Synthesis and anticancer activity of novel rapamycin C-28 containing triazole moiety compounds

Article abstract of DOI:10.1002/ardp.201800123

Rapamycin is an mTOR allosteric inhibitor with multiple functions such as immunosuppressive, anticancer, and lifespan prolonging activities. Its C-43 semi-synthetic derivatives temsirolimus and everolimus have been used as mTOR targeting anticancer drugs

Full text of DOI:10.1002/ardp.201800123

Received: 17 April 2018
Revised: 16 September 2018
Accepted: 26 September 2018
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DOI: 10.1002/ardp.201800123
FULL PAPER
Synthesis and anticancer activity of novel rapamycin C-28
containing triazole moiety compounds
Qingwen Huang1,2
Lijun Xie2
Xiaoming Chen2
Hui Yu2
Yubing Lv3
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XueHui Huang3
Jiayin Ying2
Congshen Zheng2
Yuanrong Cheng2
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Jie Huang1,2
1 Fujian University of Traditional Chinese
Medicine, Fuzhou, P. R. China
Abstract
2 Fujian Provincial Key Laboratory of
Screening for Novel Microbial Products,
Fujian Institute of Microbiology, Fuzhou,
P. R. China
Rapamycin is an mTOR allosteric inhibitor with multiple functions such as
immunosuppressive, anticancer, and lifespan prolonging activities. Its C-43 semi-
synthetic derivatives temsirolimus and everolimus have been used as mTOR targeting
anticancer drugs in the clinic. Following our previous research on antitumor rapalogs
modified on the C-43 position, 13 novel rapamycin triazole hybrids (6a–g, 7a–f) were
designed and synthesized on the C-28 position of rapamycin via Huisgen's reaction.
Anticancer assays indicated that the targeted derivatives containing phenyl and
4-methylphenyl groups showed an obvious raise in anticancer activity. On the
contrary, the compounds with methoxyl, amine, and halogen groups on the benzene
ring displayed lower anticancer activity. Compound 6c, as the most active compound,
showed a stronger inhibition effect as compared with rapamycin for almost all of the
tested cell lines (p < 0.01), except PC-3. Meanwhile, the effect of 6c on inducing
apoptosis and cell cycle arrest in A549 cells was more powerful than that of rapamycin.
In addition, 6c inhibited the phosphorylation of mTOR and its downstream key kinases
4EBP1 and p70S6K1 in A549 cells, indicating that 6c also effectively inhibits the
mTORC1 signaling pathway as rapamycin. On the basis of these findings, 6c may have
the potential to be developed as a new mTOR inhibitor against specific cancers.
3 Hangzhou Huadong Medicine Group
Pharmaceutical Research Institute Co. Ltd.,
Hangzhou, P. R. China
Correspondence
Dr. Jie Huang, Fujian University of Traditional
Chinese Medicine, 25 Jinbu Road, Cangshan
District, Fuzhou 350007, P. R. China.
Email: 251994737@qq.com
Dr. Lijun Xie, Fujian Provincial Key Laboratory
of Screening for Novel Microbial Products,
Fujian Institute of Microbiology, 25 Jinbu
Road, Cangshan District, Fuzhou 350007,
P. R. China.
Email: garrison1225@163.com
Funding information
National Natural Science Foundation of China,
Grant number: 81502935
K E Y W O R D S
anticaner activity, rapamycin, semi-synthesis, triazole
pathway by indirect binding to the FRB domain of mTOR.[3,4] As a
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1
INTRODUCTION
potent mTOR inhibitor, rapamycin has been developed as
a
The mTOR (mammalian target of rapamycin) inhibitor rapamycin
(also known as sirolimus), nitrogen-containing 36-membered
multifunctional drug with antifungal, immunosuppressive, anti-
proliferative, antitumor activities,[1,5–10] and recently, found to
improve cardic function,[11,12] slow aging as well as prolong mouse
lifespan.[13–16] Correspondingly, FDA approved rapamycin as an
immunosuppressant indicated for the prophylaxis of organ rejection
in patients receiving renal transplants, as drug-eluting stents coating
a
macrolide, was discovered as an antifungal antibiotic produced by
Streptomyces hygroscopics AY-994 in a soil sample from Easter Island
in 1975,[1,2] it combines with FKBP12 (FK506 binding protein 12) to
form
a rapamycin-FKBP12 complex which inhibits mTORC1
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Arch Pharm Chem Life Sci. 2018;e1800123.
wileyonlinelibrary.com/journal/ardp
© 2018 Deutsche Pharmazeutische Gesellschaft
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to prevent coronary restenosis following balloon angioplasty as well
as an mTOR targeted antitumor drug for the treatment of patients
with lymphangioleiomyomatosis.
that rapamycin contains two secondary-hydroxyl groups at positions
C-28 and C-43, it should be difficult to achieve a selective synthesis of
C-28 esters, but Nan et al.[24] had already overcome these difficulties
by providing a region-selective synthesis of 28-esters of rapamycin.
Therefore, we would like to use the same method to synthesize target
rapalogs which were modified with 28-esters linkage. First of all, rapa-
28-OTMS (1) was obtained by treating rapamycin with trimethylsilyl
chloride, imidazole, and H2SO4 at 0°C for 12 h. Following, compound 2
was synthesized by mixing 1 with tert-butyldimethylsilyl chloride,
imidazole at 6°C for 5 h. Secondly, compound 2 was treated with
bromoacetyl bromide using pyridine in DCM, which subsequently
reacted with 2 N H2SO4 in acetone to afford intermediate 4 as white
solids. The reaction of bromide 4 with sodium azide resulted in azido-
rapamycin (5) yields of 40.3%. Finally, the targeted compounds 6a–g,
A number of semi-synthetic derivatives of rapamycin have been
developed and some of them are in clinic. Temsirolimus, made from
rapamycin by acylation of the C-43, was approved as the first mTOR
target antitumor drug for advanced renal cell carcinoma.[17] Then,
everolimus, O-alkylation in position C-43 of rapamycin, is used not only
as a mTOR target drug to treat advanced renal cell carcinoma,
neuroendocrine tumors, renal angiomyolipoma as well as breast
cancer, etc. in clinic,[18–20] but also as an immunosuppressant and stent
coating. Other two rapalogs, biolimus A9 and zotarolimus, ethoxyethyl
ether and tetrazole derivatives of rapamycin, respectively, were also
approved as stent coating. It is observed that all rapamycin analogs
(rapalogs) applied in clinic are modified at position C-43 of rapamycin
(Figure 1).
7a–f, were obtained by Huisgen's reaction that
5 underwent
cycloaddition with 1-ethynyl-substituted benzene, propargyl alcohol,
1-diethylamino-2-propyne, using CuI as catalyzer. All intermediates
and targeted compounds were purified by column chromatography on
silica gel and the chemical structures were confirmed by IR, MS, HRMS,
1H NMR and 13C NMR, which would be explained in Section 4.
In our previous work, rapamycin was chemically modified in the
position C-43 and a number of new rapalogs with antitumor activities
were obtained. Among these new rapalogs, rapamycin triazole
derivative 9e[21] and thiazole derivative 9b[22] etc. exhibited stronger
antitumor activities compared with rapamycin.[23] To obtain more
potent rapalogs and further explore their structure–activity relation-
ship, we tried to modify the structure of rapalogs we had reported
before[21] by migrating side chain moiety from the C-43 position to
C-28 position as showed in Figure 2. It may open a new structural
space if we can develop more potent rapalogs modified at C-28
position. Prompted by the aforementioned findings, in the current
study, we hereby report the synthesis and biological evaluation of
novel rapalogs incorporating triazole moiety at C-28 position. Besides,
the mechanism of action for 6c was also preliminary elucidated.
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2.2 Biological activity
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2.2.1 Novel rapalogs modified in the position C-28
of rapamycin possessed effect against cancer cells
proliferation
As shown in Table 1, most of the tested compounds were more potent
than rapamycin against A549 cells, and all of targeted compounds
showed less potency for PC-3 cell line when compared with rapamycin.
Compound 6c, with a 4-methylphenyl moiety on the triazole ring, as
the most active compound, showed stronger inhibition effect as
compared with rapamycin on all of tested cell lines (p < 0.01), except
PC-3. Among the targeted derivatives containing different substituted
benzene group on the triazole ring, the compounds 6a (H), 6b (3-Me),
and 6c (4-Me) displayed more potent activity than rapamycin against
most of tested cell lines. On the contrary, compounds 6d (2-Cl),
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2
RESULTS AND DISCUSSION
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2.1 Chemistry
The synthesis of the C-28 triazole-based rapalogs was accomplished
according to the synthetic protocol showed in Scheme 1. As we know
FIGURE 1 Structure of rapamycin and analogs

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FIGURE 2 Design of the target compounds
6e (4-MeO), 6f (4-F), and 6g (3-NH2) showed lower activity. These
results implied that the different substituted groups on the benzene
group may be highly related with their activity. Interestingly, it is worth
to mention that the introduction of aryl-amino-methyl group on
triazole ring, such as 7a, 7c, and 7d, also led to an obvious raise in
anticancer activity while hydroxyl-alkyl (7e and 7f) caused anticancer
potency to be lowered significantly. More interestingly, we had
reported the synthesis and antitumor activities of C-43
triazole-based rapamycin derivatives, in which the side chain of
compound 4a, 4b, 5e[21] were almost the same with 6c, 6f, 7a,
respectively, but the anticancer activities of 4a, 4b, 5e were much
weaker, indicating that the anticancer activities of the rapalogs
SCHEME 1 Syntheses of compounds 6a–f and 7a–f. Reagents and conditions: (a, b) TMSCl, 0.5 N H2SO4, EA, 0°C; (c) TBSCl, imidazole,
DMF, 6°C; (d) bromoacetyl bromide, pyridine, DCM, −2°C; (e) 1 N H2SO4, acetone, 0°C; (f) NaN3, KI, DMF, 55°C; (g) CuI, DIPEA, acetonitrile,
25°C

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TABLE 1 Anti-proliferative IC50 of rapalogs on human cancer cell lines
a
IC50 (µM), mean SD
Compound no.
