Bioorganic & Medicinal Chemistry 22 (2014) 2123–2132

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier .com/locate /bmc

Novel nitroimidazole alkylsulfonamides as hypoxic cellradiosensitisers

http://dx.doi.org/10.1016/j.bmc.2014.02.0390968-0896/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: DCM, dichloromethane; E(1), one-electron reduction potential;FCS, foetal calf serum; FRT, fractionated radiotherapy; HCR, hypoxic cytotoxicityratio; NCS, N-chlorosuccinimide; SAR, structure activity relationship; SBRT, stereo-tactic body radiotherapy.⇑ Corresponding author. Tel.: +64 649 923 1190; fax: +64 949 373 7502.

E-mail address: m.hay@auckland.ac.nz (M.P. Hay).

Muriel Bonnet, Cho Rong Hong, Yongchuan Gu, Robert F. Anderson, William R. Wilson, Frederik B. Pruijn,Jingli Wang, Kevin O. Hicks, Michael P. Hay ⇑Auckland Cancer Society Research Centre, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand

a r t i c l e i n f o

Article history:Received 13 December 2013Revised 7 February 2014Accepted 18 February 2014Available online 3 March 2014

Keywords:NitroimidazoleRadiosensitiserSulfonamideHypoxia

a b s t r a c t

A novel class of nitroimidazole alkylsulfonamides have been prepared and evaluated as hypoxia-selectivecytotoxins and radiosensitisers. The sulfonamide side chain markedly influences the physicochemicalproperties of the analogues: lowering aqueous solubility and raising the electron affinity of the nitroim-idazole group. The addition of hydroxyl or basic amine groups increased aqueous solubility, with chargedamine groups contributing to increased electron affinity. The analogues covered the range of electron affin-ity for effective radiosensitisation with one-electron reduction potentials ranging from�503 to �342 mV.Cytotoxicity under normoxia or anoxia against a panel of human tumour cell lines was determined using aproliferation assay. 2-Nitroimidazole sulfonamides displayed significant hypoxia-selective cytotoxicity (6 to64-fold), while 4- and 5-nitroimidazole analogues did not display hypoxia-selective cytotoxicity. Allanalogues sensitised anoxic HCT-116 human colorectal cells to radiation at non-toxic concentrations.2-Nitroimidazole analogues provided modest sensitisation due to the relatively low concentrations usedwhile several 5-nitroimidazole analogues provided equivalent sensitisation to misonidazole and etanidazoleat similar molar concentrations.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction tial to replace surgery with an outpatient procedure, indicates sub-

Fractionated radiotherapy (FRT) is one of the main treatmentsfor cancer patients with over 50% of patients receiving FRT (typi-cally 60–70 Gy delivered in 2–3 Gy fractions over 7–8 weeks),mostly with curative intent.1 FRT is a difficult regimen for bothhealth service delivery and patient compliance and often normaltissue toxicity precludes delivery of sufficient doses of radiationto achieve local tumour control. Stereotactic body radiotherapy(SBRT) uses hypofractionated (1–5 doses) high dose (25–60 Gy to-tal dose) radiation to treat tumours.2 This new approach leveragesrecent advances in the accuracy and precision of radiation deliveryto allow dose intensification to small tumours while minimisingthe effects to adjacent normal tissue. Clinical trials using SBRT totreat various solid tumours have demonstrated comparable con-trol, toxicity and efficacy profiles to FRT.2 The reduced treatmenttime and number of patient visits, combined with emerging poten-

stantial health, social and economic advantages for SBRT and isdriving increasing use of SBRT for treating cancer.

However, evidence is emerging that SBRT accentuates the roleof hypoxia in radioresistance.3,4 Hypoxia, which is a consequenceof the inefficient vascularisation of tumours, contributes to alteredtumour metabolism,5 invasion,6 and metastasis,7 and is associatedwith poor prognosis and resistance to therapeutic agents.8 Hypoxiais prevalent in a wide range of solid tumours9,10 and patients withhypoxic tumours have significantly poorer outcomes than thosewith lower levels of hypoxia.11,12 The significance of hypoxia inresistance to cytotoxic therapy has renewed interest in targetingthese cells.13 Hypoxic cells are less sensitive to radiation-inducedDNA breakage, because oxygen is not available to oxidise radia-tion-induced DNA radicals to generate strand breaks.14

One approach to increase radiation response in hypoxic tu-mours is to use nitroimidazole radiosensitisers.14 These radiosensi-tisers are relatively non-toxic molecules which react withradiation-induced DNA radicals and cause DNA strand breaks anal-ogously to oxygen. They have to be present at the time of irradia-tion and are mechanistically distinct from hypoxia-selectivecytotoxins which are enzymatically reduced under hypoxia togenerate a cytotoxic moiety.13 Misonidazole (1) (Fig. 1) was exten-sively trialled with FRT in the clinic and despite indications of

N N

NO2

OMeOH

N N

NO2 HN

OHO

N NN

O

NO21 2 4

N N

NO2

O OH

3OH

OH

Figure 1. Clinically evaluated nitroimidazole radiosensitisers.

2124 M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132

clinical benefit in some trials, failed to provide significant improve-ment on radiotherapy alone, with delayed peripheral neuropathylimiting treatment.15 Attempts to design more polar analogues16

with increased systemic clearance to minimise neurotoxicity17

were only partially successful with etanidazole (2) failing to pro-vide benefit in head & neck cancer.18,19 A similar approach withthe more polar doranidazole (3)20 is currently being clinically eval-uated. Although a recent meta-analysis has confirmed the clinicalactivity of nitroimidazole radiosensitisers,21 only nimorazole (4)is in clinical use.22 Nonetheless, the recent description of a hypoxicgene signature,23 that in a retrospective study identifies head &neck cancer patients with hypoxic tumours and predicts the bene-fit of nimorazole in only those patients, has provided clinical vali-dation of the use of nitroimidazole radiosensitisers withradiotherapy.24 However, existing agents have no or limited intel-lectual property protection which restricts options for clinicaldevelopment.

The advent of SBRT, combined with new approaches to identifypatients with hypoxic tumours, heralds a new opportunity for theuse of novel nitroimidazole radiosensitisers. We have addressedthis opportunity and identified a class of nitroimidazole that incor-porates an alkylsulfonamide moiety as a key element, with theobjective of developing improved, novel radiosensitisers for SBRT.Here, we report the synthesis of a pilot set of compounds andin vitro appraisal of their potential as hypoxia-selective radiosensi-tisers. We also evaluate their hypoxia-selective cytotoxicity in cul-ture as a measure of their sensitivity to bioreductive activation.

2. Results

2.1. Chemistry

Our synthetic strategy was directed towards nitroimidazolesbearing an alkyl sulfonamide as a moiety conferring both novelty

NHN

NO2

NN

NO2

Cl

N

NO

6

Cl SO2Cl Cl SO

HN

OOMe

(c) (d)

(f)

(g)

5

NHNN

O2NO2N 14

13

(i)

(a), (b)

N

N

N

N

1112

Scheme 1. Reagents and conditions: (a) ClCH2Br, potassium thioacetate, MeCN; (b) iPr2N(e) NCS, 2 M aq HCl, DCM; (f) MeOCH2CH2NH2, Et3N, DCM, 16% from 6; (g) Na2SO3, aceto

and a strong modulating effect on the electron affinity of the nitro-imidazole moiety. We initially explored a strategy previously usedto prepare alkylaryl sulfonamides,25 generating chloro-methylthioacetate in situ to alkylate 2-nitroimidazole (5), but thisfailed to provide the key sulfonyl chloride 9 in our hands(Scheme 1). To avoid the use of the volatile chloromethylthioace-tate we attempted a stepwise strategy to prepare 9. Alkylation of5 with bromochloromethane under a variety of conditions was ex-plored; however formation of the dimer 8 predominated undermost conditions. Optimum results were obtained using Cs2CO3 inthe presence of an excess (20 equiv) of bromochloromethane giv-ing chloride 6 which was condensed with potassium thioacetateto give thioester 7 in modest yield. Oxidation of 7 with NCS, gavethe unstable sulfonyl chloride 9. Efforts to isolate 9 resulted in sig-nificant loss of material and so 9 was used directly in subsequentreactions. Reaction of crude sulfonyl chloride 9 with 2-methoxy-ethylamine gave sulfonamide 10 in low yield. In an effort to avoidthe unstable intermediates 7 and 9 we prepared the sulfonate salt11 and sulfonic acid 12. Attempts to prepare sulfonamides fromeither 11 or 12 with an array of coupling reagents (oxalyl chloride,EDCI, HOBt, HBTU, HATU) using multiple reaction conditions failedto provide positive results. We explored an alternative approach to9 by condensing chloromethanesulfonyl chloride with 2-methoxy-ethylamine to give sulfonamide 13 which was used to alkylate2-nitroimidazole. Although only providing a similar yield tothe oxidative route, this approach was more direct and alsoprovided access to the isomeric analogues 15 and 16, albeit inlow yield.

