A novel and practical synthesis of CAT3: A phenanthroindolizidine alkaloid with potential in treating glioblastoma

Article abstract of DOI:10.1039/c8ra04511a

CAT3, one of the (+)-deoxytylophorinine-based phenanthroindolizidine alkaloids, is a promising therapeutic agent for the treatment of hedgehog (Hh)-driven glioblastoma and is currently being evaluated in preclinical studies. In this paper, a novel and practical synthetic route for CAT3 was firstly demonstrated with 10% overall yield in 11 steps and has been successfully validated for pilot-plant scale preparation. Investigation of the substitution at the 3-position of phenanthrene revealed that the electron-donating functionality can well preserve the S configuration. In particular, the excellent enantiomeric excess of CAT3 (≥99% ee) was achieved by introducing the strongly electron-donating tert-butyldimethylsilyl (TBS) group.

Full text of DOI:10.1039/c8ra04511a

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A novel and practical synthesis of CAT3:
a phenanthroindolizidine alkaloid with potential in
Cite this: RSC Adv., 2018, 8, 29301
treating glioblastoma†
Ru-Bing Wang,
Shuang-Gang Ma, Yun-Bao Liu, Jing Qu and Shi-Shan Yu
Hai-Ning Lv, Shan-Shan Zhu, Xiao-Dong Ren,
Song Xu,
*
*
CAT3, one of the (+)-deoxytylophorinine-based phenanthroindolizidine alkaloids, is
a promising
therapeutic agent for the treatment of hedgehog (Hh)-driven glioblastoma and is currently being
evaluated in preclinical studies. In this paper, a novel and practical synthetic route for CAT3 was firstly
demonstrated with 10% overall yield in 11 steps and has been successfully validated for pilot-plant scale
preparation. Investigation of the substitution at the 3-position of phenanthrene revealed that the
electron-donating functionality can well preserve the S configuration. In particular, the excellent
enantiomeric excess of CAT3 ($99% ee) was achieved by introducing the strongly electron-donating
tert-butyldimethylsilyl (TBS) group.
Received 29th May 2018
Accepted 13th July 2018
DOI: 10.1039/c8ra04511a
rsc.li/rsc-advances
and
(13aS)-3-pivaloyloxyl-6,7-dimethoxyphenanthro[9,10-b]
Introduction
indolizidine (CAT3) was designed and synthesized as the pro-
drug of PF403 (Fig. 1). A series of preclinical studies has proven
that CAT3 is a promising therapeutic agent against hedgehog
(Hh)-driven GBM.10,12 Although the structure of CAT3 only
differs slightly from that of CAT, the synthesis of which has
been accomplished in several total syntheses,13–15 the mutigram
synthesis of CAT3 is still challenging. A main problem lies in the
completely different starting materials due to the methoxy and
pivaloyoxy group for CAT and CAT3, respectively, used in the
synthesis. When the same synthetic strategy of CAT was used to
synthesize CAT3,13–15 it did not work aer attempting multiple
times in the past. This challenge, together with its excellent
potency for GBM, stimulated us to investigate a novel and effi-
cient synthetic route for CAT3.
Glioblastoma (GBM) is one of the most common and malignant
brain tumors in the human central nervous system along with
high invasion, high mortality and poor prognoses,1,2 which ends
with an average survival of 15–17 months even with the best
available treatment.3 Temozolomide (TMZ), the principal rst-
line chemotherapeutic agent, is widely used for the treatment
of recurrent and high-grade glioma and is proven to be an
efficient inhibitor of GBM proliferation.4,5 However, due to the
emergence of intrinsic and acquired TMZ resistance in GBM
cells, its use is seriously limited.6,7 Therefore, there is an urgent
need to develop new therapeutic agents for GBM disease.
(+)-Deoxytylophorinine (CAT) was isolated from the roots of
Tylophora atrofolliculata and Tylophora ovata and showed
signicant anticancer properties in vivo with its ability to well
penetrate the blood brain barrier,8–11 while serious damage on
gastrointestine was also observed. In the study of its metabolic
products in vivo, (13aS)-3-hydroxyl-6,7-dimethoxyphenanthro
[9,10-b]indolizidine (PF403, Fig. 1) was found to possess excel-
lent antiproliferative activity with the IC50 value less than
0.01 nM in multiple cancer cell lines in vitro.10–12 However, the
excellent data in vitro cannot be reected to its data in vivo
probably due to its poor pharmacokinetic properties. To
address this issue, the prodrug strategy was employed herein
Results and discussion
Strategically, CAT3 can be easily synthesized from acylation of
PF403. Therefore, the efficient synthesis of PF403 is a key
question. Takashi and coworkers disclosed the synthesis of
PF403 starting from 4-benzyloxybenzaldehyde and 2-(3,4-dime-
thoxyphenyl)acetic acid with 10.8% yield in 13 steps,16,17 which
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union
Medical College, Beijing 100050, China. E-mail: yushishan@imm.ac.cn; qujing@
imm.ac.cn; Fax: +86-10-63017757
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra04511a
Fig. 1 The development process of the preclinical drug CAT3.
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was the rst meaningful synthetic route. However, photo-
induced electron cyclization reaction was used to construct
the phenanthrene ring, and it was not efficient for large-scale
preparation.18 PF403 was also successfully prepared in 16
steps in our previous work, but the route was very tedious.15
Moreover, it has been demonstrated that it is only applicable for
small-amount synthesis. When we tried multigram synthesis,
the yield decreased to as low as 2%, making it unacceptable for
the large-scale preparation of CAT3.
