Synthesis and antitumor evaluation of (aryl)methyl-amine derivatives of dehydroabietic acid-based B ring-fused-thiazole as potential PI3K/AKT/mTOR signaling pathway inhibitors

Article abstract of DOI:10.1007/s11030-020-10081-7

Abstract: In an attempt to search for new natural product-based antitumor agents, a series of novel (aryl)methyl-amine derivatives of dehydroabietic acid-based B ring-fused-thiazole were designed and synthesized. The primary bioassay showed that compounds

Full text of DOI:10.1007/s11030-020-10081-7

Molecular Diversity
https://doi.org/10.1007/s11030-020-10081-7
ORIGINAL ARTICLE
Synthesis and antitumor evaluation of (aryl)methyl‑amine derivatives
of dehydroabietic acid‑based B ring‑fused‑thiazole as potential PI3K/
AKT/mTOR signaling pathway inhibitors
Nai‑Yuan Chen1,2 · Yu‑Lan Xie1,2 · Guo‑Dong Lu1,2 · Fang Ye3 · Xin‑Yu Li2,3 · Yu‑Wen Huang1,2 · Ming‑Li Huang1,2
Tie‑Yu Chen4 · Cui‑Ping Li2,5
·
Received: 16 February 2020 / Accepted: 31 March 2020
© Springer Nature Switzerland AG 2020
Abstract
In an attempt to search for new natural product-based antitumor agents, a series of novel (aryl)methyl-amine derivatives of
dehydroabietic acid-based B ring-fused-thiazole were designed and synthesized. The primary bioassay showed that com-
pounds 5r and 5s presented certain inhibitory activity against cancer cells, weak cytotoxic activity against normal cells, and
inhibitory activity against PI3K/AKT/mTOR signaling pathway. The binding modes and the binding site interactions between
the active compounds and the target proteins were predicted preliminarily by the molecular docking method.
Graphic abstract
Keywords Dehydroabietic acid · Thiazole · Antitumor · Activity · Synthesis
Introduction
Nai-Yuan Chen and Yu-Lan Xie have contributed equally to this
Rosin, the distillation residue of pine resins, is an abun-
work.
dantly available natural product and a traditional medicine.
However, Rosin is used mainly as an agent in paper sizing,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s11030-020-10081-7) contains
supplementary material, which is available to authorized users.
printing inks, soup, cosmetics, adhesives, and emulsifers.
3
* Nai-Yuan Chen
School of Preclinical Medicine, Guangxi Medical University,
chennaiyuan@gxmu.edu.cn
Nanning 530021, China
4
* Cui-Ping Li
Pharmaceutical College, Guangxi Medical University,
gxcuiping@sina.com
Nanning 530021, China
5
Guangxi Key Laboratory of Oral and Maxillofacial
1
School of Public Health, Guangxi Medical University,
Rehabilitation and Reconstruction, Guangxi Key Laboratory
of Oral and Maxillofacial Surgery Disease Treatment,
Guangxi Clinical Research Center for Craniofacial
Deformity, School of Stomatology, Guangxi Medical
University, Nanning 530021, China
Nanning 530021, China
2
Guangxi Colleges and Universities Key Laboratory
of Prevention and Control of Highly Prevalent Diseases,
Guangxi Medical University, Nanning 530021, China
Vol.:(0123456789)
1 3

Molecular Diversity
Its essentially bioactive property as secondary metabolites
against potential herbivores and pathogens has not still been
sufciently developed for pharmaceutical use [1, 2]. Thus, in
order to enhance the added value of rosin, the development
of rosin components for medical utilization has been studied
continually in recent decades [3].
ring-fused-thiazole derivatives of dehydroabietic acid, a
series of (aryl)methyl-amine derivatives of dehydroabi-
etic acid-based B ring-fused-thiazole were designed and
synthesized in this study. The cytotoxic activities of target
compounds against human hepatoma cells HepG2, human
oral squamous cell SCC9, and human embryonic kidney cell
293T were detected by CCK8 method. The inhibitory activ-
ity against PI3K/AKT/mTOR signaling pathway was studied
by western blotting analysis. The possible interaction modes
between the target compounds and the signaling pathway
proteins were evaluated by molecular docking study.
Dehydroabietic acid (DHA) is one of the components of
rosin in small content, but can be readily obtained from dis-
proportionated rosin which is one of the modifed products
of rosin. Recent reports indicated that DHA and its deriva-
tives exhibited a broad spectrum of biological activities,
such as antimicrobial [4], antitumor [5], antifungal [6], anti-
viral [7], infammatory [8], antiprotozoal [9], anti-aging [10]
and BK channel opening activities [11]. Therefore, DHA
has been proved to be a promising starting material in the
synthesis of physiological or pharmacological compounds.
In our previous studies, a series of dehydroabietic acid-
based B ring-fused-thiazole amides were synthesized, some
of which showed notable antifungal activities [12]. In addi-
tion, thiazole moiety is an important scafold in many bio-
logically active compounds and has been found in many
anticancer drugs such as epothilones, ixabepilone, bleo-
mycin, and tiazofurin [13]. Some compounds with thiazole
moiety were reported as kinase inhibitors of PI3K signaling
pathway which provides strong growth and survival signals
to tumor cells [14, 15].
Results and discussion
Chemistry
The synthetic approaches adopted to aford the target com-
pounds are outlined in Scheme 1. Compounds 1, 2, and 3
were prepared according to the studies [4, 16]. Subsequently,
compound 3 was treated with bromine and then with thio-
urea in one pot to give compound 4. The yield of this step
is better than that of our previous work in which the bro-
mide formed in the reaction was purifed [12]. Then, the
target compounds 5a–5t were synthesized in good yields
by condensation reaction of compound 4 with correspond-
ing aldehydes followed by reduction reaction with sodium
borohydride in one pot.
Inspired by the above facts and in continuation
of our interest in designing and developing new B
Scheme 1 Synthetic route of (aryl)methyl-amide derivatives of dehydroabietic acid-based B ring-fused-thiazole 5a–5t
1 3

