First-in-Class Dual Mechanism Ferroptosis-HDAC Inhibitor Hybrids

Abstract

HDAC inhibitors are an attractive class of cytotoxic agents for the design of hybrid molecules. Several HDAC hybrids have emerged over the years, but none combines HDAC inhibition with ferroptosis, a combination which is being extensively studied because it leads to enhanced cytotoxicity and attenuated neuronal toxicity. We combined the pharmacophores of SAHA and CETZOLE molecules to design the first-in-class dual mechanism hybrid molecules, which induce ferroptosis and inhibit HDAC proteins. The involvement of both mechanisms in cytotoxicity was confirmed by a series of biological assays. The cytotoxic effects were evaluated in a series of cancer and neuronal cell lines. Analogue HY-1 demonstrated the best cytotoxic profile with GI₅₀ values as low as 20 nM. Although the increase in activity of the hybrids over the combinations is modest in cellular systems, they have the potential advantage of homogeneous spatiotemporal distribution in in vivo systems.

Graphical Abstract

INTRODUCTION

Chemotherapy remains one of the major approaches in cancer treatment with small molecules still dominating the market. These small organic compounds target diverse pathways causing cell cycle arrest and death of cancer cells. Due to genetic instability, tumors become more heterogeneous during the progression of the disease leading of cells with distinct molecular signatures. When this diverse population is subjected to chemotherapeutic agents, drug resistance can emerge due to chemotherapy acting as evolutionary pressure to select for cells that can grow in the presence of the drug.¹ Indeed, clinically, there is a negative correlation between the diversity of the tumor and therapeutic outcome. Despite being extensively studied, drug resistance remains a major impediment in cancer treatment.² Combinatorial treatment approaches^3,4 are adopted to decrease the chances of drug resistance and increase the chances of tumor eradication. In this approach, chemotherapy can be combined with radiotherapy or surgery, or in the alternative, a combination of two or more drugs can be used to target different cell survival pathways. More than 370 drug combinations have been approved by the food and drug administration FDA.⁵ Frei et al.⁶ first conceptualized combination therapy by showing the benefits of combining methotrexate, 6-mercaptopurine, vincristine, and prednisone (POMP regimen) to treat pediatric patients with acute lymphocytic leukemia. Since then, numerous drug combination regimens with synergistic or additive effects have been established, allowing reduced dosage requirements and therefore, fewer side effects.^7–9 Drug combinations are a common practice due to their advantages over monotherapy. However, the combined drugs remain different molecular entities with different pharmacological properties. Differences in biodistribution at both cellular and organism levels can hinder the successful administration of drug combinations. Thus, combinatorial therapy works best when drugs demonstrate similar pharmacokinetic profiles, which limits the available options. An emerging alternative is the rational design of multitargeted hybrid molecules,¹⁰ which will ensure homogenous spatiotemporal biodistribution of the individual active entities. In this approach, pharmacophoric features from the drugs that are to be combined are incorporated into a single scaffold, which maintains the pharmacodynamic effects of the individual drugs. The hybrid molecule ensures that the required activities are uniformly distributed in space and time. A potential drawback of hybrid molecules is the limitation of the fixed equimolar ratio of the two components. In addition, the design of hybrid molecules is extremely challenging because many pharmacophores do not tolerate significant structural changes. One attractive class of drugs for this approach is histone deacetylase inhibitors (HDACi).¹¹ HDACi act as epigenetic regulators by inhibiting histone deacetylases. This class of enzymes, in combination with histone acetyl transferases (HATs), controls chromatin remodeling by regulating the acetylation levels of histones. HDACs also deacetylate numerous non-histone proteins contributing to the effects of HDAC inhibitors¹² on a plethora of diverse cellular functions such as proliferation, cell death, metastasis, autophagy, metabolism, and ciliary expression.¹³ Based on phylogenetic comparison with yeast homologues, HDAC proteins are classified into four classes. Class I includes HDAC-1, HDAC-2, HDAC-3 and HDAC-8. They use Zn²⁺ as a cofactor and mainly localize in the nucleus with strong deacetylase activity toward histones. Class II HDACs use Zn²⁺ as a cofactor as well, and are further divided into two subclasses, class IIa, which includes HDAC-4, HDAC-5, HDAC-7, and HDAC-9, and class IIb which includes HDAC-6 and HDAC-10. Subclass IIa is found in both the nucleus and the cytoplasm and controls the activities of several nonhistone proteins like myocyte enhancer factor-2 (MEF2), while subclass IIb is found mainly in the cytoplasm with deacetylase activities against several interesting targets like tubulin deacetylation by HDAC-6, which regulates microtubule stability.¹⁴ Class III HDACs are NAD⁺-dependent enzymes that do not use Zn²⁺ as a cofactor and are referred to as sirtuins (SIRT 1–7). Class IV contains only HDAC-11, which uses Zn²⁺ as a cofactor and has similarities with both class I and class II HDACs. Very little is known about its biochemical function with early data suggesting roles in immune activation and tumorigenesis.¹⁵ Elevated levels of several HDAC isoforms are associated with tumor survival and progression.¹⁶ For example, prostate cancers have elevated levels of HDAC1,¹⁷ while gastric, colorectal carcinomas, cervical, and endometrial cancers all overexpress HDAC2,^18–20 when compared to the corresponding normal cells. These observations made HDACi attractive drug candidates. Four HDACi have already gained FDA approval and are important clinically used drugs.²¹ However, use of HDACi is currently limited to hematological malignancies and clinical trials are underway to use them for solid tumors in combination with other drugs.²² Dual mechanism HDAC hybrids have been the topic of extensive research in the past two decades, Figure 1A. A typical HDACi pharmacophore consists of a zinc-binding group (ZBG), a short planar aliphatic or aromatic linker and a cap group (Figure 1B). The ZBG and linker are crucial for HDAC inhibition as they are necessary to access and bind to the zinc ion in the internal cavity of the enzyme (Figure 2A). The amino acids in these regions are highly conserved among the different HDAC isoforms. The cap group binds to a less conserved area around the rim at the entrance to the active site (Figure 2A). HDACi have the ability to tolerate cap groups of diverse structures (preferably aromatic systems) without affecting the HDAC inhibitory capabilities significantly. With large cap groups, isoform selectivity may be attained, as exemplified by the SAHA analogue tubacin.²³ At the cellular level, the net effect of HDAC inhibition can be summarized as induction of apoptosis. In fact, most of hybrid molecules to date (including HDACi hybrids) combine two pharmacophores that target different cellular pathways, but both leading to apoptotic death such as hybrids of HDACs with cyclin-dependent kinases (CDK),^24,25 topoisomerase (topo),^26–28 and bromodomain (BRD) inhibitors²⁹ and DNA cross-linkers³⁰ (some examples are shown in Figure 1C–F). The lack of diversity in activating different cell death mechanisms is a major disadvantage of such hybrids molecules, because many cancer cells either have defective apoptosis pathways, or can dysregulate different steps in these pathways leading to drug resistance.³¹ The design and synthesis of hybrid molecules that combine apoptosis with other cell death machinery would be a more attractive alternative. In particular, combinations using drugs to sensitize cancer progenitor cells, which are insensitive to standard therapies, could be especially useful in improving the clinical outcome of chemotherapy.³² Dixon et al.³³ reported a novel nonapoptotic cell death mechanism, which they termed ferroptosis. The hallmark of ferroptosis³⁴ is iron-dependent lipid peroxidation, which, in combination with defective lipid peroxide repair mechanisms, leads to programmed cell death without requiring caspase activity. Synthetic agents can induce ferroptosis by inhibiting cellular components that are crucial for maintaining an intracellular reductive environment such as system Xc⁻ (erastin,³³ sorefinib³⁵) or GPX4 (RSL3,³⁶ ML210³⁷). In addition, several other processes such as ferritinophagy, epithelial-to-mesenchymal transition (EMT), glutamine, and iron metabolism modulate ferroptotic cell death. One important and unique feature of ferroptosis that makes it attractive for drug development is the enhanced sensitivity of mesenchymal cells to ferroptotic agents due to their high dependency on pathways that lead to lipid peroxidation quenching.³⁸ The mesenchymal state has been associated with drug resistance and cell migration, and thus, enhanced and selective killing of this subpopulation of cells will lead to reduced drug resistance and tumor metastasis, especially when combined with another cell death mechanism such as apoptosis. Currently, there is intense interest in the combined effects of HDACi and ferroptosis inducers, with respect to cancer treatment and reduced neurotoxicity.^39–47 For example, HDACi increase the sensitivity of cells to ferroptosis leading to synergetic killing of cancer cells.³⁹ In addition, the neuroprotective effects of HDACi can be an added benefit of such drug combinations by attenuating the neurotoxic effects of ferroptotic agents.³⁹ Expression of distinct HDAC isoforms in neurons versus cancer cells has been proposed to explain this difference.³⁹ Although, the exact mechanisms by which HDAC inhibitors enhance ferroptosis in cancer cells is yet not fully understood, significant progress was made by Oliveira et al. who reported that HDAC inhibition induces EMT and alters cellular iron homeostasis.⁴⁸ Herein, we report the first hybrid molecules that are capable of inducing ferroptosis in cancer cells while maintaining HDAC inhibitory activity, including their design, general synthetic route, in silico, in vitro, and cellular evaluation.

Figure 1.

HDAC inhibitors are an attractive class of molecules for the design of hybrid molecules. (A) Number of publications per year (1990–2021), which include the key word “hybrid HDAC inhibitors”. For this graph, the pubmed.gov was used as the search engine. (B) Tool box for the design HDAC inhibitor hybrids consisting of ZBG, linkers, and cap groups. Among these, the cap group, which typically prefers to have some aromatic components, tolerates diverse groups without loss of activity. Thus, most of the HDAC inhibitor hybrids have the additional functionality incorporated into the cap group. (C–F) Examples of HDAC inhibitor hybrids, which utilize some of the tools described in panel B. All examples cited have a hydroxamate as the ZBG, a short aliphatic chain as the linker (mimicking a SAHA-like model), and aromatic cap groups, where the additional functionality is typically installed.

Figure 2.

(A) 3D representation of active site of HDAC-8 PDB id: 1W22. A typical HDAC active site consists of a zinc-binding cavity, which is connected to an outside rim through a narrow tunnel. This active site structure dictates the design of HDAC inhibitors (including hybrid molecules), which are composed of a zinc binding group that chelates with zinc, a linker that occupies the narrow tunnel, and a cap group which covers the outside rim area. (B) Pose analysis of our designed HY-1 molecule and SAHA shows that the hybrid molecule positions its ZBG and linker in an identical way to SAHA, while the cap group has a slightly different orientation (vide infra for docking studies). (C, D) 2D representation of binding interactions for HY-1 and SAHA. In both, the ZBG demonstrates bidentate chelation with a zinc ion. In addition, hydrogen bonding interactions with HIS142 and TYR306 further facilitate the positioning of the ZBG in the zinc-containing cavity. On HDAC-8, the cap group of HY-1 is positioned toward PHE207, while in SAHA, it is positioned toward the loop containing PRO35-ILE34-Lys33. (E) Rational design for the synthesis of the amide and olefinic series. Combination of the simplified CETZOLE-1 analog (1) and SAHA analog (2) provides the hybrid molecule HY-1. Combination of CETZOLE-1 analog (3) with SAHA analog (4) by Wittig reaction provides the hybrid molecules HY-(2–5).

RESULTS AND DISCUSSION

Rational Design of Ferroptosis-HDACi Hybrid Molecules.

The design of HDACi was dictated by the characteristics of the active site (Figure 2A). We used the pan inhibitor SAHA as a model for the design of the first generation ferroptosis HDACi hybrids. Hydroxamic acid was chosen as the ZBG group, while a short aliphatic linker of comparable length to that of SAHA was used. We speculated that the cap group would be the ideal site for positioning the ferroptotic pharmacophore. Although many different pharmacophores have been reported to be capable of inducing ferroptosis, incorporation of some of them into a hybrid molecule could be challenging. For example erastin, it has a large pharmacophore with strict structure–activity-relationship (SAR)³⁵ requirements, thus rendering its incorporation into an HDAC pharmacophore without affecting HDAC and ferroptosis activities challenging. We have identified and reported a new ferroptotic agent CETZOLE-1^49–51 (Figure 2E) that has a 4-cyclopentenyl-2-ethynylthiazole scaffold (therefore, referred to as CETZOLEs). This compound induces ferroptosis but has a simpler structure, making its incorporation into hybrid molecules easier. In addition, extensive SAR studies in our laboratory⁵² reveal that the small 2-alkynyl thiazole system retains the ability to induce ferroptosis with wider group tolerance at the 5-position. We performed two different types of structural modifications; the first via a carboxylate at the 5-position appropriate for amide coupling (Figure 2E), and the second via a formyl group at this position for Wittig reactions (Figure 2E). These modifications result in analogs that have bioisosteric relationship. In addition, Wittig reaction allows the control of the geometry of the double bond as well as the degree of unsaturation of the linker, allowing us to perform a small SAR study in quest of more potent and/or selective inhibitors. The analogue synthesis via carboxylate would result in reverse amides of SAHA, which however is not expected to have any biological significance as such modifications have previously been reported as “noncritical.”⁵³ Indeed, preliminary molecular modeling data on HDAC-8 (Figure 2A–D) confirm that reversing the amide bond direction and modifying the cap group do affect the binding ability of the resulting HY-1 molecule, which accesses the active site in a similar way to SAHA (for detailed analysis on docking vide infra for in silico evaluation).

A total of 20 compounds were designed based on this pharmacophore as summarized in Figure 3. Members of group A (Figure 3A) were designed as inactive analogs that can be used as negative controls in biological studies. They lack a terminal alkyne group at the 2-position of the thiazole ring that is necessary for ferroptotic activity⁴⁹ and a hydroxamic acid metal-binding group necessary for HDAC inhibitory activity (negative controls (NC)). In addition, molecules in group A serve as convenient synthetic intermediates to access the molecules in the other groups. Figure 3B contains analogs, which are designed to induce only ferroptosis without HDAC inhibitory activity (ferroptosis controls (FC)). In contrast, Figure 3C has analogs that are designed to have only HDAC inhibitory activity without any ferroptotic effect (HDAC controls (HC)). Hybrid molecules that are expected to have both ferroptotic and HDAC inhibitory activity are shown in Figure 3D (hybrid molecules (HY)). The first row includes SAHA-like analogs, which are similar in shape and length. The second and third rows include the E/Z isomers of bioisosteric SAHA analogs. The fourth and fifth rows contain analogs, which were designed to investigate the effect of the chain length as well as the degree of unsaturation of the linker, while maintaining the general concept of “SAHA-like” compounds.

Figure 3.

Designed library of analogs consisting of: (A) negative controls NC-(1–5), (B) ferroptosis only controls FC-(1–5),. (C) HDACi only controls HC-(1–5), and (D) hybrid molecules HY-(1–5).

Chemical Synthesis.

