Design, Synthesis, Biological Evaluation and Molecular Docking of Novel F-18-Labeled Focal Adhesion Kinase Inhibitors as Potential Tumor Radiotracers

Tumor diagnosis, especially at the early stages, holds immense significance. Focal adhesion kinase (FAK) is often highly expressed across various types of tumors, making it a promising target for both therapy and diagnosis. In this study, seven novel inhibitors were designed and synthesized. The inhibitory activity of these compounds against FAK was notably potent, with an IC50 range of 1.27–1968 nM. In particular, compounds 7a and 7c, with IC50 values of 5.59 nM and 1.27 nM, respectively, were radiolabeled with F-18 and then evaluated with S-180 tumor-bearing mice. Subsequently, they exhibited moderate-to-high tumor uptake values, with [18F]7a showing 1.39 ± 0.30%ID/g at 60 min post injection and [18F]7c demonstrating 6.58 ± 0.46%ID/g at 30 min post injection. In addition, the results from docking studies revealed the binding specifics of the studied compounds. Overall, these findings hold the potential to offer valuable guidance for enhancing the development of radiotracers and enzyme inhibitors.


Introduction
The new case numbers for cancer incidence and associated death are still at high levels, especially for cancers of the lungs, liver, prostate and breast [1][2][3][4].Focal adhesion kinase (FAK), located both in tumor cells and in the tumor microenvironment, is a non-receptor tyrosine kinase that is overexpressed in most types of cancers and plays an important role in tumor survival, angiogenesis and metastasis [5][6][7], making it a consolidated drug design target for oncological therapy and diagnosis.Most studies have focused on the development of therapeutic drug developments targeting FAK, such as defactinib (VS-6063), conteltinib (CT-707) and GSK2256098, all of which have been investigated in clinical phase II trails (Figure 1).There are also other clinical Phase I and preclinical candidates, including follow-up drug combination studies [8,9].

Introduction
The new case numbers for cancer incidence and associated death are still at high levels, especially for cancers of the lungs, liver, prostate and breast [1][2][3][4].Focal adhesion kinase (FAK), located both in tumor cells and in the tumor microenvironment, is a nonreceptor tyrosine kinase that is overexpressed in most types of cancers and plays an important role in tumor survival, angiogenesis and metastasis [5][6][7], making it a consolidated drug design target for oncological therapy and diagnosis.Most studies have focused on the development of therapeutic drug developments targeting FAK, such as defactinib (VS-6063), conteltinib (CT-707) and GSK2256098, all of which have been investigated in clinical phase Ⅱ trails (Figure 1).There are also other clinical Phase Ⅰ and preclinical candidates, including follow-up drug combination studies [8,9].As is well known, the early diagnosis of cancer holds significant value for prolonging the survival time of patients.Positron diagnostic drugs such as F-18-labeled radiotracers are are the most sensitive molecules in the field of clinical imaging for tumor diagn ever, the target of the radiotracer being non-specific to tumor and tissue e widely known and used 18 F-FDG to fatal drawbacks such as false-positive re flammation and infection or false-negative results in slow-growing tumors.T study group has been committed to developing novel F-18-labeled radiotracer the tumor-specific FAK, and we have conducted considerable research to seek with high FAK affinity and the proper nature for diagnosis [10][11][12].However, room for the improvement in the inhibitory activity targeting FAK.Moreover tures should also be further optimized.As shown in Figure 2, the 2-position of t has been extensively discussed, with PEG linker-substituted aniline being valid hance hydrophilicity and provide the F-18-labeled position.Regarding the mthe arylamine, CH3, OCH3, and PEG groups have been previously investigated ther research in this area to be conducted.In contrast, the 4-position of the p particularly the para-position of the arylamine, has received less academic atte might because this position is deeper within the protein, and larger substitu could potentially impose a negative impact on the interaction between the mo the FAK protein.Through analyzing the protein conformation, a deep po enough to accommodate a six-member circle was found.This concept was firs for the whole field of the drug design targeting FAK, including the developm inhibitors and PROTACs [13,14].Encouraged by this idea, seven novel FAK were designed and synthesized in this study.Their diaminopyrimidine cores the formation of the key hydrogen bonds between the molecules and the FA which was similar to those of clinical molecules.Meanwhile, the difference w chemical space of the deep binding sites was extended to explore the effects on inhibitory and drug properties for biodistribution in vivo.