Thus it is necessary to develop a novel synthesis of PF403.
The retrosynthetic analysis was illustrated in Scheme 1. We
envisioned that PF403 could be accessible from the interme-
diate A, which could be readily made by reduction and protec-
tion from B. It was envisaged that B could be assembled from
compound C and D by a substitution reaction.
Scheme
2
A simple synthetic route firstly designed for PF403.
Reagents and conditions: (a) PPh3, imidazole, I2, DCM, r.t., then methyl
L-prolinate hydrochloride, imidazole, r.t.; (b) DIBAL-H, DCM. 0 ꢀC; 40%
for two steps.
Based on our retrosynthetic analysis, the synthesis of PF403
commenced with the known compound 1, which was obtained
using our reported method in 6 steps.15 The ester 2 was easily
prepared from the condensation of 1 with commercially avail-
able methyl L-prolinate hydrochloride. Compound 3, which was
expected to produce phenanthroindolizidine by eliminating
one molecule of H2O, was easily obtained by reducing 2 with
diisobutylaluminum hydride.19 However, multiple attempts to
construct the indolizidine ring from 3 in one step failed
according to literature,20,21 even if we tried to convert the
hydroxy group to a better leaving group such as MsO–, TfO– or
TsO–. Except compound 1, other different substitutions on 3-
position was tried, such as TBSO– and HO–, but both result were
disappointed. Thus the preparation of PF403 via this route
proved probably impracticable (Scheme 2).
We deduce that there are two probable pathways starting from
3 under the acid condition to construct phenanthroindolizidine.
Theoretically, the route should work well if the aromatic electron
at 10-position of phenanthrene ring attacks carbocation to
generate phenanthroindolizidine via pathway 1. However, iden-
tication of the chlorinated compound 5 suggests that the car-
bocation, which probably arised from transition state 4 with the
aid of the nitrogen lone-pair electrons, appears to favor the
formation of the nitrogen-containing three-membered ring
structure as a transition state via pathway 2 (Scheme 3).
acid and 4-hydroxybenzaldehyde gave compound 6 in almost
quantitative yield, which was conrmed by difference NOE
spectrum as cis-conguration (see ESI†). Compound 9 was
prepared in 50% overall yield in three steps, including the scroll
coupling in the presence of FeCl3, esterication in methanol
with con. H2SO4, substitution of 3-OH with tert-butyldime-
thylsilyl chloride (TBSCl). The key intermediate 10 was achieved
in 95% yield by the reduction of 9 with diisobutylaluminum
hydride, which was transformed to 11 in 50% yield through
a reaction sequence including iodine substitution, nucleophilic
substitution with L-glutamic acid dimethyl ester hydrochloride,
and subsequent hydrolysis. Aer formation of 12 in 80% yield
by Friedel–Cras reaction from 11, its 3-position hydroxyl group
was again protected by TBS to give 13 in 85% yield. The 14-
position carbonyl of 13 was then reduced to compound 14 in
80% yield, in which the lactam carbonyl group was further
reduced to methylene in 95% yield, and PF403 was successfully
synthesized till now. Aer PF403 in hand, we rstly tried to
Aer multiple attempts, we eventually found a novel and
practical synthetic route to produce CAT3 in 11 steps (Scheme
4). The condensation between 2-(3,4-dimethoxyphenyl)acetic
Scheme 3 A plausible mechanism explaining the failure in con-
Scheme 1 The retrosynthetic analysis of CAT3.
29302 | RSC Adv., 2018, 8, 29301–29308
structing the phenanthroindolizidine.
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Scheme 4 Reagents and conditions for the large-scale preparation route of CAT3: (a) Ac2O, Et3N, reflux; (b) FeCl3, m-CPBA, TFA, r.t.; (c) MeOH,
H2SO4, reflux; (d) TBSCl, imidazole, DCM, r.t.; 50% in four steps; (e) DIBAL-H, DCM. 0 ꢀC to r.t., 95%; (f) i: NaI, TMSCl, then L-dimethyl glutamate,
K2CO3, r.t.; ii: 5% HAc, r.t.; iii: NaOH, CH3OH, r.t., then HCl; 55%; (g) TFAA, r.t., then BF3–Et2O, reflux, 85%; (h) TBSCl, imidazole, DMF, r.t., 85%; (i)
NaBH4, DCM-MeOH, then Et3SiH, BF3–Et2O, r.t., 80%; (j) Red-Al, THF, r.t., 95%; (k) pivalic anhydride, Et3N, DMAP, DCM, r.t., 70%.
prepare CAT3 by acylation of PF403 using pivaloyl chloride. temperature used in this step. Different kinds of solvents or/and
However, a main by-product chlorinated substitution on 4- their combination were tested, such as diethylene glycol
position of CAT3 was produced, which was difficult to be dimethyl, tetrahydrofuran, methanol, ethanol, dichloro-
separated because of its polarity almost the same as CAT3. At methane and so on. Meanwhile, we tried the reaction at
last, pivaloyl chloride was replaced by pivalic anhydride and different temperatures, including ꢁ20 ꢀC, 0 ꢀC and 20 ꢀC.
