Macrocyclic Pyridyl Polyoxazoles: Structure-Activity Studies of the Aminoalkyl Side-Chain on G-Quadruplex Stabilization and Cytotoxic Activity

Pyridyl polyoxazoles are 24-membered macrocyclic lactams comprised of a pyridine, four oxazoles and a phenyl ring. A derivative having a 2-(dimethylamino)ethyl chain attached to the 5-position of the phenyl ring was recently identified as a selective G-quadruplex stabilizer with excellent cytotoxic activity, and good in vivo anticancer activity against a human breast cancer xenograft in mice. Here we detail the synthesis of eight new dimethylamino-substituted pyridyl polyoxazoles in which the point of attachment to the macrocycle, as well as the distance between the amine and the macrocycle are varied. Each compound was evaluated for selective G-quadruplex stabilization and cytotoxic activity. The more active analogs have the amine either directly attached to, or separated from the phenyl ring by two methylene groups. There is a correlation between those macrocycles that are effective ligands for the stabilization of G-quadruplex DNA (ΔTtran 15.5–24.6 °C) and cytotoxicity as observed in the human tumor cell lines, RPMI 8402 (IC50 0.06–0.50 μM) and KB3-1 (IC50 0.03–0.07 μM). These are highly selective G-quadruplex stabilizers, which should prove especially useful for evaluating both in vitro and in vivo mechanism(s) of biological activity associated with G-quaqdruplex ligands.

treated with the pyridyl polyoxazole macrocycle had a %T/C value (average tumor volumes of treated/control animals) of 27.7% which clearly demonstrated in vivo efficacy against this breast cancer xenograft. The initial structure-activity investigation as reported for the pyridy polyoxazole macrocycles suggests that a basic side-chain on the phenyl ring enhances cytotoxic activity and greatly improves the water-solubility of the macrocycle, allowing for easier formulation for in vivo evaluation [21]. In that report a 2-(N,N-dimethylamino)ethyl group was selected as the basic side-chain based on SAR obtained from the HX series of compounds [22]. The 5-position of the phenyl ring was chosen as the site of attachment based on the ease of synthesis. Herein we report the synthesis of analogs that have the side-chain attached to either the 4-or 5-position of the phenyl ring and that vary with respect to the number of spacer methylene groups connecting the tertiary amine to the ring. In addition, the preparation of analogs in which either one or two 2-(N,N-dimethylamino)ethyl groups are attached to the oxazole(s) that are more distant from the pyridine ring is also described. The effect of each of these structural changes on G-quadruplex selectivity and stabilization as well as on cytotoxic activity has been evaluated and is reported below.

Synthesis of Linkers Having a Basic Side-Chain at the 4-Position
The synthesis of diamine linkers having side-chains emanating from the 4-position of the phenyl ring is shown in Scheme 1. Those analogs having the N,N-dimethylamino group separated from the phenyl ring by two or three aliphatic carbons were prepared starting from dimethyl 4-bromoisophthalate [23]. The 2-aminoethyl analog was prepared by Suzuki reaction with potassium 2-[(tert-butoxycarbonylamino)ethyl]trifluoroborate [24] followed by lithium borohydride reduction to give diol 3a in 56% yield. For the 3-aminopropyl analog hydroboration of N-Boc allylamine with 9-borobicyclo[3.3.1]nonane (9-BBN) followed by Suzuki coupling [25,26] of the derived borane with dimethyl 4-bromoisophthalate gave, after LiBH 4 reduction, the three-carbon analog 3b in 71% yield. Both diols were converted to their diazides with diphenyl phosphorylazide (DPPA), followed by reduction of the azide groups with polymer-supported triphenylphosphine to afford the bis(aminomethyl) derivatives 4a and 4b in good overall yield. The synthesis of a 4-(N,Ndimethylamino)methyl analog however proved challenging and despite much effort was not successful. An analog having a N,N-dimethylamino group directly attached to the phenyl ring at the 4-position was prepared started from the known dimethyl 4-(N,N-dimethylamino)isophthalate 5 [27]. The ester groups were reduced and the diol was converted into 1,3-bis(aminomethyl) derivative 6 as described above. Macrocyclization of the 4-substituted linkers was achieved by condensation of pentacyclic diacid 7 [21] in the presence of EDC and HOBt. The bis(lactams) 8-10 were prepared in yields ranging from 17%-18%. The N-Boc protected 2-aminoethyl and 3-aminopropyl compounds were deprotected using HCl and then converted to the N,N-dimethylamines 11 and 12 by reductive amination using formaldehyde and sodium triacetoxyborohydride. Scheme 1. Synthesis of macrocycles having a basic side-chain at the 4-position.