A549b
PC-3b
CASKIb
769-Pb
ECA-109b
19.2 0.4**
18.6 0.2**
14.8 0.4**
23.4 0.4
29.1 0.4
n.d.c
6a
14.2 0.7**
14.6 0.3**
12.8 0.4**
18.5 0.5
17.1 0.4
16.1 0.4*
17.6 0.2
n.d.c
15.9 0.6
14.7 0.1
13.1 0.4
20.6 0.4
17.4 0.4
n.d.c
16.2 0.3
14.2 0.4*
13.1 0.4**
21.1 0.4
27.1 0.4
n.d.c
16.7 0.5**
15.4 0.2**
13.9 0.4**
24.3 0.4
28.3 0.4
25.3 0.4
25.3 0.7
n.d.c
6b
6c
6d
6e
6f
6g
17.1 0.5
15.1 0.2
16.4 0.8
15.8 0.8
16.5 0.7
18.1 0.8
17.8 0.8
12.3 0.7
25.2 0.2
20.7 0.3
22.9 0.8
20.3 0.8
18.3 0.4*
30.4 0.8
36.1 0.8
15.2 0.4
23.2 0.5*
20.4 0.1*
22.3 0.8
22.9 0.8
21.6 0.4
31.2 0.8
>50
7a
7b
12.4 0.8**
12.7 0.8**
12.8 0.5**
17.3 0.4
17.9 0.8
18.1 0.6
23.8 0.8
22.4 0.8**
21.3 0.4**
34.4 0.8
37.5 0.8
24.5 0.6
7c
7d
7e
7f
Rapamycin
21.8 0.4
aIC50 was tested by the SRB assay. Experiments were performed in triplicate and the results were presented as average values. *p < 0.05, **p < 0.01.
bA549, human non-small cell lung cancer cell line; PC-3, human prostatic cancer cell line; CASKI, human cervical cancer line; 769-P, human renal carcinoma
cell line; and ECA-109, human esophageal cancer cell line, were employed to test the anticancer activities of new rapalogs.
cn.d.: not determined.
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modified on C-28 position were stronger than that of the rapalogs
modified on C-43 position.
2.2.4 6c blocked mTOR signaling pathway in A549
mTOR pathway is an important driver of tumor development and
progression. Western blot analysis showed that 6c inhibited the
phosphorylation of mTOR at Ser 2448 which is an important
phosphorylation site for mTOR activity. Furthermore, the phosphor-
ylation of mTOR major downstream 4E-BP1 and p70S6K1 were
significantly decreased by 6c in dose-dependent manner (Figure 5).
These results indicated that 6c repressed tumor cell growth by
blocking mTOR signaling pathway, which was same as rapamycin.
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2.2.2 6c induced apoptosis in A549 cells
To determine whether 6c-induced cell death was related to induction
of apoptosis, A549 was treated with 6c in different concentration for
48 h and Annexin V staining assay was performed with flow cytometry.
As displayed in Figure 3, similar to rapamycin, 6c showed the action on
inducing A549 cells apoptosis in dose-dependent manner. As the
concentration of which from 10 to 20 µM, the apoptosis rate of A549
increased from 3.83 to 7.97%. At the same concentration, the
apoptosis rate of A549 by 6c (4.05%) was higher than that by
rapamycin (3.54%). It indicated that the effect of 6c was stronger than
of rapamycin in inducing cell apoptosis.
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2.2.5 Binding mode analysis
To further elucidate the binding mode of compounds, docking analysis
was performed by means of the MOE software and a proposed binding
mode was obtained. As shown in Figure 6, detailed analysis of the
interactions revealed the following features. First, three carbonyl
oxygen atoms of lactone in 6c formed three hydrogen-bonding
interactions with lleA56, TyrA82, and LysA52, respectively. Mean-
while, one hydrogen bond formed between the hydroxyl group of
hemiacetal and AspA37. In addition, methylene (─CH2─) of side chain
in 6c formed hydrogen bond with GlnA53. Second, the triene moiety
fitted into the hydrophobic pocket that was formed by LeuB2031,
TrpB2101, Phe2108, etc. Notably, the benzene group of side chain
was almost out of docking pocket, thus it may explain the similar
activity for these series derivatives because varied substituted groups
on the benzene ring would not have great positive effect for the
binding result.
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2.2.3 6c induced cell cycle arrest in G1 phase in
A549
To further investigate whether the growth inhibitory effect of 6c on
A549 was partly due to cell cycle arrest, A549 was exposed to various
concentrations of 6c for 48 h and the DNA content of cells was
measured by flow cytometry. As showed in Figure 4 and Table 2, 6c
caused accumulation of A549 in the G1 phase of cell cycle in dose-
dependent manner. At the same concentration of 15 µM, 6c exhibited
more effect on arrest of A549 in the G1 phase of cell cycle than that of
rapamycin.

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FIGURE 3 Effect of 6c and rapamycin on apoptosis in A549 cells. A549 was treated with 6c or rapamycin for 48 h at the indicated
concentrations. Cells were stained with Annexin V-FITC and propidium iodide and analyzed by flow cytometry
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CONCLUSIONS
4
EXPERIMENTAL
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This work presents the synthesis of rapamycin–triazole hybrids
modified on the position C-28 of rapamycin and their anticancer
activity against different cancer cell lines. To our delight, the most
promising compound 6c showed stronger effect than rapamycin not
only in anticancer activity against most of tested cancer cell lines but
also in inducing cancer cell apoptosis, cell cycle arrest, and inhibition
of mTOR pathway. In addition, conclusions regarding the structure–
activity relationships could be tentatively drawn as two aspects. (1)
Among target compounds (6a–g), the result indicated that the
introduction of hydrogen or methyl group on the benzene ring made
a good contribution to potency, with following rank order: H < Me.
Compounds with halogen or methoxyl groups on the benzene ring
lost significant potency against 769-P and ECA-109 cell lines. (2)
Moreover, the introduction of aryl-amino-methyl group on triazole
ring is advantageous to the potency while the introduction of
hydroxyl-alkyl (7e, 7f) moiety has negative impact on their anti-
cancer activities. In conclusion, based on the experimental data and
the binding mode, modification of rapamycin on C-28 position is a
promising approach to obtain more potent mTOR inhibitors and 6c
holds the potential to become a new therapeutic mTOR inhibitor
against various cancers.
4.1 Chemistry
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4.1.1 Reagents and general procedures
Unless otherwise noted, all materials were obtained from commercial
suppliers and used without further purification. All melting points were
obtained on a Buchi Melting Point M-560 (Buchi Labortechnik, Flawil,
Switzerland) and were recorded. Mass spectra (MS) were taken in ESI
mode Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA), [a]D values
were recorded on Autopol I-Rudolph (USA). High resolution mass
spectra (HRMS) were recorded on Agilent QTOF 6520 instrument.
Nuclear magnetic resonance spectroscopy was performed using
Bruker Advance, 600 MHz spectrometers (Bruker, Germany) with
TMS as an internal standard. IR spectra (KBr disks) were recorded with
a Bruker IFS 55 instrument (Bruker).
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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4.1.2 Preparation of Rapa-28-OTMS (1)
Rapamycin (10.0 g, 10.9 mmol) and imidazole (9.4 g, 137.2 mmol) were
dissolved in dry ethyl acetate (115.0 mL) and the solution was stirred at

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FIGURE 4 Effect of 6c and rapamycin on cell cycle arrest in A549. A549 cells were treated with 6c or rapamycin for 48 h at the indicated
concentrations. Cells were stained with annexin V-FITC and analyzed by flow cytometry
0°C, then a solution of trimethylsilyl chloride (9.3 g, 85.3 mmol) was
added dropwise to the reaction solution within 38 min. After the Rapa
was transformed into Rapa-28,43-bis-OTMS, a 66 mL of 0.5 N sulfuric
acid was added dropwise for 15 min and the mixture was stirred for 3 h
at 0°C. The organic layer was washed with saturated NaHCO3 aqueous
solution and brine, dried over Na2SO4, filtered, and concentrated to
afford 1 (10.3 g) as white solids in 95.6% yield; m.p.: 145.8–147.5°C;
MS (ESI) m/z: 1008.5000 (M+Na)+; [a]D −25.7 (c = 1.0, MeOH);
1H NMR (600 MHz, DMSO-d6) δ 6.47 (s, 1H), 6.41 (dd, J = 14.3,
11.5 Hz, 1H), 6.23 (dd, J = 24.6, 13.7 Hz, 1H), 6.15 (dd, J = 21.1,
10.9 Hz, 1H), 6.10 (d, J = 15.1 Hz, 1H), 5.46 (dd, J = 14.8, 9.6 Hz, 1H),
5.11 (d, J = 10.2 Hz, 1H), 5.01 (d, J = 3.6 Hz, 1H), 4.95 (d, J = 4.8 Hz, 1H),
4.68–4.62 (m, 1H), 4.02 (t, J = 9.3 Hz, 2H), 3.87 (d, J = 5.7 Hz, 1H), 3.61
(dd, J = 33.1, 18.7 Hz, 1H), 3.45 (d, J = 12.6 Hz, 1H), 3.33 (s, 3H), 3.29
(dd, J = 19.4, 8.7 Hz, 1H), 3.17 (s, 3H), 3.14 (t, J = 5.7 Hz, 2H), 3.08 (d,
J = 21.0 Hz, 3H), 2.82 (dd, J = 15.5, 8.0 Hz, 1H), 2.72 (dd, J = 33.3,
11.9 Hz, 1H), 2.50 (d, J = 12.8 Hz, 1H), 2.23 (s, 1H), 2.13 (d, J = 9.6 Hz,
1H), 2.05–2.01 (m, 1H), 1.85 (d, J = 9.3 Hz, 1H), 1.83 (s, 1H), 1.77 (s,
3H), 1.67 (s, 1H), 1.64 (s, 3H), 1.61 (s, 1H), 1.38 (d, J = 12.7 Hz, 1H), 1.30
(s, 1H), 1.24 (d, J = 11.3 Hz, 1H), 1.20 (d, J = 11.1 Hz, 1H), 0.99 (d,
J = 6.3 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H), 0.83 (d, J = 6.3 Hz, 3H), 0.78 (d,
J = 6.5 Hz, 3H), 0.74 (d, J = 6.4 Hz, 3H), 0.03–0.05 (m, 9H).