CAT3 was smoothly achieved with a yield as high as 70% Unfortunately, the results were unsatisfactory (27–93% ee,
avoiding the by-product. In general, there are several aspects of Scheme 5) in all these cases. Then we turned to modify the 3-
advantages for this route as follows: (1) the conventional design position of compound 12. Three different substitutes were
for the synthesis of 6 is broken so that no protection step is selected and compared, including the free hydroxyl group, and
required for 4-hydroxybenzaldehyde, thus leading to a shorter the hydroxyl protected with the electron-donating benzyl and
synthesis; (2) the operation and post treatment of the reactions electron-withdrawing acetyl group. The best enantiomeric
in this route are easy and convenient, which also result in a high excess was obtained when 3-position hydroxyl was protected as
overall yield (10%), and no traditional column chromatography benzyl ether (95–98% ee, Scheme 6) while the worst result ob-
is used; (3) pilot-scale preparation has been achieved for this tained when protected as acetyl ester (10–50% ee, Scheme 7).
route with cheap reagents and materials used, indicating that it Thus it can be preliminarily inferred that substitution on 3-
is probably an economical route for large-scale synthesis; (4) all position is related with the enantiomeric excess of the nal
the chiral intermediates and products are prepared in high product, and stronger electron-donating ability may further
optical purity ($99% ee), which is vital for a drug candidate.
increase the optical purity of the product. As we expect, excellent
It should be noted that compound 14 can be synthesized in enantiomeric excess was achieved when the 3-position hydroxyl
one step from 12 with high yield (Scheme 5). However, the
optical purity of CAT3 from this approach was not satisfactory.
We studied all the chiral intermediates and noticed that the
optical purity obviously decreased when the 14-position ketone
of compound 12 was reduced to the hydroxyl group. In order to
gure it out, a batch of experiments were performed. As
a common sense, we rst focused our efforts on the solvent and
Scheme 6 Reagents and conditions for the synthesis of PF403 starting
from 12 with benzyl substitution at 3-position: (a) BnBr, K2CO3,
Scheme 5 Reagents and conditions for the synthesis of PF403 starting acetone, reflux; (b) NaBH4, then Et3SiH, BF3–Et2O, r.t.; (c) Red-Al, THF,
from 12: (a) NaBH4, then Et3SiH, BF3–Et2O, r.t.; (b) Red-Al, THF, r.t.
r.t.; (d) Pd–C, H2, MeOH, r.t.
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Table 1 The ee and yield of the final product from the different
synthetic route
Substitution at 3-position of compound 12
HO
BnO
TBSO
AcO
ee
27–93%
95–98%
$99%
10–50%
Yield
13.2%
2.9%
10.0%
10.1%
(HPLC) was performed the HPLC system was equipped with
a UV-vis detector (G1315C/D), an Agilent 1200 series G1322A IV
pump and soware for process control and data handling
(Agilent ChemStation for LC 3D Systems) equipping an GL
Intersil ODS column (250 ꢂ 10 mm, 5 mm). UV spectra were
measured on a JASCO V650 spectrophotometer. IR spectra were
recorded on a Nicolet 5700 FT-IR microscope instrument (FT-IR
microscope transmission).
Scheme 7 Reagents and conditions for the synthesis of PF403 starting
from 12 with acetyl substitution at 3-position: (a) Ac2O, TEA, DMAP,
DCM, r.t.; (b) NaBH4, then Et3SiH, BF3–Et2O, r.t.; (c) NaOH, CH3OH, r.t.,
then HCl; (d) Red-Al, THF, r.t.
was protected by the strong electron-donating TBS group
($99% ee, Scheme 8). We deduce that the electron-donating
group at 3-position, which appears far from the ketone at C-
14 in the plane structure, has a remote control on the stereo-
chemistry of the phenanthroindolizidine moiety in favor of the
formation of the S-congured center. The yields and ee values
generated from different substitutions were illustrated in
Schemes 5–8 and Table 1.
(S)-(1-((3-(Benzyloxy)-6,7-dimethoxyphenanthren-9-yl)methyl)
pyrrolidin-2-yl)methanol (3)
To a stirring solution of imidazole (90 mg, 1.32 mmol) and
triphenylphosphine (267 mg, 1.02 mmol) in DCM (15 ml) was
added iodine (214 mg, 0.84 mmol). Aer stirring for 10 min at
room temperature, compound 1 (224 mg, 0.6 mmol) was added.
Aer completion of the substitution, imidazole (122 mg, 1.8
mmol) and methyl L-prolinate hydrochloride (198 mg, 1.2
mmol) was added to the stirring reaction in sequences. Aer
completion of the reaction, it was quenched by the addition of
the aqueous solution of saturated NH4Cl (20 ml). The reaction
mixture was extracted into dichloromethane CH2Cl2 (3 ꢂ 20
ml). The combined organic layer was washed with brine (2 ꢂ 30
ml), dried with anhydrous Na2SO4, and concentrated under
vacuum to furnish the residue as crude product (2). To the
cooled (ꢁ10 ꢀC) solution of the crude product in dry CH2Cl2 (30
ml) was added DIBALH (0.8 ml, 1.2 mmol, 1.5 M in toluene)
dropwise under argon, and the reaction mixture was allowed to
stir at room temperature for 12 h. Then the reaction mixture was
quenched with the solution of seignette salt along with stirring
for additional 12 h. The reaction mixture was separated as two
layers and the water layer was extracted with dichloromethane
CH2Cl2 (3 ꢂ 20 ml). The combined organic layer was washed
with brine (2 ꢂ 30 ml), dried with anhydrous Na2SO4, and
concentrated under vacuum to furnish the crude product,
which was puried by PTLC (DCM/MeOH ¼ 10/1) to provide 3
(109 mg, 0.24 mmol) as yellow oil with 40% yield in two steps.
