Marine Cytotoxin Santacruzamate A Derivatives as Potent HDAC1-3 Inhibitors and Their Synergistic Anti-Leukemia Effects with Venetoclax

Acute myeloid leukemia (AML) is a hematologic malignancy characterized by infiltration of the blood and bone marrow, exhibiting a low remission rate and high recurrence rate. Current research has demonstrated that class I HDAC inhibitors can downregulate anti-apoptotic proteins, leading to apoptosis of AML cells. In the present investigation, we conducted structural modifications of marine cytotoxin Santacruzamate A (SCA), a compound known for its inhibitory activity towards HDACs, resulting in the development of a novel series of potent class I HDACs hydrazide inhibitors. Representative hydrazide-based compound 25c exhibited concentration-dependent induction of apoptosis in AML cells as a single agent. Moreover, 25c exhibited a synergistic anti-AML effect when combined with Venetoclax, a clinical Bcl-2 inhibitor employed in AML therapy. This combination resulted in a more pronounced downregulation of anti-apoptotic proteins Mcl-1 and Bcl-xL, along with a significant upregulation of the pro-apoptotic protein cleaved-caspase3 and the DNA double-strand break biomarker γ-H2AX compared to monotherapy. These results highlighted the potential of 25c as a promising lead compound for AML treatment, particularly when used in combination with Venetoclax.


Introduction
Acute myeloid leukemia (AML) is a heterogeneous blood disease caused by genetic mutations that give myeloid blast cells a selective growth advantage and cause them to fail to differentiate into normal blood cells.This disease is one of the most common types in the adult leukemia family, and it predominantly affects the elderly.Among the therapeutic drugs of AML, the most common standard chemotherapy combination regimen is Cytarabine/Daunorubicin (3 + 7), which involves three days of oral administration of medications such as Mitoxantrone or Idarubicin, followed by seven days of intravenous infusion of maintenance chemotherapy, and it is often used in patients < 60 years who are in good health [1].Molecularly targeted drugs such as Bcl-2 inhibitors, FLT3 inhibitors and IDH inhibitors also show promising clinical efficacy [2,3].Pre-clinical and clinical studies indicate that Bcl-2 inhibitors (e.g., Venetoclax) can successfully restart the unregulated apoptosis process of malignant cells with high safety, and with good synergy when combined with drugs targeting multiple molecular pathways [4], thus showing great research potential.
Tumorigenesis factors of hematological tumor diseases are often characterized by genetic aberrations that disrupt normal cellular functions, and the pathogenesis of AML is closely related to epigenetic perturbations [4][5][6].Histone deacetylases (HDACs), a group of epigenetic anti-cancer targets, have garnered significant attention in recent years [7].So far, a total of 18 isoforms have been found in humans, and they can be divided into two categories according to different catalytic sites: Zn 2+ -dependent proteins and NAD +dependent proteins.The former consists of 11 subtypes, which are divided into three classes: class I (HDAC1, 2, 3, 8), class II (HDAC4, 5, 6, 7, 9, 10) and class IV (HDAC11).The latter consists of seven subtypes, such as class III (sirtuin 1-7) [8].Among those, class I isoforms HDAC1-3 play a key role in regulating the acetylation levels of histones, thus modifying the chromatin structure and its accessibility for gene transcription, which is more significant in solid tumors and hematological malignancies [5].
Recent studies reveal that class I HDACs are generally overexpressed in AML cells.Depletion of HDAC1/2 promotes AML cell apoptosis, and the loss of HDAC3 can inhibit cell proliferation and promote cell differentiation of AML cells [9].Previous studies have shown that class I HDAC inhibitor (HDACI) monotherapy downregulates anti-apoptotic proteins, leading to the activation of pro-caspase3 and triggering apoptosis in AML cells [10].Our previous study indicated that class I HDACIs could induce G1/S cell cycle arrest leading to cell death by a p53-dependent pathway [11].These findings demonstrate the potential of class I HDACI monotherapy in the treatment of AML.A number of class I HDACIs are currently in development, such as Entinostat [12] and Chidamide [13].It has been found that class I HDACIs can also promote the efficacy of other drugs and have a strong effect in reversing drug resistance.Chen et al. found that the downregulation of Mcl-1 and Bcl-xL by Chidamide fully activated the pro-apoptotic gene Bim to induce apoptosis.And when combined with Venetoclax, Chidamide might have a synergistic effect and overcome the effect of drug resistance for Venetoclax in AML cells [14].Moreover, Chidamide was used in the treatment of three R/R AML patients who failed to respond to the combination regimen of Venetoclax and Azacitidine, successfully achieving complete remission after relapse and greatly extending the life cycle of the patients [15].Therefore, it is very necessary to develop novel class I HDACIs and explore the combination of multiple drugs.
The ocean is regarded as an important treasure house of natural medicine.Santacruzamate A (SCA), a cytotoxin isolated from Panamanian Pacific tuft cyanobacteria, was found in previous studies to have potential inhibitory activity against HDAC2 [16].According to the virtual docking model of SCA and HDAC2 constructed by Gromek et al., the chemical structure of SCA corresponded with the pharmacophore model of traditional HDAC inhibitors, which is composed of the Cap, Linker and zinc binding group (ZBG) [17].The group interacting with the protein surface is named "Cap", the one binding Zn 2+ at the bottom of the catalytic site is called "ZBG" and the group connecting these two parts in the middle hydrophobic region is termed "Linker" [11].To a certain extent, the selectivity and potency of HDAC inhibitors are significantly dependent on the structural characteristics of their ZBGs [18].At present, the ZBGs used in class I HDACIs mainly include hydroxamic acids, phenylamides and hydrazides.The docking results indicated that SCA formed monodentate chelation with zinc ions of HDAC2 [17].In a previous study, we found that hydrazides as ZBG exhibited a higher titer, higher class I HDAC (1 and 3) selectivity and better pharmacokinetics than hydroxamic acids and phenylamides [11,19,20].It is hypothesized that modifying the ZBG to form bidentate chelates may increase the HDAC inhibitory activity of SCA.Consequently, this study aimed to use the marine-derived compound SCA as a hit compound, fix ZBG as a hydrazide structure and modify Cap and Linker to obtain a new series of class I HDACIs.