The direct oxidative approach provided low yields of 17, reflect-ing losses of material in the aqueous workup, however use of a pro-tected ethanolamine 18 gave no overall improvement in yield of 17(Scheme 2). An alternative alkylation strategy using the protectedchlorosulfonamide 19 provided a modest improvement in the yieldof 20, and subsequently 17.

N

2

S Me

O

7NN

NO2

SO2Cl

8

NN

NO2

SO

HN

OOMe

9

10

(e)

(f)

NN SHN

O OOMe

NO2

N SHN

O OOMe +

15

N

O2

N N

NO2

16

+

N

O2

SR

O O

R = O-Na+

R = OH (h)

(i)

Et, NaI, MeCN; (c) ClCH2Br, Cs2CO3, DMF, 45%; (d) potassium thioacetate, DMF, 42%;ne/H2O; (h) DOWEX50 WX8, water, 87%; (i) 5 (18%) or 14 (7% and 3%), Cs2CO3, DMF.

NN

Me

NO2

OHNN

Me

NO2

S

O

Me

NN

Me

NO2

SCl

O O

NN

Me

NO2

SNH

O OOMe

22

23

21

24

(a), (b)

(c)

(d)

NN

Me

NO2

SNH

O ON

25

NN

Me

NO2

SNH

O ON

26

(e) (f)

Scheme 3. Reagents and conditions: (a) MsCl, Et3N, DMAP, DCM; (b) potassiumthioacetate, DMF, 70% from 21; (c) NCS, 2 M aq HCl, DCM; (d) MeOCH2CH2NH2,Et3N, DCM, 50% from 22; (e) 2-(pyrrolidin-1-yl)ethanamine, Et3N, DCM, 25% from22; (f) 2-(piperidin-1-yl)ethanamine, Et3N, DCM, 68% from 22.

NN

NO2

S Me

O

7

NN

NO2

SO2Cl

9

NN

NO2

SO

HN

OOH

(a)

(d)

(b)

H2NOH

NN

NO2

SO

HN

OOTBDMS

H2NOTBDMS

(c)

(e)18

20

17

SO

HN

OOTBDMS

19

Cl

(f)

(g)

NN

NO2

(h)

Cl

6

Scheme 2. Reagents and conditions: (a) potassium thioacetate, DMF; (b) NCS, 2 M aq HCl, DCM; (c) HOCH2CH2NH2, Et3N, DCM, 10% from 6; (d) TBDMSCl, imidazole, DCM,82%; (e) 9, Et3N, DCM, 11%; (f) AcOH, THF, H2O, 73%; (g) ClCH2SO2Cl, Et3N, DCM, 88%; (h) 5, Cs2CO3, DMF, 35%.

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132 2125

Metronidazole (21) provided a convenient starting point for thepreparation of several 5-nitroimidazole analogues (Scheme 3).Mesylation and displacement with potassium thioacetate gavethe thioester 22 in good yield. The sulfonyl chloride 23 was not iso-lated and converted directly to sulfonamides 24–26 in fair to goodyields.

2.2. Physicochemical data

Aqueous solubility, lipophilicity and electron affinity are threekey physicochemical parameters that influence the efficacy of

Table 1Physicochemical parameters

Compound Solubility (mM) LogDa

Misonidazole (1) >100 �0.41Etanidazole (2) >100 �1.3710 6.5 �1.1915 23.5 �0.8316 >27 �0.6417 18.1 �1.5324 29.6 �0.8425 61.7 �1.8726 >51 �0.39

aCalculated by ACDLab logD calculator v12.5; bACD/PhysChem v12; cRef.31; dReference c

nitroimidazole radiosensitisers. Typically, nitroimidazole radiosen-sitisers need to be present at millimolar concentrations to be effec-tive sensitisers and so aqueous solubility is an importantprerequisite. A pragmatic measurement of aqueous solubility wasadopted; determining solubility by HPLC (see Section 4.1.1) forsolutions of compounds diluted from a DMSO stock solution to<1% DMSO with a-MEM and 5% added foetal calf serum (FCS)(Table 1). Although the sulfonamide group provides an increasein polar surface area (PSA), it has a negative effect on aqueoussolubility. The solubility of neutral 2-nitroimidazole sulfonamides(e.g., 10, 15–17, 24) is considerably less than either misonidazole(1) or etanidazole (2), but is still in the millimolar range. The useof basic side chains (e.g., 25, 26) provides ca. two-fold increase insolubility over 24.

LogD values at pH 7.4 were calculated using ACD LogD calcula-tor v12.5 (Advanced Chemistry Development, Inc.). Lipophilicityinfluences two important properties of nitroimidazole sensitisers:extravascular transport26 and normal tissue toxicity.16 While therole of extravascular transport remains to be determined for thesesensitisers, more lipophilic nitroimidazole sensitisers, such asmisonidazole, have demonstrated significant normal tissue toxic-ity, most markedly peripheral neuropathy.27,28 These neuropathiesare somewhat ameliorated in etanidazole. The new analogues areat least as polar as misonidazole (1) with two analogues (e.g., 17and 25) more polar than etanidazole (2).

The electron affinity of the nitroimidazole moiety dominatesthe in vitro SAR for radiosensitisation29 and cytotoxicity,30 andone-electron reduction potential [E(1)] values correlate with elec-tron affinity: so this is a key parameter for our SAR studies. Wehave determined the E(1) values of the three possible regioisomersby pulse radiolysis (Table 1). These potentials span the range forefficient reaction with DNA radicals (�503 to �342 mV) with the2-nitroimidazole more electron affinic than the corresponding

PSAb E(1) mV Reference E(1)d

93.1 �389c

113.0 �388c

127.4 �352 ± 10 BV127.4 �503 ± 7 MV127.4 �421 ± 8 MV138.3 �342 ± 8 MV127.4 �500± 8 TQ121.4 �475 ± 8 TQ121.4 nd

ompounds: BV, benzylviologen, MV, methylviologen, TQ, triquat.

1 2 10 15 16 17 24 25 26

HC

R

0.1

1

10

100HT29 H1299HCT116 PC322RV1

Figure 3. Hypoxic selectivity as hypoxic cytotoxicity ratio [HCR = IC50(normoxic)/IC50(anoxic)]. Cell lines: HT29 and HCT116 colorectal carcinomas, H1299 non-smallcell lung carcinoma, 22Rv1 and PC3 prostate carcinomas. Error bars represent theSEM for 2–3 independent experiments.

Frac

tion

10-2

10-1

100

2126 M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132

5- and 4-isomers. The sulfonamide group provides a strong elec-tron-withdrawing influence to raise the electron affinity, withthe two 2-nitroimidazoles 10 and 17 having E(1) values raised by36 and 46 mV, respectively, compared to etanidazole. Similarly,the 4-nitroimidazole 15 and the 5-nitroimidazole 16 have raisedE(1) values compared to comparable nitroimidazoles (e.g., nimo-razole (4) has an E(1) value of �457 mV, while the analogous 4-iso-mer has an E(1) of �554 mV).31 The 5-nitroimidazole 24 has aslightly reduced E(1) compared to metronidazole (21)(�480 mV)31 reflecting the reduced electronic effect over the long-er ethyl spacer unit, but a positively charged solubilising side chain(e.g., 25) offsets this to some extent.

