Phenoxyaromatic Acid Analogues as Novel Radiotherapy Sensitizers: Design, Synthesis and Biological Evaluation

Radiotherapy is a vital approach for brain tumor treatment. The standard treatment for glioblastoma (GB) is maximal surgical resection combined with radiotherapy and chemotherapy. However, the non-sensitivity of tumor cells in the hypoxic area of solid tumors to radiotherapy may cause radioresistance. Therefore, radiotherapy sensitizers that increase the oxygen concentration within the tumor are promising for increasing the effectiveness of radiation. Inspired by hemoglobin allosteric oxygen release regulators, a series of novel phenoxyacetic acid analogues were designed and synthesized. A numerical method was applied to determine the activity and safety of newly synthesized compounds. In vitro studies on the evaluation of red blood cells revealed that compounds 19c (∆P50 = 45.50 mmHg) and 19t (∆P50 = 44.38 mmHg) improve the oxygen-releasing property effectively compared to positive control efaproxiral (∆P50 = 36.40 mmHg). Preliminary safety evaluation revealed that 19c exhibited no cytotoxicity towards HEK293 and U87MG cells, while 19t was cytotoxic toward both cells with no selectivity. An in vivo activity assay confirmed that 19c exhibited a radiosensitization effect on orthotopically transplanted GB in mouse brains. Moreover, a pharmacokinetic study in rats showed that 19c was orally available.


Introduction
Hemoglobin, an essential oxygen carrier, is composed of four iron-containing subunits. Stereo conformational isomerism of hemoglobin affects its oxygen affinity, while allosteric modulators change the oxygen affinity of red blood cells by altering the three-dimensional conformation of hemoglobin [1]. Hemoglobin appears to be in a tense state (T state) in the deoxygenated form, which is conducive to the release of oxygen molecules. Alternatively, the oxygenated hemoglobin is in a relaxed state (R state), which tends to bind oxygen molecules [2,3]. The development of a series of hemoglobin allosteric effectors will be beneficial in the treatment of diseases linked to oxygen supply.
GB is one of the most malignant tumors, with a median survival time of 12-15 months [4][5][6][7]. The standard treatment for GB is maximal surgical resection combined with radiotherapy and chemotherapy [8][9][10]. However, the blood-brain barrier (BBB) prevents small molecules from entering tumor tissues to exert their pharmacological effects [11][12][13]. As a result, the necessity of BBB passage complicates the design of chemotherapeutic drugs [14][15][16]. However, allosteric modulators of hemoglobin are unaffected by the obstacles of the BBB. Based on the binding mode of efaproxiral ( Figure 2), the SARs of the reported phenoxyaromatic acid derivatives were analyzed. Interestingly, the 3,5-dimethyl aniline moiety of efaproxiral binds in the hydrophobic pocket formed by residues Val96, Lys99, Leu100, His103, Phe36, and Asn108 [35]. We can thus interpret that the substitution of 3,5dimethyl aniline with proper hydrophobic groups may maintain or even improve its activity. The carbonyl