Novel nitrogen-enriched oridonin analogues with thiazole-fused a-ring: Protecting group-free synthesis, enhanced anticancer profile, and improved aqueous solubility

Article abstract of DOI:10.1021/jm400367n

Oridonin (1), a complex ent-kaurane diterpenoid isolated from the traditional Chinese herb Isodon rubescens, has demonstrated great potential in the treatment of various human cancers due to its unique and safe anticancer pharmacological profile. Nevertheless, the clinical development of oridonin for cancer therapy has been hampered by its relatively moderate potency, limited aqueous solubility, and poor bioavailability. Herein, we report the concise synthesis of a series of novel nitrogen-enriched oridonin derivatives with thiazole-fused A-ring through an efficient protecting group-free synthetic strategy. Most of them, including compounds 7-11, 13, and 14, exhibited potent antiproliferative effects against breast, pancreatic, and prostate cancer cells with low micromolar to submicromolar IC₅₀ values as well as markedly enhanced aqueous solubility. These new analogues obtained by rationally modifying the natural product have been demonstrated not only to significantly induce the apoptosis and suppress growth of triple-negative MDA-MB-231 breast cancer both in vitro and in vivo but also effective against drug-resistant ER-positive MCF-7 clones.

Full text of DOI:10.1021/jm400367n

Article
pubs.acs.org/jmc
Novel Nitrogen-Enriched Oridonin Analogues with Thiazole-Fused
A‑Ring: Protecting Group-Free Synthesis, Enhanced Anticancer
Profile, and Improved Aqueous Solubility
Chunyong Ding,† Yusong Zhang,‡,§ Haijun Chen,† Zhengduo Yang,§ Christopher Wild,† Lili Chu,§
Huiling Liu,† Qiang Shen,*,§ and Jia Zhou*,†
†Chemical Biology Program, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas
77555, United States
‡Department of Oncology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, China
§Department of Clinical Cancer Prevention, Division of Cancer Prevention and Population Sciences, The University of Texas M.D.
Anderson Cancer Center, Houston, Texas 77030, United States
S
* Supporting Information
ABSTRACT: Oridonin (1), a complex ent-kaurane diterpenoid isolated from the traditional Chinese herb Isodon rubescens, has
demonstrated great potential in the treatment of various human cancers due to its unique and safe anticancer pharmacological
profile. Nevertheless, the clinical development of oridonin for cancer therapy has been hampered by its relatively moderate
potency, limited aqueous solubility, and poor bioavailability. Herein, we report the concise synthesis of a series of novel nitrogen-
enriched oridonin derivatives with thiazole-fused A-ring through an efficient protecting group-free synthetic strategy. Most of
them, including compounds 7−11, 13, and 14, exhibited potent antiproliferative effects against breast, pancreatic, and prostate
cancer cells with low micromolar to submicromolar IC50 values as well as markedly enhanced aqueous solubility. These new
analogues obtained by rationally modifying the natural product have been demonstrated not only to significantly induce the
apoptosis and suppress growth of triple-negative MDA-MB-231 breast cancer both in vitro and in vivo but also effective against
drug-resistant ER-positive MCF-7 clones.
a rising attention in recent years due to its extensive biological
activities.9−11 Of particular interest is its unique, safe, and
remarkable anticancer pharmacological profile.12 Increasing
clinical evidence has suggested that oridonin may greatly
improve the survival rates of cancer patients through hampering
the progression of tumor, mitigating tumor burden, and
alleviating cancer syndrome.13 Because of the relatively low
toxicity of 1, it can be used for a long period of time without
significant abdominal discomfort.5 For instance, 1 was reported
not only to induce typical mitochondrial apoptosis in acute
myeloid leukemia cells at low micromolar concentration but
also to exhibit significant antileukemia activities with a low side-
effect in murine models (15 mg/kg).14 Accumulating
mechanistic studies have revealed that 1 possesses unique but
versatile antiproliferative capabilities including cell cycle arrest,
apoptosis, and autophagy by regulating a series of transcription
factors, protein kinases, as well as pro- and/or antiapoptotic
proteins.15−25 All these findings support that oridonin
INTRODUCTION
■
Natural products have a profound impact upon both chemical
biology and drug discovery, and the great structural diversity of
natural products with various interesting biological character-
istics has always provided medicinal chemists an important
source of inspiration in their search for new molecular entities
with pharmacological activity.1−3 Among them, natural
tetracyclic diterpenoids, especially ent-kaurane diterpenoids
with exo-methylene cyclopentanone in the D-ring such as
oridonin (1), ponicidin, and phyllostachysin F (Figure 1),
constitute an important class of natural products which exhibit
considerable pharmacological activities including antitumor,
antibacterial, antituberculosis, and anti-inflammatory effects.4−7
Isodon rubescens (Chinese name “Donglingcao”) is an
important source of a traditional Chinese herbal medicine
that has been approved by State Food and Drug Administration
of China for the treatment of inflammation such as acute
tonsillitis, esophagitis, stomatitis, and gingivitis.7,8a It has also
been widely used as herbal medicine in China for the treatment
of esophageal and cardia cancer for many years.8b 1, an ent-
kaurane diterpenoid isolated from this herb, has been attracting
Received: March 12, 2013
© XXXX American Chemical Society
A
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bleomycin.36−38 Modifications of the thiazole ring have proven
highly effective with regards to improved potency and less
toxicity.39 Therefore, we have designed a series of nitrogen-
enriched oridonin derivatives in which a thiazole ring is fused at
C-1 and C-2 of the A-ring and envisioned these natural
product-like molecules may possess better anticancer potency
and aqueous solubility. Herein, we describe our efforts for the
synthesis of these novel compounds through a protecting
group-free semisynthetic approach and the discovery of
promising anticancer agents in this endeavor. It is noteworthy
that this work is the first attempt to generate new molecules
with a nitrogen-containing heterocyclic scaffold that is fused
with oridonin A-ring system.
RESULTS AND DISCUSSION
■
Chemistry. We took advantage of 1, a naturally abundant
and commercially available ent-kaurane diterpenoid, as a basic
template to synthesize the thiazole-fused oridonin derivatives.
To date, there is little evidence available for the pursuit of
chemical transformations at the A-ring of 1, probably due to its
structural complexity. Given the prevalence of the successful
application of Hantzsch synthesis of thiazoles40 and amino-
thiazoles41 in other natural products, we were inspired to apply
this reaction to the oridonin template for the synthesis of new
derivatives with variously substituted thiazole-fused A-rings.
To explore a meaningful structure−activity relationship of
substituents on the thiazole ring, we have designed a series of
diversely substituted thiazole derivatives such as oridonin
analogues 5, 7−12, and 14−15, which were endowed with
typical alkyl, amino, allylamino, alkyl-substituted amino, or
amide groups. With the aim of further improving aqueous
solubility, compounds 13 and 16 have been designed with
additional hydrophilic moieties such as piperidinyl and amidinyl
groups. As depicted in Scheme 1, following a literature
procedure,42 oxidation of 1 with Jones reagent in ice−water
Figure 1. Structures of oridonin and other typical natural ent-kaurane
diterpenoids.
represents a promising anticancer drug candidate with great
potential for bench to bedside development. However, the
clinical development of oridonin for cancer therapy has been
hampered, to a large degree, by its relatively moderate potency,
limited aqueous solubility, and bioavailability.26,27 In addition,
oridonin exerts only modest to poor inhibitory effects on highly
aggressive cancer cells such as estrogen triple-negative breast
cancer cell MDA-MB-23128 and multiple drug resistant (MDR)
breast cancer cells. All these observations combine to justify an
urgent need for developing novel oridonin analogues by
rationally modifying the natural product to improve the
anticancer activity profile and drug-like properties including
aqueous solubility.