2.3. Biology

The cytotoxicity (as IC50: the compound concentration over a4 h aerobic or anoxic exposure at 37 �C required to inhibit subse-quent cell growth by 50%) was determined using a sulforhodamineB proliferation assay in a panel of human tumour cell lines (HT29and HCT116 colorectal carcinomas, H1299 non-small cell lung car-cinoma, 22Rv1 and PC3 prostate carcinomas) which reflect a rangeof one-electron reductase capacities.32 The anoxic IC50 values of thecompounds spans a range from ca. 70 lM to ca. 24 mM (Fig.2) withthe PC3 prostate cell line being the least sensitive under anoxia,presumably reflecting low one-electron reductase activity. Nota-bly, the 2-nitroimidazoles 10 and 17 show almost a 10-fold in-crease in anoxic cytotoxic potency compared to misonidazoleand etanidazole, reflecting their increased electron affinity.Similarly, the 5-nitroimidazoles 16 and 24–26 show increased an-oxic cytotoxic potency compared to nimorazole (4) (IC50(anoxic)

9.8 ± 1.6 mM in HCT116 cells) and metronidazole (21) (IC50(anoxic)

>16.7 mM in HCT116 cells), respectively.The compounds fall into two categories: those that have modest

(ca. 6–64 fold) hypoxia-selective cytotoxicity [HCR = IC50(normoxic)/IC50(anoxic)] and those that show little or no hypoxic selectivity(Fig. 3). The 2-nitroimidazole analogues 10 and 17 with higherreduction potentials (>�380 mV) display hypoxic selectivity ofca. 10 to 20-fold and this is also seen for etanidazole and misoni-dazole. In contrast, nitroimidazoles with lower E(1) values(<�420 mV) (4- and 5-nitroimidazoles 15, 16, 24–26) show essen-tially no hypoxic selectivity (0.9 to 4-fold).

Radiosensitization was determined by clonogenic assay inHCT116 cells under anoxic conditions by treating cells at a single,non-toxic compound concentration and a range of radiation dosesusing a lead wedge to cover the 96-well plate in an anoxic cham-

1 2 10 15 16 17 24 25 26

IC50

(mM

)

0.01

0.1

1

10

100HT29H1299HCT116PC322RV1

Figure 2. Anoxic IC50 values for compounds. Cell lines: HT29 and HCT116 colorectalcarcinomas, H1299 non-small cell lung carcinoma, 22Rv1 and PC3 prostatecarcinomas. Error bars represent the SEM for 2–3 independent experiments.

ber.33 Since radiosensitisation by nitroimidazoles (or by oxygen)is a physicochemical process it is largely independent of cell type.HCT116 was chosen as a representative line which provides effi-cient colony formation. Total compound treatment time was 3 h,after which cells were trypsinized and plated out for colony forma-tion. Colonies (>50 cells) were counted after 10 days, survivingfractions calculated and compared to control radiation survivalcurves produced in the same experiment without compound treat-ment. This approach is demonstrated for etanidazole (2) and com-pound 25 and indicates the derivation of the SensitiserEnhancement Ratio (SER1% = radiation dose for 1% survivalwithout/with compound) (Fig. 4). Compounds were tested at con-

Dose (Gy)0 5 10 15 20 25 30

Surv

ivin

g

10-5

10-4

10-3

SER

Oxic Anoxic

Figure 4. Radiosensitisation of compound 25 (0.7 mM) and etanidazole (2)(1.0 mM) under normoxic and anoxic conditions in HCT116 human colorectalcarcinoma cells. Symbols: RAD + anoxia alone, d; Compound 2 + RAD + anoxia, j;Compound 25 + RAD + anoxia, N; RAD + normoxia, s; Compound 2 + RAD + nor-moxia, h; Compound 25 + RAD + normoxia, 4. Error bars represent the SEM forduplicate cultures within each experiment. Lines are fits to the linear-quadraticmodel. Compound alone resulted in no measurable cytotoxicity in either normoxic[SF (2) 1.10 ± 0.04; SF (25) 1.06 ± 0.04] or anoxic [SF (2) 1.08 ± 0.08; SF (25)0.97 ± 0.1] radiosensitisation experiments.

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132 2127

centrations corresponding to the anoxic IC50(anoxic) determinedabove; the lower temperature in the radiosensitisation assays (18vs. 37 �C) and shorter compound exposure time (3 vs. 4 h) ensuredthat the compounds were non-toxic under these conditions, whichwas confirmed in each experiment using compound-treatedunirradiated controls (See Table S1). Oxygen is an excellent radio-sensitiser with irradiation under normoxic conditions providingdose-modifying sensitisation with an oxygen enhancement ratio(OER) of 2.4 and no additional sensitisation was observed whencells were irradiated in the presence of 1 or 25 under normoxia(Fig. 4). SERs were generally dose-modifying, showing no obvioustrend with radiation dose at 10%, 1% and 0.1% survival, and hencewere compared at 1% survival (SER1%) (Table S1). The SER1% formisonidazole, also tested at a concentration corresponding to itsIC50(anoxic) (0.53 mM), was evaluated as a positive control in eachexperiment and showed consistent results with a mean SER1% of1.37 and SD of 0.04 (Fig. 5 and Table S1). A range of radiosensitisa-tion values were observed for the compound set with the 2-nitro-imidazoles 10 and 17 showing significant radiosensitisation atapproximately one fifth of the concentration (77 and 108 lM,respectively) used for misonidazole. Several 5-nitroimidazoles(25, SER = 1.54 and 26, SER = 1.71) demonstrated comparableactivity to misonidazole and etanidazole in this assay.

3. Discussion

The search for effective radiosensitisers has had a long historyin which nitroimidazoles and related heterocycles featured prom-inently.14,34 Despite setbacks due to peripheral neuropathy withmisonidazole,15 and to a lesser extent etanidazole,35 the use ofnimorazole in combination with radiotherapy in the treatment ofhead and neck cancer demonstrates the successful clinical applica-tion of a radiosensitiser.22 The increasing implementation of SBRTto replace conventional fractionated radiotherapy2,36 presents anew opportunity to use radiosensitisers with hypoxia predictedto play a larger role in treatment resistance,3,4 thus favouring theuse of a radiosensitiser to kill treatment-resistant hypoxic tumourcells. This opportunity is further expanded by the use of gene sig-natures to identify patients with hypoxic tumours.24,37 However,limited or expired patent protection, coupled with an extensivemining of the chemical space around suitable nitroheterocycles

2 10 15 16 17 24 25 26

SER

0.8

1.0

1.2

1.4

1.6

1.8

2.0Compound SERMISO (1) SER

Figure 5. Sensitiser Enhancement Ratios calculated as (radiation dose for 1%survival—compound)/(radiation dose for 1% survival + compound) for compoundsunder anoxia with misonidazole (1) at 0.53 mM as the intra-experiment control.Error bars for all compounds represent the SEM for 2–3 independent experiments.See Table S1 and Figure S2 for SF of compound alone for each experiment.

presents a significant challenge for the discovery of new radiosen-sitisers designed and optimised for SBRT. In developing syntheticmethodology to prepare nitroimidazoles bearing an alkylsulfona-mide side chain, we have identified a chemical niche with potentialto lead to novel nitroimidazoles that have been optimised for SBRT.

The preparation of nitroimidazole alkylsulfonamides as radio-sensitisers has not been reported previously and our initial syn-thetic efforts have given an insight into the absence of this classfrom the radiosensitiser literature. Our attempts to prepare nitro-imidazole sulfonyl chlorides (e.g., 9) via chloromethyl thioacetatewere unsuccessful. Although we could prepare 2-nitroimidazolesulfonate 12 and corresponding acid 13 we were unable to identifycoupling conditions to provide the corresponding sulfonamides. Astepwise elaboration of 2-nitroimidazole via chloride 6, thioester 7and subsequent chlorosulfonylation did produce the unstable sul-fonyl chloride 9 which could be trapped with various amines. How-ever, this route was capricious and a more reliable route wasidentified using chloromethanesulfonyl chloride as the startingpoint. Sulfonamide formation followed by alkylation of the nitro-imidazole, although providing only modest yields, was direct andproved to be generally applicable.

