Oligosaccharide-camptothecin conjugates as potential antineoplastic drugs: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2020.112509

Thirty novel 20 (S)–O-linked camptothecin (CPT) glycoconjugates were synthesized. They showed more potent in vitro cytotoxicities over irinotecan, but very weak direct topoisomerase I (Topo I) inhibition was observed at 100.0 μM. Oligosaccharide types, length of a PEG linker and acetyl groups exerted obvious effects on cytotoxicity, selectivity, water solubility and stability of the newly synthesized CPT glycoconjugates. Construct 40, with a bleomycin (BLM) disaccharide linked to diethylene glycol in the introduced ester moiety, demonstrated a superior antitumor activity and a distinct selectivity compared to CPT. No toxicity was detectable in animal acute toxicity intravenously (160 mg/kg). Collectively, attachment of oligosaccharides with tumor targeting to 20 (S)–OH of CPT could offer a solution to the daunting problems posed by current Topo I poisons.

Full text of DOI:10.1016/j.ejmech.2020.112509

Journal Pre-proof
Oligosaccharide-camptothecin conjugates as potential antineoplastic drugs: Design,
synthesis and biological evaluation
Maolin Li, Wenchong Ye, Kaishuo Fu, Cui zhou, Yonghui Shi, Weiping Huang,
Wenming Chen, Jiliang Hu, Zhilin Jiang, Wen Zhou
PII:
S0223-5234(20)30481-5
DOI:
https://doi.org/10.1016/j.ejmech.2020.112509
EJMECH 112509
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 April 2020
Revised Date: 20 May 2020
Accepted Date: 23 May 2020
Please cite this article as: M. Li, W. Ye, K. Fu, C. zhou, Y. Shi, W. Huang, W. Chen, J. Hu, Z. Jiang,
W. Zhou, Oligosaccharide-camptothecin conjugates as potential antineoplastic drugs: Design,
synthesis and biological evaluation, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
j.ejmech.2020.112509.
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Oligosaccharide-camptothecin
conjugates
as
potential
antineoplastic drugs: Design, synthesis and biological evaluation
1
#
1#
1#
1
2
1
Maolin Li , Wenchong Ye , Kaishuo Fu , Cui zhou , Yonghui Shi , Weiping Huang ,
3
1
4∗
1∗
Wenming Chen , Jiliang Hu , Zhilin Jiang , Wen Zhou
1
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, E. 232, University town,
Waihuan Rd, Panyu, Guangzhou, 510006, China.
2
Department of Pharmacy, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
510120, Guangdong, China
3
Department of Pharmaceutical Production Center, The First Affiliated Hospital of Hunan University of
Chinese Medicine, 95, Shaoshan Rd,Changsha, Hunan, 41007, China.
4
Puer University, Puer, 665000, Yunan, China
#
contributed equally to this work.
Thirty novel 20 (S)-O-linked camptothecin (CPT) glycoconjugates were
synthesized. The SAR indicated that oligosaccharide types, length of a PEG linker
and acetyl groups exerted obvious impacts on the antitumor activities and selectivity
of the CPT glycoconjugates. Construct 40 demonstrated good druggablity profiles.
∗
Correspondent. E-mail: zhilin_jiang@126.com
∗
Correspondent. E-mail: zhouwen60@126.com

Oligosaccharide-camptothecin
conjugates
as
potential
antineoplastic drugs: Design, synthesis and biological evaluation
1
#
1#
1#
1
2
1
Maolin Li , Wenchong Ye , Kaishuo Fu , Cui zhou , Yonghui Shi , Weiping Huang ,
3
1
4∗
1∗
Wenming Chen , Jiliang Hu , Zhilin Jiang , Wen Zhou
1
School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, E. 232, University town,
Waihuan Rd, Panyu, Guangzhou, 510006, China.
2
Department of Pharmacy, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou,
510120, Guangdong, China
3
Department of Pharmaceutical Production Center, The First Affiliated Hospital of Hunan University of
Chinese Medicine, 95, Shaoshan Rd,Changsha, Hunan, 41007, China.
4
Puer University, Puer, 665000, Yunan, China
#
contributed equally to this work.
Abstract:
Thirty novel 20 (S)-O-linked camptothecin (CPT) glycoconjugates were
synthesized. They showed more potent in vitro cytotoxicities over irinotecan, but very
weak direct topoisomerase I (Topo I) inhibition was observed at 100.0 µM.
Oligosaccharide types, length of a PEG linker and acetyl groups exerted obvious
effects on cytotoxicity, selectivity, water solubility and stability of the newly
synthesized CPT glycoconjugates. Construct 40, with a bleomycin (BLM)
disaccharide linked to diethylene glycol in the introduced ester moiety, demonstrated
a superior antitumor activity and a distinct selectivity compared to CPT. No toxicity
was detectable in animal acute toxicity intravenously (160 mg/kg). Collectively,
attachment of oligosaccharides with tumor targeting to 20 (S)-OH of CPT could offer
a solution to the daunting problems posed by current Topo I poisons.
Key words: Camptothecin; Oligosaccharides; Aqueous solubility and stability;
Anti-tumor activity; Toxicological assessment
1
. Introduction
Camptothecin (Fig. 1, CPT, 1), a plant-derived antitumor ingredient, exhibits a
wide spectrum of inhibitory activity towards cancer cells as a unique DNA Topo I
∗
Correspondent. E-mail: zhilin_jiang@126.com
∗
Correspondent. E-mail: zhouwen60@126.com

[
1-3]
poison.
The molecule includes a planar pentacyclic ring system and one chiral
center in a closed α-hydroxyl-δ-lactone (E ring). The planar ploycyclic geometry has
been demonstrated as a guiding moiety that recognizes and stabilizes the Topo I/DNA
[2]
cleavage complex. E ring able to block reversal of the complex leads to cell
apoptosis. Therefore, the planar geometry and the closed lactone are required for
[
3]
Topo I poisoning. The approval of irinotecan (2) and topotecan (3) with an intact
[
3]
[4]
planar pentacyclic structure in the treatment for colorectal cancer, liver cancer,
[
3]
and small-cell lung cancer (SCLC) highlights the privileged structure of CPT.
1
, Camptothecin (CPT)
R =R =R =R =H
1
2
3
4
O
O
R2
9
R3
7
2, Irinotecan
N
N
; R =R =H; R =CH CH
2 4
3 2 3
R1=
R1
O
E
1
0
A
B
C
N
3
, Topotecan
R =OH; R =CH N(CH ) ; R =R =H
1 2 2 3 2 3 4
N
D
O
H
HOOC
O
N
4
,
O
O
R =R =R =H; R = HO
R4O
1
3
4
2
O
O
OH
HO
HOOC
O
O
4
a,
O
R =R =R =H;
R = HO
2
3
4
1
HO
OH
H
H
N
O
Val
N
O
R =R =R =H;
R4=
N
4
b,
1
2
3
S
His H
O
O
O
OH
OH
OH
Fig.1. Structures of camptothecin (CPT) and CPT-derived agents.
CPT-derived Topo I poisons irinotecan (2) and topotecan (3) obviously suppress
the growth of tumors, but the serious adverse effects increasingly compromise the
clinical applications, including dose-limiting bone marrow suppression, grievous
diarrhea. Meantime, the rapid diffusion of CPT and some CPT-based derivatives leads
to the prolonged infusion times, and the poor aqueous solubility creates difficulty in
[
5]
formulation, and the quick inactivation of E-ring-opening in human blood plasma
[6,7]
generates the carboxylate form that readily binds to human blood proteins.
Accordingly, CPT derivatives that preserve the antitumor properties of the parent
compound CPT, and improve the water solubility, in vivo stability and the safe profile,
[
5,8]
are highly urgent to be developed. From CPT’s structural analysis, the formation
of intramolecular hydrogen bond of E ring with 20-OH favors the carboxylate form at
physiological pH or above. Modification of 20-free hydroxyl group blocks the
[
9]
participation and stabilizes the closed-lactone moiety. Coupling of oligosaccharides

with tumor targeting to 20-free hydroxyl group of CPT could offer access to solving
the above issues.
Oligosaccharides such as mono- or disaccharides characteristic with little
toxicity, high water solubility and tumor targeting are widely used in the drug design.
[
10-13]
Glucuronide prodrugs 4 and 4a, in which a glucuronic acid was coupled to
positions 10 and 9 of CPT via a self-immolating linker, respectively, exhibited good
[
1,8]
water solubility, strong antitumor activity and differential toxicity
. These
advantageous properties were ascribed to the hydrophilicity of a glucuronide moiety
[
8,14]
that was preferentially activated at tumor β-glucuronidase-overexpressing cells.
.
But fast renal clearance of prodrugs 4 and 4a caused by a highly polar glucuronide
posed the clinical limitations. Additionally, 20 (S)-O-carbonate linked tripeptide
[
15]
conjugates with a fucoside derivative (4b) displayed good druggability properties,
whereas, a tripeptide spacer was difficultly manipulated and controlled due to
instability towards enzymes, leading to no entry into clinical trials. Therefore, a
saccharide type and a linker are very crucial for oligosaccharide-based CPT inhibitors.
We envisage that an oligosaccharide with tumor targeting incorporated to 20 (S)-OH
of CPT via an appropriate linker increases plasma stability and augments the
antitumor activity.
Given the above considerations, intact CPT structure would maintain specificity
of CPT derivatives to Topo I and exert the antitumor activities. The oligosaccharide
scaffolds with tumor targeting as the hydrophilic moiety would increase water
solubility, in vivo stability and selectivity of CPT derivatives. The suitable length of
PEG (ethylene glycol)-based linkers, which were biocompatible in both hydrophobic
[
16]
and hydrophilic environments and hydrolytically stable towards various enzymes
,
would mediate a CPT moiety and a carbohydrate moiety as an integrity to enhance the
antitumor activity and lower the untoward effect. In this study, four monosaccharides
(glucose, galactose, mannose, 3-O-carbamyl mannose) and a bleomycin (BLM)
disaccharide were selected to explore the potential influence on the physicochemical
properties, Topo I inhibitions and antitumor activities of CPT glycoconjugates (Fig. 2).
Construct 40 decorated with a BLM disaccharide, which displayed the potent