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4.1.3 Preparation of Rapa-43-OTBDMS (2)
TABLE 2 Cell cycle distribution of A549 cells after 6c or rapamycin
treatment for 48 h
Rapa-28-OTMS (10.3 g, 10.5 mmol) and imidazole (24.5 g,
Cell cycle distribution (%)
360.4 mmol) were added to N,N-dimethylformamide (DMF,
110.0 mL) at 6°C,
a solution of tert-butyldimethylsilyl chloride
Compound (μM)
6c (10)
G1
S
G2
(TBDMSCl) (14.7 g, 138.3 mmol) in DMF (10.0 mL) was added within
20 min. The mixture was stirred at 6°C for 5 h. The reaction mixture
was diluted with DMF and washed thoroughly with water, 1 N HCl
aqueous solution, NaHCO3 aqueous solution and brine, dried over
Na2SO4, filtered, and concentrated. Silica gel chromatography (PE/EA
4:1) of the crude mixture afforded 2 (6.0 g, 46.3%); m.p.: 113.4–
127.73
145.12
148.12
137.12
122.24
12.56
7.53
6.28
9.13
14.16
6.03
3.24
2.79
5.18
6.23
6c (15)
6c (20)
Rapamycin (15)
Control

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FIGURE 5 Inhibition of 6c and rapamycin on the phosphorylation
of mTOR, p70S6K, 4E-BP1 in A549 cells. Cells were treated with 6c
or rapamycin for 48 h and lysed, proteins were subjected to SDS–
PAGE and transferred to nitrocellulose membranes. Bands were
visualized by using the enhanced chemiluminescence Western blot
detection system
115.6°C; MS (ESI) m/z: 1050.6000 (M+Na)+; [a]D −13.2 (c = 1.0,
MeOH); 1H NMR (600 MHz, DMSO-d6) δ 6.46 (d, J = 1.4 Hz, 1H),
6.44–6.37 (m, 1H), 6.23 (dd, J = 14.5, 10.6 Hz, 1H), 6.17–6.11 (m, 1H),
6.11 (d, J = 4.5 Hz, 1H), 5.47 (dd, J = 14.9, 9.6 Hz, 1H), 5.28 (d,
J = 4.5 Hz, 1H), 5.11 (d, J = 10.1 Hz, 1H), 5.01–4.97 (m, 1H), 4.95 (d,
J = 6.0 Hz, 1H), 4.05–3.99 (m, 2H), 3.95 (d, J = 4.6 Hz, 1H), 3.45 (d,
J = 12.7 Hz, 1H), 3.33 (s, 4H), 3.20–3.17 (m, 1H), 3.16 (s, 3H), 3.13 (s,
1H), 3.06 (s, 3H), 2.76–2.71 (m, 1H), 2.44–2.39 (m, 1H), 2.24–2.18 (m,
1H), 2.14–2.08 (m, 1H), 2.04 (dd, J = 15.0, 6.9 Hz, 1H), 1.85–1.80 (m,
1H), 1.74 (s, 4H), 1.68 (s, 1H), 1.68–1.66 (m, 1H), 1.64 (s, 4H), 1.60 (d,
J = 4.8 Hz, 1H), 1.59–1.56 (m, 2H), 1.53 (s, 2H), 1.38 (d, J = 10.5 Hz,
1H), 1.29 (d, J = 11.8 Hz, 3H), 1.25 (d, J = 11.1 Hz, 2H), 0.98 (d,
J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.86 (s, 9H), 0.83 (d, J = 6.4 Hz,
4H), 0.78 (d, J = 6.7 Hz, 3H), 0.74 (d, J = 6.7 Hz, 3H), 0.04 (d, J = 1.4 Hz,
6H).
FIGURE 6 The binding model of compound 6c with FKBP12-
FRB. (A) Overview of predicted 3D binding modes of 6c with the
FKPB12-FRB crystal structure (PDB: 1FAP); (B) predicted 2D
binding modes of 6c with the FKPB12-FRB crystal structure
J = 3.8 Hz, 1H), 6.41–6.37 (m, 1H), 6.23 (dd, J = 10.3, 4.8 Hz, 1H), 6.15
(dd, J = 12.9, 7.1 Hz, 1H), 6.12 (d, J = 7.0 Hz, 1H), 5.51–5.47 (m, 1H),
5.05 (d, J = 11.3 Hz, 1H), 5.02–4.96 (m, 2H), 4.94 (d, J = 5.4 Hz, 5H),
4.57 (s, 1H), 4.08–3.98 (m, 3H), 3.91 (d, J = 2.8 Hz, 1H), 3.63 (dd,
J = 20.6, 9.3 Hz, 1H), 3.45 (d, J = 12.6 Hz, 1H), 3.33 (dd, J = 2.6, 1.5 Hz,
3H), 3.32–3.27 (m, 1H), 3.22–3.19 (m, 3H), 3.17 (d, J = 2.2 Hz, 3H),
3.15–3.13 (m, 2H), 3.05 (d, J = 2.8 Hz, 3H), 2.88–2.80 (m, 1H), 2.67 (dd,
J = 9.2, 6.2 Hz, 1H), 2.39 (dd, J = 16.1, 9.2 Hz, 1H), 2.12 (d, J = 13.1 Hz,
1H), 2.04–1.99 (m, 1H), 1.90 (d, J = 13.1 Hz, 1H), 1.86–1.80 (m, 1H),
1.78–1.73 (m, 1H), 1.67 (s, 1H), 1.63–1.59 (m, 1H), 1.55 (d, J = 12.8 Hz,
1H), 1.53 (s, 1H), 1.27 (s, 1H), 1.26–1.22 (m, 1H), 1.21–1.16 (m, 1H),
0.98 (d, J = 2.1 Hz, 3H), 0.87 (d, J = 1.9 Hz, 3H), 0.85–0.83 (m, 12H),
0.83–0.82 (m, 3H), 0.78 (d, J = 7.8 Hz, 3H), 0.74 (dd, J = 6.5, 2.4 Hz, 3H),
0.59 (dd, J = 23.8, 11.9 Hz, 1H), −0.03 to 0.06 (m, 6H).
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4.1.4 Preparation of Rapa-28-O-(2-bromoacetyl)-43-
OTBDMS (3)
Rapa-43-OTBDMS (6.0 g, 5.8 mmol) and pyridine (17.7 g, 0.2 mol)
were added into dichloromethane (DCM, 100.0 mL) at −2°C. A solution
of bromoacetyl bromide (9.0 mL, 104.2 mmol) in DCM (15.0 mL) was
added into this mixture within 40 min and further stirred for 10 min.
The reaction mixture was diluted with DCM and washed thoroughly
with water, 1 N HCl aqueous solution, NaHCO3 aqueous solution and
brine, dried over Na2SO4, filtered, and concentrated. Silica gel
chromatography (PE/EA 4:1) of the crude mixture afforded 3 (4.8 g,
70.8%); m.p.: 94.1–96.4°C; MS (ESI) m/z: 1170.5000 (M+Na)+; [a]D
−35.5 (c = 1.0, MeOH); 1H NMR (600 MHz, DMSO-d6) δ 6.42 (d,
|
4.1.5 Preparation of Rapa-28-O-(2-bromoacetyl) (4)
Rapa-28-O-(2-bromoacetyl)-43-OTBDMS (4.8 g, 4.2 mmol) was dis-
solved in acetone (70.0 mL) and the solution was stirred at 0°C, and

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dilute sulfuric acid (2.0 mol/L, 18.0 mL) was added into the solution and
further stirred for 18 h. The reaction mixture was diluted with EtOAc
and washed thoroughly with NaHCO3 aqueous solution and brine,
dried over Na2SO4, filtered, and concentrated. Silica gel chromatogra-
phy (PE/EA 1:1) of the crude mixture afforded 4 (3.6 g, 79.5%); m.p.:
101.3–104.6°C; MS (ESI) m/z: 1056.4000 (M+Na)+; [a]D −23.8 (c = 1.0,
MeOH); 1H NMR (600 MHz, DMSO-d6) δ 6.42 (d, J = 4.5 Hz, 1H), 6.40
(d, J = 3.6 Hz, 1H), 6.23 (d, J = 4.0 Hz, 1H), 6.19–6.15 (m, 1H), 6.12 (s,
1H), 5.47 (d, J = 5.3 Hz, 1H), 5.09 (s, 1H), 4.98 (d, J = 3.8 Hz, 1H), 4.94 (s,
1H), 4.65 (s, 1H), 4.02–4.00 (m, 2H), 3.91 (d, J = 2.7 Hz, 1H), 3.62 (d,
J = 6.2 Hz, 1H), 3.44 (d, J = 2.1 Hz, 1H), 3.33 (s, 3H), 3.26 (s, 1H), 3.22 (s,
3H), 3.19 (d, J = 2.1 Hz, 1H), 3.05 (s, 3H), 2.84–2.82 (m, 1H), 2.73 (dd,
J = 8.8, 5.2 Hz, 1H), 2.51 (dt, 2H), 2.46–2.42 (m, 1H), 2.10 (d,
J = 11.8 Hz, 2H), 1.82 (s, 3H), 1.66–1.65 (m, 1H), 1.61 (s, 3H), 1.58
(s, 1H), 1.41 (s, 1H), 1.30 (s, 1H), 0.99 (d, J = 6.6 Hz, 3H), 0.88–0.86 (m,
3H), 0.84 (dd, J = 4.8, 3.8 Hz, 3H), 0.77 (d, J = 6.8 Hz, 3H), 0.74 (d,
J = 6.6 Hz, 3H).
solution and brine, dried over Na2SO4, filtered, and concentrated. Silica
gel chromatography (PE/EA 1:1) of the crude mixture afforded the
targeted compounds.
