Design, Synthesis, and Antiproliferative Activity of Selective Histone Deacetylases 6 Inhibitors Containing a Tetrahydropyridopyrimidine Scaffold

The development of selective histone deacetylase 6 inhibitors (sHDAC6is) is being recognized as a therapeutic approach for cancers. In this paper, we designed a series of novel tetrahydropyridopyrimidine derivatives as sHDAC6 inhibitors. The most potent compound, 8-(2, 4-bis(3-methoxyphenyl)-5, 8-dihydropyrido [3, 4-d]pyrimidin-7(6H)-yl)-N-hydroxy-8-oxooctanamide (8f), inhibited HDAC6 with IC50 of 6.4 nM, and showed > 48-fold selectivity over other subtypes. In Western blot assay, 8f elevated the levels of acetylated α-tubulin in a dose-dependent manner. In vitro, 8f inhibited RPMI-8226, HL60, and HCT116 tumor cells with IC50 of 2.8, 3.20, and 3.25 μM, respectively. Moreover, 8f showed good antiproliferative activity against a panel of tumor cells.

To date, a lot of synthetic sHDAC6is have been reported [25][26][27][28][29][30][31].The structure of HDAC6i typically contains three parts: (a) a zinc-binding group (ZBG) coordinating with Zn 2+ ion at the bottom of the active site, (b) a linker region embedding in the hydrophobic tunnel between the catalytic site and the outer surface, and (c) a capping group overlaying on the surface (Figure 1).The clinical ACY-1215 (1) inhibited HDAC1 and HDAC6 with Because the cap region of the HDAC6 pocket is wider and larger than that of HDAC1 [35], a more rigid and bigger capping group might improve the selectivity toward HDAC6.For HDAC6is 1-3, the common feature is apparent: a "Y" shaped and predominantly aromatic capping group with hydroxamic acid as ZBG.The 5, 6, 7, 8tetrahydropyrido [3, 4-d]pyrimidine (4) was frequently used in the development of kinase inhibitors for cancer treatment [36,37].Hence, the introduction of such a scaffold in one molecule might be beneficial for the anticancer efficacy of HDAC6is.In this paper, we replaced the N, N-diphenylpyrimidine capping group of ACY1215 with 5, 6, 7, 8tetrahydropyrido [3, 4-d]pyrimidine and retained the six-carbon linker as well as hydroxamic acid ZBG (Figure 2).Here, we reported the design, structure, and activity relationship (SAR) study and antiproliferative evaluation of these tetrahydropyridopyrimidines.

Chemistry
The synthetic route to target compounds 8a-h was initiated by the preparation of key intermediate 6a-h from commercially available material 5 and two equivalent arylboronic acids by Suzuki reaction with Pd(dppf)Cl2 as a catalyst and K2CO3 as a base (Scheme 1).For preliminary exploration, the same aryls were introduced on the C2 and C4 positions of the tetrahydropyridopyrimidine scaffold.Compounds 6a-h underwent Boc Because the cap region of the HDAC6 pocket is wider and larger than that of HDAC1 [35], a more rigid and bigger capping group might improve the selectivity toward HDAC6.For HDAC6is 1-3, the common feature is apparent: a "Y" shaped and predominantly aromatic capping group with hydroxamic acid as ZBG.The 5, 6, 7, 8-tetrahydropyrido [3, 4-d]pyrimidine (4) was frequently used in the development of kinase inhibitors for cancer treatment [36,37].Hence, the introduction of such a scaffold in one molecule might be beneficial for the anticancer efficacy of HDAC6is.In this paper, we replaced the N, N-diphenylpyrimidine capping group of ACY1215 with 5, 6, 7, 8-tetrahydropyrido [3, 4d]pyrimidine and retained the six-carbon linker as well as hydroxamic acid ZBG (Figure 2).Here, we reported the design, structure, and activity relationship (SAR) study and antiproliferative evaluation of these tetrahydropyridopyrimidines.
In a phase I study, KA2507 showed selective target engagement, no significant toxicities, and prolonged disease stabilization in a subset of patients [34].Despite great success in sHDAC6is discovery, available clinical agents are still rare, and the lack of therapeutic effect on solid tumors is another problem for HDAC inhibitors.Because the cap region of the HDAC6 pocket is wider and larger than that of HDAC1 [35], a more rigid and bigger capping group might improve the selectivity toward HDAC6.For HDAC6is 1-3, the common feature is apparent: a "Y" shaped and predominantly aromatic capping group with hydroxamic acid as ZBG.The 5, 6, 7, 8tetrahydropyrido [3, 4-d]pyrimidine (4) was frequently used in the development of kinase inhibitors for cancer treatment [36,37].Hence, the introduction of such a scaffold in one molecule might be beneficial for the anticancer efficacy of HDAC6is.In this paper, we replaced the N, N-diphenylpyrimidine capping group of ACY1215 with 5, 6, 7, 8tetrahydropyrido [3, 4-d]pyrimidine and retained the six-carbon linker as well as hydroxamic acid ZBG (Figure 2).Here, we reported the design, structure, and activity relationship (SAR) study and antiproliferative evaluation of these tetrahydropyridopyrimidines.

