Design, synthesis and anticancer activity evaluation of novel C14 heterocycle substituted epi-triptolide

Article abstract of DOI:10.1016/j.ejmech.2013.11.044

Two series of novel C14 heterocycle substituted epi-triptolide derivatives as potential anticancer agents were synthesized and tested for their cytotoxicity against SKOV-3 and PC-3 tumor cell lines. The introduction of C14β-aryl heterocycle aminomethyl su

Full text of DOI:10.1016/j.ejmech.2013.11.044

European Journal of Medicinal Chemistry 73 (2014) 46e55
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Design, synthesis and anticancer activity evaluation of novel C14
heterocycle substituted epi-triptolide
*
Hongtao Xu, Huanyu Tang, Huijin Feng, Yuanchao Li
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhangjiang Hi-Tech Park, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of novel C14 heterocycle substituted epi-triptolide derivatives as potential anticancer agents
were synthesized and tested for their cytotoxicity against SKOV-3 and PC-3 tumor cell lines. The
Received 17 April 2013
Received in revised form
31 July 2013
Accepted 25 November 2013
Available online 14 December 2013
introduction of C14
be an effective modification method to retain the potent anticancer activity. Meanwhile, the series of epi-
triptolide derivatives (21e40) with C14 -hydroxyl group, still retained the natural product’s cytotoxicity.
This is apparently challenges the classical structureeactivity relationship of triptolide that considers the
C14 -hydroxyl group to be essential for its anticancer activity.
Ó 2013 Elsevier Masson SAS. All rights reserved.
b-aryl heterocycle aminomethyl substituent to the leading compound was found to
a
b
Keywords:
Triptolide
Heterocycle
Synthesis
Anticancer
SKOV-3
PC-3
1. Introduction
epoxide and C14
ing its antitumor activity [2], and the configuration of the C14-
hydroxyl group should be orientation. Based on this principle,
for a long time, the aim of C14 modification was limited to
improve the natural compound’s water-solubility by carboxylation
of C14-hydroxyl group with water-solubility enhancing fragments.
For example, C14-succinyl triptolide sodium salt (4, Fig. 1) and
minnelide (5, Fig. 1) are both prodrugs that will convert to trip-
tolide in serum and retain all the toxicity of triptolide [21,22].
However, Takaya and Li group respectively found that substitution
b-hydroxyl group might play a key role in exert-
Triptolide (1, Fig. 1), a diterpenoid triepoxide, was originally
isolated from extracts of Tripterygium wilfordii Hook F (TWHF) [1].
It possesses potent antitumor, antiinflammatory, immunosup-
pressive, and antifertility activities [2e23]. At the cellular level,
triptolide shows strong antiproliferative activity, and it inhibits
the proliferation of all 60 cancer cell lines of US National Cancer
Institute with IC50 values in a low nanomolar range (average
IC50 ¼ 12 nM). Meanwhile, triptolide interferes with a number of
b
transcription factors including NF-
k
B, HSP70, p53, NF-AT and HSF-
of C14b-hydroxyl group with b-fluoride ((14R)-fluorotriptolide (6),
1 at the molecular level [8,19,23]. It also inhibits the growth of
xenograft models formed by different solid tumor cells [11].
Compared with some conventional chemotherapeutic drugs,
triptolide has similar or even superior anticancer activity, espe-
cially over p53 mutated or multi-drug resistant cells [14]. All the
studies mentioned above greatly support its potential develop-
ment as an antineoplastic agent. However, its clinical develop-
ment has been hindered by its severe toxicity and poor water
solubility.
Fig. 1) and a chiral epoxide group ((14S)-14, 21-epoxytriptolide (7),
Fig. 1) could also retain the cytotoxicity of the natural compound
[24e26]. So, the SAR of triptolide cannot be explained as simply as
before.
The introduction of nitrogen is usually a useful tactic in the
natural product modification. In fact, the synthetic drugs generally
contain more nitrogen than the natural products, because nitrogen
can carry a positive charge and act as a hydrogen bond acceptor or
donor that strongly influence the interaction between the medici-
nal agent and its target molecule [27e29]. In addition, the pKa
values of amines are often in the range of physiological pH, which is
essential for improving the bioavailability of drugs [30]. Moreover,
N-heterocycles are ubiquitous in a variety of natural products and
biologically active molecules [31], and they have been assigned as
privileged structures in drug development, because N-heterocyclic
Previous studies on the structureeactivity relationship (SAR) of
triptolide indicated that the hydrogen-bond between C9,C11-
* Corresponding author. Tel./fax: þ86 21 50807288.
E-mail address: ycli@mail.shcnc.ac.cn (Y. Li).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.044

H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
47
Fig. 1. Structure of triptolide, epi-triptolide, triptonide, 14-succinyl triptolide sodium salt, (14R)-fluorotriptolide, minnelide, and (14S)-14,21-epoxytriptolide.
moieties often exhibit improved solubility and can facilitate the salt
formation property, both of which are important for oral absorption
and bioavailability [32]. Based on these principle, we designed two
series of novel C14 heterocycle substituted epi-triptolide de-
rivatives by introducing of 1H-1,2,3-triazolylmethyl substituents
(compounds 17e19, Scheme 1) and aryl heterocycle aminomethyl
substituents (compounds 21e42, Schemes 2 and 3). Their anti-
cancer activities were evaluated by ovary (SKOV-3) and prostate
(PC-3) tumor cell lines.
2. Chemistry
The synthetic strategy followed for the preparation of the ana-
logs 17e19 and 21e40 is depicted in Schemes 1e3. We used trip-
tonide (3), which was extracted from TWHF of our region, as the
starting material. Treatment of 3 with (isopropoxy-dimethylsilyl)
methylmagnesium chloride in THF, and subsequently Tamao
oxidation yielded a 1:2 mixture of triol 10 (28%) and diol 11 (59%)
[25,33]. With diol 11 in hand, we then switched to the synthesis of
Scheme 1. Synthesis of compounds 17e19. Reagents and conditions: (a) (isopropoxy-dimethylsilyl)methyl chloride, Mg, THF, reflux 45 min; (b) KF, KHCO3, 30% H2O2, 0 ꢀC, 2 h, 10
(28% over 2 steps), 11 (59% over 2 steps); (c) Ph3P, DEAD, DPPA, CH2Cl2, rt, 8 h, decomposition of 11; (d) (1) SOCl2, Et3N, CH2Cl2, 0 ꢀC to rt, 2 h; (2) NaIO4, RuCl3$3H2O, CH3CN/
H2O ¼ 4:1, rt, 15 min, 86%; (e) NaN3, DMF, rt to 100 ꢀC, decomposition of 13; (f) MsCl, TEA, CH2Cl2, 0 ꢀC to rt, 1.5 h, 97%; (g) K2CO3, CH3OH, rt, 20 min, 90% or NaN3, THF, rt, 30 min,
91%; (h) NaN3, NH4Cl, 2-methoxyethanol, 80 ꢀC, 48 h, 16 (53%) and 12 (27%); (i) alkyne, sodium ascorbate, CuSO4$5H2O, t-BuOHeH2O 1:1, 50 ꢀC. Ph3P ¼ Triphenylphosphane,
DEAD ¼ diethyl diazenedicarboxylate, DPPA ¼ diphenylphosphoryl azide.

48
H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
Scheme 2. Synthesis of derivatives 21e40. Reagents and conditions: (a) TEMPO, Trichloroisocyanuric acid, CH2Cl2, 0 ꢀC, 1 h, 81%; (b) NH2R, NaBH(OAc)3, CH2Cl2, rt; TEMPO ¼ 2,2,6,6-
tetramethylpiperidine-1-oxyl.
the key azide compound 12. Unfortunately, the transformation of
diol 11 to azide 12 was found to be problematic. Initially, diol 11
treated directly with Ph3P, DEAD and DPPA failed to yield the
desired azide 12. Only decomposition of starting material was
observed under these conditions. Next, transformation of diol 11 to
the corresponding cyclic sulfate 13, followed by nucleophilic azide
reaction, also failed to produce the desired product. Further, we
tried the selective mesylation of the primary alcohol in 11, followed
by reaction with NaN3, but none of the desired azide 12 was yiel-
ded. Instead, the intramolecular cyclization product (14S)-14,21-
epoxytriptolide 7 was afforded in high yield (91%). From an in-
spection of a molecular model of 7, it appeared that the nucleophilic
substitution reaction may occur at the less-hindered C12,C13-
epoxide and C14,C21-epoxide of 7. Fortunately, upon reaction of 7
with azide sodium in 2-methoxyethanol at 80 ꢀC, a 1:2 mixture of
C12-azide 16 (53%) and C21-azide 12 (27%) were produced. Sub-
sequently, reaction of 12 with various alkyne produced 1H-1,2,3-
triazole analogs 17e19 in moderate to high yield. Then the focus
was shifted to the synthesis of aryl heterocycle aminomethyl sub-
stituent analogs 21e40. After surveying a variety of oxidation
conditions, including Dess-Martin periodinane, TPAP/NMO, PCC,
IBX, we determined that TEMPO/trichloroacetonitrile system was
the most effective condition for oxidizing diol 11 to the corre-
sponding aldehyde 20 (97%). Subsequently, the reductive amina-
tion of compound 20 with a variety of aryl heterocycle amine
yielded analogs 21e40. Further, protection of the C14-hydroxy
group and C21-amino group of compounds 25 and 29 with meth-
ylene group produced C21-amino group conformation bonding
compounds 41 and 42 in high yield.
3. Results and discussion
As shown above, we obtained two series of novel C14 hetero-
cycle substituted epi-triptolide analogs. To examine whether the
substitution affected their biological activities, we evaluated the
anticancer effects of these target compounds (17e19 and 21e42)
against two human tumor cell lines derived from ovary (SKOV-3)
and prostate (PC-3) using sulforhodamine B (SRB) assays [34]. The
result revealed that although there was no free C14
b-hydroxyl
group in our new derivatives, the series of C14 -aryl heterocycle
b
aminomethyl substituted analogs 21e40 were all effective against
these two cell lines, with IC50 values ranging from 11.7 to 2807 nM
(Table 1). Among them, compound 33 exhibited the highest po-
tency, with the lowest IC50 value (11.7 nM for SKOV-3 cells).