Molecular Diversity
Table 1 In vitro cytotoxic activities of compounds 5a–5t
The identities of the target compounds were confrmed
using physicochemical analytical methods. 1H NMR spectra
revealed that the amino-group proton exhibited a character-
istic broad single signal in the 5.90–5.32 ppm range. The
aromatic protons of the dehydroabietic acid scafold showed
signals at 7.73–7.63, 7.21–7.17, and 7.16–7.12 ppm. The
methyl ester group protons showed a single signal in the
3.66–3.62 ppm range. The protons of the methyl bound with
amino-group exhibited quartet of doublets or quartet signals
in the 4.66–4.48 ppm range. The 13C NMR spectra signals
of all the target compounds were matched with the number
of the carbon types. The high-resolution mass spectra con-
frmed the expected molecular weights of the fnal products.
Compound
IC50 (μmol/L)
HepG2
SCC9
293T
5a(R=Ph)
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
51.13 0.40 >100
60.13 0.69 >100
>100
56.28 0.28 >100
96.08 0.38 >100
>100
82.62 0.99 >100
51.93 0.61 >100
>100
>100
56.68 0.28 >100
82.72 0.24 >100
>100
>100
>100
>100
>100
5b(R=2CH3-Ph)
5c(R=3CH3-Ph)
5d(R=4CH3-Ph)
5e(R=2CH3O-Ph)
5f(R=3CH3O-Ph)
5g(R=4CH3O-Ph)
5h(R=2F-Ph)
>100
5i(R=3F-Ph)
5j(R=4F-Ph)
5k(R=2Cl-Ph)
5l(R=3Cl-Ph)
5m(R=4Cl-Ph)
5n(R=2Br-Ph)
5o(R=3Br-Ph)
5p(R=4Br-Ph)
5q(R=furan-2-)
5r(R=1H-pyrrol-2-)
5s(R=pyridin-3-)
Antitumor activity
>100
The synthesized target compounds 5a–5t were evaluated for
their in vitro cytotoxic activities against the two human can-
cer cell lines (HepG2 and SCC9) and the human normal cell
(293T) by CCK-8 assay [17]. The results are summarized
in Table 1.
>100
>100
It was found that the newly synthesized compounds
showed weak to moderate cytotoxic activities against the
two human cancer cell lines and displayed weak cytotoxic
activity against the normal cell 293T. Among the target
compounds, 5r and 5s showed the best cytotoxic activity
against HepG2 with half-inhibitory concentration (IC50)
values of 24.41 0.26 and 22.92 0.24 µM, respectively,
better than those of DHA and positive control 5-fuorouracil.
Moreover, compound 5s exhibited the best cytotoxic activity
against SCC9, better than that of DHA and positive control
5-fuorouracil. This result of IC50 indicated that DHA could
be modifed according to the strategy in this study to form
new derivatives with better antitumor activity. In addition,
the compounds with pyrrole moiety (5r) and unsubstituted
pyridine moiety (5s) displayed better cytotoxic activities
than other compounds. The compound with ortho-chloro-
substituted pyridine moiety (5t) showed much weaker cyto-
toxic activity than compound 5s. This result suggested that
the cytotoxic activities of compounds may be afected by the
electron donating ability of R group.
24.41 0.26 44.15 0.49 76.25 0.50
22.92 0.24 21.79 0.34 68.64 0.24
5t(R=6-Cl-pyridin-3-) 91.72 0.27 95.04 0.23 >100
1(DHA)
5-Fluorouracil
>100
32.74 0.17 >100
76.66 0.30 >100
>100
IC50 values are taken as means standard deviation from three experi-
ments. HepG2 is human hepatoma cells; SCC9 is human oral squa-
mous cell; 293T is human embryonic kidney cell
In the treatment group, the expression levels of the phos-
phorylated proteins, p-PI3K, p-AKT, p-mTOR, p-S6K, p-S6,
and p-4EBP1 sufered a signifcant decrease, while the total
mTOR and S6 expressions were decreased greatly and
slightly, respectively. The expression levels of total 4EBP1
and α-tubulin were almost not afected by the treatments
with compounds, compared with the untreated control. Gen-
erally, this result showed that compounds 5r and 5s could
inhibit the expressions of the phosphorylated proteins of
PI3K/AKT/mTOR signaling pathway and the phosphoryla-
tion of the downstream efectors S6 and 4EBP1.
PI3K/AKT/mTOR signaling pathway inhibitory activ-
ity of 5r and 5s
Molecular docking studies
To investigate the PI3K/AKT/mTOR signaling pathway
inhibitory activity of the two efective compounds, in the
treatment group, HepG2 cells were treated with 5r or 5s
in concentrations of 20 μM and 40 μM. The HepG2 cells
untreated were served as blank control. The protein expres-
sions of p-PI3K, p-AKT, p-mTOR, mTOR, p-S6K, p-S6,
p-4EBP1, S6, 4EBP1, and α-tubulin were detected by the
western blot method. The result is shown in Fig. 1.
In an attempt to study the possible interaction mode between
compounds (5r or 5s) and the active sites of PI3K, AKT, and
mTOR, molecular docking was conducted using AutoDock
Tools. Each docking result contained 50 conformations pre-
dicted, and the conformation with the lowest binding energy
was chosen as representative to analysis.
The representative docking results of compounds within
PI3K are shown in Fig. 2. The compound 5r binds to PI3K
1 3

Molecular Diversity
[18], the interacting residues Ala177, Val164, Met281,
Phe438, and Glu234 were found as the common interact-
ing sites. The O atom of carbonyl group forms a hydrogen
bond with Lys185. The binding energy of this conformation
is −7.88 kcal/mol. The compound 5s binds to AKT possi-
bly by interacting with 15 residues through 14 non-bonding
interactions and 1 hydrogen bond (Fig. 3c, d). Compared
with other reported ATP competitive inhibitors, the inter-
acting residues Glu234, Phe438 and Val164 were found as
the common sites. The N atom of thiazole moiety forms a
hydrogen bond with Arg4 (B). The binding energy of this
conformation is −9.15 kcal/mol.
The representative docking results of compounds within
mTOR are shown in Fig. 4. The compound 5r binds to
mTOR possibly by interacting with 17 residues through 15
non-bonding interactions and 2 hydrogen bonds (Fig. 4a, b).
Compared with other reported ATP competitive inhibitors
[18], the interacting residues Gly2238, Val2240, Met2345,
Asp2357, Ile2237, Ile2356, Leu2185, Trp2239, Asp2195,
and Glu2190 were found as the common sites. The N atom
of amino moiety and pyrrole ring form hydrogen bonds with
Glu2190 and Asp2195, respectively. The binding energy of
this conformation is − 8.39 kcal/mol. The compound 5s
binds to mTOR possibly by non-bonding interaction with
14 residues (Fig. 4c, d). Compared with other reported ATP
competitive inhibitors, the interacting residues Leu2185,
Ile2237, Tyr2225, Asp2357, Ile2356, Gly2238, Val2240,
Trp2239, and Met2345 were found as the common sites.
The binding energy of this conformation is −8.99 kcal/mol.
These results indicated that compounds 5r and 5s may
inhibit the PI3K/AKT/mTOR signaling pathway by acting
as ATP competitive inhibitor.
Fig. 1 The protein expressions afected by the treatment with 5r and
5s
possibly by interacting with 15 residues through 13 non-
bonding interactions and 2 hydrogen bonds (Fig. 2a, b).
Some adenosine triphosphate (ATP)-binding sites reported
were found in the interacting residues [18], such as Ala805,
Ser806, Asp964, Tyr867, Glu880, and Lys890. The S atom
of thiazole moiety and the N atom of pyrrole ring form
hydrogen bonds with Lys890 and Ala885, respectively. The
binding energy of this conformation is −7.04 kcal/mol. The
compound 5s binds to PI3K possibly by interacting with 14
residues through 13 non-bonding interactions and 1 hydro-
gen bond (Fig. 2c, d). Ser806, Asp950, Ala805, Asp964,
Glu880, Val882, Trp812, and Lys890 were found in the
interacting residues reported as (ATP)-binding site. The S
atom of thiazole moiety forms a hydrogen bond with Lys890.
The binding energy of this conformation is −6.81 kcal/mol.
The representative docking results of compounds within
AKT are shown in Fig. 3. The compound 5r binds to AKT
possibly by interacting with 16 residues through 15 non-
bonding interactions and 1 hydrogen bond (Fig. 3a, b).
Compared with other reported ATP competitive inhibitors
Conclusions
In summary, 20 new (aryl)methyl-amine derivatives of
dehydroabietic acid-based B ring-fused-thiazole were syn-
thesized and their in vitro cytotoxic activities were inves-
tigated by CCK-8 assay against HepG2, SCC9, and 293T.
Compounds 5r and 5s presented certain inhibitory activ-
ity against cancer cells and weak cytotoxic activity against
normal cells. The western blot assay showed that these two
compounds can inhibit the PI3K/AKT/mTOR signaling
pathway. According to the result of molecular docking study,
the compounds tested may inhibit the pathway through ATP
competition. The moieties integrated into the skeleton of
DHA, such as amino thiazole moiety and nitrogen heterocy-
cles, may play as an important role in the inhibitory activity
of the compounds. Compounds 4, 5r and 5s could be chosen
as the leading compounds for further modifcation to search
new antitumor agents.
1 3

Molecular Diversity
Fig. 2 a 3D conformation of 5r docked in PI3K. b Predicted interactions between 5r and the amino acids of PI3K. c 3D conformation of 5s
docked in PI3K. d Predicted interactions between 5s and the amino acids of PI3K
downfeld relative to TMS as an internal standard; NMR
abbreviations: s = singlet, d = doublet, t = triplet, q = quar-
tet, m = multiplet. MS spectra were obtained by means of
the electrospray ionization (ESI) on a high-resolution
mass spectrometer UPLC I-CLASS-XEVOG2-XSQTOF
(Waters, Milford, USA). Reaction products were isolated
by using column chromatography on silica gel column
(200–300 mesh). All chemicals and reagents were pur-
chased from commercial suppliers and used as received.
Materials and methods
Chemistry
The melting points (m.p.) were determined on an X-5A
digital melting point apparatus (Gongyi Kerui Instruments,
Gongyi, China). NMR spectra were recorded in CDCl3 on
a Bruker AVANCE III HD500 spectrometer (Bruker, Swit-
zerland), and chemical shifts were expressed in ppm (δ)
1 3