oxygen of the effectors formed a direct hydrogen bond with the Lys99 residue, which is essential to stabilize the T state conformation of deoxyhemoglobin. As a result, it was retained in a position similar to that in many of our newly designed compounds. As observed in Figure 2b, the methylpropionic acid tail of efaproxiral formed a few water-mediated hydrogen bonds with Lys99 and Arg541 in the central water cavity. Therefore, we designed various analogues with linker and tail region replacement to obtain stronger and more direct contact in the cavity. Inspired by all of these SAR results, we designed three series of compounds including the substitution of the benzene ring in the head region (region I) along with exploration of the linker structure (region II) and terminal region replacement (region III), as shown in Figure 1c. Based on the binding mode of efaproxiral ( Figure 2), the SARs of the reported phenoxyaromatic acid derivatives were analyzed. Interestingly, the 3,5-dimethyl aniline moiety of efaproxiral binds in the hydrophobic pocket formed by residues Val96, Lys99, Leu100, His103, Phe36, and Asn108 [35]. We can thus interpret that the substitution of 3,5-dimethyl aniline with proper hydrophobic groups may maintain or even improve its activity. The carbonyl oxygen of the effectors formed a direct hydrogen bond with the Lys99 residue, which is essential to stabilize the T state conformation of deoxyhemoglobin. As a result, it was retained in a position similar to that in many of our newly designed compounds. As observed in Figure 2b, the methylpropionic acid tail of efaproxiral formed a few watermediated hydrogen bonds with Lys99 and Arg541 in the central water cavity. Therefore, we designed various analogues with linker and tail region replacement to obtain stronger and more direct contact in the cavity. Inspired by all of these SAR results, we designed three series of compounds including the substitution of the benzene ring in the head region (region I) along with exploration of the linker structure (region II) and terminal region replacement (region III), as shown in Figure 1c. The syntheses of key intermediates 12a-12y are outlined in Scheme 1. The amide condensation between substituted aniline (10a-r) and p-hydroxybenzoic acid (11a-h) was carried out with the addition of a condensing agent EDCl, HOBT to obtain 12b, 12f-12w or with the addition of catalyst boric acid to produce 12a, 12c-12e and 12x-12y.  The syntheses of key intermediates 12a-12y are outlined in Scheme 1. The amide condensation between substituted aniline (10a-r) and p-hydroxybenzoic acid (11a-h) was carried out with the addition of a condensing agent EDCl, HOBT to obtain 12b, 12f-12w or with the addition of catalyst boric acid to produce 12a, 12c-12e and 12x-12y. The syntheses of key intermediates 12a-12y are outlined in Scheme 1. The amide condensation between substituted aniline (10a-r) and p-hydroxybenzoic acid (11a-h) was carried out with the addition of a condensing agent EDCl, HOBT to obtain 12b, 12f-12w or with the addition of catalyst boric acid to produce 12a, 12c-12e and 12x-12y.