The sulfonamide group lowers aqueous solubility relative toanalogous carboxamide 2 or hydroxyl 1 analogues and this canbe offset to some degree by the addition of basic amine groups.The sulfonamide unit also has a strong influence on the electronaffinity of the nitroimidazole ring and this raises the E(1) by upto 42 mV, leading to an increase in hypoxic cytotoxicity and hyp-oxic selectivity, for example, 10 and 17. This raises the prospectof a ‘mixed mechanism’38,39 of radiosensitisation and hypoxia-selective cytotoxicity which is also seen with misonidazole andetanidazole. Whether this also leads to significant normal tissuetoxicity remains to be determined. The addition of an extra meth-ylene spacer reduced the influence of the sulfonamide group onelectron affinity, but this is offset to some extent by the presenceof a positive charge on the side chain (e.g., 25 and 26). The 5-nitro-imidazole analogues 24–26 were less potent cytotoxins under hy-poxia and were not hypoxia-selective. These analogues, althoughless potent cytotoxins, are likely to act solely as radiosensitisersakin to nimorazole (4).

In developing a method to survey a series of analogues for theirability to radiosensitise hypoxic tumour cells we exposed cells in96-well plates in a portable anoxic box using a lead wedge as pre-viously described33 to attenuate the radiation dose. Exposure wasfollowed by harvesting cells for clonogenic assay. This system al-lowed rapid and reproducible generation of survival curves. Forcomparison of compounds we elected to use the compounds atan ‘equitoxic’ compound concentration. At concentrations corre-sponding to the anoxic IC50 value compound alone did not causesignificant cell killing under the conditions of the radiosensitisa-tion assay. All of the compounds provided radiosensitisation withthe 2-nitroimidazoles 10 and 17 providing modest sensitisationand the 5-nitroimidazoles 24–26 providing sensitisation similaror superior to misonidazole. The increased radiosensitisation seenfor the nitroimidazoles bearing basic side chains may be due to in-creased cell uptake, as seen for pimonidazole.40

We have identified a new nitroimidazole-based class of hypox-ia-selective radiosensitiser with a sulfonamide group providingnovelty. We have prepared analogues spanning the range of elec-tron affinity for effective radiosensitisation [E(1) �503 to�342 mV] and have demonstrated dose-modifying hypoxia-spe-cific radiosensitisation in vitro. Our future studies aim to optimisethe pharmacological properties of this class and to explore theirability to radiosensitise hypoxic tumour cells in vivo. This newclass could be developed in conjunction with SBRT where a combi-nation therapy has the potential to reduce side effects through

2128 M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132

improved tumour targeting, reduce health costs and improve pa-tient experience through fewer hospital visits.

4. Experimental Section

4.1. Chemistry

4.1.1. General proceduresAnalyses were carried out in the Campbell Microanalytical Lab-

oratory, University of Otago, Dunedin, NZ. Melting points weredetermined on an Electrothermal 2300 Melting Point Apparatus.NMR spectra were obtained on a Bruker Avance 400 spectrometerat 400 MHz for 1H and 100 MHz for 13C spectra. Spectra were ob-tained in (CD3)2SO unless otherwise specified, and were referencedto Me4Si. Chemical shifts and coupling constants were recorded inunits of ppm and Hz, respectively. Assignments were determinedusing COSY, HSQC, and HMBC two-dimensional experiments. Lowresolution mass spectra were gathered by direct injection of meth-anolic solutions into a Surveyor MSQ mass spectrometer using anatmospheric pressure chemical ionization (APCI) mode with acorona voltage of 50 V and a source temperature of 400 �C. High-resolution spectra were obtained on a Bruker micrOTOF-QII massspectrometer using an ionizing potential of 70 eV at nominal reso-lutions of 3000, 5000, or 10000 as appropriate. All spectra were ob-tained using electrospray ionisation (ESI) with PFK as the referenceunless otherwise stated. Solutions in organic solvents were driedwith anhydrous Na2SO4. Solvents were evaporated under reducedpressure on a rotary evaporator. Thin-layer chromatography wascarried out on aluminium-backed silica gel plates (Merck 60 F254)with visualization of components by UV light (254 nm) or exposureto I2. Column chromatography was carried out on silica gel (Merck230–400 mesh). All final products were analysed by reverse-phaseHPLC (Altima C18 5 lm column, 150 � 3.2 mm; Alltech Associated,Inc., Deerfield, IL) using an Agilent HP1100 equipped with a UV/vis-absorbance diode-array detector. Mobile phases were gradients of80% CH3CN/20% H2O (v/v) in 45 mM ammonium formate at pH 3.5and 0.5 mL/min. Purity was determined by monitoring at330 ± 100 nm. CH3CN refers to acetonitrile; DCM refers to dichloro-methane; DMF refers to dry N,N-dimethylformamide; Et2O refersto diethyl ether; Et3N refers to triethylamine; EtOAc refers to ethylacetate; HOAc refers to acetic acid; MeOH refers to methanol; NCSrefers to N-chlorosuccinimide, pet. ether refers to petroleum ether,boiling range 40–60 �C.

4.1.2. N-(2-Methoxyethyl)(2-nitro-1H-imidazol-1-yl)methanesulfonamide (10)4.1.2.1. 1-(Chloromethyl)-2-nitro-1H-imidazole (6). Bromo-chloromethane (5.7 mL, 88 mmol) was added to a stirred suspen-sion of 2-nitroimidazole (500 mg, 4.4 mmol) and Cs2CO3 (2.87 g,8.8 mmol) in anhydrous DMF (30 mL), and the reaction was stirredat 18 �C for 16 h. The reaction mixture was partitioned betweenEtOAc (200 mL) and H2O (50 mL), the organic phase was washedwith H2O (3 � 50 mL), washed with brine (50 mL), dried and thesolvent was evaporated. The crude product was suspended inEtOAc (3 mL) and the white solid was filtered off. The filtrate wasconcentrated and dried to give chloride 6 (320 mg, 45%) as a tanwax, which was used without further purification: 1H NMR d7.88 (d, J = 1.2 Hz, 1H, H-5), 7.27 (d, J = 1.2 Hz, 1H, H-4), 6.27 (s,2H, CH2); 13C NMR d 143.7, 128.3, 127.4, 54.7.

4.1.2.2. S-[(2-Nitro-1H-imidazol-1-yl)methyl] ethanethioate(7).25 Potassium thioacetate (390 mg, 3.4 mmol) was added toa stirred solution of chloride 6 (550 mg, 3.4 mmol) in anhydrousDMF (15 mL) and the mixture was stirred at 18 �C for 16 h. Thereaction mixture was partitioned between EtOAc (200 mL) and

H2O (50 mL) and the organic phase was washed with H2O(3 � 50 mL), washed with brine (50 mL), dried and the solventwas evaporated. The residue was purified by column chromatogra-phy, eluting with 30% EtOAc/pet. ether, to give thioacetate 7(285 mg, 42%) as a colourless oil: 1H NMR d 7.67 (d, J = 1.2 Hz,1H, H-5), 7.18 (d, J = 1.2 Hz, 1H, H-4), 5.79 (s, 2H, CH2), 2.41 (s,3H, CH3); 13C NMR d 190.9, 144.1, 127.9, 127.7, 46.7, 30.2.