Molecular Diversity
Fig. 3 a 3D conformation of 5r docked in AKT. b Predicted interactions between 5r and the amino acids of AKT. c 3D conformation of 5s
docked in AKT. d Predicted interactions between 5s and the amino acids of AKT
Compounds 1, 2, and 3 were prepared according to the
of compound 3 (5.0 g, 15 mmol) in glacial acetic acid
(5.0 mL) at 60 °C. Then, the mixture was continuously
stirred for 10 min followed by adding thiourea (2.25 g,
30 mmol). Later, the reacting mixture was heated to
105 °C for 12 h. After that, the mixture was evaporated in
vacuo to remove acetic acid and then dissolved in CH2Cl2
followed by washing with excess aqueous NaHCO3 solu-
tion three times. After removing CH2Cl2, the residue
was purified by column chromatography on silica gel
studies [4, 16].
Preparation of (7bS,11R)‑methyl
2‑amino‑7b,8,9,10,11,11a‑hexahydro‑5‑isopropyl‑7b,11‑di
methylphenanthro[9,10‑d]thiazole‑11‑carboxylate(4)
A mixture of glacial acetic acid (1.4 mL) and Br2
(1.1 mL, 21.4 mmol) was gradually added to the solution
1 3

Molecular Diversity
Fig. 4 a 3D conformation of 5r docked in mTOR. b Predicted interactions between 5r and the amino acids of mTOR. c 3D conformation of 5s
docked in mTOR. d Predicted interactions between 5s and the amino acids of mTOR
(petroleum ether–ethyl acetate, 5:1) to aford compound 4
borohydride was gradually added to the reaction mixture
till the color of solution became no longer lighter, and the
solution was evaporated in vacuo to remove solvent fol-
lowed by dissolving in CH2Cl2. After washing with deion-
ized water and drying with anhydrous sodium sulfate, the
resulting solution was purifed by column chromatography
on silica gel (petroleum ether–ethyl acetate, 10:1) to aford
target compounds 5a–5t.
as white powder, Yield 65%.
Synthesis of (aryl)methyl‑amine derivatives of dehydroabi‑
etic acid‑based B ring‑fused‑thiazole 5a–5t
Compound 4 (0.1 g, 0.26 mmol) and corresponding alde-
hydes (0.3 mmol) were dissolved in anhydrous metha-
nol (1.5 mL) and heated to 60 °C for 4 h. Then, sodium
1 3

Molecular Diversity
(7bS,11R)‑methyl 2‑(benzylamino)‑7b,8,9,10,11,11a‑hex‑
ahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,10‑d]
thiazole‑11‑carboxylate (5a)
(126 MHz, CDCl3) δ 178.52, 167.82, 146.98, 146.36, 144.24,
138.37, 137.71, 130.08, 128.57, 128.56, 128.42, 125.39,
124.87, 122.13, 121.83, 119.99, 52.41, 49.98, 46.67, 46.57,
39.20, 37.39, 35.36, 33.84, 24.15, 23.90, 21.75, 21.40, 18.29,
17.60. Mass spectrum (ESI), m/z: 489.2466 [M+H]+ Caled
C30H36 N2O2S 488.2497.
1
Yield 74%, white solid, m.p.79.5–80.6 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 1.6 Hz, 1H, H-14),
7.44–7.28 (m, 5H, H-Ph), 7.18 (d, J = 8.0 Hz, 1H, H-11),
7.12 (dd, J=8.0, 1.9 Hz, 1H, H-12), 5.45 (s, 1H, NH), 4.52
(ddd, J=33.9, 14.2, 5.4 Hz, 2H, H-23), 3.71 (d, J=6.8 Hz,
1H, H-5), 3.63 (s, 3H, COOCH3), 2.94 (dq, J=13.8, 6.9 Hz,
1H, H-15), 2.29 (dd, J =8.5, 5.9 Hz, 1H, H1-e), 1.91–1.75
(m, 5H, H-3, H-2, H1-a), 1.58 (s, 3H, H-19), 1.29 (dd,
J = 6.9, 2.2 Hz, 6H, H-16, H-17), 1.15 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.51, 167.79, 146.99, 146.35,
144.23, 137.80, 130.05, 128.68, 127.82, 127.69, 125.41,
122.13, 121.82, 120.04, 52.42, 49.98, 46.66, 46.57, 39.20,
37.38, 35.35, 33.83, 24.14, 23.89, 21.75, 18.28, 17.60.
Mass spectrum (ESI), m/z: 475.2240 [M + H]+ Caled
C29H34N2O2S 474.2341.
(7bS,11R)‑methyl 2‑(4‑tolylmethanamino)‑7b,8,9,10,11,11a
‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,10‑d]
thiazole‑11‑carboxylate (5d)
1
Yield 75%, white solid, m.p. 81.0–82.7 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J=1.8 Hz, 1H, H-14), 7.31 (d,
J=8.0 Hz, 2H, H-Ph), 7.18 (d, J=8.3 Hz, 3H, H-11, H-Ph),
7.12 (dd, J=8.0, 1.9 Hz, 1H, H-12), 5.37 (d, J=5.1 Hz, 1H,
NH), 4.47 (ddd, J=31.8, 13.9, 5.5 Hz, 2H, H-23), 3.72 (s,
1H, H-5), 3.64 (s, 3H, COOCH3), 2.94 (dq, J=13.8, 6.9 Hz,
1H, H-15), 2.37 (s, 3H, CH3-Ph), 2.29 (dd, J=8.5, 5.8 Hz,
1H, H1-e), 1.91–1.75 (m, 5H, H-3, H-2, H1-a), 1.58 (s, 3H,
H-19), 1.29 (dd, J=6.9, 2.0 Hz, 6H, H-16, H-17), 1.15 (s, 3H,
H-20).13C NMR (126 MHz, CDCl3) δ 178.53, 167.79, 149.14,
146.98, 146.35, 144.23, 137.39, 134.73, 130.09, 129.34,
127.83, 125.38, 122.12, 121.83, 119.97, 52.41, 49.78, 46.67,
46.57, 39.20, 37.39, 35.36, 33.83, 24.14, 23.89, 21.75, 21.13,
18.29, 17.60. Mass spectrum (ESI), m/z: 489.2734 [M+H]+
Caled C30H36 N2O2S 488.2497.
(7bS,11R)‑methyl 2‑(2‑tolylmethanamino)‑7b,8,9,10,11,11a
‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,10‑d]
thiazole‑11‑carboxylate (5b)
1
Yield 67%, white solid, m.p. 178.6–179.1 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J= 1.4 Hz, 1H, H-14), 7.39
(d, J=7.2 Hz, 1H, H-Ph), 7.27–7.20 (m, 3H, H-Ph), 7.18 (d,
J=8.0 Hz, 1H, H-11), 7.12 (dd, J=8.0, 1.9 Hz, 1H, H-12),
5.22 (s, 1H, NH), 4.49 (qd, J=13.7, 5.2 Hz, 2H, H-23), 3.73
(s, 1H, H-5), 3.65 (s, 3H, COOCH3), 2.95 (hept, J=6.9 Hz,
1H, H-15), 2.43 (s, 3H, CH3-Ph), 2.33–2.27 (m, 1H, H1-e),
1.92–1.76 (m, 5H, H-3, H-2, H1-a), 1.59 (s, 3H, H-19), 1.30
(dd, J=6.9, 1.6 Hz, 6H, H-16, H-17), 1.16 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.54, 167.63, 146.98, 146.42,
144.25, 136.59, 135.55, 130.55, 130.09, 128.65, 127.93,
126.18, 125.41, 122.12, 121.84, 119.97, 52.44, 48.03, 46.67,
46.57, 39.21, 37.41, 35.36, 33.84, 24.14, 23.90, 21.76,
19.10, 18.29, 17.62. Mass spectrum (ESI), m/z: 489.2557
[M+H]+ Caled C30H36 N2O2S 488.2497.
(7bS,11R)‑methyl 2‑(2‑methoxybenzylamino)‑7b,8,9,10,11,
11a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5e)
1
Yield 72%, white solid, m.p. 235.8–236.4 °C. H NMR
(500 MHz, CDCl3) δ 7.67 (d, J=1.7 Hz, 1H, H-14), 7.39
(dd, J=7.4, 1.5 Hz, 1H, H-Ph), 7.30 (dt, J=8.0, 1.8 Hz, 1H,
H-Ph), 7.17 (d, J=8.0 Hz, 1H, H-11), 7.11 (dd, J=8.0, 1.9 Hz,
1H, H-12), 6.94 (ddd, J=12.3, 9.4, 4.5 Hz, 2H, H-Ph), 5.60
(s, 1H, NH), 4.49 (qd, J=14.4, 6.1 Hz, 2H, H-23), 3.89 (s,
3H, CH3O-Ph), 3.71 (s, 1H, H-5), 3.65 (s, 3H, COOCH3),
2.95 (hept, J=6.9 Hz, 1H, H-15), 2.34–2.24 (m, 1H, H1-e),
1.90–1.75 (m, 5H, H-3, H-2, H1-a), 1.59 (s, 3H, H-19), 1.30
(dd, J=6.9, 1.6 Hz, 6H, H-16, H-17), 1.14 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.58, 168.15, 157.58, 146.93,
146.33, 144.22, 130.14, 129.71, 129.00, 125.94, 125.27,
122.09, 121.82, 120.41, 119.81, 110.30, 55.30, 52.41, 46.67,
46.54, 46.02, 39.18, 37.38, 35.37, 33.83, 24.16, 23.88, 21.73,
18.30, 17.61. Mass spectrum (ESI), m/z: 505.2514 [M+H]+
Caled C30H36 N2O3S 504.2247.
(7bS,11R)‑methyl 2‑(3‑tolylmethanamino)‑7b,8,9,10,11,11a
‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,10‑d]
thiazole‑11‑carboxylate (5c)
1
Yield 82%, white solid, m.p. 153.5–154.8 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J=1.4 Hz, 1H, H-14), 7.24 (dt,
J=17.8, 7.6 Hz, 3H, H-Ph), 7.18 (d, J=8.0 Hz, 1H, H-11),
7.15–7.11 (m, 2H, H-12, H-Ph), 5.43 (s, 1H, NH), 4.48 (ddd,
J=35.6, 14.1, 5.5 Hz, 2H, H-23), 3.73 (s, 1H, H-5), 3.63 (s,
3H, COOCH3), 2.95 (hept, J=6.9 Hz, 1H, H-15), 2.38 (s,
3H, CH3-Ph), 2.30 (dd, J=8.6, 5.8 Hz, 1H, H1-e), 1.91–1.75
(m, 5H, H-3, H-2, H1-a), 1.59 (s, 3H, H-19), 1.30 (dd, J=6.9,
2.1 Hz, 6H, H-16, H-17), 1.15 (s, 3H, H-20).13C NMR
1 3