Generally, electron rich aryl heterocycle amines substituted analogs
were more potent than electron deficient aryl heterocycle amines
substituted analogs, and as for the indazole aminomethyl
substituted analogs 23e31, there were no significant substituent
effect. Moreover, the conformation bonding compounds 41 and 42,
both of which were impossible to form the similar hydrogen bond
as that of triptolide, still retained the cytotoxic activity, albeit a little
less potent. This indicated that the hydrogen bond of C21-amino
Scheme 3. Synthesis of derivatives 41 and 42. Reagents and conditions: (a) 37% aqueous formaldehyde, 30% aqueous acetic acid, acetonitrile, rt.

H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
49
Table 1
In vitro anticancer activity of triptolide derivatives in SKOV-3, and PC-3 cells.
Compound
R
IC50 (nM)
SKOV-3
Compound
R
IC50 (nM)
SKOV-3
PC-3
PC-3
1
e
6
20
29
36.4
36.6
2
7
e
e
790
100
1320
270
30
31
77
99
40.8
66.1
17
>10,000
>10,000
32
38.4
54.1
84.9
18
19
>10,000
>10,000
>10,000
>10,000
33
34
11.7
85
452
21
745.4
2807
35
368.7
157
22
23
53.8
17.4
97.8
36
37
274
781
353
25.3
87.2
24
49
188
38
367.9
341
172
25
26
27
15
22.3
102
132
97
39
40
41
32.1
12.5
79.7
67.9
188
e
e
365
253
28
35.8
264
42
152

50
H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
group and C9,C11-epoxide might not play a critical role in retaining
the potent anticancer activity. But, unfortunately, C14 -1H-1,2,3-
triazolylmethyl substituted analogs 17e19 all lost their cytotox-
icity against PC-3 and SKOV-3 cell lines. On the whole, our result
showed that the introduction of C14b-aryl heterocycle amino-
methyl substituent to the leading compound was an effective
modification method to retain the potent anticancer activity.
Meanwhile, our result apparently challenges the classical struc-
few minutes. After stirring at 0 ꢀC for 2 h, the mixture was
quenched with a saturated NH4Cl solution and extracted with
EtOAc. The organic layer was washed with brine, dried over anhy-
drous Na2SO4, and concentrated under reduced pressure to give a
single adduct. To the stirred mixture of colorless crude product,
MeOH (5.0 mL), THF (8.0 mL), KHCO3 (416 mg, 4.2 mmol), and KF
(464 mg, 4.9 mmol) was added H2O2 (30%, 1.1 mL, 9.71 mmol)
dropwise at room temperature. The mixture was stirred at room
temperature until starting material disappeared. Aqueous sat.
Na2S2O3 solution (50%) was added slowly to the mixture and stirred
until a negative starch/iodide test was observed. The mixture was
extracted with EtOAc. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. The crude product was pu-
rified by chromatography on silica gel using ethyl acetate/cyclo-
hexane (1/3 to 1/2) to provide 10 as a white solid in 28% yield
(113 mg, 0.28 mmol) and 11 as a white solid in 59% yield (230 mg,
0.59 mmol).
b
tureeactivity relationship of triptolide (1) that considers the C14
hydroxyl group to be essential for its anticancer activity [2]. And
these indicate that the C14 -hydroxyl group of triptolide is not
b-
b
unchangeable even in order to generate analogs with potent anti-
cancer activity.
4. Conclusions
Two series of novel derivatives of triptolide as potential anti-
cancer agents were synthesized and tested for their cytotoxicity
10: 1H NMR (CDCl3, 300 MHz)
d
7.10 (d, J ¼ 1.7 Hz, 1H), 4.51 (s,
against two human tumor cell lines. The introduction of C14
b
-
1H), 4.24 (dd, J ¼ 11.6, 4.5 Hz, 1H), 3.84 (d, J ¼ 3.2 Hz, 1H), 3.81 (d,
J ¼ 5.6 Hz, 1H), 3.66 (dd, J ¼ 11.5, 8.8 Hz, 1H), 3.47 (d, J ¼ 3.2 Hz, 1H),
3.22 (s, 1H), 2.98 (dd, J ¼ 8.9, 4.7 Hz, 1H), 2.70 (ddd, J ¼ 12.2, 6.4,
1.4 Hz, 1H), 2.59 (dd, J ¼ 16.9, 4.7 Hz, 1H), 2.52e2.35 (m, 3H), 1.91
(dd, J ¼ 15.3, 12.4 Hz, 1H), 1.72 (s, 1H), 1.50 (dd, J ¼ 12.3, 4.0 Hz, 1H),
1.25e1.16 (m, 1H), 1.04 (s, 3H), 0.95e0.87 (m, 6H); 13C NMR (CDCl3,
heterocycle aminomethyl substituent to the natural product was
found to be an effective modification method to retain the potent
anticancer activity. Meanwhile, in our study, the series of epi-
triptolide derivatives (21e40) with C14a-hydroxyl group, still
retained the natural product’s cytotoxicity. This will add new SAR to
triptolide. And also, by introducing of nitrogen atom, the water
solubility of leading compound may be improved and also facilitate
the salt formation property, both of which are important for oral
100 MHz) d 147.7, 136.7, 125.3, 116.7, 74.7, 67.6, 65.9, 65.1, 64.8, 57.2,
56.0, 55.6, 54.6, 37.4, 36.0, 31.0, 25.8, 25.5, 20.8, 18.6, 15.7, 13.0. MS
(EI, 70 eV) m/z (%) 404 (Mþ, 90), 387 (33), 357 (72), 71 (100), 55
absorption and bioavailability. Furthermore, C14
b
-aryl heterocycle
(69); HRMS (EI) calcd. for
404.1833.
C
22H28O7 (Mþ): 404.1835, found
aminomethyl substituent analogs (21e40) generally possess strong
fluorescence, which makes the series possible as molecular fluo-
rescent probes to study the mechanism of triptolide with its target
molecule.
11: 1H NMR (CDCl3, 300 MHz)
d
4.67 (s, 2H), 4.26 (d, J ¼ 11.8 Hz,
1H), 3.87e3.80 (m, 2H), 3.64 (d, J ¼ 11.5 Hz, 1H), 3.46 (d, J ¼ 3.3 Hz,
1H), 2.76e2.64 (m, 1H), 2.45 (sept, J ¼ 6.9 Hz, 1H), 2.37e2.25 (m,
1H), 2.23e2.04 (m, 2H), 1.89 (t, J ¼ 14.1 Hz, 1H), 1.55 (dd, J ¼ 12.6,
5.2 Hz, 1H), 1.25e1.13 (m, 1H), 1.07 (s, 3H), 0.91 (d, J ¼ 6.9 Hz, 3H),
5. Experimental
0.89 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 173.2, 160.2,
5.1. Chemistry
125.4, 74.4, 70.0, 67.5, 65.3, 65.2, 65.0, 56.5, 56.1, 54.4, 40.3, 36.0,
30.1, 25.5, 23.4, 20.9, 18.6, 17.1, 13.7. MS (EI, 70 eV) m/z (%) 391,
([M þ H]þ, 2), 372 (1), 71 (100). HRMS (EI) calcd. for C22H28O7 (Mþ):
390.1679, found 390.1677. These assignments matched with those
previously reported by our group [25].
5.1.1. General
Mass spectra were obtained on Finnigan MAT 95 (EI), Thermo-
DFS (EI) or Finnigan MAT 900 (ESI) spectrometers, high resolution
mass spectra on a Finnigan MAT 95 (HR-EIMS) or on a Finnigan MAT
900 (HR-ESI-MS). 1H and 13C NMR spectra were determined on
Bruker AM-300, Bruker AM-400 instruments using tetramethylsi-
lane as internal reference. Data are presented as follows: chemical
shift, multiplicity (s ¼ singlet, brs ¼ broad singlet, d ¼ doublet,
brd ¼ broad doublet, t ¼ triplet, m ¼ multiplet), J ¼ coupling
constant in hertz (Hz). The signals of the 13C NMR were assigned
utilizing DEPT experiments and on the basis of literature data. Silica
gel 60H (200e300 mesh) manufactured by Qingdao Haiyang
Chemical Group Co. (China) was used for general chromatography.
5.1.3. (14S)-14-Spiro-14a, 21-sulfonyldioxytriptolide (13)
To a solution of compound 11 (78 mg, 0.2 mmol) in anhydrous
CH2Cl2 (6.0 mL) was added dry Et3N (0.21 mL, 1.6 mmol) dropwise.
The mixture was then cooled to 0 ꢀC. Under Ar atmosphere, SOCl2
(0.3 mL, 1.2 mmol) was added to the mixture carefully. After being
stirred for 2 h, the mixture was quenched with water and extracted
with CH2Cl2. The organic layer was washed with water and brine
and dried over anhydrous Na2SO4. After removal of the solvent
under reduced pressure, the crude product was dissolved in CH3CN
(8.0 mL), then NaIO4 (80 mg, 0.184 mmol), RuCl3$3H2O (12 mg,
0.056 mmol) and H2O (2.0 mL) were added. The mixture was stirred
at room temperature for 15 min. After removal of the solvent under
reduced pressure, the residue was diluted with ethyl acetate and
washed with water and brine and dried over anhydrous Na2SO4.