Chemistry
The synthetic route to target compounds 8a-h was initiated by the preparation of key intermediate 6a-h from commercially available material 5 and two equivalent arylboronic acids by Suzuki reaction with Pd(dppf)Cl2 as a catalyst and K2CO3 as a base (Scheme 1).For preliminary exploration, the same aryls were introduced on the C2 and C4 positions of the tetrahydropyridopyrimidine scaffold.Compounds 6a-h underwent Boc

Chemistry
The synthetic route to target compounds 8a-h was initiated by the preparation of key intermediate 6a-h from commercially available material 5 and two equivalent arylboronic acids by Suzuki reaction with Pd(dppf)Cl 2 as a catalyst and K 2 CO 3 as a base (Scheme 1).For preliminary exploration, the same aryls were introduced on the C2 and C4 positions of the tetrahydropyridopyrimidine scaffold.Compounds 6a-h underwent Boc deprotection under TFA/CH 2 Cl 2 condition and subsequent condensation reaction with 8-methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7a-h was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.
methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC 50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC 50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF 3 , -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.

Table 1. Intro inhibitory activities of target compounds 8a-h against HDAC1 and HDAC6 (IC 50 , nM).
Molecules 2023, 28, 7323 3 of 14 deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.deprotection under TFA/CH2Cl2 condition and subsequent condensation reaction with 8methoxy-8-oxooctanoic acid through HATU, yielding the ester precursors 7a-h.Then, 7ah was converted to the final hydroxamate product 8a-h using aqueous hydroxylamine under basic conditions.Different electron-withdrawing or electron-donating substituents were introduced on two phenyls present in the capping part to explore the SAR.Moreover, the phenyl group was also replaced with an aromatic heterocycle such as thienyl or furyl.

HDAC1, 6 Activities and SAR Study of the Target Compounds
The target compounds 8a-h were screened against HDAC6 with sHDAC6i ACY1215 and nonselective SAHA as the positive controls.Considering specific and redundant functions of class I HDACs in the control of proliferation as well as potential toxicity [38], HDAC1 was chosen for selectivity evaluation.As displayed in Table 1, all eight compounds demonstrated low nanomolar HDAC6 activity and two-digital selectivity against HDAC1.The most potent 8f, with meta-OMe phenyls as the capping group, inhibited HDAC6 with an IC50 value of 6.4 nM and showed 48-fold selectivity against HDAC1, better than that of ACY1215.In addition, unsubstituted 8a also had an IC50 of 16.2 nM and 35-fold selectivity.The introduction of para-OMe phenyl (8c) maintained the potency.Although -CF3, -Me, or furyl were adopted, a slight decrease in HDAC6 inhibition was observed (8b, 8d, and 8h).For thienyl derivatives 8e and 8g, the position of the sulfur atom obviously affected the HDAC6 activity (25.7 nM vs. 54 nM, respectively).It seemed that the substituent on the phenyl cap was critical for enzymatic activity.8f, with the highest potency, was chosen for a detailed screening against other HDACs, including class I HDACs (HDAC2, 3, 8), HDAC 4, 5 (class IIa), and HDAC6 (class IIb) with ACY1215, SAHA, and TMP269 (a selective class IIa inhibitor) [39] as references.As demonstrated in Table 2, 8f shows highly selective inhibition (more than 48-fold over other subtypes) toward HDAC6, and its selectivity values were higher than those of reference compound ACY1215.The IC50 values of 8f against HDAC1-3 were 308 nM, 390 8f, with the highest potency, was chosen for a detailed screening against other HDACs, including class I HDACs (HDAC2, 3, 8), HDAC 4, 5 (class IIa), and HDAC6 (class IIb) with ACY1215, SAHA, and TMP269 (a selective class IIa inhibitor) [39] as references.As demonstrated in Table 2, 8f shows highly selective inhibition (more than 48-fold over other subtypes) toward HDAC6, and its selectivity values were higher than those of reference compound ACY1215.The IC50 values of 8f against HDAC1-3 were 308 nM, 390 8f, with the highest potency, was chosen for a detailed screening against other HDACs, including class I HDACs (HDAC2, 3, 8), HDAC 4, 5 (class IIa), and HDAC6 (class IIb) with ACY1215, SAHA, and TMP269 (a selective class IIa inhibitor) [39] as references.As demonstrated in Table 2, 8f shows highly selective inhibition (more than 48-fold over other subtypes) toward HDAC6, and its selectivity values were higher than those of 8f, with the highest potency, was chosen for a detailed screening against other HDACs, including class I HDACs (HDAC2, 3, 8), HDAC 4, 5 (class IIa), and HDAC6 (class IIb) with ACY1215, SAHA, and TMP269 (a selective class IIa inhibitor) [39] as references.As demonstrated in Table 2, 8f shows highly selective inhibition (more than 48-fold over other subtypes) toward HDAC6, and its selectivity values were higher than those of reference compound ACY1215.The IC 50 values of 8f against HDAC1-3 were 308 nM, 390 nM, and 411 nM, respectively.8f showed poor activity for HDAC4, 5 and 8.The results further validate the importance of tetrahydropyridopyrimidine with bulky capping groups to yield pronounced HDAC6 selective inhibition.