4.1.2.3. N-(2-Methoxyethyl)(2-nitro-1H-imidazol-1-yl)methane-sulfonamide (10). Potassium thioacetate (495 mg, 4.3 mmol)was added to a stirred solution of chloride 6 (700 mg, 4.3 mmol) inanhydrous DMF (20 mL) and the mixture was stirred at 18 �C for16 h. The reaction mixture was partitioned between EtOAc(200 mL) and H2O (50 mL), the organic phase was sequentiallywashed with H2O (3 � 50 mL) and then brine (50 mL), dried, andthe solvent was evaporated to give crude thioacetate 7 which wasused without further purification. A solution of crude thioacetate7 in CH3CN (2 mL) was added dropwise to NCS (2.30 g, 17.2 mmol)in a mixture of aq HCl (2 M, 1.2 mL) and CH3CN (5.9 mL) at 10 �Cand the reaction was stirred at 15 �C for 30 min. The mixture waspartitioned between Et2O (200 mL) and 12% aq NaCl solution(50 mL) and the pH was then adjusted to 7 with saturated aqNaHCO3 solution. The organic phase was separated and washedwith brine (50 mL), dried and the solvent evaporated to give crudesulfonyl chloride 9. 2-Methoxyethylamine (1.8 mL, 20 mmol) andEt3N (2.8 mL, 20 mmol) were added to a solution of sulfonyl chlo-ride 9 in anhydrous DCM (20 mL) at 0 �C and the mixture allowedto warm to 18 �C and stirred for 16 h. The mixture was diluted withDCM (200 mL), washed with H2O (3 � 50 mL), washed with brine(50 mL), dried and the solvent was evaporated. The residue waspurified by column chromatography, eluting with a gradient (50–100%) of EtOAc/pet. ether, to give the sulfonamide 10 (80 mg, 16%from chloride 6) as a white solid: mp (EtOAc/pet. ether) 128–130 �C; 1H NMR d 7.90 (br t, J = 4.8 Hz, 1H, NH), 7.65 (d, J = 1.2 Hz,1H, H-5), 7.27 (d, J = 1.2 Hz, 1H, H-4), 5.86 (s, 2H, CH2), 3.35 (t,J = 5.7 Hz, 2H, CH2O), 3.26 (s, 3H, OCH3), 3.09–3.13 (m, 2H, CH2N);13C NMR d 144.2, 128.2, 127.7, 70.8, 62.4, 57.8, 41.9; MS m/z265.4 (MH+, 100%). HRMS calcd for C7H13N4O5S (MH+) m/z265.0601, found m/z 265.0595 (2.3 ppm). HPLC purity 99.7%.

4.1.3. Sodium (2-nitro-1H-imidazol-1-yl)methanesulfonate (11)A suspension of chloride 6 (566 mg, 3.5 mmol) and Na2SO3

(574 mg, 4.6 mmol) in a 3:1 mixture of acetone/H2O was heatedat 60 �C for 2 h. The solvent evaporated to give the crude methane-sulfonate salt 11 (1.2 g) as a cream solid which was used in thenext step without further purification: 1H NMR d 7.55 (d,J = 1.1 Hz, 1H, H-50), 7.09 (d, J = 1.1 Hz, 1H, H-40), 5.20 (s, 2H,H-1); 13C NMR d 144.7, 127.9, 126.4, 62.1.

4.1.4. (2-Nitro-1H-imidazol-1-yl)methanesulfonic acid (12)A suspension of chloride 6 (566 mg, 3.5 mmol) and Na2SO3

(574 mg, 4.6 mmol) in a 3:1 mixture of acetone/water was heatedat 60 �C for 2 h. The solvent was evaporated and the residue wasdissolved in H2O (30 mL), DOWEX50 WX8 ion exchange resin(3 g) was added and the mixture was stirred at 18 �C for 15 min.The resin was filtered and thoroughly washed with water, thenwashed with MeOH. The organic fraction was evaporated to givesulfonic acid 12 (713 mg, 87%): 1H NMR d 7.55 (d, J = 1.0 Hz, 1H,H-50), 7.09 (d, J = 1.0 Hz, 1H, H-40), 5.20 (s, 2H, H-1); MS m/z208.3 (MH+, 100%).

4.1.5. Alternative preparation of N-(2-methoxyethyl)(2-nitro-1H-imidazol-1-yl)methanesulfonamide (10)4.1.5.1. Chloro-N-(2-methoxyethyl)methanesulfonamide(13). A solution of chloromethanesulfonyl chloride (454 lL,5.0 mmol) in DCM (10 mL) at 0 �C was added dropwise to a

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132 2129

solution of 2-methoxyethylamine (435 lL, 5.0 mmol) and Et3N(0.70 mL, 5.0 mmol) in DCM (10 mL) at 0 �C and the reaction mix-ture allowed to warm to 18 �C and stirred for 3 h. The mixture wasdiluted with DCM (100 mL), washed sequentially with dilute aque-ous HCl (2 � 50 mL), H2O (50 mL) and brine (50 mL). The organicphase was dried and the solvent evaporated to give crude chloro-sulfonamide 13 (552 mg, 59%) as a white solid which was usedwithout further purification: 1H NMR d 7.79 (br t, J = 5.7 Hz, 1H,NH), 4.89 (s, 2H, CH2Cl), 3.37 (t, J = 5.7 Hz, 2H, CH2O), 3.26 (s, 3H,CH3), 3.13–3.18 (m, 2H, CH2N); 13C NMR d 71.5, 59.1, 55.7, 43.9.

4.1.5.2. N-(2-Methoxyethyl)(2-nitro-1H-imidazol-1-yl)methane-sulfonamide (10). A mixture of 2-nitroimidazole (340 mg,3.0 mmol), chlorosulfonamide 13 (374 mg, 2.0 mmol) and Cs2CO3

(980 mg, 3.0 mmol) in anhydrous DMF (15 mL) was stirred at70 �C for 5 h. The mixture was cooled and partitioned betweenEtOAc (200 mL) and H2O (50 mL). The organic phase was washedwith H2O (2 � 50 mL), washed with brine (50 mL), dried and thesolvent was evaporated. The residue was purified by column chro-matography, eluting with a gradient (50–100%) of EtOAc/pet. ether,to give sulfonamide 10 (93 mg, 18%) as a white solid: mp (EtOAc/pet. ether) 127–129 �C; spectroscopically identical to the sampleprepared above.

4.1.6. N-(2-Methoxyethyl)(4-nitro-1H-imidazol-1-yl)methanesulfonamide (15)

A mixture of 4-nitroimidazole (14) (825 mg, 7.3 mmol), chlo-ride 13 (550 mg, 4.9 mmol) and Cs2CO3 (2.37 g, 7.3 mmol) in anhy-drous DMF (30 mL) was stirred at 70 �C for 5 h. The mixture wascooled to 18 �C, partitioned between EtOAc (200 mL) and H2O(50 mL). The organic phase was washed with H2O (3 � 50 mL),washed with brine, dried and the solvent was evaporated. Theresidue was purified by column chromatography, eluting with agradient (50–70%) of EtOAc/pet. ether, to give a 7:3 mixture of4- and 5-nitroimidazole isomers 15 and 16 (317 mg, 25%). Theregioisomers were separated by HPLC [gradient (70–10–70%)ammonium formate pH 3.45/90% CH3CN/H2O] to give 4-nitroimi-dazole 15 (96 mg, 7%) as a white crystalline solid: mp (CH3CN/H2O) 132–134 �C; 1H NMR d 8.34 (d, J = 1.4 Hz, 1H, H-2), 7.89 (d,J = 1.4 Hz, 1H, H-5), 7.84 (br s, 1H, NH), 5.57 (s, 2H, CH2SO2), 3.38(t, J = 5.5 Hz, 2H, CH2O), 3.28 (s, 3H, CH3), 3.16 (t, J = 5.5 Hz, 2H,CH2N); 13C NMR d 146.9, 138.1, 122.1, 70.7, 61.5, 57.8, 42.0; MSm/z 265.4 (MH+, 100%). HRMS calcd for C7H13N4O5S (MH+) m/z265.0601, found m/z 265.0597 (1.6 ppm). HPLC purity 99.9%.