The crude product was purified by chromatography on silica gel
using acetate/cyclohexane (1/4) to provide 13 as a white solid in
5.1.2. (3bS,5S,5aS,6S,7S,8S,9S,9aS,9bS)-(5,5a)b,(7,8)b,(9,9a)a-
Tris(epoxy)-6-hydroxyl-7-isopropyl-9b-methyl-3b,4,5,5a,6,7,8,9,9a,
9b,10,11-dodecahydrophenanthro[2,1-c]furan-1,6-dimethanol (10)
and (14S)-14
b
-(hydroxymethyl)epitriptolide (11)
portion of a solution of (iso-
Under Ar atmosphere,
a
propoxydimethylsilyl)methyl chloride (0.72 mL, 4.0 mmol) in
anhydrous THF (7.0 mL) was added to Mg turnings (108 mg,
4.5 mmol). To the stirred mixture was added a few drops of 1,2-
dibromoethane at 60 ꢀC and an exothermic reaction started in
several minutes. The remaining solution was added dropwise over
5 min. After the addition was completed, the gray mixture was
refluxed for 45 min and then cooled to 0 ꢀC. A solution of triptonide
3 (358 mg, 1.0 mmol) in anhydrous THF (10.0 mL) was added to the
Grignard reagent (freshly prepared) at the same temperature over a
86% yield (78 mg, 0.17 mmol). 1H NMR (CDCl3, 300 MHz)
d 4.92 (d,
J ¼ 10.0 Hz, 1H), 4.72e4.63 (m, 3H), 3.87 (d, J ¼ 2.9 Hz, 1H), 3.83 (d,
J ¼ 5.6 Hz,1H), 3.65 (d, J ¼ 3.0 Hz,1H), 2.79e2.68 (m,1H), 2.51 (sept,
J ¼ 6.8 Hz, 1H), 2.39e2.07 (m, 3H), 1.98 (t, J ¼ 14.2 Hz, 1H), 1.53 (dd,
J ¼ 12.6, 5.2 Hz, 1H), 1.29e1.14 (m, 1H), 1.09 (s, 3H), 0.99 (d,
J ¼ 6.8 Hz, 3H), 0.97 (d, J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
172.9, 159.4, 125.6, 91.3, 73.4, 69.9, 66.3, 65.2, 61.9, 58.3, 55.7, 55.5,
40.3, 35.7, 30.2, 25.5, 23.2, 20.4, 18.3, 17.0, 13.4; ESIMS m/z 453.5

H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
51
[M þ H]þ; HRMS (ESI) calcd. for C21H25SO9 [M þ H]þ 453.1219,
found 453.1221. These assignments matched with those previously
reported by our group [25].
29.7, 25.4, 23.5, 20.7, 18.5, 17.0, 13.4; MS (EI, 70 eV) m/z (%) 416 (Mþ,
3), 387 (2), 372 (16), 243 (100), 71 (96); HRMS (EI) calcd. for
C
21H25N3O6 (Mþ): 415.1743, found 415.1739.
16: 1H NMR (CDCl3, 300 MHz)
d
4.70 (s, 2H), 3.90 (d, J ¼ 5.7 Hz,
5.1.4. (14S)-14
b-(Methylsulfonyloxy)epitriptolide (15)
1H), 3.77 (d, J ¼ 5.7, 1H), 3.45 (d, J ¼ 5.7, 1H), 2.90e2.51 (m, 3H), 2.32
(d, J ¼ 14.7 Hz, 1H), 2.20e2.07 (m, 2H), 1.98e1.89 (m, 2H), 1.76e1.64
(m, 2H), 1.34e1.28 (m, 1H), 1.07 (s, 3H), 0.97 (d, J ¼ 6.6 Hz, 3H), 0.93
Compound 11 (0.50 g, 1.3 mmol) was dissolved in dry pyridine
(4.0 mL, 49.3 mmol). The solution was cooled to 0 ꢀC, then MsCl
(0.61 mL, 7.7 mmol) was added to the solution dropwise and stirred
for 10 min at room temperature. After removal of the solvent under
reduced pressure, the residue was diluted with water, then
extracted with ethyl acetate, washed with brine, and dried with
anhydrous Na2SO4. After concentration, the residue was purified by
chromatography by ethyl acetate/cyclohexane (1/2) to provide 15
as a white solid in 97% yield (0.61 g, 1.3 mmol). 1H NMR (CDCl3,
(d, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 173.6, 161.2, 124.6,
75.4, 70.1, 66.3, 60.6, 58.9, 57.7, 57.4, 57.1, 47.7, 39.5, 34.9, 30.4, 27.7,
22.6, 17.2, 16.7, 16.3, 13.7; MS (EI, 70 eV) m/z (%) 416 (Mþ, 2), 387 (3),
372 (19), 71 (100), 55 (31); HRMS (EI) calcd. for C21H25N3O6 (Mþ):
415.1743, found 415.1736.
5.1.7. General procedure for the synthesis of compounds (17e19)
A solution of the azide 12 (0.1 mmol), the alkyne (0.11 mmol),
1 M aq CuSO4$5H2O (0.01 mmol) and 1 M aq. sodium ascorbate
(0.01 mmol) in a t-BuOHeH2O 1:1 mixture (3.0 mL) was heated at
50 ꢀC until TLC showed no traces of the starting azide. The solvent
was removed under reduced pressure, the residue was dispersed in
EtOAc (10 mL) and the solution was washed with H2O (3 ꢁ 15 mL),
dried over MgSO4, concentrated and purified by chromatography
on silica gel to give the target analog.
300 MHz)
d
4.70e4.57 (m, 4H), 3.80 (d, J ¼ 3.2 Hz, 1H), 3.77 (d,
J ¼ 5.6 Hz, 1H), 3.47 (d, J ¼ 3.2 Hz, 1H), 3.10 (s, 3H), 2.77e2.67 (m,
1H), 2.54 (sept, J ¼ 6.9 Hz, 1H), 2.37e2.26 (m, 1H), 2.25e2.06 (m,
1H), 1.91 (dd, J ¼ 14.8, 13.4 Hz, 1H), 1.54 (dd, J ¼ 12.9, 5.1 Hz, 1H),
1.28e1.13 (m, 2H), 1.07 (s, 3H), 0.92 (d, J ¼ 7.7 Hz, 3H), 0.89 (d,
J ¼ 7.7 Hz, 3H). 13C NMR (CDCl3, 100 MHz)
d 173.2, 159.4, 125.6, 91.3,
73.4, 69.9, 66.3, 65.2, 61.9, 58.3, 55.7, 55.5, 40.3, 35.7, 30.2, 25.5,
23.2, 20.4, 18.3, 17.0, 13.4; ESIMS m/z 469.5 [M þ H]þ; HRMS (ESI)
calcd. for C22H29SO9 [M þ H]þ 469.1532, found 469.1534. These
assignments matched with those previously reported by our group
[25].
5.1.7.1. (14S)-14
triptolide (17). 17 (white solid, 26 mg, 55%), 1H NMR (CDCl3,
300 MHz)
b-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)methylepi-
d
7.35 (s, 1H), 5.12 (d, J ¼ 13.8 Hz, 1H), 4.65 (d, J ¼ 13.8 Hz,
5.1.5. (14S)-14, 21-Epoxytriptolide (7)
1H), 4.63 (s, 2H), 3.86 (d, J ¼ 3.0 Hz, 1H), 3.71 (s, 1H), 3.52 (d,
J ¼ 3.0 Hz,1H), 3.39 (d, J ¼ 5.7 Hz,1H), 2.72e2.67 (m, 1H), 2.56 (sept,
J ¼ 6.9 Hz, 1H), 2.32 (d, J ¼ 16.5 Hz, 1H), 2.24e1.94 (m, 5H), 1.68 (t,
J ¼ 13.8 Hz, 1H), 1.58 (dd, J ¼ 12.3 Hz, J ¼ 4.8 Hz, 1H), 1.21e1.06 (m,
To a solution of 15 (504 mg, 1.1 mmol) in CH3OH (20.0 mL) was
added K2CO3 (1.33 g, 9.6 mmol). The mixture was stirred for 20 min
under room temperature. After removal of the solvent under
reduced pressure, the residue was diluted with water and then
extracted with ethyl acetate, washed with brine, and dried with
anhydrous Na2SO4. The crude product was purified via chroma-
tography on silica gel using ethyl acetate/cyclohexane (1/4) to give
7 as a white solid in 90% yield (368 mg, 0.99 mmol). 1H NMR (CDCl3,
3H), 0.98 (s, 3H), 0.97 (d, J ¼ 4.5 Hz, 3H), 0.95 (d, J ¼ 4.8 Hz, 3H); 13
C
NMR (CDCl3, 125 MHz) d 173.2, 160.2, 150.5, 125.3, 122.4, 75.1, 69.9,
67.8, 65.6, 61.4, 56.0, 55.5, 53.6, 53.3, 40.5, 36.1, 29.6, 25.7, 23.3, 20.6,
18.6, 17.1, 13.3, 7.8 ꢁ 2, 6.6; MS (EI, 70 eV) m/z (%) 481 (Mþ, 8), 359
(3), 337 (6), 94 (95), 59 (100); HRMS (EI) calcd. for C26H31N3O6
(Mþ): 481.2213, found 481.2207.
300 MHz)
d
4.67 (s, 2H), 3.88 (d, J ¼ 2.9 Hz, 1H), 3.52 (d, J ¼ 3.1 Hz,
1H), 3.40 (d, J ¼ 5.5 Hz, 1H), 2.84 (d, J ¼ 5.2 Hz, 1H), 2.80e2.69 (m,
2H), 2.37e2.25 (m, 1H), 2.21e2.05 (m, 2H), 1.91e1.80 (m, 2H), 1.57
(dd, J ¼ 12.4, 4.9 Hz, 1H), 1.29e1.17 (m, 1H), 1.06 (s, 3H), 0.86 (d,
J ¼ 6.8 Hz, 3H), 0.80 (d, J ¼ 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
5.1.7.2. (14S)-14
methylepitriptolide (18). 18 (white solid, 36 mg, 72%). 1H NMR
(CDCl3, 300 MHz)
b-(4-(Methoxycarbonyl)-1H-1,2,3-triazol-1-yl)
d
8.25 (s, 1H), 5.24 (d, J ¼ 13.8 Hz, 1H), 4.78 (d,
d
173.2, 160.2, 125.2, 69.9, 65.2, 65.0, 58.4, 56.8, 55.9, 55.6, 54.1, 47.9,
J ¼ 13.8 Hz, 1H), 4.67e4.54 (m, 2H), 3.93 (s, 3H), 3.89 (d, J ¼ 3.0 Hz,
1H), 3.54 (d, J ¼ 3.0 Hz, 1H), 3.39e3.35 (m, 2H), 2.70e2.66 (m, 1H),
2.53 (sept, J ¼ 6.9 Hz,1H), 2.15e2.06 (m, 2H),1.75e1.65 (m, 2H),1.57
(dd, J ¼ 12.3 Hz, J ¼ 4.8 Hz, 1H), 1.19e1.14 (m, 1H), 0.97 (s, 3H), 0.95
40.4, 35.6, 30.2, 23.4, 23.2, 19.8, 17.6, 17.0, 13.5; ESIMS m/z 373.4
[M þ H]þ; HRMS (ESI) calcd. for C22H25O6 [M þ H]þ 373.1651, found
373.1658. These assignments matched with those previously re-
ported by our group [25].