The amide analogs were synthesized as shown in Scheme 1 using ethyl 2-bromothiazole-4-carboxylate (1) and 7-aminoheptanoic acid (6) as the main building blocks. 2-Bromothiazole-4-carboxylic acid obtained by the hydrolysis of ester (1) and the methyl ester of 7-aminoheptanoic acid were subjected to amide coupling under Steglich conditions to obtain compound (NC-1). It served as the inactive control in biological assays and also as the precursor for the synthesis of the rest of the analogs. Sonogashira coupling of NC-1 with trimethylsilylacetylene resulted in the alkynyl ester (FC-1), which was designed to induce only ferroptosis (ferroptosis control (FC)). Hydrolysis of (NC-1) gave the carboxylate (8), which served as the precursor for the synthesis of hydroxamic acids. Direct conversion of the esters NC-1 and FC-1 to hydroxamic acid proved difficult due to the presence of electrophilic bromine or alkyne on the thiazole ring. Coupling of compound (8) with NH₂OTHP resulted in the hydroxamic acid derivative (9) in good yields. Sonogashira coupling of (9) with TMS-acetylene proceeded efficiently to give the TMS-alkyne derivative, which underwent desilylation during separation resulting directly in compound (10) in moderate yields. Removal of THP-protecting group using a stochiometric amount of 1 M HCl in MeOH resulted in the “SAHA-like” ferroptosis HDACi hybrid (HY-1).

Scheme 1.

Synthesis of Amide Analogs^a

aThis synthetic route allows the access of amide analogs NC-1, FC-1, HC-1, and HY-1.

For the synthesis of the non-amide olefinic congeners, Wittig reaction of 2-bromothiazole-4-carbaldehyde (3) (synthesized in situ as shown in Scheme S5) with ethyl 7-(triphenyl-λ⁵-phosphaneylidene)heptanoate (11) resulted in a mixture of both (E) and (Z) isomers NC-2-M (∼1:1 E/Z ratio). Slow column chromatography separation on silica allowed the separation of a small amount of pure (Z) analog (NC-2), while the rest was obtained as an E/Z mixture (1:1 ratio) (NC-2-M). We proceeded to synthesize analogues using both the pure (Z) analog as well as the E/Z mixture, using similar synthetic procedures (Scheme 2).

Scheme 2.

Synthesis of the Olefinic Analogs^a

^aThis synthesis generates a mixture of isomers (and the corresponding N/F/HC-2-M and HY-2-M analogs), as well as the pure Z-isomers (and the corresponding N/F/HC-2 and HY-2 analogs). Unfortunately, the pure E-analog could not be obtained through this process.

To access the doubly unsaturated linker, the stabilized phosphonate carbanion (15) formed in situ as shown in Scheme S6, was reacted with 2-bromothiazole-4-carbaldehyde (3) to yield predominantly the E-analog NC-4, followed by a similar synthetic route to access the rest of the analogs of the series (Scheme 3).

Scheme 3.

Synthesis of Analogs Containing Doubly Unsaturated Linker

Similarly, reaction of compound (3) with methyl 3-(triphenyl-l5-phosphaneylidene)propanoate (19) synthesized as shown in Scheme S7 gave the short unsaturated analogs NC-5, FC-5, HC-5, and HY-5 (Scheme 4).

Scheme 4.

Synthesis of the Short-Linker Analogs

As shown in Table 1 and Figures 10 and 11, there is a difference between the activity profiles of the pure Z-analogs (FC-2, HC-2, and HY-2) and the E/Z-mixtures (FC-2-M, HC-2-M, and HY-2-M). Most of E/Z analog mixtures have higher IC₅₀s on both H522 and HCT-116 cell lines. In addition, the E/Z mixture, when tested for H3 and tubulin hyperacetylation, did not show results similar to those of the pure Z-analog. The latter shows H3 hyperacetylation only at 10 μM and tubulin hyperacetylation at 5 and 10 μM. In contrast, the E/Z mixture did not show H3 hyperacetylation even at 10 μM and showed tubulin hyperacetylation only at 10 μM. We hypothesized that the different pharmacological profiles stem from different activities of E and Z isomers, with the E-analogs being less reactive than the Z-analogs. To test this hypothesis, pure E-analogs were synthesized.

Table 1.

IC₅₀ ± SD (n = 3) Values (μM) for the Designed Library of Compounds on NCI-H522 and HCT-116 Cell Lines^a

hybrid molecules
cell-line /compound	HY-1	HY-2	HY-2-M	HY-3	HY-4	HY-5	N/A
NCI-H522	0.5 ± 0.01	1.07 ± 0.10	1.67 ± 0.06	1.59 ± 0.16	0.52 ± 0.04	0.79 ± 0.04	N/A
HCT-116	0.61 ± 0.11	1.19 ± 0.07	3.42 ± 0.47	4.96 ± 0.78	5.43 ± 1.34	5.16 ± 0.76	N/A
HDACi
cell-line /compound	HC-1	HC-2	HC-2-M	HC-3	HC-4	HC-5	SAHA
NCI-H522	3.26 ± 0.26	8.94 ± 2.18	9.54 ± 2.00	20.71 ± 3.27	12.48 ± 1.07	3.23 ± 0.25	1.46 ± 0.10
HCT-116	1.09 ± 0.12	6.28 ± 0.86	11.99 ± 3.42	12.95 ± 1.95	17.20 ± 2.95	1.30 ± 0.14	0.77 ± 0.08
inducers of ferroptosis
cell-line /compound	FC-1	FC-2	FC-2-M	FC-3	FC-4	FC-5	CETZOLE-1
NCI-H522	3.33 ± 0.15	7.33 ± 1.28	13.13 ± 2.00	15.7 ± 4.51	2.80 ± 0.34	2.29 ± 0.36	5.62 ± 0.13
HCT-116	>40	>40	>40	33.01 ± 1.42	>40	13.73 ± 4.64	>40
inactive analogs
For all inactive analogs, IC50 > 40 μM for both cell lines

^aN/A: not applicable.

Figure 10.

Treatment of NCI-H522 cells with the hybrid molecules leads to hyper acetylation of histones and tubulin. (A) Western blot analysis using acetyl-H3 (K9) and an acetyl tubulin antibody. NCI-H522 cells were treated with the hybrid molecules or controls at the corresponding concentrations in the presence of Liproxstatin-1 (0.25 μM) for 3 days. For HY-1, similar results were obtained with a “pan-acetyl-lysine” antibody shown in Figure S6B.

Figure 11.

Intercellular propagation of ferroptosis is more intense with the hybrid molecules. Addition of ferroptosis inhibitor Liproxastatin-1 makes the hybrid molecules have similar antimitotic affects with SAHA. (A) Time-lapse of microscopy of NCI-H522 cells treated with DMSO, SAHA (5 μM), CETZOLE-1 (20 μM), HY-1 (5 μM), and HY-1 (5 μM) + Liproxstatin-1 (0.25 μM). The images were captured every 12 min for a total of 300 pictures. (B) Kaplan–Meier curve for the corresponding treatments of Figure 13A. For these graphs cell death at certain time intervals (1 or 0.2 h) was considered as an event. Left comparison of all treatments at time intervals of 1 h. Right comparison of HY-1 and CETZOLE-1 at time intervals of 0.2 h. (C) Flow cytometry data of fixed NCI-H522 cells stained with PI after treatment with DMSO, SAHA (5 μM), or HY-1 (5 μM) in the presence of Liproxstatin-1 (0.25 μM). The attached graph shows the % values of cells that are dead in the G1, S, G2/M, or post-G2 phase for the corresponding time after treatment.

The Wittig reaction has the inherent tendency to provide mainly the Z-isomer as the faster formed kinetic product. The nature of the counter ion of the base used can have an effect on the stereochemical outcome of the reaction.⁵⁴ The smaller and harder Lewis acidic Li⁺ ions result in mostly the E-isomer and softer counter ions such as Na⁺ lead to equimolar E/Z mixtures. Indeed, using LiOH as the base resulted in mixtures enriched with E-analogue (2.5:1 E/Z ratio), allowing for its partial separation and purification (Scheme 5). Although Horner–Wadsworth–Emmons reaction would be a good alternative, it was not undertaken due to success in obtaining the E-isomer by counter ion control.

Scheme 5.

Synthesis of the Pure E-Analogs^a

^aThe nature of the counter ion of the base used for the transformation of (23) to (24) dictates the resulting analogs. Use of Na⁺ provides E/Z mixtures, which allow the separation of some pure Z-product (NC-2). The use of Li⁺ provided E-enriched mixtures, which allowed for the partial separation of pure E-product.

Hybrid Molecules Are Potent Cytotoxic Agents.

We first investigated the cytotoxic effects of the compounds using two cell lines NCI-H522 (nonsmall lung cancer) and HCT116 (human colon carcinoma). Previous studies in our laboratory have shown that NCI-H552 cells undergo ferroptosis,⁵⁵ while the HCT116 cells were insensitive to ferroptosis induced by CETZOLEs, sulfasalazine, or simple cystine deprivation.⁵⁵ The HCT116 cells are killed by erastin; however, this is only partially explained by ferroptosis with erastin likely having other targets in this cell line. Comparing effects in NCI-H522 and HCT116 made it possible to delineate the HDAC inhibitory and ferroptotic effects. The negative control (NC) analogs were inactive against both cell lines. The compounds that are designed to induce only ferroptosis (ferroptosis control) showed low μM activity only on the NCI-H522 cell line (2–13 μM IC₅₀) but were inactive against HCT116 cells, except for analog FC-5, which showed some activity on HCT116 (13.73 μM IC₅₀) (Table 1). To investigate if this effect is due to ferroptosis or some other activity, we cotreated the cells with a ferroptosis inhibitor Liproxstatin-1 (0.25 μM), which rescued the cells (Figure S5). As expected, the HDAC inhibitors showed promising activity on both cell lines. The hybrid molecules showed enhanced killing of NCI-H522 cells (0.5–3.61 μM IC₅₀) and HCT116 cell line (0.61–8.67 μM IC₅₀). In fact, at concentrations close to 2.5 μM, most of the hybrid molecules killed NCI-H522 cells to a significantly greater extent than any of the controls used, including CETZOLE-1 and SAHA (Figures 6 and 8 and Figure S4). This suggests a potential synergistic or additive effect because the same phenomenon was not observed with HCT116 cells; where at the same concentrations, the activity of hybrid molecules was similar to that of SAHA (Figures 6 and 8 and Figure S4). In addition, a similar observation was made on cotreatment of the two types of cells with CETZOLE-1 and SAHA (Figure 5A). This is in line with literature data, which suggests synergistic effects of HDAC inhibitors and ferroptosis inducers.^39–41 The E and Z isomers have different pharmacological behavior with respect to HDAC inhibitory activity, as seen from the activity profiles of both the HDAC controls (bromo-analogs) and the hybrid molecules (Table 1). Because of the different activity profiles in the rest of the biological assays, the pure E-analogs will be used and not the mixture. Although, in this case the activity differences are minimal, it is of significant importance in medicinal chemistry and drug design to evaluate both activities and side effects of individual isomers and decide whether the convenient choice of using a mixture is appropriate or not. Driven by the promising antiproliferative activity of HY-1 (Table 1 and Figure 4), we performed a chain elongation study of the linker connecting the ferroptotic cap group with the ZBG (Schemes S1–S3, Figure S1, and Table S1). Results indicate that our initial design was optimal because HY-1 demonstrated a superior cytotoxicity profile than its shorter or longer analogs. Thus, HY-1 was used in most of the biological assays as it is the most potent analog.

Figure 6.

Hybrid molecule shows selectivity rates for normal cells over cancer cells ranging between sensitivity to SAHA and CETZOLE-1. HY-1 has superior pharmacological effects on cancer cells (enhanced sensitivity of H522) vs the normal cells. NCI-H522, HCT-116, WI-38, and RPE cells grown in FBS media are treated with the inhibitors in a dose response manner. (A) Dose response graphs for NCI-H522, HCT-116, WI-38, and RPE cells treated with HY-1, SAHA, and CETZOLE-1. (B) The cell survival bar refers to a representative concentration of approximately 2 μM. Data are mean ± SD (n = 3) and expressed as a fold decrease relative to the value for the control group (DMSO). Statistical analysis utilizing the one-way ANOVA test *P < 0.05, **P < 0.01, ***P < 0.001. (C) Table with IC₅₀ ± SD (n = 3) values (μM) for HY-1, SAHA, and CETZOLE-1. Fold change¹ refers to the ratio of mean IC₅₀ (WI38) over mean IC₅₀ (NCI-H522). Fold change² refers to the ratio of mean IC₅₀ (RPE) over mean IC₅₀ (HCT-116).

Figure 8.

Flow cytometry data utilizing the C11-BODIPY^581/591 probe for lipid peroxide quantification. NCI-H522 cells were treated with DMSO, SAHA (10 μM), HY-1 (10 μM), or HY-1 (10 μM) + Liproxstatin-1 (0.25 μM) for 6 h.

Figure 5.

Cell survival assay of NCI-H522 cells with CETZOLE-1, SAHA, equimolar mixture of CETZOLE-1+SAHA; FC-1, HC-1, equimolar mixture of FC-1+HC-1, HY-1; FC-2, HC-2, equimolar mixture of FC-2 + HC-2 and HY-2, at concentrations of 1 μM, 2 μM and 5 μM. (A) Equimolar combination of SAHA and CETZOLE-1 led to significant enhancement of cytotoxicity at a concentration of 2 μM when compared to the corresponding SAHA treatment. At lower (1 μM) or higher (5 μM) concentrations, no significant differences were observed. (B) For FC-1 and HC-1, equimolar combinations behaved exactly like a combination of SAHA and CETZOLE-1. Enhanced cytotoxicity of the equimolar mix was observed only at 2 μM, when compared to the corresponding HC-1 treatments. However, HY-1, being highly cytotoxic, showed enhanced effects even at 1 μM when compared to HC-1 or the equimolar mix. At 2 and 5 μM, HY-1 behaved like the equimolar mix. (C) For FC-2 and HC-2, a high concentration of 5 μM was required to observe the enhanced effects. In contrast, HY-2 demonstrated enhanced cytotoxicity even at 2 μM. (D) The enhanced cytotoxicity of HY-1 and the equimolar mix of SAHA and CETZOLE was not limited to the NCI-H522 cell line. MDA-MB-231 cells were more sensitive to HY-1 (2.5 μM) and SAHA+CETZOLE equimolar combination (2.5 μM) than the corresponding treatments with only SAHA (2.5 μM) or only CETZOLE-1 (2.5 μM). The IC₅₀ values for each condition on the MDA-MB-231 cells are provided in Figure S6A.

Figure 4.

Cell survival rates of NCI-522 and HCT-116 cells after treatment with HY-1, CETZOLE-1, and SAHA (2.5 μM). HY-1 combines ferroptosis and HDACi, which leads to enhanced sensitivity of the NCI-H522 cell line over the parent molecules. HCT-116 cells are more resistant to ferroptosis; thus, HY-1 has a significant effect over CETZOLE-1, but comparable to the effect with SAHA. Data are mean ±SD. (n = 3) and expressed as a fold decrease relative to the value for the control group (DMSO). Statistical analysis utilizing the one-way ANOVA test *P < 0.05, **P < 0.01, ***P < 0.001.

In Figure 4, we demonstrate the ability of our hybrid molecules to show enhanced cytotoxicity on the ferroptosis sensitive NCI-H522 cell line. To investigate how this enhancement compares with treatment with equimolar combinations of appropriate ferroptosis and HDAC controls, we performed cell survival assays at concentrations of 1, 2, and 5 μM for CETZOLE-1, SAHA, and their equimolar combinations (Figure 5A), as well as the two most active hybrid molecules HY-1 (Figure 5B) and HY-2 (Figure 5C) and equimolar combinations of the two individual components. Our results indicate that hybrid molecules demonstrate enhanced cytotoxicity (preferably at concentrations around 2 μM) in a similar way to equimolar combination of the two individual components, as in the case of HY-1 (Figure 5B). But if the corresponding controls are significantly less potent than the hybrid molecules, such as in the case of HY-2, then, the equimolar combination requires higher concentrations (5 μM) to reach cytotoxicity levels comparable to that of the hybrid molecules, (Figure 5C). Similar results were observed for HY-1 with MDA-MB-231, which is another ferroptosis sensitive cell line (Figure 5D).