Synthesis
Scheme 1 reports the synthesis routes of new derivatives 7a-7g.The six-membered heterocycles (morpholine or thiomorpholine 1,1-dioxide) were introduced to the structure by reacting with the 5-fluoro-N-methyl-2-nitrobenzamide at 50 • C. The nitro groups of 2 were then reduced to the amino group with iron filings in the presence of ammonium chloride to obtain 3. The key intermediate 4 was produced from 3 via a nucleophilic substitution at the 4-position of 5-bromo-2,4-dichloropyrimidine and then treated with different substituted aryl amines (12 and 14) to produce derivative 5 using TsOH as a catalyst.It should be noted that the silicon protective group was removed at the same time.This was because deprotection can also be implemented under faintly acidic conditions [15].The hydroxyl groups were activated by TsCl and substituted by fluoride ions to obtain the labelling precursors and the stable standards.In this way, the two structures were obtained simultaneously, which can also be considered the advantage of this synthesis route.It should be noted that the silicon protective group was removed at the same time.This was because deprotection can also be implemented under faintly acidic conditions [15].The hydroxyl groups were activated by TsCl and substituted by fluoride ions to obtain the labelling precursors and the stable standards.In this way, the two structures were obtained simultaneously, which can also be considered the advantage of this synthesis route.Scheme 2 presents the synthesis routes of the key intermediates 12a, 12b, 12d and 14.One oxhydryl of the PEG linker was protected by TBS, and the other was activated by TsCl to obtained 10.Then, 10 was introduced to the substituted p-notrophenol through a nucleophilic substitution to obtain 11.Next, 11 was reduced to 12a, 12b and 12d.As to 14, the Suzuki reaction was conducted after the formation of the new ester bond (11c), rather than before, as we have previously tried both strategies.The phenolic hydroxyl group may have a negative effect on the Suzuki reaction.
Scheme 2 presents the synthesis routes of the key intermediates 12a, 12b, 12d and 14.One oxhydryl of the PEG linker was protected by TBS, and the other was activated by TsCl to obtained 10.Then, 10 was introduced to the substituted p-notrophenol through a nucleophilic substitution to obtain 11.Next, 11 was reduced to 12a, 12b and 12d.As to 14, the Suzuki reaction was conducted after the formation of the new ester bond (11c), rather than before, as we have previously tried both strategies.The phenolic hydroxyl group may have a negative effect on the Suzuki reaction.

Radiolabeling and Stability Experiment
As shown in Scheme 3, the labeling produced in one step was implemented to produce the [ 18 F]7a and [ 18 F]7c.The free 18 F − ions were obtained using Kryptofix 2.2.2. and potassium carbonate, and then heated to 100 °C in the presence of the labeling precursor for 20 min.The overall radiochemical yields were 15.43% and 17.28%, respectively.After being purified, the two radiotracers presented a high radiochemical purity (>95%).To ensure the correctness of the radioactive products, [ 18 F]7a and [ 18 F]7c were co-injected with sTable 7a and 7c, respectively.The retention period of HPLC was within acceptable error levels, as depicted in Figure 3. Scheme 3. Method of radiolabeling: k222K 18 F, K2CO3, MeCN, 100 °C.