The highly oxygenated oridonin, structurally belonging to
7,20-epoxy-ent-kaurane-type diterpenoid, is primarily charac-
terized with its densely functionalized, stereochemistry-rich
frameworks including an α,β-unsaturated ketone moiety in the
D-ring and a 6-hydroxyl-7-hemiacetal group in the B-ring.
Previous studies on the structure−activity relationship (SAR)
of 1 indicated that the α-methylene cyclopentanone system in
D-ring was an essential active center for the antitumor activity,
and any destruction of this enone system (e.g., split ring or
saturated methylene) could lead to a total loss of bioactivity.9,29
Meanwhile, the presence of hydrogen-bonding between 6β-OH
and ketone group at C-15 was beneficial for an enhanced
antitumor activity of 1.30 To date, the reported structural
modifications to generate new oridonin derivatives have been
mainly focused on the 1-O and 14-O positions by introducing
aqueous solubility-enhancing moieties via coupling ester
appendages to its hydroxyl groups.31−33 However, ester
bonds usually suffer from poor metabolic stability in vivo due
to the enzymatic hydrolysis34,35 and such derivatives tend to act
as prodrugs. Therefore, it is imperative to develop novel
oridonin analogues with effective modifications to accelerate
the search for promising anticancer drug candidates.
Scheme 1. Synthesis of 2-Amino Thiazole-Fused Oridonin
a
Derivative 5
In the design of novel oridonin derivatives, our approach was
guided by the idea of incorporating nitrogen into the core
scaffold of oridonin that may enhance their aqueous solubility
through the formation of salts with acids while retaining key
pharmacophores of 1 such as the enone moiety. The thiazole
ring system, a nitrogen-containing aromatic heterocycle,
represents an important building block that exists in many
biologically active natural products including clinically used
anticancer drugs such as epothilones, ixabepilone, and
a
Reagents and conditions: (a) Jones reagent, acetone, rt, 30 min, 82%;
(b) Me2C(OMe)2, p-TsOH, acetone, rt, 2 h, 95%; (c) PyHBr3, THF, 0
°C, 2 h; (d) thiourea, EtOH, reflux, 5 h.
B
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a
Scheme 2. Synthesis of 2-Substituted Thiazole-Fused Oridonin Derivatives 7−16
a
Reagents and conditions: (a) EtOH, reflux, 3−6 h, 35−65%.
Table 1. Effects of New Oridonin Analogues on Proliferation of Human Breast, Pancreatic Cancer, and Prostate Cancer Cells
a
IC50 (μM)
breast cancer
pancreatic cancer
prostate cancer
DU145
MCF-7
(ER positive)
MDA-MB-231
(ER negative)
compd
AsPC1
Panc-1
1
6.6
1.0
2.6
1.3
0.8
0.6
0.9
1.2
1.0
0.2
2.0
3.4
29.4
3.2
6.9
2.1
0.3
1.1
0.8
6.8
1.8
0.2
6.8
19.3
5.6
6.5
1.8
1.4
1.4
1.7
2.3
1.1
1.1
4.8
15.6
6.1
7.8
2.3
3.3
4.0
3.7
6.3
1.5
1.1
6.7
>10
14.2
3.5
7.6
4.1
5.3
4.2
1.8
4.7
1.2
1.2
6.3
>10
5
7
8
9
10
11
12
13
14
15
16
b
>10
>10
a
Breast cancer cell lines: MCF-7 and MDA-MB-231. Pancreatic cancer cell lines: AsPC1 and Panc-1. Prostate cancer cell line: DU145. Software:
b
MasterPlex ReaderFit 2010, MiraiBio, Inc. The values are the mean SE of at least three independent experiments. If a specific compound is given
a value >10, it indicates that a specific IC50 cannot be calculated from the data points collected, meaning “no effect”.
bath selectively afforded 1-oxo-oridonin derivative 2 in 82%
yield. Initially, taking into account of the oridonin’s delicate and
complex template, particularly the reactive hydroxyl functional
groups, it was thought important to elaborate a protecting
group strategy that would avoid possible side reactions during
the subsequent steps. Thus, protection of 7,14-dihydroxyl of 2
with 2,2-dimethoxypropane catalyzed by p-TsOH in acetone
solely provided cyclic ketal 3 in 95% yield. However,
bromination of 3 with NBS in the refluxing CCl4 resulted in
a complex mixture. Alternatively, PyHBr3 was chosen as the
bromination agent to react with 3 in THF at 0 °C to give
intermediate 4 as a major product in 40% yield. Hantzsch
C
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reaction of 4 with thiourea in the refluxing ethanol directly
afforded the target oridonin derivative 5 in 20% yield, in which
cyclic ketal was simultaneously removed due to the presence of
HBr generated from the thiazole formation. Nevertheless, the
total yield of this synthetic route was only 6.2%, which was too
low to gain sufficient amount of 5 for further pharmacological
evaluation. Considering that cyclic ketal could be easily
deprotected in Hantzsch thiazole synthesis, we attempted a
protecting group-free synthetic strategy to improve the total
yield. Thus, intermediate 2 without any protecting groups was
directly utilized to react with PyHBr3 at 0 °C. To our delight,
the desired intermediate 6 was obtained in 60% yield, and the
subsequent Hantzsch reaction of 6 with thiourea yielded the
desired compound 5 in a much better yield of 53%, suggesting
that the protection of 7,14-dihydroxyl of 2 was unnecessary.
Therefore, a concise protecting group-free synthetic approach
to readily access 5 has been established through three steps in
26% total yield. To examine the antitumor effects of
substituents on a thiazole ring, a series of novel oridonin
derivatives with diversely substituted thiazole scaffolds were
generated in 35−65% yields using the Hantzsch reaction of
common building block 6 with thioacetamide or N-substituted
thioureas (Scheme 2).
Interestingly, analogue 16 with a guanidinyl group on the
thiazole only displayed a potent activity against MCF-7 cell line
but moderate to low activity against other tested cell lines.
In Vitro Growth Inhibitory Activity in Drug-Resistant
Breast Cancer Cells. Resistance to chemotherapy is a major
cause of the ultimate failure of breast cancer treatment. To
investigate whether these thiazole-fused oridonin analogues are
still effective on drug-resistant breast cancer cells, compounds
7, 8, and 14 with different substituted thiazole moieties, as well
as 1, were selected to evaluate their growth inhibitory activity
against adriamycin (ADR)-resistant MCF-7 clone at the
dosages of 1.0, 5.0, and 10.0 μM using MTT assays. As
shown in Figure 2, compound 1 displayed no growth inhibitory
In Vitro Antiproliferative Activity. The growth inhibitory
potency of synthesized novel oridonin derivatives was evaluated
in two breast cancer cell lines MCF-7 (ER-positive) and MDA-
MB-231 (ER-negative and triple-negative), two pancreatic
cancer cell lines AsPC1 and Panc-1, as well as one prostate
cancer cell line DU145 using MTT assays as described in the in
vitro screening protocol (Experimental Section). The ability of
these new analogues to inhibit the growth of cancer cells was
summarized in Table 1. 1 was tested for comparison. The
results showed that most of newly synthesized thiazole-fused
oridonin derivatives (5, 7−15) not only exhibited significantly
improved antiproliferative activity against breast cancer cell
MCF-7 relative to 1 but also displayed marked growth
inhibitory effects on other tested cancer cell lines including
highly invasive breast cancer MDA-MB-231 cells, for which 1
had only modest activity with an average IC50 value greater than
19 μM. As shown in Table 1, 2-aminothiazole derivative 5
exerted 2−9-fold more potent antitumor activity than 1 against
all tested cancer cells, indicating that introduction of thiazole at
C-1/C-2 of oridonin A-ring is tolerable. Analogue 7 with a
methyl instead of free amino group at C-2 of thiazole ring led
to a 2-fold decreased activity against breast and prostate cancer
cells compared with 5, suggesting that subsequent optimization
of the substituents on the thiazole ring may tune their
antiproliferative effects. In general, further substitution on the
primary amine with various alkyl groups was found to
significantly increase antiproliferative activities against all tested
cancer cell lines. For example, most of the N-alkyl substituted
derivatives including 8−11 and 13−14 displayed potent activity
against breast cancer cells, with IC50 values varying from the
low micromolar to submicromolar range. Particularly, com-
pound 14 with an N-allyl substituted thiazole moiety exhibited
the most potent antiproliferative activity against both MCF-7
and MDA-MB-231 cells with the same IC50 values of 0.2 μM,
which are approximately 33-fold and 147-fold more potent than
1, respectively. It is noteworthy to mention that N-methyl
analogue 8 with low micromolar potency against all the tested
cancer lines was found to display an excellent dose−response
relationship. Compound 15 with an acetamide group on the
thiazole showed a slightly lower antiproliferative activity than 5.