(s, 3H), 0.93 (s, 3H); 13C NMR (CDCl3, 125 MHz)
d 173.1, 161.0, 160.0,
139.9, 130.1, 125.3, 75.2, 69.8, 67.7, 65.5, 61.5, 56.9, 55.6, 53.7, 53.6,
52.2, 40.4, 36.0, 29.5, 25.8, 23.2, 20.4, 18.6, 17.0, 13.2; MS (EI, 70 eV)
m/z (%) 499 (Mþ, 3), 468 (7), 141 (89), 83 (100), 82 (37); HRMS (EI)
calcd. for C25H29N3O8 (Mþ): 499.1955, found 499.1950.
5.1.6. (14S)-14
12 -azido-13 -hydroxyl-14, 21-epoxytriptolide (16)
To stirred solution of 7 (372 mg, 1.0 mmol) in 2-methoxyethanol
b-Azidomethylepitriptolid (12) and (12R,13R,14S)-
b
a
(20.0 mL), H2O (2 mL), NaN3 (260 mg, 4.0 mmol), NH4Cl (107 mg,
2.0 mmol) were added, and the mixture was stirred at 80 ꢀC for
24 h, then cooled to room temperature. After removal of the solvent
under reduced pressure, the residue was diluted with water and
then extracted with ethyl acetate, washed with brine, and dried
with anhydrous Na2SO4. The crude product was purified via chro-
matography on silica gel using ethyl acetate/cyclohexane (1/8 to 1/
4) to give 12 as a white solid in 27% yield (112 mg, 0.27 mmol) and
16 as a white solid in 53% yield (220 mg, 0.53 mmol).
5.1.7.3. (14S)-14
triptolide (19). 19 (white solid, 45 mg, 89%), 1H NMR (CDCl3,
400 MHz)
7.93 (s, 1H), 7.84 (d, J ¼ 7.6 Hz, 2H), 7.44e7.40 (m, 2H),
b-(4-Phenyl-1H-1,2,3-triazol-1-yl)methylepi-
d
7.35e7.31 (m, 1H), 5.26 (d, J ¼ 13.6 Hz, 1H), 4.77 (d, J ¼ 14.0 Hz, 1H),
4.65e4.55 (m, 2H), 3.90 (d, J ¼ 3.2 Hz, 1H), 3.68 (s, 1H), 3.55 (d,
J ¼ 3.2 Hz, 1H), 3.41 (d, J ¼ 5.6 Hz, 1H), 2.71e2.67 (m, 1H), 2.65e2.58
(sept, J ¼ 6.8 Hz, 1H), 2.31 (d, J ¼ 13.8 Hz, 1H), 2.15e1.99 (m, 2H),
1.68 (t, J ¼ 14.0 Hz, 1H), 1.60 (dd, J ¼ 12.4 Hz, J ¼ 4.8 Hz, 1H), 1.22e
1.18 (m, 1H), 1.00 (d, J ¼ 2.6 Hz, 3H), 0.98 (d, J ¼ 2.9 Hz, 3H), 0.97 (s,
12: 1H NMR (CDCl3, 300 MHz)
d
4.68 (s, 2H), 4.10 (d, J ¼ 13.2,1H),
3.80e3.77 (m, 2H), 3.62 (d, J ¼ 13.2, 1H), 3.47 (d, J ¼ 13.2, 1H), 2.75e
2.57 (m, 2H), 2.44 (sept, J ¼ 6.9 Hz, 1H), 2.31 (d, J ¼ 13.8 Hz, 1H),
2.24e2.15 (m, 2H), 1.93 (t, J ¼ 14.4 Hz, 1H), 1.55 (dd, J ¼ 13.1 Hz,
J ¼ 5.1 Hz, 1H), 1.21e1.17 (m, 1H), 1.10 (s, 3H), 0.91 (d, J ¼ 6.9 Hz, 3H),
3H); 13C NMR (CDCl3, 125 MHz)
d
173.2, 160.2, 147.8, 130.4 ꢁ 2,
128.9, 128.2, 125.6 ꢁ 2, 125.2, 122.4, 75.2, 69.9, 67.8, 65.7, 61.5, 56.5,
55.6, 53.7, 53.5, 40.5, 36.1, 29.6, 25.8, 23.2, 20.5, 18.6, 17.1, 13.4; MS
(EI, 70 eV) m/z (%) 517 (Mþ, 36), 500 (8), 460 (24), 105 (100), 59 (47);
HRMS (EI) calcd. for C29H31N3O6 (Mþ): 517.2213, found 517.2204.
0.90 (d, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3, 125 MHz)
d 173.3, 160.4,
125.2, 74.4, 69.9, 68.0, 65.1, 63.1, 56.3, 56.0, 55.4, 54.3, 40.1, 35.9,

52
H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
5.1.8. (14S)-14
b
-Formylepitriptolid (20)
J ¼ 14.1 Hz, 1H), 1.57 (dd, J ¼ 12.9, 5.1 Hz, 1H), 1.22e1.18 (m, 1H), 1.11
(s, 3H), 1.02 (d, J ¼ 6.9 Hz, 3H), 0.95 (d, J ¼ 6.9 Hz, 3H); 13C NMR
To a stirred solution of 11 (420 mg, 1.08 mmol) in dichloro-
methane (15.0 mL) at 0 ꢀC, trichloroisocyanuric acid (376 mg,
1.62 mmol) and TEMPO (16 mg, 0.108 mmol) were added, and the
mixture was stirred at 0 ꢀC for 30 min, saturated Na2CO3 solution
(1.0 mL) was added to quench the reaction. Then extracted with
dichloromethane, washed with brine, and dried with anhydrous
Na2SO4. The crude product was purified via chromatography on
silica gel using ethyl acetate/cyclohexane (1/3) to give 20 as a white
solid in 81% yield (40 mg, 0.87 mmol). 1H NMR (CDCl3, 300 MHz)
(CDCl3, 100 MHz)
d 173.4, 160.5, 142.5, 135.9, 133.8, 125.3, 123.9,
119.9, 110.7, 102.3, 73.3, 70.0, 68.5, 65.3, 64.7, 56.3, 55.8, 54.6, 51.1,
40.4, 36.0, 30.0, 25.7, 23.5, 21.3, 18.9,17.1, 13.7; MS (EI, 70 eV) m/z (%)
505 (Mþ, 2), 342 (20), 327 (21), 133 (100), 86 (64) 58 (70); HRMS
(EI) calcd. for C28H31N3O6 (Mþ): 505.2213, found 505.2215.
5.1.9.4. (14S)-14
b
-(1H-Indazol-6-ylamino)methylepitriptolide (24).
24 (white solid, 25 mg, 69%), 1H NMR (CDCl3, 300 MHz)
d
10.51 (s,
d
10.03 (s, 1H), 4.76e4.59 (m, 2H), 3.97 (d, J ¼ 3.0 Hz, 1H), 3.91 (s,
1H), 7.88 (s, 1H), 7.51 (d, J ¼ 8.4 Hz, 1H), 6.71 (s, 1H), 6.62 (d, J ¼ 8.7,
1H), 4.71e4.60 (m, 3H), 3.80e3.80 (m, 4H), 3.57e3.48 (m, 3H),
2.76e2.67 (m, 1H), 2.55 (sept, J ¼ 6.6 Hz, 1H), 2.30 (d, J ¼ 17.1 Hz,
1H), 2.16e2.07 (m, 1H), 1.86 (t, J ¼ 13.8 Hz, 1H), 1.55 (dd, J ¼ 12.6,
5.1 Hz, 1H), 1.22e1.19 (m, 1H), 1.08 (s, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H),
1H), 3.75 (d, J ¼ 5.9 Hz, 1H), 3.60 (d, J ¼ 3.0 Hz, 1H), 2.79e2.67 (m,
1H), 2.38e2.26 (m, 1H), 1.87 (dd, J ¼ 14.7, 13.6 Hz, 1H), 1.58 (dd,
J ¼ 12.6, 4.0 Hz, 1H), 1.03 (s, 3H), 0.83 (d, J ¼ 6.9 Hz, 3H), 0.79 (d,
J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 198.3, 173.2, 160.1,
125.4, 81.7, 69.9, 65.9, 65.5, 62.1, 56.3, 55.8, 54.0, 40.4, 36.0, 30.2,
26.6, 23.3, 19.8, 17.3, 17.1, 13.6; MS (EI, 70 eV) m/z (%) 389 ([M þ 1]þ,
4), 388 (Mþ, 1), 343 (6), 327 (52), 299 (88), 71 (100). HRMS (EI)
calcd. for C21H24O7 (Mþ): 388.1522, found 388.1525.
0.93 (d, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 173.3, 160.4,
147.8, 141.8, 134.8, 125.4, 121.5, 117.1, 113.5, 90.4, 73.8, 70.0, 68.4,
65.3, 64.6, 56.3, 55.8, 54.6, 50.0, 40.4, 36.1, 30.0, 25.7, 23.5, 21.1, 18.8,
17.1, 13.7; MS (EI, 70 eV) m/z (%) 505 (Mþ, 3), 342 (7), 145 (60), 133
(81), 55 (100); HRMS (EI) calcd. for C28H31N3O6 (Mþ): 505.2213,
found 505.2211.
5.1.9. General procedure for the synthesis of compounds 21e40
A solution of 20 (0.1 mmol) and the amino (0.12 mmol), in
dichloromethane (3.0 mL) under argon protect was stirred at room
temperature for 30 min, then NaBH(OAc)3 (0.15 mmol)was added
and stirred at the same temperature until TLC showed no traces of
the starting aldehyde 19. The solvent was removed under reduced
pressure, the residue was dispersed in EtOAc (10 mL) and the so-
lution was washed with H2O (3 ꢁ 15 mL), dried over MgSO4 and
concentrated to give a target analog.