A salient property of a successful chemotherapeutic agent is the ability to discriminate between normal and cancer cells. A lack of selectivity is usually the cause of severe side effects. To investigate the potential of the hybrid molecules to selectively kill cancer cells over normal cells, we tested the cytotoxicity of one of the most potent hybrid analogs HY-1 on WI-38 cells (normal human lung fibroblasts) and hTERT-immortalized RPE cells (retinal pigment epithelial cells) (Figure 6A–C). The two parent molecules SAHA and CETZOLE-1 were used as controls. Based on the mean IC₅₀ values, the hybrid molecule was approximately 17-fold more selective for H522 over WI-38 and 10-fold more selective for epithelial HCT116 over RPE cell lines (Figure 6A,C). As expected, CETZOLE-1 did not show any notable effect on both cancerous HCT116 and normal RPE epithelial cells (IC₅₀ > 30 μM) (Figure 6A,C). However, it showed poor selectivity (approximately 3-fold) for NCI-H522 cells over the WI-38 (Figure 6A,C). This can be explained as due to fast and stress-induced cell death, which is an inherent characteristic of ferroptosis. In contrast, SAHA kills cells with more than 20-fold selectivity for NCI-H522 over WI-38 and 19-fold selectivity for HCT116 over RPE. It is noteworthy that the hybrid molecule, which combines characteristics from both mechanisms, has selectivity rates in-between those of the ferroptosis inducer and HDACi. Interestingly, comparison of selectivity at concentrations of therapeutic relevance (2 μM) shows an enhanced cytotoxic effect of the hybrid molecule on the NCI-H522 cell line in comparison to SAHA and CETZOLE-1, but at the same time, the survival rate of WI-38 is comparable to that of SAHA (Figure 6B). Thus, the hybrid molecule in the mesenchymal system has a significantly greater cytotoxic effect on cancer cells compared to SAHA and CETZOLE-1, while at the same time showing no toxic effects on normal cells. On the epithelial system, the sensitivity to hybrid molecule is comparable to that of SAHA with respect to killing of the cancer cells (HCT116), while no toxicity was observed on the normal cells (RPE) (Figure 6B).

Neuronal cells are inherently more sensitive to ferroptosis, as they have higher levels of polyunsaturated fatty acids (PUFAs), which serve as precursors for lipid peroxidation. In addition, due to their high metabolic activity, brain cells are particularly vulnerable to oxidative stress.⁵⁶ Indeed, ferroptosis plays an important role in a series of neurodegenerative diseases^57,58 and CNS-related toxic effects can explain the failure of ferroptotic agents in clinical applications as potent anticancer agents. Thus, there is a need to find strategies to attenuate their neurotoxic effects while retaining the beneficial anticancer effects. Recently, Zille et al.³⁹ reported that most ferroptosis inhibitors are capable of inhibiting ferroptosis in both neuronal and cancer cells, but class I histone deacetylase (HDAC) inhibitors selectively protect neurons, while enhancing ferroptosis in cancer cells. Although the pathways that lead to ferroptosis-mediated neuronal and cancer cell death are the same, they are differently regulated by HDACs. Thus, we tested the hybrid molecule HY-1 and its corresponding negative controls on the SH-SY5Y cell line (human neuroblastoma), which can serve as a model for neurodegenerative diseases. As shown in Figure 7A (and Figure S7 for the rest of the hybrids), the inactive analog NC-1 had no effect on the neuronal cells. As expected, the ferroptosis inducer FC-1 showed significant neurotoxicity. Interestingly, the HDACi HC-1 showed very mild toxicity on the neuronal cells at the highest concentration tested. Strikingly, the hybrid molecule HY-1, when tested at concentrations of therapeutic relevance (2.32 μM), had no neurotoxic effects. These results are in line with reports, which suggest that HDACi may attenuate ferroptotic neurotoxicity.³⁹ The latter findings become more important when we contrast the effects on neuronal cells with the effect on the cancer cell lines NCI-H522 and HCT116 (Figure 7B), where opposite effects are observed. In the NCI-H522 cell line, which is sensitive to both ferroptosis and HDACi, the hybrid molecule showed a significant effect compared to its ferroptosis or HDACi only controls. Since HCT116 cells are relatively resistant to ferroptotic cell death, the observed sensitivity is mostly due to HDAC inhibition. Thus, the sensitivity to hybrids and the HDACi controls are of comparable rates. (The same applies for CETZOLE-1 and SAHA, see Figure 4). The analog HY-4 (and its corresponding controls) did not show enhanced killing of the H522 cell line, while at the same time not showing attenuated neurotoxicity, suggesting the possibility that the mechanisms responsible for both effects are the same. We attribute the failure of this analog to provide beneficial effects to its poor HDAC inhibitory activity, as indicated by its relatively high HCT116 IC₅₀ values (Table 1), low levels of H3 and tubulin hyperacetylation (Figure 10) and the HDAC isoform selectivity data (Figure 12). As a result, its ferroptotic activity predominates, suggesting that a balance between ferroptosis and HDAC inhibition activities is required. A similar result with HY-4 was obtained with PC-12 (pheochromocytoma) cells as well. Nondifferentiated PC-12 cells have stem-like properties, but when differentiated by nerve growth factor, they demonstrate neuronal behavior (Figure 7C). Both these states proved to be resistant to our ferroptosis control (FC-1), indicating resistance to ferroptosis induction. As a consequence, we were unable to test the protective effect of the HDAC moiety on ferroptosis in this neuronal cell line. In fact, the hybrid HY-1 behaved like the HDAC control HC-1 (Figure 7D). These results indicate that the hybrid molecules have attenuated neuronal toxicity in a cell line-dependent manner, which requires further evaluation. The molecular crosstalk between HDACi and ferroptosis has gained great interest recently and our findings, in combination with previously reported ones, promise an exciting future for these hybrids.

Figure 7.

Hybrid molecule shows attenuated effects on neuronal cells, accompanied by significant sensitivity of the NCI-H522 and HCT-116 cell lines. (A) Viability of the SH-SY5Y cell line after 3 days treatment with the amide series analogs at concentrations of 0.5 × IC₅₀, 1 × IC₅₀, and 2 × IC₅₀ of the corresponding ferroptosis or HDAC inhibitor controls on H522 cancer cell line. For HY-1, the tested concentrations are approximately 1 × IC₅₀, 2 × IC₅₀, and 4 × IC₅₀ on the H522 cancer cell line (B) Cell viability percentages of NCI-H522 and HCT-116 cells after 3-day treatment with the amide series analogs at a concentration of 2.5 μM. The cell survival is shown as % of average of DMSO treatment after triplicate experiment. (C) Nondifferentiated PC-12 cells have stem-like properties, but when differentiated by a nerve growth factor, they demonstrate neuronal behavior. (D) Both differentiated and undifferentiated PC-12 cells proved to be resistant to ferroptosis induction. HY-1 behaved like the HDAC control. Data are mean ± SD. (n = 3) and expressed as a fold decrease relative to the value for the control group (DMSO). Statistical analysis utilizing the one-way ANOVA test *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 12.

HDAC isoform selectivity data for SAHA, Tubastatin, hybrid analogs, and their corresponding HDAC controls. IC₅₀ (nM) values were determined against HDAC 1, 2, 3, 4, 6, and 8 using recombinant HDAC proteins. Crossed boxes indicate that the values were not determined. Data in white boxes indicate IC₅₀ > 100,000 nM. Data are mean (n = 3) IC₅₀ values. Complete data mean ± standard error (n = 3) can be found in Table S4.

Hybrid Molecules Induce Ferroptosis while Maintaining HDAC Inhibitory Activity.

Next, we investigated key biomarkers to directly assess ferroptosis and HDAC inhibition by our hybrid molecules. To measure ferroptosis, we first investigated lipid peroxidation. The fluorescent dye C11-BODIPY^581/591 is an established system for the quantification of oxidation processes in membranes of living cells that lead to accumulation of lipid peroxides. Fluorescent measurements in the green area of the spectra can provide information on the levels of the oxidized form of the dye, which are proportional to the lipid peroxide levels. In 2014, Angeli et al.⁵⁹ screened a library of compounds as potential ferroptosis inhibitors and identified Liproxstatin-1 as a potent small molecule that suppresses ferroptosis in cells. Later studies revealed that Liproxstatin-1 inhibits lipid peroxidation by acting as a radical trapping antioxidant selectively on lipid bilayers.⁶⁰ Flow cytometry data (Figure 8) shows that treatment of NCI-H522 cells with the hybrid molecule (HY-1) leads to increased levels of lipid peroxides within 6 h of treatment. When HY-1 was used in combination with Lipoxstatin-1, lipid peroxidation levels decreased. It was a perfect overlap with the graph obtained with SAHA treatment. HDACi have been reported to increase reactive oxygen species (ROS) production.⁶¹ In a study performed by Xu et al., SAHA resistant cell lines failed to show accumulation of ROS, in contrast to significant increases in ROS levels in sensitive cell lines, in a caspase independent manner.⁶² Normal cells exhibited increased levels of the antioxidant protein thioredoxin, thereby revealing a potential mechanism for tumor cell selectivity of HDAC inhibitors.⁶³ Thus, the increased fluorescence in cells treated with SAHA or HY-1 + Liproxstatin-1 compared to DMSO might stem from this effect. It is worth mentioning that increased levels of ROS might serve as one of the possible origins of synergism between HDAC inhibitors and ferroptosis inducers. To investigate whether lipid peroxide accumulation is the only driving force that leads to cell death, we performed rescue experiments in which NCI-H522 cancer cells were treated with several of our inhibitors in the presence and absence of Liproxstatin-1 for either one day or four days (Figure 9). Lirpoxstatin-1 showed significant rescue from ferroptosis inducer CETZOLE-1 and our hybrid molecules, while cell death from SAHA after one-day treatment was only about 30%. However, although Lirpoxstatin-1 still rescued cells from CETZOLE-1 after four days of treatment, it failed to do so with the hybrid molecules due to activation of nonferroptotic cell death pathways arising from the potential HDAC activity of the hybrid molecules. Next, we investigated whether the hybrid molecules inhibit HDAC proteins by measuring hyperacetylation of the HDAC substrates, histones for class I inhibition and tubulin for class II (HDAC-6) (Figure 10). As expected CETZOLE-1 did not increase the acetylation levels of either H3 or tubulin, while the hybrids HY-1 and HY-2 led to hyperacetylation of both in a similar way to SAHA (pan-inhibitor). Interestingly, compound HY-5, at the concentration of 5 μM, did not show any H3 hyperacetylation, but only tubulin hyperacetylation, suggesting potential isoform selectivity. Compound HY-4 did not show significant effects at the tested concentrations.

Figure 9.

Addition of ferroptosis inhibitor Liproxstatin-1 (0.25 μM) rescues the cells after one day treatment with the hybrid molecules, but not after 4 days of treatment. (A) Cell survival after one day treatment with CETZOLE-1 (10 μM), SAHA (5 μM), HY-1 (5 μM), HY-2 (5 μM), HY-2-M (10 μM), HY-4 (20 μM), and HY-5 (20 μM) in the presence (0.25 μM) or absence of Liproxstatin-1. (B) Cell survival % after four days of treatment with CETZOLE-1 (10 μM), SAHA (5 μM), HY-1 (5 μM), HY-2 (5 μM), HY-2-M (10 μM), HY-4 (2.5 μM), and HY-5 (20 μM) in the presence (0.25 μM) or absence of Liproxstatin-1. Data are mean ± SD (n = 3) and expressed as a fold decrease relative to the value for the control group (DMSO). Statistical analysis utilizing the one-way ANOVA test *P < 0.05, **P < 0.01, ***P < 0.001.

Activation of the ferroptotic pathway by hybrid molecules was also indicated by increased levels of the transferrin receptor, which was recently identified as a selective marker for ferroptosis⁶⁴ (Figure S8). Hybrid molecules also increased the caspase-3 cleavage indicating activation of the apoptotic pathway, possibly as a result of HDAC inhibition (Figure S8). However, the pan-caspase inhibitor Z-VAD-FMK did not rescue cells from HDACi SAHA and had no effect on hybrid-induced death. This observation is consistent with a previous report⁶⁵ and can be attributed to additional noncaspase-mediated cell death induced by HDACi. In addition, other cell death inhibitors like autophagy inhibitors had no effect on hybrid-induced cell death (Figure S8). Thus, hybrid molecules increase the pleiotropic effects of HDAC inhibitors with ferroptosis providing an additional mechanism of action as a means to overcome drug resistance. Another important characteristic of ferroptosis is that cell death is fast, possibly due to self-amplifying oxidation reactions.^66,67 In contrast, HDAC inhibition has a slower mechanism of action, which through epigenetic regulation of histones, or hyper-acetylation of other proteins, arrests cells in G1 and/or G2/M,^68,69 eventually leading to cell death. Our observations with SAHA, CETZOLE-1, and the hybrid molecule HY-1 were consistent with these ideas (Figure 11A and Supporting Information Movies 1–5). NCI-H522 cells treated with SAHA were alive even 60 h after treatment. In contrast, CETZOLE-1-induced ferroptosis began approximately 12 h after treatment, and within the next 8 h, most of the cells were dead. Strikingly, the hybrid molecule induced death even faster than CETZOLE-1, and the wave-like propagation of ferroptosis is more intense. After initiation of ferroptosis 8 h after treatment, all cells were dead in the next 2 h. However, in the presence of Liproxstatin-1, the hybrid molecule behaved more like SAHA, with cells still alive even 60 h after treatment. Next, we cotreated cells with Liproxstatin-1 and HY-1 to see the prolonged effect of HDACi on the cell cycle arrest as determined by flow cytometry of cells stained with propidium iodide (PI) (Figure 11C). NCI-H522 cells treated with DMSO and Liproxstatin-1 have a distribution of cells between G1, S, and G2/M. However, when treated with SAHA and the hybrid molecules (in combination with Liproxstatin-1) at different time points, the S population of cells disappeared and the majority of cells were either dead or possibly arrested in the G1 or G2/M phase. The flow cytometry data shows once again that, if we diminish the ferroptotic activity of the hybrid molecules by the addition of a ferroptosis inhibitor like Liproxstatin-1, they behave in a similar way to SAHA, indicating their potential to inhibit HDAC proteins (similar results were obtained from combinatorial treatment of SAHA and CETZOLE-1, Figure S9).

Isoform Selectivity Data.