Figure 2. Growth inhibitory effect of 1, 7, 8, and 14 against drug-
resistant breast cancer cells. Adrimycin-resistant MCF-7/ADR clone
was treated with variable concentrations of 1, 7, 8, and 14 (1.0, 5.0,
and 10.0 μM), respectively, for 48 h. The values are the mean SE of
at least three independent experiments. * represents p < 0.05,
comparing to the effect from oridonin at the same dosage.
activity at all concentrations, while new analogues 7 and 8 have
been found to significantly inhibit the growth of drug-resistant
MCF-7 clone, and their growth inhibitory rates were even
greater than 50% at 5.0 and 10.0 μM, respectively. Particularly,
compound 14 exhibited the most potent antiproliferative
activity against MCF-7/ADR cells with an IC50 value less
than 1 μM.
Aqueous Solubility. To examine whether the synthesized
thiazole-fused analogues have better aqueous solubility than 1, a
previously reported HPLC method43 was employed to measure
the solubility of several selected analogues such as 7
(CYD0619), 8 (CYD0554), and 14 (CYD0618). One-point
calibration was performed against standards with known
concentrations of the sample compounds to determine
concentrations of the indicated compounds in samples. As
expected, incorporating a substituted thiazole-fused moiety into
oridonin not only enhanced the antiproliferative activity but
also significantly improved their aqueous solubility. For
instance, aqueous solubility of analogue 7 with 2-methyl
thiazole moiety was determined to be 4.47 mg/mL, and the N-
alkyl derivatives 8 and 14 in the form of HCl salt demonstrated
an excellent solubility, with a saturated concentration of 42.4
and 81.2 mg/mL, respectively, indicating approximately 32-fold
to 62-fold improvement in comparison with that of oridonin
(1.29 mg/mL) (Figure 3). Some other analogues such as
compounds 13 and 16 (in the HCl salt form) possess an even
superior aqueous solubility greater than 100 mg/mL.
D
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studies to determine whether the growth inhibition induced by
14 in human breast cells was attributed to apoptosis. MDA-
MB-231 and MCF-7/ADR cells were not only treated with
vehicle alone as controls but also dealt with 14 at different
concentrations (0.25, 0.5, or 1.0 μM) for 48 h and stained with
FITC-Annexin V and propidium iodide (PI). The percentages
of apoptotic MDA-MB-231 cells were determined by flow
cytometry. As shown in Figure 4, compound 14 displayed
marked effects to induce apoptosis of breast cancer cells in a
dose-dependent manner. Treatment of the MDA-MB-231 cells
with 0.25, 0.5, and 1.0 μM of compound 14 for 48 h resulted in
21.2% 1.1, 41.3% 16.3, and 61.7% 3.7 of apoptotic cells
(early and late apoptosis), respectively, as compared to 6.1%
2.0 in an untreated vehicle control. Similarly, treatment of
MCF-7/ADR cells with compound 14 also led to 10.4% 3.0,
23.1% 7.6, and 78.2% 2.5 of apoptotic cells at the same
three concentrations as above, respectively. Apparently,
compound 14 mediated apoptosis of MDA-MB-231 and
MCF-7/ADR cells, at least in part, contributes to its
antiproliferative effects.
Figure 3. Aqueous solubility of novel oridonin analogues. Compounds
7 (CYD0619), 14 (CYD0618), and 8 (CYD0554) showed
significantly improved solubility compared with 1. Compounds 14
(HCl salt) and 8 (HCl salt) are soluble in water with saturated
concentrations of 42.4 and 81.2 mg/mL, respectively, which are
approximately 32- and 62-fold better than that of 1 (1.29 mg/mL).
The values are the mean
experiments.
SE of at least three independent
Compound 14 Regulated Apoptotic Related Proteins.
Previous studies have demonstrated that 1 induces apoptosis of
cancer cells by regulating a series of transcription factors,
protein kinases, as well as pro- and/or antiapoptotic proteins
Compound 14 Induced Apoptosis of Breast Cancer
Cells. On the basis of the antiproliferative data, the most
potent compound 14 was selected for further mechanistic
Figure 4. Induction of apoptosis on MDA-MB-231 and MCF-7/ADR cells by compound 14. (A) Flow cytometry analysis of apoptotic MDA-MB-
231 cells induced by 14 at different concentrations. (B) Flow cytometry analysis of apoptotic MCF-7/ADR cells induced by 14 at different
concentrations. (C) Apoptotic ratio of different concentrations of 14 in MDA-MB-231 cells. (D) Apoptotic ratio of different concentrations of 14 in
MCF-7/ADR cells. Cells were treated with vehicle or 14 at 0.25, 0.5, and 1.0 μM concentrations, respectively, for 48 h. The values are means SE of
at least three independent experiments. * represents p < 0.05 comparing to vehicle-treated control.
E
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Figure 5. Western blot analysis of biological markers for apoptosis induction by compound 14 (CYD0618) and 1 in the MDA-MB-231 cells at
different concentrations (48 h).
such as NF-κB,23,25b,28 MAPK,15,16 Bax, and Bcl-2.17,19 To
elucidate the potential mechanisms contributed to apoptosis
induction by the new derivative 14, several proteins related to
apoptosis were determined by Western blotting. As shown in
Figure 5, treatment of MDA-MB-231 cells with compound 14
at low concentrations (0.25−1.0 μM) dose dependently led to
the down-regulation of antiapoptotic protein Bcl-2 levels and
the up-regulation of the pro-apoptotic protein Bax. In addition,
it also induced a significant decrease of NF-κB (p65) protein
expression, suggesting that NF-κB inhibition might contribute
to the reduction of Bcl-2/Bax ratio. Meanwhile, compound 14
also triggered PARP cleavage from its full-length form (116
kDa) to the cleaved form (25 kDa), as indicated by PARP
fragments appearance in a dose-dependent manner, which
could be viewed as a marker of apoptosis. Similarly, exposure to
the high dosages of 1 (10−30 μM) also led to down-regulation
of NF-κB (p65), Bcl-2 and PARP (116 kDa) and up-regulation
Figure 6. In vivo efficacy of 1 and compound 14 (CYD0618) in
of Bax and cleaved PARP (25 KDa). These preliminary data
inhibiting growth of xenograft tumors (breast cancer MDA-MB-231)
in mice (ip) at the dose of 5 mg/kg, respectively. Data are presented as
indicated that the thiazole derivative 14 might mediate the
apoptosis in MDA-MB-231 cells at low concentrations through
the mean
SE of tumor volume at each time point. Significant
similar multiple apoptotic pathways to those of 1. Other than
apoptosis, oridonin has also been found to inhibit tumor cell
proliferation and induces cancer cell death though cell cycle
arrest,17,20,28 autophagy,23−25 and necrosis.15 Therefore, more
extensive mechanism studies on the new derivative 14 are
ongoing, and the results will be reported in due course.
