Synthesis and biological evaluation of hypoxia-activated prodrugs of SN-38

Article abstract of DOI:10.1016/j.ejmech.2017.03.040

We designed new hypoxia-activated prodrugs by conjugating (1-methyl-2-nitro-1H-imidazol-5-yl)methanol with 7-ethyl-10-hydroxy camptothecin (SN-38). Initially, we improved the method of multi-gram scale synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which increased the yield to 42% compared to 8% by the original synthesis method. The improved method was used to synthesize evofosfamide (TH-302) and hypoxia-activated prodrugs of SN-38. Two different linkages between (1-methyl-2-nitro-1H-imidazol-5-yl)methanol and SN-38 were evaluated that afforded different hypoxia-selectivity and toxicity. Compound 16 (IOS), containing an ether linkage, was considered to be a promising hypoxia-selective antitumor agent.

Full text of DOI:10.1016/j.ejmech.2017.03.040

Accepted Manuscript
Synthesis and biological evaluation of hypoxia-activated prodrugs of SN-38
Chen Jin, Qiumeng Zhang, Wei Lu
PII:
S0223-5234(17)30197-6
10.1016/j.ejmech.2017.03.040
EJMECH 9300
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 December 2016
Revised Date: 1 February 2017
Accepted Date: 21 March 2017
Please cite this article as: C. Jin, Q. Zhang, W. Lu, Synthesis and biological evaluation of hypoxia-
activated prodrugs of SN-38, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.03.040.
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Graphical Abstract
In this study, we proposed a new hypoxia-activated prodrug that conjugated
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol with SN-38. Compound IOS containing
ether linkage was evaluated as a promising hypoxia-selective antitumor agent.

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of hypoxia-activated prodrugs of SN-38
Chen Jin,a Qiumeng Zhanga and Wei Lua,*
a School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road,
Shanghai 200062, P. R. China
* Corresponding authors:
E-mail: wlu@chem.ecnu.edu.cn; Tel.: +86-21-62238771
Abstract:
We
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol with 7-ethyl-10-hydroxy camptothecin (SN-38).
Initially, we improved the method of multi-gram scale synthesis of
designed
new
hypoxia-activated
prodrugs
by
conjugating
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which increased the yield to 42% compared to 8% by
the original synthesis method. The improved method was used to synthesize evofosfamide (TH-302)
and
hypoxia-activated
prodrugs
of
SN-38.
Two
different
linkages
between
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol and SN-38 were evaluated that afforded different
hypoxia-selectivity and toxicity. Compound 16 (IOS), containing an ether linkage, was considered to
be a promising hypoxia-selective antitumor agent.
Keywords: Hypoxia-activated prodrug, SN-38, 2-Nitroimidazole
1. Introduction
Natural products are an important resource in the search for new anticancer molecules. Many
compounds derived from plant metabolites are commonly used in chemotherapy, such as
camptothecin, paclitaxel, and teniposide. However, the severe side effects on normal tissues and
multidrug resistance limit their applications in clinical treatment.[1,2]
Camptothecin is a potent antitumor alkaloid that is isolated from Camptotheca acuminate, and it
specifically targets DNA topoisomerase I (Topo I). Topo I is a DNA unwinding protein.
Camptothecin inhibits the catalytic activity of Topo I and blocks the rejoining step of the
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breakage-reunion reaction of unwound DNA.[3-5] Structure–activity relationship studies of
camptothecin derivatives summarized that a hydroxyl substitution at the 10-position, such as
topotecan and irinotecan, enhanced antitumor activity.[6] Irinotecan, acting through its active
metabolite SN-38, was used in the treatment of metastatic colorectal cancer. However, non-specific
toxicity and poor water solubility were the major clinical limitations of camptothecin derivatives. To
improve these deficiencies, many efforts have been made to develop new camptothecin analogs, such
as peptidic and boronic acid prodrugs, nanodrugs, and antibody conjugates of SN-38, some of which
have been recently tested in clinical trials.[7-16] Recently, Bertozzi et al. reported that camptothecin
derivatives could impair the hypoxia-induced cell transcriptional response and reduce
hypoxia-inducible factor-1α (HIF-1α) protein expression and activity,[17] which indicated that
camptothecin could be used to treat hypoxic tumors. Thus far, there have been few studies on this
application of camptothecin. Of note, Zhang et al. reported a novel class of camptothecin derivatives
conjugated with 4-nitrobenzyl and 4-nitrofuryl alcohols at the 20-hydroxyl position for use in
hypoxia-targeting tumor chemotherapy.[18]
Hypoxia occurs in many human diseases, especially in solid tumors.[19,20] Hypoxia in solid
tumors has led to multiple contributions to chemoresistance, radioresistance, angiogenesis,
vasculogenesis, invasiveness, metastasis, and resistance to cell death.[21-24] Because of its critical role
in tumor progression and resistance to radiation and chemotherapy, hypoxia is becoming a
compelling therapeutic target.[25] Hypoxia-activated prodrugs (HAPs), which are selectively
activated by reductive enzymes with a bioreductive group, have been proposed.[26-31]
2-Nitroimidazole has been widely used in the development of hypoxia-selective agents.[32-35] One of
the most clinically advanced hypoxia-activated prodrugs, evofosfamide (TH-302), was synthesized
on the basis of the DNA cross-linking toxin bromo-isophosphoramide mustard (Br-IPM) and
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol.[36] However, TH-302 did not show effective results in
phase III trials. Patients with locally advanced unresectable or metastatic soft tissue sarcoma treated
with TH-302 in combination with doxorubicin did not demonstrate a statistically significant
improvement in overall survival compared to those observed with doxorubicin alone
(ClinicalTrials.gov Identifiers: NCT01746979; NCT01440088).
In this study, we designed new hypoxia-activated prodrugs of SN-38 for the first time. We
initially developed an improved method for multi-gram scale synthesis
of
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(1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which increased the yield to 42% compared with 8%
by the original synthetic method. This method was then used for to synthesize TH-302 and
hypoxia-activated prodrugs of SN-38. SN-38 has a phenolic hydroxyl group that can be used to
attach functional groups. Attaching antitumor drugs to the functional groups with a suitable bond
significantly affected the pharmacokinetic properties of the prodrugs. Therefore, we used ether and
bis-carbamate linkages to connect (1-methyl-2-nitro-1H-imidazol-5-yl)methanol and SN-38[37,38]
.
These prodrugs were designed to be stable in the bloodstream and normal tissues. At the target site,
the prodrugs underwent different reductive processes, causing the release of SN-38 (Figure 1). We
aimed to reduce the toxicity of SN-38 to normal tissues and increase the effectiveness against
treatment-resistant hypoxic tumor cells.
Figure 1. Proposed mechanism of prodrugs activated via enzymatic metabolism under hypoxic conditions.
2. Results and discussion
2.1. Chemistry
The original synthesis method of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol, which was
developed by Matteucci et al., started with a sarcosine methyl ester HCl salt and led to a final
product over seven steps with an 8% overall yield.[39] (Scheme 1, A). It was obvious that the first
four steps produced a very low yield. Thus, recently, O’Connor et al. reported an improved method
for this synthesis (Scheme 1, B).[40] They improved the synthesis of compound 5 with a one-pot
procedure, with yields of 48–54% over the first three steps. However, there were several problems
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when we investigated this procedure. The insolubility of the starting material and the large amount of
NaH consumed (as much as 10 equivalents) made it extremely difficult to scale up. It was initiated
with 2 g of starting material and yielded several hundred milligrams of compound 8. The amount
produced was far from sufficient.
Scheme 1 Original synthesis
Reagents and conditions: A. a) K2CO3, HCOOEt, EtOH; b) NaH, HCOOEt; c) HCl, EtOH; d) NH2CN, AcONa,
H2O, 15% over four steps; e) AcOH, NaNO2, 62%;f) NaOH, H2O, 95%; g) isobutyl chloroformate, NaBH4, THF,
88%. B. h) HCOOEt, THF, NaH; i) EtOH, conc. HCl; j) EtOH, H2O, NH2CN, 48–54% over three steps.
Scheme 2 New synthesis
Reagents and conditions: a) n-BuLi, i-PrONO2, THF; b) n-BuLi, TsN3, THF, 82%; c) HCl, MeOH, 4h,
87%; d) Pd/C, H2, MeOH, 1h, 96%; e) NaNO2, Cu, HBF4, H2O, 61%.
For the aforementioned reasons, we considered a new strategy to obtain the basic structure of
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imidazole
(Scheme
2).
It
has
been
reported
that
5-(((tert-butyldimethylsilyl)oxy)methyl)-1-methyl-1H-imidazole (compound 9) was readily obtained
by using the commercially available 1,3-dihydroxyacetone dimer in three steps upon scale of up to
tens of grams.[41-46] Compared with the original synthesis, this synthetic route was more convenient
to obtain a 1-methyl-5-hydroxymethylimidazole scaffold at a considerable yield.
Initially, we sought to introduce the nitro functional group into starting material 9 in one step.
Davis et al. reported a convenient method for the synthesis of 2-substituted imidazole that was based
on the reaction of 2-lithioimidazole with electrophiles.[47] Thus, 2-lithioimidazole was formed by the
reaction of compound 9 with n-BuLi at -78°C in anhydrous THF and was followed by dropwise
addition of i-PrONO2. However, no reaction was observed at –78°C. Then, the reaction became
complicated when the temperature was raised to -40°C. No target product was obtained. As a result,
i-PrONO2 was unsuitable for introducing the nitro group.
There were few examples of successfully introducing a nitro group directly at the C-2 position
of imidazole derivatives. Therefore, we investigated another strategy that used TsN3 to introduce the
azido group instead.[48] 2-Lithioimidazole was obtained as before, and TsN3 was added to it. The
reaction was completed rapidly and purified by silica gel to produce compound 10 with an 82% yield.
Then, compound 10 was deprotected with HCl/MeOH and purified with ether to give compound 11.
Compound 11 was reduced by Pd/C and H2 to produce amino 12. With multiple grams of amino 12
in hand, we sought to introduce a nitro functional group via diazotization, which was the most
critical process in this method. The original synthesis, which used acetic acid, provided a low yield.
We considered that diazonium acetate was less stable than diazonium tetrafluoroborate. Therefore,
HBF4 (40%) was used instead of AcOH. At the same time, Cu powder as a catalyst was necessary for
the reaction.[49] These modified conditions improved diazotization, with a moderate yield of 61%.
Compound 8 was produced with a moderate yield of 3 g.
TH-302 was synthesized according to the procedure of Duan et al.[36] The
bromo-isophosphoramide mustard was synthesized from the corresponding 2-bromoethylamine
hydrobromide salt. Then, it was reacted with compound 8 via a Mitsunobu reaction to produce
TH-302 with a 51% yield.
For the synthesis of IOS, compound 8 was reacted with TsCl in the presence of DMAP to
produce compound 15. Then, SN-38 was reacted with compound 15 in K2CO3/DMF to give
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compound 16 (IOS) with an overall yield of 44%. Compound 8 was also treated with
bis(4-nitrophenyl)carbonate followed by addition of N-trityl-N,N’-dimethylethylenediamine to
produce compound 17. The trityl protecting group was removed in 10% TFA/DCM to produce
compound 18. Then, SN-38 was treated with bis(4-nitrophenyl)carbonate, followed by addition of
the compound 18 solution to produce compound 20 (INS) with an overall yield of 41% (Scheme 3).
Scheme 3. Synthesis of IOS and INS
Reagents and conditions: a) TsCl, DMAP, DCM; b)SN-38, K2CO3, DMF, DCM; c) bis(4-nitrophenyl)carbonate,
N,N’-dimethyl-N-tritylethane-1,2-diamine, DIPEA, DCM; d) 10% TFA/DCM; e) bis(4-nitrophenyl)carbonate,
DIPEA, DCM; f) DIPEA, DCM.
2.2. Biological evaluation
2.2.1. In vitro cytochrome P450 metabolism assay
Stability is an important requirement for hypoxia-activated prodrugs of SN-38. Both compounds
were stable when treated with PBS (10 mM, pH = 7.4) at 37°C after 48 h. Another critical factor is
microsomal stability in the presence of cytochrome P450.[36] As is known, many drugs undergo
oxidative and reductive metabolism via cytochrome P450 activation. However, the prodrugs were
designed to only be reduced. Thus, we used mouse liver microsomes to assess the metabolism of the
prodrugs in vitro. When the compounds were treated with mouse liver microsomes for 6 h without
NADPH, both compounds were stable. The results indicated that neither of them was a substrate for
cytochrome P450 oxidation. Furthermore, treatment of the compounds with mouse liver microsomes
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in the presence of NADPH resulted in different stability profiles (Table 1). HPLC analysis indicated
that IOS was only reduced to SN-38, while INS was converted to other derivatives (Figure 2). We
hypothesized that IOS underwent a bioreductive metabolism, as we proposed previously. However,
INS underwent a more complicated metabolic process, which indicated that the prodrug of SN-38
based on the bis-carbamate linkage could be a substrate of other enzymes besides reductase in
cytochrome P450.
A
B
Figure 2. A. Percent of compound remaining and SN-38 releasing in 6h of incubation with mouse liver
microsomes in presence of NADPH. B. Compound metabolism with mouse liver microsomes in presence of
NADPH detected by HPLC. a IOS, t = 0h; b IOS, t = 1h; c INS, t = 0h; d INS, t = 1h; e SN-38.
Table 1. In vitro stability of prodrugs
a Percent of compound remaining after 48h of incubation with PBS. b,c Percent of compound remaining after 6h of
incubation with mouse liver microsomes in presence or absence of NADPH. d Percent of SN-38 releasing after 6h
of incubation with mouse liver microsomes in presence of NADPH.
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2.2.2. In vitro cytotoxicity assay
The compounds were evaluated for cytotoxicity under normoxic and hypoxic (94% N2/5%
CO2/1% O2) conditions using human lung cancer H460 cells and human colon cancer HT29 cells.
These cell lines were used for hypoxic/normoxic selectivity tests because they were known to
express high levels of DT-diaphorase, an oxygen-insensitive reductase. This widely expressed
enzyme has the potential to activate the prodrugs via enzymatic reduction, thereby increasing the
toxicity of the prodrugs under normoxic conditions.[36] Cells were treated with the test compounds at
various concentrations under a normoxic or hypoxic condition for 24 h and were then washed and
incubated under normoxic conditions for up to 72 hours in fresh medium. Cell viability and
proliferation were assessed by a MTT assay. The IC50 values of the tested compounds for the
inhibition of proliferation are shown in Table 2.
Table 2. In vitro cytotoxicity assay data summary.
aHypoxia cytotoxicity ratio (HCR) was determined by the differential cytotoxicity under normoxic and hypoxic
conditions: HCR= IC50(Normoxia)/IC50(Hypoxia).
The results indicated that IOS had moderate hypoxia selectivity and was much more toxic under
the hypoxic condition compared with TH-302. The toxicity of IOS was 10-fold higher than that of
TH-302. Furthermore, the toxicity of IOS was lower than that of SN-38 under a normoxic condition.
However, both hypoxia selectivity and toxicity of INS were lower than those of IOS.
We believe that the different linkages between (1-methyl-2-nitro-1H-imidazol-5-yl)methanol
and SN-38 were responsible for the significant differences in hypoxia selectivity and toxicity of the
tested compounds. IOS and INS underwent distinct enzymatic metabolism mediated by cytochrome
P450. The results of the assays indicated that IOS was only reduced to SN-38. However, INS
containing N,N'-dimethyl-1,2-ethanediamine may be a substrate of other enzymes besides the
reductase in cytochrome P450. INS was converted to other derivatives, which may cause decreased
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hypoxia selectivity and toxicity.
3. Conclusions
We developed an improved method for multi-gram scale synthesis of
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol in four steps. This new strategy was used to synthesize
compound 8 upon the basic structure of 1-methyl-5-hydroxymethylimidazole. Key steps included the
synthesis of 2-azidoimidazole based on the reaction of 2-lithioimidazole with TsN3 and modified
diazotization of 2-aminoimidazole. This procedure was more convenient than the original synthesis
method and increased the overall yield from 8% to 42%. The improved method was used for
synthesis of TH-302 and hypoxia-activated prodrugs of SN-38. Two different linkages between
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol and SN-38 were evaluated. Compound IOS containing
an ether linkage had moderate hypoxia selectivity and more toxicity compared with TH-302.
However, INS containing the N,N'-dimethyl-1,2-ethanediamine may be a substrate of other enzymes
besides the reductase in cytochrome P450, which may cause the decreased hypoxia selectivity and
toxicity. In conclusion, compound IOS was considered to be a promising hypoxia-selective antitumor
agent, and an ether linkage was considered to be effective for the development of more efficient
hypoxia-activated prodrugs.
4. Experimental sections
4.1. Materials and methods
13
1H and C nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-400 MHz
spectrometer (400 and 101 MHz, respectively) using CDCl3, or DMSO-d6 as solvents with TMS as
an internal standard. Chemical shifts were reported as d (ppm) and spin-spin coupling constants as J
(Hz) values. The mass spectra (MS) were recorded on a Finnigan MAT-95 mass spectrometer.
Melting points were taken on a SGW X-4 melting point apparatus, uncorrected and reported in
degrees Centigrade. Column chromatography was performed with silica gel (200–300 mesh).
4.2. General synthesis
2-azido-5-(((tert-butyldimethylsilyl)oxy)methyl)-1-methyl-1H-imidazole (10)
n-Butyllithium (25.5 mL,61.1 mmol, 2.4 N in hexane) was added dropwise to a solution of
compound 9 (11.5 g, 50.9 mmol) in anhydrous THF (100 mL) at −78°C. The reaction was stirred at
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−78°C for 1 h, then TsN3 (7.0 g，45.8 mmol) was added dropwise. The reaction mixture was
continued stirring at −78°C for 1 h. Then the mixture was quenched with aq. NH4Cl and extracted
with EtOAc. The organic phase was successively washed with brine and H2O, and dried over
Na2SO4. Concentration afforded the crude product as yellow oil and was purified by column
chromatography on silica gel (heptane–EtOAc 10 : 1) to afford the product 10 (10 g, 82%) as yellow
oil. 1H NMR (400 MHz, CDCl3) δ 6.73 (s, 1H), 4.55 (s, 2H), 3.40 (s, 3H), 0.87 (s, 9H), 0.04 (s, 6H).
13C NMR (101 MHz, CDCl3) δ 141.4, 130.3, 125.2, 55.5, 29.6, 25.8, 18.2, -5.3. HRMS (ESI): m/z
Calcd for C11H22N5OSi, [M+H]+: 272.1907, found: 272.1829.
(2-azido-1-methyl-1H-imidazol-5-yl)methanol (11)
Compound 10 (10.0 g, 37.5 mmol) was stirred in 1 N HCl/MeOH (100 mL) at room temperature for
4 h. Then the triethylamine was added to the reaction to adjust pH to 7. The mixture was
concentrated in vacuum and then the crude product washed with ether to afford the compound 11
1
(5.0 g, 87%) as yellow solid. mp 105-107°C. H NMR (400 MHz, CDCl3) δ 6.60 (s, 1H), 4.47 (s,
2H), 4.06 (br, 1H), 3.40 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 141.5, 130.8, 125.1, 54.2, 29.5. MS
(ESI) m/z = 154.07 [M+H]+.
(2-amino-1-methyl-1H-imidazol-5-yl)methanol (12)
Compound 11 (5.0g, 32.7mmol) was dissolved in 50 mL of MeOH, then Pd/C (0.5g of 10%Pd) was
added and the mixture was hydrogenated under hydrogen balloon for 1 h. The catalyst was removed
by filtration and the filter was evaporated by vacuum to afford compound 12 (4.0 g, 96%) as gray
solid. mp 177-179°C. 1H NMR (400 MHz, DMSO) δ 6.25 (s, 1H), 5.26 (br, 2H), 4.78 (br, 1H), 4.25
(s, 2H), 3.28 (s, 3H). 13C NMR (101 MHz, DMSO) δ 150.1, 126.5, 122.2, 53.2, 28.7. HRMS (ESI):
m/z Calcd for C5H10N3O, [M+H]+: 128.0824, found: 128.0819.
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol (8)
Compound 12 (4.0 g, 31.5 mmol) was dissolved in fluoboric acid (40% w/v, 20 mL). The solution
was cooled to -15°C, and a solution of sodium nitrite (3.3 g, 47.3 mmol) in water (10 mL) was added
dropwise. The solution was stirred at -15°C for 30 min, then added dropwise into a solution of Cu
powder (2.0 g, 119 mmol) and sodium nitrite (21.7 g, 157.5 mmol) in water (50 mL).The reaction
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mixture was stirred at room temperature for 1 h. The solution was extracted with EtOAc. The organic
extract was evaporated in vacuum, and the residue was eluted by DCM to afford compound 8 (3.0 g,
61%) as yellow solid. mp 142-144°C. 1H NMR (400 MHz, DMSO) δ 7.11 (s, 1H), 5.47 (t, J = 5.4 Hz,
1H), 4.54 (d, J = 5.4Hz, 2H), 3.92 (s, 3H). 13C NMR (101 MHz, DMSO) δ 145.7, 138.7, 126.6, 53.0,
34.1. HRMS (ESI): m/z Calcd for C5H8N3O3, [M+H]+: 158.0487, found: 158.0560.
(1-methyl-2-nitro-1H-imidazol-5-yl)-N,N–bis(2-bromoethyl) phosphordiamidate (TH-302)
To a suspension of N,N'-bis(2-bromoethyl)phosphorodiamidic acid (50 mg, 0.16 mmol),
1-methyl-2-nitroimidazole-5-methanol (50 mg, 0.32 mmol), and PPh3 (84 mg, 0.32 mmol) in THF
(15 mL) was added DIAD (0.13 mL, 0.32 mmol) at 0°C. After the addition of DIAD, the reaction
mixture was warmed to room temperature and stirred overnight. The solvent was removed, and the
residue was purified by column chromatography on silica gel (DCM : MeOH = 50:1) to afford
1
compound 14 (37 mg, 51%) as yellow gum. H NMR (400 MHz, DMSO) δ 7.25 (s, 1H), 5.05-4.99
(m, 2H), 4.98 (d, J = 7.6 Hz, 2H), 3.94 (s, 3H), 3.42 (t, J = 6.9 Hz, 4H), 3.17-3.05 (m, 4H). 13C
NMR (101 MHz, DMSO) δ 146.1, 134.1(d, J = 8.0 Hz), 128.2, 55.7 (d, J = 4.3 Hz), 42.7, 34.3, 34.1
(d, J = 5.0 Hz). HRMS (ESI): m/z Calcd for C9H17Br2N5O4P, [M+H]+: 469.9204, found: 469.9186.
5-(chloromethyl)-1-methyl-2-nitro-1H-imidazole (15)
To a stirred solution of compound 8 (47 mg, 0.3 mmol) and DMAP (73 mg,0.6 mmol) in DCM (3
mL) was added TsCl (69 mg, 0.36 mmol). The reaction was stirred for 4 h at room temperature. The
mixture was concentrated in vacuum and purified by column chromatography on silica gel (heptane :
1
EtOAc = 10 : 1) to afford compound 15 (50mg, 95%) as yellow solid. mp 96-98°C. H NMR (400
MHz, CDCl3) δ 7.19 (s, 1H), 4.62 (s, 2H), 4.07 (s, 3H). MS (ESI) m/z = 175.87 [M+H]+.
Compound 16 (IOS)
To a stirred solution of SN-38 (78 mg, 0.2 mmol) and K2CO3 (27 mg, 0.2 mmol) in anhydrous DMF
(2 mL) and DCM (2 mL) was added compound 15 (35 mg, 0.2 mmol). The mixture was stirred
overnight under nitrogen balloon at room temperature. The reaction was quenched with H2O and
extracted with EtOAc. The combined organic layers were dried over Na2SO4. Concentration was
purified by column chromatography on silica gel (DCM : MeOH = 50 : 1) to afford compound 16
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(50 mg, 47%) as yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 9.2 Hz, 1H), 7.62 (s, 1H),
7.54 – 7.44 (m, 2H), 7.33 (s, 1H), 5.75 (d, J = 16.2 Hz, 1H), 5.31 (d, J = 16.3 Hz, 1H), 5.27 (s, 2H),
5.26 (s, 2H), 4.15 (s, 3H), 3.75 (s, 1H), 3.17 (q, J = 7.5 Hz, 2H), 1.98 – 1.82 (m, 2H), 1.42 (t, J = 7.6
Hz, 3H), 1.04 (t, J = 7.4 Hz, 3H). HRMS (ESI): m/z Calcd for C27H26N5O7 (M+H+): 532.1832, found:
532.1815.
(1-methyl-2-nitro-1H-imidazol-5-yl)methyl methyl(2-(methyl(trityl)amino)ethyl)carbamate (17)
To a stirred solution of compound 8 (30 mg, 0.19 mmol) and DIPEA (49 mg, 0.38 mmol) in DCM (5
mL) was added bis(4-nitrophenyl)carbonate (64 mg, 0.21 mmol). The reaction was stirred for 4 h at
room temperature, then was added N,N’-dimethyl-N-tritylethane-1,2-diamine (94 mg, 0.29 mmol).
The mixture was stirred for another 1h. Then the solution was washed with Na2CO3 and extracted
with DCM. The combined organic layers were dried over Na2SO4. Concentration was purified by
column chromatography on silica gel (heptane : EtOAc = 2 : 1) to afford compound 17 (88 mg, 91%)
as yellow solid. mp 100-102°C. 1H NMR (400 MHz, CDCl3) δ 7.50 – 7.37 (m, 6H), 7.26 – 7.19 (m,
6H), 7.18 – 7.11 (m, 3H), 7.08 (s, 1H), 5.19 – 5.01 (m, 2H), 4.05 – 3.62 (m, 3H), 3.56 – 3.37 (m, 2H),
3.13 – 2.89 (m, 3H), 2.38 – 2.18 (m, 2H), 2.18 – 2.05 (m, 3H). MS (ESI) m/z = 514.37 [M+H]+.
Compound 20 (INS)
To a stirred solution of compound 17 (40 mg, 0.078 mmol) in DCM (2 mL) was added
trifluoroacetic acid (0.2 mL). The reaction was stirred for 1 h at room temperature, and concentrated
in vacuum to afford the crude product of compound 18. In another bottle,
bis(4-nitrophenyl)carbonate (24 mg, 0.078 mmol) was added into the DCM solution (5 mL) of
SN-38 (31 mg, 0.078 mmol) and DIPEA (20 mg, 0.156 mmol), which afforded the compound 19
after 3 h reaction. Then compound 18 was added into the solution of compound 19 without
purification. Appropriate DIPEA was added to keep pH among 7~8. The reaction was stirred at room
temperature for 3 h. The mixture was concentrated in vacuum and purified by column
chromatography on silica gel (DCM : MeOH = 50 : 1) to afford compound 20 (24 mg, 45%) as
yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.27 – 8.18 (m, 1H), 7.90 – 7.77 (m, 1H), 7.66 (s, 1H),
7.60 – 7.45 (m, 1H), 7.24 – 7.13 (m, 1H), 5.78 – 5.68 (m, 1H), 5.32 – 5.15 (m, 5H), 4.09 – 3.91 (m,
4H), 3.75 – 3.43 (m, 4H), 3.24 – 2.93 (m, 8H), 1.98 – 1.81 (m, 2H), 1.46 – 1.32 (m, 3H), 1.02 (t, J =
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7.3 Hz, 3H). HRMS (ESI): m/z Calcd for C33H36N7O10 [M+H]+: 690.2524, found: 690.2515.
4.3. General procedure for Cytochrome P450 reductase assay
Mouse liver microsomes (20 mg/mL) were purchased from Corning. NADPH was purchased from
Sigma-Aldrich.
The prodrugs measurements were made in 10 mM PBS (pH 7.4) according to the following
procedure. 1 µL of 5 mM prodrug in DMSO were diluted in 1mL PBS, followed by addition of
MgCl2 (5 µmol), NADPH (1 µmol), and 50 µL mouse liver microsomes. The incubation mixtures of
test compounds with mouse liver microsomes contained the following at the indicated final
concentrations: 10 mM PBS (pH 7.4), 5 µM test compound, 5 mM MgCl2, microsomes (1 mg/mL)
with or without 1 mM NADPH. The mixtures were preincubated at 37 °C. The metabolism reaction
was initiated by the addition of 50 µL mouse liver microsomes solution. At 0, 0.5, 1, 2, 4, 6 h, 100uL
reaction mixture was quenched by 100 µL cold methanol. The samples were centrifuged at 4 °C for 5
min at 12000 rpm. The supernatant was transferred to a vial for analysis using HPLC.
4.4. In Vitro Cell-Proliferation Assay
Human lung cancer cell line H460 and human colon cancer cell line HT29 were purchased from the
Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China).
H460 cells and HT29 cells were cultured in RPMI 1640 medium and McCoy’s 5A medium
respectively, supplemented with 10% fetal bovine serum and 1% penicillin streptomycin (Invitrogen)
in a 5%CO2 humidified environment at 37 °C. Cells were seeded in 96-wellplates and incubated for
24 h. The next day, serial dilutions of test compounds in DMSO were made in RPMI 1640 medium
or McCoy’s 5A medium, such that final concentrations of DMSO did not exceed 0.1%. The cells in
the hypoxia treatment group were incubated for 24h in the anaerobic chamber flushed with a certified
anaerobic gas mixture (94% N2/5% CO2/1% O2; Thermo). The cells in the air treatment group were
incubated for 24 h in standard cell-culture incubator. After the 24 h of treatment with test compounds,
the cells were washed with 200 µL PBS and incubated up to 72 h in 200 µL of fresh medium. After 3
days, the cells were treated with MTT and measured by a SpectraMax M5 microplate reader at an
emission wavelength of 570 nm. The 50% inhibitory concentration (IC50) of test compounds was
calculated from results (n=3).
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4.5. General HPLC Method
HPLC analysis was performed at room temperature using a Diamonsil C18 (250 mm × 4.6 mm) and a
mobile phase gradient from 5% CH3CN/buffer (0.1% TFA/H2O) to 95% CH3CN /buffer (0.1%
TFA/H2O) for 15 min, a flow rate of 1.0 mL/min, and plotted at 373 nm. This method was used to
determine the purity for the tested compounds, and also used in stability studies.
Supplementary data
Supplementary data related to this article can be found at.
References
[1] A. L. Harvey, Drug. Discov. Today 13 (2008) 894-901.
[2] D. J. Newman, G. M. Cragg, J. Nat. Prod. 70 (2007) 461-477.
[3] M. E. Wall, M. C. Wani, C. E. Cook, K. H. Palmer, A. T. McPhail, G. A. Sim, J. Am. Chem. Soc. 88
(1966) 3888-3890.
[4] Y. H. Hsiang, R. Hertzberg, S. Hecht, L. F. Liu, J. Biol. Chem. 260 (1985) 14873-14878.
[5] J. C. Wang, Annu. Rev. Biochem. 65 (1996) 635-692
[6] H. Ulukan, P. W. Swaan, Drugs (62) 2002 2039-2057.
[7] Q. Y. Li, X. Q. Deng, Y. G. Zu, H. Lv, L. Su, L. Yao, Y. Zhang, L. Li, Eur. J. Med. Chem. (45) 2010
3200-3206.
[8] Y. Jiao, H. Liu, M. Geng, W. Duan, Bioorg. Med. Chem. Lett. 21 (2011) 2071-2074.
[9] M. S. Christodoulou, F. Zunino, V. Zuco, S. Borrelli, D. Comi, G. Fontana, M. Martinelli, J. B. Lorens,
L. Evensen, M. Sironi, S. Pieraccini, L. Dalla Via, O. M. Gia, D. Passarella, ChemMedChem. 7 (2012)
2134-2143.
[10] Y. Q. Liu, W. Dai, C. Y. Wang, S. L. Morris-Natschke, X. W. Zhou, L. Yang, X. M. Yang, W. Q. Li, K.
H. Lee, Bioorg. Med. Chem. Lett. 22 (2012) 7659-7661.
[11] L. Zhu, C. Zhuang, N. Lei, Z. Guo, C. Sheng, G. Dong, S. Wang, Y. Zhang, J. Yao, Z. Miao, W. Zhang,
Eur. J. Med. Chem. 56 (2012) 1-9.
[12] R. Cincinelli, L. Musso, S. Dallavalle, R. Artali, S. Tinelli, D. Colangelo, F. Zunino, M. De Cesare, G. L.
Beretta, N. Zaffaroni, Eur. J. Med. Chem. 63 (2013) 387-400.
14

ACCEPTED MANUSCRIPT
[13] Z. Miao, L. Zhu, G. Dong, C. Zhuang, Y. Wu, S. Wang, Z. Guo, Y. Liu, S. Wu, S. Zhu, K. Fang, J. Yao,
J. Li, C. Sheng, W. Zhang, J. Med. Chem. 56 (2013) 7902-7910.
[14] G. Rodriguez-Berna, M. J. D. Cabañas, V. Mangas-Sanjuán, M. Gonzalez-Alvarez, I. Gonzalez-Alvarez,
I. Abasolo, S. Schwartz, M. Bermejo, A. Corma, ACS Med, Chem. Lett. 4 (2013) 651-655.
[15] L. Wang, S. Xie, L. J. Ma, Y. Chen, W. Lu, Eur. J. Med. Chem. 116 (2016) 84-89.
[16] X. Zhang, K. Tang, H. Wang, Y. Liu, B. Bao, Y.Fang, X. Zhang, W. Lu, Bioconjugate Chem. 27 (2016)
1267–1275.
[17] D
．Bertozzi, J. Marinello, S. G. Manzo, F. Fornari, L. Gramantieri, G. Capranico, Mol. Cancer. Ther. 13
(2014) 239-248.
[18] Z. Zhang, K. Tanabe, H. Hatta, S. Nishimoto, Org. Biomol. Chem
[19] W. R. Wilson, M. P. Hay, Nat Rev Cancer 11 (2011) 393-410.
[20] J. M. Brown, Cancer Biol. Ther. 1 (2002) 453-458.
. 3 (2005) 1905-1910.
[21] R. A. Cairns, I. S. Harris, T. W. Mak, Nature Rev. Cancer 11 (2011) 85-95.
[22] Y. Wang, M. Ohh, J. Cell. Mol. Med. 14 (2010) 496-503.
[23] R. D. Guzy, B. Hoyos, E. Robin, H. Chen, L. P. Liu, K. D. Mansfield, M. C. Simon, U. Hammerling, P.
T. Schumacker, Cell. Metab. 1 (2005) 401-408.
[24] J. M. Brown, W. R. Wilson, Nat. Rev. Cancer 4 (2004) 437-447.
[25] G. O. Ahn, M. Brown, Front. Biosci. 12 (2007) 3483-3501.
[26] R. M. Phillips, Cancer Chemother Pharmacol. 77 (2016) 441-457.
[27] T. Thambi, J. H. Park, D. S. Lee, Chem. Commun. 52 (2016) 8492-8500.
[28] A. J. Lin, L. A. Cosby, C. W. Shansky, A. C. Sartorelli, J. Med. Chem. 15 (1973) 1247-1252.
[29] S. R. McKeown, R. L. Cowen, K. J. Williams, Clin. Oncol. 19 (2007) 427-442.
[30] R. P. Mason, J. L. Holtzman, Biophys. Res. Commun. 67 (1975) 1267-1274.
[31] E. Nieto, M. Delgado, M. Sobrado, M. L. Ceballos, R. Alajarín,L. G. García, J. Kelly, I. Lizasoain, M. A.
Pozo, Eur. J. Med. Chem. 101 (2015) 604-615.
[32] C. Karnthaler
‐Benbakka, D. Groza, B. Koblmüller, A. Terenzi, K. Holste, M. Haider, D. Baier, W.
Berger, P. Heffeter, C. R. Kowol, B. K. Keppler, Chemmedchem. 11 (2016) 2410-2421.
[33] F. Zhou, S. Zanganeh, I. Mohammad, C. Dietz, A. Abuteen, M. B. Smith, Q, Zhu, Org. Biomol. Chem.
13 (2015) 11220-11227.
15

ACCEPTED MANUSCRIPT
[34] M. P. Hay, R. F. Anderson, D. M. Ferry, W. R. Wilson, W. A. Denny, J. Med. Chem. 46 (2003)
5533-5545
.
[35] M. P. Hay, W. R. Wilson, W. A. Denny, Bioorg. Med. Chem. 13 (2005) 4043-4055.
[36] J. X. Duan, H. Jiao, J. Kaizerman, T. Stanton, J. W. Evans, L. Lan, G. Lorente, M. Banica, D. Jung, J.
Wang, H. Ma, X. Li, Z. Yang, R. M. Ho
51 (2008) 2412-2420.
ffman, W. S. Ammons, C. P. Hart, M. Matteucci, J. Med. Chem.
[37] G. Anna, P. Neta, S. Marina, R. Christoph, S. Doron, Angew. Chem. Int. Ed. 42 (2003) 327-332.
[38] A. Gopin, S. Ebner, B. Attali, D. Shabat, Bioconjug. Chem. 17 (2006) 1432-1440.
[39] M. Matteucci, J. X. Duan, H. Jiao, J. Kaizerman, S. Ammons, M. H. Hopkins, US Patent Office, WO
2007/002931 A2.
[40] L. J. O’Connor, C. Cazares-Körner, J. Saha, C. N. G. Evans, M. R. Stratford, L. E. M. Hammondb, S.
Conway, J. Org. Chem. Front. 2 (2015) 1026-1029.
[41] J. P. Collman, M. Zhong, S. Costanzo, C. Zhang, J. Org. Chem. 66 (2001) 8252-8256.
[42] M. Baumann, I. R. Baxendale, Org. Lett. 16 (2014) 6076-6079.
[43] H. Sarkarzadeh, R. Miri, O. Firuzi, M. Amini. N. Razzaghi-Asl, N. Edraki, A. Shafiee, Arch. Pharm. Res.
36 (2013) 436-447.
[44] P. Aulaskari, M. Ahlgrén, J. Rouvinen, P. Vainiotalo, E. Pohjala, J. Vepsäläinen, J. Heterocyclic Chem.
33 (1996) 1345-1354.
[45] J. J. Lee, L. F. Huang, K. Zaw, L. Bauer, J. Heterocyclic Chem. 35 (1998) 81-89.
[46] RM de Figueiredo, L. Coudray, J. Dubois, Org. Biomol. Chem. 5 (2007) 3299-3309.
[47] M. P. Hay, W. A. Denny, Tetrahedron Lett. 38 (1997) 8425-8428.
[48] D. P. Davis, K. L. Kirk, L. A. Cohen, J. Heterocyclic Chem. 19 (1982) 253-256.
[49] I. Parveen, D. P. Naughton, W. J. D. Whish, M. D. Threadgill, Bioorg. Med. Chem. Lett. 9 (1999)
2031-2036.
Figure 1. Proposed mechanism of prodrugs activated via enzymatic metabolism under hypoxic
conditions.
Figure 2. A. Percent of compound remaining and SN-38 releasing in 6h of incubation with mouse
liver microsomes in presence of NADPH. B. Compound metabalism with mouse liver microsomes in
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b
c
d
e
presence of NADPH detected by HPLC. a IOS, t = 0h; IOS, t = 1h; INS, t = 0h; INS, t = 1h;
SN-38.
a
Table 1. In Vitro Stability of Prodrugs. Percent of compound remaining after 48h of incubation
with PBS. b,c Percent of compound remaining after 6h of incubation with mouse liver microsomes in
presence or absence of NADPH. d Percent of SN-38 releasing after 6h of incubation with mouse liver
microsomes in presence of NADPH.
a
Table 2. In vitro cytotoxicity assay data summary. Hypoxia cytotoxicity ratio (HCR) was
determined by the differential cytotoxicity under normoxic and hypoxic conditions: HCR=
IC50(Normoxia)/IC50(Hypoxia).
Scheme 1. Original synthesis. Reagents and conditions: A. a) K2CO3, HCOOEt, EtOH; b) NaH,
HCOOEt; c) HCl, EtOH; d) NH2CN, AcONa, H2O, 15% over four steps; e) AcOH, NaNO2, 62%;f)
NaOH, H2O, 95%; g) isobutyl chloroformate, NaBH4, THF, 88%. B. h) HCOOEt, THF, NaH; i)
EtOH, conc. HCl; j) EtOH, H2O, NH2CN, 48–54% over three steps.
Scheme 2 New synthesis. Reagents and conditions: a) n-BuLi, i-PrONO2, THF; b) n-BuLi, TsN3,
THF, 82%; c) HCl, MeOH, 4h, 87%; d) Pd/C, H2, MeOH, 1h, 96%; e) NaNO2, Cu, HBF4, H2O,
61%.
Scheme 3. Synthesis of IOS and INS. Reagents and conditions: a) TsCl, DMAP, DCM; b)SN-38,
K2CO3, DMF, DCM; c) bis(4-nitrophenyl)carbonate, N,N’-dimethyl-N-tritylethane-1,2-diamine,
DIPEA, DCM; d) 10% TFA/DCM; e) bis(4-nitrophenyl)carbonate, DIPEA, DCM; f) DIPEA, DCM.
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Highlights
ò We
proposed
new
hypoxia-activated
prodrugs
that
conjugated
(1-methyl-2-nitro-1H-imidazol-5-yl)methanol with SN-38.
ò We improved the synthesis of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol,
which increased the yield from 8% to 42%.
ò Compound IOS was evaluated to be a promising hypoxia-selective antitumor
agent.