Synthesis of Linkers Having a Basic Side-Chain at the 5-Position
Scheme 2 depicts the synthesis of macrocycles having a basic side-chain attached to the 5-position of the phenyl ring. For the synthesis of a linker molecule having a tertiary amine directly attached to the phenyl ring N,N-dimethyl 3,5-bis(bromomethyl)aniline [28] was treated with sodium azide to give a bis(azidomethyl) derivative 13 that was then reduced to diamine 14 using triphenylphosphine in aqueous THF. Synthesis of the 5-(N,N-dimethylaminomethyl) analog began by displacement of dimethyl 5-bromomethylisophthalate [29] by dimethylamine to afford 15 in 97% yield. The ester groups were then reduced with LiBH 4 , converted into the diazide derivative and reduced with triphenylphosphine to give the 1,3-bis(aminomethyl) derivative 16. Scheme 2. Synthesis of macrocycles having a basic side-chain at the 5-position. Analog 2 possessing a 5-[2-(N,N-dimethylamino)ethyl] side chain has been synthesized previously [21]. Preparation of the 3-aminopropyl analog began with synthesis of potassium [3-(tertbutoxycarbonyl)amino)propyl]trifluoroborate, which was prepared in quantitative yield from N-Boc allylamine using the procedure detailed by Molander for the ethyl derivatives [24]. This was coupled with dimethyl 5-[(trifluoromethanesulfonyl)oxy]isophthalate [30] in the presence of palladium acetate, RuPhos, and cesium carbonate to give the N-Boc 3-aminopropyl derivative 17 in 71% yield. The conversion of 17 into diamine 18 was achieved by first reducing the ester groups to alcohols. The subsequent reaction with DPPA was not clean and therefore the diol was converted instead into a dimesylate derivative. Displacement with sodium azide afforded the diazide that was then reduced to give 18. Macrocyclization of 14, 16, and 18 was performed by condensation with pentacyclic diacid 7 in the presence of EDC and HOBt to afford bis(lactams) 19, 20, and 21 in yields of 10%, 4%, and 43%. Compound 21 was treated with TFA to remove the Boc protecting group and the resulting amine was subjected to reductive amination as described above to give analog 22.