Compound 14 Suppressed Growth of Xenograft
Tumors in Nude Mice. In our pilot in vivo studies, analogue
14 was further evaluated for its anticancer activity in
suppression of tumor growth in the triple-negative breast
cancer MDA-MB-231 xenograft model. As shown in Figure 6,
mice treated with 5.0 mg/kg of compound 14 via ip showed a
much better effect in inhibiting tumor growth as compared to
the mice treated with the same dose of oridonin (p < 0.0001).
Meanwhile, compound 14 was found to be tolerated during the
experiments and showed no significant loss of body weight
(data not shown). These findings suggest that compound 14
(CYD0618) is a promising anticancer drug candidate with
potent antitumor activity and excellent aqueous solubility for
further clinic development.
differences between compound 14 treatment group, oridonin
treatment group, and control were determined using one way
ANOVA. p < 0.0001.
demonstrated that most of these new molecules not only
exhibited significantly enhanced antiproliferative activity against
breast cancer MCF-7 clone relative to 1 but also displayed
marked growth inhibitory effects on the other oridonin-
insensitive cancer cell lines including highly invasive triple-
negative breast cancer MDA-MB-231 cells with low micromolar
to submicromolar IC50 values. Particularly, compound 14 with
an N-allyl substituted thiazole moiety exhibited the most potent
antiproliferative activity against both MCF-7 and MDA-MB-
231 cells with the IC50 values of 0.2 μM, which are
approximately 33-fold and 147-fold more potent than 1,
respectively. Meanwhile, these new analogues such as 7, 8, and
14 with diversely substituted thiazole moieties remained to be
effective against adriamycin-resistant MCF-7 clone, for which 1
displayed no effect at the same doses. Moreover, they also have
significantly improved aqueous solubility in comparison with
that of the natural product oridonin. In our pilot mechanism
studies, compound 14 was found to significantly induce
apoptosis of MDA-MB-231 and MCF-7/ADR cells at low
concentrations in a dose-dependent manner and likely mediate
apoptosis through similar multiple pathways to those of 1. In
nude mice bearing breast tumor xenografts, compound 14 at 5
CONCLUSIONS
■
For the first time, an efficient and concise protecting group-free
synthetic approach has been established to readily access a
series of novel thiazole-fused oridonin analogues starting from
the natural product oridonin. In vitro pharmacological studies
F
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1.21 (s, 3H), 0.98 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 206.4,
mg/kg significantly suppressed MDA-MB-231 xenograft tumor
growth in vivo and was found more efficacious than oridonin.
These new molecules with a nitrogen-containing heterocyclic
scaffold that is fused with oridonin ring system open new
avenues to explore the therapeutic potential of oridonin-based
derivatives and develop promising natural product-like drug
candidates for the treatment of cancer.
202.6, 150.6, 122.3, 98.0, 72.9, 72.2, 65.6, 61.7, 58.1, 52.2, 49.8, 48.4,
1
45.1, 42.7, 33.8, 29.8, 29.4, 22.3, 18.1. Minor isomer: H NMR (600
MHz, CDCl3): δ 6.26 (s, 1H), 5.98 (d, 1H, J = 12.0 Hz), 6.00 (br s,
1H), 5.66 (s, 1H), 4.87 (s, 1H), 4.80 (m, 1H), 4.39 (d, 1H, J = 10.8
Hz4), 4.06 (d, 1H, J = 10.8 Hz), 3.80 (m, 1H), 3.08 (d, 1H, J = 9.0
Hz), 2.67 (m, 1H), 2.36 (d, 1H, J = 5.4 Hz), 2.33 (d, 1H, J = 5.4 Hz),
2.12 (m, 1H), 1.90 (m, 1H), 1.65 (m, 1H), 1.25 (m, 1H), 1.22 (s, 3H),
1.05 (m, 1H), 1.04 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 206.2,
202.6, 150.5, 122.6, 98.0, 72.9, 71.9, 64.5, 61.2, 58.7, 51.4, 49.8, 48.9,
45.1, 42.6, 34.8, 30.4, 29.3, 24.9, 18.9. HRMS (ESI) calcd for
C20H26BrO6 [M + H]+ 441.0907; found 441.0909.
(5aR,6S,7S,7aR,10S,12aS,12bR,15R)-2-Amino-6,7,15-trihy-
droxy-5,5-dimethyl-9-methylene-5,5a,6,7,10,11,12,12a-octa-
hydro-4H-7,12b-(epoxymethano)-7a,10-methanocyclohepta-
[7,8]naphtho[1,2-d]thiazol-8(9H)-one (5). To a solution of 6 (50
mg, 0.11 mmol) in ethanol (4 mL) was added thiourea (12 mg, 0.16
mmol) at rt. The reaction mixture was heated under reflux for 3 h.
After cooling and basifying with saturated NaHCO3 aqueous solution,
the mixture was concentrated in vacuo to give an oily residue. The
residue was purified by silica gel column; elution with 50% EtOAc in
hexane afforded the desired product 5 (25 mg, 53%) as an amorphous
gel; [α]D25 +190 (c 0.10, CHCl3); HPLC purity 97.0% (tR = 9.0 min).
1H NMR (600 MHz, CDCl3/CD3OD = 5:1): δ 6.16 (s, 1H), 5.59 (s,
EXPERIMENTAL SECTION
■
General. All commercially available starting materials and solvents
were reagent grade and used without further purification. Reactions
were performed under a nitrogen atmosphere in dry glassware with
magnetic stirring. Preparative column chromatography was performed
using silica gel 60, particle size 0.063−0.200 mm (70−230 mesh,
flash). Analytical TLC was carried out employing silica gel 60 F254
plates (Merck, Darmstadt). Visualization of the developed chromato-
grams was performed with detection by UV (254 nm). NMR spectra
were recorded on a Bruker-600 (1H, 600 MHz; 13C, 150 MHz)
1
spectrometer. H and 13C NMR spectra were recorded with TMS as
an internal reference. Chemical shifts downfield from TMS were
expressed in ppm, and J values were given in Hz. High-resolution mass
spectra (HRMS) were obtained from Thermo Fisher LTQ Orbitrap
Elite mass spectrometer. Parameters include the following: nano ESI
spray voltage was 1.8 kV, capillary temperature was 275 °C, and the
resolution was 60000; ionization was achieved by positive mode.
Melting points were measured on a Thermo Scientific Electrothermal
digital melting point apparatus and uncorrected. Purity of final
compounds was determined by analytical HPLC, which was carried
out on a Shimadzu HPLC system (model: CBM-20A LC-20AD SPD-
20A UV/vis). HPLC analysis conditions: Waters μBondapak C18
(300 mm × 3.9 mm), flow rate 0.5 mL/min, UV detection at 270 and
254 nm, linear gradient from 30% acetonitrile in water (0.1% TFA) to
100% acetonitrile (0.1% TFA) in 20 min, followed by 30 min of the
last-named solvent. All biologically evaluated compounds are >95%
pure.
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-5,6,14-Trihydroxy-4,4-di-
methyl-8-methylenedecahydro-1H-6,11b-(epoxymethano)-
6a,9-methanocyclohepta[a]naphthalene-1,7(8H)-dione (2). To
a stirring solution of oridonin (500 mg, 1.37 mmol) in acetone (40
mL) was added Jones reagent (0.6 mL) dropwise at ice−water bath.