Molecular Diversity
(7bS,11R)‑methyl 2‑(3‑methoxybenzylamino)‑7b,8,9,10,11,
11a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5f)
H-2, H1-a), 1.58 (s, 3H, H-19), 1.30 (dd, J=6.9, 2.6 Hz, 6H,
H-16, H-17), 1.14 (s, 3H, H-20).13C NMR (126 MHz, CDCl3)
δ 178.50, 167.45, 162.04, 160.08, 146.96, 146.40, 144.20,
130.15, 129.44, 125.44, 125.08, 124.17, 122.11, 121.81,
120.21, 115.54, 52.42, 46.65, 46.58, 43.66, 39.19, 37.37,
35.34, 33.82, 24.14, 23.89, 21.75, 18.27, 17.59. Mass spec-
trum (ESI), m/z: 493.2198 [M+H]+ Caled C29H33ClN2O2S
492.2247.
1
Yield 86%, white solid, m.p. 116.7–117.6 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 1.8 Hz, 1H, H-14),
7.30–7.27 (m, 1H, H-Ph), 7.18 (d, J = 8.0 Hz, 1H, H-11),
7.12 (dd, J = 8.0, 1.9 Hz, 1H, H-12), 6.99 (dd, J = 9.4,
4.9 Hz, 2H, H-Ph), 6.86 (dd, J = 8.2, 2.4 Hz, 1H, H-Ph),
5.45 (s, 1H, NH), 4.50 (ddd, J = 33.2, 14.2, 5.4 Hz, 2H,
H-23), 3.82 (s, 3H, CH3O-Ph), 3.72 (s, 1H, H-5), 3.63 (s,
3H, COOCH3), 2.94 (dq, J=13.8, 6.9 Hz, 1H, H-15), 2.29
(dd, J=8.6, 5.9 Hz, 1H, H1-e), 1.92–1.75 (m, 5H, H-3, H-2,
H1-a), 1.58 (s, 3H, H-19), 1.29 (dd, J=6.9, 2.0 Hz, 6H, H-16,
H-17), 1.15 (s, 3H, H-20).13C NMR (126 MHz, CDCl3) δ
178.51, 167.76, 159.90, 146.99, 146.36, 144.23, 139.41,
130.06, 129.71, 125.41, 122.14, 121.83, 120.07, 120.05,
113.36, 113.18, 55.25, 52.42, 49.96, 46.66, 46.57, 39.20,
37.39, 35.35, 33.84, 24.14, 23.89, 21.75, 18.28, 17.60. Mass
spectrum (ESI), m/z: 505.2535 [M + H]+ Caled C30H36
N2O3S 504.2247.
(7bS,11R)‑methyl 2‑(3‑fuorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5i)
1
Yield 93%, white solid, m.p. 132.8–133.6 °C. H NMR
(500 MHz, CDCl3) δ 7.67 (d, J=1.8 Hz, 1H, H-14), 7.33
(td, J=7.9, 5.9 Hz, 1H, H-Ph), 7.23–7.09 (m, 4H, H-Ph,
H-12, H-11), 7.00 (td, J=8.3, 2.1 Hz, 1H, H-Ph), 5.56 (d,
J=5.1 Hz, 1H, NH), 4.53 (ddd, J=34.3, 14.8, 5.5 Hz, 2H,
H-23), 3.71 (s, 1H, H-5), 3.62 (s, 3H, COOCH3), 2.94 (hept,
J=6.9 Hz, 1H, H-15), 2.29 (dd, J=8.6, 5.9 Hz, 1H, H1-e),
1.91–1.73 (m, 5H, H-3, H-2, H1-a), 1.57 (s, 3H, H-19), 1.29
(dd, J=6.9, 2.9 Hz, 6H, H-16, H-17), 1.15 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.49, 167.56, 163.98, 162.02,
147.00, 146.39, 144.22, 140.61, 130.20, 125.48, 123.27,
122.16, 121.81, 120.24, 114.73, 114.44, 52.41, 49.32, 46.65,
46.58, 39.19, 37.36, 35.34, 33.82, 24.13, 23.88, 21.75, 18.27,
17.59. Mass spectrum (ESI), m/z: 493.2198 [M+H]+ Caled
C29H33ClN2O2S 492.2247.
(7bS,11R)‑methyl 2‑(4‑methoxybenzylamino)‑7b,8,9,10,11,
11a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5g)
1
Yield 77%, white solid, m.p. 72.3–72.8 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (s, 1H, H-14), 7.33 (d, J=8.4 Hz,
2H, H-Ph), 7.18 (d, J=8.0 Hz, 1H, H-11), 7.12 (dd, J=8.0,
1.8 Hz, 1H, H-12), 6.90 (d, J=8.4 Hz, 2H, H-Ph), 5.46 (d,
J=28.9 Hz, 1H, NH), 4.43 (ddd, J=31.1, 13.8, 5.2 Hz, 2H,
H-23), 3.82 (s, 3H, CH3O-Ph), 3.72 (s, 1H, H-5), 3.64 (s,
3H, COOCH3), 2.99–2.90 (m, 1H, H-15), 2.35–2.24 (m,
1H, H1-e), 1.91–1.76 (m, 5H, H-3, H-2, H1-a), 1.58 (s, 3H,
H-19), 1.29 (dd, J=6.9, 2.1 Hz, 6H, H-16, H-17), 1.15 (s,
3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.52, 167.76,
159.16, 146.97, 146.32, 144.24, 130.08, 129.84, 129.20,
125.38, 122.12, 121.82, 119.95, 114.04, 55.30, 52.42, 49.47,
46.67, 46.57, 39.20, 37.40, 35.36, 33.83, 24.15, 23.90,
21.75, 18.29, 17.61. Mass spectrum (ESI), m/z: 505.2535
[M+H]+ Caled C30H36 N2O3S 504.2247.
(7bS,11R)‑methyl 2‑(4‑fuorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5j)
1
Yield 90%, white solid, m.p. 75.1–76.3 °C. H NMR
(500 MHz, CDCl3) δ 7.67 (d, J=1.8 Hz, 1H, H-14), 7.41–7.35
(m, 2H, H-Ph), 7.18 (d, J=8.0 Hz, 1H, H-11), 7.12 (dd,
J=8.0, 1.9 Hz, 1H, H-12), 7.08–7.01 (m, 2H, H-Ph), 5.57
(s, 1H, NH), 4.48 (qd, J=14.3, 5.2 Hz, 2H, H-23), 3.71 (s,
1H, H-5), 3.62 (s, 3H, COOCH3), 2.94 (dq, J=13.8, 6.9 Hz,
1H, H-15), 2.34–2.25 (m, 1H, H1-e), 1.90–1.76 (m, 5H, H-3,
H-2, H1-a), 1.57 (s, 3H, H-19), 1.29 (dd, J=6.9, 2.7 Hz, 6H,
H-16, H-17), 1.14 (s, 3H, H-20).13C NMR (126 MHz, CDCl3)
δ 178.49, 167.62, 163.26, 161.31, 146.99, 146.35, 144.23,
133.63, 130.00, 129.52, 125.47, 121.79, 120.12, 115.57, 52.41,
49.18, 46.65, 46.56, 39.19, 37.37, 35.34, 33.83, 24.14, 23.88,
21.75, 18.27, 17.59. Mass spectrum (ESI), m/z: 493.2303
[M+H]+ Caled C29H33ClN2O2S 492.2247.
(7bS,11R)‑methyl 2‑(2‑fuorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5h)
1
Yield 89%, white solid, m.p. 127.6–128.1 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J=1.9 Hz, 1H, H-14), 7.50 (td,
J=7.6, 1.6 Hz, 1H, H-Ph), 7.33–7.27 (m, 1H, H-Ph), 7.17 (d,
J=8.0 Hz, 1H, H-11), 7.16–7.07 (m, 3H, H-12, H-Ph), 5.45
(s, 1H, NH), 4.59 (qd, J=14.8, 5.7 Hz, 2H, H-23), 3.71 (s,
1H, H-5), 3.64 (s, 3H, COOCH3), 2.99–2.91 (m, 1H, H-15),
2.29 (dd, J=8.6, 5.9 Hz, 1H, H1-e), 1.91–1.75 (m, 5H, H-3,
1 3