4.1.7. N-(2-Methoxyethyl)(5-nitro-1H-imidazol-1-yl)methanesulfonamide (16)

Also isolated was 5-nitroimidazole 16 (40 mg, 3%) as a white so-lid: mp (CH3CN/H2O) 87–88 �C; 1H NMR d 8.18 (d, J = 1.0 Hz, 1H, H-2), 8.12 (d, J = 1.0 Hz, 1H, H-4), 7.90 (br s, 1H, NH), 5.80 (s, 2H, CH2-

SO2), 3.36 (t, J = 5.7 Hz, 2H, CH2O), 3.27 (s, 3H, CH3), 3.11 (t,J = 5.7 Hz, 2H, CH2N); 13C NMR d 143.8, 138.1, 133.4, 70.9, 60.5,57.9, 42.0; MS m/z 265.4 (MH+, 100%). HRMS calcd for C7H13N4O5S(MH+) m/z 265.0601, found m/z 265.0592 (3.5 ppm). HPLC purity99.9%.

4.1.8. N-(2-Hydroxyethyl)(2-nitro-1H-imidazol-1-yl)methanesulfonamide (17)

Potassium thioacetate (495 mg, 4.3 mmol) was added to a stir-red solution of chloride 6 (700 mg, 4.3 mmol) in anhydrous DMF(20 mL) and the reaction mixture was stirred at 18 �C for 16 h.The reaction mixture was partitioned between EtOAc (200 mL)and H2O (50 mL), the organic phase was washed with H2O(3 � 50 mL), washed with brine (50 mL), dried and the solventwas evaporated to give crude thioacetate 7 which was used with-out further purification. A solution of crude thioacetate 7 in CH3CN

(2 mL) was added dropwise to NCS (2.30 g, 17.2 mmol) in a mix-ture of aq HCl (2 M, 1.2 mL) and CH3CN (6 mL) at 10 �C and thereaction was stirred at 15 �C for 30 min. The reaction mixturewas partitioned between Et2O (200 mL) and 12% NaCl solution(50 mL) and the pH was adjusted to 7 with saturated aq NaHCO3

solution. The organic phase was separated and washed with brine(50 mL), dried and the solvent was evaporated to give sulfonylchloride 9. Ethanolamine (1.2 mL, 20 mmol) and Et3N (2.8 mL,20 mmol) were added to a stirred solution of sulfonyl chloride 9in anhydrous DCM (20 mL) at 0 �C and the reaction mixture wasallowed to warm to 18 �C and stirred for 16 h. The mixture wasdiluted with DCM (200 mL), washed with H2O (3 � 50 mL), washedwith brine (50 mL), dried and the solvent was evaporated. Theresidue was purified by column chromatography, eluting with agradient (0–10%) of MeOH/EtOAc, to give a solid which was tritu-rated with EtOAc, to give sulfonamide 17 (47 mg, 10% from chlo-ride 6) as a white solid: mp (EtOAc/MeOH) 131–133 �C; 1H NMRd 8.00 (br s, 1H, NH), 7.70 (d, J = 1.1 Hz, 1H, H-5), 7.27 (d,J = 1.1 Hz, 1H, H-4), 5.87 (s, 2H, CH2SO2), 4.88 (t, J = 5.4 Hz, 1H,OH), 3.42 (q, J = 5.8 Hz, 2H, CH2O), 3.01 (br t, J = 5.9 Hz, 2H,CH2N); 13C NMR d 144.2, 128.1, 127.7, 62.4, 60.3, 44.8; MS m/z251.3 (MH+, 100%). HRMS calcd for C6H11N4O5S (MH+) m/z251.0445, found m/z 251.0444 (0.2 ppm). HPLC purity 100.0%.

4.1.9. Alternate preparation of N-(2-hydroxyethyl)(2-nitro-1H-imidazol-1-yl)methanesulfonamide (17)4.1.9.1. 2-(tert-Butyldimethylsilyloxy)ethanamine (18). Asolution of TBDMSCl (3.15 g, 21 mmol) in anhydrous DCM(10 mL) was added dropwise over 3 min to a stirred solution ofethanolamine (1.2 mL, 20 mmol) and imidazole (2.72 g, 40 mmol)in DCM (20 mL) at 18 �C and the mixture was stirred for 1 h. Themixture was washed with H2O (50 mL). The aqueous phase was ex-tracted with DCM (3 � 50 mL), the combined organic phase wasdried and the solvent was evaporated to give amine 1841 (2.88 g,82%) as a pale yellow oil which was used in the next step withoutfurther purification: 1H NMR d 3.64 (t, J = 5.3 Hz, 2H, CH2OSi), 2.79(t, J = 5.3 Hz, 2H, CH2N), 0.91 [s, 9H, SiC(CH3)3], 0.07 [s, 6H,Si(CH3)2].

4.1.9.2. N-[2-(tert-Butyldimethylsilyloxy)ethyl](2-nitro-1H-imi-dazol-1-yl)methanesulfonamide (20). A solution of thioace-tate 7 (1.0 g, 5.0 mmol) in CH3CN (2 mL) was added dropwise to amixture of NCS (2.65 g, 19.9 mmol) in CH3CN and 2 M aq. HCl (4:1,16.2 mL) at 10 �C and the reaction mixture was stirred at 15 �C for30 min. The mixture was partitioned between EtOAc (200 mL) andaq NaHCO3 solution (50 mL). The organic phase was separated,washed with brine (50 mL), dried and the solvent was evaporated.The crude sulfonyl chloride was dissolved in anhydrous DCM(20 mL) and the solution was cooled to 0 �C, and then amine 18(5.0 g, 23 mmol) and Et3N (3.2 mL, 23 mmol) were added. The reac-tion mixture was allowed to warm to 18 �C and stirred for 16 h.The mixture was diluted with DCM (200 mL), washed with H2O(3 � 50 mL), washed with brine (50 mL), dried and the solventwas evaporated. The residue was purified by column chromatogra-phy, eluting with a gradient (50–100%) of EtOAc/pet. ether, to givesulfonamide 20 (160 mg, 11%) as an oil: 1H NMR d 7.85 (s, 1H, NH),7.67 (d, J = 1.2 Hz, 1H, H-5), 7.27 (d, J = 1.1 Hz, 1H, H-4), 5.87 (s, 2H,CH2SO2), 3.57 (t, J = 6.3 Hz, 2H, CH2OSi), 3.02 (t, J = 6.3 Hz, 2H,CH2N), 0.87 [s, 9H, SiC(CH3)3], 0.05 [s, 6H, Si(CH3)2].

4.1.9.3. N-(2-Hydroxyethyl)(2-nitro-1H-imidazol-1-yl)methane-sulfonamide (17). A solution of sulfonamide 20 (140 mg,0.4 mmol) in a mixture of HOAc/THF/H2O (3:1:1, 25 mL) was stir-red at 18 �C for 16 h. The solvent was evaporated and the residuewas purified by column chromatography, eluting with a gradient(0–5%) of MeOH/EtOAc to give sulfonamide 17 (73 mg, 73%) as a

2130 M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132

tan powder: mp (EtOAc/MeOH) 129–132 �C; spectroscopicallyidentical to the sample prepared above.

4.1.10. Alternate preparation of N-[2-(tert-butyldimethylsilyloxy)ethyl](2-nitro-1H-imidazol-1-yl)methanesulfonamide (20)4.1.10.1. N-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)(chloro)methanesulfonamide (19). A solution of amine 18 (2.61 g,14.9 mmol) and Et3N (2.1 mL, 14.9 mmol) in anhydrous DCM(30 mL) was cooled to 0 �C in an ice-water bath. A solution of chlo-romethanesulfonyl chloride (1.35 mL, 14.9 mmol) in DCM (20 mL)was then added dropwise at 0 �C; the mixture was allowed towarm to 18 �C and stirred for 2.5 h. The mixture was then dilutedwith DCM (200 mL), washed with H2O (3 � 50 mL), washed withbrine (50 mL), dried and the solvent was evaporated to give crudesulfonamide 19 (3.81 g, 88%) as a yellow oil which was used with-out further purification: 1H NMR d 4.98 (br s, 1H, NH), 4.53 (s, 2H,CH2SO2), 3.74–3.77 (m, 2H, CH2O), 3.28–3.33 (m, 2H, CH2N), 0.91[s, 9H, C(CH3)3], 0.08 [s, 6H, Si(CH3)2]; 13C NMR d 62.2, 55.4, 46.3,26.0 (3), 18.0, �5.3 (2).