cytotoxicity and high selectivity, was further performed toxicological assessment.
O
N
N
CPT responsible for
O
O
antitumor effects.
O
20
O
O
N
O
HO
H
n
Responsible for stability
O
Oligosaccharides responsible
for water solubilityand targeting
n=0, 1, 2
Fig. 2 Design of 20 (S)-O-linked CPT glycoconjugates.
2
. Results and discussion.
2
.1 Chemistry synthesis
A series of CPT glycoconjugates were achieved by attaching five
tumor-targeting simple sugars, such as glucose, galactose, mannose, 3-O-carbamyl
mannose and BLM disaccharide, to the highly hydrophobic CPT scaffold. Prior to
syntheses of CPT glycoconjugates, the general synthetic routes for activated CPT 5
and benzoxycarbonyl (Cbz)-protected linkers 8 and 9 were depicted in Scheme 1. For
the challenging acylation of the tertiary and unreactive 20 (S)-hydroxyl group, we
used activated CPT 5 as a key intermediate, which was obtained by CPT with
4
-nitrophenyl carbonochloridate in the presence of dimethylaminopyridine (DMAP)
[
11]
in an 86.3% yield . In the syntheses of Cbz-protected linkers 8 and 9, treatment of
ethanolamine 6 and its derivative 7 with benzyl carbonochloridate easily afforded
[
11]
them in 85.2% and 87.5% yields, respectively .
o
Scheme 1. Reagents and conditions: a) 4-nitrophenyl carbonochloridate, DMPA, CH Cl , 0 C-r.t., 3h, 86.3%; b)
2
2
benzyl carbonochloridate, N(Et) , THF, r.t. 1h, 85.2% for 8, 87.5% for 9.
3
CPT glycoconjugates (22a-d, 23a-d) and the corresponding peracetylated ones
18a-d, 19a-d) were illustrated in scheme 2. Peracetylated 11a-c served as starting
(
materials, which were prepared from the corresponding commercially available
[
17]
monosaccharides (10a-c).
The selective deacetylation of constructs 11a-c and

subsequent nucleophilic addition reaction of compounds 12a-c with trichloro-
acetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as the catalyst base
[
18]
yielded trichloroacetimidates 13a-c in high yields, respectively . Noticeably,
synthesis of peracetylated unnatural 3-O-carbamyl mannose 12d included a six-step
[
11]
reaction starting from mannose in an overall 69.1% yield , and the activation of
acetylated 12d
o
Scheme 2. Reagents and conditions: a) Ac O, 3% con. H SO ,, r.t; b) AcOHN H , DMF, 50 C; c) For 13a-c,
2
2
4
2
4
o
o
Cl CCN, 0.1 eq. DBU, CH Cl , 0 C; For 13d, PO(OPh) Cl, n-BuLi, THF, -78℃ ; d) -5 C-r.t., linker 8 or 9,
3
2
2
2
TMSOTf; e) H , 10% Pd/C, AcOEt, r.t.; f) CH ONa, CH OH, Dowex50 resin, r.t.; g) 5, CH Cl , DIPEA, r.t..
2
3
3
2
2
with PO(OPh) Cl and n-BuLi as a base afforded diphenyl phosphate 13d in a high
2
[
19, 20]
yield
. Then treatment of compounds 13a-d with Cbz-protected linkers 8 and 9
yielded linker-coupled derivatives 14a-d and 15a-d under the catalysis of TMSOTf,
1
respectively. H NMR analysis demonstrated coupling constants of H-H at the

reducing end of sugar residues in 14a-b and 15a-b were 8.0 Hz, indicating glucose
and galactose were coupled to linkers 8 and 9 in a β-configured manner. By contrast,
α-linkages for mannose and 3-O-carbamyl mannose connected to likers 8 and 9 were
deduced in constructs 14c-d and 15c-d due to H-H coupling constants of 4 Hz at the
[20]
reducing end. Nucleophilic substitution of activated 5 with amines 16a-d and 17a-d
favorably yielded protected CPT glycol-conjugates 18a-d and 19a-d in the presence
of N,N-diisopropylethylamine (DIPEA), respectively. Deprotection of constructs
1
6a-d and 17a-d with CH ONa in anhydrous methanol followed by similar reactions
3
with activated 5 afforded the corresponding CPT glycoconjugates 22a-d and 23a-d.
No trace of 22a-d and 23a-d was detectable when directly deacetylated from the
corresponding precursors 18a-d and 19a-d.
However, the synthesis of CPT conjugates (26a-d, 27a-d) bearing longer-chain
linkers (n=2) was accomplished by another strategy (Scheme 3). Coupling of 13a-d
and 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol and subsequent azide substitution of
chlorinated 24a-d produced derivatives 25a-d, which were reduced with 10% Pd/C
and hydrogen followed by the similar reaction conditions as compounds 22a-d and
1
8a-d to yield target CPT conjugates 26a-d and 27a-d respectively. Analogously,
deacetylation of compounds 27a-d failed to generate the corresponding conjugates
2
6a-d. The linkage modes of saccharides coupled to linkers in compounds 26a-d and
7a-
2
Scheme 3. Reagents and conditions: a) 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol, TMSOTf, anhydrous CH Cl , -5
2
2

o
o
C; b) NaN , DMF, 70 C; c) i) CH ONa, anhydrous CH OH, Dowex50 resin, ii) H , 10% Pd/C, 1.0 atm, iii) 5,
3
3
3
2
o
o
DIPEA, 0 C, overnight; d) i) H , 10% Pd/C, 1.0 atm, ii) 5, DIPEA, 0 C, overnight, e) NaOCH , anhydrous
2
3
CH OH.
3
1
d were in complete accordance with those of compounds 24a-d. The signals from H
NMR of 24a-d clearly supported a β -glycosidic bond for glucose and galactose
(J=8.0 Hz) but an α-linkage for mannose and 3-O-carbamyl mannose (J= 4.0 Hz) in
the coupling reaction.
BLM disaccharide consisting of L-gulose and 3-O-carbamyl-D-mannose, a
[
12,13]
tumor-seeking moiety of BLM
, was selected as a representative for exploring the
effect of disaccharides on CPT. The appropriately designed route for the synthesis of
BLM disaccharide-based CPT derivatives 39-44 was demonstrated in scheme 4.
Construct 28 was activated with PO(OPh) Cl and n-BuLi, yielding the only
2
[11]
α-glycosyl diphenyl phosphate 29 in 70.0% yield.
between construct 29 and Cbz-protected linkers 8 and 9
BLM disaccharides 30 and 31 respectively, which were subjected to a hydrogenolysis
The nucleophilic substitution
[19]
yielded linker-coupled
[
19]
procedure followed by the in situ nucleophilic reaction with activated 5 to afford
the corresponding conjugates 42 and 43. Similarly, treatment of construct 29 with
2
-(2-(2-chloroethoxy)ethoxy)ethan-1-ol in the presence of TMSOTf followed by
adize substitution yielded linker-coupled compound 32. Then coupling reaction of
amine 35, which was derived from azide 32 under the catalysis of 10% Pd/C and H2,
and compound 5 afforded conjugate 44. Linker-coupled disaccharides 33-35 were first
deacylated with CH ONa in anhydrous CH OH followed by direct amidation with 5
3
3
to provide gylcoconjugates 39-41 in acceptable yields.

Scheme 4. Reagents and conditions: a) PO(OPh) Cl, n-BuLi, THF, -78℃ , 70.0%; b) linker 8 or 9, TMSOTf,
2
anhydrous CH Cl ; c) i) 2-(2-(2-chloroethoxy)ethoxy)ethan-1-ol, TMSOTf, anhydrous CH Cl , ii)NaN , DMF, 70
2
2
2
2
3
o
o
C; d) H , 10% Pd/C, r.t.; e) CH ONa, anhydrous CH OH; h) 5, DIPEA, 0 C, overnight.
2
3
3
All the newly synthesized CPT glycoconjugates were obtained according to the
synthetic routes illustrated as Schemes 1-3, respectively, and the yields were generally
acceptable ranging from 23.7% to 73.3% (Table 1). Although the yields of these CPT
derivatives lacked the comparative basis due to the different synthetic routes, some
tendency was still observed. For instance, CPT modified with acetyl-capped
oligosaccharides had higher yields than CPT with the corresponding oligosaccharides
except for 18c and 22c, 19b and 23b, 19c and 23c, this was possibly ascribed to better
separation and less reaction of CPT gylcoconjugates capped with acetyl groups. As
for compounds 18a-d, 19a-d, 22a-d and 23a-d following the procedure listed in
Scheme 1, the yields was largely dependent on sugar type and had little connection
with length of a linker. However, the yields of compounds 39-44 prepared as the
procedure shown in Scheme 3 were closely associated with length of a liner, and
compounds 40 and 43 with diethylene glycol had the lowest yields.
Table 1. The yields of newly synthesized CPT glycoconjugates
Compds R1
m
n
Yield (%)
Compds
R1
m
n
Yield (%)
a
a
1
1
1
1
2
2
2
8a
8b
8c
8d
2a
2b
2c
Ac-
Ac-
Ac-
Ac-
H-
1
1
1
1
1
1
1
0
0
0
0
0
0
0
41.7
23d
27a
27b
27c
27d
26a
26b
H-
1
1
1
1
1
1
1
1
2
2
2
2
2
2
24.5
a
b
27.8
Ac-
Ac-
Ac-
Ac-
H-
73.3
a
b
25.8
45.3
a
b
50.3
48.4
a
b
23.8
41.3
a
b
H-
24.5
27.0
a
b
H-
49.1
H-
32.3