Synthesis of Macrocycles Having the Basic Side-Chain(s) Located on Oxazole(s)
The synthesis of PyPX analogs having either a single or two 2-(N,N-dimethylamino)ethyl side chains on the oxazoles closer to the phenyl linker required the preparation of a suitable 5-substituted oxazole building block. This is detailed in Scheme 3 shown below. Starting from N-Cbz-β-alanine, treatment with oxalyl chloride gave the acid chloride which was reacted with ethyl isocyanoacetate in the presence of DBU to give oxazole 23 [31]. At this point a change in amine protecting group was deemed prudent, due to the difficulty in removing Cbz groups from intact macrocycles that we have observed in the PyPX series of compounds. Hydrogenolysis of the Cbz group in the presence of di-tert-butyl dicarbonate afforded the corresponding Boc-protected amine in high yield. Deprotonation of the remaining oxazole proton with LiHMDS, transmetallation to the zinc derivative and treatment with iodine occurred in one pot to give 2-iodooxazole 24 in 95% yield. Stille coupling with tributyl(vinyl)tin yielded the 2-vinyloxazole 25, which was dihydroxylated using AD-mix-β [32]. The stereochemistry of the secondary alcohol is irrelevant since this stereocenter eventually becomes part of an oxazole ring, but the AD-mix procedure was more convenient and higher yielding than simple treatment with OsO 4 . The primary alcohol was selectively protected as TBS ether 26 and the remaining alcohol was converted into mesylate 27 in quantitative yield. Displacement of the mesylate with azide and reduction with polymer-supported triphenylphosphine completed the synthesis of alkylaminooxazole 28. Elaboration of aminoalkyloxazole 28 into pyridyl tetraoxazole dicarboxylic acids having either one or two 2-(N,N-dimethylamino)ethyl side chains is outlined in Scheme 4. For the analog having a single aminoethyl side-chain pyridine-2,6-dicarboxylic acid was condensed with 0.6 equivalents of aminooxazole 29 [18] to limit the amount of diamide formed. The remaining carboxylic acid group was then condensed under the same conditions with 1 equivalent of aminooxazole 28 to give unsymmetrical diamide 30. For the analog having two side-chains the pyridine dicarboxylic acid was condensed with 2 equivalents of oxazole 28 to give the symmetrical diamide 31. In both cases the silyl ethers were removed by treatment with pyridine-HF complex and the resulting alcohols were treated with DAST and then BrCCl 3 [33,34] to give the pyridyl tetraoxazoles 32 and 33 which were then hydrolyzed and macrocyclized with 1,3-bis(aminomethyl)benzene in the presence of MnSO 4 to give 34 and 35. We had found MnSO 4 to sometimes have a beneficial templating effect on such macrocyclizations, although in this case the yields were in the 20%-28% range. The macrocycles were treated with TFA to remove the Boc protecting groups and the resulting amines were subjected to reductive amination to give the corresponding N,N-dimethyl amines 36 and 37. Scheme 4. Synthesis of macrocycles having one or two aminoethyl side chains.

Evaluation of G-Quadruplex Stabilization and Selectivity
Each compound was evaluated for its ability to selectively bind and stabilize G-quadruplex DNA in the presence of K + ions (150 mM). Salmon testes (ST) DNA was employed as a model of duplex DNA and the human telomeric sequence d[T 2 G 3 (T 2 AG 3 ) 3 A], denoted as hTel, was used as a model of quadruplex DNA. This sequence has been shown by Patel and co-workers, to exist as an intramolecular (3 + 1) G-quadruplex in which three strands are oriented in one direction and the fourth is oriented in the opposite direction in K + solution [35]. The first-derivative forms of the UV melting profiles for ST DNA and hTel DNA were recorded in the absence and presence of the various macrocycles. The ligand-induced changes, if any, in the transition temperature (T tran ) corresponding to the maxima (for quadruplex DNA) or minima (for duplex DNA) of these first-derivative melting profiles are listed in Table 1 for each macrocycle. With the exception of 8 which has a slight (<1 °C) destabilizing effect, none of the other macrocycles alters the thermal stability of ST duplex DNA to any significant extent and any observed changes in T tran are within the experimental uncertainty. This observation is consistent with little or no duplex DNA binding by these macrocycles. Similar behavior has been observed for other macrocyclic pyridyl polyoxazoles [21].
The results observed with the hTel quadruplex DNA however, stand in stark contrast with the ST duplex DNA results. In the series of three 4-phenyl substituted analogs compound 8 and 11 strongly stabilize G-quadruplex DNA by 20.5 and 24.6 °C respectively. Compound 8 has the tertiary amine directly connected to the phenyl while in 11 the amine is separated from the phenyl ring by two methylene groups. In contrast, the 3-dimethylaminopropyl analog 12 stabilizes G-quadruplex DNA to a much lesser extent. When the side-chain is attached to the 5-position of the phenyl ring the results are quite dramatic with the arylamine 19, the previously-reported 2-(dimethylamino)ethyl analog 2 [21], and the propyl analog 22 all displaying strong stabilization of G-quadruplex DNA with T tran values of 15.5, 20.5 and 28.6 °C respectively. In striking contrast however is the dimethylaminomethyl analog 20 which has no significant stabilization (T tran = 0.1 °C) of G-quadruplex DNA. Quadruplex stabilization is also considerably less efficient when the side chain(s) are moved away from the phenyl ring and onto one or two of the oxazole rings. A 2-(dimethylamino)ethyl chain attached to one oxazole ring provides for weak G-quadruplex stabilization (T tran = 4.6 °C) while two such side-chains result in an even lower degree of stabilization (T tran = 1.6 °C). Most of these 4-and 5-phenyl substituted macrocyclic pyridyl polyoxazole analogs are stronger G-quadruplex stabilizers than HXDV (T tran = 11.5 °C) [21].
Notes: a T tran reflects the change in transition temperature (T tran ) of the target nucleic acid induced by the presence of the substrate. Values of T tran were determined from the maxima or minima of first-derivative UV melting profiles. The uncertainty in the T tran values is ± 0.5 °C. b Values from ref. [21]. na = not applicable.