Compound Design
As reported in the original document, SCA has good inhibitory activity against class I HDACs, especially HDAC2, with an IC50 value of 0.112 nM [16].However, currently designed and synthesized SCA derivatives did not significantly enhance the inhibitory

Compound Design
As reported in the original document, SCA has good inhibitory activity against class I HDACs, especially HDAC2, with an IC 50 value of 0.112 nM [16].However, currently designed and synthesized SCA derivatives did not significantly enhance the inhibitory activity and selectivity of HDACs, and they regrettably showed proliferation inhibition ability at the micromolar level in a variety of cancer cell lines [17,21].Gromek et al. developed a docking model of HDAC2 and SCA and discovered that the modification of the carbamate structure of SCA might potentially enhance its HDAC inhibitory activity [16].Specifically, the presence of a terminal oxygen in SCA limited its ability to form optimal chelation interactions, hindering activity promotion.Additionally, the planar structure of the terminal urethane extension might impact the binding conformation.Furthermore, SCA might not form hydrogen bonds with key residues like Asp104, His146 and His145 in the model [17].Therefore, the substitution of the end ZBG of SCA is crucial for the initial optimization of the compounds.As mentioned earlier, considering the hydrazide group has shown higher activity and selectivity against HDAC1/3, we conducted a docking of SCA and HDAC3 to further verify the above conclusions (Figure 1).The results were consistent, so we decided to replace the ZBG of the SCA with a hydrazide group.Our group previously explored the structure-activity relationships (SARs) of N-substituted hydrazidebased HDACIs and found that three-carbon linker chain monosubstitution hydrazide showed the best selectivity and inhibitory effect [11].In this study, we designed compounds with N-propyl acetyl hydrazine.Since the modification of Cap and Linker groups could potentially improve the inhibitory activity and selectivity, our initial approach tried to compare the effect of the amide order in Cap and Linker on activity and then optimize the Cap groups and Linker groups (Figure 1).These newly designed compounds were expected to have the following improvements: (1) increased selectivity for class I HDACs and (2) enhanced activity against class I HDACs and anti-leukemia activity compared to the parent molecule SCA.The initial step involves an examination of the impact of the sequence of amide bonds connecting the Cap to the Linker and the optimal Linker length, which is analyzed through two distinct scenarios.Compound 9b is achieved by directly modifying the ethyl carbamate of SCA with N-propyl acetyl hydrazine.Subsequent variations in the length of the Linker alkyl chain, while keeping the Cap and ZBG constant, result in the synthesis of compounds 9a and 9c-9f (Table 1).Furthermore, altering the sequence of amide bonds within the Cap region, while maintaining the alkyl chain length, leads to compound 14b, while adjusting the length of the Linker alkyl chain results in compounds 14a and 14c-14f (Table 1).Due to the pronounced enhancement effect of the hydrazide group on the activity of HDAC1-3, especially HDAC1 and HDAC3, recombinant HDAC1/3 enzymes were The initial step involves an examination of the impact of the sequence of amide bonds connecting the Cap to the Linker and the optimal Linker length, which is analyzed through two distinct scenarios.Compound 9b is achieved by directly modifying the ethyl carbamate of SCA with N-propyl acetyl hydrazine.Subsequent variations in the length of the Linker alkyl chain, while keeping the Cap and ZBG constant, result in the synthesis of compounds 9a and 9c-9f (Table 1).Furthermore, altering the sequence of amide bonds within the Cap region, while maintaining the alkyl chain length, leads to compound 14b, while adjusting the length of the Linker alkyl chain results in compounds 14a and 14c-14f (Table 1).Due to the pronounced enhancement effect of the hydrazide group on the activity of HDAC1-3, especially HDAC1 and HDAC3, recombinant HDAC1/3 enzymes were utilized in the screening of the newly designed compounds for initial enzymatic inhibitory activity.The initial screening concentrations were 100 nM and 10 nM, respectively.In terms of the hydrazide sequence, phenethylamine-capped compounds 9a-9f generally exhibited superior enzymatic activity compared to phenacetyl-capped compounds (14a-14f).The overall enzymatic inhibition activity of both series of compounds increased initially with the lengthening of the Linker, followed by a decrease.Notably, compounds 9e and 14e displayed the highest activity when the Linker was an alkyl chain with eight carbons.In addition, in our enzyme inhibitory activity determination, SCA showed no activity in the corresponding system, which was inconsistent with the original literature reports [16].However, some literature reports later confirmed that the inhibitory activity of synthetic SCA was relatively weak [17,22].The initial step involves an examination of the impact of the sequence of amide bonds connecting the Cap to the Linker and the optimal Linker length, which is analyzed through two distinct scenarios.Compound 9b is achieved by directly modifying the ethyl carbamate of SCA with N-propyl acetyl hydrazine.Subsequent variations in the length of the Linker alkyl chain, while keeping the Cap and ZBG constant, result in the synthesis of compounds 9a and 9c-9f (Table 1).Furthermore, altering the sequence of amide bonds within the Cap region, while maintaining the alkyl chain length, leads to compound 14b, while adjusting the length of the Linker alkyl chain results in compounds 14a and 14c-14f (Table 1).Due to the pronounced enhancement effect of the hydrazide group on the activity of HDAC1-3, especially HDAC1 and HDAC3, recombinant HDAC1/3 enzymes were utilized in the screening of the newly designed compounds for initial enzymatic inhibitory activity.The initial screening concentrations were 100 nM and 10 nM, respectively.In terms of the hydrazide sequence, phenethylamine-capped compounds 9a-9f generally exhibited superior enzymatic activity compared to phenacetyl-capped compounds (14a-14f).The overall enzymatic inhibition activity of both series of compounds increased initially with the lengthening of the Linker, followed by a decrease.Notably, compounds 9e and 14e displayed the highest activity when the Linker was an alkyl chain with eight carbons.In addition, in our enzyme inhibitory activity determination, SCA showed no activity in the corresponding system, which