1H NMR (500 MHz, DMSO-d6) d 8.15 (s, 1H), 8.03 (s, 1H), 7.93 (s,
1H), 7.81 (d, J ¼ 8.7 Hz, 1H), 7.59–7.53 (m, 3H), 7.43 (d, J ¼
7.5 Hz, 1H), 7.41 (d, J ¼ 7.5 Hz, 1H),7.36–7.34 (m, 1H), 7.26 (dd, J
¼ 8.7, 1.7 Hz, 1H), 5.36 (s, 2H), 4.70–4.67 (m, 1H), 4.62–4.59 (m,
1H), 4.03 (s, 3H), 3.93 (s, 3H), 3.66–3.61 (m, 1H), 3.51–3.45 (m,
2H), 2.66–2.59 (m, 2H), 2.25–2.22 (m, 1H), 1.91–1.87 (m, 1H),
1.61–1.50 (m, 3H). 13C NMR (125 MHz, DMSO-d6) d 156.9, 148.8,
148.7, 137.3, 131.5, 130.6, 129.8, 128.8, 128.5, 128.1, 127.9,
126.5, 125.3, 124.3, 116.1, 106.4, 105.4, 104.2, 69.7, 65.3, 64.9,
59.0, 55.9, 55.3, 54.5, 28.0, 22.5. HRESIMS, m/z calculated for
Experimental
General methods
All the reactions sensitive to air or moisture were carried out
under argon atmosphere in anhydrous solvents. The purchased
extra dry Tetrahydrofuran (THF) with the resealable package
was used directly. Thin layer chromatography (TLC) was per-
formed using glass precoated silica gel GF254 plates. Spots were
visualized under 254 nm or 365 nm UV light or colored by 1%
KMnO4 in 5% NaCO3 aqueous solution. NMR spectra were ob-
tained on Bruker-400 MHz and Bruker-500 MHz spectrometers.
Chemical shis are reported in d (ppm) relative to solvent
peaks. High-resolution electrospray ionization mass spectra
(HRESIMS) data were obtained on micromass Autospec-Ultima
ETOF spectrometer. High performance liquid chromatography
Scheme
8 Reagents and conditions for the synthesis of PF403
starting from 12 with TBS substitution at 3-position: (a) TBSCl, imid-
azole, DMF, r.t.; (b) NaBH4, then Et3SiH, BF3–Et2O, r.t.; (c) Red-Al, THF,
r.t.
C
29H32NO4 [M + H]+: 458.2331. Found: 458.2338.
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solution of 7 (800 g) in methanol (2500 ml) was added
concentrated sulfuric acid (125 ml), and the reaction was
reuxed for 12 h. Aer completion of the reaction, it was
quenched by the addition of imidazole (306 g) and was stirred
for another 30 min. Then the mixture was concentrated under
vacuum to furnish the residue as crude product (8). To the
stirring solution of the crude product and imidazole (238 g, 3.5
mol) in dichloromethane (2000 ml) was added TBSCl (530 g, 3.5
mol). Aer stirring for 12 h, the produced solid was ltered and
the ltrate was washed by brine (2 ꢂ 2000 ml), dried with
anhydrous Na2SO4, and concentrated under vacuum to furnish
the residue, which was puried by fast silica column chroma-
tography (PE/EtOAc, 5 : 1) to give 9 (213 g, 0.5 mol) as yellow
solid with 50% yield in four steps. Mp 144–146 ꢀC; 1H NMR (500
MHz, CDCl3) d 8.65 (s, 1H), 8.45 (s, 1H), 7.86 (s, 1H), 7.86 (d, J ¼
2.2 Hz, 1H), 7.82 (d, J ¼ 8.6 Hz, 1H), 7.13 (dd, J ¼ 8.6, 2.2 Hz,
1H), 4.12 (s, 3H), 4.09 (s, 3H), 4.02 (s, 3H), 1.06 (s, 9H), 0.32 (s,
6H), 0.07 (s, 4H). 13C NMR (125 MHz, CDCl3) d 168.3, 156.6,
149.9, 148.9, 133.6, 131.9, 131.4, 125.0, 124.9, 124.7, 121.9,
120.8, 111.3, 106.9, 103.2, 55.95, 55.90, 52.1, 25.9, 18.5, ꢁ4.0. IR
(KBr) nmax ¼ 3120, 3004, 2951, 2857, 1708, 1617, 1520, 1469,
(S)-1-((3-(Benzyloxy)-6,7-dimethoxyphenanthren-9-yl)methyl)-
2-(chloromethyl)pyrrolidine (5)
To the cooled (0 ꢀC) solution of 3 (55 mg, 0.12 mmol) in dry
CH2Cl2 (10 ml) was added pyridine (21 mg, 0.26 mmol), DMAP
(1.2 mg, 0.01 mmol) and methanesulfonyl chloride. Aer stir-
ring for 12 h at room temperature, the reaction was quenched
by the addition of the saturated sodium bicarbonate aqueous.
The reaction mixture was separated as two layers and the water
layer was extracted with dichloromethane CH2Cl2 (2 ꢂ 20 ml).