Evaluation of Cytotoxic Activity
Each N,N-dimethylamino-substituted macrocycle was also evaluated for cytotoxic activity against a human lymphoblastoma RPMI 8402 and a human epidermoid carcinoma KB3-1 cell line ( Table 2). The results from these assays are informative about the relationship of structure to cytotoxic activity among the PyPX macrocyclic G-quadruplex stabilizers. Of greatest significance, especially from a synthetic viewpoint, is that for any given side-chain (8 vs. 19 and 11 vs. 2) attachment at the 5-position of the phenyl ring provides superior cytotoxic activity than attachment at the 4-position. An explanation for this observation is not clear at this time. The three-carbon analogs 12 and 22 have nearly equivalent activity at both positions, but these longer-chain analogs are generally lower in activity than the two-carbon analogs. Attachment of the dimethylamino group directly to the phenyl ring (8 and 19) leads to compounds having the greatest cytotoxic potency at their respective points of attachment. Unfortunately, these compounds are significantly less water-soluble than those analogs in which the amine is separated from the phenyl ring by one or more methylene groups. In general analogs that have an even number (0 or 2) of methylene spacer groups are more cytotoxic than those with an odd number. The one-carbon analog at the 5-position (20), displays especially poor cytotoxic potency. Moving the side-chain away from the phenyl ring and onto an oxazole ring also fails to improve cytotoxic activity. As we have noted previously, a compound having a single water-solubilizing 2-(dimethylamino)ethyl chain 36 is superior to an analog having two such substituents 37 [22,36]. While 36 has reasonable cytotoxic potency and good water-solubility, the synthesis of this analog was considerably more involved than compound 2.
The results from these assays suggest that for the macrocyclic pyridyl polyoxazoles those compounds that are inefficient at stabilizing G-quadruplex DNA are in general the least cytotoxic against KB3-1 and RPMI 8402 cells. These results are consistent with those recently reported for a series of 24-membered oxazole-containing macrocycles with a biphenyl scaffold [37]. With the exception of 22 those analogs that strongly stabilize G-quadruplex DNA exhibit cytotoxic activity against KB3-1 cells with IC 50 values ≤ 70 nM while their activity against RPMI 8402 is also good, but more variable. An earlier investigation of the effect of side-chain length on quadruplex stabilization and cytotoxic activity among hexaoxazole (HX) analogs showed a similar pattern of results. While the propyl (n = 3) analog was a slightly more efficient G-quadruplex stabilizer than the ethyl analog, the 2-(dimethylamino)ethyl analog was more cytotoxic. A lysinyl (n = 4) analog was neither stabilizing towards G-quadruplexes not cytotoxic (IC 50 > 3 μM) [22]. The aryl amine analogs 8 and 19 and the 2-(dimethylamino)ethyl analogs 11 and 2 are strongly cytotoxic with excellent G-quadruplex stabilization. The lower water-solubility of the aryl amines however, makes them less suitable than the 2-(dimethylamino)ethyl analogs for possible in vivo evaluation.