In Vitro Red Blood Cell Evaluation and SAR Analysis
In the first stage of our research, three series of analogues were designed and synthesized to explore refinement of the hydrophobic parts of 19f, 19m, 24a and 24b; hinge areas of 18g, 19b and 19y; and tail regions of 15c, 15d and 16c, respectively. We used the Softron Analyzer to evaluate the effect of these compounds on the half-blood oxygen saturation(P50) value of red blood cells. The most promising phenoxyaromatic acid compound, efaproxiral, was used as a positive control. As is shown in Tables 1-3, compound 18g (∆P50 = 35.71 mmHg), 19m (∆P50 = 33.05 mmHg) and 19y (∆P50 = 38.78 mmHg) exhibited a similar effect compared to efaproxiral (∆P50 = 36.40 mmHg). The results reminded us that the modification of the hydrophobic part or linker extension is beneficial to improve an allosteric effect.

In Vitro Red Blood Cell Evaluation and SAR Analysis
In the first stage of our research, three series of analogues were designed and synthesized to explore refinement of the hydrophobic parts of 19f, 19m, 24a and 24b; hinge areas of 18g, 19b and 19y; and tail regions of 15c, 15d and 16c, respectively. We used the Softron Analyzer to evaluate the effect of these compounds on the half-blood oxygen saturation(P 50 ) value of red blood cells. The most promising phenoxyaromatic acid compound, efaproxiral, was used as a positive control. As is shown in Tables 1-3, compound 18g (∆P 50 = 35.71 mmHg), 19m (∆P 50 = 33.05 mmHg) and 19y (∆P 50 = 38.78 mmHg) exhibited a similar effect compared to efaproxiral (∆P 50 = 36.40 mmHg). The results reminded us that the modification of the hydrophobic part or linker extension is beneficial to improve an allosteric effect.
Next, a total of 26 compounds were designed and synthesized. Further evaluation showed that 19i (∆P 50 = 41.37 mmHg), 19m-1 (∆P 50 = 36.43 mmHg), 19n-1 (∆P 50 = 38.73 mmHg), 24d (∆P 50 = 38.31 mmHg) and 19c (∆P 50 = 45.50 mmHg) were as good as or even better than efaproxiral; of these, 19c (∆P 50 = 45.50 mmHg) had the highest ∆P 50 (Table 2). In vitro analysis revealed that the most successful exploration of the linker part was the chain structure composed of three carbon atoms (Table 2). Moreover, the halogenation or introduction of a group that could increase the conjugated system of the benzene ring of the hydrophobic part increased the activity of the effectors (Tables 1-3). Compared with para substitution products 19o (∆P 50 = 33.37 mmHg), 19m (∆P 50 = 33.05 mmHg) and19n (∆P 50 = 19.15 mmHg), meta substitution products 19p (∆P 50 = 35.91 mmHg), 19m-1 (∆P 50 = 36.43 mmHg) and 19n-1 (∆P 50 = 38.73 mmHg) were more effective (Table 1).   Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P 50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P 50 C is the control value of P 50 in the presence of 1% DMSO. ∆P 50 = (P 50 − P 50 C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.
Molecules 2022, 27, 2428 9 of 36 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P 50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P 50 C is the control value of P 50 in the presence of 1% DMSO. ∆P 50 = (P 50 − P 50 C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Series II
Molecules 2022, 27, 2428 10 of 36 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P50C is the control value of P50 in the presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Series III
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4). Table 3. Results of in vitro red blood cell studies.

Compound
Region III P 50 (mmHg) ∆P 50 (mmHg) P 50 /P 50 C K 15a Molecules 2022, 27, 2428 10 of 36 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P50C is the control value of P50 in the presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Series III
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well ( Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P50C is the control value of P50 in the presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4). 57 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P50C is the control value of P50 in the presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well ( Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (  Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4).
47.11 ± 8.08 5.27 ± 3.27 1.12 ± 0.06 2.39 ± 0.16 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P 50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P 50 C is the control value of P 50 in the presence of 1% DMSO. ∆P 50 = (P 50 − P 50 C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P 50 = 44.38 mmHg), with a robust allosteric effect as well ( Table 4).
presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD. Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4). presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Series IV
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4). presence of 1% DMSO. ∆P50 = (P50-P50C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Series IV
Finally, we combined the selected hydrophobic structure and linker structure obtained in the first two stages for better effectors. In vitro evaluation showed that most of the compounds designed by this strategy produced negative results. However, we obtained a potential compound, 19t (∆P50 = 44.38 mmHg), with a robust allosteric effect as well (Table 4).

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3, 19t was cytotoxic to both HEK293 cells (TC50 = 165 μM) and U87MG cells (IC50 = 183 μM) without selectivity (selection index = 1.1). Compound 19t showed cytotoxicity to HEK293 and U87MG cells at a concentration of 10 μm. Meanwhile, there was no cytotoxic effect observed for 19c on the HEK293 and U87MG cells at a concentration of 5 mM.
56.76 ± 2.93 18.85 ± 2.93 1.50 ± 0.08 1.79 ± 0.01 Red blood cell analyses were carried out at a final effector concentration of 2 mM. All stock solutions were prepared in DMSO at a concentration of 200 mM. P 50 represents the partial pressure of oxygen when 50% hemoglobin is saturated in the presence of effectors. P 50 C is the control value of P 50 in the presence of 1% DMSO. ∆P 50 = (P 50 − P 50 C) in mmHg. K is the Hill coefficient of the oxygen dissociation curve when the red blood cells are 50% saturated. Data are displayed as mean ± SD.