was inconsistent with the original literature reports [16].However, some literature reports later confirmed that the inhibitory activity of synthetic SCA was relatively weak [17,22].The initial step involves an examination of the impact of the sequence of amide bonds connecting the Cap to the Linker and the optimal Linker length, which is analyzed through two distinct scenarios.Compound 9b is achieved by directly modifying the ethyl carbamate of SCA with N-propyl acetyl hydrazine.Subsequent variations in the length of the Linker alkyl chain, while keeping the Cap and ZBG constant, result in the synthesis of compounds 9a and 9c-9f (Table 1).Furthermore, altering the sequence of amide bonds within the Cap region, while maintaining the alkyl chain length, leads to compound 14b, while adjusting the length of the Linker alkyl chain results in compounds 14a and 14c-14f (Table 1).Due to the pronounced enhancement effect of the hydrazide group on the activity of HDAC1-3, especially HDAC1 and HDAC3, recombinant HDAC1/3 enzymes were utilized in the screening of the newly designed compounds for initial enzymatic inhibitory activity.The initial screening concentrations were 100 nM and 10 nM, respectively.In terms of the hydrazide sequence, phenethylamine-capped compounds 9a-9f generally exhibited superior enzymatic activity compared to phenacetyl-capped compounds (14a-14f).The overall enzymatic inhibition activity of both series of compounds increased initially with the lengthening of the Linker, followed by a decrease.Notably, compounds 9e and 14e displayed the highest activity when the Linker was an alkyl chain with eight carbons.In addition, in our enzyme inhibitory activity determination, SCA showed no activity in the corresponding system, which was inconsistent with the original literature reports [16].However, some literature reports later confirmed that the inhibitory activity of synthetic SCA was relatively weak [17,22].Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.α Inhibition rate values are the mean of three independent experiments ± the standard error of the mean (SEM).
Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.α Inhibition rate values are the mean of three independent experiments ± the standard error of the mean (SEM).
Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.α Inhibition rate values are the mean of three independent experiments ± the standard error of the mean (SEM).
Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.
Subsequently, given the high flexibility of the Linker of the octyldiacyl group, we endeavored to create compounds 9g-9m through a rigid modification of 9e, incorporating benzene rings and double bonds as structural constraints.In this series, the inhibitory activity of 9j-9l, modified by an isoelectronic body, ware nearly double that of 9e, with an inhibition rate toward HDAC3 exceeding 50% at 10 nM.However, the impact of double bonds and sequence on enzyme activity was deemed insignificant, as demonstrated by 9j and 9k.Based on the retention of the benzene ring and double bond in compound 9k, the original amide bond was substituted with an amino group to obtain compound 25c.Remarkably, the activity of compound 25c surpassed that of the previously described compounds, with an inhibition rate exceeding 95% toward HDAC3 at a concentration of 10 nM.This finding underscored the essential role of the amino group in enzyme inhibition.Consequently, the structural framework of compound 25c was retained while the Cap group was modified (Table 2).Consequently, the structural framework of compound 25c was retained while the Cap group was modified (Table 2).Consequently, the structural framework of compound 25c was retained while the Cap group was modified (Table 2).Consequently, the structural framework of compound 25c was retained while the Cap group was modified (Table 2).Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately 67.9 ± 1. 4 23 Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately Initially, compound 25a, featuring an aniline group as the Cap group, exhibited inferior enzyme inhibition activity compared to compounds 25b and 25c, which respectively featured benzylamine and phenylethylamine groups as Cap groups.Additionally, it was observed that excessively elongating the Cap group led to undesired flexible transformations, underscoring the importance of maintaining an optimal Cap group length.In order to investigate the impact of a larger Cap group volume, the phenyl groups in compounds 25a and 25b were substituted with naphthalene groups to yield compounds 25f and 25g, respectively.However, the inhibitory activity of the latter was approximately might not enhance the compound's activity.The synthesis of compounds 25e and 30 through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).might not enhance the compound's activity.The synthesis of compounds 25e and 30 through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).might not enhance the compound's activity.The synthesis of compounds 25e and 30 through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).might not enhance the compound's activity.The synthesis of compounds 25e and 30 through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).might not enhance the compound's activity.The synthesis of compounds 25e and 30 through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).through cyclization of the phenylethylamine group of 25c resulted in lesser variance in activity for the former compound, while the latter activity had significantly reduced.Furthermore, a comparison of compounds 19a-19d, obtained by altering the Cap group of 9k, reiterated that a bulky Cap group was not ideal for enzyme binding.These findings hold significant implications for the strategic development of hydrazine HDACIs (Table 3).Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed rela- Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed rela- Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed rela- Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed rela- Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC 50 s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.Based on the preliminary screening results mentioned above, we comprehensively selected 10 compounds (9j, 9k, 9l, 25a, 25b, 25c, 25d, 25e, 25g and 30) to determine their IC50s toward HDAC1-3 (Table 4).The results were largely as anticipated and consistent with the findings from the initial screening.Intuitively, 9j, 25b, 25c and 25e showed relatively higher HDAC1-3 activity.Finally, compounds 25b, 25c, 25e and 25g were chosen for subsequent cell-based activity screening.