a
a
a
a
a
a
a
a
b
b
2
1
1
1
1
2
2
2d
9a
9b
9c
9d
3a
3b
3c
H-
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
34.3
41.0
24.4
28.2
44.4
29.6
34.3
44.3
26c
26d
39
H-
1
1
2
2
2
2
2
2
2
2
0
1
2
0
1
2
23.7
41.4
38.5
25.7
42.8
60.5
48.9
60.3
Ac-
Ac-
Ac-
Ac-
H-
H-
c
H-
c
c
c
c
c
40
H-
41
H-
42
Ac-
Ac-
Ac-
H-
43
2
H-
44
a
b
means the yield calculated from 13a-d according to the synthetic route shown in Scheme 1; means
c
the yield calculated from 13a-d according to the synthetic route shown in Scheme 2; means the yield
calculated from construct 29 according to the synthetic route shown in Scheme 3
2
2
.2 In vitro and in vivo evaluation
.2.1 Cytotoxicity assay.
The in vitro antitumor activities of all the newly synthesized CPT gylcoconjugates
were assessed in comparison with the marketed irinotecan and the parent compound
CPT against three cancer cell lines including hepatocellular carcinoma cell line
(HepG2), colorectal carcinoma cell line (HCT116) and pancreatic carcinomas cell line
(SW1990) and one normal cell line HEK-293 (Table 2). Clinically, CPT derivatives
[3,4]
treated the patients suffering from intestinal cancer or hepatocellular carcinoma
.
Irinotecan was an approved anticancer agent by FDA, constituting the first-line
[
21]
therapy for colon cancer. The in vitro cytotoxicity was expressed as a half-maximal
inhibitory concentration (IC , the concentration of a compound needed to inhibit half
5
0
growth of cells).
All the CPT gylcoconjugates (18a-d, 19a-d, 22a-d, 23a-d, 26a-d, 27a-d and
9-44) decorated with five oligosaccharides and the corresponding acetyl-capped ones
3
exhibited potent or moderate anti-proliferative activities against three cancer cells
with IC values ranging from 0.3 to 32.8 µM. Against three cancerous cell lines, the
5
0
CPT derivatives tested were more sensitive towards HCT116 and HepG2 over
SW1990. Compared to CPT, all the derivatives showed comparable or inferior
antitumor activities and differential toxicities to normal cells HEK-293, implying that

acetyl groups, oligosaccharide type and length of ethylene glycol-based likers had
considerable but complicated impacts on the cytotoxicity and selectivity. On the
contrary, compared to the positive control irinotecan (>100 µM), the CPT
glycoconjugates demonstrated more potent antitumor activity. This was probably
ascribed to the action mechanisms different from that of prodrug irinotecan that was
[
22]
activated by the carboxylesterase to release free drug CPT
.
Compounds 22a-b, in which the carbohydrate moieties were GLUT-4-mediated
[
23]
[24]
glucose
and ASGPR-directed galactose
,
respectively, showed good
cytotoxicities against tree cancer cells (10.3 µM >IC >3.8 µM) but displayed no
5
0
selectivity to HEK-293 (IC =2.2 µM for 22b, 3.5 µM for 22a). When glucose (22a)
5
0
[
25]
was replaced with mannose receptor (MR) -mediated mannose (22c) and
[13]
3
-O-carbamyl mannose with unknown mechanism
(22d), respectively, the
decreased tendency of the antitumor activities was observed, except that 22d
exhibited a slight increase in inhibiting HCT116 growth (IC50 =2.2 µM). The
introduction of acetyl groups to sugar moieties (18a-b and 18d) resulted in an obvious
loss of the in vitro potency against cancer cells. 18b with an acetyl-capped galactose
(
IC =60.2 µM) indicated 12 times less active than 22b bearing a galactose (IC =
5
0
5
0
4
.9 µM) in inhibiting ASGPR-overexpressing HepG2 cells, suggesting that
galactose-mediated targeting outbalanced the lipophilicity of acetyl groups. By
contrast, acetylation of a mannose scaffold (18c) doubled the antitumor activity
towards HepG2 (IC =10.3 µM (22c) vs 4.3 µM (18c)) and SW1990 (IC =20.4 µM
5
0
50
(22c) vs 9.7 µM (18c)). The selectivity of 18a-d towards HEK-293 was not
significantly affected.
Compounds 23a-d with diethylene glycol chains were more effective in the
antiproliferative activities compared to compounds 22a-d having monoethylene
glycol chains, accompanying with less toxicity to HEK-293. 23a carrying a glucose
exhibited 8.6 times and 3.0 times more potent than the corresponding 22a in the
inhibition of HCT116 (IC =0.5 µM (23a) vs 4.3 µM (22a)) and HepG2 (IC = 1.2
50
50
µM (23a) vs 3.8 µM (22a)), respectively, however, no detectable changes for
SW1990 was observed. Noticeably, 23c with a mannose moiety displayed the most

obvious increase in the potency of antitumor activity against HepG2 (IC =0.3 µM
5
0
(23c) vs 10.3 µM (22c)). Similar to constructs 22a-d, attachment of the acetyl groups
to a carbohydrate moiety (19a-d) led to a decrease with varying degrees, and
acetylation had little impacts on the selectivity towards HEK-293. When diethylene
glycol was elongated to triethylene glycol (26a-d), the antitumor activities (26a-d,
1
9.9 µM >IC50 >0.5 µM) decreased a little compared to 23a-d (10.6 µM >
IC50 >0.3µM). Surprisingly, the selectivity of 26a-d completely disappeared. This
hinted that length of a PEG linker was too long to mediate the selectivity of CPT via a
carbohydrate moiety with tumor targeting. Therefore, the appropriate linker length
turned to be of particular importance in the potency and selectivity of CPT
glycoconjugates as antitumor agents.
The synthesis of glycoconjugates 39-41 was used to explore the effects of BLM
[
12,13]
disaccharide, which had been demonstrated to be a tumor-seeking scaffold
and to
possess higher water-solubility, on the antitumor activity and selectivity of CPT.
Compounds 42-44 were synthesized to investigate the effects of acetyl groups on the
cytotoxicity and selectivity. With length of a PEG linker was increased from
monomer to trimer, the antitumor activities of compounds 39-41 remained small
change, whereas compound 40 with a diethylene glycol chain exhibited a high
selectivity between HCT116 cells (IC =0.8 µM) and HEK-293 cells (IC >100 µM).
5
0
50
Acetylation fail to lead to any significant effects on the antitumor activity (compounds
2-44), and no selectivity was observed. Taken together, oligosaccharide-CPT
4
conjugates were sensitive to HepG2 cells and HCT116 cells. CPT modified with
galactose and BLM disaccharide via exhibited higher selectivity, which were possibly
associated with sugar moieties introduced. Diethylene glycol that was the optimal
linker to assemble CPT and oligosaccharides improved the antitumor activity and
selectivity of CPT glycoconjugates. Acetylation disfavored the antitumor activity,
which possibly affected a cellular uptake of the CPT glycoconjugates mediated by
[
15]
oligosaccharides with tumor targeting
.
Table 2. The cytotoxicities of CPT glycoconjugates against hepatocellular carcinoma cells
HepG2), colorectal carcinoma cells (HCT116), pancreatic carcinoma cells (SW1990) and kidney
(

cells (HET-293).
a
Compds
R1
m
n
IC values (µM)
50
HepG2
29.9
60.2
4.3
HCT-116
15.0
29.5
7.5
SW1990
24.8
37.5
9.7
HEK-293
29.5
8.6
1
1
1
1
2
2
2
2
1
1
1
1
2
2
2
2
2
2
2
2
2
2
8a
8b
8c
8d
2a
2b
2c
2d
9a
9b
9c
9d
3a
3b
3c
3d
7a
7b
7c
7d
6a
6b
Ac-
Ac-
Ac-
Ac-
H-
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2.3
18.5
3.8
10.8
4.3
20.6
10.3
9.3
3.9
3.5
H-
4.9
4.1
2.2
H-
10.3
11.6
2.1
8.2
20.4
32.8
10.3
15.7
10.3
7.6
5.8
H-
2.3
3.2
Ac-
Ac-
Ac-
Ac-
H-
4.2
12.3
>100
20.6
50.2
24.6
43.5
22.4
52.1
0.6
5.6
3.5
4.8
4.4
3.7
3.6
1.2
0.5
9.6
H-
2.8
2.6
10.6
3.2
H-
0.3
5.6
H-
0.9
1.2
5.2
Ac-
Ac-
Ac-
Ac-
H-
0.7
0.8
4.5
0.7
0.9
8.3
0.8
1.6
0.6
28.9
31.4
12.5
19.9
3.4
1.8
4.6
3.4
1.3
4.8
0.7
H-
1.2
8.2
0.7