Analysis of Cytotoxicity for Preliminary Safety Evaluation
For further in vivo evaluation of newly synthesized compounds, 19c and 19t were selected as candidates. Hemoglobin allosteric effectors require a relatively high blood concentration to be effective. Cytotoxicity evaluation was conducted out of consideration for medication safety. HEK293 and U87MG cells were used to evaluate the cytotoxicity. As revealed in Figure 3

The Influence of Compound 19c on the Bohr Effect of Red Blood Cells and the Analysis of Its Dose-Response Relationship
The existence of the Bohr effect preserves the regulation of hemoglobin oxygen affinity by endogenous allosteric agents 2,3 DPG and Cl -. Therefore, it is essential to illustrate whether or not it still exists in the presence of an exogenous effector. As a result, the influence of 19c on the Bohr effect of mouse red blood cells was analyzed. The results showed that a change in acidity would lead to an apparent oxygen equilibrium curve (OEC) shift (Figure 4a). This phenomenon indicates that 19c will not offset the Bohr effect of hemoglobin.
Research on the in vitro dose-response relationship of 19c is essential for evaluating the potential of this compound. It will provide crucial support for in vivo antitumor evaluation. As shown in Figure 4b, compound 19c dose-dependently altered the oxygen affinity of hemoglobin. This suggests that in the in vivo radiosensitization experiments, the oxygen partial pressure in the hypoxic region of the tumor can be improved by increasing the dose of 19c.

The Influence of Compound 19c on the Bohr Effect of Red Blood Cells and the Analysis of Its Dose-Response Relationship
The existence of the Bohr effect preserves the regulation of hemoglobin oxygen affinity by endogenous allosteric agents 2,3 DPG and Cl − . Therefore, it is essential to illustrate whether or not it still exists in the presence of an exogenous effector. As a result, the influence of 19c on the Bohr effect of mouse red blood cells was analyzed. The results showed that a change in acidity would lead to an apparent oxygen equilibrium curve (OEC) shift (Figure 4a). This phenomenon indicates that 19c will not offset the Bohr effect of hemoglobin.

The Influence of Compound 19c on the Bohr Effect of Red Blood Cells and the Analysis of Its Dose-Response Relationship
The existence of the Bohr effect preserves the regulation of hemoglobin oxygen affinity by endogenous allosteric agents 2,3 DPG and Cl -. Therefore, it is essential to illustrate whether or not it still exists in the presence of an exogenous effector. As a result, the influence of 19c on the Bohr effect of mouse red blood cells was analyzed. The results showed that a change in acidity would lead to an apparent oxygen equilibrium curve (OEC) shift (Figure 4a). This phenomenon indicates that 19c will not offset the Bohr effect of hemoglobin.
Research on the in vitro dose-response relationship of 19c is essential for evaluating the potential of this compound. It will provide crucial support for in vivo antitumor evaluation. As shown in Figure 4b, compound 19c dose-dependently altered the oxygen affinity of hemoglobin. This suggests that in the in vivo radiosensitization experiments, the oxygen partial pressure in the hypoxic region of the tumor can be improved by increasing the dose of 19c. Research on the in vitro dose-response relationship of 19c is essential for evaluating the potential of this compound. It will provide crucial support for in vivo antitumor evaluation. As shown in Figure 4b, compound 19c dose-dependently altered the oxygen affinity of hemoglobin. This suggests that in the in vivo radiosensitization experiments, the oxygen partial pressure in the hypoxic region of the tumor can be improved by increasing the dose of 19c.

Docking Results
To illustrate the differences between 19c and efaproxiral at a molecular level, the docking results were carefully analyzed. Compared to efaproxiral (Figure 2), linker extension changed the binding mode of methylpropionic acid tail in the central water cavity. Compound 19c formed more water-mediated hydrogen bounds, and it preserved all binding positions in the hydrophobic region (Figure 5b). Compared to efaproxiral, 19c fitted deeper into the central water cavity and formed water-mediated hydrogen bonds with Lys127, Thr534, Thr537, Ser538 and Thr540 as well as Arg541. These results explained the increased allosteric activity of 19c (Figure 5a).

Docking Results
To illustrate the differences between 19c and efaproxiral at a molecular level, the docking results were carefully analyzed. Compared to efaproxiral (Figure 2), linker extension changed the binding mode of methylpropionic acid tail in the central water cavity. Compound 19c formed more water-mediated hydrogen bounds, and it preserved all binding positions in the hydrophobic region (Figure 5b). Compared to efaproxiral, 19c fitted deeper into the central water cavity and formed water-mediated hydrogen bonds with Lys127, Thr534, Thr537, Ser538 and Thr540 as well as Arg541. These results explained the increased allosteric activity of 19c (Figure 5a).

