therapeutic efficacy.11 In this case, a branched polydisperse
PEGs with high molecular weight and complicated
geometry was used. Although polydisperse PEGs (regular
PEGs) are overwhelmingly used in pharmaceutical industry
and biomedical research, their heterogeneity issue
complicates their application and brings a range of problems
in purification, characterization, quality control, and clinic
application.12-14 Fortunately, these problems could be
avoided by replacing regular PEGs with monodisperse PEGs
(M-PEGs). However, M-PEGs are difficult to prepare and
expensive. Recently, a macrocyclic sulfate-based strategy
was developed for the convenient synthesis of M-PEGs15-18
and M-PEGs were successfully employed to modify clinical
used drugs, including Polidocanol and Propofol.19,20 It was
found that M-PEGs modification is crucial for achieving
optimal physicochemical properties (e.g, water solubility
and logP), therapeutic efficacy, drug safety, etc.
(Figure 2k). As polydisperse PEGs are mixture of oligomers,
polydisperse PEGs modified HCPT would give a HPLC
chromatogram like Figure 2k and the obvious heterogeneity
would complicate the quality control, downstream
accurately quantitative solubility, therapeutic efficacy and
safety study.
a) SOCl2, Et3N, DMAP, DCM, 0 oC
O O
S
b) NaIO4, RuCl3 3H2O,
H2O-CH3CN-DCM, 0 oC to rt
O
O
H(OCH2CH2)nOH
n-1
O
6f
5a
, n = 4;
5b
, n = 5;
5c
, n = 6;
5d
, n = 7;
5e
, n = 8;
5f
, n = 9
6a
6b
6c
6d
6e
, n = 4, 47%; , n = 9, 42%
5g
, n = 10
, n = 11
, n = 12
6g
6h
6i
, n = 5, 40%;
, n = 6, 43%;
, n = 10, 40%
, n = 11, 31%
, n = 7, 49%; , n = 12, 42%
, n = 8, 31%
5h
5i
O
HCPT, K2CO3, DMF, then,
H2SO4, H2O, THF, reflux
O
N
Here, low molecular weight linear M-PEGs were
employed to modify CPT, HCPT and SN38 into the
corresponding prodrugs 1-4 in order to achieve high water
solubility, prolong in vivo half time, and maintain high
therapeutic efficacy (Figure 1). We envision that, through
manipulating the M-PEGs length (4-24 ethylene glycol units)
and the way of conjugation (ether and carbonate), the
physicochemical and biological properties of CPT, HCPT
and SN38 prodrugs could be accurately fine-tuned and
promising anticancer drug candidate would be discovered.
To fine-tune the physicochemical and biological properties, a
series of M-PEGs modified prodrugs of CPT, HCPT and SN38
were then synthesized (Scheme 1). First, after macrocyclization
of M-PEG 5a-5i of 4 to 12 ethylene glycol units with thionyl
chloride, the M-PEGs macrocyclic sulfates 6a-6i smoothly
reacted with HCPT in the presence of potassium carbonate to
give 9 ether derivatives of HCPT 1a-1i with high efficacy.
Second, monomethylated M-PEGs 7g-7p of 8 to 24 ethylene
glycol units were conjugated to HCPT and SN38 through
O
N
OH
O(CH2CH2O)mH
6f
6g
, m = 10, from , 85%
6h
, m = 11, from , 87%
1a
1b
1c
1d
1e
6a
1f
1g
1h
1i
, m = 4, from , 89%; , m = 9, from
6b
, m = 5, from , 88%;
6c
, m = 6, from , 81%;
,
88%
6d
6i
,
, m = 7, from , 83%; , m = 12, from
6e
84%
, m = 8, from , 75%
6a,
NaH, THF, then
H2SO4, H2O, THF, reflux
Me(OCH2CH2)mOH
7a
Me(OCH2CH2)mOH
7d
7e
7f
7g
7h
, from , 75%;
7b
, from , 82%;
7c
from , 98%;
7d
, from , 98%;
7e
, from , 85%;
from
from , 92%;
7a
7k
7h
, from , 91%
, m = 0; 7h
, m = 10
m = 11
m = 12
m = 15
, m = 16
, m = 20
7b
7c
7d
7e
7l
7m
7n
7o
7p
7i
,
, m = 2; 7i
, m = 3; 7j
, m = 4; 7l
,
from
, from
, from
, from
93%
94%
82%
,
,
,
,
7j
7l
,
,
, m = 6; 7m
7m
, 83%
7i
7j
7f
,
7g
,
,
83%;
7f
7o
, from , 84%
, m = 7; 7o
7g
, m = 8;
4-Nitrophenyl chloroformate,
Et3N, rt, DCM
O
O2N
O
O(CH2CH2O)mMe
8f 7l
8a
7g
, m = 8, from , 78%; , m = 15, from
8b, m = 10, from 7h, 72%; 8g, m = 16, from 7m, 75%
, m = 11, from
, m = 12, from
, m = 14, from , 67%; , m = 24, from
, 76%
8c
8d
8e
7i
8h
7n
7o
7p
,
67%;
, m = 19, from
,
,
,
68%
76%
66%
7j
8i
65%; , m = 20, from
,
carbonates
formation
on
the
10-hydroxyl
groups.
7k
8j
Monomethylated M-PEGs 7g-7p were prepared through iterative
ring opening reaction on macrocyclic sulfate 6a with methanol
7a and commercially available alcohol 7b-7c. After many
unsuccessful attempts on carbonate formation, M-PEGs 7g-7p
were first transformed into the corresponding 4-nitrophenyl
carbonates 8a-8j which then underwent transesterification with
HCPT and SN38 to give the corresponding prodrugs 2a-2j, 4a
and 4b in moderate yields, respectively. Third, 10 carbonates
prodrugs of CPT 3a-3j were conveniently synthesized through
carbonated formation between the 20-hydroxyl group of CPT and
monomethylated M-PEGs 7g-7p in the presence of triphosgene
and 4-(dimethylamino)pyridine (DMAP).
The prodrugs of HCPT 1a-1i and 2a-2j, CPT 3a-3j, SN38 4a
and 4b were prepared on multi-hundred milligram scales and
fully characterized with 1H/13C NMR and mass spectra. The
benefit of M-PEGs modification was first exhibited by their
unambiguous spectra data, which indicated a single high
pure component in these prodrugs, respectively. The purities
of these prodrugs were further evaluated with HPLC (Figure
2, ethers 1a-1i; Figure S1 in supporting information,
carbonates 2a-2j and 3a-3j). High purities of the M-PEGs
modified prodrugs were showed in Figure 2 and S1 as a
single HPLC peak, respectively. Comparing to CPT and
HCPT, their prodrugs gave longer retention times in HPLC.
It is interesting to point out that the length of M-PEGs solely
determined the HPLC retention times of the prodrugs and
even the difference of 1 ethylene glycol unit could be
detected by HPLC, which was clearly illustrated by HPLC
chromatogram of HCPT and its prodrugs 1a-1i mixture
HCPT, Et3N,
DMF, 60 oC
O
O
N
8a-8j
O
O
N
OH
O
O(CH2CH2O)mMe
2a
2b
2c
2d
2e
8a
2f
8f
, m = 8, from , 47%; , m = 15, from , 57%
8b 2g 8g
, m = 10, from , 42%; , m = 16, from , 43%
8c
2h
8h
, m = 11, from , 48%; , m = 19, from , 48%
, m = 12, from , 54%; , m = 20, from , 50%
, m = 14, from , 68%; , m = 24, from
8d 8i
2i
8e
2j
8j
,
62%
O
O
N
CPT, Triphosgene,
DMAP,DCM
O
N
O
7g-7p
O
Mem(OH2CH2C)O
3a
3b
3c
3d
3e
7g
3f
7l
,
, m = 8, from , 55%; , m = 15, from
7h 3g
43%
7m
, m = 10, from , 44%;
, m = 16, from
, 61%
7i
7j
7k
3h
, 49%;
7n
, m = 19, from , 50%
, m = 11, from
, m = 12, from
, m = 14, from , 48%; , m = 24, from , 43%
3i
3j
7o
64%; , m = 20, from , 56%
,
7p
O
SN38, Et3N,
DMF, 60 o
O
N
C
8f 8j
&
O
O
N
OH
O
O(CH2CH2O)mMe
8f 4b 8j
, m = 15, from , 52%; , m = 20, from , 55%
4a
Scheme 1. Synthesis of M-PEGs modified CPT, HCPT and SN38 prodrugs.
Water solubility of these M-PEGs modified prodrugs was then
measured with an ultraviolet spectrophotometry method (Figure
3).21 Comparing to poorly soluble CPT, HCPT and SN38, the M-