Rapa-28-O-(2-(4-(phenyl)-1H-1,2,3-trizole-1-yl)acetyl) (6a)
This compound was obtained as white solids in 69.2% yield; m.p.:
116.3–118.3°C; MS (ESI) m/z: 1098.6000 (M+Na)+; [a]D −20.7 (c 1.0,
MeOH); IR (KBr) cm−1: 3428.0, 2940.5, 1728.3, 1641.7, 1450.7,
1372.9, 1095.3, 990.3, 766.6. 1H NMR (600 MHz, DMSO-d6) δ 8.43 (s,
1H), 7.86 (d, J = 1.0 Hz, 1H), 7.84 (s, 1H), 7.46 (d, J = 7.4 Hz, 1H), 7.43 (s,
1H), 7.33 (dd, J = 13.3, 5.9 Hz, 1H), 6.44 (d, J = 1.1 Hz, 1H), 6.37 (dd,
J = 14.3, 11.4 Hz, 1H), 6.22 (dd, J = 17.0, 6.4 Hz, 1H), 6.19–6.11 (m,
1H), 6.10 (d, J = 4.1 Hz, 1H), 5.63–5.48 (m, 1H), 5.37 (dd, J = 23.2,
10.0 Hz, 2H), 5.09 (d, J = 7.3 Hz, 1H), 5.03–4.94 (m, 2H), 4.57–4.52 (m,
1H), 4.35 (d, J = 3.6 Hz, 1H), 4.31–4.10 (m, 1H), 4.06–3.96 (m, 1H), 3.65
(dd, J = 33.1, 11.7 Hz, 1H), 3.46 (d, J = 13.5 Hz, 1H), 3.29 (d, J = 6.6 Hz,
3H), 3.27–3.22 (m, 1H), 3.21 (s, 3H), 3.19–3.07 (m, 2H), 3.03 (d,
J = 16.7 Hz, 3H), 2.85–2.75 (m, 1H), 2.72–2.60 (m, 1H), 2.40 (dd,
J = 17.5, 8.2 Hz, 1H), 2.35–2.27 (m, 1H), 2.16 (dd, J = 27.4, 14.1 Hz,
1H), 2.02 (dd, J = 15.4, 7.9 Hz, 1H), 1.90 (d, J = 12.0 Hz, 1H), 1.83 (d,
J = 13.9 Hz, 1H), 1.74–1.64 (m, 3H), 1.62–1.49 (m, 6H), 1.41 (dd,
J = 27.3, 13.6 Hz, 2H), 1.24 (dd, J = 13.6, 7.8 Hz, 2H), 1.20–1.10 (m,
3H), 1.10–1.00 (m, 2H), 0.98 (t, J = 6.4 Hz, 3H), 0.91–0.79 (m, 6H),
0.79–0.69 (m, 6H), 0.56 (dd, J = 23.7, 11.9 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 208.7, 206.8, 198.6, 169.2, 166.8, 165.5,
146.3, 139.0, 137.9, 132.3, 131.7, 130.5, 128.7, 127.8, 126.9, 125.6,
125.1, 122.6, 98.9, 83.7, 82.1, 81.7, 77.8, 73.9, 73.1, 66.1, 57.1, 56.6,
55.4, 50.9, 50.3, 45.2, 43.6, 40.8, 40.6, 40.0, 39.8, 39.6, 39.5,
39.3, 39.1, 39.0, 37.9, 35.1, 35.0, 34.6, 33.7, 32.8, 32.5, 31.3, 29.7,
28.6, 26.1, 24.4, 22.4, 21.4, 20.4, 19.4, 18.9, 15.5, 15.2, 14.9,
14.1, 13.0, 11.1, 10.4; HRMS (ESI): calcd. for C61H86N4NaO14
[M+Na]+ = 1098.6115. Found = 1098.6141.
|
4.1.6 Preparation of Rapa-28-O-(2-azidopropiomyl)
(5)
Rapa-28-O-(2-bromoacetyl) (3.6 g, 3.5 mmol) was dissolved in DMF
(50.0 mL) and the solution was stirred at 55°C, catalytic quantity KI and
NaN3 (0.9 g, 13.8 mmol) was added into the solution and further stirred
for 1 h. The reaction mixture was diluted with EtOAc and washed
thoroughly with water, 1 N HCl aqueous solution, NaHCO3 aqueous
solution and brine, dried over Na2SO4, filtered, and concentrated. Silica
gel chromatography (PE/EA 1:1) of the crude mixture afforded 5 (1.4 g,
40.3%); m.p.: 100.3–103.7°C; MS (ESI) m/z: 1019.5000 (M+Na)+; [a]D
−13.7 (c = 1.0, MeOH); 1H NMR (600 MHz, DMSO-d6) δ 6.47 (s, 1H),
6.40 (dd, J = 14.6, 11.3 Hz, 1H), 6.24 (dd, J = 14.5, 10.7 Hz, 1H), 6.15
(dd, J = 15.7, 5.3 Hz, 1H), 6.12 (d, J = 4.6 Hz, 1H), 5.47 (dd, J = 15.0,
9.7 Hz, 1H), 5.34 (t, J = 5.9 Hz, 1H), 5.05 (d, J = 10.1 Hz, 1H), 4.98–4.93
(m, 2H), 4.61 (s, 1H), 4.36 (d, J = 3.8 Hz, 1H), 4.09–3.97 (m, 2H), 3.95 (d,
J = 9.8 Hz, 1H), 3.63 (dd, J = 17.2, 5.6 Hz, 1H), 3.46 (d, J = 13.9 Hz, 1H),
3.33 (s, 3H), 3.30–3.27 (m, 1H), 3.27–3.24 (m, 1H), 3.22 (s, 3H), 3.13–
3.08 (m, 1H), 3.05 (s, 3H), 2.86–2.82 (m, 1H), 2.67 (dd, J = 17.5, 2.7 Hz,
1H), 2.52–2.49 (m, 1H), 2.44–2.36 (m, 2H), 2.24 (s, 2H), 2.13 (d,
J = 12.5 Hz, 1H), 1.85 (s, 1H), 1.82 (s, 3H), 1.62 (s, 3H), 1.59–1.55 (m,
4H), 1.28 (s, 1H), 1.00 (d, J = 6.5 Hz, 6H), 0.89–0.86 (m, 6H), 0.84–0.82
(m, 1H), 0.77 (d, J = 6.7 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H).
Rapa-28-O-(2-(4-(3-methylphenyl)-1H-1,2,3-trizole-1-yl)acetyl)
(6b)
This compound was obtained as white solids in 38.2% yield; m.p.:
121.7–123.9°C; MS (ESI) m/z: 1112.6000 (M+Na)+; [a]D −32.2 (c 1.0,
MeOH); IR (KBr) cm−1: 3449.0, 2936.4, 1725.0, 1646.0, 1450.2,
1375.8, 1249.8, 1091.4, 989.2, 784.3. 1H NMR (600 MHz, DMSO-d6)
δ 8.40 (d, J = 6.2 Hz, 1H), 7.67 (s, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.33 (dd,
J = 14.0, 6.5 Hz, 1H), 7.15 (d, J = 7.2 Hz, 1H), 5.45 (dd, J = 22.4, 12.7 Hz,
1H), 5.38–5.31 (m, 2H), 5.15–5.05 (m, 1H), 5.03–4.93 (m, 2H), 4.55 (t,
J = 6.2 Hz, 1H), 4.35 (d, J = 3.5 Hz, 1H), 4.27 (d, J = 3.9 Hz, 1H), 4.06–
3.95 (m, 1H), 3.65 (dd, J = 32.2, 11.9 Hz, 1H), 3.46 (d, J = 13.5 Hz, 1H),
3.29 (d, J = 7.7 Hz, 3H), 3.27–3.23 (m, 1H), 3.19 (d, J = 13.9 Hz, 3H),
3.12 (dd, J = 15.7, 10.7 Hz, 2H), 3.03 (d, J = 16.4 Hz, 3H), 2.84–2.76 (m,
1H), 2.66 (d, J = 15.3 Hz, 1H), 2.43–2.37 (m, 1H), 2.36 (s, 3H), 2.33–
2.29 (m, 1H), 2.19–2.11 (m, 1H), 2.02 (dd, J = 15.0, 7.6 Hz, 1H), 1.86
(dd, J = 30.5, 14.0 Hz, 3H), 1.74–1.66 (m, 3H), 1.54 (d, J = 14.8 Hz, 5H),
1.45–1.36 (m, 2H), 1.34–1.27 (m, 2H), 1.22 (d, J = 13.9 Hz, 2H), 1.20–
1.12 (m, 3H), 1.04 (dd, J = 18.7, 6.3 Hz, 3H), 0.97 (d, J = 6.3 Hz, 3H),
0.87 (t, J = 9.6 Hz, 4H), 0.77–0.70 (m, 8H), 0.55 (dd, J = 23.8, 11.9 Hz,
1H). 13C NMR (151 MHz, DMSO-d6) δ 208.7, 206.8, 198.7, 169.2,
|
4.1.7 General procedure for the preparation of
compounds 6a–g, 7a–f
Mix of azidorapamycin (0.3 g, 0.3 mmol), 1-ethynyl-substituted
benzene or other types of alkynes (3.0 equiv), catalytic quantity CuI
and ethyldiisopropylamine (0.1 mL) in acetonitrile (10.0 mL). The
heterogeneous mixture was stirred vigorously overnight at room
temperature. TLC analysis indicated complete consumption of the
azidorapamycin within 2–24 h, depending on the substrate and steric
hindrance. The reaction mixture was diluted with EtOAc and washed
thoroughly with water, 1 N HCl aqueous solution, NaHCO3 aqueous

HUANG ET AL.
9 of 13
|
166.8, 165.6, 146.4, 139.0, 137.8, 132.3, 131.8, 130.4, 128.6, 128.5,
126.9, 125.7, 122.5, 122.3, 110.0, 98.9, 83.7, 82.1, 81.7, 77.8, 73.9,
73.1, 66.1, 57.1, 56.6, 55.4, 50.9, 50.3, 45.2, 43.6, 40.9, 40.6, 40.0,
39.8, 39.6, 39.5, 39.3, 39.1, 39.0, 37.8, 35.1, 34.9, 34.6, 33.8,
32.8, 32.5, 31.3, 29.7, 26.1, 24.4, 21.4, 20.9, 20.4, 15.5, 15.2,
14.9, 14.1, 13.0, 10.4; HRMS (ESI): calcd. for C62H88N4NaO14
[M+Na]+ = 1112.6279. Found = 1112.6297.