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selec-

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selec-

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selective inhibitor Entinostat was chosen as a positive control.

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selective inhibitor Entinostat was chosen as a positive control.

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selective inhibitor Entinostat was chosen as a positive control.Table 5 summarized the IC50 of 37.6 ± 2.6 3.8 ± 1.1 77.9 ± 1.6 α IC 50 values are the mean of three independent experiments ± SEM.

Cell Proliferation Inhibition
We first examined the anti-proliferative activity of 25b, 25c, 25e and 25g against four AML-associated cell lines (MV4-11, THP-1, HL60 and Kasumi-1 cells).The cell viability after compound treatment was evaluated by resorufin intensity.The class I HDACs selective inhibitor Entinostat was chosen as a positive control.Table 5 summarized the IC 50 of the selected compounds in four cell lines.The selected compounds could inhibit the growth of the above four kinds of cells.Among the tested compounds, 25c displayed the best anti-proliferative activity, with IC 50 values of 337.6-480.3nM.However, no inhibitory activity was detected in the synthesized SCA (Table 5).α IC 50 values are the mean of three independent experiments ± SEM.

HDAC Isoform Selectivity of 25c
Enzymatic and cell-based assays indicate that 25c was the most potent HDACI among all of the compounds we designed.Thus, we chose 25c for further biological study.To assess the HDAC isoforms selectivity of 25c, we determined its IC 50 value for other HDAC subtypes (HDAC4, 5, 6, 7, 8, 9 and 11) (Table 6).As there were no appropriate substrates for HDAC10, the inhibitory activity toward this isotype was not determined.The inhibitory activity of compound 25c for HDAC4, 5, 6, 7, 9 and 11 exceeded 10,000 nM, and its inhibitory activity for HDAC8 was 4109 nM, so 25c was a class I HDAC selective inhibitor.We also conducted Western blot analysis to validate the selectivity of 25c.The results indicated that compound 25c significantly elevated the level of acetylated histone H3 (AcHH3) and acetylated histone H4 (AcHH4), aligning with the inhibitory effects on HDAC1, 2 and 3. Surprisingly, the level of acetyl-tubulin increased even though 25c did not inhibit HDAC6, a protein responsible for the deacetylation of tubulin.Our hypothesis conjectured that the upregulation of Ac-tubulin, a substrate of HDAC6, could be attributed to the transcriptional inhibition of HDAC6 (Figure 2).Class II b HDAC6 >10,000 Class IV HDAC11 >10,000 α IC50 values are the mean of three independent experiments ± SEM.