2
2
3
4
4
4
4
4
6c
6d
9
H-
1
1
2
2
2
2
2
2
2
2
0
1
2
0
1
2
0.8
0.8
2.5
1.2
2.6
4.2
1.6
1.9
0.4
>100
0.5
16.3
18.2
30.1
30.5
32.6
25.1
11.3
28.6
0.2
0.6
H-
0.9
0.4
H-
13.2
0.8
6.2
0
H-
>100
30.7
1.6
1
H-
18.3
6.7
2
Ac-
Ac-
Ac-
3
1.3
1.5
4
23.3
0.4
2.3
CPT
0.6
Irinotecan
>100
>100
>100
a
indicates the average value of the three independent experiments.
2
.2.2 DNA Topo I inhibition assays
To determine whether the antiproliferation activity against three cancer cell lines
of the newly synthesized CPT glycoconjugates originated from the inhibition of DNA
Topo I, a cell-free assay was conducted using purified recombinant human Topo I
(Fig.3). In this assay, supercoiled plasmid DNA was relaxed and nicked by
recombinant Topo I. With supercoiled plasmid DNA only plus recombinant human
Topo I as the vehicle control, relaxed and nicked DNA were detectable. As the parent
compound CPT was unable to reach a tested concentration of 100.0 µM due to poor
aqueous solubility, 10-hyroxyl camptothecin (10-HCPT) was instead of CPT to act as
a positive control. Irinotecan was another positive control. As demonstrated in Fig 3A
and Fig S153A-B, 10-HCPT strongly inhibited Topo I activity at 100.0 µM in the cell
[
26]
free assay.
Whereas, the tested CPT derivatives showed very weaker inhibitory
activity towards DNA Topo I, which were slightly superior or comparable to the
positive control irinotecan. When a tested concentration was decreased to 50.0 µM,
Topo I inhibition of the CPT glycoconjugates completely disappeared (Fig.3B and
Fig. S153C). Unlike a sulfonylamidine group in the induced ester moiety desirable to
[
27]
better the inhibitory e℃ ect on Topo I,
modification with simple oligosaccharides
had a devastating effect on the Topo I activity irrespective of sugar type and linker
length, probably blocking recognition of E-ring to the interface of Top1/DNA

complex. Although the CPT glycoconjugates were similar to irinotecan in the
inhibition of Topo I activity, the antiproliferation activity was entirely different
(Table 1). Accordingly, we speculated that the antiproliferation activities of the CPT
glycoconjugates were not due to direct anti-Topo I activity, hinting that new
mechanisms of antitumor action were possibly associated with a carbohydrate moiety.
[
15]
Fig. 3 The CPT gylcoconjugaes directly inhibit Topo I in a cell-free system. (A) Recombination human Topo I was
incubated with the vehicle (DNA+Topo I), 100 µM Iri (Irinotecan), 100 µM 10-HCPT, or 100 µM the newly
synthesized CPT glycoconjugates (19b, 23d, 26b, 27b, 40, 42-44) followed by incubation with supercoiled
plasmid DNA. (B) Recombination human Topo I was incubated with the vehicle (DNA+Topo I), 50 µM Iri
(Irinotecan), 50 µM 10-HCPT, or 50 µM the CPT glycoconjugates (19b, 23d, 40, 43) followed by incubation with
supercoiled plasmid DNA. Plasmid DNA was separated by agarose gel and stained with ethidium bromide. NK
means nicked, RLX means relaxed, SC means supercoiled.
2
.2.3 In vitro evaluation of water solubility and stability
Water solubility and in vivo stability were major reasons for the side effects and
[5,7]
the ineffectiveness of CPT and its derivatives.
To ascertain whether the
introduction of oligosaccharides and acetyl-capped oligosaccharides to 20 (S)-OH
increased the aqueous solubility and stability, 19b, 23d and 40 with the more potent
cytotoxicity and higher selectivity were selected as model compounds for the assays.
According to the procedures for aqueous solubility and in vitro stability reported
[
1,28]
previously
, all the data were illustrated in Table 3. CPT was very poorly soluble
in aqueous solution at pH=7.4 (0.06 mg/mL). The incorporation of an acetyl-capped
monosaccharide residue (19b, 0.5 mg/mL) resulted in an 8-fold increase in aqueous
solubility. In contrast, 40 with a BML disaccharide unit (21.2 mg/mL) and 23d
carrying a 3-O-carbamyl mannose residue (16.3 mg/mL) were 3533-fold and
2
717-fold more soluble than the lead CPT, respectively. Incubation of CPT or 19b or

2
3d or 40 in PBS (phosphate-buffered saline,
Table 3. Aqueous solubility and in vitro stability of CPT and compounds 19b, 23d, 40 at pH 7.4
a,c
b,c
Compds
CPT
Solubility (mg/mL)
PBS stability (carboxylate/lactone)
0.06
0.5
93/5
3/91
5/92
0/94
1
2
9b
3d
16.3
21.2
4
0
a
Aqueous solubility was measured in mg/mL of a compound tested in 0.1M Na HPO solution (pH=7.4, and 37
2
4
o
b
C); Stability was determined as the ratio of carboxylate and lactone of a compound tested in 10% phosphate
o
c
buffer solution at pH=7.4 for 48 h at 37 C. The mean value of triplicate determinations was shown.
pH 7.4) revealed that the glycococonjugates were stable for 48 h but CPT due to its
unstable lactone was absolutely converted into the carboxylate form in 50 min. 40 was
found to be highly stable in PBS with 94% presence after 48 h without the generation
of the carboxylate from. The prolonged stability of compounds 19b, 23d and 40 in
PBS at neutral pH indicated that the PEG-coupled carbamate linker was resistant to
pH-mediated cleavage, confirming that oligosaccharides attached to 20 (S)-OH were
conductive to existence of the lactone ring. Thus, such glycoconjugates with excellent
water solubility, just like 23d and 40, should be sufficiently stable in culture medium
and in serum.
2
.2.4 In vivo toxicological evaluation.
Since 40 was unable to directly inhibit Topo I activity but possessed strong in
vitro cytotoxicities against HCT116 and HepG2 and a higher selectivity towards
HEK-293, suggesting that it was a novel antitumor agent with lower in vivo toxicity,
we further performed the primary toxicological assessment of 40. Acute toxicity of 40
in the ICR mouse was evaluated pathologically. Thirty-two 6-week-old female ICR
mice were randomized into four groups (n = 8) to receive 0 (control), 85, 160, or 320
mg/kg of 40 intravenously on day 0. All the treated animals displayed no allergic
responses, or significant body weight loss (Fig. 4A) and were as healthy as the
untreated animals, indicating significantly reduced toxicity compared to that of

[
29]
irinotecan (LD =85.1 mg/kg, i.v) . On day 14, animals were euthanized, and tissues
5
0
from the liver, lung, kidney and spleen were evaluated histopathologically according
[
30]
to the guidelines , and symptomatic lesions, including inflammatory cell infiltration,
and focal necrosis in the liver, kidney, spleen and lung, were graded. A few
microscopic lesions were detectable in liver, lung and kidney from 40-treated group.
As demonstrated in Fig. 4B and 4C, the animals clearly tolerated treatment with 160
mg/kg of 40, portending an acceptable safety profile. Noticeably, although
inflammatory cell infiltration in lung was moderate at 320 mg/kg, no mice died during
the experimental period, hinting maximum tolerated dose (MTD) was more than 320
mg/kg. We postulated that the toxicological improvement on normal tissues might be
associated with introduction of a BLM disaccharide in the 20 (S) -OH of 40. Further
studies including metabolic and pharmacokinetic evaluations are currently underway
to address this hypothesis.

Fig. 4. Pathological evaluation of toxicity of 40 in mice. (A) No significant effects on body weight. Thirty-two
ICR mice were randomly divided into four groups, and 8 animals in each group received an i.v. injection of 40 at 0
(Control), 85, 160, or 320 mg/kg. (B) Histopathological examination of hepatotoxicity. Formalin-fixed livers from
animals treated with vehicle, 85 mg/kg, 160 mg/kg or 320 mg/kg, were embedded in paraffin. Tissue sections were
stained with hematoxylin and eosin (H&E). Stained sections were evaluated histopathologically for focal necrosis,
and inflammatory cell infiltration. Central vein (C) and portal tract (P) were indicated (upper right panel).
Inflammatory cell infiltration and focal necrosis were labeled as arrows. (C) Summary of histopathological
evaluation of toxicity. Tissues from the kidney, spleen, lung, and liver were fixed with formalin and embedded in
paraffin. Tissue sections were evaluated histopathologically as indicated after staining with H&E.
3
. Conclusion
In summary, we have designed and synthesized thirty new CPT glycoconjugates
for assessing the aqueous solubility, stability, in vitro antitumor activity, Topo I
inhibition and in vivo toxicological assessment. Such 20 (S)-O-linked CPT
glycoconjugates were conductive to the significant increase in aqueous solubility and
sufficient hydrolytic stability of the lactone ring. Oligosaccharide type, length of a
PEG linker and acetyl group had major impacts on the antitumor activity and
selectivity. All the newly synthesized CPT glycoconjugates had no direct inhibition
against Topo I. In vivo animal models demonstrated that MTD of 40 was more than
3
20 mg/kg, portending a more safety profile compared to irinotecan. Collectively, the
compelling evidence advocated CPT glycoconjugates as potential targeted drugs.
. Experimental section
.1. Chemistry.
4
4