The HDAC inhibitory ability of the hybrid molecules and their HDAC controls was further evaluated using recombinant HDAC proteins utilizing in vitro bioluminescence activity assay. The IC₅₀ of all compounds was determined with HDAC 1, 2, 3, 4, 6, and 8 as shown in Figure 12 (figures S10–S16 and Tables S4–S11). The FDA-approved HDAC inhibitor SAHA and the HDAC 6-selective inhibitor Tubastatin have been previously tested in the same activity assay and were used as standards for comparison.^70,71 Consistent with the biological data, HY-1 proved to be the most potent inhibitor demonstrating an activity profile similar to SAHA, with a remarkable 1.2 ± 0.1 nM IC₅₀ for HDAC-6. This pattern of activity is of particular importance for combinations of ferroptosis and HDACi because it involves the inhibition both Class I HDAC proteins and HDAC-6 with enhanced killing of cancer cells and attenuated neurotoxicity associated with ferroptosis while having no or minimal effect on other HDAC proteins like HDAC-4 and HDAC-8. The corresponding control HC-1 demonstrated an activity profile similar to HY-1. Although, the E/Z isomers (HY-3, HC-3/HY-2, and HC-2) have different biological profiles in this in vitro assay, they demonstrated similar cytotoxic activity, with both series being less active than their amide counter parts (HY-1 and HC-1), indicating the possibility of binding interactions originating from the amide part of the pharmacophore. In addition, these results prove the double bond as a poor bioisosteric change for the design of HDAC inhibitors. Consistent with our biological evaluations, HY-4 and HC-4 showed poor HDAC inhibitory activity. For HY-5, we observed only tubulin hyperacetylation with no hyperacetylation of histones. The isoform selectivity data suggests that this compound has the potential to act as a more HDAC-6-selective inhibitor because it shows moderate activity (160 ± 20 nM) against HDAC-6 and relatively poor activity (890–1100 nM) Class I HDACs. Most of the tested compounds showed high HDAC inhibitory ability in the in vitro HDAC activity assay at very low (nM) concentrations. Although this assay cannot be extrapolated to behavior in cells as most HDACs exist in multimolecular complexes, it demonstrates once again that the designed molecules have the potential to inhibit HDAC proteins.

In Silico Evaluation.

We performed molecular docking studies of the designed analogs to estimate their potential to bind to HDAC proteins. We focused our docking study on class I and class II HADCs using Maestro v10.6 computational software. Initially, a library of 22 compounds, including the 20 analogs shown in Figure 3, and CETZOLE-1 and SAHA as controls was generated. X-Ray crystallographic data of the HDAC-isoforms cocrystalized with appropriate inhibitors downloaded from the PDB were used. Rotation of side chains was allowed only for the amino acids within the active site. Extra precision docking of our compounds to selected HDAC-isoforms provided us with the docking scores of each compound as summarized in Figure 13 (and Table S2).

Figure 13.

Docking scores for the designed library of analogs as well as for the controls SAHA and CETZOLE-1 on several HDAC isoforms. (A) The x-axis represents the entry ID based on the docking score, e.g., the compound with the lowest docking score is entry 1. The y-axis represents the docking score, which degreases from top to bottom. As a result of this representation, we can draw a diagonal from top right to bottom left, a direction in which the binding ability increases. Thus, the best compounds for each HDAC isoform will be accumulated more toward the bottom left of each graph. (B–I) Docking scores for seven different HDAC enzyme crystal structures downloaded from PDB were used. HDAC-1 PDB id: 5ICN, HDAC-2 PDB id: 4LXZ, HDAC-8 PDB id: 1W22, HDAC-4 PDB id: 2VQM, HDAC-7 PDB id: 3C10, HDAC-6-CD1 PDB id: 6UO2, HDAC-6-CD2 PDB id: 5G0H, HDAC-10 PDB id: 6WBQ. SAHA and HY-1 (colored red) showed similar behavior and constantly ranked among the best compounds (bottom left of the graphs). Each data point is colored based on the value of the docking score. Red represents the highest docking score, while blue represents the lowest docking score in each data set. For HDACs 1,2,6(CD2) and 10, no good binding poses of CETZOLE-1 were found. Thus, no docking scores are reported. All HDAC proteins used in this in silico study are of human origin, except HDAC-6 CD1, CD2, and HDAC 10 which originate from zebrafish.

The docking scores suggested that the hybrid molecules as well as the HDACi controls have the ability to bind to the HDAC enzymes in a similar way as SAHA (Figures 2 and 13 and Table S2). In addition, a slight preference for the class I proteins was observed when compared with class II. An exception to this observation is HDAC-6, for which the hybrid analogs had good docking scores for both catalytic domains (CD1 and CD2). In contrast, analogs that were designed as inactive controls or only as ferroptotic agents showed poor binding scores, although they have similar shape and form. This is expected as they lack the ZBG. Pose analysis of binding (Figure 14 for HY-1 (red) and HY-2 (blue)) reveals that the hybrid analogs retain the ability to bind in a similar binding motif like SAHA (as previously shown in Figure 2B). The hydroxamic acid chelates with the zinc ion by positioning the linker in the narrow tunnel and the thiazole cap group in the rim area of the HDAC proteins. The surface representation is provided to emphasize the complementary shape of our hybrids to the active site (“key into lock”). In Figures S2 and S3 are provided 2D and 3D representations of HY-1 and SAHA on representative HDAC isoforms with emphasis on interacting residues.

Figure 14.

3D representation of the binding motifs for two representative hybrid analogs on 7 different HDAC proteins. Compounds HY-1 (orange) and HY-2 (turquoise) bind to the active site as expected for HDACi, with the hydroxamate binding to the zinc ion, the linker placed in the narrow tunnel, and the cap group placed at the rim area. HDAC-1 PDB id 5ICN: HDAC-2 PDB id: 4LXZ, HDAC-8 PDB id: 1W22, HDAC-4 PDB id: 2VQM, HDAC-7 PDB id: 3C10, HDAC-6-CD1 PDB id: 6UO2, HDAC-6-CD2 PDB id: 5G0H, and HDAC-10 PDB id: 6WBQ.

NCI-60 One-Dose and Five-Dose Assays.

The cytotoxicity of hybrid analogs were additionally evaluated in the NCI-60 human tumor cell lines screen by the national cancer institute (NCI) through the development therapeutics program (DTP), initially at a single dose of 10 μM to determine mean inhibition and then in a five-dose assay to determine growth inhibition (GI₅₀) values. The results for both one-dose and five-dose assays are summarized in Figure 15 where the mean values from approximately sixty cell lines are included. Figure S17–S34 provide per cell line information for the same values. As previous studies demonstrated that compounds, which consisted of only the ferroptosis component, had poor activity profiles in the NCI 60 cell line assay, the ferroptosis positive controls were not tested in this assay (e.g., Figure S34 data for CETZOLE-1). Consistent with cytotoxicity evaluations performed in our laboratories, data obtained by the NCI-60 DTP confirmed that HY-1 demonstrated the best cytotoxicity profile when compared to other hybrids or HDAC controls and was even more active than SAHA. Many cell lines across multiple cancer types showed enhanced sensitivity to HY-1 (Figure 16). Taking into consideration that the HDAC inhibitory abilities of HY-1 and SAHA are comparable, we attribute the higher cytotoxic activity of HY-1 to the combination of HDAC inhibition with ferroptosis. For the leukemia cell lines, HY-1 showed an enhanced effect on all tested cell lines with the largest difference being observed with the CCRF-CEM cell line (Growth %: 18.40 for SAHA and −41.42 for HY-1). Similarly, HY-1 showed enhanced effects on the NSC-lung cancer cell lines, with the most noticeable difference being observed with the NCI-H23 cell line (Growth % 3.30 for SAHA and −74.60 for HY-1). HY-1 and SAHA demonstrated similar activity profiles with the colon cancer cell lines, indicating poor contribution by ferroptosis in this type of cancer. SF-539 and SNB-75 are two CNS cancer cell lines with the most noticeable enhancement of HY-1-induced cytotoxicity (SAHA Growth % 16.80 for SF-539 and 18.00 for SNB-75, HY-1 −80.14 for SF-539 and −65.15 for SNB-75). With respect to melanoma cells, HY-1 has substantially greater activity on the LOX IMVI cell line (Growth % −84.63) when compared to SAHA (Growth % −3.4). The same effect was observed with the ovarian cancer cell line IGROV-1 (Growth % 23.7 for SAHA and −72.55 for HY-1). Strikingly, most renal cancer cells were outstandingly more sensitive to all hybrid molecules, when compared to their HDAC controls or SAHA, suggesting greater therapeutic benefits arising from combination of ferroptosis and HDAC inhibition on this type of cancer cells. Of the two prostate cancer cells tested, PC-3 cells showed similar levels of sensitivity HY-1 and SAHA (Growth % 10.4 for SAHA and 14.28 for HY-1), but DU-145 cells were much more sensitive to HY-1 (Growth % 3.5 for SAHA and −51.12 for HY-1). Finally, the robust triple-negative breast cancer (TNBC) cell line MDA-MB-231 showed enhanced sensitivity to HY-1 (Growth % −71.82%) when compared to SAHA (Growth % 16.4). This is of particular importance because TNBC represents approximately 10–15% of all breast cancers, and patients with TNBC have a poor outcome when compared to the other subtypes of breast cancers.⁷² Our own testing on this cell line further confirms these results (Figure 5). In contrast to HY-1, analog HY-4 demonstrated the worst cytotoxicity profile. This analog is a poor HDAC inhibitor and its ferroptosis activity predominates. The other hybrid compounds (HY-2,3 and 5) demonstrated cytotoxicity profiles in between those of HY-1 and HY-4. Most of the HDAC controls demonstrated activity profiles similar to that of SAHA. This is of particular importance because the HDAC inhibitory ability of hybrid analogs and HDAC controls are similar. Thus, any difference in cytotoxicity stems from the combination of ferroptosis with HDAC inhibition.

Figure 15.

Summary of data from the one-dose and five-dose assays performed by NCI-60 DTP. (A) Mean inhibition (%) values for hybrids and HDAC controls. Information for individual cell lines is provided in Figure S17–S34. (B) Average GI₅₀ values for the compounds selected by NCI-60 DTP to be tested on the five-dose assay. Information for individual cell lines is provided in figure x and y. Both one-dose and five-dose assays are performed by NCI-60 DTP as single experiments and not in multiple replicates.

Figure 16.

NCI-60 one-dose data for HDAC controls, HY-(1–5), and SAHA. Most of the tested cell lines showed enhanced sensitivity to the hybrid molecules over the HDAC controls. HY-1 demonstrated the best profile. A plethora of cell lines showed remarkable sensitivity to HY-1 over SAHA. Data for SAHA was obtained from the public data base of NCI. SAHA (NSC 759852) NSC Cancer Chemotherapy National Service Center number assigned by NCI. Data are Growth % values.

Analogs that demonstrated promising cytotoxicity in the one-dose assay were tested in the five-dose assay to determine the GI₅₀ values (Figure 17). These included all hybrid molecules (except HY-4), HC-1, and HC-5. HY-1 showed the best cytotoxicity profile with an average GI₅₀ of 0.85 μM, which is lower than the 1.21 μM average GI₅₀ for SAHA. Surprisingly, HY-2,3 and 5 showed attenuated effects when compared to SAHA with an average GI₅₀ of 2.18, 3.39, and 2.47 μM, respectively. Consistent with our own cytotoxicity evaluation and inhibition of purified HDACs results, HC-1 demonstrated lower activity than SAHA with an average GI₅₀ 1.51 μM, demonstrating the contribution to cytotoxicity by ferroptosis. Figure 17 summarizes the dose response for the selected compounds. All leukemia cell lines showed greater sensitivity to HY-1 over SAHA and corresponding HDAC controls. Although in the one-dose assay, the CCRF-CEM cell line showed the highest difference in sensitivity to HY-1 and SAHA, in the five-dose assay RPMI-8226 demonstrated the highest difference due to the combined effect of ferroptosis and HDAC inhibition (HY-1 GI₅₀ 0.35 μM, SAHA GI₅₀ 1.5 μM). Consistent with the results of the one-dose assay at 10 μM, the NSC lung cancer cell line NCI-H23 showed enhanced sensitivity to HY-1 with GI₅₀ 0.78 μM over SAHA with GI₅₀ 2 μM. The insensitivity of the colon cancer cell lines to ferroptosis was once again confirmed by similar GI₅₀ values obtained for HY-1 and SAHA against most of the colon cancer cell lines (Figures S17–S34). Contrary to the results of the one-dose data, of the CNS cancer cell lines, only the SF-539 cell line showed a significant difference in sensitivity to HY-1 and SAHA (HY-1 GI₅₀ 1 μM over SAHA GI₅₀ 2 μM). Of the melanoma cell lines, LOXIMVI showed remarkable sensitivity to the hybrid molecules. HY-1 demonstrated GI₅₀ of 0.02 μM, which is significantly lower than a GI₅₀ value of 1.25 μM for SAHA. Of the ovarian cancer cell lines, once again, IGROV-1 demonstrated enhanced sensitivity to hybrid molecules (GI₅₀ 0.57 μM for HY-1 compared to GI₅₀ 2 μM for SAHA). Although the differences were slightly lower when compared to the results of the one-dose assay, the renal cancer cell lines (e.g., 786–0 cell line) were more sensitive to our hybrid molecules (Figures S17–S34). Of the two prostate cancer cell lines, PC-3 was slightly more sensitive to SAHA (1.58 μM) compared to HY-1 (GI₅₀ 2 μM), while DU-145 demonstrated opposite effects with GI₅₀ 0.36 μM for HY-1 and GI₅₀ 1.25 μM for SAHA. The breast cancer cell line HS578 showed enhanced sensitivity to HY-1 with GI₅₀ 0.39 μM over 2 μM for SAHA. Analysis of the data in the one-dose and five-dose assays identifies two basic types of cell lines; those that are significantly more sensitive to hybrid molecules than the HDAC controls and those that have similar effects. We hypothesize that the enhanced cytotoxicity results from the combination of ferroptosis with HDAC inhibition. From a potential therapeutic point of view, it will be useful to find molecular markers that can be used to identify the types of cell lines that can be treated with these hybrid molecules. Our future efforts will be focused in this direction.

Figure 17.

Heat map of GI₅₀ values (μM) for HY-1, HY-2, HY-3, HY-5, SAHA, HC-1, and HC-5 in the NCI-60 five-dose assay. Crossed boxes denote data not determined. White boxes indicate GI₅₀ > 4 μM and are not included for better resolution. Exact Growth % and GI₅₀ values for one- and five-dose assays can be found at Figures S17–S34 on the Supporting Information.

CONCLUSIONS

In this work, we report the first-in-class dual mechanism hybrid molecules capable of inducing ferroptosis and inhibiting HDAC proteins simultaneously. The HDAC inhibitory component of the hybrid molecules was based on a “SAHA”-like model in which hydroxamates were used as the ZBG, while a short aliphatic chain constituted the linker. A 2-alkynyl thiazole moiety, which is the warhead of previously reported CETZOLE ferroptosis inducers, was used as the cap group. The design of the first-generation hybrid molecules focused mainly on the length and nature of the linker. Hybrid molecules that lacked either the hydroxamate ZBG or ferroptosis-inducing thiazole moiety or both were used as appropriate controls. Small SAR study was performed focusing on the length of the linker as well as the unsaturation levels. Our results proved the double bond to be a poor bioisosteric change for the amide in case of the HDAC inhibitors because it provided analogs with attenuated cytotoxicity. In silico studies suggested that the hybrid molecules are capable of binding to HDAC proteins in a similar binding motif as SAHA, with the ZBG occupying the active site and chelating with the zinc ion, and the cap group occupying the outer rim of the proteins. The cytotoxicity of the library of analogues was initially determined on two cell lines; the ferroptosis-sensitive NCI-H522 and the ferroptosis-”resistant” HCT-116 cell lines. This combination allowed the delineation of the HDAC inhibitory and ferroptosis-inducing potential of the molecules. Most of the hybrid molecules showed enhanced cytotoxicity on the NCI-H522 cell line when compared to SAHA, CETZOLE-1, or the corresponding HDAC and ferroptosis controls, suggesting potential synergism by the combination of the two mechanisms. In addition, hybrid molecules showed attenuated neurotoxicity. At concentrations capable of showing cytotoxic effects on the NCI-H522 cell line, hybrid molecules showed a minimal effect on the neuronal cell line SH-SY5Y, in contrast to the enhanced neuronal toxicity of the ferroptosis inducers and the moderate effects of HDAC controls. These preliminary data suggest the potential for using these hybrid molecules as anticancer agents capable of inducing ferroptosis with minimized neuronal side effects. However, this effect seems to be cell line dependent and requires further evaluation. HY-1 demonstrated the best cytotoxicity profile with low nM GI₅₀ values on many cancer cell lines. Our first-generation hybrid molecules incorporated a pan-HDAC inhibitor component, which demonstrated no isoform selectivity. Future endeavors will focus on the design and synthesis of isoform-selective hybrids that incorporate alternative zinc-binding groups, aromatic linkers, and versatile cap groups. In addition, we plan to test the enhanced cytotoxicity and attenuated neuronal effects of the hybrid molecules in animal models and identify selective biomarkers that will allow the characterization of cancer types that will benefit from such combination.