6H), 0.76–0.70 (m, 3H), 0.56 (dd, J = 23.9, 12.0 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 208.8, 207.1, 199.1, 185.0, 182.3, 169.6,
167.4, 142.9, 139.6, 139.2, 138.3, 138.1, 132.8, 132.2, 130.7, 130.7,
129.8, 129.4, 127.9, 127.4, 126.1, 99.4, 84.2, 82.5, 78.2, 74.3, 73.6,
71.8, 66.5, 57.5, 57.1, 55.9, 51.4, 50.6, 45.5, 44.0, 40.4, 40.2,
40.1, 39.9, 39.8, 39.7, 39.5, 38.4, 35.6, 33.3, 33.0, 31.8, 30.0,
28.6, 26.8, 24.9, 22.0, 21.4, 19.7, 16.0, 15.7, 15.4, 14.4, 13.6, 10.9,
9.9. HRMS (ESI): calcd. for C61H85ClN4NaO14 [M+Na]+ = 1155.5643.
Found = 1155.5622.
Rapa-28-O-(2-(4-(4-methylphenyl)-1H-1,2,3-trizole-1-yl)acetyl)
(6c)
This compound was obtained as white solids in 62.5% yield; m.p.:
124.1–127.3°C; MS (ESI) m/z: 1112.6000 (M+Na)+; [a]D −40.3 (c 1.0,
MeOH); IR (KBr) cm−1: 3459.5, 2930.5, 1721.2, 1638.8, 1454.5,
1247.7, 1091.4, 999.5. 1H NMR (600 MHz, DMSO-d6) δ 8.36 (s, 1H),
7.73 (d, J = 7.0 Hz, 2H), 7.27 (t, J = 17.0 Hz, 2H), 6.43 (s, 1H), 6.41–6.30
(m, 1H), 6.30–6.15 (m, 1H), 6.15–6.03 (m, 2H), 5.54–5.41 (m, 1H), 5.36
(d, J = 23.1 Hz, 2H), 5.05 (d, J = 39.3 Hz, 1H), 4.97 (d, J = 11.1 Hz, 2H),
4.30 (d, J = 41.9 Hz, 2H), 4.01 (s, 1H), 3.62 (d, J = 11.0 Hz, 1H), 3.46 (d,
J = 11.5 Hz, 1H), 3.28 (s, 3H), 3.20 (s, 3H), 3.18–3.10 (m, 2H), 3.04 (s,
3H), 2.79 (s, 1H), 2.66 (d, J = 16.8 Hz, 1H), 2.44–2.38 (m, 1H), 2.33 (s,
3H), 2.15 (d, J = 13.3 Hz, 1H), 2.01 (d, J = 6.6 Hz, 1H), 1.86 (dd, J = 33.4,
12.8 Hz, 3H), 1.78 (s, 1H), 1.67 (d, J = 8.8 Hz, 3H), 1.50 (d, J = 27.5 Hz,
5H), 1.46–1.35 (m, 2H), 1.23 (s, 2H), 1.20–1.10 (m, 3H), 1.05 (d,
J = 7.4 Hz, 3H), 0.97 (d, J = 5.4 Hz, 3H), 0.83 (dd, J = 17.8, 5.8 Hz, 6H),
0.74 (s, 6H), 0.60–0.53 (m, 1H). 13C NMR (151 MHz, DMSO-d6) δ
208.3, 206.6, 198.6, 169.2, 166.8, 165.6, 146.4, 139.0, 137.9, 137.1,
132.3, 131.7, 130.4, 129.3, 127.7, 126.9, 125.6, 125.1, 122.2, 108.2,
98.9, 83.7, 82.1, 81.7, 77.8, 73.9, 73.1, 66.1, 57.1, 56.6, 55.4, 50.9,
50.3, 45.2, 40.8, 40.6, 40.0, 39.8, 39.6, 39.5, 39.3, 39.1, 39.0, 37.9,
35.1, 35.0, 34.6, 33.7, 32.8, 32.5, 31.3, 29.7, 26.1, 24.4, 21.4, 20.7,
20.4, 15.5, 15.2, 14.9, 14.1, 13.0, 10.4. HR-MS(ESI): calcd. for
Rapa-28-O-(2-(4-(4-methoxylphenyl)-1H-1,2,3-trizole-1-yl)-
acetyl) (6e)
This compound was obtained as yellow solids in 15.4% yield; m.p.:
113.9–115.8°C; MS (ESI) m/z: 1128.6000 (M+Na)+; [a]D −33.5 (c 1.0,
MeOH); IR (KBr) cm−1: 3455.0, 2931.3, 1723.8, 1639.4, 1449.6,
1246.9, 1095.1, 993.7. 1H NMR (600 MHz, DMSO-d6) δ 8.32 (d,
J = 5.5 Hz, 1H), 7.85–7.74 (m, 2H), 7.12–6.99 (m, 2H), 6.47 (d,
J = 8.0 Hz, 1H), 6.38 (dd, J = 14.5, 11.4 Hz, 1H), 6.21 (dd, J = 14.4,
10.9 Hz, 1H), 6.11 (dd, J = 18.5, 10.6 Hz, 2H), 5.43 (dd, J = 15.0, 9.8 Hz,
1H), 5.39–5.32 (m, 2H), 5.13–5.05 (m, 1H), 5.02–4.92 (m, 2H), 4.59 (d,
J = 4.0 Hz, 1H), 4.37 (d, J = 3.2 Hz, 1H), 4.31–4.11 (m, 1H), 4.03 (d,
J = 10.9 Hz, 1H), 3.79 (s, 3H), 3.63 (dd, J = 32.8, 21.3 Hz, 1H), 3.47 (d,
J = 12.9 Hz, 1H), 3.29 (s, 3H), 3.21 (s, 3H), 3.17–3.13 (m, 2H), 3.05 (s,
3H), 2.80 (dd, J = 15.6, 8.3 Hz, 1H), 2.66 (dd, J = 31.8, 16.8 Hz, 1H), 2.40
(dd, J = 17.3, 8.2 Hz, 2H), 2.24–2.13 (m, 1H), 2.10 (d, J = 23.6 Hz, 1H),
2.05–1.97 (m, 1H), 1.87 (dd, J = 38.0, 13.4 Hz, 3H), 1.75–1.65 (m, 3H),
1.55–1.40 (m, 5H), 1.33 (d, J = 34.9 Hz, 2H), 1.25 (d, J = 14.3 Hz, 2H),
1.20–1.12 (m, 3H), 1.06 (ddd, 3H), 0.98 (d, J = 6.3 Hz, 3H), 0.90–0.80
(m, 6H), 0.76–0.70 (m, 6H), 0.58–0.52 (m, 1H). 13C NMR (151 MHz,
DMSO-d6)
δ 207.8, 200.4, 198.4, 185.3, 181.7, 180.7, 174.1,
169.7, 166.3, 159.5, 152.8, 147.9, 135.8, 126.8, 114.8, 99.6, 84.0,
73.5, 64.5, 56.8, 55.6, 46.1, 40.4, 40.2, 40.1, 39.9, 39.8, 39.7, 39.5,
33.1, 21.7, 16.0, 15.5, 15.4, 14.0, 11.2, 10.5; HRMS (ESI): calcd. for
C
62H88N4NaO14 [M+Na]+ = 1112.6269. Found = 1112.6297.
Rapa-28-O-(2-(4-(2-chlorophenyl)-1H-1,2,3-trizole-1-yl)acetyl)
(6d)
C
62H88N4NaO15 [M+Na]+ = 1128.6238. Found = 1128.6246.
This compound was obtained as yellow solids in 36.4% yield; m.p.:
111.2–114.3°C; MS (ESI) m/z: 1155.5000 (M+Na)+; [a]D −12.8 (c 1.0,
MeOH); IR (KBr) cm−1: 3452.0, 2939.1, 1722.0, 1640.6, 1454.2,
1195.3, 1100.2, 991.2. 1H NMR (600 MHz, DMSO-d6) δ 8.63 (d,
J = 13.4 Hz, 1H), 8.12 (t, J = 8.6 Hz, 1H), 7.60 (dd, J = 31.5, 7.7 Hz, 1H),
7.55–7.44 (m, 1H), 7.40 (t, J = 7.1 Hz, 1H), 6.47 (s, 1H), 6.39 (dd,
J = 26.1, 14.6 Hz, 1H), 6.28 (dd, J = 36.5, 27.6 Hz, 1H), 6.18 (dd,
J = 21.6, 10.2 Hz, 1H), 6.11 (d, J = 10.9 Hz, 1H), 5.47–5.40 (m, 1H), 5.37
(d, J = 14.6 Hz, 1H), 5.03 (d, J = 9.9 Hz, 1H), 4.96 (d, J = 18.6 Hz, 1H),
4.58 (dd, J = 15.6, 11.9 Hz, 1H), 4.30 (dd, J = 30.2, 11.6 Hz, 1H), 4.23–
4.10 (m, 1H), 4.00 (t, J = 19.6 Hz, 1H), 3.65 (dd, J = 37.1, 12.7 Hz, 1H),
3.46 (d, J = 11.3 Hz, 2H), 3.29 (s, 3H), 3.25 (dd, J = 12.4, 4.7 Hz, 1H),
3.20 (s, 1H), 3.13 (dd, J = 27.6, 13.4 Hz, 3H), 3.05 (s, 3H), 2.81 (dd,
J = 15.3, 8.2 Hz, 1H), 2.68 (t, J = 16.1 Hz, 1H), 2.40 (dd, J = 17.3, 8.1 Hz,
1H), 2.19 (s, 1H), 2.12 (t, J = 14.2 Hz, 1H), 2.02 (d, J = 7.2 Hz, 1H), 1.89–
1.81 (m, 3H), 1.75–1.65 (m, 3H), 1.59–1.50 (m, 5H), 1.45–1.33 (m, 2H),
1.25 (dd, J = 40.0, 14.1 Hz, 2H), 1.16 (dd, J = 21.8, 7.9 Hz, 2H), 1.04 (dd,
J = 18.6, 6.3 Hz, 2H), 1.00–0.95 (m, 3H), 0.94 (s, 3H), 0.88–0.81 (m,
Rapa-28-O-(2-(4-(4-fluorinphenyl)-1H-1,2,3-trizole-1-yl)acetyl)
(6f)
This compound was obtained as white solids in 30.3% yield; m.p.:
118.7–120.3°C. MS (ESI) m/z: 1116.6000 (M+Na)+; [a]D −15.6 (c 1.0,
MeOH); IR (KBr) cm−1: 3456.2, 2941.4, 1717.0, 1638.4, 1453.1,
1374.0, 1193.2, 1096.0. 1H NMR (600 MHz, DMSO-d6) δ 8.42 (d,
J = 4.9 Hz, 1H), 7.88 (dd, J = 8.5, 5.5 Hz, 2H), 7.29 (t, J = 8.8 Hz, 2H),
6.44 (s, 1H), 6.37 (dd, J = 14.4, 11.3 Hz, 1H), 6.24–6.16 (m, 1H), 6.12 (t,
J = 12.6 Hz, 1H), 6.08 (d, J = 10.7 Hz, 1H), 5.46–5.41 (m, 1H), 5.33 (s,
2H), 5.07 (d, J = 22.9 Hz, 1H), 4.98 (d, J = 10.9 Hz, 2H), 4.59–4.52 (m,