Molecular Diversity
(7bS,11R)‑methyl 2‑(2‑chlorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5k)
H-19), 1.29 (dd, J = 6.9, 2.8 Hz, 6H, H-16, H-17), 1.14 (s,
3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.48, 167.60,
146.99, 146.35, 144.22, 136.42, 133.41, 129.97, 129.14,
128.77, 125.49, 122.17, 121.78, 120.18, 52.42, 49.17,
46.64, 46.56, 39.19, 37.36, 35.33, 33.82, 24.14, 23.88,
21.75, 18.26, 17.59. Mass spectrum (ESI), m/z: 509.2021
[M + H]+ Caled C29H33ClN2O2S 508.1951.
1
Yield 87%, white solid, m.p. 166.8–167.4 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 1.8 Hz, 1H, H-14),
7.58–7.53 (m, 1H, H-Ph), 7.43–7.40 (m, 1H, H-Ph),
7.28–7.25 (m, 2H, H-Ph), 7.17 (d, J = 8.0 Hz, 1H, H-11),
7.12 (dd, J = 8.0, 1.9 Hz, 1H, H-12), 5.57 (d, J = 5.7 Hz,
1H, NH), 4.63 (qd, J = 15.1, 5.9 Hz, 2H, H-23), 3.70 (s,
1H, H-5), 3.63 (s, 3H, COOCH3), 2.94 (hept, J = 6.9 Hz,
1H, H-15), 2.29 (dd, J =8.6, 6.1 Hz, 1H, H1-e), 1.90–1.75
(m, 5H, H-3, H-2, H1-a), 1.57 (s, 3H, H-19), 1.30 (dd,
J = 6.9, 2.5 Hz, 6H, H-16, H-17), 1.14 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.50, 167.45, 146.96, 146.41,
144.20, 135.42, 133.70, 130.05, 130.03, 129.63, 129.00,
126.90, 125.45, 122.12, 121.81, 120.16, 52.43, 47.58, 46.65,
46.59, 39.19, 37.37, 35.34, 33.82, 24.15, 23.89, 21.75,
18.27, 17.60. Mass spectrum (ESI), m/z: 509.2016 [M+H]+
Caled C29H33ClN2O2S 508.1951.
(7bS,11R)‑methyl 2‑(2‑bromobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5n)
1
Yield 84%, white solid, m.p. 170.9–171.4 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 1.9 Hz, 1H, H-14), 7.58
(ddd, J = 16.0, 7.8, 1.3 Hz, 2H, H-Ph), 7.31 (td, J = 7.5,
1.2 Hz, 1H, H-Ph), 7.21–7.16 (m, 2H, H-11, H-Ph), 7.12
(dd, J = 8.0, 1.9 Hz, 1H, H-12), 5.60 (t, J = 5.8 Hz, 1H,
NH), 4.62 (qd, J = 15.1, 5.9 Hz, 2H, H-23), 3.70 (s, 1H,
H-5), 3.63 (s, 3H, COOCH3), 2.95 (hept, J = 6.9 Hz, 1H,
H-15), 2.33–2.27 (m, 1H, H1-e), 1.90–1.73 (m, 5H, H-3,
H-2, H1-a), 1.57 (s, 3H, H-19), 1.30 (dd, J = 6.9, 2.6 Hz,
6H, H-16, H-17), 1.14 (s, 3H, H-20).13C NMR (126 MHz,
CDCl3) δ 178.50, 167.35, 146.96, 146.42, 144.20, 137.04,
132.91, 130.24, 130.04, 129.26, 127.52, 125.45, 123.77,
122.12, 121.81, 120.17, 52.44, 49.91, 46.65, 46.59, 39.19,
37.38, 35.34, 33.82, 24.15, 23.89, 21.76, 18.27, 17.60.
Mass spectrum (ESI), m/z: 553.1537; 555.1518 [M + H]+
Caled C29H33BrN2O2S 552.1446; 554.1426.
(7bS,11R)‑methyl 2‑(3‑chlorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5l)
1
Yield 89%, white solid, m.p. 166.5–167.1 °C. H NMR
(500 MHz, CDCl3) δ 7.67 (d, J = 1.9 Hz, 1H, H-14),
7.42 (s, 1H, H-Ph), 7.29 (d, J = 6.2 Hz, 3H, H-Ph), 7.18
(d, J = 8.0 Hz, 1H, H-11), 7.12 (dd, J = 8.0, 1.9 Hz, 1H,
H-12), 5.90–5.65 (m, 1H, NH), 4.50 (ddd, J= 39.6, 14.9,
5.0 Hz, 2H, H-23), 3.72 (d, J = 6.1 Hz, 1H, H-5), 3.62 (s,
3H, COOCH3), 3.00–2.86 (m, 1H, H-15), 2.35–2.25 (m,
1H, H1-e), 1.89–1.75 (m, 5H, H-3, H-2, H1-a), 1.57 (s, 3H,
H-19), 1.29 (dd, J=6.9, 3.5 Hz, 6H, H-16, H-17), 1.14 (s,
3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.48, 167.56,
147.01, 146.36, 144.22, 140.08, 134.50, 129.97, 129.90,
127.87, 127.77, 125.85, 125.48, 122.17, 121.81, 120.22,
52.42, 49.23, 46.65, 46.58, 39.19, 37.36, 35.34, 33.83,
24.15, 23.88, 21.76, 18.27, 17.59. Mass spectrum (ESI),
m/z: 509.2231 [M+H]+ Caled C29H33ClN2O2S 508.1951.
(7bS,11R)‑methyl 2‑(3‑bromobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5o)
1
Yield 88%, white solid, m.p. 157.7–158.5 °C. H NMR
(500 MHz, CDCl3) δ 7.67 (d, J = 1.9 Hz, 1H, H-14), 7.57
(d, J = 14.6 Hz, 1H, H-Ph), 7.44 (t, J = 7.2 Hz, 1H, H-Ph),
7.32 (dd, J = 19.4, 7.6 Hz, 1H, H-Ph), 7.26–7.21 (m, 1H,
H-Ph), 7.18 (d, J = 8.0 Hz, 1H, H-11), 7.14–7.10 (m, 1H,
H-12), 5.59 (d, J = 5.7 Hz, 1H, NH), 4.50 (ddd, J = 41.0,
14.8, 5.1 Hz, 2H, H-23), 3.71 (s, 1H, H-5), 3.63 (s, 3H,
COOCH3), 2.94 (hept, J = 6.9 Hz, 1H, H-15), 2.34–2.25
(m, 1H, H1-e), 1.91–1.74 (m, 5H, H-3, H-2, H1-a), 1.57 (s,
3H, H-19), 1.29 (dd, J = 6.9, 3.0 Hz, 6H, H-16, H-17),
1.14 (s, 3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.48,
167.49, 147.01, 146.37, 144.22, 140.32, 130.79, 130.73,
130.20, 129.96, 126.34, 125.50, 122.71, 122.16, 121.81,
120.26, 52.44, 49.18, 46.65, 46.58, 39.20, 37.36, 35.34,
33.83, 24.14, 23.90, 21.76, 18.27, 17.59. Mass spec-
trum (ESI), m/z: 553.1536; 555.1519 [M + H]+ Caled
C29H33BrN2O2S 552.1446; 554.1426.
(7bS,11R)‑methyl 2‑(4‑chlorobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5m)
1
Yield 87%, white solid, m.p. 77.3–78.8 °C. H NMR
(500 MHz, CDCl3) δ 7.66 (d, J = 1.9 Hz, 1H, H-14),
7.37–7.30 (m, 4H, H-Ph), 7.18 (d, J = 8.0 Hz, 1H, H-11),
7.12 (dd, J = 8.0, 2.0 Hz, 1H, H-12), 5.60 (d, J = 4.7 Hz,
1H, NH), 4.49 (qd, J = 14.6, 5.0 Hz, 2H, H-23), 3.71 (d,
J = 6.5 Hz, 1H, H-5), 3.62 (s, 3H, COOCH3), 2.93 (dq,
J = 13.8, 6.9 Hz, 1H, H-15), 2.29 (dd, J = 8.7, 5.9 Hz,
1H, H1-e), 1.91–1.75 (m, 5H, H-3, H-2, H1-a), 1.57 (s, 3H,
1 3