EXPERIMENTAL SECTION

Materials and Methods.

All chemicals and solvents were purchased from commercial sources and used without further purification, unless stated otherwise. Anhydrous tetrahydrofuran was freshly distilled from sodium and benzophenone before use. ¹H and ¹³C NMR spectra were recorded on Brucker Avance 600 MHz, INOVA 600 MHz, and Varian VXRS 400 MHz NMR spectrometers in deuterated solvents using residual undeuterated solvents as an internal standard. High-resolution mass spectra (HRMS) were recorded on a Waters Synapt high-definition mass spectrometer (HDMS) equipped with a nano-ESI source. Melting points were determined using a Fisher–Johns melting-point apparatus. Purification of crude products was performed by either flash chromatography on silica gel (40–63 μ) from Sorbent Technologies or on a Teledyne ISCO CombiFlash Companion chromatography system on RediSep prepacked silica cartridges. Thin-layer chromatography (TLC) plates (20 cm × 20 cm) were purchased from Sorbent Technologies (catalog #4115126) and were viewed under a Model UVG-54 mineral light lamp UV-254 nm. A Shimadzu Prominence HPLC with an LCT20AT solvent delivery system coupled to a Shimadzu Prominence SPD 20AV Dual wavelength UV/vis absorbance detector, a Shimadzu C₁₈ column (1.9 m, 2.1 mm × 50 m), and HPLC grade solvents (MeOH, H₂O with 0.1% formic acid) were used to determine the purity of compounds by HPLC. All compounds are >95% pure by HPLC analysis.

Synthesis.

General Method for Sonogashira Coupling.

A mixture of the corresponding thiazole-bromide, TMS-acetylene (1.5 equiv), PdCl₂(PPh₃)₂ (5 mol %), CuI (6 mol %), Et₃N (2 equiv), and DCE (5 mL/mmol) was heated under reflux at 83 °C for 1 h. The reaction was quenched by the addition of brine, followed by DCM. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The product was purified by flash chromatography on silica in EtOAC/hexanes (0 to >100% EtOAc) to yield the pure product.

General Method for Ester Hydrolysis.

To a solution of the corresponding ester in freshly distilled THF (2 mL/mmol) and H₂O (1 mL/mmol) was added LiOH (3 equiv). The resulting mixture was stirred for 2 h at r.t. Most of the THF was removed under reduced pressure; the remaining aqueous solution was washed with EtOAc, acidified with conc. HCl, and extracted with EtOAc. The organic extract was dried over Na₂SO₄, filtered, and concentrated under reduced pressure to yield the pure product.

General Method for NH₂OTHP Coupling.

To a stirred suspension of the corresponding carboxylic acid in anhydrous DCM (3 mL/mmol) was added EDC-HCl (1.5 equiv) and followed by the addition of THP-O-NH₂ (1.1 equiv). The resulting mixture was stirred for 8 h at r.t. The reaction mixture was diluted with brine and extracted with EtOAc. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica in EtOAC/DCM (0 to >100% EtOAc) to yield the pure compound.

General Method for THP Deprotection.

To a stirred solution of corresponding THP-protected hydroxamic acid in MeOH (3 mL/mmol) was added 1 M HCl (4 equiv), and the resulting mixture was stirred at r.t for 3 h. After removal of most of the MeOH under reduced pressure, the residue was diluted with brine and extracted with EtOAC. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica in DCM/MeOH (0 to >7% MeOH) to yield the pure compound.

2-Bromothiazole-5-carboxylic Acid (5).

Synthesized according to general hydrolysis conditions (10 mmol scale, 96% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 13.33 (br, 1H), 8.47 (s, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 161.6, 147.6, 137.1, 133.6.

Methyl 7-Aminoheptanoate Hydrochloride Salt (7).

To a stirred solution of 7-aminoheptanoic acid (1. 45 g, 10 mmol) in MeOH (30 mL) at 0 °C was added SOCl₂ (7 mL). The resulting mixture was allowed to warm to r.t and stirred at that temperature for 3 h. The volatiles were removed under reduced pressure to yield the pure product (30) (1.96 g, 100%) as a white solid, which was directly used in the next step without any purification. ¹H NMR (600 MHz, DMSO-d₆) δ 8.11 (br, 3H), 3.58 (s, 3H), 2.72 (t, J = 7.2 Hz, 2H), 2.30 (t, J = 7.4 Hz, 2H), 1.59–1.46 (m, 4H), 1.34–1.19 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.8, 51.7, 39.1, 33.6, 28.4, 27.2, 26.0, 24.7.

Methyl 7-(2-Bromothiazole-4-carboxamido)heptanoate (NC-1).

To a stirred suspension of 2-bromothiazole-5-carboxylic acid (5) (416 mg, 2 mmol) in anhydrous DCM (10 mL) were added EDC-HCl (575 mg, 3 mmol, 1.5 equiv) and DMAP (10 mol %) and followed by methyl 7-aminoheptanoate hydrochloric salt (7) (391 mg, 2 mmol, 1 equiv) and Et₃N (0.28 mL, 2 mmol, 1 equiv). The resulting mixture was stirred overnight at r.t; after which, it was diluted with brine and DCM. The two layers were separated, and the organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica in EtOAC/hexanes (0 to >100% EtOAc) to yield the pure product (NC-1) (435 mg, 1.24 mmol, 62%) (>99% HPLC purity) as a colorless oil. ¹H NMR (600 MHz, CDCl₃) δ 8.05 (s, 1H), 7.22 (br, 1H), 3.67 (s, 3H), 3.42 (dt, J = 7.2, 3.6 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.68–1.55 (m, 4H), 1.42–1.33 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 174.2, 159.6, 150.1, 135.7, 126.6, 51.5, 39.4, 34.0, 29.4, 28.7, 26.6, 24.9, 28.7, 26.6, 24.9. HRMS calcd for C₁₂H₁₇BrN₂NaO₃S (M + Na) 371.0040; found 371.0041.

Methyl 7-(2-Ethynylthiazole-4-carboxamido)heptanoate (FC-1).

Synthesized according to general Sonogashira coupling conditions (1 mmol scale, 65% yield) (>99% HPLC purity). ¹H NMR (600 MHz, CDCl₃) δ 8.13 (s, 1H), 7.33 (br, 1H), 3.67 (s, 3H), 3.56 (s, 1H), 3.44 (dd, J = 13.4, 7.0 Hz, 2H), 2.31 (t, J = 7.5 Hz, 2H), 1.68–1.58 (m, 4H), 1.45–1.32 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 174.2, 160.2, 150.7, 147.2, 124.9, 83.1, 75.7, 51.5, 39.3, 34.0, 29.4, 28.8, 26.6, 24.8. HRMS calcd for C₁₄H₁₈N₂NaO₃S (M + Na) 317.0935; found 317.0934.

7-(2-Bromothiazole-4-carboxamido)heptanoic Acid (8).

Synthesized according to general hydrolysis conditions (0.27 mmol scale, 93% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 11.98 (br, 1H), 8.51 (t, J = 5.7 Hz, (NH), 1H), 8.25 (s, 1H), 3.21 (dd, J = 13.7, 6.6 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.53–1.44 (m, 4H), 1.30–1.24 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.0, 159.6, 150.4, 136.6, 128.7, 39.2, 34.1, 29.4, 28.7, 26.6, 24.9.

2-Bromo-N-(7-oxo-7-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-heptyl)thiazole-4-carboxamide (9).

Synthesized according to the general NH₂OTHP coupling conditions (1.85 mmol scale, 85% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.22 (br, 1H), 8.23 (s, 1H), 7.96 (br, 1H), 4.93 (s, 1H), 3.97 (t, J = 10.6 Hz, 1H), 3.42 (dd, J = 13.6, 6.7 Hz, 2H), 2.10 (t, J = 7.1 Hz, 2H), 1.78–1.47 (m, 10H), 1.38 (d, J = 3.0 Hz, 4H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.5, 159.3, 150.6, 135.6, 127.4, 101.3, 61.4, 39.0, 32.6, 29.5, 28.6, 28.0, 26.4, 25.3, 25.1, 18.4.

2-Bromo-N-(7-(hydroxyamino)-7-oxoheptyl)thiazole-4-carboxamide (HC-1).

Synthesized according to the general THP deprotection conditions (0.23 mmol scale, 68% yield) (>99% HPLC purity). ¹H NMR (600 MHz, DMSO-d₆) δ 10.34 (s, 1H), 8.67 (s, 1H), 8.51 (t, J = 5.8 Hz, 1H), 8.25 (s, 1H), 3.21 (dd, J = 13.6, 6.7 Hz, 2H), 1.93 (t, J = 7.4 Hz, 2H), 1.52–1.44 (m, 4H), 1.30–1.18 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 169.6, 159.6, 150.4, 136.7, 128.8, 39.1, 32.7, 29.5, 28.8, 26.6, 25.6. HRMS (ESI) calcd for C₁₁H₁₆BrN₃NaO₃S (M + Na) 371.9993; found 371.9991.

2-Ethynyl-N-(7-oxo-7-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-heptyl)thiazole-4-carboxamide (10).

Synthesized according to general Sonogashira coupling conditions (0.5 mmol scale, 60% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.10 (br, 1H), 8.27 (s, 1H), 7.94 (br, 1H), 4.92 (s, 1H), 4.43 (s, 1H), 3.96 (t, J = 10.6 Hz, 1H), 3.51 (d, J = 11.0 Hz, 1H), 3.43 (dd, J = 13.6, 6.7 Hz, 2H), 2.13–2.04 (m, J = 8.9, 6.3, 4.4 Hz, 3H), 1.79–1.46 (m, 10H), 1.44–1.34 (m, J = 3.1 Hz, 4H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.4, 159.7, 151.4, 147.1, 125.2, 101.2, 84.2, 75.7, 61.4, 38.9, 32.5, 29.4, 27.9, 26.4, 25.2, 25.0, 18.4.

2-Ethynyl-N-(7-(hydroxyamino)-7-oxoheptyl)thiazole-4-carboxamide (HY-1).

Synthesized according to the general THP deprotection conditions (0.26 mmol scale, 61% yield) (>99% HPLC purity). ¹H NMR (600 MHz, acetone-d₆) δ 10.01 (br, 1H), 8.27 (s, 1H), 8.16 (br, J = 28.9, 17.3 Hz, 1H), 7.94 (br, 1H), 4.43 (s, 1H), 3.42 (dd, J = 13.6, 6.8 Hz, 2H), 2.11 (dd, J = 10.3, 4.4 Hz, 2H), 1.67–1.58 (m, 4H), 1.44–1.33 (m, 4H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.8, 159.7, 151.3, 147.1, 125.2, 84.2, 75.7, 38.9, 32.2, 29.4, 26.4, 25.1. HRMS (ESI) calcd for C₁₃H₁₇N₃NaO₃S (M + Na) 318.0888 found 318.0889.

Ethyl (Z)-8-(2-Bromothiazol-4-yl)oct-7-enoate (NC-2).

To a round-bottom flask containing ethyl 7-bromoheptanoate (S23) (6 g, 35.3 mmol) and triphenyl phosphine (6.6 g, 25.3 mmol, 1 equiv) was added toluene (55 mL). The resulting mixture was refluxed for 24 h. Toluene was evaporated under reduced pressure, and the residue was washed with hexanes and diethyl ether and dried under vacuum to obtain the phosphonium bromide salt (S24), which was used in the next step without further purification.

To a stirred solution of 2-bromothiazole-4-carbaldehyde (2) (prepared as shown in Scheme 1) (2.5 g, 13 mmol) and (7-ethoxy-7-oxoheptyl)triphenylphosphonium bromide (S24) (6.6 g, 13 mmol, 1 equiv) in DCM (20 mL) was added dropwise a solution of NaOH (8 mL of 50% solution in H₂O). The resulting biphasic mixture was stirred at r.t for 2 h and partitioned between DCM and brine. The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product mixture was separated on silica in EtOAc/Hex (0 to > 20% EtOAc), providing some fractions with the pure Z-isomer (NC-2) (1.9 g, 5.85 mmol, 45%). ¹H NMR (600 MHz, CDCl₃) δ 7.03 (s, 1H), 6.38 (dt, J = 11.7, 1.6 Hz, 1H), 5.77 (dt, J = 11.7, 7.3 Hz, 1H), 4.15 (q, J = 7.1 Hz, 2H), 2.55 (qd, J = 7.4, 1.7 Hz, 2H), 2.34–2.30 (m, 2H), 1.68 (dt, J = 15.2, 7.5 Hz, 2H), 1.51 (dd, J = 15.1, 7.6 Hz, 2H), 1.44–1.38 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 173.8, 153.9, 135.6, 134.7, 121.2, 119.3, 60.2, 34.3, 29.1, 28.8, 24.8, 14.3.

Some fractions with the E/Z mixture (1.3 g, 3.9 mmol, 30%) were obtained as well and were used to obtain a mixture of E/Z analogs.

Ethyl (Z)-8-(2-Ethynylthiazol-4-yl)oct-7-enoate (FC-2).

Synthesized according to general Sonogashira coupling conditions (2 mmol scale, 62.5% yield) (>99% HPLC purity). ¹H NMR (600 MHz, CDCl₃) δ 7.12 (s, 1H), 6.45 (dt, J = 11.7, 1.7 Hz, 1H), 5.81 (dt, J = 11.7, 7.2 Hz, 1H), 4.14 (q, J = 7.1 Hz, 2H), 2.57 (qd, J = 7.4, 1.7 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 1.67 (dt, J = 15.2, 7.6 Hz, 2H), 1.52 (dd, J = 15.1, 7.6 Hz, 2H), 1.45–1.38 (m, 2H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 173.9, 154.2, 146.2, 135.7, 121.5, 117.9, 81.8, 76.7, 60.2, 34.3, 29.2, 29.0, 28.8, 24.8, 14.3.

(Z)-8-(2-Bromothiazol-4-yl)-N-hydroxyoct-7-enamide (HC-2).

Synthesized according to the general THP deprotection conditions (0.1 mmol scale, 76% yield). ¹H NMR (600 MHz, acetone-d₆) δ 9.94 (s, 1H), 7.86 (s, 1H), 7.46 (s, 1H), 6.38 (d, J = 11.7 Hz, 2H), 5.80–5.74 (m, 1H), 2.61 (q, J = 7.4 Hz, 2H), 2.11 (t, J = 7.3 Hz, 2H), 1.64 (dt, J = 14.9, 7.5 Hz, 2H), 1.50 (dt, J = 14.9, 7.3 Hz, 2H), 1.39 (dt, J = 15.2, 7.6 Hz, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.6, 154.0, 134.9, 134.3, 122.3, 120.9, 32.3, 29.5, 29.0, 28.6, 25.2. HRMS (ESI) calcd for C₁₁H₁₆BrN₂O₂S (M + H) 319.0115 found 319.0122.