Radiolabeling and Stability Experiment
As shown in Scheme 3, the labeling produced in one step was implemented to produce the [ 18 F]7a and [ 18 F]7c.The free 18 F − ions were obtained using Kryptofix 2.2.2. and potassium carbonate, and then heated to 100 • C in the presence of the labeling precursor for 20 min.The overall radiochemical yields were 15.43% and 17.28%, respectively.After being purified, the two radiotracers presented a high radiochemical purity (>95%).To ensure the correctness of the radioactive products, [ 18 F]7a and [ 18 F]7c were co-injected with stable 7a and 7c, respectively.The retention period of HPLC was within acceptable error levels, as depicted in Figure 3.

Radiolabeling and Stability Experiment
As shown in Scheme 3, the labeling produced in one step was implemented to produce the [ 18 F]7a and [ 18 F]7c.The free 18 F − ions were obtained using Kryptofix 2.2.2. and potassium carbonate, and then heated to 100 °C in the presence of the labeling precursor for 20 min.The overall radiochemical yields were 15.43% and 17.28%, respectively.After being purified, the two radiotracers presented a high radiochemical purity (>95%).To ensure the correctness of the radioactive products, [ 18 F]7a and [ 18 F]7c were co-injected with sTable 7a and 7c, respectively.The retention period of HPLC was within acceptable error levels, as depicted in Figure 3.    Furthermore, the stability of the two radiotracers in murine serum and saline was studied.As shown in Figure 4, the radiochemical purities were all greater than 95%, indicating their high in vitro stability.

Experiment on Octanol/Water Partition Coefficient Determination
The partition coefficients of the radiotracers [ 18 F]7a and [ 18 F]7c were determined to evaluate the octanol/water partition properties.The logD7.4s of the two radiotracers were 0.96 and 0.74, respectively, which might be attributable to the possession of the sulfonyl and the pyrazol groups.Furthermore, the stability of the two radiotracers in murine serum and saline was studied.As shown in Figure 4, the radiochemical purities were all greater than 95%, indicating their high in vitro stability.Furthermore, the stability of the two radiotracers in murine serum and saline was studied.As shown in Figure 4, the radiochemical purities were all greater than 95%, indicating their high in vitro stability.

Experiment on Octanol/Water Partition Coefficient Determination
The partition coefficients of the radiotracers [ 18 F]7a and [ 18 F]7c were determined to evaluate the octanol/water partition properties.The logD7.4s of the two radiotracers were 0.96 and 0.74, respectively, which might be attributable to the possession of the sulfonyl and the pyrazol groups.

Experiment on Octanol/Water Partition Coefficient Determination
The partition coefficients of the radiotracers [ 18 F]7a and [ 18 F]7c were determined to evaluate the octanol/water partition properties.The logD 7.4 s of the two radiotracers were 0.96 and 0.74, respectively, which might be attributable to the possession of the sulfonyl and the pyrazol groups.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC 50 ) in the range of 1.27~1968 nM.Among them, the IC 50 s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC 50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC 50 : 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC50) in the range of 1.27~1968 nM.Among them, the IC50s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC50: 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC50) in the range of 1.27~1968 nM.Among them, the IC50s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC50: 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC50) in the range of 1.27~1968 nM.Among them, the IC50s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC50: 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC50) in the range of 1.27~1968 nM.Among them, the IC50s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC50: 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

FAK Inhibitory Assay
The inhibitory activities of the 7a-7c against FAK were tested using a homogeneous time-resolved fluorescence methodology-based kinase assay.To ensure accuracy, VS-6063 was taken as a positive control under the same conditions.As shown in Table 1, all of the novel compounds demonstrated enzyme inhibition values (IC50) in the range of 1.27~1968 nM.Among them, the IC50s of 7a and 7c were less than 10 nmol (5.59 and 1.27 nM, respectively), which were compared to that of VS-6063 (1.26 nM).The 5-bromo pyrimidine was previously demonstrated to be an attractive core for developing a promising FAK radiotracer.Thus, some pharmacophores containing a hydroxyl group were introduced here to improve the hydrophilicity and the FAK inhibition.It could be clearly observed that the inhibitors containing sulfonyl groups and morpholine showed better inhibition compared to others (IC50 of 1.27, 5.59, 10.02, and 24.6 nM vs. 643, 1238, and 1968 nM).This could be attributed to the six-membered aliphatic cyclic structure, which might facilitate suitable penetration into the protein pocket.Interestingly, when comparing 7f and 7b (IC50: 10.02 vs. 24.6 nM), the morpholine moiety also significantly contributed to the enhanced activity.This could stem from the favorable conformation achieved when the morpholine was positioned at the 4-position of the diaminopyrimidine, while the acetylpiperazine was at the 2-position.To further increase the hydrophilicity, the PEG linker was chosen to form ether with the aniline, which also maintained the high inhibition.To this end, compounds 7a and 7c, with high inhibition and good hydrophilicity were chosen for biodistribution studies in S-180 tumor-bearing Kunming mice.