5.1.9.5. (14S)-14
triptolide (25). 25 (white solid, 37 mg, 72%), 1H NMR (CDCl3,
300 MHz)
7.82 (s, 1H), 7.26 (d, J ¼ 8.7 Hz, 1H), 6.98e6.93 (m, 2H),
b-(1-Methyl-1H-indazol-5-ylamino)methylepi-
d
4.68 (s, 2H), 4.15 (s, 1H), 4.02 (s, 3H), 3.93 (d, J ¼ 13.2 Hz, 1H), 3.87
(d, J ¼ 5.6 Hz, 1H), 3.83 (d, J ¼ 3.1 Hz, 1H), 3.51 (d, J ¼ 3.0 Hz, 1H),
3.44 (d, J ¼ 13.1 Hz, 1H), 2.71 (brs, 1H), 2.52 (sept, J ¼ 6.8 Hz, 1H),
2.31 (d, J ¼ 14.3 Hz, 1H), 2.24e2.09 (m, 2H), 1.90 (t, J ¼ 14.4 Hz, 1H),
1.56 (dd, J ¼ 12.4, 4.7 Hz, 1H), 1.23e1.16 (m, 1H), 1.10 (s, 3H), 1.01 (d,
J ¼ 6.9 Hz, 3H), 0.95 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
5.1.9.1. (14S)-14
b
-(Pyridin-3-ylamino)methylepitriptolide
(21).
21 (white solid, 30 mg, 65%), 1H NMR (CDCl3, 300 MHz)
d
8.14 (s,
d 173.3, 160.5, 142.1, 135.9, 131.4, 125.3, 124.5, 119.4, 109.8, 102.6,
1H), 8.03 (s, 1H), 7.15 (dd, J ¼ 8.4, 4.8 Hz, 1H), 7.06 (d, J ¼ 8.4 Hz, 1H),
4.68 (s, 2H), 3.83e3.90 (m, 3H), 3.51 (d, J ¼ 3.3 Hz, 1H), 3.46 (d,
J ¼ 13.2 Hz, 1H), 2.72 (d, J ¼ 12.9 Hz, 1H), 2.48 (sept, J ¼ 6.9 Hz, 1H),
2.32 (d, J ¼ 15.6 Hz, 1H), 2.14e2.23 (m, 2H), 1.90 (t, J ¼ 14.1 Hz, 1H),
1.56 (dd, J ¼ 12.3, 4.8 Hz, 1H), 1.23e1.18 (m, 1H), 1.09 (s, 3H), 0.98 (d,
J ¼ 7.2 Hz, 3H), 0.92 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
73.2, 70.0, 68.3, 65.3, 64.7, 56.2, 55.8, 54.5, 51.2, 40.3, 36.0, 35.6,
30.0, 25.7, 23.5, 21.2, 18.8, 17.1, 13.7; ESIMS m/z 520.6 [M þ H]þ;
HRMS (ESI) calcd. for C29H34N3O6 [M þ H]þ 520.2448, found
520.2445.
5.1.9.6. (14S)-14
triptolide (26). 26 (white solid, 26 mg, 50%), 1H NMR (CDCl3,
300 MHz)
b-(2-Methyl-2H-indazol-5-ylamino)methylepi-
d
173.3, 160.3, 144.3, 139.7, 136.8, 125.4, 123.9, 120.6, 73.8, 70.0, 68.1,
65.3, 64.5, 56.2, 55.8, 54.6, 49.6, 40.4, 36.1, 30.0, 25.8, 23.5, 21.1, 18.8,
17.2, 13.7; MS (EI, 70 eV) m/z (%) 466 (Mþ, 3), 107 (100), 78 (14);
HRMS (EI) calcd. for C26H30N2O6 (Mþ): 466.2104, found 466.2095.
d
7.68 (s, 1H), 7.54 (d, J ¼ 9.2 Hz, 1H), 6.85 (dd, J ¼ 9.2,
2.1 Hz,1H), 6.76 (d, J ¼ 2.1 Hz,1H), 4.65 (s, 2H), 4.21 (brs,1H), 4.15 (s,
3H), 4.04 (s, 1H), 3.90e3.84 (m, 2H), 3.82 (d, J ¼ 3.1 Hz, 1H), 3.50 (d,
J ¼ 3.1 Hz,1H), 3.44 (d, J ¼ 13.0 Hz,1H), 2.69 (d, J ¼ 13.1 Hz,1H), 2.52
(sept, J ¼ 6.9 Hz, 1H), 2.29 (d, J ¼ 16.5 Hz, 1H), 2.20e2.09 (m, 2H),
1.87 (t, J ¼ 14.1 Hz 1H), 1.54 (dd, J ¼ 12.4, 4.5 Hz, 1H), 1.19e1.16 (m,
5.1.9.2. (14S)-14
b
-(1H-Indol-4-ylamino)methylepitriptolide
(22).
8.23 (s,
22 (white solid, 40 mg, 80%), 1H NMR (CDCl3, 300 MHz)
d
1H), 7.14 (t, J ¼ 2.7 Hz, 1H), 7.10 (t, J ¼ 8.1 Hz, 1H), 6.93 (d, J ¼ 8.1 Hz,
1H), 6.53 (m, 1H), 6.42 (d, J ¼ 7.5 Hz, 1H), 4.68 (s, 2H), 4.02 (d,
J ¼ 12.9 Hz, 1H), 3.92 (s, 1H), 3.88 (d, J ¼ 5.7 Hz, 1H), 3.84 (d,
J ¼ 3.3 Hz, 1H), 3.66 (d, J ¼ 13.2 Hz, 1H), 3.52 (d, J ¼ 3.3 Hz, 1H), 2.71
(s, 1H), 2.60 (sept, J ¼ 6.9 Hz, 1H), 2.32 (d, J ¼ 14.1 Hz, 1H), 2.14e2.24
(m, 2H), 1.92 (t, J ¼ 13.8 Hz, 1H), 1.57 (dd, J ¼ 12.3, 5.7 Hz, 1H), 1.22e
1.19 (m, 1H), 1.11 (s, 3H), 1.03 (d, J ¼ 6.9 Hz, 3H), 0.94 (d, J ¼ 6.9 Hz,
1H), 1.08 (s, 3H), 1.00 (d, J ¼ 6.9 Hz, 3H), 0.93 (d, J ¼ 6.9 Hz, 3H); 13
C
NMR (CDCl3, 100 MHz)
d 173.3, 160.5, 145.6, 142.4, 125.2, 122.5,
122.0, 121.2, 118.3, 98.4, 73.3, 70.0, 68.4, 65.3, 64.7, 56.2, 55.8, 54.5,
50.4, 40.3, 40.1, 36.0, 30.0, 25.7, 23.4, 21.3, 18.8, 17.1, 13.7; ESIMS m/z
520.6 [M þ H]þ; HRMS (ESI) calcd. for C29H34N3O6 [M þ H]þ
520.2448, found 520.2444.
3H); 13C NMR (CDCl3, 100 MHz)
d
174.0, 161.3, 140.8, 136.3, 124.9,
5.1.9.7. (14S)-14
methylepitriptolide) (27). 27 (white solid, 35 mg, 63%), 1H NMR
(CDCl3, 300 MHz)
8.05e8.02 (m, 2H), 7.04 (dd, J ¼ 9.0 Hz,
b-((1-(Methoxycarbonyl)-1H-indazol-5-ylamino)
122.7, 122.4, 117.8, 102.7, 100.1, 98.0, 73.7, 70.23, 68.4, 65.2, 64.4,
56.1, 55.6, 54.5, 49.2, 40.2, 35.9, 29.7, 25.5, 23.3, 21.0, 18.5, 16.9, 13.5;
MS (EI, 70 eV) m/z (%) 504 (Mþ, 26), 342 (21), 59 (100); HRMS (EI)
calcd. for C29H32N2O6 (Mþ): 504.2260, found 504.2268.
d
J ¼ 2.4 Hz, 1H), 6.97 (d, J ¼ 2.1 Hz, 1H), 4.68 (s, 2H), 4.50e4.48 (m,
1H), 4.10 (s, 3H), 3.87e3.83 (m, 2H), 3.76 (s, 1H), 3.61 (d, J ¼ 7.2 Hz,
1H), 3.51 (d, J ¼ 3.0, 1H), 2.72 (d, J ¼ 12.6 Hz, 1H), 2.50 (sept,
J ¼ 6.6 Hz, 1H), 2.34e2.14 (m, 4H), 1.90 (t, J ¼ 13.5 Hz, 1H), 1.57 (dd,
J ¼ 12.3, 5.4 Hz, 1H), 1.22e1.19 (m, 1H), 1.10 (s, 3H), 1.00 (d,
J ¼ 6.9 Hz, 3H), 0.93 (d, J ¼ 6.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
5.1.9.3. (14S)-14
b
-(1H-Indazol-5-ylamino)methylepitriptolide (23).
10.02 (s,
23 (white solid, 43 mg, 85%), 1H NMR (CDCl3, 300 MHz)
d
1H), 7.94 (s, 1H), 7.36 (d, J ¼ 9.0 Hz, 1H), 7.02 (s, 1H), 6.96 (d, J ¼ 8.7,
1.5 Hz, 1H), 4.69 (s, 2H), 4.21 (dd, J ¼ 10.5, 4.2 Hz, 1H), 4.12 (s, 1H),
3.94 (dd, J ¼ 12.9, 4.2 Hz, 1H), 3.88 (d, J ¼ 5.7, 1H), 3.84 (d, J ¼ 3.0 Hz,
1H), 3.52 (d, J ¼ 3.0, 1H), 3.46 (t, J ¼ 12.6 Hz, 1H), 2.71 (s, 1H), 2.51e
2.61 (m, 1H), 2.32 (d, J ¼ 15.0 Hz, 1H), 2.22e2.12 (m, 2H), 1.91 (t,
d
173.3, 160.4, 151.0, 145.1, 139.7, 134.5, 126.8, 125.3, 120.3, 115.0,
102.4, 73.5, 70.0, 68.2, 65.3, 64.6, 56.2, 55.8, 54.5, 54.3, 50.6, 40.4,
36.1, 30.0, 25.7, 23.5, 21.2, 18.8, 17.1, 13.7; ESIMS m/z 564.6, HRMS
(ESI) calcd. for C30H34N3O8 [M þ H]þ 564.2346, found 564.2351.

H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
53
5.1.9.8. (14S)-14
methylepitriptolide) (28). 28 (white solid, 40 mg, 71%), 1H NMR
(CDCl3, 300 MHz)
b
-((3-(Methoxycarbonyl)-1H-indazol-5-ylamino)
d
8.40 (s, 1H), 6.74e6.71 (m, 2H), 6.63 (dd, J ¼ 8.4, 1.6 Hz, 1H), 4.68
(s, 1H), 4.04 (s, 1H), 3.88e3.82 (m, 3H), 3.50e3.48 (m, 3H), 3.36 (d,
J ¼ 13.3 Hz, 1H), 2.72 (d, J ¼ 13.3 Hz, 1H), 2.47 (sept, J ¼ 6.9 Hz, 1H),
2.32 (m, 1H), 2.22e2.13 (m, 2H), 1.89 (t, J ¼ 13.8 Hz, 1H), 1.55 (dd,
J ¼ 12.3, 4.7 Hz, 1H), 1.19e1.17 (m, 1H), 1.09 (s, 3H), 0.98 (d,
J ¼ 6.9 Hz, 3H), 0.93 (d, J ¼ 6.7 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
11.08 (s, 1H), 7.48 (d, J ¼ 9.0 Hz, 1H), 7.45 (d,
J ¼ 1.8 Hz, 1H), 7.00 (dd, J ¼ 9.0, 2.4 Hz, 1H), 4.69 (s, 2H), 4.47 (d,
J ¼ 5.4 Hz, 1H), 4.03 (s, 3H), 3.97 (d, J ¼ 13.5, 1H), 3.86 (m, 3H), 3.56
(m, 2H), 2.72 (s, 1H), 2.58 (sept, J ¼ 6.9 Hz, 1H), 2.32 (d, J ¼ 13.8 Hz,
1H), 2.20 (m, 2H), 1.93 (t, J ¼ 13.8 Hz, 1H), 1.58 (dd, J ¼ 14.4, 4.5 Hz,
1H), 1.22e1.19 (m, 1H), 1.11 (s, 3H), 1.05 (d, J ¼ 6.9 Hz, 3H), 0.96 (d,
d
177.2, 173.3, 160.4, 143.7, 135.4, 126.6, 125.3, 114.3, 112.8, 110.1,
73.2, 70.0, 68.3, 65.3, 64.6, 56.2, 55.8, 54.6, 51.3, 40.4, 36.5, 36.0,
30.0, 25.7, 23.5, 21.2, 18.8, 17.1, 13.7; MS (EI, 70 eV) m/z (%) 520 (Mþ,
20), 342 (63), 327 (42), 148 (100), 72 (55); HRMS (EI) calcd. for
J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 173.4, 163.6, 160.5,
144.5, 137.1, 134.6, 125.3, 123.7, 120.0, 112.2, 101.9, 73.5, 70.0, 68.4,
65.3, 64.7, 56.3, 55.9, 54.6, 51.9, 50.5, 40.4, 36.1, 30.0, 25.8, 23.50,
21.3,18.9,17.2,13.8; MS (EI, 70 eV) m/z (%) 564 (Mþ, 2), 342 (23), 327
(21), 203 (76), 191 (89), 84 (100); HRMS (EI) calcd. for C30H33N3O8
(Mþ): 563.2268, found 563.2274.
C
29H32N2O7 (Mþ): 520.2210, found 520.2213.
5.1.9.13. (14S)-14
epitriptolide (33). 33 (white solid, 34 mg, 60%), 1H NMR (CDCl3,
300 MHz)
b-(1,3,3-Trimethyl-2-oxoindolin-5-ylamino)methyl-
d
6.72 (d, J ¼ 2.0 Hz, 1H), 6.70e6.67 (m, 2H), 4.68 (s, 2H),
5.1.9.9. (14S)-14
methylepitriptolide (29). 29 (white solid, 50 mg, 81%), 1H NMR
(CDCl3, 300 MHz)
7.82 (s, 1H), 7.28 (d, J ¼ 9.7 Hz, 1H), 6.97e6.90
b
-(1-(2-Morpholinoethyl)-1H-indazol-5-ylamino)
4.07 (s, 2H), 3.90e3.85 (m, 2H), 3.83 (d, J ¼ 3.2 Hz, 1H), 3.50 (d,
J ¼ 3.1 Hz, 1H), 3.37 (d, J ¼ 13.3 Hz, 1H), 3.17 (s, 3H), 2.79e2.66 (m,
1H), 2.48 (sept, J ¼ 6.9 Hz, 1H), 2.30 (d, J ¼ 13.8 Hz, 1H), 2.24e2.09
(m, 2H), 1.90 (t, J ¼ 14.7, 13.6 Hz, 1H), 1.55 (dd, J ¼ 12.4, 4.2 Hz, 1H),
1.34 (s, 6H), 1.22e1.14 (m, 1H), 1.09 (s, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H),
d
(m, 2H), 4.65 (s, 2H), 4.44 (t, J ¼ 6.9 Hz, 2H), 3.89 (d, J ¼ 13.2 Hz, 1H),
3.84 (d, J ¼ 5.6 Hz, 1H), 3.81 (d, J ¼ 3.2 Hz, 1H), 3.67e3.61 (m, 4H),
3.49 (d, J ¼ 3.1 Hz, 1H), 3.43 (d, J ¼ 9.6 Hz, 1H), 2.84 (dd, J ¼ 7.9,
5.9 Hz, 2H), 2.69 (d, J ¼ 11.2 Hz, 1H), 2.56e2.39 (m, 5H), 2.28 (d,
J ¼ 17.6 Hz, 1H), 2.21e2.04 (m, 2H), 1.87 (t, J ¼ 14.4 Hz, 13.6 Hz, 1H),
1.53 (dd, J ¼ 12.5, 4.4 Hz, 1H), 1.20e1.17 (m, 1H), 1.07 (s, 3H), 0.99 (d,
J ¼ 6.9 Hz, 3H), 0.92 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
0.93 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 180.8, 173.3,
160.4, 144.1, 137.1, 135.7, 125.3, 113.3, 111.4, 108.4, 73.1, 70.0, 68.3,
65.3, 64.7, 56.3, 55.8, 54.6, 51.3, 44.5, 40.4, 36.0, 30.0, 26.2, 25.7,
þ
24.4 ꢁ 2, 23.5, 21.2, 18.8, 17.1, 13.7; ESIMS m/z 563.7 [M þ H]
;
HRMS (ESI) calcd. for C32H39N2O7 [M þ H]þ 563.2757, found
d
173.3, 160.5, 142.2, 135.4, 131.9, 125.2, 124.5, 119.4, 109.8, 102.4,
563.2750.
73.2, 69.9, 68.3, 66.7 ꢁ 2, 65.2, 64.6, 57.6, 56.2, 55.8, 54.5, 53.6 ꢁ 2,
51.0, 46.5, 40.29, 36.0, 29.9, 25.6, 23.4, 21.2, 18.8, 17.1, 13.7; ESIMS m/
z 619.7 [M þ H]þ; HRMS (ESI) calcd. for C34H43N4O7 [M þ H]þ
619.3132, found 619.3130.
5.1.9.14. (14S)-14
b
-(Quinolin-6-ylamino)methylepitriptolide
(34).
8.66
34 (light yellow solid, 45 mg, 88%), 1H NMR (CDCl3, 300 MHz)
d
(d, J ¼ 4.2 Hz, 1H), 7.92 (m, 2H), 7.29 (dd, J ¼ 8.4, 4.2 Hz, 1H), 7.20
(dd, J ¼ 9.0, 2.4 Hz, 1H), 6.86 (s, 1H), 4.78 (d, J ¼ 9.3, 1H) 4.67 (s, 2H),
3.95 (dd, J ¼ 13.2, 3.9 Hz, 1H), 3.87 (s, 1H), 3.86 (s, 1H), 3.57 (m, 3H),
2.70 (s,1H), 2.57 (sept, J ¼ 6.9 Hz,1H), 2.32 (d, J ¼ 14.7 Hz,1H), 2.13e
2.22 (m, 2H), 1.91 (t, J ¼ 15.6 Hz, 1H), 1.57 (dd, J ¼ 12.0, 4.8 Hz, 1H),
1.25e1.23 (m, 1H), 1.11 (s, 3H), 1.03 (d, J ¼ 6.9 Hz, 3H), 0.94 (d,
5.1.9.10. (14S)-14
methylepitriptolide (30). 30 (white solid, 37 mg, 60%), 1H NMR
(CDCl3, 300 MHz)
7.79 (s, 1H), 7.57 (d, J ¼ 9.0 Hz, 1H), 6.86 (dd,
b-(2-(2-Morpholinoethyl)-2H-indazol-5-ylamino)
d
J ¼ 9.2, 2.0 Hz, 1H), 6.75 (d, J ¼ 1.8 Hz, 1H), 4.65 (s, 2H), 4.48 (t,
J ¼ 6.3 Hz, 2H), 3.91e3.83 (m, 2H), 3.82 (d, J ¼ 3.2 Hz, 1H), 3.71 (s,
4H), 3.50 (d, J ¼ 3.1 Hz, 1H), 3.47e3.41 (m, 1H), 2.95 (t, J ¼ 6.3 Hz,
2H), 2.67 (brs, 1H), 2.61 (s, 1H), 2.58e2.41 (m, 4H), 2.28 (d,
J ¼ 18.6 Hz, 1H), 2.20e2.08 (m, 2H), 1.87 (t, J ¼ 14.4 Hz, 1H), 1.54 (dd,
J ¼ 12.3, 4.4 Hz, 1H), 1.19e1.16 (m, 1H), 1.08 (s, 3H), 0.99 (d,
J ¼ 6.9 Hz, 3H), 0.92 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 173.3, 160.3, 146.9,
146.0, 143.7, 134.1, 130.4, 129.7, 125.4, 122.0, 121.5, 105.1, 74.0, 70.0,
68.2, 65.3, 64.6, 56.2, 55.8, 54.6, 49.7, 40.4, 36.1, 30.0, 25.8, 23.5,
21.2, 18.8, 17.2, 13.7; MS (EI, 70 eV) m/z (%) 516 (Mþ, 5), 157 (65), 144
(100), 128 (54); HRMS (EI) calcd. for C30H32N2O6 (Mþ): 516.2260,
found 516.2268.
d
173.3, 160.5, 145.3, 142.5, 125.3, 122.1, 121.9, 121.7, 118.3, 98.2, 73.4,
70.0, 68.3, 66.7 ꢁ 2, 65.3, 64.6, 58.5, 56.2, 55.8, 54.5, 53.7 ꢁ 2, 50.6,
50.3, 40.3, 36.0, 30.0, 25.7, 23.4, 21.3,18.8,17.1,13.7; ESIMS m/z 619.7
[M þ H]þ; HRMS (ESI) calcd. for C34H43N4O7 [M þ H]þ 619.3132,
found 619.3128.