(Z)-8-(2-Ethynylthiazol-4-yl)-N-hydroxyoct-7-enamide (HY-2).

Synthesized according to the general THP deprotection conditions (0.1 mmol scale, 75% yield). ¹H NMR (600 MHz, acetone-d₆) δ 9.96 (s, 1H), 8.01 (s, 1H), 7.53 (s, 1H), 6.44 (d, J = 11.7 Hz, 1H), 5.80 (dt, J = 11.7, 7.3 Hz, 1H), 4.30 (s, 1H), 2.64 (dd, J = 13.8, 7.1 Hz, 2H), 2.11 (t, J = 7.3 Hz, 2H), 1.67–1.61 (m, 2H), 1.50 (dt, J = 15.1, 7.5 Hz, 2H), 1.38 (dd, J = 14.9, 7.0 Hz, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.7, 154.4, 146.1, 135.1, 121.2, 119.1, 83.0, 76.6, 32.3, 29.1, 28.8, 28.7, 25.2. HRMS (ESI) calcd for C₁₃H₁₇N₂O₂S (M + H) 265.1010 found 265.1017.

Methyl (2E,4E)-5-(2-Bromothiazol-4-yl)penta-2,4-dienoate (NC-4).

To a round-bottom flask containing methyl (E)-4-bromobut-2-enoate (S28) (3.8 g, 21.7 mmol, 1 equiv) was added triethyl phosphite (3. 77 mL, 21.7 mmol, 1 equiv). The resulting mixture was heated at 160 °C for 30 min. After which point, it was allowed to cool to room temperature. The prepared methyl (E)-4-(diethoxyphosphoryl)but-2-enoate (S29) was added to a stirred solution of 2-bromothiazole-4-carbaldehyde (4.1 g, 21.7 mmol) in DME (50 mL), followed by the dropwise addition of freshly prepared 1 M MeONa in MeOH (21.7 mL, 1 equiv). The resulting mixture was stirred at r.t for 30 min, transferred to a flask containing iced water, and stirred for an additional 15 min. EtOAc was added, and the organic layer was collected, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product mixture was purified by silica gel chromatography in EtOAc/hexanes (0 to >100% EtOAc) to yield pure compound (NC-4) (3.7 g, 13.4 mmol, 62%). ¹H NMR (600 MHz, CDCl₃) δ 7.42 (ddd, J = 15.2, 11.5, 0.5 Hz, 1H), 7.24–7.19 (m, 1H), 7.19 (s, 1H), 6.78 (d, J = 9.3 Hz, 1H), 6.07 (d, J = 15.3 Hz, 1H), 3.79 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 167.2, 153.1, 143.7, 136.8, 130.5, 129.8, 122.9, 120.8, 51.8.

Methyl (2E,4E)-5-(2-Ethynylthiazol-4-yl)penta-2,4-dienoate (FC-4).

Synthesized according to general Sonogashira coupling conditions (0.66 mmol scale, 60% yield) (99% HPLC purity). ¹H NMR (600 MHz, CDCl₃) δ 7.49–7.40 (m, 1H), 7.33–7.25 (m, 2H), 6.85 (d, J = 15.2 Hz, 1H), 6.08 (d, J = 15.2 Hz, 1H), 3.80 (s, 3H), 3.53 (s, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 167.4, 153.6, 147.9, 143.9, 130.9, 130.1, 122.7, 119.8, 82.8, 76.2, 51.7. HRMS (ESI) calcd for C₁₁H₁₀NO₂S (M + H) 220.0432 found 220.0441.

(2E,4E)-5-(2-Bromothiazol-4-yl)penta-2,4-dienoic Acid (16).

Synthesized according to general hydrolysis conditions (0.11 mmol scale, 88% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 12.37 (s, 1H), 7.81 (s, 1H), 7.32 (dd, J = 15.1, 11.3 Hz, 1H), 7.14 (dd, J = 15.1, 11.3 Hz, 1H), 7.03 (d, J = 15.1 Hz, 1H), 6.11 (d, J = 15.2 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.9, 153.2, 143.9, 137.5, 131.4, 129.5, 124.5, 124.1.

(2E,4E)-5-(2-Bromothiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)-oxy)penta-2,4-dienamide (17).

Synthesized according to the general NH₂OTHP coupling conditions (0.1 mmol scale, 93% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.38 (s, 1H), 7.66 (s, 1H), 7.40–7.34 (m, 1H), 7.18 (t, J = 12.3 Hz, 1H), 6.97 (d, J = 15.0 Hz, 1H), 6.20 (d, J = 15.0 Hz, 1H), 4.99 (s, 1H), 4.00 (t, J = 10.5 Hz, 1H), 3.54 (d, J = 10.9 Hz, 1H), 1.88–1.65 (m, 3H), 1.66–1.48 (m, 3H). ¹³C NMR (151 MHz, acetone-d₆) δ 153.7, 139.5, 136.2, 129.8, 123.2, 122.3, 101.5, 61.5, 28.0, 25.0, 18.4.

(2E,4E)-5-(2-Bromothiazol-4-yl)-N-hydroxypenta-2,4-dienamide (HC-4).

Synthesized according to the general THP deprotection conditions (0.07 mmol scale, 50% yield) (>99% HPLC purity). ¹H NMR (600 MHz, MeOH-d₄) δ 7.54 (s, 1H), 7.36–7.30 (m, J = 14.5 Hz, 1H), 7.19–7.13 (m, J = 14.8, 11.5 Hz, 1H), 6.92–6.85 (m, J = 15.1 Hz, 1H), 6.09–6.03 (m, J = 12.8 Hz, 1H). ¹³C NMR (151 MHz, MeOH-d₄) δ 164.9, 153.5, 139.5, 136.8, 129.6, 129.4, 121.8. HRMS (ESI) calcd for C₈H₈BrN₂O₂S (M + H) 274.9489 found 274.9479.

(2E,4E)-5-(2-Ethynylthiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)-oxy)penta-2,4-dienamide (18).

Synthesized according to general Sonogashira coupling conditions (0.35 mmol scale, 70% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.82 (br, 1H), 7.55–7.42 (m, 1H), 7.30–7.27 (m, 1H), 6.83 (d, J = 15.1 Hz, 1H), 5.94 (br, J = 69.6 Hz, 1H), 5.01 (s, 1H), 4.05–3.94 (m, 1H), 3.79–3.74 (m, 1H), 3.66 (ddd, J = 11.1, 5.5, 4.1 Hz, 1H), 1.87–1.82 (m, 3H), 1.67–1.61 (m, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 207.7, 157.0, 153.3, 153.0, 147.8, 130.5, 130.2, 127.5, 121.3, 119.5, 100.8, 82.5, 76.2, 62.7, 28.1, 24.7, 18.7.

(2E,4E)-5-(2-Ethynylthiazol-4-yl)-N-hydroxypenta-2,4-dienamide (HY-4).

Synthesized according to the general THP deprotection conditions (0.063 mmol scale, 63% yield) using TFA/DCM instead of HCl/MeOH (>99% HPLC purity). ¹H NMR (600 MHz, acetone-d₆) δ 10.36 (s, 1H), 8.11 (s, 1H), 7.73 (s, 1H), 7.35 (t, J = 13.3 Hz, 1H), 7.27–7.21 (m, 1H), 7.00 (d, J = 14.9 Hz, 1H), 6.18 (d, J = 14.7 Hz, 1H), 4.37 (s, 1H). ¹³C NMR (151 MHz, acetone-d₆) δ 163.2, 154.0, 147.4, 138.9, 130.0, 129.9, 122.6, 120.2, 83.6, 76.2. HRMS (ESI) calcd for C₁₀H₉N₂O₂S (M + H) 221.0384 found 221.0962.

Methyl (E)-3-(2-Bromothiazol-4-yl)acrylate (NC-5).

To a stirred solution of triphenyl phosphine (2.3 g, 9 mmol, 1.05 equiv) in EtOAc (14 mL) was slowly added a solution of bromomethyl acetate (S30) (1.3 g, 8.5 mmol) in EtOAc (2.5 mL). The resulting mixture was stirred at r.t for 12 h. The precipitated white solid was collected by filtration, washed with diethyl ether, and air-dried overnight. It was resuspended in 1 N NaOH (16 mL) and stirred for 20 min; at which point, DCM was added. The organic layer was washed with brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum to provide ylide (19), which was used in the next step without further purification.

The ylide (19) synthesized as described above (9 mmol) and 2-bromothiazole-4-carbaldehyde (2) (1.7 g, 9 mmol, 1 equiv) were dissolved in THF (52 mL) and stirred at r.t for 12 h. Brine and DCM were added, and the organic layer was separated, dried over anhydrous Na₂SO₄, filtered, and concentrated under vacuum. The crude product was purified by silica gel chromatography in EtOAc/hexanes (0 to >100% EtOAc) to provide the pure product (NC-5) (2 g, 8.1 mmol, 90%) (>99% HPLC purity). ¹H NMR (600 MHz, CDCl₃) δ 7.55–7.49 (m, 1H), 7.37 (s, 1H), 6.77 (d, J = 15.5 Hz, 1H), 3.80 (s, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 167.2, 151.7, 137.4, 134.5, 124.8, 121.5, 52.4. HRMS (ESI) calcd for C₇H₇BrNO₂S (M + H) 247.9380 found 247.9382.

Methyl (E)-3-(2-Ethynylthiazol-4-yl)acrylate (FC-5).

Synthesized according to general Sonogashira coupling conditions (0.415 mmol scale, 83% yield) (>99% HPLC purity). ¹H NMR (600 MHz, CDCl₃) δ 7.60 (d, J = 15.6 Hz, 1H), 7.46 (s, 1H), 6.84 (d, J = 15.6 Hz, 1H), 3.82 (s, 3H), 3.54 (s, 1H). ¹³C NMR (151 MHz, CDCl₃) δ 167.3, 152.0, 148.2, 135.4, 122.8, 121.6, 82.8, 76.0, 51.8. HRMS (ESI) calcd for C9H8NO2S (M + H) 194.0275 found 194.0279.

(E)-3-(2-Bromothiazol-4-yl)acrylic Acid (20).

Synthesized according to general hydrolysis conditions (2.17 mmol scale, 99% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 12.54 (s, 1H), 8.10 (s, 1H), 7.53 (d, J = 15.5 Hz, 1H), 6.48 (d, J = 15.5 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 167.8, 151.4, 138.0, 135.7, 128.3, 121.8.

(E)-3-(2-Bromothiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-acrylamide (21).

Synthesized according to the general NH₂OTHP coupling conditions (2.11 mmol mmol scale, 88% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.42 (s, 1H), 7.86 (s, 1H), 7.53 (d, J = 15.3 Hz, 1H), 6.79 (t, J = 13.7 Hz, 1H), 5.02 (s, 1H), 4.02 (t, J = 10.5 Hz, 1H), 3.58–3.54 (m, 1H), 1.81–1.74 (m, 3H), 1.63–1.56 (m, 3H). ¹³C NMR (151 MHz, acetone-d₆) δ 162.6, 152.5, 136.6, 133.4, 131.4, 101.4, 67.4, 61.5, 27.9, 27.3, 25.0, 25.0, 22.8, 18.3.

(E)-3-(2-Bromothiazol-4-yl)-N-hydroxyacrylamide (HC-5).

Synthesized according to the general THP deprotection conditions (0.29 mmol scale, 97% yield) (>99% HPLC purity). ¹H NMR (600 MHz, DMSO-d₆) δ 10.83 (S, 1H), 9.12 (d, J = 1.7 Hz, 1H), 7.95 (s, 1H), 7.40 (d, J = 15.3 Hz, 1H), 6.63 (d, J = 15.3 Hz, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.6, 151.9, 137.6, 130.8, 126.6, 121.9. HRMS (ESI) calcd for C₆H₆BrN₂O₂S (M + H) 248.9333 found 248.9345.

(E)-3-(2-Ethynylthiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-acrylamide (22).

Synthesized according to general Sonogashira coupling conditions (1.16 mmol scale, 83% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.45 (s, 1H), 7.96 (s, 1H), 7.61 (d, J = 15.8 Hz, 2H), 6.90 (d, J = 15.8 Hz, 1H), 5.05 (s, 1H), 4.41 (s, 1H), 4.06 (s, 1H), 3.59 (s, 1H), 1.80 (m, 3H), 1.62 (m, 3H). ¹³C NMR (151 MHz, acetone-d₆) δ 162.6, 152.6, 147.8, 131.7, 128.6, 123.1, 121.7, 101.5, 83.9, 76.1, 61.5, 28.0, 25.1, 18.4.

(E)-3-(2-Ethynylthiazol-4-yl)-N-hydroxyacrylamide (HY-5).

Synthesized according to the general THP deprotection conditions (0.29 mmol scale, 96% yield) (>99% HPLC purity). ¹H NMR (600 MHz, DMSO-d₆) δ 10.85 (s, 1H), 9.12 (s, 1H), 8.05 (s, 1H), 7.44 (d, J = 15.3, 6.9 Hz, 1H), 6.68 (d, J = 15.3 Hz, 1H), 5.02 (s, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 162.8, 152.5, 147.7, 131.0, 124.1, 122.3, 87.1, 76.6. HRMS (ESI) calcd for C₈H₇N₂O₂S (M + H) 195.0228 found 195.0222.

Ethyl (E)-8-(2-Bromothiazol-4-yl)oct-7-enoate (NC-3).

Synthesized as previously described for the synthesis of compound (14) using LiOH as the base instead of NaOH (1.41 mmol scale, 70% yield). ¹H NMR (600 MHz, CDCl₃) δ 6.88 (s, 1H), 6.63–6.56 (m, 1H), 6.31 (d, J = 14.6 Hz, 1H), 4.17–4.11 (m, 2H), 2.35–2.28 (m, 2H), 2.26–2.17 (m, 2H), 1.70–1.62 (m, J = 6.9 Hz, 2H), 1.54–1.47 (m, 2H), 1.42–1.35 (m, 2H), 1.30–1.24 (m, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 173.8, 154.8, 135.8, 135.3, 122.0, 116.3, 60.2, 34.3, 32.4, 28.7, 28.6, 24.8, 14.3. HRMS (ESI) calcd for C₁₃H₁₉BrNO₂S (M + H) 334.0319 found 334.0311.

Ethyl (E)-8-(2-Ethynylthiazol-4-yl)oct-7-enoate (FC-3).

Synthesized according to general Sonogashira coupling conditions (0.268 mmol scale, 67% yield). ¹H NMR (600 MHz, CDCl₃) δ 6.99 (s, 1H), 6.68–6.62 (m, 1H), 6.39 (dd, J = 15.6, 1.4 Hz, 1H), 4.17–4.12 (m, 2H), 3.47 (s, 1H), 2.31 (dd, J = 9.7, 5.4 Hz, 2H), 2.26–2.22 (m, 2H), 1.66 (dt, J = 15.4, 7.6 Hz, 2H), 1.51 (dt, J = 15.2, 7.6 Hz, 2H), 1.44–1.36 (m, 3H), 1.27 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 173.8, 155.0, 147.0, 135.4, 122.3, 114.9, 81.9, 76.6, 60.2, 34.3, 32.5, 29.7, 28.7, 28.6, 24.8, 14.3. HRMS (ESI) calcd for C₁₅H₂₀NO₂S (M + H) 278.1214 found 278.1242.

(E)-8-(2-Bromothiazol-4-yl)-N-hydroxyoct-7-enamide (HC-3).