25c and Venetoclax Synergistically Inhibit Cell Proliferation
Venetoclax is a drug used to treat specific types of leukemia and lymphoma, known as a Bcl-2 inhibitor.Bcl-2 is a protein that plays a key role in regulating apoptosis.In some types of leukemia and lymphoma, cancer cells evade the natural mechanism of apoptosis by overexpressing Bcl-2, leading to the oversurvival of cancer cells [23].During Venetoclax treatment, acquired drug resistance is a common phenomenon, and the compensatory high expression of Mcl-1 may be one of the causes of drug resistance in AML cells [24].According to previous reports, both class I HDAC inhibitors and Venetoclax could induce apoptosis and perform a synergistic effect [24].As the IC 50 of 25c measured in the above experiments was 340.1 ± 82.5 nM in the MV4-11 cell line, showing the more obvious effect among the four cell lines, we selected the MV4-11 cell line for the combination cell assay and analyzed the data according to the Chou-Talalay method [25].The results indicated that the inhibitory effect of the combination of 25c with Venetoclax was higher than the inhibitory effect of the single agents.When the concentration of 25c was higher than 0.13 µM, the CI values of Venetoclax were all less than 1, indicating that the two drugs had a strong synergistic effect (Figure 3).and analyzed the data according to the Chou-Talalay method [25].The results indicated that the inhibitory effect of the combination of 25c with Venetoclax was higher than the inhibitory effect of the single agents.When the concentration of 25c was higher than 0.13 µM, the CI values of Venetoclax were all less than 1, indicating that the two drugs had a strong synergistic effect (Figure 3).

Venetoclax and 25c Synergistically Promote Apoptosis in AML Cell
According to previous studies and our experiments, both 25c and Venetoclax could induce apoptosis.In order to further explore the synergistic effect of combination drugs, we performed flow cytometry to detect apoptosis of single and combination drugs by analyzing Annexin V levels and propidium iodide permeability.MV4-11 cells were exposed to 25c and Venetoclax individually and in combination for a duration of 24 h.The results indicated a notable decrease in the apoptosis of MV4-11 cells when treated with the combined treatment compared to either drug alone.The observed rise in apoptosis rate was attributed to the synergistic effect of the drug combination, rather than the sum effect of the individual drugs (Figure 4).
Next, we exposed the MV4-11 cell line to the same drug concentrations and time as the apoptosis experiment and then measured the cell cycle distribution using flow cytometry.Numerous studies have confirmed that HDACIs induce cell cycle arrest in the G0/G1

Venetoclax and 25c Synergistically Promote Apoptosis in AML Cell
According to previous studies and our experiments, both 25c and Venetoclax could induce apoptosis.In order to further explore the synergistic effect of combination drugs, we performed flow cytometry to detect apoptosis of single and combination drugs by analyzing Annexin V levels and propidium iodide permeability.MV4-11 cells were exposed to 25c and Venetoclax individually and in combination for a duration of 24 h.The results indicated a notable decrease in the apoptosis of MV4-11 cells when treated with the combined treatment compared to either drug alone.The observed rise in apoptosis rate was attributed to the synergistic effect of the drug combination, rather than the sum effect of the individual drugs (Figure 4).
Next, we exposed the MV4-11 cell line to the same drug concentrations and time as the apoptosis experiment and then measured the cell cycle distribution using flow cytometry.Numerous studies have confirmed that HDACIs induce cell cycle arrest in the G0/G1 phase in vitro [26].As can be seen from Figure 5, the proportion of the S phase and G2/M phase decreased in cells treated with 25c, while the proportion of the G0/G1 phase increased.The results showed that 25c could block MV4-11 cells in the G0/G1 phase and exert the effect of cell cycle arrest and cell proliferation inhibition.However, the combination of the two drugs could induce sub-G1 cell death and apoptosis significantly (Figure 5).phase in vitro [26].As can be seen from Figure 5, the proportion of the S phase and G2/M phase decreased in cells treated with 25c, while the proportion of the G0/G1 phase increased.The results showed that 25c could block MV4-11 cells in the G0/G1 phase and exert the effect of cell cycle arrest and cell proliferation inhibition.However, the combination of the two drugs could induce sub-G1 cell death and apoptosis significantly (Figure 5).