General
All reactions were conducted under an atmosphere of dry nitrogen in oven-dried glassware unless otherwise noted. THF was dried by distillation from sodium-benzophenone. Toluene, CH 2 Cl 2 , 2,6-lutidine, Et 3 N, pyridine, DBU, and CH 3 CN were freshly distilled from CaH 2 . Anhydrous DMF was obtained by stirring overnight over anhydrous CuSO 4 followed by distillation under reduced pressure. All starting materials and reagents were commercially available and were used as received with the exception of 5, 7, and 29 which were prepared as described previously [18,21,27]. Flash chromatography was conducted using 230-400 mesh silica gel obtained from Dynamic Adsorbents, Inc. Melting points were obtained on a Thomas-Hoover apparatus and are uncorrected. Proton (400 MHz) and carbon (125 MHz) NMR spectra were recorded on a Bruker Avance III spectrometer in CDCl 3 unless otherwise noted. Chemical shifts are reported as δ units relative to internal tetramethylsilane. IR spectra were recorded on a Thermo-Nicolet Avatar 360 FT instrument as thin films on NaCl unless otherwise noted. High resolution mass spectra were provided by the Washington University Mass Spectrometry Resource, St. Louis, MO.
The reaction mixture was then cooled to room temperature and saturated aqueous NH 4 Cl was added. The mixture was extracted with CH 2 Cl 2 and the organic layer was dried over MgSO 4 and then the solvent was evaporated under reduced pressure. The crude product was purified by flash chromatography eluting with 0%-20% ethyl acetate in hexane to give 229 mg of a colorless oil that proved to be an inseparable mixture of the desired product and 2,2',4,4'-tetra(carbomethoxy)biphenyl. This mixture was carried on to the next step.
Step B. The mixture from above was dissolved in anhydrous THF (10 mL) and cooled to 0 °C under N 2 and treated with LiBH 4 (80 mg, 6 mmol) followed by EtOH (1 mL). The reaction was allowed to warm to room temperature. After 24 h the reaction mixture was poured into water and extracted with EtOAc. The organic layer was washed with brine, dried over Na 2 SO 4 and concentrated. Purification was effected by flash chromatography eluting with 0%-5% MeOH/CH 2 Cl 2 to give 3a as a colorless oil; 157 mg, 56% (two steps); 1 H-NMR δ 7.14 (s, 1H), 7.02 (d, 1H, J = 8), 6
Step A. Prepared from 5 [27] using the procedure detailed above for 3a, Step B. Flash chromatography eluting with 0%-4% MeOH/CH 2 Cl 2 gave the diol as a yellow oil; 1.

Pyridyl tetraoxazole macrocycle with a 4-[2-(N,N-dimethylamino)ethyl] group on the phenyl ring (11).
Step A. N-Boc derivative 9 (7 mg, 0.0103 mmol) was suspended in 20% HCl (1 mL) and stirred at room temperature for 2 h. The solution was evaporated under reduced pressure to give the amine salt that was used directly for the next step.
Step A. Prepared from 10 using the procedure detailed above for 11, Step A. The product from this reaction was taken directly to the next step without purification.  (13). N,N-Dimethyl-3,5-bis(bromomethyl)aniline [28] (100 mg, 0.33 mmol) was dissolved in anhydrous DMF (10 mL) and sodium azide (128 mg, 1.97 mmol) was added. The reaction mixture was placed under argon and heated to 90 °C overnight. After cooling to room temperature, the reaction was poured into water and extracted with CH 2 Cl 2 . The combined organic extracts were dried with Na 2 SO 4 and evaporated under reduced pressure to give diazide 13 as a pale orange oil; 71 mg, 73%; 1 H-NMR δ 6.59 (s, 3H), 4.29 (s, 4H), 2.98 (s, 6H); 13 (14). Prepared using the procedure detailed above for 4a,
Step B. The salt was converted into 22 using the procedure detailed above for 11 Step B. White solid; 16 mg, 48%; 1 H-NMR  (23). Cbz-β-alanine (6.41 g, 28.7 mmol) was dissolved in anhydrous CH 2 Cl 2 (20 mL) and cooled to 0 °C in an ice bath. It was then treated with oxalyl chloride (5 mL) and stirred at 0 °C for 30 min. The reaction was next warmed to room temperature and stirred for 2.5 h. Removal of solvents in vacuo gave the acid chloride as a colorless oil. This was dissolved in anhydrous DMF (15 mL) and added to a solution of ethyl isocyanoacetate (2.4 mL, 22.1 mmol) and DBU (5 mL, 33.2 mmol) in anhydrous DMF (15 mL) under argon. The dark brown solution was heated to 80 °C for 4.5 h and was then poured into saturated NaHCO 3 . This was extracted with EtOAc and the combined organic layers were washed with 5% HCl, and brine. After concentration, the resulting brown oil was flash chromatographed on SiO 2 with 15%-40% EtOAc in hexane. Oxazole 23 was isolated as a pale orange oil; 3.01 g, 43%; 1 H-NMR   (24).