Molecular Docking
Molecular docking studies were conducted to further clarify the binding details between the radiotracers and the FAK protein.As described above, all seven of the new seven pyrimidine derivatives were successfully docked into the FAK protein pocket.Compounds 7a and 7c were taken as examples to show the details in Figure 5. Two strong hydrogen bonds were formed between CYS502 and 7c, with one of the pyrimidine-N and the arylamine-NH acting as H-bond acceptors and H-bond donors, respectively (distance: 2.13 and 2.54 Å, respectively).Meanwhile, amide-O acted as a H-bond acceptor for the NH of ASP564 (distance: 2.01 Å).In addition, 7c could form one more hydrogen bond between ARG436 and the pyrazole group compared to 7a (distance: 2.06 Å).Interestingly, 7a could form only two hydrogen bonds with CYS502 and ARG426.These molecular docking results are consistent with the findings of the FAK inhibitory assays and the biodistribution experiments.

Biodistributions of [ 18 F]7a and [ 18 F]7c in S-180 Tumor-Bearing Mice
Table 2 presents the biodistributions of [ 18 F]7a and [ 18 F]7c.In general, after injection in mice, the two new F-18-labeled compounds showed increased tumor uptake.The peak uptakes of the two radiotracers were observed at 60 min and 30 min post injection, respectively (1.39 ± 0.30%ID/g and 6.58 ± 0.46%ID/g, respectively); the tumor/blood and tu-

Molecular Docking
Molecular docking studies were conducted to further clarify the binding details between the radiotracers and the FAK protein.As described above, all seven of the new seven pyrimidine derivatives were successfully docked into the FAK protein pocket.Compounds 7a and 7c were taken as examples to show the details in Figure 5. Two strong hydrogen bonds were formed between CYS502 and 7c, with one of the pyrimidine-N and the arylamine-NH acting as H-bond acceptors and H-bond donors, respectively (distance: 2.13 and 2.54 Å, respectively).Meanwhile, amide-O acted as a H-bond acceptor for the NH of ASP564 (distance: 2.01 Å).In addition, 7c could form one more hydrogen bond between ARG436 and the pyrazole group compared to 7a (distance: 2.06 Å).Interestingly, 7a could form only two hydrogen bonds with CYS502 and ARG426.These molecular docking results are consistent with the findings of the FAK inhibitory assays and the biodistribution experiments.

Biodistributions of [ 18 F]7a and [ 18 F]7c in S-180 Tumor-Bearing Mice
Table 2 presents the biodistributions of [ 18 F]7a and [ 18 F]7c.In general, after injection in mice, the two new F-18-labeled compounds showed increased tumor uptake.The peak uptakes of the two radiotracers were observed at 60 min and 30 min post injection, respectively (1.39 ± 0.30%ID/g and 6.58 ± 0.46%ID/g, respectively); the tumor/blood and tu-