1H), 4.35 (d, J = 3.3 Hz, 1H), 4.28 (s, 1H), 4.05–3.97 (m, 1H), 3.62 (d,
J = 12.3 Hz, 1H), 3.46 (d, J = 13.5 Hz, 1H), 3.28 (s, 3H), 3.26–3.22 (m,
1H), 3.20 (s, 3H), 3.17 (d, J = 6.3 Hz, 2H), 3.04 (s, 3H), 2.80 (dd, J = 15.6,
8.3 Hz, 1H), 2.66 (d, J = 14.9 Hz, 1H), 2.39 (dd, J = 17.4, 8.2 Hz, 1H),
2.15 (d, J = 13.5 Hz, 1H), 2.03–1.98 (m, 1H), 1.86 (dd, J = 31.1, 14.9 Hz,
3H), 1.74–1.65 (m, 3H), 1.54 (d, J = 15.5 Hz, 5H), 1.43 (dd, J = 24.6,
12.6 Hz, 2H), 1.22 (d, J = 15.2 Hz, 2H), 1.19–1.13 (m, 3H), 1.04 (dd,
J = 14.9, 9.0 Hz, 3H), 0.97 (d, J = 6.4 Hz, 3H), 0.85 (d, J = 6.4 Hz, 6H),

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0.75–0.70 (m, 6H), 0.55 (d, J = 11.7 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 208.7, 206.5, 198.7, 169.2, 166.9, 165.5, 160.7, 145.4,
139.0, 137.9, 132.3, 131.7, 130.4, 127.2, 127.1, 122.5, 115.8, 115.6,
98.9, 83.7, 82.1, 81.7, 77.8, 73.9, 73.1, 66.1, 57.0, 56.6, 55.4, 50.9,
50.3, 45.2, 43.6, 40.8, 40.5, 40.0, 39.8, 39.6, 39.5, 39.3, 39.1, 39.0,
37.9, 35.1, 35.0, 34.6, 33.7, 32.8, 32.5, 31.3, 26.2, 24.4, 21.4, 20.4,
15.5, 15.2, 14.9, 14.1, 12.9, 10.4; HRMS(ESI): calcd. for
J = 28.6 Hz, 1H), 2.17–2.11 (m, 1H), 2.05–1.97 (m, 1H), 1.89 (dt,
J = 25.7, 14.6 Hz, 3H), 1.68 (s, 3H), 1.59–1.47 (m, 5H), 1.45–1.35 (m,
2H), 1.27 (dd, J = 25.5, 13.6 Hz, 2H), 1.22–1.13 (m, 3H), 1.08–1.03 (m,
2H), 0.98 (d, J = 6.3 Hz, 3H), 0.94 (d, J = 7.0 Hz, 1H), 0.86 (d, J = 6.4 Hz,
3H), 0.85–0.82 (m, 1H), 0.80 (d, J = 6.3 Hz, 3H), 0.74 (t, J = 6.4 Hz, 6H),
0.57 (dd, J = 23.7, 11.9 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ
209.3, 207.2, 199.2, 169.7, 167.4, 166.1, 145.4, 143.2, 139.5, 138.4,
132.9, 132.2, 130.9, 128.6, 128.1, 127.4, 126.5, 124.7, 119.7, 118.8,
112.9, 107.4, 99.4, 84.2, 82.6, 82.2, 78.1, 74.2, 73.7, 71.8, 66.6, 57.6,
57.2, 55.9, 51.4, 50.6, 45.6, 44.1, 41.0, 40.4, 40.2, 40.1, 39.9, 39.8,
39.7, 39.5, 38.7, 35.6, 35.2, 34.1, 33.3, 33.0, 31.8, 30.0, 26.5, 25.1,
22.0, 20.9, 19.5, 16.0, 15.7, 15.4, 14.4, 13.7, 10.9 HRMS(ESI): calcd. for
C
61H85FN4NaO14 [M+Na]+ = 1116.6030. Found = 1116.6046.
Rapa-28-O-(2-(4-((3-aminophenyl)aminomethylene)-1H-1,2,3-
trizole-1-yl)acetyl) (6g)
This compound was obtained as white solids in 79.1% yield; m.p.:
132.3–135.8°C; MS (ESI) m/z: 1136.6000 (M+Na)+; [a]D −17.6 (c 1.0,
MeOH); IR (KBr) cm−1: 3377.9, 2933.9, 1728.3, 1642.2, 1447.2,
1198.1, 1093.8, 993.8. 1H NMR (600 MHz, DMSO-d6) δ 8.27 (s, 1H),
7.12 (s, 1H), 7.09–7.04 (m, 1H), 6.95 (t, J = 7.2 Hz, 1H), 6.54 (dd, J = 7.9,
1.5 Hz, 1H), 6.47 (s, 1H), 6.39 (dd, J = 14.6, 11.4 Hz, 1H), 6.21 (dd,
J = 14.5, 10.8 Hz, 1H), 6.11 (dd, J = 14.5, 11.0 Hz, 2H), 5.43 (dd,
J = 14.9, 9.7 Hz, 1H), 5.35 (dd, J = 10.3, 7.4 Hz, 2H), 5.16 (s, 2H), 5.10 (d,
J = 9.1 Hz, 1H), 5.01–4.95 (m, 2H), 4.61–4.57 (m, 1H), 4.34 (d,
J = 3.7 Hz, 1H), 4.31–4.25 (m, 1H), 4.02 (t, J = 8.1 Hz, 1H), 3.63 (d,
J = 12.9 Hz, 1H), 3.47 (d, J = 12.8 Hz, 1H), 3.30 (s, 3H), 3.27 (s, 1H), 3.21
(s, 3H), 3.05 (d, J = 3.9 Hz, 3H), 2.84–2.79 (m, 1H), 2.70–2.65 (m, 1H),
2.42 (dd, J = 17.5, 8.2 Hz, 1H), 2.20 (s, 1H), 2.16 (d, J = 13.0 Hz, 1H),
2.02 (dd, J = 14.7, 7.2 Hz, 1H), 1.88–1.81 (m, 3H), 1.79 (s, 3H), 1.68 (d,
J = 11.2 Hz, 3H), 1.61 (d, J = 6.4 Hz, 3H), 1.60–1.50 (m, 5H), 1.46–1.36
(m, 2H), 1.31–1.23 (m, 2H), 1.22–1.13 (m, 3H), 1.10–1.01 (m, 3H), 0.98
(d, J = 6.5 Hz, 3H), 0.86 (t, J = 5.7 Hz, 3H), 0.77 (d, J = 6.5 Hz, 3H), 0.74
(t, J = 6.9 Hz, 6H), 0.57 (d, J = 11.8 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 209.3, 207.4, 199.3, 169.7, 167.4, 166.1, 149.4, 147.5,
139.5, 138.4, 132.8, 132.2, 131.4, 130.9, 129.7, 127.5, 122.7, 114.0,
113.5, 110.9, 99.4, 84.2, 82.6, 78.2, 74.4, 73.6, 66.6, 57.6, 57.1, 55.9,
55.3, 51.4, 50.7, 45.7, 44.1, 41.1, 40.7, 40.4, 40.2, 40.1, 39.9,
39.8, 39.7, 39.5, 38.4, 35.6, 35.5, 35.2, 34.2, 33.3, 33.0, 31.8,
30.2, 30.1, 26.8, 24.9, 22.0, 20.9, 16.0, 15.8, 15.4, 14.5, 13.6, 10.9;
C
62H86Cl2N5O14 [M−H]− = 1195.5608. Found = 1195.5627.
Rapa-28-O-(2-(4-(methene-(N,N-diethyl))-1H-1,2,3-trizole-1-yl)-
acetyl) (7b)
This compound was obtained as white solids in 67.6% yield; m.p.:
105.5–108.3°C; MS (ESI) m/z: 1108.6000 (M+H)+; [a]D −34.2 (c 1.0,
MeOH); IR (KBr) cm−1: 3442.5, 2943.6, 1726.6, 1643.0, 1456.6,
1209.4, 1106.6, 999.2. 1H NMR (600 MHz, DMSO-d6) δ 7.91 (s, 1H),
6.45 (d, J = 22.6 Hz, 1H), 6.39 (dd, J = 14.3, 11.5 Hz, 1H), 6.21 (dd,
J = 24.1, 9.5 Hz, 1H), 6.16–6.12 (m, 1H), 6.11 (d, J = 9.9 Hz, 1H), 5.47–
5.41 (m, 1H), 5.32 (s, 2H), 5.00 (d, J = 9.9 Hz, 1H), 4.95 (d, J = 3.7 Hz,
2H), 4.59 (d, J = 27.7 Hz, 1H), 4.34 (d, J = 3.4 Hz, 1H), 4.27 (d,
J = 18.1 Hz, 1H), 4.00 (dd, J = 12.6, 9.0 Hz, 1H), 3.77 (s, 2H), 3.63 (d,
J = 11.6 Hz, 1H), 3.46 (d, J = 12.8 Hz, 1H), 3.30 (d, J = 7.7 Hz, 3H), 3.28
(d, J = 8.1 Hz, 1H), 3.20 (s, 3H), 3.16 (d, J = 12.6 Hz, 2H), 3.05 (s, 3H),
2.84–2.78 (m, 1H), 2.65 (d, J = 15.7 Hz, 1H), 2.53–2.46 (m, 4H), 2.39
(dd, J = 17.5, 8.3 Hz, 1H), 2.23 (s, 1H), 2.14 (d, J = 18.0 Hz, 1H), 2.04–
1.99 (m, 1H), 1.85 (t, J = 11.8 Hz, 3H), 1.79 (s, 3H), 1.68 (dd, J = 18.4,
9.8 Hz, 3H), 1.62 (s, 3H), 1.56 (dd, J = 18.9, 12.3 Hz, 5H), 1.41 (dd,
J = 21.6, 12.6 Hz, 2H), 1.31–1.24 (m, 2H), 1.17 (dd, J = 18.5, 11.2 Hz,
3H), 1.04 (t, J = 6.9 Hz, 9H), 0.99 (d, J = 6.4 Hz, 3H), 0.87 (d, J = 6.3 Hz,
3H), 0.82 (d, J = 6.4 Hz, 3H), 0.74 (dd, J = 5.9, 3.5 Hz, 6H), 0.56 (d,
J = 11.8 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 207.4, 199.1, 191.7,
169.6, 167.0, 166.0, 144.9, 138.1, 132.2, 127.5, 123.8, 99.4, 84.2,
82.2, 77.9, 74.2, 73.2, 66.5, 60.8, 57.2, 55.9, 51.4, 50.2, 45.6, 40.4,
40.2, 40.1, 39.9, 39.8, 39.7, 39.5, 35.4, 32.9, 31.7, 29.8, 29.5, 22.0,
16.0, 15.7, 15.4, 14.3, 13.4, 10.8; HRMS (ESI): calcd. for C60H94N5O14
[M+H]+ = 1108.6792. Found = 1108.6787.