All chemicals were of reagent grade quality or better, which were obtained from
commercial suppliers and used without further purification. Solvents were used as
received or dried over molecular sieves. All the preparations were performed using
standard Schlenk techniques. Column chromatography was conducted on silica gel
(100-200 mesh) from Qingdao Ocean Chemical Factory. TLC (HSGF 254) from
Yantai Jiangyou Silica Gel Development Co. LTD (Yantai, China) was monitored the
1
reaction process. All the key intermediates and products were determined by HNMR
1
3
1
13
and CNMR, recorded in a Bruker Avance 400 ( H at 400 MHz, C at 100 MHz),
and chemical shifts were reported in parts per million using the residual solvent peaks
1
13
as internal standards (CDCl =7.26 ppm for HNMR and 77.16 ppm for CNMR,
3
1
13
CD SOCD =2.50 ppm for HNMR and 39.6 ppm for CNMR). Some key
3
3
intermediates and products were also confirmed by electrospray ionization high
resolution mass spectrometry (ESI-HRMS), recorded on AB Sciex triple TOF 5600+
system. The purity was more than 95%, which was determined with C -column (250
1
8
x 4.6 mm, 5 µm, Agilent Eclipse Plus) run on Agilent Technologies 1260 infinity II.
.1.1 Synthesis of (S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]
4
indolizino[1,2-b]quinolin-4-yl (4-nitrophenyl) carbonate (5)
To a solution of CPT (5.0 g, 14.3 mmoL) dissolved in anhydrous DCM (100 mL)
4
-nitrophenyl chloroformate (28.6 mmoL) and 4-dimethylaminopyridine (85.8 mmoL)
were added dropwisely at 0℃ , and the reaction mixture was heated to 40℃ and
proceeded for 4 h. After completion, the solution was diluted with 500 mL of ethyl
acetate and was adjusted to be neutral with 0.1 M HCl, and the organic layer was
washed 100 mL of brine and dried with anhydrous Na SO . The residue obtained by
2
4
evaporating organic solvent was recrystallized with n-hexane to give 6.3 g of
activated CPT 5 as a yellowish white solid.
1
Yield, 86.0 %; HNMR (400 MHz, CDCl ) δ 8.44 (s, 1H), 8.26 (dd, J = 8.0 Hz,
3
1
H), 8.22 (dd, J = 8.0 Hz, 2H), 7.98 (d, J = 8.0 Hz, 1H), 7.89 (t, J = 8.0 Hz, 1H), 7.72
(t, J = 8.0 Hz, 1H), 7.44 (s, 1H), 7.4 (d, J = 9.0 Hz, 2H), 5.74 (d, J = 16.0 Hz, 1H),
5
.44 (d, J = 16.0 Hz, 1H), 5.32 (d, J = 4.0 Hz, 2H), 2.39-2.34 (m, 1H), 2.28-2.21 (m,
1
3
1
H), 1.07 (t, J = 8.0 Hz, 3H); CNMR (100 MHz, CDCl ) δ 166.98, 157.37, 155.17,
3

1
1
7
4
52.21, 151.38, 149.01, 146.95, 145.71, 145.08, 131.55, 131.08, 129.67, 128.57,
28.43, 126.36, 125.41, 121.84, 120.48, 115.81, 95.78, 79.44, 67.32, 50.25, 32.02,
.80.
.1.2. Synthesis of benzyl (2-hydroxyethyl)carbamate (8) and benzyl
(2-(2-hydroxyethoxy)ethyl)carbamate (9)
1.0 g of 2-aminoethan-1-ol (6, 16.39 mmoL) was dissolved in 15 mL of
o
tetrahydrofuran and 16 mL of trimethylamine at 0 C, and then 3.36 g of benzyl
chloroformate (19.67 mmoL) was dropwisely added at the same temperature. The
reaction was allowed to proceed for 1 h at r.t. The reaction progress was monitored
with TLC. Ethyl acetate (50 mL) and water (20 mL) were poured into the reaction
mixture，and the organic layer was washed with brine, and dried by anhydrous
Na SO , and then concentrated under reduced pressure to obtain the residue, which
2
4
was purified by silica gel column chromatography with ethyl acetate and petroleum
ether (V/V=1:1) as an eluent to afford 2.88 g of compound 8 as a white solid.
1
Yield, 85.2%. HNMR (400 MHz, CDCl ): δ 7.39-7.27 (m, 5H), 5.45 (brs, 1H),
3
13
5
.08 (s, 2H), 3.65 (d, J = 8.0 Hz, 2H), 3.30 (d, J = 8.0 Hz, 2H), 2.87 (s, 1H). CNMR
(
100 MHz, CDCl ): δ 157.18, 136.36, 128.55, 128.19, 128.09, 66.91, 61.96, 43.47.
3
Compound 9 was synthesized as the similar procedure of compound 8, only
differing
in
starting
material
2-aminoethan-ol
(6)
replaced
by
2
-(2-aminoethoxy)ethan-1-ol (7).
1
Yield, 87.5%. HNMR (400 MHz, CDCl ): δ 7.35-7.29 (m, 5H), 5.37 (brs, 1H),
3
5
2
7
4
.10 (s, 2H), 3.76-3.69 (t, J = 4.0 Hz, 2H), 3.54 (t, J = 4.0 Hz, 4H), 3.39 (d, J = 4.0 Hz,
1
3
H), 2.40 (s, 1H). CNMR (100 MHz, CDCl ): δ 156.70, 136.52, 128.51, 128.12,
3
2.28, 70.08, 66.74, 61.60, 40.87.
.1.3 General procedures of compounds 14a-d and 15a-d.
Compounds 13a-d (1.0 mmoL) were dissolved in anhydrous DCM (7 mL), and
then compound 8 (1.2 mmoL) and trimethylsilyl trifluoromethanesulfonate (1.0
mmoL) were added at -5℃. The mixture was stirred for 2 h under nitrogen atmosphere
at the same temperature. After the completion of the reaction, the mixture was
quenched with adequate trimethylamine. Ethyl acetate (30 mL) was added to the

resultant mixture. Then the organic layer was washed with brine (10 mL), dried by
anhydrous Na SO and concentrated under reduced pressure to give the crude residue,
2
4
which was purified by silica gel column chromatography with ethyl acetate and
petroleum ether (V/V=1:1) as an eluent to give the corresponding compounds 14a-d
as colorless transparent liquid. Similarly, when reagent 8 was replaced with
compound 9, compounds 15a-d were obtained
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
tetrahydro-2H-pyran-3,4,5-triyl triacetate (14a): Yield, 62.4%. HNMR (400 MHz,
CDCl ): δ 7.34-7.31 (m, 5H), 5.20 (t, J = 8.0 Hz, 2H), 5.08 (d, J = 12.0 Hz, 3H), 4.96
3
(t, J = 8.0 Hz, 1H), 4.48 (d, J = 8.0 Hz, 1H), 4.22 (dd, J = 8.0, 4.0 Hz, 1H), 4.14 (d, J
=
12.0 Hz, 1H), 3.87-3.83 (m, 1H), 3.72-3.64 (m, 2H), 3.44-3.33 (m, 2H), 2.04 (s, 3H),
1
3
2
1
6
.01 (s, 3H), 1.99 (s, 6H). CNMR (100 MHz, CDCl ) δ 170.67, 170.24, 169.49,
3
69.41, 156.36, 136.44, 128.53, 128.18, 128.14, 100.05, 72.64, 71.88, 71.25, 69.54,
8.26, 66.76, 61.83, 40.89, 20.70, 20.60.
(2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
tetrahydro-2H-pyran-3,4,5-triyl triacetate (14b): Yield, 57.2%. HNMR (400 MHz,
CDCl ): δ7.35-7.27 (m, 5H), 5.37 (d, J = 4.0 Hz, 1H), 5.22 (dd, J = 16.0, 8.0 Hz, 2H),
3
5
.08 (s, 2H), 5.01 (m, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.13 (d, J = 8.0 Hz, 2H), 3.88 (t,
J = 8.0 Hz, 2H), 3.70-3.65 (m, 1H), 3.44-3.33 (m, 2H), 2.14 (s, 3H), 2.02 (s, 3H), 1.99
1
3
(
s, 3H), 1.97 (s, 3H). CNMR (100 MHz, CDCl ) δ 170.50, 170.33, 170.22, 169.75,
3
1
6
56.45, 136.52, 128.62, 128.26, 101.61, 70.87, 70.85, 69.55, 68.91, 67.06, 66.86,
1.42, 40.97, 20.78, 20.67.
(2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
tetrahydro-2H-pyran-3,4,5-triyl triacetate (14c): Yield, 62.5%. HNMR (400 MHz,
CDCl ): δ 7.36-7.30 (m, 5H), 5.32-5.28 (m, 1H), 5.25-5.23 (m, 2H), 5.11 (s, 2H), 4.82
3
(d, J = 4.0 Hz, 1H), 4.27 (dd, J = 8.0, 4.0 Hz, 1H), 4.09 (dd, J = 12.0, 8.0 Hz, 1H),
3
3
1
6
.98-3.94 (m, 1H), 3.80-3.75 (m, 1H), 3.57-3.54 (m, 1H), 3.47-3.38 (m, 2H), 2.15 (s,
1
3
H), 2.08 (s, 3H), 2.03 (s, 3H), 1.99 (s, 3H). CNMR (100 MHz, CDCl ): δ 169.76,
3
69.10, 169.07, 168.80, 155.47, 135.47, 127.66, 127.30, 127.28, 96.86, 68.49, 68.06,
7.83, 66.84, 66.00, 65.19, 61.60, 39.78, 19.99, 19.80.