The combined organic layer was washed with brine (2 ꢂ 20 ml),
dried with anhydrous Na2SO4, and concentrated under vacuum
to furnish the crude product, which was puried by PTLC (ethyl
acetate/petroleum ether ¼ 1/1) to provide 5 (33 mg, 0.07 mmol)
1
as light yellow oil with 60% yield. H NMR (500 MHz, CDCl3)
d 7.93 (d, J ¼ 2.2 Hz, 1H), 7.92 (s, 1H), 7.83 (s, 1H), 7.76 (d, J ¼
8.7 Hz, 1H), 7.56 (d, J ¼ 2.2, 1H), 7.54 (s, 1H), 7.48 (s, 1H), 7.43
(d, J ¼ 7.5 Hz, 1H), 7.41 (d, J ¼ 7.5 Hz, 1H),7.38–7.33 (m, 1H),
7.25 (dd, J ¼ 8.7, 2.2 Hz, 1H), 5.29 (s, 2H), 4.10 (s, 3H), 4.05 (s,
3H), 3.98–3.92 (m, 1H), 3.85–3.80 (m, 1H), 3.07–3.02 (m, 1H),
2.70–2.65 (m, 1H), 2.42–2.39 (m, 1H), 2.26–2.21 (m, 1H), 2.12–
2.08 (m, 1H), 1.82–1.77 (m, 1H), 1.70–1.53 (m, 2H), 1.26–1.23
(m, 1H). HRESIMS, m/z calculated for C29H31ClNO3 [M + H]+:
476.1992. Found: 476.1996.
1421, 1258, 1208, 1113, 1033, 992, 938, 910, 832, 778 cmꢁ1
.
HRESIMS, m/z calculated for C24H30O5Si [M + H]+: 427.1938.
Found: 427.1946.
(E)-3-(4-Acetoxyphenyl)-2-(3,4-dimethoxyphenyl)acrylic acid
(6)
(3-((tert-Butyldimethylsilyl)oxy)-6,7-dimethoxyphenanthren-9-
yl)methanol (10)
To a stirring solution of 3, 4-dimethoxyphenyl acetic acid (196 g,
1 mol) and 4-hydroxybenzaldehyde (123 g, 1 mol) in acetic
anhydride (400 ml) was added triethylamine (200 ml) dropwise
and the mixture was reuxed overnight under argon. Aer
completion of the reaction, it was cooled to room temperature
and 800 ml water was added. The reaction was stirred for 1 h
and furnished 6 (341 g, 1 mol) as yellow solid aer a sequence of
ltration, water washing and drying. 1H NMR (500 MHz,
CD3OD) d 7.78 (s, 1H), 7.08 (d, J ¼ 8.7 Hz, 2H), 6.93 (d, J ¼ 8.2 Hz,
1H), 6.90 (d, J ¼ 8.7 Hz, 2H), 6.73 (s, 1H), 6.72 (dd, J ¼ 8.2,
2.0 Hz, 1H), 3.82 (s, 3H), 3.69 (s, 3H), 2.20 (s, 3H). 13C NMR (125
MHz, CD3OD) d 171.1, 170.8, 152.6, 150.6, 150.3, 140.1, 134.2,
133.9, 132.6, 129.9, 123.6, 122.6, 114.7, 112.97, 56.4, 56.3, 49.5,
49.3, 49.2, 49.0, 48.8, 48.7, 48.5, 20.9. IR (KBr) nmax ¼ 3072, 2992,
2838, 2630, 1768, 1680, 1612, 1514, 1419, 1456, 1369, 1021, 912,
848 cmꢁ1. HRESIMS, m/z calculated for C19H18O6Na [M + Na]+:
365.0996. Found: 365.0986.
To a cooled (0 ꢀC) solution of 9 (110 g, 0.26 mol) in dry CH2Cl2
(1000 ml) was added DIBALH (378 ml, 0.57 mol, 1.5 M in
toluene) dropwise under argon, and the reaction mixture was
allowed to stir at room temperature for 12 h. Then the reaction
mixture was quenched with 3 M HCl (550 ml) and extracted with
CH2Cl2 (2 ꢂ 500 ml). The combined organic layer was washed
with brine (1000 ml), dried with anhydrous Na2SO4, and
concentrated under vacuum to furnish the product 10 (98 g,
0.25 mol) with 95% yield, which was pure enough for the next
step. 1H NMR (500 MHz, CDCl3) d 7.84 (s, 1H), 7.82 (d, J ¼
2.2 Hz, 1H), 7.67 (d, J ¼ 8.6 Hz, 1H), 7.53 (s, 1H), 7.47 (s, 1H),
7.08 (dd, J ¼ 8.6, 2.2 Hz, 1H), 5.03 (s, 2H), 4.10 (s, 3H), 4.02 (s,
3H), 1.06 (s, 9H), 0.30 (s, 6H). 13C NMR (125 MHz, CDCl3)
d 154.5, 149.4, 148.9, 131.7, 131.4, 130.1, 125.9, 125.7, 124.9,
124.6, 120.4, 111.4, 104.8, 103.8, 64.8, 56.0, 25.9, 18.5, ꢁ4.1. IR
(KBr) nmax ¼ 3368, 2954, 2932, 2896, 2857, 1606, 1524, 1508,
1423, 1390, 1361, 1268, 1204, 1159, 1112, 1078, 1033, 982, 921,
893, 835, 780 cmꢁ1. HRESIMS, m/z calculated for C23H30O4SiNa
[M + Na]+: 421.1806. Found: 421.1823.