Effects of 25c and Venetoclax on Transcriptomes in Multiple Genes and Signaling Pathways in MV4-11 Cells
No substantial alterations were noted in the level of cleaved-caspase3 at concentrations of 30 nM and 90 nM in the 25c monotherapy group, but the level in the Venetoclax phase in vitro [26].As can be seen from Figure 5, the proportion of the S phase and G2/M phase decreased in cells treated with 25c, while the proportion of the G0/G1 phase increased.The results showed that 25c could block MV4-11 cells in the G0/G1 phase and exert the effect of cell cycle arrest and cell proliferation inhibition.However, the combination of the two drugs could induce sub-G1 cell death and apoptosis significantly (Figure 5).

Effects of 25c and Venetoclax on Transcriptomes in Multiple Genes and Signaling Pathways in MV4-11 Cells
No substantial alterations were noted in the level of cleaved-caspase3 at concentrations of 30 nM and 90 nM in the 25c monotherapy group, but the level in the Venetoclax  No substantial alterations were noted in the level of cleaved-caspase3 at concentrations of 30 nM and 90 nM in the 25c monotherapy group, but the level in the Venetoclax monotherapy group was slightly higher; however, a heightened level of cleaved-caspase3 was observed in the combined treatment group which was higher than any single drug effect, suggesting that the combination led to an increased apoptotic effect.γ-H2AX protein is recognized as a specific biomarker following DNA double-strand breaks, thereby suggesting an escalation in DNA damage subsequent to drug combination.The levels of γ-H2AX protein in the two monotherapy groups surpassed those of the control group, leading to enhanced efficacy in the combined treatment group.Furthermore, the expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in the Bcl-2 protein family was significantly re-Mar.Drugs 2024, 22,250 duced in the combination treatment cohort, which may be involved in apoptosis induction and cell resistance mechanisms (Figure 6).monotherapy group was slightly higher; however, a heightened level of cleaved-caspase3 was observed in the combined treatment group which was higher than any single drug effect, suggesting that the combination led to an increased apoptotic effect.γ-H2AX protein is recognized as a specific biomarker following DNA double-strand breaks, thereby suggesting an escalation in DNA damage subsequent to drug combination.The levels of γ-H2AX protein in the two monotherapy groups surpassed those of the control group, leading to enhanced efficacy in the combined treatment group.Furthermore, the expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in the Bcl-2 protein family was significantly reduced in the combination treatment cohort, which may be involved in apoptosis induction and cell resistance mechanisms (Figure 6).

Study on Metabolic Stability of 25c In Vitro
After verifying the in vitro antitumor activity of 25c and its synergistic effect in combination with Venetoclax, we conducted in vitro stability studies.25c remained relatively stable in artificial gastric juice, and degradation occurred in artificial intestinal juice and rat plasma, beginning after about 8 h, with degradation of 12.52% and 35.60%, respectively (Figure 7).

Study on Metabolic Stability of 25c In Vitro
After verifying the in vitro antitumor activity of 25c and its synergistic effect in combination with Venetoclax, we conducted in vitro stability studies.25c remained relatively stable in artificial gastric juice, and degradation occurred in artificial intestinal juice and rat plasma, beginning after about 8 h, with degradation of 12.52% and 35.60%, respectively (Figure 7).
leading to enhanced efficacy in the combined treatment group.Furthermore, the expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in the Bcl-2 protein family was significantly reduced in the combination treatment cohort, which may be involved in apoptosis induction and cell resistance mechanisms (Figure 6).

Study on Metabolic Stability of 25c In Vitro
After verifying the in vitro antitumor activity of 25c and its synergistic effect in combination with Venetoclax, we conducted in vitro stability studies.25c remained relatively stable in artificial gastric juice, and degradation occurred in artificial intestinal juice and rat plasma, beginning after about 8 h, with degradation of 12.52% and 35.60%, respectively (Figure 7).