Ethyl 5-[2-[(tert-Butoxycarbonyl)amino]ethyl]-2-[[(tert-butyldimethylsilyl)oxy]-1-hydroxyethyl]oxazole-4carboxylate (26).
Step A. AD-mix-β (17 g) and methanesulfonamide (458 mg, 4.81 mmol) were dissolved in a mixture of t-BuOH (150 mL) and water (150 mL) and stirred at room temperature until clear. Then a solution of 25 (1.49 g, 4.81 mmol) in t-BuOH (25 mL) was added. The reaction stirred at room temperature for 16 h and then additional AD-mix-β (3 g) and methanesulfonamide (458 mg, 4.81 mmol) were added and the reaction stirred at room temperature for another 24 h. Then Na 2 SO 3 (22 g) was added and the reaction stirred for 30 minutes. It was next poured into a separatory funnel and the layers were separated. The aqueous layer was extracted with EtOAc and the combined aqueous layers were dried with Na 2 SO 4 . The solvent was removed in vacuo to give a pale yellow oil which was purified by flash chromatography eluting with 2%-4% MeOH in CH 2 Cl 2 . The diol was obtained as a colorless oil; 901 mg, 55%; 1 H-NMR Step B. The diol (900 mg, 2.62 mmol) and imidazole (356 mg, 5.23 mmol) were dissolved in anhydrous DMF (10 mL) and placed under argon. The reaction mixture was cooled to 0 °C and a solution of tert-butyldimethylsilyl chloride (434 mg, 2.88 mmol) in DMF (2 mL) was added dropwise. This was allowed to slowly warm to room temperature and stirred for 24 h. Additional tert-butyldimethylsilyl chloride (120 mg, 0.8 mmol) was added and the reaction stirred at room temperature for 6 h. This was then poured into 5% HCl and extracted with CH 2 Cl 2 . The organic extracts were dried with Na 2 SO 4 and concentrated in vacuo to give a colorless oil. This was flash chromatographed on SiO 2 with 20%-40% EtOAc in hexane and product 26 was isolated as a colorless oil; 942 mg, 79%; 1 H-NMR   (27). Prepared using the procedure detailed above for 18, Step B.   (30).