(2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(2-(((benzyloxy)carbonyl)amino)ethoxy)-4-
1
(
(
(
carbamoyloxy)tetrahydro-2H-pyran-3,5-diyl diacetate(14d): Yield, 62.3%. HNMR
400 MHz, CDCl ): δ 7.37-7.27 (m, 5H), 5.35-5.33 (m, 1H), 5.28-5.20 (m, 3H), 5.10
3
s, 2H), 4.84 (s, 2H), 4.82 (d, J = 4.0 Hz, 1H), 4.26 (dd, J = 8.0, 4.0 Hz, 1H),
4
.09-4.05 (m, 1H), 3.98-3.94 (m, 1H), 3.79-3.74 (m, 1H), 3.59-3.41(m, 1H), 3.41-3.35
1
3
(
m, 2H), 2.13 (s, 3H), 2.07 (s, 3H), 2.04 (s, 3H); CNMR (100 MHz, CDCl ): δ
3
1
70.76, 170.13, 170.08, 156.50, 155.46, 136.50, 128.63, 128.26, 128.24, 97.79, 70.02,
9.97, 68.81, 66.96, 66.31, 62.60, 40.79, 21.01, 20.84, 20.80.
6
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15a): Yield, 71.6%. HNMR (400
MHz, CDCl ): δ 7.34-7.28 (m, 5H), 5.34 (brs, 1H), 5.17 (t, J = 8.0 Hz, 1H), 5.07 (s,
3
2
4
2
9
1
4
H), 5.03 (d, J = 8.0 Hz, 1H), 4.97 (dd, J = 8.0, 4.0 Hz, 1H), 4.55 (d, J = 8.0 Hz, 1H),
.21 (m, 1H), 4.09-4.07 (m, 1H), 3.87-385 (m, 1H), 3.66-3.83 (m, 2H), 3.56-3.51 (m,
H), 3.49-3.48 (m, 2H), 3.33-3.32 (m, 2H), 2.03 (d, J = 4.0 Hz, 3H), 1.99-1.94 (m,
1
3
H). C NMR (100 MHz, CDCl ): δ 170.66, 170.23, 169.42, 156.49, 136.65, 128.50,
3
28.15, 128.08, 100.74, 72.75, 71.82, 71.29, 70.15, 69.96, 68.85, 68.39, 66.64, 61.92,
0.90, 20.72, 20.62, 20.59.
(2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
ethoxy) tetrahydro-2H-pyran-3,4,5-triyl triacetate (15b): Yield, 62.0%. H NMR (400
MHz, CDCl ): δ 7.35-7.29 (m, 5H), 5.35 (s, 1H), 5.31 (s, 1H), 5.19 (t, J = 8.0 Hz, 1H),
3
5
3
2
.09 (s, 2H), 5.01 (t, J = 12.0 Hz, 1H), 4.52 (d, J = 8.0 Hz, 1H), 4.26-4.00 (m, 2H),
.92-3.87 (m, 2H), 3.69 (d, J = 8.0 Hz, 1H), 3.59 (m, 2H), 3.54-3.45 (m, 2H), 3.36 (m,
1
3
H), 2.11 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.96 (s, 3H). CNMR (100 MHz,
CDCl ): δ 170.49, 170.34, 170.21, 169.64, 156.56, 136.68, 128.59, 128.24, 128.19,
3
1
01.36, 70.92, 70.78, 70.21, 70.07, 68.90, 67.10, 66.76, 61.38, 40.98, 20.82, 20.78,
0.73, 20.68.
2
(2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)
1
ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (15c): Yield, 61.0%. HNMR (400
MHz, CDCl ): δ 7.33-7.27 (m, 5H), 5.40-5.39 (m, 1H), 5.33 (d, J = 8.0 Hz, 1H),
3
5
.26-5.22 (m, 2H), 5.07 (s, 2H), 4.86 (s, 1H), 4.26-4.21 (m, 1H), 4.06 (dd, J = 16.0,

4
.0 Hz, 2H), 3.76 (m, 1H), 3.62 (dd, J = 16.0, 4.0 Hz, 3H), 3.51 (d, J = 4.0 Hz, 2H),
.35 (d, J = 8.0 Hz, 2H), 2.10 (s, 3H), 2.06 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H).
3
1
3
CNMR (100 MHz, CDCl ): δ 170.78, 170.22, 170.00, 169.87, 156.59, 136.67,
3
1
4
28.53, 128.10, 97.58, 70.33, 70.00, 69.68, 69.03, 68.47, 67.15, 66.69, 66.29, 62.64,
1.01, 20.93, 20.80, 20.74, 20.72.
(2R,3R,4S,5S,6R)-2-(acetoxymethyl)-6-(2-(2-(((benzyloxy)carbonyl)amino)ethoxy)
ethoxy)-4-(carbamoyloxy)tetrahydro-2H-pyran-3,5-diyl diacetate (15d): Yield, 58.0%.
1
HNMR (400 MHz, CDCl ): δ 7.34-7.27 (m, 5H), 5.45 (s, 1H), 5.24 (d, J = 4.0 Hz,
3
3
H), 5.08 (s, 2H), 4.89 (d, J = 8.0 Hz, 3H), 4.30-4.23 (m, 1H), 4.13 (dd, J = 8.0, 4.0
Hz, 1H), 3.80-3.73 (m, 1H), 3.70-3.59 (m, 3H), 3.55 (d, J = 8.0 Hz, 3H), 3.38 (d, J =
1
3
8
1
1
2
4
.0 Hz, 2H), 2.10 (s, 3H), 2.07 (s, 3H), 2.00 (s, 3H). C NMR (100 MHz, CDCl ): δ
3
70.80, 170.22, 170.14, 156.63, 155.46, 136.73, 128.60, 128.55, 128.21, 128.15,
28.11, 97.58, 70.36, 70.19, 70.04, 69.91, 68.50, 67.20, 66.71, 66.41, 62.67, 41.04,
0.99, 20.82, 20.79.
.1.4. General procedures for compounds 18a-d and 19a-d.
To a solution of compounds 14a-d or 15a-d (0.396 mmoL) dissolved in ethyl
acetate (5 mL) catalytic amounts of 10 % Pd/C (80 mg) were added, and the mixture
was stirred for 1 h at room temperature under hydrogen atmosphere. After completion,
the reaction mixture was filtered with celite and concentrated under reduced pressure
to provide the residues 16a-d or 17a-d, respectively, for next reaction without
purification. To a solution of compound 5 (0.48 mmoL) dissolved in DCM (5 mL)
crude residues 16a-d or 17a-d and diisopropylethylamine (0.1 mmoL) were added at
0
℃ . The mixture was heated to r.t. and was stirred overnight. After completion of the
reaction, DCM (100 mL) and statured ammonium chloride solution (20 mL) were
added to dilute the reaction mixture and were shaken for several minutes. The organic
layer was washed by brine, dried by anhydrous Na SO , and then concentrated under
2
4
reduced pressure to give the crude residue, which was purified by silica gel column
chromatography with ethyl acetate and DCM (V/V=1:1) as an eluent to afford the
corresponding compounds 18a-d or 19a-d as white solids.
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tet

rahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl) oxy) carbonyl)amino)
1
ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (18a): Yield, 66.8%. HNMR (400
MHz, DMSO): δ 8.70 (s, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.10 (dd, J = 8.0, 4.0 Hz, 1H),
7
.84 (t, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.37 (s, 1H), 5.32 (s, 2H), 4.97 (t, J =
.0 Hz, 1H), 4.77 (t, J = 4.0 Hz, 1H), 4.72-4.66 (m, 3H), 4.62 (d, J= 8.0 Hz, 1H), 4.27
4
(t, J = 8.0 Hz, 1H), 3.90 (t, J = 4.0 Hz, 2H), 3.85-3.58 (m, 5H), 2.65-2.56 (m, 1H),
2
8
1
1
6
+
.37-2.28 (m, 1H), 2.02 (s, 3H), 1.90 (s, 3H), 1.64 (s, 3H), 1.42 (s, 3H), 0.93 (t, J =
1
3
.0 Hz, 3H). CNMR (100 MHz, DMSO): δ 172.58, 170.07, 169.28, 169.08, 168.75,
60.78, 153.71, 152.41, 148.19, 144.24, 143.92, 131.57, 130.34, 130.25, 130.22,
29.39, 128.53, 128.19, 127.68, 99.68, 96.93, 88.83, 71.75, 70.70, 70.44, 67.83, 64.90,
1.96, 55.18, 50.75, 31.08, 29.91, 20.79, 20.72, 20.56, 19.32, 7.84. TOF-MS, m/z: [M
+
+
Na ], calcd for C H N O Na , 788.2273, found, 788.2318.
3
7
39
3
15
(2R,3R,4S,5S,6R)-2-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyran
o[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy) carbonyl) amino)
ethoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (18b): Yield,
1
4
8.6%. H NMR (400 MHz, CDCl ): δ 8.42 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.96 (d,
3
J = 8.0 Hz, 1H), 7.85 (t, J = 8.0 Hz, 1H), 7.75 (s, 1H), 7.69-7.65 (m, 1H), 5.40-5.33
(m, 3H), 5.11-5.02 (m, 3H), 4.62 (d, J=8.0 Hz, 1H), 4.44 (d, J=8.0 Hz, 1H), 4.17-4.02
(m, 3H), 3.95-3.76 (m, 4H), 2.49-2.40 (m, 3H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H),
1
3
1
1
1
7
1
7
.85 (s, 3H), 1.07 (t, J = 8.0, 3H). C NMR (100 MHz, CDCl ): δ 172.38, 170.48,
3
70.29, 170.05, 169.58, 162.30, 153.70, 152.37, 148.91, 144.65, 144.56, 131.42,
31.06, 130.83, 130.34, 129.74, 128.98, 128.39, 128.23, 101.10, 98.06, 89.17, 70.92,
0.75, 68.37, 67.01, 65.72, 64.22, 61.27, 50.47, 32.08, 29.91, 20.51, 20.30, 20.05,
+
+
9.63, 7.54. TOF-MS, m/z: [M + Na ], calcd for C H N O Na , 788.2273, found,
37
39
3
15
88.2263.
2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tet
(
rahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)amino)
1
ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (18c): Yield, 41.2%. HNMR (400
MHz, DMSO): δ 8.69 (s, 1H), 8.18 (d, J = 12.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H),
8
.07 (t, J = 8.0 Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.04 (s, 1H),