Discussion
In this study, we modified the structure of SCA through the substitution of the original ZBG with hydrazide group and modifications to the Cap and Linker components.The designed and synthesized compounds exhibited significantly improved HDAC inhibitory activity, with compound 25c demonstrating the highest potency in both HDAC enzyme inhibition and cell-based antitumor assays.Compound 25c was identified as a class I HDAC selective inhibitor, displaying IC 50 values of 28.1 nM, 134.9 nM and 2.4 nM against HDAC1, 2 and 3, respectively.Furthermore, the IC 50 value of 25c against four AML cell lines ranged from 337.6 to 480.3 nM, indicating its potential anti-AML activity in vitro.Flow cytometry and Western blot analysis revealed that 25c facilitated the cleavage of caspase 3, resulting in apoptosis and inhibition of cell proliferation in MV4-11 cells.
Additionally, the co-administration of Venetoclax demonstrated a synergistic enhancement of anti-proliferative activity in AML cell lines, as evidenced by the calculated CI value at specific concentrations.When used in combination with Venetoclax, 25c demonstrated potentially and synergistically enhanced cytotoxicity.Additionally, Figures 4 and 5 clearly show an increase in the sub-G1 cell population, indicating that the combined treatment of the two drugs synergistically induced apoptosis, particularly promoting late apoptosis in the MV4-11 cell line.Furthermore, the expression levels of γH2AX, Mcl-1 and Bcl-xL decreased following the combination of 25c and Venetoclax.The former was associated with DNA damage, while the latter was linked to the inhibition of apoptosis; however, the specific underlying mechanism remained unclear.
Furthermore, prior research has indicated that elevated levels of Mcl-1/Bcl-xL may serve as a contributing factor to drug resistance to Venetoclax.It has been observed that 25c has the potential to counteract this resistance, thus implying that 25c could potentially mitigate the issue of drug resistance to Venetoclax.

General Chemistry
All chemical solvents and reagents were purchased from chemical vendors, such as Bidepharm, Aladdin, Macklin, Energy and Leyan, and used as received without further purification unless otherwise noted. 1 H NMR and 13 C NMR data were collected in DMSOd 6 /CDCl 3 using a JMTC-400 MHz (Japan Superconductor Technology, Inc, Takatsukdai, Japan) instrument or an Agilent One Probe-500 MHz spectrometer (Agilent Technologies UK Ltd, Oxford, England) with TMS as an internal standard.Chemical shifts (δ) and coupling constants (J) were reported in parts per million (ppm) and hertz (Hz), respectively.Mass spectral data were collected on a Waters Acquity QDa mass detector (Waters, Singapore and Ireland).All target compounds were >95% pure by HPLC analysis, performed on an Agilent 1260 Infinity II HPLC instrument (Agilent Technologies, Waldbronn, Germany) using an Agilent 5 TC-C18 (2) C18 column (5 µm, 4.6 mm × 250 mm) using an equivalent method of potassium phosphate buffer/acetonitrile.

Microdilution Checkerboard Assay
The inhibition effect of 25c combined with Venetoclax on the proliferation of MV4-11 cells was determined by microdilution checkerboard assay.CompuSyn software was used to calculate the combined action index of the two compounds, and the cell inhibition rates of the two compounds acting alone and the combined action were input to calculate the combination index (CI) to evaluate the synergistic effect of the drugs.A CI value less than 1 indicates a synergistic effect, a CI value equal to 1 indicates an additive effect and a CI value greater than 1 indicates an antagonistic effect.MV4-11 cells were inoculated in a 12-well plate at a density of 1 × 10 6 cells per well.After incubation overnight, cells were treated with different concentrations of 25c and Venetoclax alone or in combination.After 24 h, cells were collected, washed once with 1 mL PBS, re-suspended with 300 µL 1× binding buffer, and filtered through a cell filter to prepare a single-cell suspension.The cells were stained with AnnexinV-FITC 5 µL and propidium iodide (Absin, China) 5 µL and incubated at room temperature for 15 min, and apoptosis was analyzed by BD Accuri C6 Plus flow cytometry (BD Accuri C6, Becton Dickinson, USA).Data were analyzed by FlowJo for Windows V10.8.1 software.

Western Blot Analysis
MV4-11 cells were inoculated into 12-well plates at a density of 800,000 cells/mL.After incubation overnight, each well was treated with different concentrations of 25c and Venetoclax alone or in combination.After treatment for 24 h, the cells were collected and washed with pre-cooled PBS.A protease inhibitor (cocktail) and RIPA buffer were added for cell lysis.The decomposed solution was centrifuged at 12,000 rpm for 15 min at 4 • C, and the extracted protein was located in the supernatant.The proteins in each sample were denatured by heating at 100 • C in loading buffer (5×) for 10 min.The same amount of proteins in each sample was separated by SDS-PAGE and then electrotransferred to a PVDF membrane (Merck Millipore, Billerica, MA, USA) and enclosed in 5% skim milk powder for 1 h.The primary antibody diluted with an immune response booster solution was incubated in a shaking bed at 4 • C overnight, and the horseradish (HRP)-conjugated second antibody diluted with 5% skim milk powder was incubated at room temperature for 1 h on the second day.After the PVDF membrane was washed with TBST, the target protein bands could be detected with chemiluminescent reagents (Millipore, Billerica, MA, USA) and visualized in the Tanon 5200 + fluorescence imager.

Molecular Docking against HDAC3
Docking sites were defined using Schrodinger, and the simulations used a rigid receptor and triangle matcher methodology for ligand site placement with Schrodinger scoring.One hundred fifteen poses were calculated per binding site.Structural files included PDB 4A69 for HDAC3.