Western Blot Assay
To further determine the intracellular target specificity of 8f, human MM cell line RPMI-8226 was treated at concentrations of 1, 5, and 10 µM, along with the reference HDAC6i ACY1215 and pan-inhibitor SAHA at 10 µM (Figure 3).8f was able to increase the levels of acetylated α-tubulin in a dose-dependent manner while inducing only modest changes in the levels of acetylated histone 3 (H3), similar to those found for the reference HDAC6i ACY-1215 at 10 µM.As expected, the pan-active HDACi SAHA increased levels of both acetylated α-tubulin and acetylated histone H3 significantly compared to the vehicle.

Molecular Simulation
The representative 8a was docked into the human HDAC6 protein complex to elucidate the interaction model between these tetrahydropyridopyrimidines and the target protein.As outlined in Figure 4A, hydroxamate-Zn 2+ coordination was modeled with bidentate geometry, and the Zn 2+ −O distances are 2.4 and 1.8 Å for the OH and C=O groups, respectively.The side chain of His610 additionally accepted a hydrogen bond from the hydroxamate OH group.The aliphatic chain linker embeds into the channe between Phe620 and Phe680.Moreover, two phenyl substituents of 8a in the cap region were oriented into the crevice formed by Met682, Asp567, and Ser564 Tetrahydropyridopyrimidine scaffold as a proper connecting unit made the capping group of 8a match well with amino acids on the rim of the binding tunnel (Figure 4B).Fo

Molecular Simulation
The representative 8a was docked into the human HDAC6 protein complex to elucidate the interaction model between these tetrahydropyridopyrimidines and the target protein.
As outlined in Figure 4A, hydroxamate-Zn 2+ coordination was modeled with bidentate geometry, and the Zn 2+ −O distances are 2.4 and 1.8 Å for the OH and C=O groups, respectively.The side chain of His610 additionally accepted a hydrogen bond from the hydroxamate OH group.The aliphatic chain linker embeds into the channel between Phe620 and Phe680.Moreover, two phenyl substituents of 8a in the cap region were oriented into the crevice formed by Met682, Asp567, and Ser564.Tetrahydropyridopyrimidine scaffold as a proper connecting unit made the capping group of 8a match well with amino acids on the rim of the binding tunnel (Figure 4B).For compound 8f, its polar meta-OMe group improved the HDAC6 activity.For comparison, ACY-1215 was also docked into the HDAC6 crystallographic structure, and the superimposition of ACY-1215 and 8a was disclosed in Figure 4C.Both compounds occupied the same pocket and showed similar binding modes.
target protein.As outlined in Figure 4A, hydroxamate-Zn coordination was modeled with bidentate geometry, and the Zn 2+ −O distances are 2.4 and 1.8 Å for the OH and C=O groups, respectively.The side chain of His610 additionally accepted a hydrogen bond from the hydroxamate OH group.The aliphatic chain linker embeds into the channel between Phe620 and Phe680.Moreover, two phenyl substituents of 8a in the cap region were oriented into the crevice formed by Met682, Asp567, and Ser564.Tetrahydropyridopyrimidine scaffold as a proper connecting unit made the capping group of 8a match well with amino acids on the rim of the binding tunnel (Figure 4B).For compound 8f, its polar meta-OMe group improved the HDAC6 activity.For comparison, ACY-1215 was also docked into the HDAC6 crystallographic structure, and the superimposition of ACY-1215 and 8a was disclosed in Figure 4C.Both compounds occupied the same pocket and showed similar binding modes.