4.1.10.2. N-(2-{[tert-Butyl(dimethyl)silyl]oxy}ethyl)(2-nitro-1H-imidazol-1-yl)methanesulfonamide (20). A mixture of 2-nitroimidazole (5) (1.49 g, 13.2 mmol), chloride 19 (3.81 g,13.2 mmol) and Cs2CO3 (4.3 g, 13.2 mmol) in anhydrous DMF(120 mL) was heated at 85 �C for 2.5 h. The reaction mixture wascooled down to 18 �C and was partitioned between EtOAc(300 mL) and H2O (50 mL). The organic fraction was washed withH2O (2 � 50 mL), washed with brine (50 mL), dried and the solventwas evaporated. The residue was purified by column chromatogra-phy, eluting with 40% EtOAc/pet. ether, to give sulfonamide 20(1.7 g, 35%) as a white solid: mp (EtOAc/pet. ether) 107–110 �C;1H NMR d 7.85 (br s, 1H, NH), 7.67 (d, J = 1.2 Hz, 1H, H-5), 7.26(d, J = 1.2 Hz, 1H, H-4), 5.86 (s, 2H, CH2SO2), 3.57 (t, J = 6.3 Hz,2H, H-20), 3.01 (t, J = 6.3 Hz, 2H, H-10), 0.87 [s, 9H, C(CH3)3], 0.05[s, 6H, Si(CH3)2]; 13C NMR d 144.3, 128.3, 127.8, 62.3, 62.2, 44.6,25.8 (3), 17.9, �5.4 (2); MS m/z 365.3 (MH+, 100%).

4.1.11. N-(2-Methoxyethyl)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanesulfonamide (24)4.1.11.1. S-[2-(2-Methyl-5-nitro-1H-imidazol-1-yl)ethyl] ethan-ethioate (22). Methanesulfonyl chloride (0.25 mL, 3.2 mmol)was added dropwise to a stirred solution of metronidazole (21)(500 mg, 2.9 mmol), Et3N (0.81 mL, 5.8 mmol) and DMAP (35 mg,0.3 mmol) in anhydrous DCM (20 mL) at 0 �C. The reaction mixturewas allowed to warm to 18 �C and stirred for 16 h. The reactionmixture was diluted with DCM (100 mL), washed with H2O(3 � 50 mL) and washed with brine (50 mL), the organic phasewas dried and the solvent was evaporated to give the crude mesy-late which was used without further purification: 1H NMR d 8.06 (s,1H, H-4), 4.65 (t, J = 4.9 Hz, 2H, CH2N), 4.55 (t, J = 4.9 Hz, 2H, CH2O),3.15 (s, 3H, CH3S), 2.45 (s, 3H, CH3); MS m/z 250.3 (MH+, 100%).Potassium thioacetate (331 mg, 2.9 mmol) was added to a solutionof mesylate in anhydrous DMF (20 mL) at 18 �C and stirred for 16 h.The mixture was then heated at 60 �C for 6 h. The mixture wascooled and partitioned between EtOAc (200 mL) and H2O(50 mL). The organic phase was washed with H2O (3 � 50 mL),washed with brine (50 mL), dried and the solvent was evaporatedto give thioacetate 22 (460 mg, 70%) as an off-white powder whichwas used in the next step without further purification: 1H NMR d8.01 (s, 1H, H-4), 4.45 (t, J = 6.6 Hz, 2H, NCH2), 3.27 (t, J = 6.6 Hz,2H, CH2SO), 2.46 (s, 3H, CH3), 2.29 (s, 3H, CH3CO).

4.1.11.2. N-(2-Methoxyethyl)-2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethanesulfonamide (24). A solution of thioacetate 22(460 mg, 2.0 mmol) in CH3CN (2 mL) was added dropwise to NCS

(1.07 g, 8.0 mmol) in a mixture of CH3CN and 2 M aq HCl (4:1,6.6 mL) at 10 �C and the mixture was stirred at 15 �C for 30 minand at 18 �C for 10 min. The mixture was partitioned betweenEtOAc (200 mL) and aq NaHCO3 solution (50 mL), the organic phasewas washed with brine (50 mL), dried and the solvent was evapo-rated to give crude sulfonyl chloride 23: 1H NMR d 8.64 (s, 1H, H-4),4.62 (t, J = 6.4 Hz, 2H, CH2N), 2.91 (t, J = 6.4 Hz, 2H, CH2S), 2.73 (s,3H, CH3). 2-Methoxyethylamine (0.87 mL, 10 mmol) and Et3N(1.4 mL, 10 mmol) were added to a solution of sulfonyl chloride23 in anhydrous DCM (40 mL) at 0 �C and the mixture was allowedto warm to 18 �C and stirred for 16 h. The mixture was diluted withDCM (200 mL), washed with H2O (3 � 50 mL) and brine (50 mL),dried and the solvent was evaporated. The residue was purifiedby column chromatography, eluting with a gradient 80–100% ofEtOAc/pet. ether then 0–5% of MeOH/EtOAc, to give sulfonamide24 (290 mg, 50%) as an off-white powder: mp (MeOH/EtOAc)102–105 �C; 1H NMR d 8.04 (s, 1H, H-4), 7.46 (t, J = 5.9 Hz, 1H,NH), 4.59 (t, J = 7.2 Hz, 2H, CH2N), 3.56 (dd, J = 6.7, 7.7 Hz, 2H,CH2SO2), 3.38 (t, J = 5.6 Hz, 2H, CH2O), 3.13 (q, J = 5.7 Hz, 2H,CH2NH), 3.25 (s, 3H, OCH3), 2.49 (s, 3H, CH3); 13C NMR d 151.4,138.3, 133.0, 71.2, 57.9, 50.0, 41.9, 40.6, 13.8; MS m/z 293.4(MH+, 100%). HRMS calcd for C9H17N4O5S (MH+) m/z 293.0914,found m/z 293.0914 (0.0 ppm). HPLC purity 100.0%.

4.1.12. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-N-[2-(1-pyrrolidinyl)ethyl] ethanesulfonamide (25)

A solution of thioacetate 22 (2.23 g, 9.7 mmol) in CH3CN(30 mL) was added dropwise to a mixture of NCS (5.19 g,38.8 mmol) in CH3CN and 2 M aq HCl (4:1, 32.0 mL) at 10 �C andthe mixture was stirred at 15 �C for 30 min and at 18 �C for10 min. The mixture was partitioned between EtOAc (600 mL)and aq NaHCO3 solution (100 mL), the organic phase was washedwith brine (50 mL), dried and the solvent was evaporated to givethe sulfonyl chloride 23 (2.47 g, 100%) as a white solid whichwas used without further purification. 2-(Pyrrolidin-1-yl)ethan-amine (1.1 mL, 8.5 mmol) and Et3N (1.2 mL, 8.5 mmol) were addedto a solution of sulfonyl chloride 23 (430 mg, 1.7 mmol) in anhy-drous DCM (20 mL) at 0 �C and the mixture was allowed to warmto 18 �C and stirred for 16 h. The mixture was diluted with DCM(200 mL), washed with H2O (3 � 50 mL), washed with brine(50 mL), dried and the solvent was evaporated. The residue waspurified by column chromatography, eluting with a gradient(2–3%) of Et3N/(5–10%) of MeOH/EtOAc, to give sulfonamide 25(140 mg, 25%) as a yellow oil: 1H NMR (CDCl3) d 8.04 (s, 1H,H-40), 7.30 (br s, 1H, NH), 4.60 (t, J = 7.2 Hz, 2H, CH2CH2SO2), 3.59(t, J = 7.2 Hz, 2H, CH2CH2SO2), 3.07 (t, J = 6.8 Hz, 2H, CH2NH),2.48–2.52 (m, 2H, CH2CH2NH), 2.49 (s, 3H, CH3), 2.42–2.45 (m, 4H, H-200, H-500), 1.66 (q, J = 3.3 Hz, 4H, 2 � CH2); 13C NMR (CDCl3)d 151.4, 138.3, 133.0, 55.5, 53.4 (2), 49.9, 41.4, 40.6, 23.1 (2),13.8; MS m/z 332.4 (MH+, 100%). Anal. calcd for C12H21N5O4S: C,43.49; H, 6.39; N, 21.13. Found: C, 43.70; H, 6.37; N, 20.83. HPLCpurity 99.2%.