Molecular Docking
Molecular docking studies were conducted to further clarify the binding details between the radiotracers and the FAK protein.As described above, all seven of the new seven pyrimidine derivatives were successfully docked into the FAK protein pocket.Compounds 7a and 7c were taken as examples to show the details in Figure 5. Two strong hydrogen bonds were formed between CYS502 and 7c, with one of the pyrimidine-N and the arylamine-NH acting as H-bond acceptors and H-bond donors, respectively (distance: 2.13 and 2.54 Å, respectively).Meanwhile, amide-O acted as a H-bond acceptor for the NH of ASP564 (distance: 2.01 Å).In addition, 7c could form one more hydrogen bond between ARG436 and the pyrazole group compared to 7a (distance: 2.06 Å).Interestingly, 7a could form only two hydrogen bonds with CYS502 and ARG426.These molecular docking results are consistent with the findings of the FAK inhibitory assays and the biodistribution experiments.

Biodistributions of [ 18 F]7a and [ 18 F]7c in S-180 Tumor-Bearing Mice
Table 2 presents the biodistributions of [ 18 F]7a and [ 18 F]7c.In general, after injection in mice, the two new F-18-labeled compounds showed increased tumor uptake.The peak uptakes of the two radiotracers were observed at 60 min and 30 min post injection, respectively (1.39 ± 0.30%ID/g and 6.58 ± 0.46%ID/g, respectively); the tumor/blood and tu-

Molecular Docking
Molecular docking studies were conducted to further clarify the binding details between the radiotracers and the FAK protein.As described above, all seven of the new seven pyrimidine derivatives were successfully docked into the FAK protein pocket.Compounds 7a and 7c were taken as examples to show the details in Figure 5. Two strong hydrogen bonds were formed between CYS502 and 7c, with one of the pyrimidine-N and the arylamine-NH acting as H-bond acceptors and H-bond donors, respectively (distance: 2.13 and 2.54 Å, respectively).Meanwhile, amide-O acted as a H-bond acceptor for the NH of ASP564 (distance: 2.01 Å).In addition, 7c could form one more hydrogen bond between ARG436 and the pyrazole group compared to 7a (distance: 2.06 Å).Interestingly, 7a could form only two hydrogen bonds with CYS502 and ARG426.These molecular docking results are consistent with the findings of the FAK inhibitory assays and the biodistribution experiments.

Molecular Docking
Molecular docking studies were conducted to further clarify the binding details between the radiotracers and the FAK protein.As described above, all seven of the new seven pyrimidine derivatives were successfully docked into the FAK protein pocket.Compounds 7a and 7c were taken as examples to show the details in Figure 5. Two strong hydrogen bonds were formed between CYS502 and 7c, with one of the pyrimidine-N and the arylamine-NH acting as H-bond acceptors and H-bond donors, respectively (distance: 2.13 and 2.54 Å, respectively).Meanwhile, amide-O acted as a H-bond acceptor for the NH of ASP564 (distance: 2.01 Å).In addition, 7c could form one more hydrogen bond between ARG436 and the pyrazole group compared to 7a (distance: 2.06 Å).Interestingly, 7a could form only two hydrogen bonds with CYS502 and ARG426.These molecular docking results are consistent with the findings of the FAK inhibitory assays and the biodistribution experiments.

Biodistributions of [ 18 F]7a and [ 18 F]7c in S-180 Tumor-Bearing Mice
Table 2 presents the biodistributions of [ 18 F]7a and [ 18 F]7c.In general, after injection in mice, the two new F-18-labeled compounds showed increased tumor uptake.The peak uptakes of the two radiotracers were observed at 60 min and 30 min post injection, respectively (1.39 ± 0.30%ID/g and 6.58 ± 0.46%ID/g, respectively); the tumor/blood and tu-