Molecular Diversity
(7bS,11R)‑methyl 2‑(4‑bromobenzylamino)‑7b,8,9,10,11,1
1a‑hexahydro‑5‑isopropyl‑7b,11‑dimethylphenanthro[9,1
0‑d]thiazole‑11‑carboxylate (5p)
2.37–2.29 (m, 1H, H1-e), 1.92–1.77 (m, 5H, H-3, H-2, H1-a),
1.57 (s, 3H, H-19), 1.34–1.31 (m, 6H, H-16, H-17), 1.17 (s,
3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.47, 167.89,
147.05, 145.79, 144.25, 130.09, 129.94, 125.67, 122.34,
121.52, 120.65, 118.01, 107.73, 106.80, 52.51, 46.65, 46.61,
41.77, 39.24, 37.33, 35.33, 33.78, 24.14, 23.89, 21.80,
18.26, 17.59. Mass spectrum (ESI), m/z: 464.2389 [M+H]+
Caled C27H33N3O2S 463.2293.
1
Yield 83%, white solid, m.p. 78.4–78.6 °C. H NMR
(500 MHz, CDCl3) δ 7.65 (d, J= 1.9 Hz, 1H, H-14), 7.48
(d, J = 8.3 Hz, 2H, H-Ph), 7.29 (t, J = 4.1 Hz, 2H, H-Ph),
7.18 (d, J = 8.0 Hz, 1H, H-11), 7.12 (dd, J = 8.0, 1.9 Hz,
1H, H-12), 5.59 (d, J=5.4 Hz, 1H, NH), 4.47 (qd, J=14.7,
5.2 Hz, 2H, H-23), 3.71 (s, 1H, H-5), 3.62 (s, 3H, COOCH3),
2.93 (hept, J=6.9 Hz, 1H, H-15), 2.29 (dd, J=8.7, 5.9 Hz,
1H, H1-e), 1.90–1.75 (m, 5H, H-3, H-2, H1-a), 1.57 (s, 3H,
H-19), 1.29 (dd, J=6.9, 2.7 Hz, 6H, H-16, H-17), 1.14 (s,
3H, H-20).13C NMR (126 MHz, CDCl3) δ 178.48, 167.57,
146.99, 146.35, 144.22, 136.95, 131.73, 129.96, 129.47,
125.50, 122.17, 121.78, 121.49, 120.20, 52.42, 49.21, 46.64,
46.57, 39.19, 37.36, 35.33, 33.82, 24.14, 23.89, 21.75,
18.26, 17.59. Mass spectrum (ESI), m/z: 553.1539; 555.1528
[M+H]+ Caled C29H33BrN2O2S 552.1446; 554.1426.
(7bS,11R)‑methyl 2‑((pyridin‑3‑yl)
methylamino)‑7b,8,9,10,11,11a‑hexahydro‑5‑isopropyl‑7
b,11‑dimethylphenanthro[9,10‑d]thiazole‑11‑carboxylate
(5s)
1
Yield 52%, yellow solid, m.p. 83.7–84.3 °C. H NMR
(500 MHz, CDCl3) δ 8.69 (d, J=25.2 Hz, 1H, H-Py), 8.55
(dd, J = 4.7, 1.4 Hz, 1H, H-Py), 7.76 (t, J = 7.6 Hz, 1H,
H-Py), 7.66 (d, J = 1.9 Hz, 1H, H-14), 7.31–7.27 (m, 1H,
H-Py), 7.17 (d, J = 8.0 Hz, 1H, H-11), 7.12 (dt, J = 8.0,
3.9 Hz, 1H, H-12), 5.76 (s, 1H, NH), 4.66–4.48 (m, 2H,
H-23), 3.73–3.68 (m, 1H, H-5), 3.63 (s, 3H, COOCH3), 2.92
(dq, J=13.7, 6.9 Hz, 1H, H-15), 2.35–2.25 (m, 1H, H1-e),
1.92–1.74 (m, 5H, H-3, H-2, H1-a), 1.56 (s, 3H, H-19), 1.28
(dd, J=6.9, 3.3 Hz, 6H, H-16, H-17), 1.14 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.47, 167.33, 149.36, 149.07,
147.00, 146.29, 144.21, 135.56, 133.64, 129.88, 125.57,
123.53, 122.18, 121.79, 120.28, 52.45, 47.17, 46.63, 46.57,
39.20, 37.35, 35.32, 33.82, 24.13, 23.89, 21.75, 18.25,
17.59. Mass spectrum (ESI), m/z: 476.2391 [M+H]+ Caled
C28H33N3O2S 475.2293.
(7bS,11R)‑methyl 2‑((furan‑2‑yl)
methylamino)‑7b,8,9,10,11,11a‑hexahydro‑5‑isopropyl‑7
b,11‑dimethylphenanthro[9,10‑d]thiazole‑11‑carboxylate
(5q)
1
Yield 72%, yellow solid, m.p. 77.5–78.0 °C. H NMR
(500 MHz, CDCl3) δ 7.68 (d, J = 1.9 Hz, 1H, H-14),
7.42–7.38 (m, 1H, H-furan), 7.18 (d, J=8.0 Hz, 1H, H-11),
7.12 (dd, J = 8.0, 2.0 Hz, 1H, H-12), 6.37–6.32 (m, 2H,
H-furan), 5.36 (s, 1H, NH), 4.53 (qd, J=15.2, 5.0 Hz, 2H,
H-23), 3.72 (s, 1H, H-5), 3.66 (s, 3H, COOCH3), 2.95 (hept,
J=6.9 Hz, 1H, H-15), 2.34–2.26 (m, 1H, H1-e), 1.90–1.74
(m, 5H, H-3, H-2, H1-a), 1.58 (s, 3H, H-19), 1.29 (dd,
J = 6.9, 1.8 Hz, 6H, H-16, H-17), 1.14 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.50, 167.11, 151.11, 146.98,
146.33, 144.19, 142.31, 130.02, 125.45, 122.12, 121.83,
120.40, 110.40, 107.87, 52.44, 46.65, 46.56, 42.68, 39.19,
37.37, 35.35, 33.83, 24.13, 23.89, 21.74, 18.27, 17.59.
Mass spectrum (ESI), m/z: 465.2224 [M + H]+ Caled
C27H32N2O3S 464.2134.
(7bS,11R)‑methyl 2‑((6‑chloropyridin‑3‑yl)
methylamino)‑7b,8,9,10,11,11a‑hexahydro‑5‑isopropyl‑7
b,11‑dimethylphenanthro[9,10‑d]thiazole‑11‑carboxylate
(5t)
1
Yield 64%, yellow solid, m.p. 84.5–85.7 °C. H NMR
(500 MHz, CDCl3) δ 8.44 (s, 1H, H-Py), 7.78–7.72 (m, 1H,
H-Py), 7.63 (t, J=2.7 Hz, 1H, H-14), 7.31 (d, J= 8.2 Hz,
1H, H-Py), 7.18 (d, J=8.0 Hz, 1H, H-11), 7.13 (dd, J=8.0,
1.9 Hz, 1H, H-12), 5.80 (s, 1H, NH), 4.56 (q, J=15.0 Hz,
2H, H-23), 3.70 (s, 1H, H-5), 3.64 (s, 3H, COOCH3),
2.99–2.88 (m, 1H, H-15), 2.29 (t, J = 7.7 Hz, 1H, H1-e),
1.89–1.75 (m, 5H, H-3, H-2, H1-a), 1.56 (s, 3H, H-19),
1.29–1.28 (m, 6H, H-16, H-17), 1.13 (s, 3H, H-20).13C
NMR (126 MHz, CDCl3) δ 178.46, 167.02, 150.72, 149.14,
147.02, 146.25, 144.19, 138.47, 132.80, 129.77, 125.68,
124.22, 122.22, 121.75, 120.43, 52.48, 46.61, 46.57, 46.28,
39.20, 37.34, 35.30, 33.81, 24.12, 23.89, 21.75, 18.23,
17.59. Mass spectrum (ESI), m/z: 508.1620 [M-H]− Caled
C28H32ClN3O2S 509.1904.
(7bS,11R)‑methyl 2‑((1H‑pyrrol‑2‑yl)
methylamino)‑7b,8,9,10,11,11a‑hexahydro‑5‑isopropyl‑7
b,11‑dimethylphenanthro[9,10‑d]thiazole‑11‑carboxylate
(5r)
1
Yield 74%, yellow solid, m.p. 87.4–89.5 °C. H NMR
(500 MHz, CDCl3) δ 9.61 (d, J = 59.0 Hz, 1H, NH-pyr-
role), 7.73 (s, 1H, H-14), 7.21 (d, J = 8.0 Hz, 1H, H-11),
7.16 (d, J=8.1 Hz, 1H, H-12), 6.76 (td, J=2.6, 1.6 Hz, 1H,
H-pyrrole), 6.14 (dt, J =6.9, 2.2 Hz, 2H, H-pyrrole), 5.32
(s, 1H, NH), 4.57 (d, J = 4.3 Hz, 2H, H-23), 3.73 (s, 1H,
H-5), 3.66 (s, 3H, COOCH3), 3.02–2.93 (m, 1H, H-15),
1 3