Synthesized according to the general THP deprotection conditions (0.04 mmol scale, 77% yield). ¹H NMR (600 MHz, MeOH-d₄) δ 7.22 (s, 1H), 6.58–6.51 (m, 1H), 6.38 (d, J = 15.6 Hz, 1H), 3.66 (s, 1H), 2.25 (q, J = 6.9 Hz, 2H), 2.12 (t, J = 7.4 Hz, 2H), 1.67 (dt, J = 15.0, 7.5 Hz, 3H), 1.52 (dd, J = 15.0, 7.5 Hz, 2H), 1.41 (dd, J = 15.3, 8.3 Hz, 3H), 0.92 (t, J = 6.8 Hz, 2H). ¹³C NMR (151 MHz, MeOH-d₄) δ 171.5, 154.6, 135.8, 134.4, 122.0, 117.1, 32.3, 32.0, 28.4, 28.2, 25.2. HRMS (ESI) calcd for C₁₁H₁₆BrN₂O₂S (M + H) 319.0115 found 319.0122.

(E)-8-(2-Ethynylthiazol-4-yl)-N-hydroxyoct-7-enamide (HY-3).

Synthesized according to the general THP deprotection conditions (0.105 mmol scale, 75% yield). ¹H NMR (600 MHz, acetone-d₆) δ 9.95 (s, 1H), 7.86 (s, 1H), 7.42 (s, 1H), 6.66–6.61 (m, 2H), 6.48 (d, J = 15.6 Hz, 2H), 4.29 (s, 1H), 2.24 (q, J = 6.9 Hz, 3H), 2.12 (dd, J = 12.0, 4.6 Hz, 3H), 1.64 (dt, J = 15.1, 7.5 Hz, 4H), 1.50 (dd, J = 15.0, 7.5 Hz, 4H), 1.38 (dt, J = 15.0, 7.6 Hz, 4H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.6, 155.1, 146.7, 134.8, 122.6, 115.8, 83.0, 76.5, 32.2, 28.6, 28.5, 25.1. HRMS (ESI) calcd for C₁₃H₁₇N₂O₂S (M + H) 265.1010 found 265.1034.

5-Methoxy-5-oxopentan-1-aminium Chloride (S2).

Synthesized following a similar protocol for the synthesis of compound (7) (100% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 7.99 (s, 3H), 3.58 (s, 3H), 2.75 (s, 2H), 2.34 (s, 2H), 1.56 (s, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.6, 51.8, 38.8, 33.1, 26.8, 21.8.

Methyl 5-(2-Bromothiazole-4-carboxamido)pentanoate (S3).

Synthesized following a similar protocol for the synthesis of (NC-1) (36% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.03 (s, 1H), 7.24 (d, J = 5.6 Hz, 1H), 3.67 (s, 3H), 3.44 (dd, J = 13.1, 6.8 Hz, 2H), 2.36 (t, J = 7.3 Hz, 2H), 1.75–1.60 (m, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 173.8, 159.7, 150.0, 135.8, 126.7, 51.6, 39.0, 33.6, 29.1, 22.2.

5-(2-Bromothiazole-4-carboxamido)pentanoic Acid (S4).

Synthesized according to general hydrolysis conditions (92% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 8.54 (t, J = 6.0 Hz, 1H), 8.20 (s, 1H), 3.26–3.18 (m, 2H), 2.21 (t, J = 6.9 Hz, 2H), 1.50–1.45 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.3, 160.0, 150.0, 136.9, 128.8, 38.8, 33.7, 28.9, 22.2.

2-Bromo-N-(5-oxo-5-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-pentyl)thiazole-4-carboxamide (S5).

Synthesized according to the general NH₂OTHP coupling conditions (72% yield). ¹H NMR (600 MHz, CDCl₃) δ 9.19 (s, 1H), 8.07 (s, 1H), 7.38 (s, 1H), 4.97 (s, 1H), 3.97 (t, J = 9.9 Hz, 1H), 3.65–3.58 (m, 1H), 3.45 (dd, J = 12.8, 6.3 Hz, 2H), 2.23 (dd, J = 20.5, 13.0 Hz, 2H), 1.84–1.66 (m, 10H). ¹³C NMR (151 MHz, CDCl₃) δ 170.3, 160.0, 149.8, 136.0, 127.0, 102.4, 94.5, 62.4, 38.7, 32.5, 28.8, 28.0, 25.0, 22.5, 18.5.

2-Ethynyl-N-(5-oxo-5-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-pentyl)thiazole-4-carboxamide (S6).

Synthesized according to general Sonogashira coupling conditions (58% yield). ¹H NMR (400 MHz, CDCl₃) δ 8.81 (s, 1H), 8.10 (s, 1H), 7.40 (s, 1H), 4.94 (s, 1H), 3.94 (t, J = 9.6 Hz, 1H), 3.65–3.55 (m, 1H), 3.53 (s, 1H), 3.49–3.37 (m, 4H), 1.81–1.58 (m, 10H). ¹³C NMR (151 MHz, CDCl₃) δ 170.2, 160.5, 150.5, 147.2, 125.1, 102.3, 83.2, 75.7, 62.4, 38.6, 32.5, 29.7, 28.9, 28.0, 25.0, 22.6, 18.4.

2-Ethynyl-N-(5-(hydroxyamino)-5-oxopentyl)thiazole-4-carboxamide (S7).

Compound S7 was synthesized using the general THP deprotection conditions (14% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.04 (s, 1H), 8.27 (s, 1H), 8.23 (s, 1H), 7.95 (s, 1H), 4.43 (s, 1H), 3.47–3.41 (m, 2H), 2.17 (t, J = 6.9 Hz, 2H), 1.73–1.60 (m, 4H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.8, 159.8, 151.2, 147.1, 125.3, 84.2, 75.7, 38.5, 31.9, 29.0, 22.6.

6-Methoxy-6-oxohexan-1-aminium Chloride (S9).

Synthesized following a similar protocol for the synthesis of compound (7) (100% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 8.11 (s, 3H), 3.58 (s, 3H), 2.76–2.67 (m, 2H), 2.34–2.22 (m, 2H), 1.61–1.45 (m, 4H), 1.34–1.25 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.3, 51.3, 38.5, 33.1, 26.6, 25.3, 23.9.

Methyl 6-(2-Bromothiazole-4-carboxamido)hexanoate (S10).

Synthesized following a similar protocol for the synthesis of (NC-1) (53% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.03 (s, 1H), 7.22 (s, J = 10.8 Hz, 1H), 3.66 (s, 3H), 3.43 (dd, J = 13.6, 6.8 Hz, 2H), 2.32 (t, J = 7.5 Hz, 2H), 1.71–1.65 (m, 2H), 1.65–1.59 (m, 2H), 1.44–1.37 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 174.2, 159.8, 150.2, 135.9, 126.8, 51.7, 39.4, 34.0, 29.4, 26.5, 24.7.

6-(2-Bromothiazole-4-carboxamido)hexanoic Acid (S11).

Synthesized according to general hydrolysis conditions (92% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 11.99 (s, 1H), 8.51 (t, J = 5.8 Hz, 1H), 8.25 (s, 1H), 3.21 (dd, J = 13.5, 6.7 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.54–1.45 (m, 4H), 1.30–1.23 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.0, 159.6, 150.4, 136.7, 128.8, 39.1, 34.0, 29.3, 26.4, 24.7.

2-Bromo-N-(6-oxo-6-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-hexyl)thiazole-4-carboxamide (S12).

Synthesized according to the general NH₂OTHP coupling conditions (79% yield). ¹H NMR (600 MHz, CDCl₃) δ 9.20 (s, 1H), 8.04 (s, 1H), 7.31 (s, 1H), 4.93 (s, 1H), 3.93 (t, J = 9.8 Hz, 1H), 3.57 (d, J = 11.1 Hz, 1H), 3.38 (dd, J = 13.2, 6.6 Hz, 2H), 2.10 (s, J = 6.5 Hz, 2H), 1.80–1.54 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) δ 170.4, 159.8, 149.9, 135.9, 126.9, 102.4, 62.4, 39.3, 33.0, 29.2, 28.0, 25.0, 18.6.

2-Ethynyl-N-(6-oxo-6-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-hexyl)thiazole-4-carboxamide (S13).

Synthesized according to general Sonogashira coupling conditions (55% yield). ¹H NMR (400 MHz, CDCl₃) δ 9.21 (s, 1H), 8.11 (s, 1H), 7.39 (s, 1H), 4.92 (s, 1H), 3.92 (t, J = 9.0 Hz, 1H), 3.59–3.52 (m, 2H), 3.38 (dd, J = 13.2, 6.6 Hz, 2H), 2.10 (s, 2H), 1.77–1.51 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) δ 170.5, 160.3, 150.5, 147.3, 125.2, 102.2, 83.5, 75.6, 62.3, 39.3, 33.0, 29.2, 28.0, 26.4, 25.0, 18.5.

2-Ethynyl-N-(6-(hydroxyamino)-6-oxohexyl)thiazole-4-carboxamide (S14).

Compound S14 was synthesized using the general THP deprotection conditions (24% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.02 (s, 1H), 8.27 (s, 1H), 8.27 (s, 1H), 7.95 (s, 1H), 4.43 (s, 1H), 3.42 (dd, J = 13.5, 6.7 Hz, 2H), 2.13 (t, J = 7.3 Hz, 2H), 1.71–1.59 (m, 4H), 1.45–1.36 (m, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 169.8, 159.7, 151.3, 147.1, 125.2, 84.2, 75.7, 38.8, 32.2, 26.2, 25.0.

8-Methoxy-8-oxooctan-1-aminium Chloride (S16).

Synthesized following a similar protocol for the synthesis of compound (7) (100% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 7.80 (s, 3H), 3.58 (s, 3H), 2.76–2.72 (m, 2H), 2.31–2.27 (m, 2H), 1.56–1.46 (m, 4H), 1.31–1.21 (m, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 173.8, 51.7, 39.2, 33.7, 28.7, 28.6, 27.4, 26.1, 24.8.

Methyl 8-(2-Bromothiazole-4-carboxamido)octanoate (S17).

Synthesized following a similar protocol for the synthesis of NC-1 (40% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.03 (s, J = 1.0 Hz, 1H), 7.20 (s, 1H), 3.66 (s, 3H), 3.41 (dd, J = 13.5, 6.9 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 1.66–1.55 (m, 4H), 1.41–1.27 (m, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 174.3, 159.6, 150.1, 135.8, 126.6, 51.5, 39.5, 34.1, 29.5, 29.0, 28.9, 26.8, 24.9.

8-(2-Bromothiazole-4-carboxamido)octanoic Acid (S18).

Synthesized according to general hydrolysis conditions (83% yield). ¹H NMR (600 MHz, DMSO-d₆) δ 8.50 (t, J = 5.9 Hz, 1H), 8.23 (s, 1H), 3.20 (dd, J = 13.6, 6.7 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.50–1.43 (m, 4H), 1.25 (s, J = 2.1 Hz, 6H). ¹³C NMR (151 MHz, DMSO-d₆) δ 175.1, 159.6, 150.3, 136.7, 128.7, 39.2, 34.1, 29.5, 29.0, 28.9, 26.7, 24.9.

2-Bromo-N-(8-oxo-8-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-octyl)thiazole-4-carboxamide (S19).

Synthesized according to the general NH₂OTHP coupling conditions (72% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.88 (s, 1H), 8.08 (t, J = 15.9 Hz, 1H), 7.26 (s, J = 14.1 Hz, 1H), 4.94 (s, 1H), 3.94 (s, 1H), 3.62–3.59 (m, 1H), 3.44–3.38 (m, 2H), 2.13 (m, 2H), 1.85–1.76 (m, 4H), 1.66–1.45 (m, 12H). ¹³C NMR (151 MHz, CDCl₃) δ 170.7, 159.8, 149.9, 135.9, 102.5, 62.6, 39.2, 38.8, 29.4, 28.5, 28.0, 27.8, 24.9, 18.7.

2-Ethynyl-N-(8-oxo-8-(((tetrahydro-2H-pyran-2-yl)oxy)amino)-octyl)thiazole-4-carboxamide (S20).

Synthesized according to general Sonogashira coupling conditions (34% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.70 (s, 1H), 8.16 (s, 1H), 7.34 (s, 1H), 4.95 (s, 1H), 3.95 (t, J = 9.0 Hz, 1H), 3.61 (ddd, J = 9.8, 4.8, 3.5 Hz, 1H), 3.54 (s, 1H), 3.43 (dd, J = 13.4, 6.8 Hz, 2H), 2.10 (s, 2H), 1.84–1.54 (m, 10H), 1.34 (s, 6H). ¹³C NMR (151 MHz, CDCl₃) δ 160.3, 150.7, 147.2, 125.1, 102.5, 83.1, 75.7, 62.6, 39.1, 33.1, 29.4, 28.7, 28.4, 28.0, 26.4, 25.0, 18.7.

2-Ethynyl-N-(8-(hydroxyamino)-8-oxooctyl)thiazole-4-carboxamide (S21).

Compound S21 was synthesized using the general THP deprotection conditions (62% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.07 (s, 1H), 8.40 (s, 1H), 8.29 (s, 1H), 7.96 (s, 1H), 4.43 (s, 1H), 3.43 (dd, J = 13.4, 6.8 Hz, 2H), 2.11 (dd, J = 9.4, 5.3 Hz, 2H), 1.67–1.56 (m, 4H), 1.42–1.25 (m, 6H). ¹³C NMR (151 MHz, acetone-d₆) δ 170.0, 159.8, 151.3, 147.1, 125.3, 84.3, 75.7, 38.9, 38.7, 32.2, 29.5, 28.8, 28.7, 26.5, 25.2.

(Z)-8-(2-Bromothiazol-4-yl)oct-7-enoic Acid (S25).

Synthesized according to general hydrolysis conditions (0.27 mmol scale, 92% yield). ¹H NMR (600 MHz, acetone-d₆) δ 10.47 (br, 1H), 7.46 (s, 1H), 6.38 (dt, J = 11.7, 1.5 Hz, 1H), 5.81–5.75 (m, 1H), 2.65–2.60 (m, 2H), 2.33–2.28 (m, 2H), 1.68–1.61 (m, 2H), 1.55–1.49 (m, 2H), 1.46–1.40 (m, 2H). ¹³C NMR (151 MHz, acetone-d₆) δ 173.7, 154.0, 134.9, 134.2, 120.9, 33.2, 29.0, 28.6, 28.6, 24.6.

(Z)-8-(2-Bromothiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-oct-7-enamide (S26).

Synthesized according to the general NH₂OTHP coupling conditions (0.9 mmol scale, 90% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.53 (s, 1H), 7.02 (s, 1H), 6.36 (d, J = 11.7 Hz, 1H), 5.75 (dt, J = 11.8, 7.3 Hz, 1H), 4.96 (s, 1H), 3.95 (d, J = 8.8 Hz, 1H), 3.63 (dtd, J = 11.2, 4.2, 1.6 Hz, 2H), 2.52 (q, J = 6.8 Hz, 3H), 2.20–2.02 (m, 2H), 1.87–1.76 (m, 4H), 1.73–1.56 (m, 9H), 1.50 (dt, J = 14.5, 7.2 Hz, 4H), 1.42 (dt, J = 13.1, 6.5 Hz, 4H). ¹³C NMR (151 MHz, CDCl₃) δ 170.6, 153.8, 135.5, 134.8, 121.2, 119.4, 102.5, 62.6, 33.3, 29.1, 28.8, 28.0, 25.2, 25.0, 18.6.

(Z)-8-(2-Ethynylthiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-oct-7-enamide (S27).