In Vivo Radiosensitization Effect on Brain Tumor
In order to evaluate in vivo antitumor effects, luciferase-labeled U87MG was implanted into Balb/c nude mice brains. A small animal bioluminescence imaging system was applied to trace the tumor formation and dynamic changes in GB size. Cobalt 60 was used for fractionated-dose whole-brain irradiation therapy (IR). The tumor was irradiated with a total dose of 12 Gy in four fractions of 3 Gy at a dose rate of 0.3Gy/min. Efaproxiral or 19c was administrated at a dosage of 150mg/kg by intraperitoneal injection (ip) every four days over a period of sixteen days. IR was conducted 15 min after ip.
As shown in (Figure 6a,b), compared to vehicle group, Efa + IR, IR and 19c + IR all significantly inhibited tumor proliferation with p values of 0.0270, 0.0255 and 0.0211, respectively. Compared to IR alone, 19c combined with IR significantly inhibited tumor progression (p = 0.0449); however, efaproxiral combined with IR was statistically ineffective compared with IR therapy alone (p = 0.3462). Consistent with Figure 6, the results for the survival period of tumor-bearing mice showed that IR alone or combined with 19c or efaproxiral could improve the overall survival time of tumor-bearing mice compared to vehicle control (Figure 7a,b). The survival time obtained with 19c combined with IR was significantly longer than that obtained with IR alone (p = 0.0286). Since we did not directly detect the changes in oxygenation in the tumor area after compound 19c administration

In Vivo Radiosensitization Effect on Brain Tumor
In order to evaluate in vivo antitumor effects, luciferase-labeled U87MG was implanted into Balb/c nude mice brains. A small animal bioluminescence imaging system was applied to trace the tumor formation and dynamic changes in GB size. Cobalt 60 was used for fractionated-dose whole-brain irradiation therapy (IR). The tumor was irradiated with a total dose of 12 Gy in four fractions of 3 Gy at a dose rate of 0.3 Gy/min. Efaproxiral or 19c was administrated at a dosage of 150 mg/kg by intraperitoneal injection (ip) every four days over a period of sixteen days. IR was conducted 15 min after ip.
As shown in (Figure 6a,b), compared to vehicle group, Efa + IR, IR and 19c + IR all significantly inhibited tumor proliferation with p values of 0.0270, 0.0255 and 0.0211, respectively. Compared to IR alone, 19c combined with IR significantly inhibited tumor progression (p = 0.0449); however, efaproxiral combined with IR was statistically ineffective compared with IR therapy alone (p = 0.3462). Consistent with Figure 6, the results for the survival period of tumor-bearing mice showed that IR alone or combined with 19c or efaproxiral could improve the overall survival time of tumor-bearing mice compared to vehicle control (Figure 7a,b). The survival time obtained with 19c combined with IR was significantly longer than that obtained with IR alone (p = 0.0286). Since we did not directly detect the changes in oxygenation in the tumor area after compound 19c administration during the in vivo radiosensitization experiments, in order to better illustrate the radiosensitization effect of 19c, we prepared a hypothetical illustration. The hypothesized radiosensitization mechanism of 19c is shown in Figure 6c. As shown in the schematic diagram, 19c administration would increase tumor oxygenation in GB. Reoxygenation of the hypoxic area sensitized IR treatment.

