Synthesis and Biochemical Evaluation of Biotinylated Conjugates of Largazole Analogues: Selective Class I Histone Deacetylase Inhibitors

Abstract

The synthesis of biotinylated conjugates of synthetic analogues of the potent and selective histone deacetylase (HDAC) inhibitor largazole is reported. The thiazole moiety of the parent compound’s cap group was derivatized to allow the chemical conjugation to biotin. The derivatized largazole analogues were assayed across a panel of HDACs 1–9 and retained potent and selective inhibitory activity towards the class I HDAC isoforms. The biotinylated conjugate was further shown to pull down HDACs 1, 2, and 3.

Graphical abstract

1. Introduction

The marine natural product largazole was isolated from marine cyanobacteria Symploca sp. off of the Florida coastline by Luesch and co-workers.^[1] Largazole’s sixteen-membered cyclodepsipeptide structure (Figure 1), containing both a thioester tail and (S)-3-hydroxy-7-mercaptohept-4-enoic acid moieties, constitutes the structural basis of a promising antitumor prodrug.^[2] Research efforts have explored the therapeutic use of largazole towards not only cancer therapy but also corneal angiogenesis, rheumatoid arthritis, neuro-degenerative diseases, osteoporosis, and numerous other disease areas.^[3] The cyclodepsipeptide natural product and histone deacetylase inhibitor (HDACi) romidepsin (FK228) is currently an FDA-approved therapeutic for the treatment of cutaneous T-cell lymphoma (CTCL).^[4] Vorinostat (SAHA), a simple hydroxamate, was the first HDACi approved by the FDA for human clinical use and is a non-selective “pan” inhibitor of all the HDAC isoforms.^[5] Panobinostat, the pan- HDACi LBH-589 developed by Novartis, has been approved for multiple myeloma combination therapy as well.^[6] Largazole, FK228, and the spiruchostatins,^[7] which all contain the same (S)-3-hydroxy-7-mercaptohept-4-enoic acid moiety, are all potent biochemical inhibitors of the class I HDACs and exert their HDAC inhibitory activity through binding of the sulfhydryl residue to the active site zinc ion of the class I HDACs. Our laboratory and others have incorporated the (S)-3-hydroxy-7-mercaptohept-4-enoic acid moiety into numerous synthetic analogues, which in most cases provides selective and potent class I HDAC inhibitors.^[8]

Figure 1.

Largazole (1), the parent natural product, and largazole peptide isostere (2).

Selective HDAC isoform inhibition is essential for the development of a safe and efficient therapeutic.^[9] This has also been the most challenging aspect in the development of HDAC inhibitors to date. Eighteen HDACs have been isolated, the Schreiber laboratory spearheading this effort.^[10] The HDACs of general therapeutic interest are within classes I and II; specifically, class I includes four HDACs (1, 2, 3, and 8) that are much smaller and primarily located in the nucleus, compared to class II HDACs (4, 5, 6, 7, and 9), which each contain ~1000 amino acid residues and are expressed in both the cytoplasm and nucleus.^[11] Adverse side effects have been observed in currently marketed pan-HDAC inhibitors, which has been partially attributed to the lack of selectivity towards HDAC isoforms.^[12]

To date we have developed syntheses of more than sixty analogues of largazole by modifying three main motifs of the parent natural product.^[8,13] We have interrogated structural changes to (1) the zinc-binding domain, (2) the surface recognition unit or so-called “cap group” (thiazole–thiazoline fragment), and (3) the depsipeptide versus the corresponding peptide isosteres (Figure 1).^[13a]

Herein we describe synthetic routes to access both depsipeptide and peptide isostere analogues of the parent natural product largazole with a functional handle projecting from the thiazole moiety of the cap group that permits the chemical conjugation of other molecular entities that might prove valuable for cell-specific targeting and as probes for biochemical and cellular function (Figure 2). Our synthetic route utilizes our original strategic four-component convergent disconnection deployed in the efficient total synthesis of largazole reported by this laboratory in 2008.^[8a]

Figure 2.

Thiazole-derivatized depsipeptides (3), thiazole-derivatized peptide isosteres (4), depsipeptide–biotin conjugate (5), and peptide isostere–biotin conjugate (6).