The resulting mixture was stirred at 0 °C for 20 min, and isopropyl
alcohol was added to quench excess Jones reagent. Then the mixture
was diluted with water and extracted with dichloromethane. The
extract was washed with brine, dried over anhydrous Na2SO4, filtered,
and evaporated to give a solid crude product. The crude residue was
recrystallized from acetone−hexane to give 2 as a white solid (410 mg,
82%); mp 219−220 °C (Lit.42 mp 219−221 °C). 1H NMR (600 MHz,
(CD3)2CO): δ 6.52 (br s, 1H), 6.10 (s, 1H), 5.62 (s, 1H), 5.41 (d, 1H,
J = 10.8 Hz), 5.24 (s, 1H), 4.91 (s, 1H), 4.22 (d, 1H, J = 10.2 Hz),
3.92 (d, 1H, J = 10.8 Hz), 3.69 (m, 1H), 3.31 (br s, 1H), 3.01 (d, 1H, J
= 9.6 Hz), 2.76 (m, 5H), 2.46 (m, 1H), 2.36 (m, 1H), 2.19 (m, 1H),
1.92 (m, 3H), 1.68 (m, 1H), 1.61 (m, 1H), 1.19 (m, 1H), 1.14 (s, 3H),
0.97 (s, 3H).
1H), 5.01 (s, 1H), 4.37 (d, 1H, J = 10.2), 3.96 (d, 1H, J = 9.6 Hz), 3.80
(d, 1H, J = 3.0 Hz), 3.32 (s, 1H), 3.01 (d, 1H, J = 3.6 Hz), 2.52 (m,
2H), 2.31 (d, 1H, J = 15.6 Hz), 2.12 (m, 1H), 1.94 (dd, 1H, J = 4.8 Hz,
13.8 Hz), 1.83 (m, 1H), 1.69 (d, 1H, J = 9.0 Hz), 1.58 (m, 1H), 1.25
(s, 3H), 0.99 (s, 3H). 13C NMR (150 MHz, CDCl3/CD3OD = 5:1): δ
206.8, 166.0, 151.4, 141.7, 120.4, 120.2, 97.5, 72.7, 72.5, 64.9, 61.9,
59.1, 53.1, 43.2, 40.5, 38.6, 34.6, 30.2, 29.8, 20.2, 20.1. HRMS (ESI)
calcd for C21H26N2O5S [M + H]+ 419.1635; found 419.1638.
(6S,7S,7aR,10R,12bR,15R)-6,7,15-Trihydroxy-2,5,5-trimethyl-
9-methylene-5,5a,6,7,10,11,12,12a-octahydro-4H-7,12b-(ep-
oxymethano)-7a,10-methanocyclohepta[7,8]naphtho[1,2-d]-
thiazol-8(9H)-one (7). Compound 7 (16 mg) was prepared in 35%
yield by a procedure similar to that used to prepare compound 5. The
25
title compound was obtained as a colorless amorphous gel; [α]D
+184 (c 0.10, CHCl3); HPLC purity 97.6% (tR = 12.4 min). 1H NMR
(600 MHz, CDCl3): δ 6.18 (s, 1H), 5.81 (d, 1H, J = 12.6 Hz), 5.57 (d,
1H, J = 0.6 Hz), 5.24 (br s, 1H), 5.16 (br s, 1H), 4.96 (d, 1H, J = 1.2
Hz), 4.72 (s, 1H), 4.36 (dd, 1H, J = 0.6 Hz, 10.2 Hz), 4.08 (dd, 1H, J =
10.2 Hz), 3.79 (m, 1H), 3.03 (d, 1H, J = 9.6 Hz), 2.45 (m, 1H), 2.25
(d, 1H, J = 15.0 Hz), 2.04 (d, 1H, J = 14.4 Hz), 1.99 (m, 1H), 1.92 (s,
3H), 1.66 (d, 1H, J = 3.6 Hz), 1.59 (m, 2H), 1.21 (s, 3H), 1.00 (s,
3H). 13C NMR (150 MHz, CDCl3): δ 206.7, 192.7, 162.0, 151.6,
121.3, 100.6, 98.1, 73.2, 72.0, 66.5, 62.4, 58.4, 51.6, 46.4 (2C), 42.5,
32.9, 30.5, 30.2, 21.7, 20.7, 20.1. HRMS (ESI) calcd for C22H27NO5S
[M + H]+ 418.1683; found 418.1685.
(5aR,6S,7S,7aR,10S,12aS,12bR,15R)-6,7,15-Trihydroxy-5,5-
dimethyl-2-(methylamino)-9-methylene-5,5a,6,7,10,11,12,12a-
o c t a h y d r o - 4 H - 7 , 1 2 b - ( e p o x y m e t h a n o ) - 7 a , 1 0 -
methanocyclohepta[7,8]naphtho[1,2-d]thiazol-8(9H)-one (8).
Compound 8 (30 mg) was prepared in 65% yield by a procedure
similar to that used to prepare compound 5. The title compound was
25
obtained as a colorless amorphous gel; [α]D +146 (c 0.10, CHCl3);
(4aR,5S,6S,6aR,9S,11aS,11bS,14R)-2-Bromo-5,6,14-trihy-
droxy-4,4-dimethyl-8-methylenedecahydro-1H-6,11b-(epoxy-
methano)-6a,9-methanocyclohepta[a]naphthalene-1,7(8H)-
dione (6). To a solution of 2 (100 mg, 0.27 mmol) in THF (4 mL)
was added PyHBr3 (88 mg, 0.27 mmol) at rt. The reaction mixture was
stirred at rt for 4 h and then poured into water and extracted with
CH2Cl2 (30 mL × 3). The combined organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated in vacuo to give
an oily residue. The residue was further purified by silica gel column;
elution with 50% EtOAc in hexane afforded the desired product 6 (80
mg, 66%) as a colorless amorphous gel and a mixture of two isomers.
HPLC purity 95.6% (tR = 15.7 min). 1H NMR (600 MHz, CDCl3): δ
6.80 (br s, 1H), 6.17 (s, 1H), 5.97 (d, 1H, J = 12.0 Hz), 5.80 (br s,
1H), 5.56 (s, 1H), 5.03 (br s, 2H), 4.65 (d, 1H, J = 10.2 Hz), 3.87 (d,
1H, J = 10.2 Hz), 3.77 (dd, 1H, J = 9.6 Hz, 12.0 Hz), 3.05 (d, 1H, J =
9.0 Hz), 2.86 (s, 3H), 2.50 (m, 2H), 2.32 (d, 1H, J = 16.2 Hz), 2.15
(m, 1H), 2.04 (s, 3H), 1.88 (m, 1H), 1.82 (m, 1H), 1.69 (d, 1H, J =
9.0 Hz), 1.53 (m, 1H), 1.22 (s, 3H), 0.93 (s, 3H). 13C NMR (150
MHz, CDCl3): δ 206.7, 168.5, 151.9, 143.1, 120.9, 118.8, 97.9, 73.5,
72.1, 65.1, 62.6, 58.0, 53.2, 42.7, 41.2, 39.0, 35.0, 32.3, 30.5, 30.2, 20.9,
20.4. HRMS (ESI) calcd for C22H28N2O5S [M + H]+ 433.1792; found
433.1795.