Synthesized according to general Sonogashira coupling conditions (0.06 mmol scale, 65% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.38 (s, 1H), 7.12 (s, 1H), 6.44 (d, J = 11.7 Hz, 1H), 5.80 (dt, J = 11.7, 7.3 Hz, 1H), 4.96 (s, 1H), 3.98–3.93 (m, 1H), 3.66–3.62 (m, 1H), 2.55 (q, J = 6.9 Hz, 2H), 2.16–2.11 (m, 2H), 1.87–1.77 (m, 2H), 1.73–1.58 (m, 6H), 1.51 (dd, J = 14.7, 7.4 Hz, 2H), 1.42 (dd, J = 14.6, 7.5 Hz, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 170.6, 154.2, 146.2, 135.7, 121.5, 118.0, 102.5, 81.9, 76.7, 62.6, 33.3, 29.0, 28.9, 28.8, 28.0, 25.1, 25.0, 18.6.

(E)-8-(2-Bromothiazol-4-yl)oct-7-enoic Acid (S31).

Synthesized according to general hydrolysis conditions (1.3 mmol scale, 92% yield). ¹H NMR (600 MHz, MeOH-d₄) δ 7.22 (s, 1H), 6.58–6.51 (m, 1H), 6.38 (dt, J = 15.5, 1.3 Hz, 1H), 2.32 (t, J = 7.4 Hz, 2H), 2.24 (tt, J = 7.0, 3.4 Hz, 2H), 1.65 (dt, J = 15.1, 7.5 Hz, 2H), 1.53 (dt, J = 14.9, 7.4 Hz, 2H), 1.44–1.39 (m, 2H). ¹³C NMR (151 MHz, MeOH-d₄) δ 176.2, 154.6, 135.8, 134.5, 121.9, 117.0, 33.5, 32.1, 28.5, 28.4, 24.5.

(E)-8-(2-Bromothiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-oct-7-enamide (S32).

Synthesized according to the general NH₂OTHP coupling conditions (530 mg, 1.27 mmol, 90%). ¹H NMR (600 MHz, CDCl₃) δ 8.26 (s, 1H), 6.90 (s, 1H), 6.62–6.55 (m, 1H), 6.32 (d, J = 15.5 Hz, 1H), 4.96 (s, 1H), 3.99–3.92 (m, 1H), 3.65 (m, 1H), 2.23 (q, J = 7.0 Hz, 2H), 2.12 (S, 2H), 1.88–1.80 (m, 4H), 1.71–1.62 (m, 6H), 1.53–1.48 (m, 2H), 1.42–1.38 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 156.7, 154.7, 135.8, 122.1, 116.3, 100.5, 33.4, 32.4, 29.1, 28.6, 28.0, 25.3, 25.0, 20.3, 18.6.

(E)-8-(2-Ethynylthiazol-4-yl)-N-((tetrahydro-2H-pyran-2-yl)oxy)-oct-7-enamide (S33).

Synthesized according to general Sonogashira coupling conditions (0.167 mmol scale, 67% yield). ¹H NMR (600 MHz, CDCl₃) δ 8.23 (br, J = 20.4 Hz, 1H), 7.00 (s, 1H), 6.64 (dt, J = 14.4, 7.0 Hz, 1H), 6.39 (d, J = 15.6, 1.3 Hz, 1H), 4.96 (s, 1H), 3.95 (t, J = 7.5 Hz, 1H), 3.67–3.63 (m, 1H), 2.24 (dd, J = 13.9, 6.9 Hz, 2H), 2.12 (s, 2H), 1.87–1.78 (m, 4H), 1.71–1.59 (m, 6H), 1.51 (dt, J = 14.7, 7.2 Hz, 2H), 1.44–1.37 (m, 2H). ¹³C NMR (151 MHz, CDCl₃) δ 170.6, 155.0, 147.1, 135.3, 122.4, 115.0, 102.5, 82.0, 76.6, 62.6, 33.4, 32.5, 29.7, 28.7, 28.0, 25.2, 25.0, 18.5.

Docking.

The docking analysis was accomplished using a Glide tool on maestro version 10.6. A library of the compounds was generated utilizing the ligand prep tool allowing possible ionization states at pH = 7 ± 2. Protein structures were obtained from protein data bank (PDB), and their structures were prepared using the protein preparation wizard. The prepared protein structures were used to generate the receptor grid using the receptor grid generation tool. The active site was determined by utilizing the cocrystalized ligand. Metal coordination to the zinc ion was implemented as a constrain. Rotation of amino acid side chains was allowed only within the determined active site. Then, the library of the compounds was screened with the obtained receptors utilizing the ligand docking tool using extra precision and an OPLS3 force field. The results were analyzed with the XP visualizer tool, and pose analysis was performed with the pose viewer tool. For the pose analysis, the Maestro version 13.1 for academic use was used.

Cell Culture and Cell Viability Assays for NCI-H522, HCT-116, WI38, and RPE.

HCT116 human colorectal carcinoma cells, NCI-H522 human lung cancer cells, WI38 diploid human cell line, and human retinal pigment epithelial RPE cells were maintained in the Dulbecco’s modified Eagle’s medium (Mediatech, Inc.) supplemented with 10% calf serum (Atlanta Biologicals) or 10% fetal bovine serum (Gemini Bio-Products # 100–106) and 1000 U/mL of both penicillin and streptomycin (Mediatech, Inc.) at 37 °C with 5% CO_2. Cell viability was assessed using methylene blue staining: cells were plated at 5000/well in 96-well plates (n = 3), respectively, and treated the next day. Three days after treatment, cells were fixed/stained in methylene blue saturated in 50% ethanol for 30 min at r.t.. Plates were washed with excess water to wash off extra dye. Retained dye was dissolved in 0.1 N HCl, and absorbance was measured at 668 nm.

Cell Culture and Cell Viability Assays for SH-SY5Y Cells.

Human neuroblastoma cells (SH-SY5Y) were grown on poly-d-lysine-coated plates and cultured in a DMEM/F12 medium (HyClone, Thermo Scientific) containing 10% fetal bovine serum and 1% penicillin–streptomycin. Cells were maintained in a 37 °C incubator with a 5% CO₂ atmosphere. Cells were seeded at a density of 15,000 cells in 96-well plates for 36 h before treatment of drugs.

MTT Cell Proliferation and Viability Assay.

Cell viability was assessed using the MTT reagent 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Promega Corporation USA, Ref no.G4102) according to the manufacturer’s instructions. Cells were treated with amide series analogs for 24 h. Briefly, the cells were incubated with MTT reagent for 2 h at 37 °C incubator. Then the solubilization/stop solution was added to solubilize the formazan products, incubated for 1 h, and absorbance at 570 was measured using micro-plate reader (SynergyH1, BioTek, USA). DMSO is used a vehicle control. The experimental data represent the means ± SD (*P < 0.05) of triplicates from three separate experiments. The statistical significance was determined by one-way ANOVA and significant differences P < 0.05, P < 0.01, P < 0.001, and P < 0.0001 are symbolized as *, **, ***, and ****, respectively.

Cytotoxicity Evaluation on Undifferentiated PC-12 Cells.

Pheochromocytoma (PC-12) cells were grown on poly-D-lysine coated plates and cultured in DMEM/F12 medium (HyClone, Thermo Scientific) containing 5% fetal bovine serum, 5% horse serum, and 1% penicillin–streptomycin. Cells were maintained in a 37 °C incubator with a 5% CO₂ atmosphere. Cells were seeded at a density of 15,000 cells per well in 96-well plates for 36 h before treatment with the test compounds. The MTT assay was performed after 24 h of treatment.

Cytotoxicity Evaluation on Differentiated PC-12 Cells.

Pheochromocytoma (PC-12) cells were seeded on poly-D-lysine coated plates containing neuron differentiation medium (100 ng nerve growth factor, DMEM/F12, 5% fetal bovine serum, 5% horse serum, and 1% penicillin–streptomycin) and grown for 5 days to differentiate to neurons. Cells were maintained in a 37 °C incubator with a 5% CO₂ atmosphere. Cells were trypsinized, and seeded at a density of 15,000 cells/well in 96-well plates with neuron differentiation medium for 36 h before treatment with the test compounds. The MTT assay was performed after 24 h of treatment.

Microscopy.

For time lapse imaging, NCI-H522 cells were plated at ∼70% density and let overnight to adhere and pre-equilibrate to 10% CO₂. In the next day, cells were treated with DMSO or inhibitors, and the flask was maintained sealed. Flasks were placed on a 37 °C heated stage on an inverted microscope. Images were captured every 12 min and for a total of 300 images using a ×40 microscope objective and an Olympus C740 digital camera controlled by AmScope software. Image analysis was done using ImageJ. For the Kaplan–Maier graphs, at least 100 cells were counted and the event of cell death was measured on a time frame of either 1 or 0.2 h.

Flow Cytometry.

C11-BODIPY Lipid Peroxidation Assay. NCI-H522 cells (∼70% density) were plated on 7 cm dishes and let overnight to adhere. In the next day, cells were treated with DMSO, inhibitors (10 μM) [with (0.25 μM) or without Liproxstatin-1]. Bodipy 581/591 C11 (1 μM) (ThermoFischer) was added at the time of treatment. After 6 h, cells were washed with 1×PBS and collected by trypsinization and centrifugation. The cells were washed once with 1×PBS and resuspended in PBS containing 2% FBS. Cells were analyzed using a BD LSR Fortessa FACScanner and FlowJo software. For each sample, 20 × 10³ cells were analyzed.

Cell Cycle Analysis.

NCI-H522 cells (5 × 10⁵ cells per condition) were plated on 7 cm dishes and let overnight to adhere. After 12, 24, or 48 h of treatment with DMSO or inhibitors (in the presence of Liproxstatin-1 0.25 μM), floating and attached cells were collected by trypsinization and centrifugation. Cells were then washed once with 1×PBS and fixed by prechilled ethanol (70% final concentration). After fixation, cells were collected by centrifugation and resuspended in PBS and followed by treatment with RNase H (10 μg/mL) for 30 min at 37 °C. Cells again were collected by centrifugation and resuspended in PBS containing 2% FBS and propidium Iodide. Cells were analyzed using a BD LSR Fortessa FACScanner and FlowJo software. For each sample, 20 × 10³ cells were analyzed.

Western Blotting.

NCI-H522 cell pellets obtained after the corresponding treatment conditions were lysed using lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, and 0.5% NP-40 (supplemented with 1 μg/mL aprotinin, 2 μg/mL leupeptin, 1 μg/mL pepstatin A, 1 mM DTT, and 0.1 M PMSF) for 30 min on ice and centrifuged at 13 × 10³g for 25 min at 4 °C. The protein levels of the obtained lysates were normalized using a BCA Protein Assay Kit (Pierce) and separated by SDS-polyacrylamide gel electrophoresis (12.5% acrylamide). Transfer to polyvinylidene difluoride membranes (Millipore) was followed by blocking of membranes with blocking buffer containing 5% (w/v) nonfat dry milk dissolved in PBST [1×PBS containing 0.05% (v/v) Tween 20] for 1 h at room temperature. Membranes were then incubated with corresponding primary antibodies overnight at 4 °C. The membranes were then washed (3 × 15 min each) with PBST and incubated with secondary antibodies conjugated to horse-radish peroxidase, obtained from Biorad and used at a dilution of 1:10,000. Bound antibodies were detected using enhanced chemiluminescence (Biorad).

Isoform Selectivity.

Inhibitor Testing with HDAC Classes I, IIa, and IIb.

In a half-area 96-well white opaque plate (Corning), recombinant HDAC1 (1 μL; 3 ng/μL, BPS Bioscience), HDAC2 (1 μL; 1 ng/μL, BPS Bioscience), HDAC3 (1 μL; 30 ng/μL, BPS Bioscience), HDAC6 (1 μL; 35 ng/μL, BPS Bioscience), and HDAC8 (1 μL; 70 ng/μL, BPS Bioscience) were added to HDAC-Glo buffer (43 μL) provided by the manufacturer (Promega). In the case of HDAC4, recombinant HDAC4 (1 μL; 2 ng/μL, BPS Bioscience) was added to HDAC-Glo IIa buffer (43 μL) provided by Promega. For negative controls, either the HDAC-Glo or the HDAC-Glo IIa buffer (44 μL) alone was used. Serial dilutions or single concentrations of inhibitors (1 μL in DMSO, concentrations shown in Tables S4–S9) or DMSO alone (1 μL) were added to the enzyme solution and followed by 3 h incubation at room temperature. For HDAC1, 2, 3, 6, and 8, the HDAC-Glo reagent was prepared using the premeasured lyophilized HDAC-Glo I/II substrate (Promega) and dissolving in the buffer provided (10 mL). To activate the HDAC-Glo I/IIsubstrate, the developer reagent was added (1 μL for every 1 mL of HDAC-Glo I/II substrate solution). For HDAC4, the HDAC-Glo IIa reagent was prepared using the premeasured lyophilized HDAC-Glo IIa luciferase (Promega) and dissolving in the HDAC-Glo IIa buffer provided (10 mL). To activate, the HDAC-Glo IIa substrate (7 μL for every 1 mL of buffer luciferase solution) was added to the luciferase buffer solution and incubated at 37 °C for 1 h. The developer reagent was added (1 μL for every 1 mL of HDAC-Glo IIa substrate/luciferase solution). The HDAC-Glo reagent (5 μL) was added to each reaction containing HDAC1, 2, 3, 5, and 8, whereas the HDAC-Glo IIa reagent (5 μL) was added to each reactions containing HDAC4. Luminescent signal was measured every 3 min over the course of 30 min using an M-Plex Infinite 200 Pro (Tecan). To determine IC₅₀, the luminescent signal at peak reading was first background corrected with the signal from a background control reaction where HDAC was excluded. The background corrected luminescence signal of each inhibitor-containing reaction was divided by the signal of the reaction without an inhibitor for each HDAC enzyme to generate a percent deacetylase activity remaining value. IC₅₀ values were calculated by fitting the percent deacetylase activity remaining as a function of inhibitor concentration to a sigmoidal dose response curve$\left(y=100/{\left(1+x/{\text{IC}}_{50}\right)}^z\right)$, where y = percent deacetylase activity and x = inhibitor concentration) using nonlinear regression with KaleidaGraph 4.1.3 software.

ABRREVIATIONS USED

BDR

bromodomain

CD

catalytic domain

CDK

cyclin dependent kinases

CNS

central nervous system

d

doublet (spectral)

DCM

dichloromethane

e.g.

for example (exempli gratia)

EMT

epithelial to mesenchymal transition

ESI

electrospray ionization

et al.

and others

FC

ferroptosis control

FDA

Food and Drug Administration

GPX4

glutathione peroxidase 4

HAT

histone acetyl transferase

HC

HDAC control

HDAC

histone deacetylase

HDACi

histone deacetylase inhibitor

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass Spectra

HY

hybrid molecules

MEF2

myocyte enhancer factor-2

NAD

nicotinamide adenine dinucleotide

NC

negative control

NCI

National Cancer Institute

NMR

nuclear magnetic resonance

PDB

Protein Data Bank

PI

propidium iodide

PUFAs

polyunsaturated fatty acids

q

quartet (spectral)

ROS

reactive oxygen species

RPE

retinal pigment epithelial

s

singlet (spectral)

SAR

structure–activity relationship

SD

standard deviation

t

triplet (spectral)

THP

tetrahydropyran

THP

tetrahydropyran-2-yl

TLC

thin-layer chromatography

TMS

trimethylsilyl

TNBC

triple-negative breast cancer

Topo

topoisomerase

UV

ultraviolet

ZBG

zinc-binding group

Z-VAD-FMK

N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone