Next-Generation Hypoxic Cell Radiosensitizers: Nitroimidazole Alkylsulfonamides

Article abstract of DOI:10.1021/acs.jmedchem.7b01678

Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the advent of radio-immuno-oncology, herald new opportunities for classical oxygen-mimetic radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer, and there is a dearth of new radiosensitizers in development. Here we present a survey of novel nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase aqueous solubility and to improve tumor drug delivery. A 2-nitroimidazole and a 5-nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo assays of surviving clonogens or tumor regrowth delay.

Full text of DOI:10.1021/acs.jmedchem.7b01678

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Article
Next-generation hypoxic cell radiosensitizers:
nitroimidazole alkylsulfonamides
Muriel Bonnet, Cho R. Hong, Way W Wong, Lydia P Liew, Avik Shome, Jingli Wang,
Yongchuan Gu, Ralph J Stevenson, Wen Qi, Robert F. Anderson, Frederik B. Pruijn,
William R. Wilson, Stephen M. F Jamieson, Kevin O Hicks, and Michael Patrick Hay
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01678 • Publication Date (Web): 18 Dec 2017
Downloaded from http://pubs.acs.org on December 19, 2017
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Nextꢀgeneration hypoxic cell radiosensitizers: nitroimidazole
alkylsulfonamides
†
†
†
†#
†
Muriel Bonnet, Cho Rong Hong, Way Wua Wong, Lydia P. Liew, Avik Shome,
†
† #
†
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ǂ†#
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Jingli Wang, Yongchuan Gu, Ralph J. Stevenson, Wen Qi, Robert F. Anderson,
†#
†#
†§#
†#
Frederik B. Pruijn, William R. Wilson, Stephen M.F. Jamieson, Kevin O. Hicks,
†#*
Michael P. Hay
†
Auckland Cancer Society Research Centre, School of Medical Sciences, Faculty of Medical
and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand
ǂ
School of Chemical Sciences, Faculty of Science, University of Auckland, Private Bag
92019, Auckland, New Zealand
§
Department of Pharmacology and Clinical Pharmacology, School of Medical Sciences,
Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019,
Auckland, New Zealand
#
Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, 3 Symonds St,
Auckland Central, New Zealand
Key words:
Nitroimidazole, radiosensitizer, sulfonamide, hypoxia, tumor microenvironment
*Corresponding author. Address: Auckland Cancer Society Research Centre, Faculty of
Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland 1142,
New Zealand. Tel: +649 923 1190; Fax: +649 373 7502. Email address:
m.hay@auckland.ac.nz
ORCID 0000ꢀ0002ꢀ7087ꢀ9087
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ABSTRACT
Innovations in the field of radiotherapy such as stereotactic body radiotherapy, along with the
advent of radioꢀimmunoꢀoncology, herald new opportunities for classical oxygenꢀmimetic
radiosensitizers. The role of hypoxic tumor cells in resistance to radiotherapy and in
suppression of immune response continues to endorse tumor hypoxia as a bona fide, yet
largely untapped, drug target. Only nimorazole is used clinically as a radiosensitizer and
there is a dearth of new radiosensitizers in development. Here we present a survey of novel
nitroimidazole alkylsulfonamides and document their cytotoxicity and ability to
radiosensitize anoxic tumor cells in vitro. We use a phosphate prodrug approach to increase
aqueous solubility and to improve tumor drug delivery. A 2ꢀnitroimidazole and a 5ꢀ
nitroimidazole analogue demonstrated marked tumor radiosensitization in either ex vivo
assays of surviving clonogens or tumor regrowth delay.
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INTRODUCTION
Hypoxia is a cardinal component of the tumor microenvironment and plays a key role
in tumor cell resistance to therapy, disease progression and avoidance of immune
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surveillance. Hypoxia contributes to resistance to therapy through a variety of mechanisms.
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The clearest evidence for hypoxiaꢀmediated resistance to therapy has been observed with
5
6ꢀ9
radiation therapy in cervical carcinoma and head and neck squamous cell carcinoma,
where patients with more hypoxic tumors fare poorly in terms of overall survival or
progression free survival. Hypoxia exerts a pernicious role in disease progression with
10
11
influences on angiogenesis , vasculogenesis, the activation of a glycolytic shift in
12,13
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15ꢀ17
metabolism,
and enhancing invasion and metastasis.
Hypoxia also promotes
18
genomic instability through increased production of reactive oxygen species and
1
9,20
suppression of DNA repair processes,
and exerts complex and contradictory influences on
21ꢀ23
immune cell responses to tumors.
Efforts to modify hypoxia to improve treatment response have a long history.
4
,24ꢀ28
2
9
30
Three main approaches have been explored. Hyperbaric oxygen or carbogen breathing
have provided increased oxygen delivery to tumors. Oxygenꢀmimetic nitroimidazoles acted
31ꢀ33
to “fix” radiation damage in hypoxic tumor cells
and these agents evolved into a third
approach where hypoxic cell cytotoxins were designed to selectively kill hypoxic tumor
4
,34,35
cells.
While many individual trials failed to meet statistical endpoints, when taken
2
7
together in a retrospective analysis, these interventions have been shown to provide benefit.
Oxygenꢀmimetic radiosensitizers are most commonly nitroimidazoles where the
electronꢀaffinic nitro group is designed to react with DNA radicals produced by ionizing
31
radiation in a manner similar to oxygen. Formation of these adducts leads to DNA strand
36
breaks and subsequent cell death. Early clinical trials with misonidazole (1, Figure 1)
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3
demonstrated considerable antiꢀtumor activity, but this was limited by delayed peripheral
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neuropathies when combined with fractionated radiotherapy.
More polar analogues such
as etanidazole (2) demonstrated improved therapeutic indices, but clinical application was
33,39
again limited in combination with fractionated radiotherapy.
Doranidazole (3) was
designed to be more polar than 1 and 2 in order to limit normal tissue exposure and
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underwent investigation in nonꢀsmall cell lung cancer and pancreatic cancer in conjunction
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with intraꢀoperative RT where it displayed a small survival advantage. The most successful
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2,43
example of a nitroimidazole radiosensitizer is nimorazole (4).
It is in clinical use in head
2
4
and neck cancer in combination with standard fractionated radiotherapy (FRT) and is
undergoing further evaluation in head and neck cancer (ClinicalTrials.gov: NCT01950689,
44,45
NCT01880359).
One important aim of one of the trials is the prospective validation of a
hypoxic gene signature (ClinicalTrials.gov: NCT01950689) shown to predict benefit for
43,46
nimorazole in a retrospective study.
47,48
While FRT is one of the main treatments for cancer patients,
a new approach,
stereotactic body radiotherapy (SBRT), is demonstrating comparable control, toxicity and
efficacy profiles with improved cost and compliance, relative to daily FRT in many
49ꢀ51
indications
. SBRT leverages recent advances in the accuracy and precision of radiation
delivery to provide hypofractionated (1–5 doses), high dose (25–60 Gy total dose) radiation
to small tumors while minimizing the effects to adjacent normal tissue. However, evidence is
52ꢀ54
emerging that SBRT may accentuate the impact of hypoxia on radioresistance.
The
advance of SBRT, along with the development of suitable biomarkers for assessing the extent
55
56
of hypoxia in individual tumors, provides new opportunities for radiosensitizers.
57ꢀ59
There are few recent examples of novel oxygenꢀmimetic sensitizers
, and these do
not appear to be in clinical development. 2ꢀNitroimidazole radiosensitizers, such as 1, 2 and
, also display hypoxiaꢀselective cytotoxicity by virtue of their higher electron affinity. Oneꢀ
3
electron reduction by bioreductive enzymes produces a nitro radical anion, which may be
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reoxidized in the presence of oxygen, or further reduced to nucleophilic species that induce
cytotoxicity. This mechanism is distinct from radiosensitization and might be associated with
the toxicity displayed by these compounds. One recent example of a nitroimidazoleꢀbased
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radiosensitizer is the sulfamide 5 which is a potent inhibitor of CAIX. Although containing
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a metronidazole motif, 5 sensitizes tumor cells to radiation by inhibition of CAIX, through
interaction of the sulfamide unit with the Zn in the catalytic center, and interruption of tumor
cell pH homeostasis at drug concentrations considerably lower than those required for 5ꢀ
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nitroimidazoleꢀbased oxygenꢀmimetic radiosensitization.
62,63
We recently identified a novel class of nitroimidazole sulfonamide radiosensitizer.
Here, we report our efforts to more fully characterize this class. We have explored
compounds that span a wide range of electron affinity to explore the hypothesis that more
electronꢀaffinic compounds with a dual mechanism of action may be optimized for SBRT
where cumulative neurotoxicity will be minimized. The contrasting premise is that we may
also identify less electronꢀaffinic compounds that are less toxic and are well tolerated with
FRT. We evaluated hypoxiaꢀselective cytotoxicity and radiosensitization in vitro for
compounds 6–38 and identified lead compounds that provide substantial hypoxic cell
radiosensitization in vivo, at least comparable to that seen with 2 and 4.
CHEMISTRY
A series of 2ꢀnitroimidazoles bearing sulfonamideꢀcontaining side chains were
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prepared using novel chemistry established in our lab. Examples of neutral (6, 7), basic (8,
9
) and acidic (10, 11) side chains were prepared through condensation of halomethylsulfonyl
chloride 43 with the appropriate amine to give 44, 46, 48–50, and 52, with subsequent
alkylation of 2ꢀnitroimidazole and deprotection if required (Scheme 1). We initially used the
commercially available chloromethylsulfonyl chloride 43a to prepare sulfonamides, but
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subsequently found that the readily prepared bromomethylsulfonyl chloride 43b gave
superior yields in the synthesis of sulfonamides (e.g. 7). Attempts to prepare sulfonamides of
secondary amines (54, 55) via this approach were not successful. Elaboration of the
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chloromethyl 2ꢀnitroimidazole 56 to the thioester 57 allowed access to the intermediate
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nitroimidazole methylsulfonyl chloride, and condensation with morpholine was required to
prepare morpholide 12. Higher ethylsulfonamide homologues were accessed from the 2ꢀ
nitromidazole ethanol 60, derived by deprotection of THPꢀ or TBDMSꢀethers 59 or 64,
respectively (Scheme 2). Elaboration of alcohol 60 to thioester 62 and conversion to the
sulfonyl chloride in situ allowed condensation with amines to give 13–15. Attempts to
directly alkylate 2ꢀnitroimidazole with the corresponding ethenyl sulfonamides in a Michael
reaction were unsuccessful.
Examples of 4ꢀ and 5ꢀnitroimidazole methylsulfonamides (16–21) were prepared by
alkylation of 4ꢀnitroimidazole with the appropriate halomethyl sulfonamides (44, 46, 48)
(Scheme 3). These reactions gave mixtures of 4ꢀ and 5ꢀnitro isomers in ratios of ca. 4:1 which
could be separated by preparative RPꢀHPLC. Examples of ethylsulfonamides were accessed
by alkylation of 4ꢀnitroimidazole with bromide 63 to give a mixture of 4ꢀ and 5ꢀ
nitroimidazole silyl ether isomers 68 and 69 which were separable by column
chromatography (Scheme 4). Deprotection of 68 gave alcohol 70 which was converted, via
thioester 71, to the sulfonyl chloride in situ, with subsequent reaction with amines to give
sulfonamides 22 and 72, which was subsequently deprotected to give 24. A similar sequence
with the 4ꢀnitroimidazole silyl ether 69 gave the sulfonamides 23 and 25.
A series of 2ꢀmethylꢀ5ꢀnitroimidazole methyl sulfonamides were prepared from
metronidazole (76) (Scheme 5). Thioesterification of 76 gave the thioester 77 which was
converted to the sulfonyl chloride in situ and this was reacted with a series of amines to
provide sulfonamides 26–38.
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Oxygenꢀmimetic radiosensitizers need to be present at relatively high (often mM)
concentrations at the time of irradiation to be effective sensitizers and so high aqueous
solubility is an important prerequisite for a successful radiosensitizer. Solubility of the
compounds in culture medium containing 5% fetal calf serum (FCS) was determined as a
practical guide for cell culture studies (Table 1). A direct comparison of the effect of
exchanging sulfonamide for carboxamide groups (i.e., 2 vs 7) shows ca. 10ꢀfold reduction in
solubility. Other neutral nitroimidazole sulfonamides (e.g., 6, 16, and 17) are considerably
less soluble than either 1 or 2, but solubility could be increased by homologation of the linker
chain (e.g., 6 vs 13 and 7 vs 14). The use of basic amine side chains also provided substantial
increases in solubility over corresponding neutral compounds (e.g., 8 vs 6, 30 vs 27).
Although the aqueous solubility was sufficient for in vitro experiments, concern that the
relatively low aqueous solubility of the neutral alkylsulfonamides compared to 2 would limit
drug delivery prompted us to consider a phosphate prodrug strategy. Phosphate prodrugs 39–
42 of alcohols 7, 14, 19 and 24 were prepared via the corresponding tertꢀbutyl esters 78–81,
respectively (Scheme 6). Although we used the traditional tetrazoleꢀmediated
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phosphorylation in the preparation of 78, we found imidazole/imidazole.HCl mediated
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methodology to be a more convenient and efficient procedure, producing improved yields
of 79–81. The phosphate prodrugs 39–42 displayed increased aqueous solubility (220 to >600
mM) in saline containing 2 equivalents of NaHCO (Table S1).
3
The lipophilicities of the compounds were calculated as logD values at pH 7.4
(Advanced Chemistry Development, Inc. Toronto, Canada) (Table 1). The sulfonamides
series spanned a range of 0.11 to ꢀ4.75 compared to ꢀ0.41 for 1 and ꢀ1.37 for 2. Generally, the
neutral analogues fell within the range bounded by 1 and 2 with the sulfonamide group
providing slightly more polarity than the corresponding carboxamide (e.g., compare 7 (ꢀ1.53)
with 2 (ꢀ1.37)). Compounds bearing acidic side chains were the most polar compounds (ꢀ
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4.38, ꢀ4.75) with strongly basic amine side chains providing significant influence (e.g., 9, 31–
34), spanning the range ꢀ1.55 to ꢀ2.21. Weaker bases such as morpholine were close to
neutral in their influence, providing increased aqueous solubility, without lowering the logD
below the value for 2.
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We determined the oneꢀelectron reduction potentials, E(1), for representative
compounds of the series since electron affinity has been defined as the key parameter in the
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in vitro structureꢀactivity relationships (SAR) for radiosensitization and toxicity for a series
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of nitroimidazole radiosensitizers. By using each of the 2ꢀ, 4ꢀ and 5ꢀnitroimidazole
regioisomers, in combination with methyl or ethyl linker side chains to modulate the effect of
the strongly electronꢀwithdrawing sulfonamide moiety, the analogues effectively span the
range of electron affinity for efficient reaction with DNA radicals (as defined by their E(1)
values; ꢀ538 to ꢀ342 mV) (Table 1). 2ꢀNitroimidazoles with a C1 linker (e.g., 6, 7) were more
electronꢀaffinic (ꢀ352 and ꢀ342 mV, respectively) than 2 (ꢀ388 mV), reflecting the increased
electronic demand from the sulfonamide group. This effect diminished with increased side
chain length (e.g., 13, 14 at ꢀ391 mV). As expected, the 5ꢀnitroimidazole 17 displayed lower
electron affinity (ꢀ421 mV) compared to the corresponding 2ꢀnitroimidazole analogue 6 and a
further reduction in affinity (ꢀ458 mV) was observed with increasing side chain length, (e.g.,
22). The electronꢀdonating influence of the 2ꢀmethyl substituent further reduced electron
affinity (e.g., ꢀ500 mV for 26) although this could be countered somewhat by the presence of
a positive charge on the side chain (e.g., ꢀ475 mV for 34). The 4ꢀnitroimidazole analogues
(
16 and 23) were less electronꢀaffinic than their corresponding 5ꢀnitroimidazole analogues
17 and 22).
(
RESULTS AND DISCUSSION
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We have prepared a novel series of nitroimidazole sulfonamides that span a wide range
of aqueous solubility, lipophilicity and electron affinity and have examined their in vitro
hypoxic selectivity and radiosensitization properties to identify lead analogues suitable for inꢀ
depth preclinical investigation.
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In vitro cytotoxicity. We determined the cytotoxicity (as IC ) of each compound
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using a sulforhodamine B (SRB) proliferation assay in HCT116 human colorectal carcinoma
cells following a 5 day regrowth after 4 h drug exposure under oxic or anoxic conditions. The
hypoxic cytotoxicity ratio (HCR) was determined as HCR = IC50(oxic)/IC50(anoxic)
.
The 2ꢀnitroimidazoles 6–15 displayed oxic cytotoxicity in the low mM range (IC50
values 0.38–4.8 mM) and anoxic potency of these compounds spanned a range from ca. 10
µM to ca. 4 mM (Table 1 and Figure 2). The relatively high anoxic potency displayed by 6–9
reflects the increased electron affinity due to the strong electronꢀwithdrawing effect from the
side chain with the E(1) value for 7 being 46 mV higher than for 2. 2ꢀNitroimidazoles 6–9
with neutral and basic side chains displayed considerable hypoxic selectivities (HCR 40–96)
while examples with acidic side chains (10, 11) displayed lower hypoxic selectivity, due to
decreased anoxic potency as a result of reduced electronic withdrawal due to the presence of
the anionic side chain (Table 1 and Figure 2). Extension of the side chain by one methylene
unit (13–15) also reduced hypoxic selectivity (HCR 5–10) through decreased anoxic potency,
reflecting the reduced influence of the sulfonamide group on electron affinity [E(1) for 14 = ꢀ
391 mV].
The 4ꢀ and 5ꢀnitroimidazoles (16–38) showed similar oxic potency to the 2ꢀ
nitroimidazoles 6–15, but all of these displayed little to no hypoxic selectivity. As expected,
the electron affinity was considerably lower for the 5ꢀnitroimidazole analogues with E(1) for
17 being 69 mV lower than for the corresponding 2ꢀnitroimidazole isomer 6. This resulted in
a substantial drop in anoxic potency and hypoxic selectivity (HCR 1.4–3.7) for compounds
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19 and 21 compared to 7 and 8, respectively. Curiously, 17 displayed unexpected oxic
cytotoxicity, leading to a loss in hypoxic selectivity. Homologation of the side chain led to a
reduction of 29 mV in E(1) for 22 compared to 17 and this was accompanied by a loss of
anoxic potency and low HCR values. Similarly, addition of an electronꢀdonating methyl
substituent at the 2ꢀ position further reduced the electron affinity of analogues 26–38 [E(1)
for 22: ꢀ450 mV; for 34: ꢀ475 mV], resulting in anoxic cytotoxicity in the low mM range and
low HCR values. The 4ꢀnitroimidazoles 16, 18 and 20 had slightly less electron affinity to the
2ꢀmethylꢀ5ꢀnitroimidazole analogues [E(1) for 16: ꢀ503 mV] and homologation (23, 25)
resulted in a further drop in electron affinity [E(1) for 23: ꢀ538 mV]. It is clearly evident that
analogues with E(1) values below ca. ꢀ420 mV do not display hypoxic selectivity (Figure 2).
This difference in hypoxic selectivity reveals two broad categories of compound. In the first,
the more electronꢀaffinic 2ꢀnitroimidazole analogues (6–15) are likely to deliver both
radiosensitization and bioreductive enzymeꢀmediated cytotoxicity in hypoxic cells as seen for
1 and 2. In contrast, 4ꢀ and 5ꢀnitroimidazole analogues with lower electron affinity, reflected
by E(1) values below ca. ꢀ420 mV, show little hypoxic selectivity and are expected to be pure
radiosensitizers, similarly to 4.
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Assigning E(1) values to all compounds based on the measured E(1) for each chemical
subclass allowed examination of SAR by multiple linear regression. Anoxic IC50 was found
to be related to the physiochemical properties (E(1), pKa and logP), while no relationship was
found for the oxic IC :
50
ꢀ
ꢁꢂꢃꢄꢅ ꢈ = ꢉ + ꢊꢋꢊꢌ1 + ꢍꢊꢎꢏꢉ + ꢐꢊꢀꢁꢂꢑ
(1)
ꢆꢇ
where a = ꢀ3.08 ± 0.79 (p = 0.00056), b = ꢀ0.0083 ± 0.0017, (p = 2.9eꢀ05), c = ꢀ0.109 ±
2
0
.052673 (p = 0.045) and d = ꢀ0.348 ± 0.165 (p = 0.044280). R = 0.4859.
A stronger relationship was found for the HCR:
1
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logꢃꢒꢅꢓꢈ = ꢉ + ꢊꢋꢊꢌ1 + ꢍꢊꢎꢏꢉ
(2)
where a = 3.32 ± 0.51 (p = 5.12eꢀ07), b = 0.00782 ± 0.00111 (p = 1.32eꢀ07) and c = 0.0822 ±
2
0.0366 (p = 0.0328), R = 0.6499.
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In vitro radiosensitization: Survival ratios. We evaluated radiosensitization by the
compounds by measuring clonogenic survival in HCT116 cells under anoxic conditions
(
Table 1). HCT116 cells were treated with compounds at a single nonꢀtoxic drug
concentration and a single radiation dose (15 Gy) in a customꢀdesigned metal anoxic chamber
62,63,67
apparatus.
The drug concentration equivalent to the anoxic IC50 value was used in this
assay and was nonꢀtoxic due to the shorter drug exposure time (1 h) and the exposure at room
temperature (21 °C) rather than 37 °C for the IC50 assay (Figure S1). Cells were exposed to
drug for 1 h, irradiated at 15 Gy in 96ꢀwell plates and plated for clonogenic survival. Survival
ratios at 15 Gy (SR15) were calculated as: SR15 = (cell survival with radiation)/(cell survival
with drug + radiation).
SR15 is a measure of the efficacy of the radiosensitizer at a single high dose of radiation
and provides a relatively simple screening assessment of hypoxic cell radiosensitization and
6
3
allows triage of compounds at this stage. The appropriate radiation dose was determined
empirically for HCT116 cells as 15 Gy, which alone induces ca. 1 log of cell kill (Figure S1),
and we used the anoxic IC50 value to compare the compounds at an equitoxic drug
concentration, rather than compare compounds at an equimolar concentration. No significant
drugꢀalone toxicity was observed across the compound set (p = 0.39, oneꢀway ANOVA) at
these drug concentrations (see Figure S1). This approach allowed us to quickly evaluate
radiosensitization for compounds with widely varying solubility and hypoxic cytotoxicities.
It became clear that in trying to make an equitoxic comparison the SR15 values were strongly
influenced by the drug concentration in the assay. For example, the weakly electronꢀaffinic 4
appears to be a good radiosensitizer compared to both 1 and 2, but was used at ca. 10ꢀ or 20ꢀ
1
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fold higher concentration than 2 and 1, respectively. This cytotoxicity/radiosensitization
conundrum underpins any consideration of radiosensitizers with mixed mechanisms of action
and can only be resolved by consideration of the achievable concentrations at the target site
in efficacy studies in vivo. It is clear that a number of the more electronꢀaffinic compounds
0
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(e.g., 6ꢀ9, 12, 17) have high molar potencies as radiosensitizers, and the lack of drugꢀalone
toxicity (Figure S1) suggests this was due to radiosensitization alone. We had previously
reported a steep concentration dependence of radiosensitization for the strongly electronꢀ
6
3
affinic 6. This observation encouraged us to advance 6 and similar electronꢀaffinic
compounds, alongside representative compounds from each nitroimidazole subclass, to a full
radiation doseꢀresponse assay in comparison to 1, 2 and 4. For each subclass of
radiosensitizer, the SR15 was related to the drug concentration and HCR:
logꢃꢔꢓ ꢈ = ꢉ + ꢊꢋꢊꢊ + ꢍꢊꢅꢁꢖꢍ + ꢐꢊꢒꢅꢓ
(3)
ꢕꢆ
where a = 0.3123 ± 0.1497 (p = 0.046), b = 1.0855 ± 0.177 ((for 5ꢀnitroimidazoles 17, 19, 21,
ꢀ6
22 and 24), p = 1.14 × 10 ), or b = 0.350 ± 0.154 ((for 2ꢀMe 5ꢀnitroimidazoles 26–38), p =
ꢀ5
2
0
.031), c = 0.519 ± 0.103 (p = 2.11 × 10 ), d = 0.583, (p = 0.000352), R = 0.720. The
relationship to HCR is stronger than to nominal E(1) and HCR provides a rapid continuous
measure of electron affinity in drug screening.
In vitro radiosensitization: Sensitizer enhancement ratios. We determined
radiosensitization for selected compounds at concentrations equivalent to the anoxic IC50 as
above, but using a lead wedge above the anoxic chamber in order to generate a range of
6
3,67
radiation doses (6–29 Gy) in the 96ꢀwell plates.
HCT116 cells were exposed to drug for
1
h as above, irradiated, and then dissociated and plated for clonogenic survival. Sensitizer
enhancement ratios (SER) were calculated as the ratio of radiation dose for 1% survival
without or with the drug (See Figure S2). Each radiosensitization experiment included 1 as a
control and the SER data were normalized (SERnorm) against the activity of 1 (Table 2). All
1
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compounds demonstrated radiosensitization with 4, 19, 24 and 30 showing clearly increased
sensitization compared to 1.
2
ꢀ4
SER correlated well with the logSR15 (R = 0.683, p = 6 × 10 , Figure 4b) while the
2
logSR from the SER experiments was also highly correlated with the logSR (R = 0.764, p
15
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ꢀ5
=
6 × 10 , Figure 4a). This correlation could be improved using multiple regression to
normalize for the effects of cell kill by radiation alone (SFrad), since SR is radiation dose
dependent, whereas the SER is usually considered constant for a defined drug concentration.
This allowed prediction of the SER at the IC50 drug concentration, from data collected in the
screening process. The correlation was also improved by the addition of SERMISO to the
2
multiple regression allowing for experimental variability with a final R of 0.802.
ꢔꢌꢓ = ꢊꢉ logꢃꢔꢓ ꢈ + ꢋ logꢃꢔꢗꢘꢙꢚꢈ + ꢍꢊꢔꢌꢓꢛꢜꢝꢞ
(4)
ꢕꢆ
ꢀ5
where a = ꢀ0.335 ± 0.060, (p = 2.46 × 10 ), b = 0.6588 ± 0.157 (p = 0.000545) and c = 0.994
±
0.40263 (p = 0.0238); SF (surviving fraction). The theoretical relationship between SR and
SER is:ꢊ
ꢢ
ꢢ
ꢢ
− logꢃꢔꢓꢈ = ꢟꢔꢌꢓꢠ + ꢊꢡꢔꢌꢓ ꢠ − ꢟꢠ − ꢊꢡꢠ ꢊ
However, as the SR data were determined at a constant dose of 15 Gy, the relationship is
approximately linear. SER was primarily determined by the radiosensitizer concentration
(though the SR) and a single term involving SFrad. This provides evidence that the
appropriately determined SR, at a nonꢀtoxic concentration, can be used as a measure of
radiosensitization for screening compounds.
Tumor radiosensitization. A maximum tolerated dose (MTD) was determined in nonꢀ
tumor bearing mice. In those cases where an MTD could not be determined because of
solubility limitations a maximum achievable dose (MAD) was defined. Drug plasma and
tumor pharmacokinetic profiles were determined following single i.v. doses at the MTD or
MAD to determine achievable drug concentrations and the optimal time for irradiation (Tmax
)
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(Table 3). Oxygenꢀmimetic radiosensitizers act at the time of radiation damage and so
maximizing the drug concentration in hypoxic tumor cells at the time of irradiation is critical.
Compounds were compared with 2 and 4 for in vivo radiosensitization (12.5 Gy) of hypoxic
cells in HCT116 human tumor xenografts using tumor excision 18 h later and clonogenic
survival as the endpoint. Radiosensitization was determined as the ratio of surviving fraction
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(SF) after radiation without and with radiosensitizer present (the in vivo SR). The advantage
of this in vivo/ex vivo assay is that it integrates radiosensitizer tumor microꢀpharmacokinetics
and extravascular transport to the radioꢀresistant hypoxic fraction, and explicitly assesses
impact on clonogenic cell survival.
We had previously explored the activity of 6 and 7 in combination with radiation
63
against HCT116 colorectal tumor xenografts in comparison with 2. We had found that 2
was well tolerated with a MAD of 2.20 mmol/kg leading to plasma and tumor drug
concentrations of 3.64 mM and 0.80 mM, respectively (Table 3). As expected, 2 was active
demonstrating an in vivo SR of 4.0. Plasma and tumor concentrations of 6 and 7 were 6–10
fold lower than for 2 as a result of poorer solubility and greater toxicity, with 7 showing an in
vivo SR of 2.4 (Table 3 and Figure 5). We had explored the potential of lipophilic amines that
showed increased aqueous solubility, with 15 achieving a higher MAD and plasma and tumor
concentrations than 6 and 7, but this did not translate into significant radiosensitization (in
63
vivo SR 2, P = 0.096).
In the present study, we confirmed the activity of 2 as an effective radiosensitizer (in
vivo SR 8) (Table 3, Figure 5). We also explored the activity of 4 in the HCT116 xenograft
model since 4 is the clinical benchmark, although it is used orally in patients. In our hands we
found 4 to be relatively toxic when given i.v. and could only achieve a MTD of 1.24
mmol/kg. When given at 75% of MTD 4 did demonstrate significant radiosensitization with
an in vivo SR of 4.
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We sought to increase aqueous solubility and improve drug delivery through the use of
phosphate prodrugs. We prepared the phosphate prodrug 39 of alcohol 7 and observed a
substantial increase in aqueous solubility (e.g., solubility of 39 in culture medium is 185 mM
compared to 18 mM for 7). Phosphate 39 was not toxic when administered at 2.20 mmol/kg
1
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(equimolar with 2) and was rapidly converted to alcohol 7 in vivo (Figure S3), leading to a ca.
2ꢀ and 4ꢀfold increase in plasma and tumor drug concentrations, respectively, of the alcohol 7
compared to direct administration of 7 (Table 3). This increase resulted in significant
radiosensitization with an in vivo SR of 10. Expansion of the phosphate prodrug approach led
to the preparation of phosphates 40–42 based on the corresponding alcohols 14, 19 and 24,
respectively. Although 14 displayed modest sensitization in vitro (SERnorm 0.92), both 19 and
2
4 were good sensitizers (SERnorm 1.28 and 1.23, respectively) (Table 2). Moreover, the three
alcohols along with 7 represented structural subclasses explored in this study. Phosphates 40–
2 all demonstrated excellent aqueous solubility (Table S1) and were able to be administered
4
without any observable toxicity at 2.20 mmol/kg (equimolar with 2). Phosphates 40 and 41
were also rapidly converted to the corresponding alcohols 14 and 19 achieving plasma and
tumor concentrations similar to those of 7 when administered as 39 (Table 3 and Figure S3).
The radiosensitization demonstrated by 40 and 42 was modest, whereas 41 provided the
largest effect with an in vivo SR of 13.
The antitumor activity of phosphates 39 and 41 in combination with a single dose of
radiation (12.5 Gy) was also compared with 2 and 4 using tumor growth inhibition as an
endpoint. All compounds were well tolerated at their MAD (2.20 mmol/kg for 2, 39 and 41)
or 75% of MTD (0.93 mmol/kg for 4) with minimal body weight loss (Figure 6a).
Compounds 39 and 41 showed similar radiosensitization activity to 2 and 4 at equitoxic
3
doses, appearing to delay the time taken for HCT116 tumors to triple in size (RTV , relative
tumour volume 3×) compared to radiation alone, although these differences did not reach
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statistical significance for any of the treatments (LogꢀRank with HolmꢀSidak multiple
comparison analysis) (Figure 6b).
CONCLUSIONS
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We have identified two novel radiosensitizers 7 and 19 with significant in vivo activity
against hypoxic tumor cells when delivered as the phosphate prodrugs 39 and 41,
respectively. The phosphate prodrug strategy improved aqueous solubility to allow equimolar
comparison with etanidazole (2) and improved drug delivery, resulting in increased peak
tumor drug concentrations, compared to administration of the corresponding alcohols.
Although these peak tumor drug concentrations were still lower than for 2, both 39 and 41
demonstrated substantial radiosensitization of hypoxic tumor cells in vivo and were equally
active as either 2 or 4 at inhibiting tumor growth in combination with radiation. The 2ꢀ
nitroimidazole 7 is similar to 2 in so far as it is highly electronꢀaffinic and displays hypoxiaꢀ
selective cytotoxicity in addition to radiosensitization in vitro. Etanidazole (2) was an
68
39, 69
effective radiosensitizer preclinically, but failed to provide benefit in cancer patients.
Although designed to be more polar and more readily cleared than misonidazole 1, the
70ꢀ72
toxicity was still limiting in a FRT setting
and could be associated with bioreductive
mechanisms. Compound 7 is similar in polarity to 2 (logD 1.53 and 1.37, respectively), but is
more electronꢀaffinic and displays increased hypoxiaꢀselective cytotoxicity compared to 2,
identifying 7 (as its prodrug 39) as a compound more suited to use in a SBRT setting. In
contrast, compound 19 is more akin to nimorazole (4) in its properties. It is slightly more
electronꢀaffinic than 4 with an E(1) likely to be around ꢀ420 mV and displays little hypoxic
cytotoxicity (HCR 4) suggesting potential for prodrug 41 in the context of FRT. We plan to
further explore the potential of both 39 and 41 as representatives of each end of the
mechanistic spectrum of these nitroimidazole oxygenꢀmimetic radiosensitizers.
1
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EXPERIMENTAL SECTION
Chemistry. General experimental details are described in the Supporting Information.
All final products were analyzed by RPꢀHPLC (Altima C18 5 ꢁm column, 150 mm × 3.2
mm; Alltech Associated, Inc., Deerfield, IL) using an Agilent HP1100 equipped with a diodeꢀ
1
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2
array detector. Mobile phases were gradients of 80% MeCN/20% H O (v/v) in 45 mM
ammonium formate at pH 3.5 and 0.5 mL/min. Purity was determined by monitoring at 330
(± 50 nm) and was >95% with the exception of compound 10 (90.4%) (Table S1). The
solubility of the final products was determined by HPLC analysis after incubation of
saturated solutions of drug in αMEM containing 5% FCS and 1% DMSO at 37 °C to mimic
cell culture conditions (Table S1). Stability of the same solutions after 24 h incubation at 37
°C was measured by HPLC analysis and was found to be >95% (Table S1).
Example of Synthetic Methods. See Supporting Information for full experimental
details.
Method A. Preparation of halomethyl sulfonamides. A solution of alkyl sulfonyl
chloride (1.0 eq.) in dry DCM (2 mL/mmol) was added drop wise to a stirred solution of
amine (1.2 eq.) and iPr NEt (1.3) in dry DCM (10 mL/mmol) at 0 °C and the solution stirred
2
at 20 °C for 16 h. The solution was diluted with DCM (50 mL/mmol) and washed with water
(3 × 20 mL/mmol), washed with brine (20 mL/mmol), dried and the solvent evaporated. The
residue was purified by chromatography, eluting with appropriate mixtures of EtOAc/pet.
ether, to give the sulfonamide.
Method B. Alkylation of nitroimidazoles. A mixture of nitroimidazole (1.5 eq.),
2 3
haloalkyl sulfonamide (1.0 eq.), Cs CO (1.1 eq.) and NaI (1.0 eq.) in dry DMF (10
mL/mmol) was stirred at 80 °C for 3 h. The solvent was evaporated and the residue
suspended in EtOAc (100 mL/mmol) and washed with water (3 × 30 mL/mmol), washed with
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brine (20 mL/mmol), dried and the solvent evaporated. The residue was purified by
chromatography, eluting with appropriate mixtures of EtOAc/pet. ether, to give the
nitroimidazole sulfonamide.
Method C. Deprotection of nitroimidazole sulfonamides. A solution of silyl ether
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(1.0 eq.) in HOAc/water/THF (3:1:1, 10 mL/mmol) was stirred at 20 °C for 48 h. The solvent
was evaporated and the residue suspended in toluene (3 × 20 mL) and the solvent evaporated.
The residue was triturated with pet. ether or purified by chromatography, eluting with
appropriate mixtures of EtOAc/pet. ether, to give the alcohol.
Method D. Preparation of nitroimidazole thioesters. Potassium thioacetate (1.5
eq.) was added to a stirred solution of nitroimidazole alkyl chloride or mesylate (1.0 eq.) in
anhydrous DMF (5 mL/mmol), and the mixture was stirred at 20 °C for 16 h, then heated at
80 °C for 4 h. The mixture was cooled and partitioned between EtOAc (30 mL/mmol) and
water (30 mL/mmol). The organic layer was washed with water (3 × 10 mL/mmol) then
washed with brine (10 mL/mmol), dried and the solvent was evaporated. The residue could
be purified by column chromatography, eluting with a gradient (50–100%) of EtOAc/pet.
ether, to give thioacetate or used directly as the crude product.
Method E. Conversion of thioesters to sulfonamides. A solution of thioacetate (1
3
eq.) in CH CN (2 mL/mmol) was added drop wise to a mixture of NCS (4.0 eq.) in aqueous
HCl (2 M, 12 mL/mmol) and CH CN (3 mL/mmol) at 10 °C. The mixture was stirred at 10–
3
15 °C for 30 min, and allowed to warm to 20 °C over 10 min. The mixture was partitioned
3
between EtOAc (50 mL/mmol) and aq NaHCO (25 mL/mmol). The organic phase was
washed with aqueous NaCl (3 × 25 mL/mmol), washed with brine (20 mL/mmol), dried and
the solvent was evaporated to give the crude sulfonyl chloride which could be used without
further purification. Amine (1.1 eq.) and iPr NEt (1.3 eq.) were successively added to a
2
solution of the crude sulfonyl chloride in anhydrous DCM (10 mL/mmol) at 0 °C. The
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mixture was allowed to warm to 20 °C and stirred for 16 h. The mixture was diluted with
DCM (30 mL/mmol), washed with water (3 × 15 mL/mmol), washed with brine (15
mL/mmol), dried and the solvent was evaporated. The residue was purified by column
chromatography, eluting with appropriate mixtures of EtOAc/pet. ether, to give the
sulfonamide.
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2 2
Method F. Preparation of phosphate prodrugs. iPr NP(OtBu) (1.5 eq.) was added
drop wise to a stirred solution of alcohol (1.0 eq.), imidazole (1.0 eq.) and imidazole
hydrochloride (1.5 eq.) in DMF (5 mL/mmol) at 20 °C and the solution was stirred at 20 °C
for 1 h. The solution was cooled to 0 °C and aqueous H O solution (30%, 2.5 eq.) was added
2
2
drop wise. The mixture was allowed to warm to 20 °C over 1 h. The mixture was diluted with
EtOAc (100 mL/mmol) and washed with cold water (3 × 50 mL/mmol), washed with cold
brine (50 mL/mmol), dried and the solvent evaporated. The residue was purified by
chromatography, eluting with a gradient (0–6%) of MeOH/DCM, to give the phosphate ester.
A solution of HCl in MeOH (1.25 M, 10 eq.) was added drop wise to a stirred solution of the
phosphate ester (1.0 eq.) in DCM (20 mL/mmol) and the solution was stirred at 20 °C for 3
days. The solvent was evaporated and the residue triturated with DCM (3 × 10 mL/mmol)
and dried to produce a white soft solid. The residue was dissolved in water (5 mL/mmol) and
freeze dried to give the phosphate.
Oneꢀelectron reduction potentials. Pulse radiolysis studies were carried out using
the University of Auckland’s Dynaray 4 (4 MeV) linear accelerator (200 ns pulse length with
73
a customꢀbuilt optical radical detection system) as previously described. The oneꢀelectron
•ꢀ
reduction potentials of the compounds, E(A/A ), vs. NHE, were determined at pH 7.0 (2.5
mM phosphate buffer) by establishing redox equilibria within 20 ꢀs between three mixtures
of the oneꢀelectron reduced compounds and the reference compounds benzylviologen
2
+
+
2+
•+
(E(BV /BV ) = ꢀ380 ± 10 mV), methylviologen (E(MV /MV ) = ꢀ447 ± 7 mV) or triquat
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2
+
•+
e
(E(TQ /TQ ) = ꢀ548 ± 7 mV) and calculating ꢂE values from the equilibrium constants, K ,
7
4
using the Nernst equation. Data were obtained at three concentration ratios at room
temperature (22 ± 2 °C).
In vitro cytotoxicity testing. HCT116 cells were authenticated by short tandem
repeat profiling at DNA Diagnostics Ltd, Auckland, NZ. Cells were grown in αMEM
medium with 5% fetal calf serum (FCS), without antibiotics, and were confirmed
mycoplasmaꢀfree using a PCRꢀELISA method (Roche Diagnostics). Logꢀphase cells were
seeded into 96ꢀwell plates at 700 cells/well, allowed to attach for 2 h, then test compounds
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were added by dilution from DMSO stocks to give a top concentration in <1% DMSO before
serial 3ꢀfold dilution in the plates. After 4 h, cultures were washed 3 times with fresh
medium and grown for a further 5 days before staining with sulforhodamine B to determine
7
5
IC50 values as previously. For hypoxic exposure to compounds, cells were pelleted by
centrifugation, transferred to a Pdꢀcatalyst anaerobic chamber (BactronꢀII, Shel Lab),
resuspended in anoxic medium and exposed to drugs as above, but using medium and plates
that had been equilibrated in the chamber for at least 3 days. After drug washout, cells were
grown and stained as for the oxic IC50 assays. The hypoxic cytotoxicity ratio HCR was
determined as HCR = IC50(oxic)/IC50(anoxic)
.
5
Radiosensitization studies. Cells were seeded onto 96ꢀwell plates at 10 cells per
well and allowed to attach for 2 hours. Replicates were treated with compound at the
respective anoxic IC concentration 30 min prior to irradiation. For anoxic irradiation, plates
5
0
were transferred to a customꢀbuilt, airꢀtight, stainless steel, portable box (13 × 16.5 × 3 cm)
within the anaerobic chamber, then sealed and transported to the radiation machine. The
60
plates were irradiated (Eldorado 78 Co teletherapy unit, ~2 Gy/min) with 15 Gy under
anoxia at room temperature for SR experiments. A metal wedge placed on the top of the
metal chamber was used to achieve a graduated radiation dose across the plate varying from 6
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to 29 Gy, calibrated by Fricke dosimetry with ammonium thiocyanate as previously
63,67
described
for SER experiments. The control plate (compound alone, no radiation) was left
inside the anaerobic chamber at room temperature during the irradiation period. After
treatment, the cells were trypsinized and suspended in αMEM + 5% FCS + 1% penicillinꢀ
streptomycin, and 10ꢀfold serial dilutions were plated for clonogenic survival. After 10 days
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plates were stained with methylene blue (2 g/L in EtOH:H O, 1:1 v/v) and colonies with
more than 50 cells were counted. The surviving fraction (SF) was determined as: SF =
PE(irradiated) / PE(control) where the plating efficiency (PE) = (No. of colonies) / (No. of cells
plated). SF was plotted against radiation dose. Survival ratios (SR) were calculated: SR =
(
cell survival with radiation) / (cell survival with drug + radiation). Sensitizer enhancement
ratios (SER) were calculated: SER = (radiation dose for 1% survival without compound) /
radiation dose for 1% survival with compound). Misonidazole (1) at its hypoxic IC50 (0.5
(
mM) was used as an intraꢀexperiment control.
In vivo toxicity. A maximum tolerated dose (MTD) was determined for each
compound in nonꢀtumor bearing specific pathogenꢀfree NIHꢀIII female mice (∼20 g). Freshly
prepared solutions of compounds were given i.v. to mice at 10 mL/kg body weight, using
1.33ꢀfold dose increments. The MTD was defined as the highest dose causing no drugꢀrelated
deaths, body weight loss of more than 15%, or severe morbidity in a group of three to six
animals, with an observation time of up to 14 days. A maximum achievable dose (MAD) was
defined as the maximum dose able to be dissolved in the injection volume. Drugs were
administered as solutions in 0.9% saline containing 1% DMSO or for 39–42 as solutions in
3
0.9% saline containing 2 molar equivalents of NaHCO .
6
Pharmacokinetics. Female NIHꢀIII mice were inoculated subcutaneously with 10
3
HCT116 cells in 100 µl media. Once tumor size reached ca. 300 mm mice were injected
with a single i.v. dose at the MAD or 75% of the MTD for each compound. At multiple time
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points after dosing, blood and tumor tissue were sampled by cardiac puncture and tumor
2
excision under terminal CO anaesthesia. Blood was immediately centrifuged (3,000 × g, 3
minutes), and plasma and tumor stored at −80 °C for HPLC. Tumor samples were prepared
63,76,77
and analysed by HPLC using standard protocols established in our laboratory.
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Noncompartmental PK parameters were determined by Phoenix WinNonLin v 6.2 (Pharsight
Corp.).
Tumor radiosensitization: ex vivo clonogenic assay. Compounds were evaluated for
in vivo radiosensitization of hypoxic cells in HCT116 human tumor xenografts using
78
clonogenic survival as an endpoint using previously report methods. HCT116 tumors were
6
grown subcutaneously on the flanks of NIHꢀIII mice by injecting 10 cells. Once tumors
3
reached a volume of approximately 300 mm , mice were randomly assigned to 4 treatment
groups and treated with: A) vehicle control; (B) 12.5 Gy RAD only; (C) sensitizer only; (D)
sensitizer given before 12.5 Gy RAD. Drugs were administered as a single i.v. dose at the
60
MAD or 75% of the MTD. Mice were injected with drug and irradiated (Eldorado 78 Co
teletherapy unit, ~2 Gy/min, whole body irradiation at 12.5 Gy) 10 minutes later. Tumors
were excised 18 h after treatment, and tumor cells were dissociated enzymatically and plated
78
to determine the number of surviving (clonogenic) cells per gram of tumor tissue. SF and
SR were calculated as described above. Statistical significance of drug effects was tested
using oneꢀway ANOVA with HolmꢀSidak test for multiple comparisons vs radiation alone
(Table 3).
Tumor radiosensitization: tumor regrowth delay. Compounds were evaluated for
their ability to inhibit tumor growth in combination with a single dose of radiation (12.5 Gy)
in HCT116 tumor xenografts on the backs of NIHꢀIII mice. Mice were randomly assigned to
4
treatment groups and treated with: A) vehicle control; (B) 12.5 Gy RAD only; (C) sensitizer
only; (D) sensitizer given 10 min before 12.5 Gy RAD. Drugs were administered as a single
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i.v. dose at the MAD or 75% of the MTD. Treatment commenced when the average tumor
3
size reached (300 to 400 mm ) and tumor size was measured until study endpoint. Mice were
placed in a customꢀbuilt leadꢀshielded jig that allowed irradiation of the whole tumor, but
shielded the mouse from radiation. Tumor growth inhibition was presented as the time taken
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for tumors to increase in relative tumor volume to 3× tumor size at the time of treatment
3
(
RTV ). The significance of differences in activity between the groups was assessed by Logꢀ
Rank with HolmꢀSidak multiple comparison analysis. All animal experiments were
conducted under the auspices of the University of Auckland Animal Ethics Committee
(Approval 1781).
ASSOCIATED CONTENT
Supporting information. . The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmedchem.
Experimental details and characterization data for compounds 6–42 and intermediates 43–81.
1
13
Tables of in vitro data for compounds 6–42 and associated figures. H, C NMR and HPLC
spectra for compounds 6–42. Molecular formula strings (CSV).
AUTHOR INFORMATION
Corresponding Author
*Eꢀmail: m.hay@auckland.ac.nz
Notes
The authors declare no competing interests.
ACKNOWLEDGEMENTS
The authors would like to thank Dr Thorsten Melcher (J&J Innovation) for helpful
discussions and thank Dr Adrian Blaser, Ms Karen Tan and Mr Sisira Kumara for technical
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assistance. The authors acknowledge support from the Australian Institute for Nuclear
Sciences and Engineering, the University of Auckland’s Biopharma Thematic Research
Initiative, UniServices Investment / Ministry of Business, Innovation and Employment Preꢀ
Seed Accelerator Fund, the Cancer Society of New Zealand (14/17) and the Health Research
Council of NZ (16/120).
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ABBREVIATIONS USED
DCM, dichloromethane; E(1), oneꢀelectron reduction potential; FCS, foetal calf serum; FRT,
fractionated radiotherapy; HCR, hypoxic cytotoxicity ratio, αMEM, minimum essential
media; NCS, Nꢀchlorosuccinimide; SAR, structureꢀactivity relationship; SBRT, stereotactic
body radiotherapy; SF, surviving fraction; SR, survival ratio; SER, sensitizer enhancement
ratio.
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