Antiproliferative Activities of Representative Compounds
Hematological tumors such as lymphoma, multiple myeloma, and chronic myeloid leukemia are more sensitive to HDAC inhibitors.Therefore, HL60 and RPMI-8226 tumor cells were used for antiproliferative biological tests of our compounds.Moreover, colon cancer cell HCT116 was also added to evaluate the antiproliferative effect for solid tumors of these tetrahydropyridopyrimidines.IC50 values of three representative compounds 8a, 8c, and 8f toward HL60 and RPMI-8226 cells range from 2.80 to 16.3 μM, which indicated that these tetrahydropyridopyrimidines tested kept the cell-based activity (Table 3).For solid tumor cells HCT116, all three analogs exhibited promising efficacy, especially for 8c

Antiproliferative Activities of Representative Compounds
Hematological tumors such as lymphoma, multiple myeloma, and chronic myeloid leukemia are more sensitive to HDAC inhibitors.Therefore, HL60 and RPMI-8226 tumor cells were used for antiproliferative biological tests of our compounds.Moreover, colon cancer cell HCT116 was also added to evaluate the antiproliferative effect for solid tumors of these tetrahydropyridopyrimidines.IC 50 values of three representative compounds 8a, 8c, and 8f toward HL60 and RPMI-8226 cells range from 2.80 to 16.3 µM, which indicated that these tetrahydropyridopyrimidines tested kept the cell-based activity (Table 3).For solid tumor cells HCT116, all three analogs exhibited promising efficacy, especially for 8c and 8f, with IC 50 s of 4.72 and 3.25 µM.This result rendered these new inhibitors valuable hits for applications beyond multiple myeloma.Then, 8f was submitted to NCI for antiproliferative evaluation against 59 different tumor cell lines.The cancer types of the NCI-60 program include leukemia, non-small cell lung cancer (NSCLC), colon cancer, CNS cancer, melanoma, ovary cancer, renal cancer, prostate cancer, and breast cancer.As shown in Table 4, 8f had an overall antiproliferative profile with percent inhibitions of 56 cell lines > 80% at 10 µM concentration.

6 IC50
values for enzymatic inhibition of HDAC1 and HDAC6 enzyme.We ran experiments in duplicate, SD < 15%.Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).

6 IC
50 values for enzymatic inhibition of HDAC1 and HDAC6 enzyme.We ran experiments in duplicate, SD < 15%.Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).

Molecules 2023, 28 , 7323 5 of 1 Figure 3 .
Figure 3. 8f increases the levels of acetylated α-tubulin in a dose-dependent manner in RPMI-8226 cells-densitometric analyses of Ac-H3 and Ac-α-tubulin.Cells were treated for 24 h with compounds, and then Western blotting analysis was performed.** p < 0.01, the *** p < 0.001 indicate comparison with the control group.

Figure 3 .
Figure 3. 8f increases the levels of acetylated α-tubulin in a dose-dependent manner in RPMI-8226 cells-densitometric analyses of Ac-H3 and Ac-α-tubulin.Cells were treated for 24 h with compounds, and then Western blotting analysis was performed.** p < 0.01, the *** p < 0.001 indicates comparison with the control group.

Figure 4 .
Figure 4. (A) Binding model of 8a (yellow) in the catalytic pocket of human HDAC6 (PDB code: 5EDU).(B) Surface map of 8a in the catalytic pocket of HDAC6 (grey).(C) The superimposition of ACY-1215 and 8a in HDAC6.Key residues were labeled in green.The hydrogen bonds were labeled in blue.Zinc ion was shown in brown.

Figure 4 .
Figure 4. (A) Binding model of 8a (yellow) in the catalytic pocket of human HDAC6 (PDB code: 5EDU).(B) Surface map of 8a in the catalytic pocket of HDAC6 (grey).(C) The superimposition of ACY-1215 and 8a in HDAC6.Key residues were labeled in green.The hydrogen bonds were labeled in blue.Zinc ion was shown in brown.
IC50 values for enzymatic inhibition of HDAC1 and HDAC6 enzyme.We ran experiments in duplicate, SD < 15%.Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).
IC 50 values for enzymatic inhibition of HDAC enzymes.We ran experiments in duplicate, SD < 15%.Assays were performed by Reaction Biology Corporation (Malvern, PA, USA).ND = not determined.