2. Results and Discussion

2.1 Design

Prior molecular modeling studies of largazole, performed in collaboration with the Wiest laboratory, revealed that the cap group of largazole has the unique ability to interact with the protein surface of the HDAC enzyme and projects away from the active site in unencumbered space, providing an opportunity for chemical functionalization.^[13a,14] We reasoned that thiazole derivatization would be chemically tractable and would allow for the installation of functionality that would not hinder the Zn^{2 +} binding arm of this scaffold from interaction with the Zn^{2 +} atom ensconced at the active site of the enzyme. With the dual objective of not abrogating the Zn²⁺ binding role of the sulfhydryl-containing side chain^[15] and incorporating a chemically flexible handle for conjugating cancer-cell-specific targeting motifs and other reporter molecules, we modified our convergent synthetic route, utilizing previously established disconnections.^[8b] This design was probed using our previously established^[16] molecular dynamics (MD) protocol for the study of HDAC inhibitors. Figure 3 shows the model of 6 bound to the crystal structure of HDAC8 (PDB code: 4RN0).^[14] As envisioned in the original design, the biotin and the amide linker protrude away from the binding site, leaving all contacts of the protein with the cap group and the zinc binding domain intact. It is therefore expected that the binding of 5 and 6 will mimic largazole and its amide isostere, respectively. Although no crystal structure of largazole derivatives to HDAC1-3 is currently available, these findings should also apply to these HDACs due the previously described similarities in binding modes.^[13a,14]

Figure 3.

MD refined model of the complex of 6 bound to HDAC8.

We chose to evaluate the installation of both hydroxyl group handles and amino group handles for chemical conjugation studies (Figure 2). The amine handle allows for extensive conjugation across a series of cancer-relevant biological molecules via robust amide linkages, which might prove resistant to proteolytic cleavage in vitro and in vivo, whereas the hydroxyl residue allows for chemical conjugation through more labile ester linkages.^[15] Biotin conjugation has been widely used to study and identify small-molecule drug targeting to cellular proteins and has been used in the context of numerous pull-down experiments.^[16]

2.2 Synthesis

Scheme 1 shows the synthetic strategy we have successfully used to access largazole derivatives and we have applied this to the preparation of compounds 3 and 4.^[8c] The two halves, depsipeptide or peptide isostere fragments (9) and thiazole-thiazoline moiety (10), are utilized to access the pre-macrocyclization species. Macrocyclization to S-trityl-protected precursors is accessed through standard peptide coupling techniques. The two halves can then be further dissected into the β-amino acid or β-hydroxy acid fragments (12, X = NH₂, OH, respectively), protected L-valine (11), α-methyl cysteine (13), and the substituted thiazole cyano precursor (14). As shown in Scheme 2, commercially available acrolein (15) was reacted with trityl mercaptan followed by a Wittig olefination to yield aldehyde 16. Alcohol stereochemistry was established through the use of a Crimmins-type aldol condensation, the product of which was then cleaved using 2- (trimethylsilyl)ethanol. Amino acid EDCI coupling between the free alcohol of 18 and L-valine was successful in providing compound 19.

Scheme 1.

Retrosynthetic pathway for cap group derivatization of the thiazole fragment of parent compounds of largazole (X= O or NH).

Scheme 2.

Synthesis of compound 19 for depsipeptide series. Reagents and conditions: (a) TrtSH, Et₃N, CH₂Cl₂; (b) (formyl-methylene)triphenylphosphorane, PhH, 80°C, 77 % over two steps; (c) (R)-1-(4-benzyl-2-thioxothiazolidin-3-yl)ethanone, TiCl₄, DIPEACH₂Cl₂, −78 °C, 76 %; (d) 2-(trimethylsilyl)ethanol, imidazole, CH₂Cl₂, 83 %; (e) N-Fmoc-Val-OH, EDCI, DIPEA, DMAP, CH₂C_l2, 77%.

As illustrated in Scheme 3, the corresponding peptide isostere precursor was accessed via Boc-protected amino acid derivative 20.^[8b] Reduction of the carboxylic acid (20 to 21), Swern oxidation, and subsequent Wittig olefination provided terminal alkene 22. Grubb’s cross-metathesis with 3-buten-1- ol was employed to provide compound 23. The yield at this stage was very modest and alternative routes were explored but were either unsuccessful or resulted in epimerization of the amino group.^[7,17] Tosylation and reaction with trityl thiol provided protected thiol 24, which then underwent methyl ester formation to provide compound 25. N-t-Boc deprotection using TFA exposed the free amine group, which was then coupled to N-t-Boc protected l-valine through the use of PyBOP, furnishing fragment 26.