Methyl 3-((tert-butyldimethylsilyl)oxy)-6,7-dimethoxy
phenanthrene-9-carboxylate (9)
(S)-1-((3-Hydroxy-6,7-dimethoxyphenanthren-9-yl)methyl)-5-
oxopyrrolidine-2-carboxylic acid (11)
To a solution of 6 (341 g, 1 mol) and ferric chloride in tri-
uoroacetic acid (1400 ml) was added dropwise a solution of 3-
chloroperbenzoic acid (189.8 g, 1.1 mol) in triuoroacetic acid To the stirring mixture of alcohol 10 (98.0 g, 0.246 mmol), NaI
(1700 ml) below 0 ꢀC and then stirred for 12 h at room (8.14 g, 53.3 mmol), 1,4-dioxane (400 ml), and MeCN (600 ml)
temperature. Aer completion of the reaction, a solution of was added TMSCl (46.8 ml, 0.387 mmol) dropwise below 20 ꢀC.
sodium thiosulfate pentahydrate (20 g in 100 ml water) was Aer stirring 1 h at room temperature, anhydrous K2CO3 and
added and then stirred for 30 min. The reaction was evaporated dimethyl L-glutamate hydrochloride was added. When the
under reduced pressure to afford crude product 7 (800 g), which reaction nished, it was ltered and acetic acid was added to
was used for the next step without further purication. To the the ltration. Aer completion of the reaction, the reaction was
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evaporated under vacuum to furnish the residue. The obtained J ¼ 9.2 Hz, 1H), 7.86 (d, J ¼ 2.5 Hz, 1H), 7.85 (s, 1H), 7.29 (s, 1H),
residue was dissolved in methanol (600 ml) and dichloro- 7.20 (dd, J ¼ 9.2, 2.5 Hz, 1H), 5.70 (d, J ¼ 17.8 Hz, 1H), 4.65 (d, J
methane (100 ml), and 4 M NaOH (240 ml) was added to the ¼ 17.8 Hz, 1H), 4.44–4.41 (m, 1H), 4.15 (s, 3H), 4.09 (s, 3H),
solution dropwise until the pH was stable around 11–12. Aer 2.63–2.51 (m, 4H), 1.05 (s, 9H), 0.30 (s, 6H). 13C NMR (125 MHz,
completion of the hydrolysis, most methanol was evaporated CDCl3) d 195.5, 174.2, 154.6, 151.7, 149.9, 137.4, 131.4, 129.1,
under reduced pressure. The obtained crude residue was mixed 127.7, 123.1, 122.8, 122.6, 121.6, 111.4, 104.3, 103.6, 61.2, 56.3,
with 500 ml water and then extracted with dichloromethane (3 56.1, 40.7, 30.2, 25.9, 21.0, 18.5, ꢁ4.01, ꢁ4.02. IR (KBr) nmax
ꢂ 500 ml). The aqueous phase was carefully acidied with 4 M 3106, 2953, 2934, 2856, 1691, 1612, 1582, 1521, 1462, 1419,
HCl to pH 2 at 0–5 ꢀC and off-white solid precipitated out of the 1391, 1260, 1209, 1177, 1120, 1025, 953, 886, 825, 785 cmꢁ1
¼
.
reaction mixture. Simple ltration and baking of the solid HRESIMS, m/z calculated for C25H34NO9 [M + H]+: 492.2228.
afforded 11 (53.5 g, 0.135 mol) with 55% yield. Mp 190–192 ꢀC; Found: 492.2241. HPLC (Daicel Chiralpak OD-H, 4.6 mm ꢂ 250
1H NMR (500 MHz, CD3OD) d 7.92 (s, 1H), 7.83 (d, J ¼ 2.1 Hz, mml, 10 mm, hexane/iPrOH ¼ 50 : 50, ow rate ¼ 1 ml minꢁ1, l
1H), 7.69 (d, J ¼ 8.6 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 7.08 (dd, J ¼ 264, 355 nm): tR ¼ 25.9 min; 99.5% ee.
¼ 8.6, 2.1 Hz, 1H), 5.49 (d, J ¼ 14.6 Hz, 1H), 4.30 (d, J ¼ 14.6 Hz,
1H), 4.03 (s, 3H), 3.95 (s, 3H), 3.78 (dd, J ¼ 9.4, 3.3 Hz, 1H), 2.62–
(S)-3-Hydroxy-6,7-dimethoxy-12,13,13a,14-tetrahydrodibenzo
2.55 (m, 1H), 2.46–2.40 (m, 1H), 2.19–2.13 (m, 1H), 2.04–1.98
(m, 1H). 13C NMR (125 MHz, CD3OD) d 177.5, 175.0, 157.8,
150.9, 150.2, 133.2, 131.2, 129.0, 127.0, 126.5, 126.1, 125.9,
117.5, 106.7, 106.1, 105.3, 60.0, 56.5, 56.3, 45.6, 30.95, 23.8. IR
(KBr) nmax ¼ 3240, 2994, 2966, 2838, 1742, 1664, 1634, 1612,
1581, 1532, 1511, 1443, 1391, 1359, 1320, 1159, 1122, 1064,
1030, 984, 880, 859, 841, 796 cmꢁ1. HRESIMS, m/z calculated for
[f,h]pyrrolo[1,2-b]isoquinolin-11(9H)-one (14)
To the cooled solution of 13 (48.2 g, 0.098 mol) in dichloro-
methane (200 ml) and methanol (200 ml) was slowly added
NaBH4 (11.1 g, 0.294 mol). When the reaction nished, 1 M HCl
was added to the reaction to make sure pH 7–8. Aer stirring for
additional 1 h, the reaction mixture was extracted into CH2Cl2 (3
ꢂ 200 ml). The combined organic layer was concentrated under
vacuum to furnish the intermediate. To a solution of the
intermediate was added triethylsilane (120 ml) and boron
uoride ethyl ether (120 ml). Aer completion of the reaction,
lots of off-white solid precipitated. Simple ltration, washing by
C
22H20NO6 [M ꢁ H]ꢁ: 394.1296. Found: 394.1308.