1
Major isomer: H NMR (600 MHz, CDCl3): δ 6.26 (s, 1H), 6.09 (d,
1H, J = 11.4 Hz), 6.00 (br s, 1H), 5.65 (s, 1H), 4.91 (s, 1H), 4.72 (br
s, 1H), 4.31 (m, 2H), 3.97 (d, 1H, J = 10.8 Hz), 3.80 (m, 1H), 3.08 (d,
1H, J = 9.0 Hz), 2.59 (dd, 1H, J = 4.8 Hz, 13.2 Hz), 2.24 (d, 1H, J =
8.4 Hz), 2.12 (m, 1H), 1.90 (m, 1H), 1.65 (m, 1H), 1.43 (m, 1H),
(5aR,6S,7S,7aR,10S,12aS,12bR,15R)-6,7,15-Trihydroxy-2-(iso-
p r o p y l a m i n o ) - 5 , 5 - d i m e t h y l - 9 - m e t h y l e n e -
5,5a,6,7,10,11,12,12a-octahydro-4H-7,12b-(epoxymethano)-
7a,10-methanocyclohepta[7,8]naphtho[1,2-d]thiazol-8(9H)-
G
dx.doi.org/10.1021/jm400367n | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
one (9). Compound 9 (20 mg) was prepared in 44% yield by a
procedure similar to that used to prepare compound 5. The title
compound was obtained as a colorless amorphous gel; [α]D25 +161 (c
7a,10-methanocyclohepta[7,8]naphtho[1,2-d]thiazol-8(9H)-
one (13). Compound 13 (24 mg) was prepared in 51% yield by a
procedure similar to that used to prepare compound 5. The title
compound was obtained as a colorless amorphous gel; [α]D25 +138 (c
1
0.10, CHCl3); HPLC purity 98.0% (tR = 12.3 min). H NMR (600
1
0.10, CHCl3); HPLC purity 98.5% (tR = 10.5 min). H NMR (600
MHz, CDCl3): δ 6.64 (br s, 1H), 6.17 (s, 1H), 5.93 (d, 1H, J = 12.6
Hz), 5.56 (s, 2H), 5.02 (s, 2H), 4.64 (d, 1H, J = 10.2 Hz), 3.89 (d, 1H,
J = 10.2 Hz), 3.79 (dd, 1H, J = 9.6 Hz), 3.47 (m, 1H), 3.06 (d, 1H, J =
9.6 Hz), 2.50 (m, 2H), 2.30 (d, 1H, J = 16.2 Hz), 2.15 (m, 1H), 1.88
(m, 1H), 1.83 (m, 1H), 1.69 (d, 1H, J = 9.6 Hz), 1.53 (m, 1H), 1.25
(s, 3H), 1.24 (s, 3H), 1.23 (s, 3H), 0.95 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 206.7, 166.3, 151.9, 142.6, 120.9, 118.4, 97.9, 73.5, 72.1,
65.1, 62.6, 58.0, 53.3, 48.2, 42.7, 41.2, 39.0, 35.0, 30.6, 30.2, 22.8, 22.6,
21.0, 20.5. HRMS (ESI) calcd for C24H32N2O5S [M + H]+ 461.2105;
found 461.2111.
MHz, CDCl3): δ 6.18 (s, 1H), 6.06 (d, 1H, J = 12.0 Hz), 5.74 (br s,
1H), 5.57 (s, 1H), 5.03 (s, 1H), 4.47 (d, 1H, J = 10.2 Hz), 3.99 (d, 1H,
J = 10.2 Hz), 3.85 (dd, 1H, J = 9.0 Hz, 12.0 Hz), 3.26 (t, 2H, J = 5.4
Hz), 3.05 (d, 1H, J = 9.6 Hz), 2.57 (m, 2H), 2.48 (m, 6H), 2.33 (d,
1H, J = 15.6 Hz), 2.14 (m, 1H), 1.92 (m, 2H), 1.71 (d, 1H, J = 8.4
Hz), 1.59 (m, 5H), 1.46 (m, 2H), 1.27 (s, 3H), 0.99 (s, 3H). 13C
NMR (150 MHz, CDCl3): δ 206.7, 166.1, 151.8, 143.0, 120.9, 119.1,
97.8, 73.5, 72.2, 65.6, 62.6, 58.1, 57.2, 54.4 (2C), 53.2, 42.8, 42.1, 41.0,
39.0, 35.0, 30.5, 30.3, 25.6 (2C), 24.2, 21.0, 20.3. HRMS (ESI) calcd
for C28H39N3O5S [M + H]+ 530.2683; found 530.2687.
(5aR,6S,7S,7aR,10S,12aS,12bR,15R)-2-(Butylamino)-6,7,15-
trihydroxy-5,5-dimethyl-9-methylene-5,5a,6,7,10,11,12,12a-
o c t a h y d r o - 4 H - 7 , 1 2 b - ( e p o x y m e t h a n o ) - 7 a , 1 0 -
methanocyclohepta[7,8]naphtho[1,2-d]thiazol-8(9H)-one (10).
Compound 10 (22 mg) was prepared in 48% yield by a procedure
similar to that used to prepare compound 5. The title compound was
(5aR,6S,7S,7aR,10S,12aS,12bR,15R)-2-(Allylamino)-6,7,15-tri-
hydroxy-5,5-dimethyl-9-methylene-5,5a,6,7,10,11,12,12a-oc-
t a h y d r o - 4 H - 7 , 1 2 b - ( e p o x y m e t h a n o ) - 7 a , 1 0 -
methanocyclohepta[7,8]naphtho[1,2-d]thiazol-8(9H)-one (14).
Compound 14 (21 mg) was prepared in 45% yield by a procedure
similar to that used to prepare compound 5. The title compound was
25
obtained as a colorless amorphous gel; [α]D +142 (c 0.10, CHCl3);
25
HPLC purity 98.0% (tR = 13.7 min). 1H NMR (600 MHz, CDCl3): δ
6.80 (br s, 1H), 6.17 (s, 1H), 5.94 (d, 1H, J = 12.0 Hz), 5.74 (br s,
1H), 5.56 (s, 1H), 5.02 (s, 1H), 4.64 (d, 1H, J = 10.2 Hz), 3.88 (d, 1H,
J = 10.2 Hz), 3.78 (m, 1H), 3.10 (d, 1H, J = 4.2 Hz), 3.05 (d, 1H, J =
9.0 Hz), 2.50 (m, 2H), 2.31 (d, 1H, J = 16.2 Hz), 2.15 (m, 1H), 1.88
(dd, 1H, J = 4.8 Hz, 13.8 Hz), 1.81 (m, 1H), 1.69 (d, 1H, J = 9.6 Hz),
1.61 (m, 2H), 1.54 (m, 1H), 1.39 (m, 2H), 1.24 (s, 3H), 0.93 (m, 6H).
13C NMR (150 MHz, CDCl3): δ 206.8, 167.6, 151.9, 142.8, 120.9,
obtained as a colorless amorphous gel; [α]D +85 (c 0.10, CHCl3);
HPLC purity 98.5% (tR = 11.8 min). 1H NMR (600 MHz, CDCl3): δ
6.30 (br s, 1H), 6.17 (s, 1H), 5.98 (d, 1H, J = 11.4 Hz), 5.88 (m, 1H),
5.67 (br s, 1H), 5.56 (s, 1H), 5.27 (d, 1H, J = 16.8 Hz), 5.18 (d, 1H, J
= 9.6 Hz), 5.02 (s, 1H), 4.91 (br s, 1H), 4.57 (d, 1H, J = 10.2 Hz),
3.91 (d, 1H, J = 10.2 Hz), 3.80 (m, 3H), 3.05 (d, 1H, J = 9.6 Hz), 2.48
(m, 2H), 2.31 (d, 1H, J = 15.6 Hz), 2.13 (m, 1H), 1.86 (m, 2H), 1.69
(d, 1H, J = 9.0 Hz), 1.54 (m, 1H), 1.24 (s, 3H), 0.95 (s, 3H). 13C
NMR (150 MHz, CDCl3): δ 206.7, 166.7, 151.8, 142.8, 133.7, 121.0,
119.3, 117.2, 97.9, 73.5, 72.1, 65.2, 62.6, 58.0, 53.2, 48.4, 42.7, 41.1,
39.0, 35.0, 30.5, 30.2, 21.0, 20.4. HRMS (ESI) calcd for C24H30N2O5S
[M + H]+ 459.1948; found 459.1952.