HRMS (ESI): calcd. for
Found = 1136.6132.
C
61H87N5NaO14 [M+Na]+ = 1136.6142.
Rapa-28-O-(2-(4-((2,4-dichlorophenyl)aminomethylene)-1H-
1,2,3-trizole-1-yl)acetyl) (7a)
This compound was obtained as white solids in 42.9% yield; m.p.:
110.2–113.8°C; MS (ESI) m/z: 1195.5000 (M−H)−; [a]D −16.9 (c 1.0,
MeOH); IR (KBr) cm−1: 3425.6, 2933.6, 1722.3, 1641.0, 1425.3,
1096.6, 989.3. 1H NMR (600 MHz, DMSO-d6) δ 7.91–7.87 (m, 1H),
7.36 (t, J = 6.7 Hz, 1H), 7.14 (dt, J = 7.0, 3.5 Hz, 1H), 6.81 (d, J = 8.9 Hz,
1H), 6.47 (s, 1H), 6.39 (dd, J = 14.3, 11.5 Hz, 1H), 6.20 (dd, J = 23.1,
8.5 Hz, 1H), 6.16–6.08 (m, 2H), 6.06 (d, J = 5.9 Hz, 1H), 5.49 (dd,
J = 21.0, 10.7 Hz, 1H), 5.36–5.25 (m, 2H), 5.08 (d, J = 7.4 Hz, 1H), 5.00
(d, J = 9.9 Hz, 1H), 4.96 (d, J = 4.1 Hz, 1H), 4.61 (d, J = 4.0 Hz, 1H), 4.44
(d, J = 5.9 Hz, 2H), 4.29 (d, J = 3.5 Hz, 1H), 4.05–3.94 (m, 2H), 3.93 (d,
J = 7.1 Hz, 1H), 3.63 (d, J = 12.0 Hz, 1H), 3.46 (d, J = 12.8 Hz, 1H), 3.34
(s, 3H), 3.29–3.20 (m, 2H), 3.19 (d, J = 5.5 Hz, 3H), 3.04 (d, J = 18.5 Hz,
3H), 2.85–2.79 (m, 1H), 2.72–2.60 (m, 1H), 2.44–2.31 (m, 2H), 2.24 (d,
Rapa-28-O-(2-(4-((2-fluorinphenyl)aminomethylene)-1H-1,2,3-
trizole-1-yl)acetyl) (7c)
This compound was obtained as white solids in 69.7% yield; m.p.:
82.5–85.5°C; MS (ESI) m/z: 1168.6000 (M+Na)+; [a]D −22.7 (c 1.0,
MeOH); IR (KBr) cm−1: 3422.6, 2933.7, 1723.4, 1649.1, 1444.3,
1197.6, 1100.1, 988.3. 1H NMR (600 MHz, DMSO-d6) δ 7.89 (s, 1H),
7.20–7.12 (m, 1H), 7.02–6.98 (m, 1H), 6.93 (d, J = 8.0 Hz, 1H), 6.82–
6.77 (m, 1H), 6.57–6.52 (m, 1H), 6.47 (s, 1H), 6.39 (dd, J = 14.7, 11.3 Hz,
1H), 6.25–6.19 (m, 1H), 6.17–6.12 (m, 1H), 6.12–6.09 (m, 1H), 5.43 (dd,
J = 14.8, 9.8 Hz, 1H), 5.31 (d, J = 5.4 Hz, 2H), 5.09 (d, J = 4.1 Hz, 1H),
5.00 (d, J = 10.0 Hz, 1H), 4.96 (d, J = 5.0 Hz, 1H), 4.61 (dd, J = 6.4,
4.4 Hz, 1H), 4.39 (d, J = 5.8 Hz, 2H), 4.01 (dd, J = 19.4, 9.2 Hz, 1H), 3.95

HUANG ET AL.
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(dd, J = 6.3, 1.1 Hz, 1H), 3.63 (d, J = 12.8 Hz, 1H), 3.45 (t, J = 14.0 Hz,
1H), 3.31 (s, 3H), 3.29 (d, J = 3.9 Hz, 1H), 3.19 (t, J = 5.6 Hz, 3H), 3.05 (s,
3H), 2.85–2.80 (m, 1H), 2.66 (d, J = 15.6 Hz, 1H), 2.43–2.37 (m, 2H),
2.24–2.20 (m, 1H), 2.16–2.12 (m, 1H), 2.02 (dd, J = 14.6, 7.1 Hz, 1H),
1.85 (t, J = 13.1 Hz, 3H), 1.70–1.64 (m, 3H), 1.59–1.50 (m, 5H), 1.40
(dd, J = 27.2, 13.9 Hz, 2H), 1.27 (dd, J = 14.2, 9.2 Hz, 2H), 1.22–1.16 (m,
3H), 1.05 (ddd, J = 16.3, 10.9, 9.1 Hz, 3H), 0.99 (d, J = 6.4 Hz, 3H), 0.87
(d, J = 6.4 Hz, 3H), 0.82 (d, J = 6.5 Hz, 3H), 0.74 (dd, J = 6.4, 3.1 Hz, 6H),
0.58 (d, J = 11.8 Hz, 1H). 13C NMR (151 MHz, DMSO-d6) δ 209.1,
207.4, 199.2, 169.6, 167.4, 166.2, 152.2, 150.2, 146.0, 143.6, 139.6,
138.4, 136.6, 132.9, 132.2, 130.9, 127.4, 125.1, 121.8, 118.5, 116.2,
114.7, 112.6, 99.4, 84.2, 82.6, 79.5, 78.2, 76.3, 74.3, 74.0, 73.7, 71.8,
66.6, 57.6, 57.2, 55.9, 51.4, 50.4, 45.6, 44.1, 40.4, 40.2, 40.1, 39.9,
39.8, 39.7, 39.5, 38.5, 35.6, 35.2, 33.3, 33.0, 31.8, 30.0, 28.6,
26.6, 22.0, 21.4, 20.9, 19.7, 16.0, 15.7, 15.4, 14.4, 13.7, 10.9, 10.0;
1H), 5.07 (d, J = 3.3 Hz, 1H), 4.99–4.93 (m, 2H), 4.61 (d, J = 4.3 Hz, 1H),
4.31 (d, J = 3.9 Hz, 1H), 4.30–4.24 (m, 1H), 4.04–3.97 (m, 1H), 3.63 (d,
J = 13.3 Hz, 1H), 3.46 (d, J = 12.5 Hz, 1H), 3.31 (s, 3H), 3.28 (d,
J = 4.4 Hz, 1H), 3.20 (s, 3H), 3.15 (d, J = 4.5 Hz, 2H), 3.05 (s, 3H), 2.84–
2.80 (m, 1H), 2.67 (dd, J = 17.7, 2.8 Hz, 1H), 2.43 (dd, J = 17.7, 8.3 Hz,
2H), 2.21 (d, J = 8.6 Hz, 1H), 2.12 (s, 1H), 2.04–2.01 (m, 1H), 1.88–1.81
(m, 3H), 1.77 (s, 3H), 1.69–1.65 (m, 3H), 1.62 (s, 3H), 1.60–1.50 (m, 5H),
1.47 (s, 6H), 1.44–1.35 (m, 2H), 1.22–1.14 (m, 3H), 1.10–1.01 (m, 3H),
0.99 (d, J = 6.5 Hz, 3H), 0.88 (d, J = 6.5 Hz, 3H), 0.84 (d, J = 6.5 Hz, 3H),
0.74 (t, J = 6.0 Hz, 6H), 0.58 (d, J = 11.9 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6) δ 209.5, 207.4, 199.1, 169.3, 167.3, 166.2, 156.2, 138.2,
132.1, 127.5, 122.3, 99.3, 84.2, 73.6, 67.4, 57.7, 57.2, 55.9, 51.1, 50.5,
45.5, 40.4, 40.2, 40.1, 40.0, 39.8, 39.7, 39.5, 35.6, 32.8, 31.0, 29.9,
26.5, 22.0, 20.9, 16.0, 15.8, 13.9, 10.8; HRMS (ESI): calcd. for
C
58H88N4NaO15 [M+Na]+ = 1080.6228. Found = 1080.6246.
HRMS (ESI): calcd. for
Found = 1168.6184.
C
62H88FN5NaO14 [M+Na]+ = 1168.6204.
Rapa-28-O-(2-(4-(hydroxyethyl)-1H-1,2,3-trizole-1-yl)acetyl)
(7f)
Rapa-28-O-(2-(4-((2,6-difluorinphenyl)aminomethylene)-1H-
1,2,3-trizole-1-yl)acetyl) (7d)
This compound was obtained as white solids in 39.7% yield; m.p.:
81.6–83°C; MS (ESI) m/z: 1066.6000 (M+Na)+; [a]D −39.8 (c 1.0,
MeOH); IR (KBr) cm−1: 3423.9, 2933.7, 1728.8, 1637.3, 1449.1,
1192.8, 1096.3, 986.8. 1H NMR (600 MHz, DMSO-d6) δ 7.77 (s, 1H),
6.47 (d, J = 1.4 Hz, 1H), 6.39 (dd, J = 14.7, 11.3 Hz, 1H), 6.25–6.19 (m,
1H), 6.15–6.12 (m, 1H), 6.11 (d, J = 10.5 Hz, 1H), 5.50–5.43 (m, 1H),
5.31 (t, J = 4.7 Hz, 2H), 5.27 (s, 1H), 5.11–5.06 (m, 1H), 5.00–4.93 (m,
2H), 4.71–4.67 (m, 2H), 4.62–4.57 (m, 1H), 4.34 (d, J = 3.7 Hz, 1H), 4.26
(dd, J = 18.8, 3.8 Hz, 1H), 4.01 (t, J = 9.9 Hz, 1H), 3.66–3.60 (m, 1H),
3.49–3.43 (m, 1H), 3.32–3.29 (m, 3H), 3.29 (d, J = 4.4 Hz, 1H), 3.20 (s,
3H), 3.16 (dd, J = 6.6, 3.9 Hz, 2H), 3.06 (d, J = 4.7 Hz, 3H), 2.84–2.80 (m,
1H), 2.77 (dd, J = 12.7, 5.6 Hz, 2H), 2.66 (dd, J = 17.6, 2.6 Hz, 1H), 2.40
(dd, J = 17.6, 8.4 Hz, 1H), 2.21 (d, J = 8.8 Hz, 1H), 2.17–2.12 (m, 2H),
2.02 (dd, J = 15.4, 7.4 Hz, 1H), 1.85 (t, J = 12.3 Hz, 3H), 1.79 (s, 3H),
1.71–1.65 (m, 3H), 1.62 (s, 3H), 1.60–1.50 (m, 5H), 1.42 (dd, J = 26.9,
14.2 Hz, 2H), 1.29–1.23 (m, 2H), 1.22–1.13 (m, 3H), 1.10–1.01 (m, 3H),
0.99 (d, J = 6.5 Hz, 3H), 0.86 (d, J = 6.5 Hz, 3H), 0.81 (d, J = 6.5 Hz, 3H),
0.74 (d, J = 6.6 Hz, 6H), 0.57 (dd, J = 23.8, 12.0 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 207.4, 199.1, 191.7, 169.6, 167.0, 166.0,
144.9, 138.1, 132.2, 127.5, 123.8, 99.4, 84.2, 82.2, 77.9, 74.2, 73.2,
66.5, 60.8, 57.2, 55.9, 51.4, 50.2, 45.6, 40.4, 40.2, 40.1, 39.9,
39.8, 39.7, 39.5, 35.4, 32.9, 31.7, 29.8, 29.5, 22.0, 16.0, 15.7,
15.4, 14.3, 13.4, 10.8; HRMS (ESI): calcd. for C57H86N4NaO15
[M+Na]+ = 1066.6058. Found = 1066.6090.
This compound was obtained as white solids in 61.9% yield; m.p.:
85.7–88.4°C; MS (ESI) m/z: 1186.6000 (M+Na)+; [a]D −28.9 (c 1.0,
MeOH); IR (KBr) cm−1: 3383.1, 2932.4, 1723.6, 1647.0, 1459.9,
1196.3, 1090.5, 999.3. 1H NMR (600 MHz, DMSO-d6) δ 7.84 (s, 1H),
7.07–6.94 (m, 2H), 6.91 (d, J = 7.7 Hz, 1H), 6.66 (s, 1H), 6.47 (s, 1H),
6.43–6.35 (m, 1H), 6.24–6.17 (m, 1H), 6.12 (d, J = 8.1 Hz, 2H), 5.47–
5.38 (m, 1H), 5.28 (d, J = 24.9 Hz, 2H), 5.12 (d, J = 31.5 Hz, 1H), 5.03–
4.92 (m, 2H), 4.59 (t, J = 16.4 Hz, 1H), 4.48 (d, J = 6.0 Hz, 2H), 4.42–
4.25 (m, 1H), 3.96 (dd, J = 30.0, 24.2 Hz, 1H), 3.65 (t, J = 20.7 Hz, 1H),
3.46 (d, J = 11.9 Hz, 1H), 3.31 (s, 3H), 3.29 (s, 1H), 3.19 (s, 3H), 3.15–
3.09 (m, 2H), 3.05 (s, 3H), 2.82 (s, 1H), 2.65 (d, J = 16.2 Hz, 1H), 2.39
(dd, J = 17.5, 8.8 Hz, 1H), 2.22 (s, 1H), 2.13 (s, 1H), 2.04–1.97 (m, 1H),
1.85 (s, 3H), 1.77 (s, 3H), 1.68 (s, 3H), 1.62 (s, 3H), 1.59–1.50 (m, 5H),
1.42 (d, J = 10.4 Hz, 2H), 1.27 (s, 2H), 1.17 (dd, J = 22.2, 8.5 Hz, 3H),
1.05 (d, J = 12.6 Hz, 3H), 0.99 (d, J = 5.1 Hz, 3H), 0.89–0.84 (m, 3H),
0.82 (s, 3H), 0.74 (d, J = 5.2 Hz, 6H), 0.57 (d, J = 11.3 Hz, 1H). 13C NMR
(151 MHz, DMSO-d6) δ 209.1, 207.4, 199.4, 169.3, 166.2, 153.6,
152.2, 146.5, 139.6, 138.4, 132.8, 132.1, 130.9, 127.4, 125.6, 124.3,
117.5, 112.8, 112.2, 102.1, 99.4, 84.3, 82.6, 82.2, 78.0, 74.2, 73.7,
71.8, 66.6, 57.6, 57.2, 55.9, 51.3, 50.4, 45.6, 41.0, 40.4, 40.2, 40.1,
40.0, 39.8, 39.7, 39.5, 35.5, 35.1, 33.0, 31.8, 30.3, 28.6, 26.6, 24.9,
22.0, 19.6, 16.0, 15.7, 15.4, 14.4, 13.8, 10.9, 10.0; HRMS (ESI): calcd.
for C62H87F2N5NaO14 [M+Na]+ = 1186.6110. Found = 1186.6088.
|
4.2 Biological assays
Rapa-28-O-(2-(4-(2-hydroxylisopropyl)-1H-1,2,3-trizole-1-yl)-
acetyl) (7e)
|
4.2.1 Reagents
This compound was obtained as white solids in 37.7% yield; m.p.:
94.7–96.8°C; MS (ESI) m/z: 1080.6000 (M+Na)+; [a]D −12.8 (c 1.0,
MeOH); IR (KBr) cm−1: 3452.6, 2939.7, 2355.6, 1722.6, 1642.2,
1454.8, 1374.9, 1196.2, 1089.6, 991.3. 1H NMR (600 MHz, DMSO-d6)
δ 7.81 (s, 1H), 6.47 (d, J = 1.1 Hz, 1H), 6.40 (dd, J = 14.5, 11.3 Hz, 1H),
6.22 (dd, J = 14.7, 10.7 Hz, 1H), 6.14 (dd, J = 26.7, 12.2 Hz, 2H), 5.42
(dd, J = 17.3, 7.7 Hz, 1H), 5.34 (d, J = 3.0 Hz, 1H), 5.12 (d, J = 6.4 Hz,
Rapamycin (Fujian Kerui Pharmaceutical Co. Ltd.) and new
rapalogs were dissolved in dimethyl sulfoxide (DMSO) to prepare
a 10.0 mg/mL stock solution and stored at −20°C. Sulforhod-
amine B (SRB, Sigma) was dissolved in 1% glacial acetic acid to
prepare
a 0.4% solution. Antibodies included those against
S6K1, 4E-BP1, mTOR, phospho-S6K1 (Thr389), phospho-4E-

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HUANG ET AL.
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BP1 (Thr70), phospho-mTOR (Ser2448) were purchased from Cell
Signaling, USA.
4.2.6 Western blotting analysis
Western blotting analysis was performed as described previously.[17]
Briefly, cells were lysed in 2% SDS. Equal amounts of lysate protein
were subjected to SDS–PAGE and transferred onto nitrocellulose
membranes (Bio-Rad Laboratories, USA). The target protein carrying
membranes were blocked with 5% milk and incubated with the primary
antibody at 4°C overnight. The membranes were then washed in TBST
buffer three times and incubated with the secondary antibody for 1 h.
Wash the membrane as previous procedure. Bands were visualized by
using the enhanced chemiluminescence Western blotting detection
system (GE, USA).
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4.2.2 Cell culture
All cancer cells were purchased from Cell Bank of Chinese Academy of
Sciences, Shanghai Institute of Cell Biology (Shanghai, China). Renal
cell adenocarcinoma 769-P and esophageal squamous carcinoma ECA-
109 were maintained in RPMI-1640 medium (Gibco, USA). Lung
carcinoma A549 was maintained in F12 medium (Gibco). Cervical
cancer cell line CASKI cells were maintained in RPMI medium (Gibco,
USA), prostate adenocarcinoma PC-3 was maintained in F12 medium
(Gibco). All media were supplemented with 10% fetal bovine serum
(Gibco) and gentamycin (80.0 U/mL), and all cells were grown in a
humid incubator (37°C, 5% CO2).
ACKNOWLEDGMENT
This study was supported by grants from the National Natural Science
Foundation of China (81502935).
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4.2.3 Anticancer activities
The anticancer activities of the compounds were evaluated against
A549, PC-3, CASKI, 769-P, ECA-109 cancer cell lines by sulforhod-
amine B (SRB, Sigma) assay in vitro, as described previously.[21]
Results were expressed as mean standard deviation (SD) of
independent experiments, and statistical analysis was performed using
Student's t-test. All statistical analyses were performed using SPSS 20
statistical analysis software. Values with p < 0.05 were considered to
indicate statistical significance.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
ORCID
Qingwen Huang
http://orcid.org/0000-0001-8999-1215
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4.2.4 Apoptotic cell staining
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Additional supporting information may be found online in the
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How to cite this article: Huang Q, Xie L, Chen X, et al.
Synthesis and anticancer activity of novel rapamycin C-28
containing triazole moiety compounds. Arch Pharm Chem Life
Sci. 2018;1–13. https://doi.org/10.1002/ardp.201800123
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