(S)-3-Hydroxy-6,7-dimethoxy-13,13a-dihydrodibenzo[f,h]
pyrrolo[1,2-b]isoquinoline-11,14(9H,12H)-dione (12)
To the solution of 11 (52.0 g, 0.132 mol) in triuoroacetic dichloromethane and baking of the solid afforded 14 (28.3 g,
anhydride (300 ml) was added boron uoride ethyl ether (420 0.078 mol) with 80% yield. Mp 257–259 ꢀC; 1H NMR (500 MHz,
ml) and the reaction was stirred for 30 min at 45 ꢀC. Aer DMSO-d6) d 7.97 (d, J ¼ 2.4 Hz, 1H), 7.95 (s, 1H), 7.88 (d, J ¼
completion of the reaction, it was poured to ice water (1500 ml) 9.0 Hz, 1H), 7.20 (s, 1H), 7.12 (dd, J ¼ 9.0, 2.4 Hz, 1H), 5.09 (d, J
and stirred for 1 h with light green precipitating out. Simple ¼ 17.2 Hz, 1H), 4.48 (d, J ¼ 17.2 Hz, 1H), 4.00 (s, 3H), 3.97 (s,
ltration and baking of the solid afforded 12 (43.7 g, 0.116 mol) 3H), 3.94–3.85 (m, 1H), 3.45 (dd, J ¼ 16.2, 4.1 Hz, 1H), 2.80 (dd, J
1
with 85% yield. Mp 300 C (decomposed); H NMR (500 MHz, ¼ 16.2, 10.9 Hz, 1H), 2.48–2.35 (m, 3H), 1.99–1.85 (m, 1H). 13
C
ꢀ
DMSO-d6) d 9.92 (s, 1H), 9.13 (d, J ¼ 9.2 Hz, 1H), 8.02 (d, J ¼ NMR (125 MHz, DMSO-d6) d 173.2, 155.8, 149.3, 148.4, 130.5,
2.5 Hz, 1H), 8.00 (s, 1H), 7.48 (s, 1H), 7.16 (dd, J ¼ 9.2, 2.5 Hz, 125.7, 125.2, 124.2, 123.4, 122.7, 121.4, 116.4, 106.6, 104.1,
1H), 5.53 (d, J ¼ 18.1 Hz, 1H), 4.80 (d, J ¼ 18.1 Hz, 1H), 4.57 (t, J 103.2, 55.53, 55.51, 52.4, 32.2, 29.4, 24.5. IR (KBr) nmax ¼ 3446,
¼ 6.7 Hz, 1H), 4.07 (s, 3H), 4.03 (s, 3H), 2.48–2.29 (m, 4H). 13C 2946, 2835, 1660, 1618, 1519, 1470, 1441, 1358, 1272, 1202,
NMR (125 MHz, DMSO-d6) d 196.2, 173.1, 156.2, 151.5, 149.7, 1169, 941, 864, 670 cmꢁ1. HRESIMS, m/z calculated for
137.4, 131.1, 128.6, 126.9, 122.8, 122.3, 120.8, 117.5, 106.5,
C
22H22NO4 [M + H]+: 364.1543. Found: 364.1543. HPLC (Daicel
105.2, 104.0, 60.8, 55.84, 55.79, 29.5, 20.4. IR (KBr) nmax ¼ 3167, Chiralpak OD-H, 4.6 mm ꢂ 250 mml, 10 mm, hexane/iPrOH ¼
3000, 2925, 2835, 1708, 1663, 1613, 1519, 1457, 1401, 1353, 50 : 50, ow rate ¼ 1 ml minꢁ1, l ¼ 258, 286 nm): tR ¼ 11.9 min;
1245, 1207, 1178, 1117, 1029, 940, 924, 844, 806, 760 cmꢁ1
.
99.5% ee.
HRESIMS, m/z calculated for C22H20NO5 [M + H]+: 378.1336.
Found: 378.1335.