5
4
2
8
1
1
5
.44 (d, J= 4.0 Hz, 2H),5.29 (s, 2H), 5.18-5.09 (m, 2H), 5.09 (m, 1H), 4.85 (s, 1H),
.15-4.10 (m, 1H), 4.03-3.99 (m, 2H), 3.61-3.43 (m, 2H), 3.23-3.09 (m, 2H),
.15-2.12 (m, 2H), 2.08 (s, 3H), 2.00 (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H), 0.90 (t, J =
1
3
.0 Hz, 3H). C NMR (100 MHz, DMSO): δ 170.07, 169.64, 169.58, 168.10, 156.72,
54.64, 152.48, 147.97, 146.22, 145.88, 131.70, 130.43, 129.88, 129.06, 128.62,
28.09, 127.77, 119.10, 97.01, 94.93, 74.87, 68.80, 68.72, 67.87, 66.40, 65.56, 61.92,
+
0.29, 30.86, 20.68, 20.53, 20.47, 7.62. TOF-MS, m/z: [M + H ], calcd for
+
C H N O , 766.2454, found, 766.2436.
37
40
3
15
(2R,3R,4S,5S,6S)-2-(acetoxymethyl)-4-(carbamoyloxy)-6-(2-(((((S)-4-ethyl-3,14-
dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2b] quinolin-4-yl)oxy)
carbonyl)amino)ethoxy)tetrahydro-2H-pyran-3,5-diyl diacetate (18d): Yield, 81.3%.
1
HNMR (400 MHz, DMSO): δ 8.67 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 8.0
Hz, 1H), 7.88 (t, J = 7.5 Hz, 1H), 7.72 (t, J = 7.5 Hz, 1H), 7.41 (s, 1H), 6.68 (brs, 1H),
6
1
8
0
1
1
6
.54 (brs, 1H), 5.26 (s, 2H), 5.11-5.06 (m, 1H), 5.04 (d, J = 8.0 Hz, 1H), 4.98-4.93 (m,
H), 4.91 (d, J = 8.0 Hz, 2H), 4.82 (t, J = 8.0 Hz, 2H), 4.09-3.96 (m, 3H), 3.87 (d, J =
.0 Hz, 3H), 3.78-3.69 (m, 2H), 2.41-2.33 (m, 1H), 2.06 (s, 3H), 2.00-1.98 (m, 6 H),
1
3
.99 (t, J = 8.0 Hz, 3H). CNMR (100 MHz, DMSO): δ 172.63, 170.14, 169.65,
69.35, 160.63, 155.36, 153.85, 152.52, 148.02, 144.31, 144.10, 131.69, 130.44,
29.95, 129.74, 129.12, 128.59, 128.04, 127.77, 96.98, 96.53, 88.67, 69.17, 68.41,
8.05, 65.95, 62.98, 62.05, 54.98, 51.63, 31.25, 20.75, 20.59, 20.55, 7.67. TOF-MS,
+
+
m/z: [M + Na ], calcd for C H N O Na , 789.2226, found, 789.2257.
36
38
4
15
(2R,3R,4S,5R,6S)-2-(acetoxymethyl)-6-(2-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,
1
4-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)
amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19a): Yield, 57.3%.
1
HNMR (400 MHz, DMSO): δ 8.69 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.14 (d, J = 8.0
Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.74-7.70 (m, 1H), 7.03 (s,
1
H), 5.45 (d, J = 4.5 Hz, 2H), 5.30 (s, 2H), 5.25 (t, J = 12.0 Hz, 1H), 4.91 (t, J = 12.0
Hz, 1H), 4.77-4.69 (m, 2H), 4.15-4.13 (m, 1H), 3.99-3.88 (m, 2H), 3.74-3.71 (m, 1H),
3
.61-3.54 (m, 1H), 3.62-3.56 (m, 1H), 3.53-3.03 (m, 3H), 3.09-3.04 (m, 2H),
2
.15-2.10 (m, 2H), 1.98 (s, 3H), 1.97 (s, 3H), 1.95 (s, 3H), 1.91 (s, 3H), 0.89 (t, J =

1
3
8
1
1
6
.0 Hz, 3H). C NMR (100 MHz, DMSO): δ 170.14, 169.65, 169.39, 169.16, 168.15,
56.76, 154.54, 152.58, 147.92, 146.46, 145.77, 131.75, 130.53, 129.92, 129.06,
28.67, 128.12, 127.82, 119.00, 99.60, 94.97, 74.77, 72.16, 70.95, 70.61, 69.37, 69.05,
8.59, 68.24, 66.33, 61.76, 50.32, 31.26, 29.93, 20.58, 20.48, 20.45, 20.38, 7.65.
+
+
TOF-MS, m/z: [M + Na ], calcd for C H N O Na , 832.2536, found, 832.2548.
3
9
43
3
16
(2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-t
etrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl)amino)
ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19b): Yield, 39.4%.
1
HNMR (400 MHz, CDCl ): δ 8.42 (s, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 8.0
3
Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.79 (s, 1H), 7.69 (t, J = 8.0 Hz, 1H), 5.35 (s, 1H),
5
.31 (s, 2H), 5.18-5.02 (m, 3H), 4.97 (d, J = 12.0 Hz, 1H), 4.80 (d, J = 4.0 Hz, 1H),
.47 (d, J = 4.0 Hz, 1H), 4.13 (t, J = 8.0 Hz, 2H), 3.92 (d, J = 4.0 Hz, 1H), 3.89-3.84
4
(m, 3H), 3.73-3.71 (m, 2H), 3.65-3.60 (m, 3H), 2.51-2.41 (m, 2H), 2.14 (s, 3H), 2.04
13
(
s, 6H), 1.98 (s, 3H), 0.95 (t, J = 8.0 Hz, 3H). CNMR (100 MHz, CDCl ): δ 172.39,
3
170.55, 170.43, 170.31, 168.66, 162.27, 153.90, 152.37, 148.96, 144.82, 144.48,
131.39, 131.06, 130.88, 130.29, 129.86, 128.59, 128.29, 128.25, 101.37, 98.28, 89.35,
70.99, 70.70, 70.04, 69.09, 68.91, 67.17, 66.81, 65.72, 61.34, 50.33, 32.04, 29.45,
+
2
0.87, 20.84, 20.81, 20.73, 7.79. TOF-MS, m/z: [M + Na ], calcd for C H N O Na
3
9
43
3
16
+
,
832.2536, found, 832.2531.
2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(2-(2-(((((S)-4-ethyl-3,14-dioxo-3,4,12,14-tet
rahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)carbonyl) amino)
(
ethoxy)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (19c): Yield, 46.4%.
1
HNMR (400 MHz, DMSO): δ 8.69 (s, 1H), 8.17 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.0
Hz, 1H), 7.87-7.82 (m, 2H), 7.73-7.69 (m, 1H), 7.04 (s, 1H), 5.44 (d, J = 2.2 Hz, 2H),
5
3
6
1
1
7
.27 (s, 2H), 5.07 (m, 3H), 4.81 (s, 1H), 4.12-3.87 (m, 3H), 3.68-3.66 (m, 1H),
.57-52 (m, 3H), 3.40-3.35 (m, 2H), 3.09-3.03 (m, 2H), 2.14-2.09 (m, 5H), 1.99 (s,
1
3
H), 1.92 (s, 3H), 0.91 (t, J =8.0 Hz, 3H). CNMR (100 MHz, DMSO): δ 170.39,
70.10, 169.70, 169.50, 168.10, 156.73, 154.52, 152.47, 147.96, 146.47, 145.75,
31.71, 130.47, 129.88, 129.02, 129.86, 128.63, 128.08, 127.77, 118.93, 96.68, 94.92,
4.75, 69.05, 68.89, 68.72, 67.82, 66.52, 66.29, 65.41, 61.98, 50.29, 30.72, 20.67,