5-[2-[(tert-Butoxycarbonyl)amino]ethyl]-2'-[6-[4-carboxy-[2,4'-bioxazol]-2'-yl]pyridine-2-yl]-[2,4'bioxazole]-4-carboxylic acid
Step A. 30 (245 mg, 0.26 mmol) was dissolved in anhydrous THF (10 mL) and pyridine (1 mL) and HF-pyridine complex (0.3 mL) was added. The reaction was stirred at room temperature overnight and was then poured into saturated sodium bicarbonate solution. This was extracted with CH 2 Cl 2 and dried with Na 2 SO 4 . Removal of solvent under vacuum gave 174 mg of the diol as a colorless oil, 100%; 1  Step D. The diester (71 mg, 0.13 mmol) was suspended in a mixture of THF (30 mL) and water (3 mL) and lithium hydroxide (12 mg, 0.29 mmol) was added. The reaction was refluxed for 30 min and then stirred at room temperature overnight. THF was removed under vacuum and 5% HCl was added to the remaining solution. A white solid precipitated and was filtered and washed with water. The solid was dried by azeotroping with toluene 3 times to give 50 mg of diacid 32, as a white solid, 67%; mp 225-226 °C; HRMS (ESI) m/z calcd for C 26   Temperature-Dependent Spectrophotometry. Temperature-dependent absorption experiments were conducted on an AVIV Model 14DS Spectrophotometer (Aviv Biomedical, Lakewood, NJ, USA) equipped with a thermoelectrically controlled cell holder. Quartz cells with a path length of 1.0 cm were used for all the absorbance studies. Temperature-dependent absorption profiles were acquired at either 260 nm (for ST duplex DNA) or 295 nm (for hTel quadruplex DNA) with a 5 s averaging time. The temperature was raised in 0.5 °C increments, and the samples were allowed to equilibrate for 1 min at each temperature setting. In the quadruplex melting studies, the hTel concentration was 5 µM in strand (120 μM in nucleotide). When present in these quadruplex studies, the drug concentrations were 20 µM. In the duplex melting studies the ST DNA concentrations were 15 µM base pair (30 μM in nucleotide) and, when present, the drug concentrations were 15 µM. The buffer for all the UV melting experiments contained 10 mM potassium phosphate (pH 7.5) and sufficient KCl (132 mM) to bring the total K + concentration to 150 mM. Prior to their use in the UV melting experiments, all nucleic acid solutions were preheated at 90 °C for 5 min and slowly cooled to room temperature over a period of 4 hr.

Cytotoxicity Assays
Cytotoxicity was determined using the MTT-microtiter plate tetrazolinium assay (MTA). The human lymphoblast RPMI 8402 cell line was provided by Dr. Toshiwo Andoh (Aichi Cancer Center Research Institute, Nagoya, Japan) [38]. The KB3-1 cell line was obtained from K.V. Chin (The Cancer Institute of New Jersey, New Brunswick, NJ, USA) [39]. The cytotoxicity assay was performed using 96-well microtiter plates. Cells were grown in suspension at 37 °C in 5% CO 2 and maintained by regular passage in RPMI medium supplemented with 10% heat inactivated fetal bovine serum, L-glutamine (2 mM), penicillin (100 U/mL), and Streptomycin (0.1 mg/mL). For determination of IC 50 values, cells were exposed continuously for four days to varying concentrations of drug, and MTT assays were performed at the end of the fourth day. Each assay was performed with a control that did not contain any drug. All assays were performed at least twice in six replicate wells.

Conclusions
The results from this structure-activity investigation of macrocyclic pyridyl polyoxazoles indicate that analogs that have either a dimethylamino group directly attached to, or separated from the phenyl ring at the 4-or 5-positions by two methylene groups strongly and selectively stabilize G-quadruplex DNA. These same analogs are also highly cytotoxic against KB3-1 cells with IC 50 values ≤ 70 nM. A dimethylaminomethyl group at the 5-position of the phenyl is essentially devoid of G-quadruplex stabilizing and cytotoxic activity. Extending the side-chain by one methylene group to form a propyl chain at either the 4-or 5-position of the phenyl ring fails to improve cytotoxic activity over the corresponding ethyl analogs, although the 5-substituted analog does strongly stabilize G-quadruplex DNA. Attaching a 2-(dimethylamino)ethyl chain to an oxazole instead of the phenyl ring results in an analog with moderate cytotoxic activity but low G-quadruplex stabilizing capability. It is conceivable that this compound might have affinity for other types of G-quadruplexes, perhaps RNA, which might account for its modest cytotoxic activity. Upon attaching a second such side chain onto another oxazole ring quadruplex stabilization and cytotoxic activity are both diminshed. These studies suggest that when selective G-quadruplex stabilization, cytotoxic activity, water-solubility, and ease of synthesis are all taken into account, the previously-reported 5-[2-(dimethylamino)ethyl]phenyl analog 2 represents one of the better compounds for further development.