5.1.9.15. (14S)-14
b
-(Quinoxalin-6-ylamino)methylepitriptolide (35).
8.65
35 (light yellow solid, 40 mg, 77%), 1H NMR (CDCl3, 300 MHz)
d
(d, J ¼ 1.7 Hz,1H), 8.54 (d, J ¼ 1.7 Hz,1H), 7.84 (d, J ¼ 9.1 Hz,1H), 7.21
(dd, J ¼ 9.1, 2.4 Hz, 1H), 7.04 (d, J ¼ 2.2 Hz, 1H), 5.25 (dd, J ¼ 8.3,
3.2 Hz, 1H), 4.66 (s, 2H), 3.95 (dd, J ¼ 13.1, 3.4 Hz, 1H), 3.87 (d,
J ¼ 3.1 Hz, 1H), 3.84 (d, J ¼ 5.6 Hz, 1H), 3.65 (dd, J ¼ 13.0, 8.6 Hz, 1H),
3.54 (d, J ¼ 2.9 Hz, 1H), 3.29 (brs, 1H), 2.73 (d, J ¼ 14.2 Hz, 1H), 2.58
(sept, J ¼ 6.8 Hz, 1H), 2.30 (d, J ¼ 15.0 Hz, 1H), 2.22e2.09 (m, 2H),
1.90 (t, J ¼ 14.4 Hz, 1H), 1.57 (dd, J ¼ 12.3, 4.6 Hz, 1H), 1.21e1.19 (m,
1H), 1.20 (m, 1H), 1.09 (s, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H), 0.91 (d,
5.1.9.11. (14S)-14
ylamino)methylepitriptolide (31). 31 (white solid, 30 mg, 49%), 1H
NMR (CDCl3, 300 MHz)
b-(1-(2-(Piperidin-1-yl)ethyl)-1H-indazol-5-
d
7.79 (s, 1H), 7.27 (d, J ¼ 8.8 Hz, 1H), 6.93e
6.90 (m, 2H), 4.62 (s, 2H), 4.42 (t, J ¼ 7.2 Hz, 2H), 4.22 (brs, 1H),
3.88e3.83 (m, 2H), 3.78 (d, J ¼ 3.0 Hz, 1H), 3.47 (d, J ¼ 3.0 Hz, 1H),
3.42 (brs, 1H), 2.76 (t, J ¼ 7.2 Hz, 2H), 2.66 (d, J ¼ 12.4 Hz, 1H), 2.51
(sept, J ¼ 13.7, 1H), 2.43e2.40 (m, 4H), 2.25 (d, J ¼ 14.5 Hz, 1H),
2.17e2.06 (m, 2H), 1.83 (t, J ¼ 14.0 Hz, 1H), 1.58e1.46 (m, 5H), 1.42e
1.34 (m, 2H), 1.18e1.11 (m, 1H), 1.05 (s, 3H), 0.97 (d, J ¼ 6.9 Hz, 3H),
J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 173.2, 160.2, 149.2,
145.1, 144.9, 140.8, 138.3, 130.1, 125.40, 123.0, 104.7, 74.5, 70.0, 68.1,
65.3, 64.5, 56.2, 55.8, 54.6, 49.2, 40.3, 36.1, 29.9, 25.8, 23.5, 21.1, 18.7,
17.1, 13.7; ESIMS m/z 518.6 [M þ H]þ; HRMS (ESI) calcd. for
0.91 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d
173.2, 160.4,
C
29H32N3O6 [M þ H]þ 518.2291, found 518.2287.
142.1, 135.3, 131.6, 125.1, 124.4, 119.2, 109.9, 102.2, 73.1, 69.9, 68.2,
65.1, 64.5, 58.0, 56.1, 55.7, 54.6 ꢁ 2, 54.4, 51.0, 46.7, 40.2, 35.9, 29.8,
25.7 ꢁ 2, 25.6, 24.0, 23.3, 21.1, 18.8, 17.0, 13.6; ESIMS m/z 617.7
[M þ H]þ; HRMS (ESI) calcd. for C35H45N4O6 [M þ H]þ 617.3339,
found 617.3335.
5.1.9.16. (14S)-14
b
-(Quinazolin-6-ylamino)methylepitriptolide (36).
9.16
36 (light yellow solid, 36 mg, 69%), 1H NMR (CDCl3, 400 MHz)
d
(s, 1H), 9.06 (s, 1H), 7.82 (d, J ¼ 9.1 Hz, 1H), 7.36 (d, J ¼ 9.0 Hz, 1H),
6.83 (s, 1H), 5.18 (dd, J ¼ 7.8, 3.3 Hz, 1H), 4.67e4.63 (m, 2H), 3.89e
3.82 (m, 3H), 3.62e3.52 (m, 2H), 2.71 (brs, 1H), 2.56 (sept,
J ¼ 6.9 Hz, 1H), 2.31 (d, J ¼ 14.6 Hz, 1H), 2.22e2.15 (m, 2H), 1.91 (t,
J ¼ 14.4 Hz,1H),1.57 (dd, J ¼ 12.2, 4.7 Hz,1H),1.20e1.18 (m,1H),1.09
5.1.9.12. (14S)-14
b-(2-Oxoindolin-5-ylamino)methylepitriptolide
(32). 32 (light yellow solid, 39 mg, 75%), 1H NMR (CDCl3, 300 MHz)

54
H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
(s, 3H), 1.00 (d, J ¼ 6.9 Hz, 3H), 0.92 (d, J ¼ 6.6 Hz, 3H); 13C NMR
327 (17), 162 (100); HRMS (EI) calcd. for C29H31NO8 (Mþ): 521.2050,
found 521.2053.
(CDCl3, 125 MHz)
d 173.2, 160.2, 157.8, 152.1, 147.0, 145.2, 129.1,
126.7, 126.6, 125.4, 102.4, 74.6, 69.9, 68.1, 65.4, 64.4, 56.3, 55.9, 54.6,
49.4, 40.3, 36.1, 29.9, 25.8, 23.5, 21.0, 18.7, 17.1, 13.7; ESIMS m/z 518.6
[M þ H]þ; HRMS (ESI) calcd. for C29H32N3O6 [M þ H]þ 518.2291,
found 518.2293.
5.1.10. General procedure for the synthesis of compounds 41 and 42
A solution of the amine (0.1 mmol), 37% aqueous formaldehyde
(1.0 mmol) and 30% aqueous acetic acid (1.0 mmol) in acetonitrile a
(3.0 mL) was stirred at room temperature until TLC showed no
traces of the starting amine. Then the solvent was removed under
reduced pressure, the residue was dispersed in EtOAc (10 mL) and
the solution was washed with H2O (3 ꢁ 15 mL), dried over MgSO4,
concentrated and purified by chromatography on silica gel to give
the target analog.
5.1.9.17. (14S)-14
triptolide (37). 37 (white solid, 38 mg, 72%), 1H NMR (CDCl3,
300 MHz)
7.61 (d, J ¼ 9.6 Hz, 1H), 7.19 (d, J ¼ 9.0 Hz, 1H), 6.96 (dd,
b-(2-Oxo-2H-chromen-6-ylamino)methylepi-
d
J ¼ 9.0 Hz, 2.7 Hz, 1H), 6.78 (d, 2.7 Hz, 1H), 6.38 (d, J ¼ 9.6, 1H), 4.68
(s, 2H), 4.46 (dd, J ¼ 9.6 Hz, 4.2 Hz, 1H), 3.91e3.84 (m, 3H), 3.62e
3.57 (m, 3H), 3.52 (d, J ¼ 3.3 Hz, 1H), 2.69 (d, J ¼ 12.9 Hz, 1H), 2.49
(sept, J ¼ 6.9 Hz,1H), 2.34e2.07 (m, 2H),1.90 (t, J ¼ 14.4 Hz,1H),1.56
(dd, J ¼ 12.3, 4.8 Hz, 1H), 1.21e1.18 (m, 1H), 1.06 (s, 3H), 0.99 (d,
J ¼ 6.9 Hz, 3H), 0.93 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
5.1.10.1. (14S)-30-(1-Methyl-1H-indazol-5-yl)spiro[triptolide-14,50-
oxazolidine](41). 41 (white solid, 50 mg, 95%), 1H NMR (300 MHz,
CDCl3)
d
7.82 (s, 1H), 7.30 (d, J ¼ 9.0 Hz, 1H), 6.83 (dd, J ¼ 9.0, 1.9 Hz,
d
173.3, 161.1, 160.3, 147.6, 144.9, 143.3, 125.3, 119.5, 119.2, 117.5,
1H), 6.75 (d, J ¼ 1.9 Hz, 1H), 5.21 (s, 1H), 4.65 (s, 2H), 4.57 (s, 1H),
4.02 (s, 3H), 3.93 (d, J ¼ 5.5 Hz, 1H), 3.81 (d, J ¼ 3.1 Hz, 1H), 3.76 (d,
J ¼ 9.9 Hz, 1H), 3.57e3.48 (m, 2H), 2.72 (brd, J ¼ 12.7 Hz, 1H), 2.48
(sept, J ¼ 6.9 Hz, 1H), 2.29 (brd, J ¼ 17.5 Hz, 1H), 2.21e2.05 (m, 2H),
1.86 (t, J ¼ 10.4 Hz, 1H), 1.54 (dd, J ¼ 12.4, 4.7 Hz, 1H), 1.27e1.13 (m,
116.9, 110.6, 73.7, 70.0, 68.2, 65.3, 64.5, 56.3, 55.8, 54.5, 50.5, 40.3,
36.0, 30.0, 25.7, 23.4, 21.1, 18.8, 17.1, 13.7; MS (EI, 70 eV) m/z (%) 533
(Mþ, 3), 342 (11), 133 (100), 77 (52); HRMS (EI) calcd. for C30H31NO8
(Mþ): 533.2050, found 533.2044.