118.4, 97.9, 73.5, 72.1, 65.1, 62.6, 58.1, 53.3, 46.2, 42.7, 41.2, 39.0,
35.0, 31.3, 30.5, 30.2, 20.9, 20.5, 20.1, 13.8. HRMS (ESI) calcd for
C25H34N2O5S [M + H]+ 475.2261; found 475.2264.
(6S,7S,7aR,10R,12bR,15R)-2-(Cyclohexylamino)-6,7,15-trihy-
droxy-5,5-dimethyl-9-methylene-5,5a,6,7,10,11,12,12a-octa-
hydro-4H-7,12b-(epoxymethano)-7a,10-methanocyclohepta-
[7,8]naphtho[1,2-d]thiazol-8(9H)-one (11). Compound 11 (24
mg) was prepared in 52% yield by a procedure similar to that used to
prepare compound 5. The title compound was obtained as a colorless
amorphous gel; [α] 25D +132 (c 0.10, CHCl3); HPLC purity 98.9% (tR
= 22.2 min). 1H NMR (600 MHz, CDCl3): δ 6.84 (br s, 1H), 6.19 (s,
1H), 5.95 (d, 1H, J = 12.0 Hz), 5.74 (br s, 1H), 5.58 (s, 1H), 5.04 (s,
2H), 4.67 (m, 1H), 3.89 (d, 1H, J = 10.2 Hz), 3.80 (m, 1H), 3.08 (d,
2H, J = 9.0 Hz), 2.52 (m, 2H), 2.31 (d, 1H, J = 9.6 Hz), 2.19 (m, 1H),
2.07 (m, 1H), 2.02 (m, 1H), 1.89 (dd, 1H, J = 4.8 Hz, 13.8 Hz), 1.80
(m, 2H), 1.71 (d, 1H, J = 9.0 Hz), 1.65 (d, 1H, J = 12.6 Hz), 1.56 (m,
1H), 1.28 (m, 6H), 1.27 (s, 3H), 0.97 (s, 3H). 13C NMR (150 MHz,
CDCl3): δ 206.7, 166.5, 152.0, 142.6, 120.8, 118.1, 97.9, 73.5, 72.1,
65.0, 62.6, 58.1, 55.7, 53.3, 42.7, 41.2, 39.1, 35.0, 32.9 (2C), 30.5, 30.2,
29.6, 25.5, 25.0, 21.0, 20.4. HRMS (ESI) calcd for C27H36N2O5S [M +
H]+ 501.2418; found 501.2423.
N-((6S,7S,7aR,10R,12bR,15R)-6,7,15-Trihydroxy-5,5-dimeth-
yl-9-methylene-8-oxo-5,5a,6,7,8,9,10,11,12,12a-decahydro-
4H-7,12b-(epoxymethano)-7a,10-methanocyclohepta[7,8]-
naphtho[1,2-d]thiazol-2-yl)acetamide (15). Compound 15 (17
mg) was prepared in 36% yield by a procedure similar to that used to
prepare compound 5. The title compound was obtained as a colorless
amorphous gel; [α]D25 +152 (c 0.10, CHCl3); HPLC purity 99.2% (tR
= 13.5 min). 1H NMR (600 MHz, CDCl3): δ 10.29 (s, 1H), 7.96 (br s,
1H), 6.23 (d, 1H, J = 12.6 Hz), 6.22 (s, 1H), 5.63 (s, 1H), 5.17 (s,
1H), 5.09 (s, 1H), 4.96 (d, 1H, J = 10.2 Hz), 3.76 (m, 2H), 3.12 (d,
1H, J = 9.6 Hz), 2.56 (m, 2H), 2.45 (d, 1H, J = 15.6 Hz), 2.30 (s, 3H),
2.24 (m, 1H), 1.93 (dd, 1H, J = 3.6 Hz, 13.8 Hz), 1.79 (d, 1H, J = 9.0
Hz), 1.73 (br s, 1H), 1.57 (m, 2H), 1.26 (s, 3H), 0.82 (s, 3H). 13C
NMR (150 MHz, CDCl3): δ 206.8, 168.9, 156.6, 151.5, 140.8, 125.9,
121.7, 98.0, 73.8, 72.0, 64.4, 62.4, 58.1, 52.9, 42.6, 41.0, 38.6, 34.9,
30.4, 29.9, 23.0, 20.7, 20.6. HRMS (ESI) calcd for C23H28N2O6S [M +
H]+ 461.1741; found 461.1747.
1-((5aR,6S,7S,7aR,10S,12aS,12bR,15R)-6,7,15-Trihydroxy-
5,5-dimethyl-9-methylene-8-oxo-5,5a,6,7,8,9,10,11,12,12a-
d e c a h y d r o - 4 H - 7 , 1 2 b - ( e p o x y m e t h a n o ) - 7 a , 1 0 -
methanocyclohepta[7,8]naphtho[1,2-d]thiazol-2-yl)guanidine
(16). Compound 16 (23 mg) was prepared in 50% yield by a
procedure similar to that used to prepare compound 5. The title
(6S,7S,7aR,10R,12bR,15R)-2-(Azepan-1-yl)-6,7,15-trihy-
droxy-5,5-dimethyl-9-methylene-5,5a,6,7,10,11,12,12a-octa-
hydro-4H-7,12b-(epoxymethano)-7a,10-methanocyclohepta-
[7,8]naphtho[1,2-d]thiazol-8(9H)-one (12). Compound 12 (19
mg) was prepared in 41% yield by a procedure similar to that used to
prepare compound 5. The title compound was obtained as a colorless
amorphous gel; [α]D25 +148 (c 0.10, CHCl3); HPLC purity 97.8% (tR
25
compound was obtained as a colorless solid; [α]D +108 (c 0.10,
CHCl3/CH3OH = 4:1); HPLC purity 95.1% (tR = 5.7 min). 1H NMR
(600 MHz,CDCl3/CD3OD = 5:1): δ 6.19 (s, 1H), 5.62 (s, 1H), 5.06
(s, 1H), 4.44 (d, 1H, J = 9.6 Hz), 4.00 (d, 1H, J = 9.0 Hz), 3.85 (d, 1H,
J = 8.4 Hz), 3.05 (d, 1H, J = 9.0 Hz), 2.66 (d, 1H, J = 16.2 Hz), 2.55
(m, 2H), 2.24 (m, 1H), 2.09 (dd, 1H, J = 4.8 Hz, 13.2 Hz), 1.81 (d,
1H, J = 8.4 Hz), 1.72 (m, 1H), 1.62 (m, 1H), 1.30 (s, 3H), 1.01 (s,
3H). 13C NMR (150 MHz, CDCl3/CD3OD = 5:1): δ 206.7, 156.5,
154.4, 151.1, 142.9, 126.6, 120.9, 97.5, 72.5, 64.8, 61.7, 58.8, 52.5, 43.1,
40.2, 38.0, 34.7, 30.0, 29.7, 29.3, 20.1, 19.6. HRMS (ESI) calcd for
C22H28N4O5S [M + H]+ 461.1853; found 461.1856.
1
= 24.3 min). H NMR (600 MHz, CDCl3): δ 6.17 (s, 1H), 6.05 (d,
1H, J = 12.0 Hz), 5.56 (s, 1H), 5.18 (br s, 1H), 5.01 (d, 1H, J = 1.2
Hz), 4.71 (s, 1H), 4.44 (dd, 1H, J = 1.2 Hz, 10.2 Hz), 4.00 (dd, 1H, J =
10.2 Hz), 3.86 (dd, 1H, J = 9.0 Hz, 12.0 Hz), 3.49 (m, 2H), 3.39 (m,
2H), 3.04 (d, 1H, J = 9.6 Hz), 3.48 (m, 2H), 2.32 (d, 1H, J = 15.6 Hz),
2.05 (m, 2H), 1.91 (m, 1H), 1.72 (m, 6H), 1.55 (m, 5H), 1.25 (s, 3H),
0.99 (s, 3H). 13C NMR (150 MHz, CDCl3): δ 206.7, 166.3, 151.7,
143.3, 121.1, 117.6, 97.8, 73.6, 72.2, 65.8, 62.7, 57.6, 53.2, 50.2 (2C),
42.7, 40.9, 38.8, 35.0, 30.4, 29.6, 27.9 (2C), 27.7 (2C), 21.1, 20.2.
HRMS (ESI) calcd for C27H36N2O5S [M + H]+ 501.2418; found
501.2422.
In Vitro Determination of Effects of Synthesized Com-
pounds on Cancer Cell Proliferation. Cancer cells (breast cancer
cell lines MCF-7 and MDA-MB-231, pancreatic cancer cell lines
AsPC-1 and Panc-1, as well as the prostate cancer cell line DU145)
(6S,7S,7aR,10R,12bR,15R)-6,7,15-Trihydroxy-5,5-dimethyl-9-
m e t h y l e n e - 2 - ( ( 2 - ( p i p e r i d i n - 1 - y l ) e t h y l ) a m i n o ) -
5,5a,6,7,10,11,12,12a-octahydro-4H-7,12b(epoxymethano)-
H
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were seeded in 96-well plates at a density of 1 × 104 cells/well and
treated with DMSO, 0.01, 0.1, 1, 5, 10, and 100 μM of individual
compound for 48 h. Proliferation was measured by treating cells with
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) in a CellTiter 96t AQueous Non-Radioactive Cell
Proliferation Assay kit (Promega, Madison, WI, USA). Absorbance
of all wells was determined by measuring OD at 550 nm after 1 h
incubation at 37 °C on a 96-well iMark microplate absorbance reader
(BioRad, Hercules, CA). Each individual compound was tested in
quadruplicate wells for each concentration.
Determination of Aqueous Solubility. Aqueous solubility for 7,
8 (HCl salt), 14 (HCl salt), and 1 was determined by HPLC analysis
according to a previously published protocol.43 First, 2−4 mg of 7, 8
(HCl salt), 14 (HCl salt), and oridonin were weighed and added to 1
mL of water, respectively. The suspensions were shaken at 25 °C for
24 h and then centrifuged, and the supernatants were filtered. Aliquots
(10 μL) of the supernatants were injected into the HPLC system
equipped with a C18 reverse-phase column under the same condition
which was described in the general Experimental Section. One-point
calibration44 was done by injecting 10 μL aliquots of the
corresponding buffer solutions of 7, 8 (free base), 14 (free base), or
1 with known concentrations.
Cell Apoptosis Assay. Breast cancer MDA-MB-231 cells were
incubated in 6-well plates (2.5 × 105 cells/well). Cells were then
treated with DMSO, oridonin, or new compounds at different
concentrations for 48 h, and then both adherent and floating cells
were collected, washed once with PBS. Resuspended cells were
incubated with 100 μL of PBS containing 1% BSA and 100 μL of
Annexin V and dead cell detection reagent at room temperature for 20
min. Apoptosis was measured immediately using the Muse cell
analyzer with the Muse apoptosis kit (catalogue no. MCH100105).
Western Blot Analysis. Breast cancer MDA-MB-231 cells were
treated with DMSO, oridonin, or compound 14, respectively. After 48
h of treatment, cells were harvested and lysed. Protein concentrations
were quantified by the method of Bradford with bovine serum albumin
as the standard. Equal amounts of total cellular protein extract (30 μg)
was separated by electrophoresis on SDS-polyacrylamide gels and
transferred to PVDF membranes. After blocking with 5% nonfat milk,
the membrane was incubated with the desired primary antibody
overnight at the following dilution: anti-Bcl-2 (1:200), anti-Bax
(1:1000), anti-PARP (1:10000), anti-NF-κB (1:2000), and β-actin
(1:20000). Subsequently, the membrane was incubated with
appropriate secondary antibody. The immunoreactive bands were
visualized by enhanced chemiluminescence as recommended by the
manufacturer.
ASSOCIATED CONTENT
■
S
* Supporting Information
Growth inhibitory effects of adriamycin (ADR) on MCF-7 and
1
ADR-resistant MCF-7 cells. H and 13C NMR spectra for the
compounds described in this paper. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*For J.Z.: phone, 409-772-9748; fax, 409-772-9818; E-mail,
jizhou@utmb.edu. For Q.S.: phone, 713-834-6357; fax, 713-
834-6350; E-mail, qshen@mdanderson.org.
■
Author Contributions
C.D. and Y.Z. contributed equally to this work. The manuscript
was written through contributions of all authors. All authors
have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants P50CA097007,
P30DA028821, R21MH093844 (J.Z.) from the National
Institutes of Health, R. A. Welch Foundation Chemistry and
Biology Collaborative Grant from Gulf Coast Consortia (GCC)
for Chemical Genomics, John Sealy Memorial Endowment
Fund (J.Z.), and Startup Fund from MD Anderson Cancer
Center (Q.S.). We thank Dr. Tianzhi Wang at the NMR core
facility of UTMB for the NMR spectroscopy assistance.
ABBREVIATIONS USED
■
SFDA, State Food and Drug Administration; SAR, structure−
activity relationships; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; IC50, half-maximal inhibitory
concentration; PI, propidium iodide; HRMS, high-resolution
mass spectrometry; HPLC, high-performance liquid chroma-
tography; TFA, trifluoroacetic acid; DMSO, dimethyl sulfoxide;
TLC, thin layer chromatography; NMR, nuclear magnetic
resonance; TMS, tetramethylsilane; THF, tetrahydrofuran;
EtOAc, ethyl acetate; p-Ts, 4-toluenesulfonyl; Py, pyridine;
PBS, phosphate-buffered saline; BCA, bicinchoninic acid; BSA,
bovine serum albumin; SDS, sodium dodecyl sulfate; PVDF,
polyvinylidene difluoride
In Vivo Antitumor Activity Determination. All procedures
including mice and in vivo experiments were approved by the
Institutional Animal Care and Use Committee (IACUC) of UT M.D.
Anderson Cancer Center (MDACC). Nineteen female nude mice
were obtained from MDACC and were used for orthotopic tumor
studies at 4−6 weeks of age. The mice were maintained in a barrier
unit with 12 h light−dark switch. Freshly harvested MDA-MB-231
cells (2.5 × 106 cells per mouse, resuspended in 100 μL of PBS) were
injected into the third mammary fat pad of the mice and then
randomly assigned into three groups. The mice were treated daily with
5 mg/kg of compound 14, oridonin, or vehicle through intraperitoneal
injection, when the tumor volume reached 200 mm3. All drugs were
dissolved in 50% DMSO with 50% polyethylene glycol for in vivo
administration. Body weights and tumors volume were measured daily,
and tumor volume was calculated according to the formula V = 0.5 × L
× W2, where L = length (mm) and W = width (mm).
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