+
+
2
0.57, 20.50, 20.48, 7.62. TOF-MS, m/z: [M + Na ], Calcd. for C H N O Na ,
39 43 3 16
8
32.2536, found, 832.2561.
(2R,3R,4S,5S,6R)-2-(acetoxymethyl)-4-(carbamoyloxy)-6-(2-(2-(((((S)-4-ethyl-3,14-di
oxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]quinolin-4-yl)oxy)car
bonyl)amino)ethoxy)ethoxy)tetrahydro-2H-pyran-3,5-diyl diacetate (19d): Yield,
1
7
6.5%. HNMR (400 MHz, DMSO): δ 8.66 (s, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.10 (d,
J = 8.0 Hz, 1H), 7.86-7.83 (m, 1H), 7.71-7.67 (m, 1H), 7.40 (s, 1H), 6.71 (brs, 1H),
6
.60 (brs, 1H), 5.25 (s, 2H), 5.11-4.97 (m, 3H), 4.89 (t, J = 8.0 Hz, 1H), 4.85-4.75 (m,
H), 4.13 (dd, J = 12.0, 4.0 Hz, 1H), 3.99-3.88 (m, 2H), 3.78-3.54 (m, 8 H), 2.50-2.43
3
(m, 1H), 2.40-2.31 (m, 1H), 2.09 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 0.98 (t, J = 8.0 Hz,
1
3
3
1
1
5
H). CNMR (100 MHz, DMSO): δ 172.59, 170.11, 169.61, 169.45, 160.64, 155.50,
53.90, 152.47, 147.98, 144.44, 144.09, 131.65, 130.47, 129.91, 129.78, 129.06,
28.57, 128.02, 127.75, 96.88, 96.80, 88.66, 69.31, 68.98, 66.41, 66.00, 62.19, 55.04,
+
5.01, 50.60, 31.45, 21.78, 20.61, 20.59, 7.45. TOF-MS, m/z: [M + Na ], calcd for
+
C H N O Na , 833.2488, found, 833.2510.
38
42
4
16
4
.1.5 General procedures for compounds 22a-d and 23a-d.
To a solution of compounds 14a-d or 15a-d (1 mmoL) dissolved in ethyl
acetate (5 mL) catalytic amounts of 10 % Pd/C (80 mg) were added, and then under
hydrogen atmospheres, the reaction mixture was stirred for 1 h at room temperature.
After the completion of the reduction reaction, the mixture was filtered and
concentrated under reduced pressure to obtain crude residues 16a-d or 17a-d,
respectively. The residues 16a-d or 17a-d were re-dissolved in anhydrous methanol (5
mL), and sodium methoxide (0.1 equiv) was added and the resultant solution was
+
stirred for 1 h. Dowex H resin (60 mg) was added to adjust pH, After shaken for
another 10 min, the solution was filtered and concentrated under reduced pressure to
obtain the corresponding compounds 20a-d or 21a-d as colorless oil for the next
reaction without purification. To a solution of compound 5 (1.2 mmoL) dissolved in 5
mL of anhydrous DMF crude compounds 20a-d or 21a-d and diisopropylethylamine
(0.1 mmoL) were added at 0℃ , and the reaction mixture was heated to room

temperature and stirred overnight. After consumption of compounds 20a-d or 21a-d
monitored with TLC, DCM (100 mL) and saturated ammonium chloride solution (20
mL) were poured. The crude residues obtained were purified by silica gel column
chromatography with methanol and DCM (V/V=1:20) as an eluent to afford the
corresponding compounds 22a-d and 23a-d as white solids.
(S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-b]
quinolin-4-yl(2-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-
1
pyran-2-yl)oxy)ethyl)carbamate (22a): Yield, 38.1%. HNMR (400 MHz, DMSO): δ
9.2 (s, 1H), 8.69 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H), 7.86 (t, J =
8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz, 1H), 7.40 (d, J = 4.0 Hz, 1 H), 5.45 (d, J = 4.0 Hz,
1H), 5.28 (d, J = 4.0 Hz, 2H), 4.96-4.84 (m, 1H), 4.81-4.66 (m, 2H), 4.56-4.45 (m,
1H), 4.39-4.33 (m, 1H), 4.11-4.06 (m, 1H), 3.87 (t, J = 4.0 Hz, 1H), 3.71 (s, 2H), 3.60
(d, J = 8.0 Hz, 2H), 3.51-3.14 (m, 3H), 3.12 (d, J = 8.0 Hz, 2H), 2.38-2.32 (m, 1H),
1
3
2
1
1
.14-2.10 (m, 1H), 0.95-0.87 (m, 3H). CNMR (100 MHz, DMSO): δ 172.64, 160.61,
54.61, 153.91, 152.56, 148.37, 148.00, 144.21, 131.74, 130.49, 129.97, 129.12,
28.60, 128.09, 127.72, 119.03, 97.32, 94.98, 88.58, 75.35, 73.53, 70.43, 67.99, 63.81,
+
6
0.42, 53.44, 50.62, 31.39, 7.65. TOF-MS, m/z: [M + Na ], calcd for C H N O Na
29
31
3
11
+
,
620.1851, found, 620.1855.
S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano
(
[3',4':6,7]
indolizino[1,2-b]quinolin-4-yl(2-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxyl
1
methyl)tetrahydro-2H-pyran-2-yl) oxy)ethyl)carbamate (22b): Yield, 42.6%. HNMR
(400 MHz, DMSO): δ 9.13 (s, 1H), 8.67 (d, J = 4.0 Hz, 1H), 8.20 (m, 1H), 8.11 (m,
1
H), 7.85 (m, 1H), 7.71 (dd, J =8.0, 4.0 Hz, 1H), 7.38 (s, 1H), 5.45 (d, J = 4.0 Hz,
H), 4.92-4.84 (m, 2H),4.79-4.67 (m, 2H), 4.56-4.53 (m, 1H), 4.38 (dd, J = 8.0, 4.0
1
Hz, 1H), 4.13-4.05 (m, 1H), 3.91-3.84 (m, 1H), 3.68-3.65 (m, 2H), 3.62-3.57 (m, 2H),
3
3
6
.50-3.41 (m, 2H), 3.33-3.24 (m, 2H), 3.15-3.09 (m, 2H), 2.38-1.88 (m, 2H), 0.96 (m,
+
+
H). TOF-MS, m/z: [M + Na ], calcd for C H N O Na , 620.1851, found,
29
31
3
11
20.1866.
S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]
indolizino[1,2-b]quinolin-4-yl(2-(((2S,3S,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethy
(

1
l)tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamate (22 c): Yield, 78.6%. HNMR (400
MHz, DMSO) δ 8.69 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 8.10 (d, J = 8.0 Hz, 1H), 7.86
(
t, J = 8.0 Hz, 1H), 7.70 (t, J = 8.0 Hz, 1H), 7.40 (s, 1H), 5.25 (s, 2H), 4.91 (t, J = 4.0
Hz, 2H), 4.80 (t, J = 4.0 Hz, 2H), 4.70 (d, J = 4.0 Hz, 2H), 4.63 (s, 1H), 4.53 (d, J =
.0 Hz, 1H), 4.42 (t, J = 4.0 Hz, 1H), 3.84-3.80 (m, 2H), 3.68-3.55 (m, 3H), 3.50 (d, J
4.0 Hz, 1H), 3.43-3.30 (m, 2H), 3.22-3.18 (m, 1H), 2.53-2.33 (m, 2H), 0.96 (t, J =
4
=
1
3
8
1
9
7
6
.0 Hz, 3H). CNMR (100 MHz, DMSO) δ 172.62, 160.64, 153.85, 152.51, 147.99,
44.38, 144.13, 131.65, 130.45, 129.93, 129.67, 129.11, 128.56, 128.02, 127.74,
9.52, 96.78, 88.56, 74.17, 70.85, 70.18, 66.77, 61.88, 61.09, 55.02, 50.60, 31.51,
+
+
.60. TOF-MS, m/z: [M + Na ], calcd for C H N O Na , 620.1851, found,
29
31
3
11
20.1844.
S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]
indolizino[1,2-b]quinolin-4-yl(2-(((2R,3S,4S,5R,6R)-4-(carbamoyloxy)-3,5-dihydroxy
(
-6-(hydroxyl-methyl)tetrahydro-2H-pyran-2-yl)oxy)ethyl)carbamate (22d): Yield,
1
5
5.1%. HNMR (400 MHz, DMSO): δ 8.69 (s, 1H), 8.22 (d, J = 8.0 Hz, 1H), 8.13 (d,
J = 8.0 Hz, 1H), 7.95 (d, J = 4.0 Hz, 1H), 7.88 (t, J = 8.0 Hz, 1H), 7.73 (t, J = 8.0 Hz,
1
3
H), 7.05 (s, 1H), 6.42 (s, 2H), 5.45 (s, 2H), 5.29 (s, 2H), 4.68-4.60 (m, 2H),
.93-3.31 (m, 10H), 3.24-3.07 (m, 2H), 2.14-2.09 (m, 2H), 0.89 (t, J = 8.0 Hz, 3H).
CNMR (100 MHz, DMSO): δ 168.14, 156.73, 154.61, 152.47, 147.96, 146.32,
45.79, 143.92, 131.73, 130.51, 129.90, 129.12, 128.60, 128.08, 126.55, 119.01,
00.32, 95.00, 74.82, 74.32, 73.79, 68.12, 66.38, 65.57, 64.35, 60.99, 50.31, 30.84,
1
3
1
1
7
6
+
+
.63. TOF-MS, m/z: [M + Na ], calcd for C H N O Na , 663.1909, found,
30
32
4
12
63.1903.
S)-4-ethyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3',4':6,7]indolizino[1,2-
b]quinolin-4-yl(2-(2-(((2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)
(
1
tetrahydro-2H-pyran-2-yl)oxy)ethoxy)ethyl)carbamate (23a): Yield, 41.3%. HNMR
400 MHz, DMSO): δ 8.68 (s, 1H), 8.21 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 8.0 Hz, 1H),
.88 (t, J = 8.0 Hz, 1H), 7.72 (t, J = 8.0 Hz, 1H), 7.40 (s, 1H), 5.27 (s, 2H), 4.98-4.86
m, 3H), 4.81 (t, J = 8.0 Hz, 1H), 4.49 (t, J = 8.0 Hz, 1H), 4.12-4.03 (m, 1H),
.86-3.47 (m, 8H), 3.46-3.41 (m, 2H), 3.17 (d, J = 4.0 Hz, 1H), 3.12-2.87 (m, 4H),
(
7
(
3