1H), 1.04 (s, 3H), 0.97 (d, J ¼ 6.9 Hz, 3H), 0.88 (d, J ¼ 6.9 Hz, 3H); 13
C
5.1.9.18. (14S)-14
triptolide (38). 38 (white solid, 23 mg, 43%), 1H NMR (CDCl3,
300 MHz)
b-(4-Oxo-4H-chromen-6-ylamino)methylepi-
NMR (100 MHz, CDCl3)
d 173.2, 160.3, 139.6, 135.2, 131.4, 125.2,
124.6, 116.4, 109.7, 102.2, 85.5, 84.3, 69.9, 68.0, 65.1, 64.6, 57.8, 55.8,
55.5, 55.0, 40.5, 35.7, 35.5, 30.0, 25.6, 23.6, 20.6, 18.7, 17.0, 13.4;
ESIMS m/z 532.6 [M þ H]þ; HRMS (ESI) calcd. for C30H34N3O6
[M þ H]þ 532.2448, found 532.2442.
d
7.80 (d, J ¼ 5.9 Hz, 1H), 7.36 (d, J ¼ 2.7 Hz, 1H), 7.32 (d,
J ¼ 9.0 Hz, 1H), 7.11 (dd, J ¼ 9.0, 2.7 Hz, 1H), 6.28 (d, J ¼ 5.9 Hz, 1H),
4.79 (d, J ¼ 5.5 Hz, 1H), 4.67 (s, 2H), 3.90 (dd, J ¼ 13.0, 3.9 Hz, 1H),
3.85e3.83 (m, 2H), 3.56e3.49 (m, 2H), 2.79e2.69 (m, 2H), 2.52
(sept, J ¼ 6.9 Hz, 1H), 2.31 (d, J ¼ 16.3 Hz, 1H), 2.23e2.09 (m, 2H),
1.89 (t, J ¼ 14.7 Hz, 1H), 1.56 (dd, J ¼ 11.6, 5.0 Hz, 1H), 1.20e1.18 (m,
5.1.10.2. (14S)-30-(1-(2-Morpholinoethyl)-1H-indazol-5-yl)spiro
[triptolide-14,50-oxazolidine](42). 42 (white solid, 58 mg, 92%), 1H
1H), 1.08 (s, 3H), 0.99 (d, J ¼ 6.9 Hz, 3H), 0.92 (d, J ¼ 6.9 Hz, 3H); 13
C
NMR (300 MHz, CDCl3)
d
7.83 (s, 1H), 7.33 (d, J ¼ 9.0 Hz, 1H), 6.82
NMR (CDCl3, 100 MHz)
d 177.6, 173.3, 160.4, 154.9, 150.4, 145.6,
(dd, J ¼ 9.0, 2.1 Hz, 1H), 6.73 (d, J ¼ 1.7 Hz, 1H), 5.20 (s, 1H), 4.65 (s,
2H), 4.56 (s, 1H), 4.45 (t, J ¼ 6.9 Hz, 2H), 3.93 (d, J ¼ 5.5 Hz, 1H), 3.80
(d, J ¼ 3.2 Hz, 1H), 3.75 (d, J ¼ 9.9 Hz, 1H), 3.66e3.60 (m, 4H), 3.54e
3.50 (m, 2H), 2.82 (t, J ¼ 6.9 Hz, 2H), 2.72 (brd, J ¼ 12.8 Hz, 1H),
2.52e2.41 (m, 5H), 2.28 (brd, J ¼ 18.1 Hz, 1H), 2.21e2.01 (m, 2H),
1.91e1.79 (m, 1H), 1.53 (dd, J ¼ 12.5, 4.6 Hz, 1H), 1.23e1.14 (m, 1H),
1.03 (s, 3H), 0.96 (d, J ¼ 6.9 Hz, 3H), 0.87 (d, J ¼ 6.9 Hz, 3H); 13C NMR
125.5, 125.3, 122.4, 119.1, 111.9, 105.8, 74.0, 70.0, 68.2, 65.3, 64.5,
56.2, 55.8, 54.6, 49.9, 40.3, 36.0, 29.9, 25.7, 23.4, 21.0, 18.8, 17.1, 13.8;
MS (EI, 70 eV) m/z (%) 533 (Mþ, 7), 342 (23), 327 (25), 189 (53), 161
(100), 79 (49); HRMS (EI) calcd. for C30H31NO8 (Mþ): 533.2050,
found 533.2059.
5.1.9.19. (14S)-14
(39). 39 (light yellow solid, 29 mg, 55%), 1H NMR (CDCl3, 300 MHz)
7.21 (d, J ¼ 2.4 Hz, 1H), 6.98 (dd, J ¼ 8.8, 2.6 Hz, 1H), 6.86 (d,
b-(4-Oxochroman-6-ylamino)methylepitriptolide
(100 MHz, CDCl3)
d 173.2, 160.3, 139.6, 134.8, 131.8, 125.2, 124.7,
116.4, 109.8, 102.3, 85.5, 84.3, 69.9, 68.0, 66.8 ꢁ 2, 65.1, 64.6, 57.8,
57.6, 55.7, 55.5, 55.0, 53.6 ꢁ 2, 46.6, 40.5, 35.7, 30.0, 25.6, 23.6, 20.6,
18.7, 17.0, 13.4; ESIMS m/z 631.7 [M þ H]þ; HRMS (ESI) calcd. for
d
J ¼ 8.8 Hz, 1H), 4.67 (s, 2H), 4.47 (t, J ¼ 6.4 Hz, 2H), 4.21 (dd, J ¼ 10.1,
3.8 Hz, 1H), 3.90e3.83 (m, 4H), 3.50 (d, J ¼ 2.7 Hz, 1H), 3.44e3.30
(m, 1H), 2.80e2.59 (m, 3H), 2.47 (sept, J ¼ 6.9 Hz, 1H), 2.30 (d,
J ¼ 16.9 Hz, 1H), 2.22e2.13 (m, 2H), 1.89 (t, J ¼ 14.1 Hz, 1H), 1.55 (dd,
J ¼ 12.3, 4.6 Hz, 1H), 1.22e1.16 (m, 1H), 1.08 (s, 3H), 0.98 (d,
J ¼ 6.9 Hz, 3H), 0.93 (d, J ¼ 6.9 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
C
35H43N4O7 [M þ H]þ 631.3132, found 631.3135.
5.2. Biology
5.2.1. Cell lines and cell culture
d
192.0, 173.3, 160.4, 155.8, 142.2, 125.3, 124.8, 121.3, 118.8, 110.1,
Human prostate cancer PC-3, and ovarian cancer SKOV-3 cell
lines were from Japanese Foundation of Cancer Research (Tokyo,
Japan). These cell lines were maintained in RPMI 1640 medium
(Gibco, Grand Island, NE) supplemented with 10% (v/v) heat-
inactivated fetal bovine serum (Gibco), 2 mmol/L glutamine,
73.4, 70.0, 68.2, 67.0, 65.2, 64.6, 56.2, 55.8, 54.6, 50.6, 40.3, 37.8,
36.0, 30.0, 25.7, 23.4, 21.1, 18.8, 17.1, 13.7; MS (EI, 70 eV) m/z (%) 535
(Mþ, 4), 342 (25), 327 (25), 175 (100), 163 (93), 79 (74); HRMS (EI)
calcd. for C30H33NO8 (Mþ): 535.2206, found 535.2204.
100 IU/mL penicillin, 100 mg/mL streptomycin, and 10 mmol/L HEPS
5.1.9.20. (14S)-14
ylepitriptolide (40). 40 (white solid, 26 mg, 50%), 1H NMR (CDCl3,
300 MHz)
b-(2-Oxo-2,3-dihydrobenzofuran-5-ylamino)meth-
(pH 7.4) in a humidified atmosphere of 95% air with 5% CO2 at 37 ꢀC.
d
6.93 (d, J ¼ 8.6 Hz, 1H), 6.72 (s, 1H), 6.67 (dd, J ¼ 8.6,
2.5 Hz, 1H), 4.67 (s, 2H), 4.23 (brs, 1H), 3.92e3.73 (m, 4H), 3.68 (s,
2H), 3.49 (d, J ¼ 3.1 Hz, 1H), 3.38 (d, J ¼ 13.3 Hz, 1H), 2.72 (d,
J ¼ 13.3 Hz, 1H), 2.46 (sept, J ¼ 6.9 Hz, 1H), 2.29 (d, J ¼ 14.0 Hz, 1H),
2.23e2.09 (m, 2H), 1.89 (dd, J ¼ 14.7, 13.6 Hz, 1H), 1.55 (dd, J ¼ 12.5,
4.2 Hz, 1H), 1.21e1.16 (m, 1H), 1.08 (s, 3H), 0.97 (d, J ¼ 7.0 Hz, 3H),
5.2.2. Cytotoxicity assays
The cytotoxicity of triptolide derivatives was examined using a
panel of human tumor cell lines with the methods described before
[35]. Briefly, cells in 96-well plates were treated in triplicate with
gradient concentrations of tested agents at 37 ꢀC for 72 h, then
assessed by the sulforhodamine B (Sigma) assay. The cytotoxicity
was expressed as an IC50, defined as the concentration required for
50% inhibition of cell growth compared with control cells and
calculated with the logit method.
0.92 (d, J ¼ 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz)
d 174.4, 173.3,
160.4, 147.9, 145.0, 125.3, 124.0, 114.9, 111.3, 111.1, 73.3, 70.0, 68.2,
65.3, 64.6, 56.2, 55.8, 54.5, 51.0, 40.3, 36.0, 33.5, 30.0, 25.7, 23.4,
21.2, 18.8, 17.1, 13.7; MS (EI, 70 eV) m/z (%) 521 (Mþ, 36), 342 (19),

H. Xu et al. / European Journal of Medicinal Chemistry 73 (2014) 46e55
55
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