4.1.13. 2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-N-[2-(1-piperidinyl)ethyl] ethanesulfonamide (26)

A solution of thioacetate 22 (341 mg, 1.5 mmol) in CH3CN(3 mL) was added dropwise to a mixture of NCS (795 mg,6.0 mmol) in (4:1) aq HCl (2 N, 5 mL) and CH3CN (1.2 mL) at10 �C. The mixture was stirred at 10–15 �C for 30 min, and allowedto warm to 18 �C over 10 min. The mixture was partitioned be-tween EtOAc (150 mL) and aq NaHCO3 (50 mL). The organic phasewas washed with brine (40 mL), dried and the solvent was evapo-rated to give the crude sulfonyl chloride 23 which was used with-out further purification. 2-(Piperidin-1-yl)ethanamine (0.26 mL,1.8 mmol) and then Et3N (0.63 mL, 4.5 mmol) were successivelyadded to a solution of the crude sulfonyl chloride 23 in anhydrous

M. Bonnet et al. / Bioorg. Med. Chem. 22 (2014) 2123–2132 2131

DCM (20 mL) at 0 �C. The mixture was allowed to warm to 18 �Cand stirred for 16 h. The mixture was diluted with DCM(200 mL), washed with H2O (3 � 50 mL), washed with brine(50 mL), dried and the solvent was evaporated. The residue waspurified by column chromatography, eluting with 1.5% aqueousNH3/5% MeOH/EtOAc, to give sulfonamide 26 (349 mg, 68%) as acolourless oil: 1H NMR (CDCl3) d 7.98 (s, 1H, H-4000), 4.73 (t,J = 6.8 Hz, 2H, H-2), 3.49 (t, J = 6.8 Hz, 2H, H-1), 3.14–3.17 (m, 2H,H-10), 2.59 (s, 3H, CH3), 2.45–2.48 (m, 2H, H-20), 2.38 (br s, 4 H,H-200, H-600), 1.55 (quint, J = 5.5 Hz, 4H, H-300, H-500), 1.44 (br d,J = 5.2 Hz, 2H, H-400), NH not observed; MS m/z 346.3 (MH+,100%). Anal. calcd for C13H23N5O4S: C, 45.20; H, 6.71; N, 20.28.Found: C, 45.47; H, 6.89; N, 19.98. The hydrochloride salt was pre-pared by treating 26 with 1.25 M anhydrous HCl solution in MeOHand evaporation of the solvent to give a white hygroscopic foam:1H NMR (CDCl3) d 7.98 (s, 1H, H-4000), 4.77–4.81 (m, 2H, H-2),3.47–3.53 (m, 4H, H-1, H-10), 3.02–3.05 (m, 6H, H-20, H-200, H-600),2.58 (s, 3H, CH3), 1.97 (br s, 5H, NH, H-300, H-500), 1.63 (br s, 3H,H-400, NH); 13C NMR (CDCl3) d 151.3, 138.5, 133.9, 58.8, 54.8 (2),51.3, 41.3, 38.2, 23.5 (2), 22.6, 14.5; MS m/z 346.3 (MH+, 100%).Anal. calcd for C13H24ClN5O4S: C, 40.89; H, 6.33; N, 18.34. Found:C, 41.18; H, 6.54; N, 18.06. HPLC purity 98.2%.

4.2. Pulse Radiolysis

Pulse radiolysis experiments were performed on the Universityof Auckland Dynaray 4 (4 MeV) linear accelerator (200 ns pulselength with a custom-built optical radical detection system).42

The one-electron reduction potentials of the compounds, E(A/A�),versus NHE, were determined at pH 7.0 (5 mM phosphate buffer)by utilizing the e�aq and establishing redox equilibria betweenmixtures of the one-electron reduced compounds and the refer-ence compounds benzylviologen (E(BV2+/BV+) = �380 ± 10 mV),methylviologen (E(MV2+/MV+) = �447 ± 7 mV) or triquat (E(TQ2+/TQ+) = �548 ± 7 mV). The DE values were calculated from the equi-librium constants, Ke corrected for ionic strength effects, using theNernst equation as described in the literature.31 Data were ob-tained at three concentration ratios at room temperature(22 ± 2 �C).

4.3. IC50 assays

Cytotoxicity (IC50) of each compound was determined by Sulfo-rhodamine B proliferation assay. Compound exposures were per-formed in 96-well plates using either a 37 �C, humidifiedincubator (20% O2, 5% CO2) or the incubator compartment (37 �C)of a Bactron anaerobic chamber (Sheldon Manufacturing Inc.)where palladium catalyst scrubbed gas (90% N2, 5% H2, 5% CO2) en-sures strict anoxia (<0.001% O2). All plates and media were equili-brated in the anoxic chamber and cells taken in as pellets and re-suspended in anoxic medium. Cell cultures were grown in aMEM(Invitrogen) containing 5% heat inactivated FCS and maintainedin exponential growth phase. All studies were performed usingcells cultured for <3 month from frozen stocks. For each experi-ment an appropriate number of cells were seeded [HT-29 (1100),H1299 (400), PC3 (1200), 22Rv1 (4000), and HCT116 (700) onto96 well plates in aMEM + 10% FCS + 10 mM added glu-cose + 100 lM 20-deoxycytidine (20-dCyd), and allowed to attachfor 2 h. Replicates were then exposed to compounds, using 3-foldserial dilutions in triplicate, for a further 4 h. Subsequently, cellswere washed free of compound using complete media, grown aer-obically for 5 days, stained with sulforhodamine B to measure totalcells. The IC50, defined as the compound concentration reducingstaining to 50% of controls on the same plate, was determined bynon-linear regression using proprietary software supplied by theplate reader manufacturer (Biotek EL808, BioTek Instruments,

Inc.). Hypoxic cytotoxic ratios (HCRs) were calculated for eachexperiment as IC50(normoxic)/IC50(anoxic) and averaged to give an over-all HCR for each compound in each cell line.

4.4. Radiosensitisation assays

Cells were seeded onto 96-well plates (105 cells/well) and al-lowed to attach for 2 h. Replicates were treated with compoundat the respective anoxic IC50 compound concentration 30 min priorto irradiation. For anoxic irradiation, plates were transferred to acustom-built, air-tight, stainless steel, portable box(13 � 16.5 � 3 cm), within the anaerobic chamber, then sealedand transported to the radiation machine. The plates were irradi-ated (Eldorado G 60Co teletherapy unit) under anoxia at room tem-perature for 2.5 h with a metal wedge on the top of the metalchamber to achieve a graduated radiation dose across the platevarying from 7.1 to 27.1 Gy, calibrated by Fricke dosimetry withammonium thiocyanate as previously described.33 The controlplate (compound alone, no radiation) was left inside the anaerobicchamber at room temperature during the irradiation period. Aftertreatment the cells were trypsinized and suspended in aMEM + 5%FCS, and 10-fold serial dilutions were plated for clonogenic sur-vival. After 10 days plates were stained with methylene blue(2 g/L in EtOH/H2O, 1:1 v/v) and colonies with more than 50 cellswere 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 radiationdose (See Fig. S1) and Sensitiser Enhancement Ratios (SER) werecalculated as (radiation dose for 1% survival without compound)/(radiation dose for 1% survival with compound).

Acknowledgements

The authors would like to thank Dr. Thorsten Melcher (J&J Inno-vation) for helpful discussions and thank Dr Shannon Black, Ms.Karen Tan and Mr. Sisira Kumara for technical assistance. Theauthors acknowledge support from the University of Auckland’sBiopharma Thematic Research Initiative, the Australian Instituteof Nuclear Sciences and Engineering, and UniServices Investmentand Ministry of Business, Innovation and Employment Pre-SeedAccelerator Fund.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.039.

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