/molecules25194491
www.mdpi.com/journal/molecules

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Figure 1. Kinkeloids and retrosynthetic analysis.
2. Results and Discussion
Here, we report on two synthetic methods that would be used for the preparation of kinkeloids.
Both synthetic routes are based on the Mannich reaction, which links the flavan moiety and the
piperidine moiety (Figure 1). To the best of our knowledge, only flavone and flavanone classes have
been reported from the family of flavonoids in the synthesis of their corresponding flavonoid alkaloids
through a reaction with cyclic imine [7–9]. The chemical properties between different classes of
flavonoids vary greatly and affect reaction results. In our study, the Mannich reaction between a flavan
molecule and cyclic imine was explored for the first time. As reported, the other flavonoid alkaloids
usually have low solubility in alcohol and even in DMSO, and the 8-substitued groups generally are
less soluble than the 6-substitued ones. In contrast with other flavonoid alkaloids, the synthesized
flavan alkaloids are very soluble in alcohol, including methanol and ethanol. Therefore, the solvent
selected to perform the flavan Mannich reaction was 100% methanol. Due to the comparatively
higher flexibility of flavan molecules, the substitution on the 6-,8-positions had a lack of selectivity,
even when different solvent systems were tried including MeOH, EtOH, H2O/MeOH, H2O/THF. Yet,
this substitution selectivity was observed and reported in flavonone and flavone groups, indicating
that carbonyl groups at position 4 can impact the nucleophilic activities of positions 6 and 8 [9,10].
The synthesis of cyclic imine started from commercially available compound piperidine, which was
converted to N-chloropiperidine
of the N-chloropiperidine formed with potassium hydroxide in ethanol provided the corresponding
synthetic intermediate . In this step, low yields were observed when excessive heating
1-piperidein
was used for solvent removal because of the low boiling point of compound
1-piperidein was then
crystalized out in its trimeric form as tripiperidein 9 in cold acetone (Scheme 1) [11].
7 through the addition of N-chlorosuccinimide (NCS). Treatment
∆
8
8. ∆

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O
Cl
N
H
N
a
N
b
c
N
N
+
N
Cl
N
O
5
6
7
8
8a
Scheme 1. The synthesis of
∆
1-piperidein intermediate. Reaction conditions and reagents: (
a) ether,
RT; (b) 20% KOH, ethanol, 0 ◦C to RT; (c) 80% acetone, −20 ◦C.
The two synthetic methods presented here differ from each other in the synthesis of flavan
moiety [12 13]. Synthetic method I was applied for the synthesis of the kinkeloid B series (Scheme 2),
,
while method II was used for the A series (Scheme 3). Synthetic method I started from the
commercially available compounds 3,4-dihydroxybenzaldehyde and 2,4,6-trihydroxyacetophone,
which were protected with MOM groups to provide compounds 11 and 12. MOM groups are an
ideal selection due to its stability in the basic condition and its facile removal in an acidic condition.
The ethanol mixture of 11 and 12 was then basified with 40% KOH solution in an ice bath and
gradually warmed up to room temperature to provide a 62% yield of fully protected chalcone 13 [14].
De-protection of MOM groups using 10% HCl in methanol under an elevated temperature of 50 ◦C
yielded exclusively the compound eriodictyol 14, which was cyclized from de-protected chalcone
intermediate. The phenolic acid groups were then fully protected with trimethylsilyl (TMS) groups
before the step of reductive de-oxygenation using mild reducing reagent NaBH3CN. NaBH3CN, as a
reducing agent, has remarkable stability in acidic environments, whose hydrolysis rate is 10−8 that
of sodium borohydride [15]. A wide range of acids including BF3
HCl, were compared in the reaction and it was found that 10% HCl delivered the best conversion
(60% yield) without opening the A ring at the 2 position [16 17]. The TMS protecting groups were
also removed in the reaction by hydrolysis. Flavan 16 reacted with
1-piperidein and yielded the
·
OEt2, ZnCl2, and concentrated
,
∆
kinkeloid B series with a 20% yield. Low yields may have been due to the formation of a byproduct,
6,8-di-piperidine-substituted flavan, and a low conversion percentage.
Synthetic method II involved the application of o-quinone methide and an inverse electron-demand
Diels–Alder reaction. The starting material 2,4,6-trihydroxybenzaldehyde was reacted with di-tert-butyl
dicarbonate to form the ortho o-boc compound 18, which is thermally unstable on silica column
and can decompose to the di-boc-substituted compound 18a at room temperature. The use of a
◦
cold solvent system (ethyl acetate/hexane) from a
−
20 C freezer for chromatographic separation
prevents its decomposition and keeps its tri-substituted status. Compound 18 was then used to
generate o-quinone methides in combination with magnesium bromide and lithium aluminum
hydride [18]. The generation of o-quinone methides through Grignard regents requires extreme
dryness. Subsequently, the 1,4-conjugate addition of o-quinone methide with electron-rich olefins
through an inverse Diels–Alder reaction yielded the protected flavan products. A low yield was
observed in our experiment, which we attributed to the product’s insufficient dryness during the
process. Higher yields of aldehydes, alkenes, and other materials have been reported when kept
extremely dry, using sodium through Kugelrohr distillation [19], and this inverse Diels–Alder reaction
has previously been applied to the synthesis of modified flavonoid molecules [19]. The t-butyl and
Boc groups were subsequently removed using ZnBr2 at room temperature, and 40,5,7-trihydroxyl
flavan was achieved [20]. Kinkeloids A was synthesized through a reaction between flavan 21 and
∆
1-piperidein, with a yield of 25%. Compared with the kinkeloid B series, the A series compounds
are comparatively less stable and could be easily oxidized to purple compound when exposed to
ambient air within just a few hours. This phenomenon correlates very well with the relative amount
found in the leaf extract, where the kinkeloid B series is very abundant, while the kinkeloid A series
is at trace levels. Both synthesized kinkeloids A and B were further positively confirmed through a
comparison with compounds contained in kinkeliba leaf extract on LC/UV/MS, and it was found that

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the synthesized compounds shared identical UV profiles, mass spectrums and retention times with the
ones extracted from the kinkeliba leaves.
In summary, this study presents the first synthesis of kinkeloid molecules, which are a newly
discovered flavonoid alkaloids. The synthetic approach in the preparation of such flavan alkaloids
allows for the procurement of large quantities that are needed for in vivo efficacy and toxicity studies.
Such synthetic derivatives also contribute to the in vivo disposition, metabolism and pharmacokinetic
studies through the instillation of stable radioisotopes.
Scheme 2. Route one for the synthesis of kinkeloids B. Reaction conditions and reagents: (
MOMCl, DMAP, CH2Cl2, 0 ◦C to RT (88% for 11, 74% for 12); ( ) 40% KOH, ethanol, 0 ◦C to RT (62%);
) 10% HCl, methanol, reflux (60%); ( ) TMSCl, HMDS, pyridine ( ) LiBH4, NaBH3CN, THF, 10% HCl,
RT (65%); (f) ∆1-piperideine, methanol, 70 ◦C (20%).
a) DIPEA,
b
(c
d
e

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Scheme 3. Route two for the synthesis of kinkeloids A. Reaction conditions and reagents: (
g
) Boc2O,
DIPEA, DMAP, CH2Cl2, RT, overnight (46.5%); (
h) MgBr2
·
OEt2, LiAlH4, ether, i) ZnBr2,
−
78 ◦C to RT; (
CH2Cl2, (9.7%); (e) ∆1-piperideines, methanol, 70 ◦C (25%).
3. Materials and Methods
3.1. General Experimental Procedures
1H and 13C-NMR spectra (Supplementary Materials) were performed on a Bruker Avance 400 MHz
spectrometer (Billerica, MA, USA). Analytical LC-MS was performed on a Hewlett-Packard Agilent
1100 series HPLC-MSD (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary
pump system, a degasser, an auto-sampler, a DAD detector, an MSD trap with an electrospray ion
source (ESI). Column chromatography was performed using silica gel (230–400 mesh; Selecto Scientific,
Suwanee, GA, USA) and Sephadex LH-20 (25–100 µ, Sigma-Aldrich, St. Louis, MO, USA). All synthetic
materials including the solvents were purchased from Sigma Aldrich.
3.2. ∆1-Piperidein
To a suspension solution of N-Chlorosuccinimide 6 (5.9 g, 45.0 mmol) in ether (50 mL), piperidine 5
(2.5 mL, 25.25 mmol) was added drop-wise over 30 min. After being stirred for 3 h at room temperature,
the suspension was filtered and washed with ether (20 mL). The combined organic solvent was washed
with water (20 mL), brine (20 mL), then dried using anhydrous sodium sulfate. The solvent was
removed under reduced pressure, yielding intermediate N-chloropiperidine 7, which was added
drop-wise into a solution of potassium hydroxide (3 g) in ethanol (15 mL) followed by stirring for
another 5 h at room temperature. After the reaction, diethyl ether (25 mL) was added to the solution,
which was washed with water and brine, then dried using anhydrous sodium sulfate. Solvent ether
was then removed under reduced pressure without heating, which yielded the product
(1.30 g, 62%). A low yield was observed if the solvent was removed under heating.
∆
∆
1-piperidein
1-piperidein
was crystalized as tripiperidein in cold acetone at
monomeric and trimeric forms. Monomeric form (some peaks overlapped with the trimeric form):
7.77–7.86 (m, 1H, N=C-H), 3.52–3.60 (m, 2H, -CH2-), 2.33 (m, 2H, -CH2-), 2.13 (m, 2H, -CH2-). Trimeric
form: 3.07–3.11 (m, 1H, -CH-), 2.76–2.78 (m, 1H, -CH2-), 1.95–1.99 (m, 1H, -CH2-), 1.65–1.69 (m, 3H, 2
-CH2-), 1.52–1.54 (m, 2H, -CH2-), 1.12–1.29 (m, 2H, -CH2-). 13C-NMR (100 MHz CDCl3) monomeric
form (some peaks overlapped with the trimeric form): 163.2 (CH, -N=CH-), 49.35 (CH2, -CH2-N=),
−
20 ◦C. 1H-NMR (400 MHz CDCl3), a mixture of
δ
×
δ
28.85 (CH2, -CH2-CH=), 18.80 (CH2, -CH2-); trimeric forms: 82.05 (CH, -N-CH (CH2)-N-); 46.48 (CH2,
-CH2-N-), 29.27 (CH2, -CH2-), 25.87 (CH2, -CH2-), 22.41 (CH2, -CH2-).

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3.3. 3,4-bis(methoxymethoxy)benzaldehyde (11)
To an ice-cooled suspension of 3,4-dihydroxybenzaldehyde (500 mg, 3.62 mmol) in 10 mL of
dichloromethane, N,N-diisopropylethylamine (6.0 mL) and DMAP (5 crystals) were added. Then,
MOMCl (chloromethyl methyl ether, 642 mg, 7.96 mmol) was added drop-wise at 0 ◦C, and the reaction
was gradually warmed up to room temperature and stirred overnight. After the disappearance of
the starting material on TLC, the reaction was poured into water (10 mL) and extracted with organic
solvent dichloromethane (2
dried over anhydrous sodium sulfate. The solvent was then removed under reduced pressure and the
×
20 mL). The combined organic phase was washed with water, brine and
residue was further purified on silica column using hexane/ethyl acetate (9:1) to yield compound 11 as
1
a white solid (720 mg, 88%). H-NMR (400 MHz CDCl3)
δ
3.51 (3H, s, -OCH3), 3.52 (3H, s, -OCH3),
5.28 (2H, s, -OCH2O-), 5.32 (2H, s, -OCH2O-), 7.27 (1H, d, J = 8.4 Hz, H-50), 7.50 (1H, dd, J = 8.4 and 1.9,
H-60), 7.67 (1H, d, J = 1.9 Hz, H-20), 9.86 (1H, s, -CHO); 13C-NMR (100 MHz CDCl3) 56.5 (CH3, -OCH3),
56.6 (CH3, -OCH3), 95.1 (CH2, -OCH2O-), 95.5 (CH2, -OCH2O-), 115.6 (CH, C-50), 116.1 (CH, C-20),
126.4 (CH, C-60), 131.3 (C, C-10), 147.6 (C, C-30), 152.8 (C, C-40), 190.9 (C, CHO).
3.4. 1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)ethan-1-one (12)
2,4,6-trihydroxyacetophone (500 mg, 2.97 mmol) was suspended in ice-old dichloromethane
(10 mL) followed by the addition of N,N-diisopropylethylamine (6.0 mL) and DMAP (5 crystals).
After around 10 min, MOMCl (515 mg, 6.40 mmol) was added drop-wise and the mixture was stirred
overnight. After the disappearance of starting material shown on TLC, the mixture was poured into
water (10 mL), and extracted with dichloromethane (2
washed with water (10 mL) and brine (10 mL), and dried using anhydrous sodium sulfate. The solvent
was then removed under reduced pressure and the residue was purified on silica gel column using
×
20 mL) two times. The organic phase was
hexane/ethyl acetate (9:1) to yield compound 12 (560 mg, 73.4%) as a colorless oil that was crystalized
1
as a white solid. H-NMR (400 MHz CDCl3)
δ 2.66 (s, 3H, -COCH3), 3.47 (s, 3H, -OCH3), 3.51 (3H,
s, -OCH3), 5.17 (2H, s, -OCH2O-), 5.25 (2H, s, -OCH2O-), 6.24 (1H, d, J = 2.3 Hz, Ar-H), 6.27 (1H, d,
J = 2.3 Hz, Ar-H); 13C-NMR (100 MHz CDCl3)
33.1 (CH3, -COCH3), 56.6 (CH3, -OCH3), 56.9 (CH3,
δ
-OCH3), 94.2 (CH2, 2
×
-OCH2O-), 94.7 (CH, C-50), 97.4 (CH, C-30), 107.1 (C, C-10), 160.5 (C, C-60),
163.6 (C, C-40), 167.0 (C, C-20), 203.4 (C=O, C-1).
3.5. 3-(3,4-bis(methoxymethoxy)phenyl)-1-(2-hydroxy-4,6-bis(methoxymethoxy)phenyl)prop-2-en-1-one (13)
Compounds 11 (160 mg, 0.71 mmol) and 12 (150 mg, 0.59 mmol) were co-suspended in an ice-cold
solution of ethanol (5 mL) followed by the addition of 40% KOH solution (1 mL). The mixture was
gradually warmed to room temperature and stirred overnight. After that, the reaction was poured
into water (10 mL) and extracted with dichloromethane (3
washed with water (10 mL) and brine (10 mL), and dried using anhydrous Na2SO4. The solvent was
×
20 mL). The combined organic phase was
then purified on silica column using hexane/ethyl acetate (5:1) to obtain compound 13 (170 mg, 62.0%)
1
as a yellow solid. H-NMR (400 MHz CDCl3)
δ
3.48 (3H, s, -OCH3), 3.52 (3H, s, -OCH3), 3.53 (3H, s,
-OCH3), 3.54 (3H, s, -OCH3), 5.18 (2H, s, -OCH2O-), 5.27 (2H, s, -OCH2O-), 5.28 (2H, s, -OCH2O-), 5.30
(2H, s, -OCH2O-), 6.27 (1H, d, J = 2.3 Hz, H-30), 6.31 (1H, d, J = 2.3 Hz, H-50), 7.17–7.22 (2H, m, H-5, 6),
7.51 (1H, d, J = 1.7 Hz, H-2). 7.74 (1H, d, J = 15.5, H-
α), 7.86 (1H, d, J = 15.5, H-β), 13.92 (1H, s, -OH);
13C-NMR (100 MHz CDCl3)
δ
56.4 (CH3, -OCH3), 56.5 (CH3, -OCH3), 56.6 (CH3, -OCH3), 57.0 (CH3,
-OCH3), 94.2 (CH2, -OCH2O-), 94.9 (CH2, -OCH2O-), 95.2 (CH2, -OCH2O-), 95.3 (CH2, -OCH2O-), 95.7
(CH, C-50), 97.7 (CH, C-30), 107.7 (C, C-10), 115.5 (CH, C-2), 116.4 (CH, C-5), 124.3 (CH, C-6), 126.1 (CH,
C-
(C, C-40), 192.9 (C, C=O).
α), 130.1 (C, C-1), 142.5 (CH, C-β
), 147.7 (C, C-4), 149.3 (C, C-3), 160.1 (C, C-20), 163.5 (C, C-60), 167.5

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3.6. 2-(3,4-dihydroxyphenyl)-5,7-dihydroxychroman-4-one (14)
Compound 13 (70 mg, 0.15 mmol) was suspended in methanol (5 mL) followed by the addition of
37% HCl solution (1 mL). The mixture was heated up and refluxed for 5 h. After the reaction, the solution
was cooled down and poured into water, then extracted with EtOAc (3
×
10 mL). The combined organic
phases were washed with water and brine, then dried using anhydrous Na2SO4. Then solvent was
removed under reduced pressure. The residue was dissolved in methanol (0.5 mL) and purified over
Sephadex LH-20 column using 100% methanol to yield compound 14 (30.5 mg, 71%) as a white solid.
This compound could also be purified on silica gel column using solvent hexane/ethyl acetate (4:1).
1H-NMR (400 MHz CDCl3)
δ 2.67 (1H, dd, J = 3.0 Hz and 17.1 Hz, H-3), 3.19 (1H, dd, J = 17.1 and 12.5
Hz, H-3), 5.37 (1H, dd, J = 12.5 and 3.0 Hz, H-2), 5.87 and 5.88 (2H, d, H-6 and 8), 6.74 (2H, s, H-50 and
60), 6.87 (1H, s, H-2); 13C-NMR (100 MHz CDCl3)
(CH, C-8), 101.8 (C, C-10), 114.3 (CH, C-20), 115.3 (CH, C-50), 117.9 (CH, C-60), 129.4 (C, C-10), 145.2 (C,
C-40), 145.7 (C, C-30),162.9 (C, C-5), 163.4 (C, C-9), 166.6 (C, C-7), 196.3 (C, C-4).
δ 42.1 (CH, C-3), 78.4 (CH, C-2), 94.9 (CH, C-6), 95.7
3.7. 2-(3,4-dihydroxyphenyl)chromane-5,7-diol (16)
To a solution of compound 14 (288 mg, 1.0 mmol) in dry pyridine (1 mL) was added
hexamethyldisilazane (1 mL) and trimethylchlorosilane (0.6 mL). The mixture was then further
stirred at room temperature for 1 h. The volatile constituents were distilled out under reduced pressure;
the residue was suspended in toluene and the precipitate was then filtered out. Solvent toluene solution
was then distilled out under reduced pressure and the residue was dissolved in THF (2.5 mL) followed
by the addition of LiBH4 (11 mg, 0.5 mmol). The mixture was further stirred at room temperature
for half an hour. Next, 0.5 mg of methyl orange and sodium cyanoborohydride (63 mg, 1.0 mmol)
were added into the solution. The mixture was slowly titrated using hydrochloric acid solution (1
N) with the release of hydrogen until the color of the solution turned red. The color of the solution
was controlled in red by the slow, continuous addition of hydrochloric acid. After approximately 2 h,
an extra amount of hydrochloric acid (1 to 1.5 mL) was added to make the color permanent, and the
reaction was stirred overnight at room temperature. After this, the solvent THF was distilled out under
reduced pressure and the residue was extracted with ethyl acetate and washed with 1 N hydrochloric
acid solution, distilled water, and brine. The organic solution was dried using anhydrous sodium
sulfate and purified on silica column using 10–20% ethyl acetate in hexane as eluent in gradient with a
1
65% yield. H-NMR (400 MHz, MeOD-d4)
δ 1.88 (1H, m, H-3), 2.08 (1H, m, H-3), 2.61 (2H, m, H-4),
4.77 (1H, dd, H-2), 5.84 (1H, d, J = 2.3 Hz, H-8), 5.90 (1H, d, J = 2.3 Hz, H-6), 6.67 (2H, m, H-50,60), 6.85
(1H, d, J = 1.84, H-20); 13C-NMR (100 MHz, MeOD-d4)
δ 20.35 (C-4), 30.89 (C-3), 78.848 (C-2), 96.00
(C-8), 96.08 (C-6) 102.6 (C-10), 114.47 (C-50), 116.17 (C-20), 118.83 (C-60), 135.14 (C-10), 145.93 (C-30),
146.24 (C-40), 157.31, 157.49, 157.97 (C-5,7,9).
3.8. Kinkeloids B
The synthesized flavan compound 16 (40 mg, 0.15 mmol) was dissolved in methanol (4 mL)
followed by the addition of ∆1-piperideines 8 (12.1 mg, 0.15 mmol). The mixture was stirred at 70 ◦C
under nitrogen atmosphere for 4 h. After the reaction, the solvent was removed under reduced pressure
and the residue was purified on Sephadex column LH-20 using methanol to produce a mixture of
compound 2a and 2b (10 mg, 20%, the low yield was due to the formation of di-piperidine-substituted
1
products). Compound 2a: H-NMR (400 MHz, DMSO-d6):
δ 1.30–1.72 (6H, m, H-4”, 5”,6”), 1.72–1.88
(1H, m, H-3), 2.00 (1H, m, H-3), 2.48 (2H, m, H-4) 2.60 (1H, m, H-3”), 3.08 (1H, m, H-3”), 4.10 (1H,
m, H-1”), 4.73 (1H, m, H-2), 5.78 (1H, S, H-8), 6.61–6.72 (2H, m, H-50,60), 6.76 (1H, d, J = 1.92, H-20);
13C-NMR (100 MHz, DMSO-d6)
δ
19.0 (C-4), 24.0, 24.5 (C-4”,5”), 29.0 (C-3), 30.3 (C-6”), 45.7 (C-30’) 52.8
(C-1”), 76.4 (C-2), 93.9 (C-8), 101.4 (C-10), 107.1 (C-6), 113.7 (C-20), 115.3 (C-50), 117.1 (C-60), 132.7 (C-10),
144.8, 145.1 (C-30,40) 153.2, 154.6, 155.9 (C-5,7,9). Compound 2b: H-NMR (400 MHz DMSO-d6):
δ
1
1.30–1.72 (6H, m, H-4”,5”,6”), 1.72–1.88 (1H, m, H-3), 2.00 (1H, m, H-3), 2.50 (2H, m, H-4) 2.70 (1H,

Molecules 2020, 25, 4491
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m, H-3”), 3.16 (1H, m, H-3”), 4.20 (1H, m, N-H), 4.86 (1H, m, H-2), 5.97 (1H, S, H-6), 6.61–6.8 (3H, m,
H-50,60,20); 13C-NMR (100 MHz, DMSO-d6)
δ 18.7 (C-4), 23.3 23.5 (C-4”,5”), 28.6 (C-3), 29.6 (C-6”), 45.5
(C-3”) 52.5 (C-1”), 76.4 (C-2), 95.3 (C-6), 100.2 (C-10), 104.0 (C-8), 113.4 (C-20), 115.4 (C-50), 116.5 (C-60),
132.7 (C-10), 144.8, 145.2 (C-30,40) 152.7, 155.3, 155.5 (C-5,7,9).
3.9. Tri-tert-butyl (2-formylbenzene-1, 3, 5-triyl) tricarbonate (18)
2,4,6-trihydroxybenzaldehyde (1.0 g, 6.50 mmol) was placed in an oven-dried flask filled with
nitrogen, followed by dissolution using solvent CH2Cl2. Next, N,N-diisopropylethylamine (DIPEA)
(0.7 mL), catalyst DMAP (four crystals), and Boc2O (5 mL, 21.8 mmol) were added, and the mixture
was stirred at room temperature overnight. The reaction was stopped using excessive 1M NH4Cl
and was extracted by dichloromethane (2
×
20 mL). The combined organic solvent was washed with
water (10 mL) and brine (10 mL), then dried using anhydrous MgSO4. The solution was filtered and
dried under reduced pressure. The residue was purified on silica column using refrigerated/cold
hexane/ethyl acetate (95:5), yielding compound 18 (1.37 g, 46.5%) as a colorless glue. Cold solvents are
necessary for purification due to the instability of tri-Boc protected material, which can decompose to
1
di-Boc protected side product (18a) at an elevated temperature. H-NMR (400 MHz, CDCl3)
s, 3 × -CH3), 1.56 (18H, s, 6 × -CH3), 7.10 (2H, s, 2 × Ar-H), 10.20 (1H, s, -CHO).
δ 1.55 (9H,
3.10. 2-(4-hydroxyphenyl)chromane-5,7-diol (21)
The synthesized intermediate compound 18 (100 mg, 0.22 mmol) was dissolved in anhydrous ether
and added to a flask containing 1-tert-butoxy-4-vinylbenzene (4 mL, 21 mmol) and magnesium bromide
ethyl etherate MgBr2
·
OEt2 (58 mg, 0.22 mmol) at
78 ◦C with the protection of argon. The mixture was
−
added to LiAlH4 (9.0 mg, 0.24 mmol) solution in anhydrous ether through a cannula. The reaction was
gradually warmed to room temperature and stirred for another 3 h at room temperature. The reaction
was quenched using 1M NaHCO3 and extracted using ether (3
×
10 mL). The combined organic phase
was washed with water (5 mL) and brine (5 mL), then dried using anhydrous Na2SO4. The solvent
was removed and the residue was re-dissolved in the DCM (5 mL) followed by the addition of ZnBr2
(250 mg, 2.2 mmol). The mixture was stirred at room temperature overnight. The reaction was
quenched with 1M HCl solution followed by extraction with EtOAc (3
×
10 mL). The combined organic
phase was dried under reduced pressure and purified on silica column with hexane/ethyl acetate (3:1)
to yield compound 16 (5.5 mg, 9.7%) as a white solid. Due to the reaction’s extreme water sensitivity,
1
a low yield was observed. H-NMR (400 MHz, MeOD-d4) δ 7.22 (2H, d, J = 8.6 Hz, H-30,50), 6.77 (2H,
d, J = 8.6 Hz, H-20,60), 5.90 (1H, d, J = 2.3, H-6), 5.83 (1H, d, J = 2.3, H-8), 4.82 (1H, br, d, H-2), 2.54–2.71
(2H, m, H-4), 1.87–2.13 (2H, m, H-3). 13C-NMR (100 MHz, MeOD-d4)
δ 158.09, 157.99, 157.49, 157.33
(C-5,7,9,40), 134.34 (C-10), 128.47 (C-30,50), 116.05 (C-20,60), 96.02 (C-6), 95.94 (C-8), 78.84 (C-2), 30.88
(C-3), 20.43 (C-4).
3.11. Kinkeloids A
The synthesized flavan compound 21 (200 mg, 0.78 mmol) was dissolved in methanol followed by
the addition of ∆
1-piperideines (77 mg, 0.93 mmol). The mixture was stirred at 70 ◦C under nitrogen
atmosphere for 5 h. After the reaction, the solvent was removed under reduced pressure and the
residue was purified on silica column using methanol/dichloromethane (15:85) to yield a mixture
of compound 1a and 1b. Further purification on silica gel with methanol/dichloromethae (1:5) and
Sephadex column LH-20 with methanol yielded compound 1a and 1b as a white solid (25%). It was
observed that compounds 1a and 1b would be oxidized to purple compounds after a lengthy exposure
1
to the air. Compound 1a: H-NMR (400 MHz, DMSO-d6)
δ
1.60 (2H, m, H-5” and H-4”), 1.88 (4H, m,
H-3,4”,5”,6”), 2.09 (2H m, H-3,6”), 2.62 (2H, m, H-4), 2.90 (1H, m, H-3”), 3.30 (1H, d, H-30’), 4.42 (1H,
m, H-1”), 4.88 (1H, m, H-2), 6.03 (1H, S, H-8), 6.80 (2H, d, H-20,60), 7.20 (2H, d, H-30,50); 13C-NMR
(400 MHz, DMSO-d6)
δ 19.58 (C-4), 21.8, 22.75 (C-4”,5”) 28.56 (C-6”), 28.54 (C-3), 45.40 (C-3”) 57.65
(C-1”), 76.30 (C-2), 95.35 (C-6), 101.78 (C-10), 104.85 (C-8), 114.98 (C-20,60), 127.20 (C-30,50), 157.50,

Molecules 2020, 25, 4491
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1
155.53, 154.10, 153.90 (C-5,7,9,40). Compound 1b: H-NMR (400 MHz, DMSO-d6)
δ
1.60 (2H, m, H-5”
and H-4”), 1.88 (4H, m, H-3,4”,5”,6”), 2.09 (2H m, H-3,6”), 2.62 (2H, m, H-4), 2.90 (1H, m, H-3”), 3.00
(1H, m, N-H), 4.32 (1H, m, H-1”), 5.00 (1H, m, H-2), 6.25 (1H, s, H-6), 6.80 (2H, d, H-20,60), 7.20 (2H, d,
H-30,50), 13C-NMR (100 MHz, DMSO-d6) 19.58 (C-4), 21.8, 22.75 (C-4”,5”) 28.56 (C-60’), 28.54 (C-3),
δ
45.40 (C-3”) 57.65 (C-10”), 76.30 (C-2), 95.35 (C-6), 101.78 (C-10), 104.80 (C-6), 114.98 (C-20,60), 127.20
(C-30,50), 157.50, 155.53, 154.10, 153.90 (C-5,7,9,40).
Supplementary Materials: The following are available online, for related NMR spectra.
Author Contributions: J.Z., Q.W. and J.E.S. conceived the need to synthesize novel compounds inspired by the
natural kinkeloids A and B.J.Z. and Q.W. conceived and designed the experiment; J.Z. performed the experiments
and analyzed the data; J.Z., Q.W. and J.E.S. wrote the paper. All authors have read and agreed to the published
version of the manuscript.
Funding: We thank the New Jersey Agricultural Experiment Station, Hatch Projects 1005685 and NJ12158,
the School of Environmental and Biological Sciences at Rutgers and the New Use Agriculture and National Plant
Products Program for funding and support.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).