Preliminary Pharmacokinetic Assessment
Based on the potential radiosensitization effects in vivo, we performed an in vivo pharmacokinetic (PK) study to preliminarily evaluate the druggability of 19c at a dosage of 100 mg/kg by intraperitoneal injection (ip) or oral administration (po). The corresponding PK parameters are summarized in Table 5. After ip or po administration of 19c, the values of AUCinf were 612,428 ng/mL or 410,402 ng/mL, respectively, which indicated

Preliminary Pharmacokinetic Assessment
Based on the potential radiosensitization effects in vivo, we performed an in vivo pharmacokinetic (PK) study to preliminarily evaluate the druggability of 19c at a dosage of 100 mg/kg by intraperitoneal injection (ip) or oral administration (po). The corresponding PK parameters are summarized in Table 5. After ip or po administration of 19c, the values of AUCinf were 612,428 ng/mL or 410,402 ng/mL, respectively, which indicated

Preliminary Pharmacokinetic Assessment
Based on the potential radiosensitization effects in vivo, we performed an in vivo pharmacokinetic (PK) study to preliminarily evaluate the druggability of 19c at a dosage of 100 mg/kg by intraperitoneal injection (ip) or oral administration (po). The corresponding PK parameters are summarized in Table 5. After ip or po administration of 19c, the values of AUCinf were 612,428 ng/mL or 410,402 ng/mL, respectively, which indicated moderate systemic exposure. In addition, 19c showed a half-life (T1/2) of 2.55 h and 2.78 h, respectively. These results indicated that 19c showed moderate PK properties via ip or po, which provided a foundation for further development. As is shown in Figure S1, the plasma concentration of 19c after oral administration was more stable than that after intraperitoneal injection, and the pharmacokinetic parameters were similar to those of intraperitoneal injection, which suggests that oral administration may be preferred.

Conclusions
GB is one of the most lethal tumors. However, due to the hindrance of the BBB, drug development has lagged [38][39][40]. It is particularly important to overcome radioresistance influenced by the presence of hypoxia. The existing research shows that phenoxyacetic acid derivatives represented by efaproxiral will increase the tumor oxygen supply. Thus, a series of novel phenoxyacetic acid derivatives were designed, synthesized, and evaluated. The SAR results demonstrated that a linker of three carbon atoms in the hinge area is the optimum structure for phenoxyacetic acid derivatization. Moreover, thioether and aromatic ring substitution of the hydrophobic region was shown to improve the activity of the newly synthesized compounds.
Additionally, the activity of a meta-substituted benzene ring in the hydrophobic region is better than that achieved through para-substitution. In the in vitro activity evaluation stage, we confirmed that both 19c and 19t induced in vitro hemoglobin allosteric activities that were significantly higher than that of the positive control. However, a preliminary safety evaluation revealed that 19t was cytotoxic to both normal and glioma cells at low micromolar levels without selectivity. Furthermore, in vitro activity evaluation showed that 19c exerts an allosteric effect on hemoglobin at a micromolar level. Moreover, 19c preserved the Bohr effect of hemoglobin, which means it will not interfere with the endogenous hemoglobin effectors. The in vivo antitumor results indicated that 19c is an effective GB radiosensitizer.
In conclusion, our preliminary work confirmed that 19c is an effective radiosensitizer. To obtain more potent effectors, further optimization of compound 19c based on SAR analysis is under investigation.

General Chemistry
All solvents and reagents were obtained from commercial suppliers and used directly. Silica gel TLC plates (GF254) were applied to monitor the reactions. Silica gel (200−300 mesh) was used for chromatography. The 1 H and 13 C NMR spectra were recorded on a JEOLECA400 spectrometer, with TMS as an internal standard at ambient temperature.
All chemical shifts are reported in parts per million (ppm). All coupling constants are reported in hertz. The ESI-MS was recorded on an Agilent TOF G6230A mass spectrometer.

Procedure for the Syntheses of Intermediates 12a-12z
To a stirring solution of 3,5-dimethylaniline 10a (2.7 g, 15.6 mmol) in toluene (25 mL) were add 2-(4-hydroxyphenyl)acetic acid 11a (2.0 g, 13 mmol) and boric acid (0.08 g, 0.13 mmol) at room temperature. The reaction mixture was refluxed at 140°C with a water separator for 12 h. Upon completion, the mixture was cooled to room temperature and filtered, and the filter cake was washed with toluene (3 × 20 mL) and water (3 × 20 mL), respectively. Then, the filter cake was dried in a vacuum drying oven to give 12a as a white solid