(S)-6,7-Dimethoxy-9,11,12,13,13a,14-hexahydrodibenzo[f,h]
pyrrolo[1,2-b]isoquinolin-3-ol (PF403)
(S)-3-((tert-Butyldimethylsilyl)oxy)-6,7-dimethoxy-13,13a-
dihydrodibenzo[f,h]pyrrolo[1,2-b]isoquinoline-11,14(9H,12H)-
dione (13)
To the cooled solution of 14 (28.3 g, 0.078 mol) in THF (200 ml)
was added a solution of Red-Al (78 ml, 3.5 M in toluene) in
toluene under argon, and the reaction was stirred at room
To the stirring solution of 12 (43.7 g, 0.116 mol) and N,N-dii- temperature until it nished. Then the reaction was cooled to
sopropylethylamine (45.0 g, 0.348 mol) in DMF (300 ml) was 0 ꢀC and the solution of seignette salt (78 g in 500 ml water) was
added TBSCl (34.9 g, 0.232 mol). Aer completion of the reac- added along with stirring for additional 12–24 h. The precipi-
tion, water (800 ml) was carefully added to the reaction drop- tation was ltered and baked to afford PF403 (25.8 g, 0.074 mol)
wise. Aer it was stirred for additional 1 h at ice water bath, as white solid with 95% yield. Mp 176–178 ꢀC; 1H NMR (500
yellow solid precipitated. Simple ltration, washing by meth- MHz, DMSO-d6) d 9.72 (s, 1H), 7.95 (d, J ¼ 2.2 Hz, 1H), 7.92 (s,
anol and baking of the solid afforded 13 (48.2 g, 0.098 mol) with 1H), 7.84 (d, J ¼ 8.9 Hz, 1H), 7.18 (s, 1H), 7.10 (dd, J ¼ 8.9,
85% yield. Mp 212–214 ꢀC; 1H NMR (500 MHz, CDCl3) d 9.27 (d, 2.2 Hz, 1H), 4.53 (d, J ¼ 15.0 Hz, 1H), 3.99 (s, 3H), 3.93 (s, 3H),
29306 | RSC Adv., 2018, 8, 29301–29308
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3.64–3.57 (m, 1H), 3.50 (d, J ¼ 15.0 Hz, 1H), 2.75 (dd, J ¼ 15.6, the strong electron-donating TBS group, the excellent enantio-
10.6 Hz, 1H), 2.36–2.31 (m, 2H), 2.22–2.09 (m, 1H), 1.91–1.79 meric excess was achieved for CAT3 ($99% ee) which is critical
(m, 2H), 1.78–1.73 (m, 1H), 1.68–1.56 (m, 1H). 13C NMR (125 for its further preclinical investigations. This interesting nding
MHz, DMSO-d6) d 155.6, 149.1, 148.0, 130.3, 126.6, 125.0, 124.8, not only represents a big breakthrough in the synthesis of CAT3,
124.5, 123.8, 122.6, 116.3, 106.4, 104.0, 103.4, 59.9, 55.5, 55.4, but also greatly inspires the construction and studies of chiral
54.6, 53.4, 33.1, 30.9, 21.3. IR (KBr) nmax ¼ 3565, 3092, 2965, phenanthroindolizidine or other similar structures which is
2825, 1626, 1585, 1535, 1511, 1481, 1461, 1437, 1414, 1386, highly important for researchers in diverse elds, including
1359, 1323, 1256, 1237, 1150, 1033, 1009, 920, 844, 788 cmꢁ1
.
natural product synthesis, organic chemistry, and medicinal
HRESIMS, m/z calculated for C22H24NO3 [M + H]+: 350.1751. chemistry.
Found: 350.1760. HPLC (Daicel Chiralpak AD-H, 4.0 mm ꢂ 10
mml, 5 mm, hexane (with 0.2% TEA)/iPrOH ¼ 82 : 18, ow rate ¼
Conflicts of interest
1 ml minꢁ1, l ¼ 258, 287 nm): tR ¼ 14.6 min; 99.6% ee.
There are no conicts to declare.
(13aS)-3-Pivaloyloxyl-6,7-dimethoxyphenanthro[9,10-b]-
Acknowledgements
indolizidine (CAT3)
To the cooled solution of PF403 (25.8 g, 0.077 mol), triethyl-
amine (34.5 ml, 0.23 mol) and DMAP (0.94 g, 7.7 mmol) in
dichloromethane (300 ml) was added trimethylacetic anhydride
(28.1 g, 0.15 mol). Aer completion of the reaction, it was
quenched by water. The reaction mixture was extracted into
dichloromethane CH2Cl2 (3 ꢂ 200 ml). The combined organic
layer was washed with brine (3 ꢂ 200 ml), dried with anhydrous
Na2SO4, and concentrated under vacuum to furnish the residue,
which was recrystallized by anhydrous ethanol (400 ml) to
afford CAT3 (22.4 g, 0.052 mol) as off-white crystal with 70%
yield. Mp 200.4–200.8 C; H NMR (500 MHz, DMSO-d6) d 8.41
(s, 1H), 8.07 (s, 1H), 8.01 (d, J ¼ 8.9 Hz, 1H), 7.29 (d, J ¼ 8.0 Hz,
1H), 7.22 (s, 1H), 4.58 (d, J ¼ 15.3 Hz, 1H), 4.02 (s, 3H), 3.94 (s,
3H), 3.53 (d, J ¼ 15.3 Hz, 1H), 3.38–3.32 (m, 2H), 2.87–2.73 (m,
1H), 2.37–2.31 (m, 2H), 2.16–2.13 (m, 1H), 1.85–1.81 (m, 2H),
1.68–1.59 (m, 1H), 1.40 (s, 9H). 13C NMR (125 MHz, DMSO-d6)
d 176.9, 149.5, 148.8, 148.6, 129.6, 128.0, 127.8, 126.4, 124.9,
124.8, 122.9, 120.5, 114.9, 104.6, 103.4, 59.8, 55.9, 55.5, 54.5,
53.4, 38.7, 33.1, 30.8, 27.0, 21.3. IR (KBr) nmax ¼ 3097, 2965,
2830, 1740, 1619, 1530, 1510, 1468, 1427, 1415, 1367, 1339,
1256, 1205, 1164, 1135, 1034, 899, 841, 821, 786 cmꢁ1. HRE-
SIMS, m/z calculated for C27H32NO4 [M + H]+: 434.2331. Found:
434.2332. HPLC [Daicel Chiralpak OZ-H, 4.6 mm ꢂ 250 mml, 5
mm, hexane (with 0.2% TEA)/iPrOH ¼ 90 : 10, ow rate ¼ 1
ml minꢁ1, l ¼ 259, 281 nm]: tR ¼ 11.8 min; 99.6% ee.
The authors are grateful to the Department of Instrumental
Analysis of our institute for the acquisition of UV, IR, NMR, and
MS spectra. This work was supported by grants from National
Mega-project for Innovative Drugs (No. 2014ZX09304306-001),
from CAMS Innovation Fund for Medical Sciences (No. 2016-
I2M-1-010) and from the Natural Science Foundation of China
(No. 81602996).
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