Figure 1 .
Figure 1.Basis of compound design.(a) Docking pose of SCA (green) in the binding site of HDAC3 (PDB: 4A69); (b) chemical structures of the designed analogs of SCA.

Figure 1 .
Figure 1.Basis of compound design.(a) Docking pose of SCA (green) in the binding site of HDAC3 (PDB: 4A69); (b) chemical structures of the designed analogs of SCA.

Figure 2 .
Figure 2. Western blot analysis of the enzyme substrates that interact with 25c in MV4-11 cells.(a) Western blot analysis of AcHH3(Lys27), AcHH4, Ac-tubulin, HDAC6 and α-tubulin in MV4-11 cells after treatment of compound 25c and SCA at the concentrations of 0.2, 0.4 and 200 µM for 24 h; (b) The expression levels of AcHH3(Lys27), AcHH4, Ac-tubulin and α-tubulin were quantified.Data are shown as mean ± SEM from three independent experiments.

Figure 2 .
Figure 2. Western blot analysis of the enzyme substrates that interact with 25c in MV4-11 cells.(a) Western blot analysis of AcHH3(Lys27), AcHH4, Ac-tubulin, HDAC6 and α-tubulin in MV4-11 cells after treatment of compound 25c and SCA at the concentrations of 0.2, 0.4 and 200 µM for 24 h; (b) The expression levels of AcHH3(Lys27), AcHH4, Ac-tubulin and α-tubulin were quantified.Data are shown as mean ± SEM from three independent experiments.

Figure 3 .
Figure 3. Combination index (CI) values for 25c and Venetoclax after treatment for 72 h.(a) Inhibition rate and CI for the combination of 25c and Venetoclax after treatment for 72 h.(b) Fraction affected and CI for 25c and Venetoclax after treatment for 72 h.Data were analyzed using CompuSyn Software.CIs of <1, =1 and >1 indicate synergism, additive effect and antagonism, respectively.

Figure 3 .
Figure 3. Combination index (CI) values for 25c and Venetoclax after treatment for 72 h.(a) Inhibition rate and CI for the combination of 25c and Venetoclax after treatment for 72 h.(b) Fraction affected and CI for 25c and Venetoclax after treatment for 72 h.Data were analyzed using CompuSyn Software.CIs of <1, =1 and >1 indicate synergism, additive effect and antagonism, respectively.

2. 7 .
Effects of 25c and Venetoclax on Transcriptomes in Multiple Genes and Signaling Pathways in MV4-11 Cells

Figure 6 .
Figure 6.Changes in Bcl-2 family apoptotic regulatory proteins and apoptotic pathway proteins detected by Western blot.

Figure 7 .
Figure 7. Stability of compound 25c in rat plasma, artificial gastroenteric fluid and artificial intestinal fluid within 24 h.

Figure 6 .
Figure 6.Changes in Bcl-2 family apoptotic regulatory proteins and apoptotic pathway proteins detected by Western blot.

Figure 6 .
Figure 6.Changes in Bcl-2 family apoptotic regulatory proteins and apoptotic pathway proteins detected by Western blot.

Figure 7 .
Figure 7. Stability of compound 25c in rat plasma, artificial gastroenteric fluid and artificial intestinal fluid within 24 h.

Figure 7 .
Figure 7. Stability of compound 25c in rat plasma, artificial gastroenteric fluid and artificial intestinal fluid within 24 h.

4. 2 . 4 .
Cell Cycle and Apoptosis StudyMV4-11 cells were plated into six-well plates at a density of 2.5 × 10 6 per well.After incubation overnight, cells were treated with different concentrations of 25c and Venetoclax alone or in combination for 24 h.Then, cells were harvested, washed once with 1 mL PBS and fixed with 75% pre-cooled ethanol at 4 • C overnight.The next day, fixed cells were washed twice with PBS, and incubated with DNase-free Rnase A and propidium iodide using a Cell Cycle Detection Kit (Absin, Shanghai, China).The cell cycle distribution was analyzed by flow cytometry (BD Accuri C6, Becton Dickinson, NJ, USA).Data were analyzed by FlowJo for Windows V10.8.1 software.

Table 1 . Cont. Compound No. Structure Inhibition Rate of HDAC1 (%) α Inhibition Rate of HDAC3 (%) α 100 nM 10 nM 100 nM 10 nM 14d 9f
α Inhibition rate values are the mean of three independent experiments ± the standard error of the mean (SEM).
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α Inhibition rate values are the mean of three independent experiments ± SEM.
α IC50 values are the mean of three independent experiments ±SEM.

Table 5
α IC50 values are the mean of three independent experiments ±SEM.

Table 5
α IC50 values are the mean of three independent experiments ±SEM.

Table 6 .
The HDAC inhibitory activity of 25c in vitro.
α IC 50 values are the mean of three independent experiments ± SEM.