Biodistributions of [ 18 F]7a and [ 18 F]7c in S-180 Tumor-Bearing Mice
Table 2 presents the biodistributions of [ 18 F]7a and [ 18 F]7c.In general, after injection in mice, the two new F-18-labeled compounds showed increased tumor uptake.The peak uptakes of the two radiotracers were observed at 60 min and 30 min post injection, respectively (1.39 ± 0.30%ID/g and 6.58 ± 0.46%ID/g, respectively); the tumor/blood and tumor/muscle ratios for [ 18 F]7c were 2.40 and 1.88, consistent with the FAK inhibitory experiment results, indicating that the [ 18 F]7a and [ 18 F]7c were mainly specific to FAK in vivo.Moreover, the two radiotracers showed good retention and moderate elimination in tumor tissue, and this good nature for diagnosis might result from the hydrophilicity (LogD 7.4 : 0.96 and 0.74, respectively).However, possibly because the two radiotracers were mainly metabolized through the liver and lungs, the uptakes of non-targets tissues like liver and lungs were observed to be high, indicating that they were not suited for the diagnoses of liver and lung tumors.

Discussion
The FAK protein acts as a hub in the complex network of signaling pathways [16].Thus, as described above, many researchers have developed therapeutic drugs targeting FAK, such as inhibitors and PROTACs.This also explains the frequent presence of FAK inhibitors in drug combination projects.While remarkable successes have been achieved, none of the candidates have entered the market yet.Concerning the characteristics of FAK as a drug target and the advantages of nuclear scintigraphy techniques, as well as the drawbacks of 18 F-FDG [17], the research attention of the present study was shifted from therapy to diagnosis targeting FAK.
A total of seven new inhibitors were successfully synthesized.The results of the kinase inhibition assays demonstrated the feasibility of the design strategy described above.On the whole, thiomorpholine 1,1-dioxides and morpholine were suitable for adding to the o-position of the aniline, for the relevant FAK inhibitory were better than others (1.27~24.6 vs. 643~1968 nM).Additionally, one of these molecules (7c) possessed essentially the same inhibitory activity targeting FAK as that of the VS-6063 (IC 50 : 1.27 vs. 1.26 nmol).
The molecular docking research also demonstrated the same result: 7c could form two more H-bonds than 7a.In the future, we can design more novel inhibitors and other related molecules possessing thiomorpholine 1,1-dioxides targeting FAK.
The uptake of the two F-18-labeled molecules in the tumor tissue was obvious, especially for 7c, and the ID%/g reached 6.58 ± 0.46 at 30 min post injection.This was due to the high FAK affinity of those molecules, indicating the targeting and selection of FAK.Furthermore, the retention and the elimination were acceptable for diagnosis.Interestingly, these two radiotracers ([ 18 F]7a and [ 18 F]7c) might undergo significant metabolism through the liver and lungs, as the ID%/g values of these two tissues were high.Although not fit for liver and lung cancer diagnosis, they could still be used for other cancers, e.g., brain, breast, prostate, ovarian, etc. [18][19][20].Further studies designing radiotracers with intracellular targets should prioritize modulating the logP to minimize the uptake in non-target issues.Nevertheless, the findings of this work will still provide valuable insights that are relevant in this field.

Synthesis
The general organic synthesis route is shown in Schemes 1 and 2. All the analyticalgrade chemical regents used in this study were purchased from commercial sources and used without further purification.Nuclear magnetic resonance spectroscopy (NMR) was performed on a JEOL spectrometer (600 and 400 MHz).Tetramethylsilane (TMS) was used as an internal standard, and chemical shifts are given in (δ) for 1 H NMR and 13 C NMR.The mass spectra were recorded on a Waters Quattro Micro Quadrupole Mass Spectrometer.

Figure 1 .
Figure 1.The clinical phase II molecules.

Molecules 2024 ,
29, 1224 3 of 19 were then reduced to the amino group with iron filings in the presence of ammonium chloride to obtain 3. The key intermediate 4 was produced from 3 via a nucleophilic substitution at the 4-position of 5-bromo-2,4-dichloropyrimidine and then treated with different substituted aryl amines (12 and 14) to produce derivative 5 using TsOH as a catalyst.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.

Table 1 .
In vitro enzyme activity of compound 7 and reference compound VS-6063.
* Data are expressed as the percentage of injected dose per gram (% ID/g), mean ± SD, n = 3.