Molecular Diversity
anti-phospho-S6(S235/236), anti-phospho-S6K(t389), anti-
mTOR, anti-phospho-mTOR, anti-Akt(S473), anti-phospho-
PI3K, and anti-α-tubulin, which were purchased from Cell
Signaling Technology, Boston, USA.
Antitumor activity
HepG2, SCC9, and 293T cells (Shanghai Institute of Cell
Biology, Chinese Academy of Sciences, Shanghai, China)
were cultured in Dulbecco’s modifcation Eagle’s medium
(Invitrogen, Carlsbad, USA) supplemented with 10% fetal
bovine serum (Hyclone, Logan, USA), 100 U/mL penicillin,
and 0.1 mg/mL streptomycin (Invitrogen). The cells were
incubated at 37 °C in a humidifed atmosphere with 5% CO2.
Cell proliferation was measured with the Cell Counting
Kit-8 (CCK-8) assay kit (Dojindo Corp, Kumamoto, Japan)
[17]. Cells were harvested during logarithmic growth phase,
seeded in 96-well plates at a density of 5× 103 cells/well,
and cultured at 37 °C in a humidifed incubator (5% CO2)
for 24 h, followed by exposure to various concentrations
of compounds for 24 h. Subsequently, 10 μL of CCK-8
(Dojindo) was added to each well; the cells were then incu-
bated for an additional 1 h at 37 °C. Cell growth inhibition
was determined by measuring the optical density value (OD)
at λ=450 nm using a microplate reader. Three independent
experiments were performed. The cell growth inhibition was
calculated according to the following equation:
Molecular docking studies
All docking procedures were performed using AutoDock
4.2.6 software (ADT) according to the reported paper [19].
The structures of PI3K, AKT, and mTOR were obtained
from the Protein Data Bank (PDB) with PDB IDs 3L54,
3MVH, and 4JT6, respectively [18]. In the PDB fle of
mTOR, there are 4 chains, and two of them marked as A and
B both containing an original ligand whose binding modes
to those two chains show distinctive similarity. Thus, only
the chain A was maintained to carry out molecular docking.
All the proteins in the PDB fles were cleaned by remov-
ing small molecules, ions, and original ligand in the crystal
of the proteins. Then, polar hydrogen atoms were added,
Gasteiger charges were computed, and the atoms were set
as “Assign AD4 type” in ADT. The compounds for docking
program were drawn using ChemDraw 8.0 and automatically
set the torsional bonds by the AUTOTORS module in ADT.
The docked sites of the protein were set in the place of the
original ligand. The grid map was 40×40×40 (center of the
grid box, PI3K: x=22.0, y=15.0, z=22.0; AKT: x=19.0,
y=−3.0, z=28.0; mTOR: x=52.0, y=1.0, z=−47.0). The
binding energy between the docked compound and the pro-
tein was calculated using the AutoGrid program with a grid
spacing of 0.375 Å by the Lamarckian genetic algorithm as
a searching method. The interaction between compounds
and the amino acid residues of the proteins was found by
LigPlot+ 2.1 and visualized by PyMOL 2.4.0a0 software.
(
)
OD of control − OD of treatment
OD of control − OD of blank
Growth inhibition =
× 100%
The IC50 values were obtained from linear regression
analysis of the concentration–response curves plotted for
each tested compound.
Western blot analysis of PI3K/AKT/mTOR signaling
pathway inhibitory activity of 5r and 5s
HepG2 cells were seeded at a density of 3 × 106 cells per
dish and attached for 8 h and then treated with compounds
in concentrations of 0, 20, and 40 μM for 24 h. After that,
cells were collected, washed with cold PBS, and lysed with
lysis bufer (100 mM Tris–HCl, pH 6.8, 4% SDS, 20% glyc-
erol) on ice for 30 min. Protein concentrations were detected
using the BCA method (Beyotime Institute of Biotechnol-
ogy, Haimen, China). Proteins were electrophoresed using
sodium dodecyl sulfate/polyacrylamide gel electrophoresis
(SDS-PAGE Bio-Rad, CA, USA) and transferred electro-
phoretically to membranes. The membranes were blocked
with 5% nonfat milk at room temperature for 1 h and were
incubated with primary antibodies overnight at 4 °C. The
next day, membranes were washed and incubated with the
appropriate fuorescence-conjugated secondary antibod-
ies at room temperature for 1 h. Finally, membranes were
washed and developed by the addition of ECL substrate
(Thermo Fisher Scientifc, Rockford, USA). Proteins were
visualized using the intelligent gel imaging system iBright
FL1000 (Thermo Fisher, Rockford, USA). Primary antibod-
ies were as follows: anti-EBP1, anti-phospho-EBP1, anti-S6,
Acknowledgements This research was supported by Guangxi Natural
Science Foundation under Grant Nos. 2018GXNSFAA138023 and
2019GXNSFAA245057, the Guangxi Distinguished Professor Grant
(2018), Youth Science Foundation of Guangxi Medical University (No.
GXMUYSF201703), and Open Research Project from Key Labora-
tory of High Incidence Diseases Prevention and Control of Guangxi
Universities and Colleges, No. 02402214003-1705.
Compliance with ethical standards
Conflict of interest The authors declare that they have no confict of
interest.
References
1. Tanaka R, Tokuda H, Ezaki Y (2008) Cancer chemopreventive
activity of “rosin” constituents of Pinus spez. and their deriva-
tives in two-stage mouse skin carcinogenesis test. Phytomedicine
15:985–992. https://doi.org/10.1016/j.phymed.2008.02.020
2. Luo S, Jiang L, Wang Y (2015) Review on the structural modifca-
tion of rosin’s active components. Nat Prod Res Dev 27:737–741
1 3

Molecular Diversity
12. Chen N, Duan W, Lin G, Liu L, Zhang R, Li D (2016) Synthesis
and antifungal activity of dehydroabietic acid-based B ring-fused-
thiazole-amide compounds. Fine Chem 33:811–819. https://doi.
org/10.13550/j.jxhg.2016.07.016
3. Cui Y, Rao X, Shang S, Song J (2012) Recent advances on bio-
logic activity of dehydroabietic acid derivatives. Chem Ind For
Prod 36:99–106
4. Gu W, Wang S (2010) Synthesis and antimicrobial activities
of novel 1H-dibenzo[a, c]carbazoles from dehydroabietic acid.
Eur J Med Chem 45:4692–4696. https://doi.org/10.1016/j.ejmec
h.2010.07.038
13. Chhabria MT, Patel S, Modi P, Brahmkshatriya PS (2016) Thia-
zole: a review on chemistry, synthesis and therapeutic importance
of its derivatives. Curr Top Med Chem 16:2841–2862. https://doi.
org/10.2174/1568026616666160506130731
5. Fei BL, Tu SY, Wei ZZ, Wang PP, Qiao CH, Chen ZF (2019)
Optically pure chiral copper(II) complexes of rosin derivative as
attractive anticancer agents with potential anti-metastatic and anti-
angiogenic activities. Eur J Med Chem 176:175–186. https://doi.
org/10.1016/j.ejmech.2019.05.030
14. Majeed R, Hamid A, Qurishi Y, Qazi A, Hussain A, Ahmed M,
Najar R, Bhat J, Saxena S (2012) Therapeutic targeting of can-
cer cell metabolism: role of metabolic enzymes, oncogenes and
tumor suppressor genes. J Cancer Sci Ther 4:281–291. https://doi.
org/10.4172/1948-5956.1000156
6. Chen N, Duan W, Lin G, Liu L, Zhang R, Li D (2016) Synthesis
and antifungal activity of dehydroabietic acid-based 1,3,4-thiadi-
azole-thiazolidinone compounds. Mol Divers 20:897–905. https
://doi.org/10.1007/s11030-016-9691-x
15. Sharmaa PC, Bansala KK, Sharmaa A, Sharmaa D, Deep A (2019)
Thiazole-containing compounds as therapeutic targets for cancer
therapy. Eur J Med Chem 188:112–116. https://doi.org/10.1016/j.
ejmech.2019.112016
7. Fonseca T, Gigante B, Marques MM, Gilchrist TL, Clercq ED
(2004) Synthesis and antiviral evaluation of benzimidazoles,
quinoxalines and indoles from dehydroabietic acid. Bioorg Med
Chem 12:103–112. https://doi.org/10.1016/j.bmc.2003.10.013
8. Kim E, Kang YG, Kim YJ, Lee TR, Yoo BC, Jo M, Kim JH, Kim
JH, Kim D, Cho JY (2019) Dehydroabietic acid suppresses infam-
matory response via suppression of Src-, Syk-, and TAK1-medi-
ated pathways. Int J Mol Sci 20:E1593. https://doi.org/10.3390/
ijms20071593
16. Peng QH, Duan WG, Xu XT, Fang HX, Shen CM (2007) Micro-
wave-assisted extraction of dehydroabietic acid. Appl Chem Ind
36(860–862):868
17. Chen H, Xu F, Xu BB, Xu JY, Shao BH, Huang BY, Yuan M
(2016) Design, synthesis and biological evaluation of novel
arylpiperazine derivatives on human prostate cancer cell lines.
Chin Chem Lett 27:277–282. https://doi.org/10.1016/j.cclet
.2015.09.016
18. Rehan M, Bajouh OS (2018) Virtual screening of naphthoquinone
analogs for potent inhibitors against the cancer-signaling PI3K/
AKT/mTOR signaling pathway. J Cell Biochem 2018:1–12. https
://doi.org/10.1002/jcb.27100
9. Vahermo M, Krogerus S, Nasereddin A, Kaiser M, Brun R, Jafe
CL, Yli-Kauhaluoma J, Moreira VM (2016) Antiprotozoal activity
of dehydroabietic acid derivatives against Leishmania donovani
and Trypanosoma cruzi. MedChemComm 7:457–463. https://doi.
org/10.1039/c5md00498e
19. Lin G, Bai X, Duan W, Cen B, Huang M, Lu S (2019) High value-
added application of sustainable natural forest product α-pinene:
synthesis of myrtenal oxime esters as potential KARI inhibitors.
ACS Sustain Chem Eng 7:7862–7868. https://doi.org/10.1021/
acssuschemeng.9b00254
10. Kim J, Kang YG, Lee JY, Choi DH, Cho YU, Shin JM, Park
JS, Lee JH, Kim WG, Seo DB, Lee TR, Miyamoto Y, No KT
(2015) The natural phytochemical dehydroabietic acid is an
anti-aging reagent that mediates the direct activation of SIRT1.
Mol Cell Endocrinol 412:216–225. https://doi.org/10.1016/j.
mce.2015.05.006
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
11. Cui YM, Liu XL, Zhang WM, Lin HX, Ohwada T, Ido K,
Awada K (2016) The synthesis and BK channel-opening activ-
ity of N-acylaminoalkyloxime derivatives of dehydroabietic acid.
Bioorg Med Chem Lett 26:283–287. https://doi.org/10.1016/j.
bmcl.2015.12.038
1 3