Scheme 3.

Synthesis of fragment 26 for peptide isostere macrocycle. Reagents and conditions: (a) 4-methylmorpholine, isobutyl chloroformate, THF, − 40 °C; NaBH₄, MeOH, −20 °C, 66%; (b) oxalyl chloride, DMSO, DIPEA, CH₂Cl₂, 65 °C; (c) methyltriphenylphosphonium bromide, KHMDS, THF, 78 °C, 80% over two steps; (d) 3-buten-1-ol, Grubbs catalyst, 2nd generation, CH₂Cl₂, 50 °C, 25%;(e) TsCl, Et₃N, DMAP, CH₂Cl₂, 82%; (f) TrtSH, KO^tBu, THF, 87 %; (g) LiOH, THF, MeOH, 50 8C, 96 %; (h) MeOH, EDCI, DIPEA, DMAP, CH₂Cl₂, 77%; (i) TFA, CH₂Cl₂; (j) N-Boc-Val-OH, PyBOP, DIPEA, CH₂Cl₂, 88% over two steps.

Functionalization of the thiazole ring is depicted in Scheme 4.^[18] Compound 30 with the hydroxymethyl handle was accessed via cyano-thiazole compound 27. LDA deproto-nation followed by iodination provided cross-coupling pre-cursor 28. Suzuki coupling with boronic acid 29 provided compound 30. It is important to note that additional aromatic boronic acids have been used to access derivatives successfully and will be reported elsewhere.

Scheme 4.

Synthesis of fragment 30 for cap group assembly. Reagents and conditions: (a) LDA, I₂, THF, −78°C, 74%; (b) tetrakis(triphenylphosphine)palladium(0), sodium bicarbonate, DME H₂O (3 : 1), reflux, 68 %.

Scheme 5 illustrates the depsipeptide macrocycle assembly. The free alcohol of 30 could either be protected with TBDPS for direct conjugation via ester linkages or subjected to Mitsunobu reaction using tert-butyl ((4-nitrophenyl) sulfonyl)carbamate, which provided compound 31. Cyclo-condensation of 31 with a-methyl cysteine gave carboxylic acids 32 : 33 as a 1 : 1 mixture that was carried forward as a mixture.^[19] Peptide coupling of fragment 19 provided 34 + 35 as a 1 : 1 mixture that was deprotected and cyclized to furnish the desired species 36.

Scheme 5.

Synthesis of depsipeptide macrocycle compound 36. Reagents and conditions: (a) tert-butyl ((4-nitrophenyl)sulfonyl) carbamate, diethyl azodicarboxylate, triphenylphosphine, THF, 90 %; (b) α-methyl cysteine, sodium carbonate, triethylamine, MeOH, reflux, 59%; (c) 19, MeCN-diethylamine (10:1); (d) PyBOP, DIPEA, DCM, 58 % over two steps; (e) DCM TFA (5 : 1); (f) HOBt, HATU, DIPEA, MeCN, 20 % over two steps.

For the assembly of the corresponding peptide isostere, 37 was reacted with LiOH–H₂O to provide a free carboxylic acid that underwent intramolecular HOBt and HATU coupling with the deprotected Boc amino group after subjection to TFA to provide compound 39 (Scheme 6).

Scheme 6.

Synthesis of peptide isostere macrocycle compound 39. Reagents and conditions: (a) 26, DCM-TFA (10 : 1); (b) PyBOP, DIPEA, 1 4 DCM; (c) lithium hydroxide - H₂O, THF-MeOH-H₂O (2 : 2 : 1); (d) DCM–TFA (10 : 1); (e) HOBt, HATU, DIPEA, MeCN, 12 % over five steps.

Compounds 36 and 39 were each subjected to treatment with thiophenol and 2 M KOH in acetonitrile to remove the nosyl protecting group and expose the free amine for coupling to biotin.^[20] NaHCO₃ was used to promote the amide coupling of biotin for both the depsipeptide and peptide largazole derivatives. Biotin linker 40 was used due to the stability of the amide bond, reliable synthesis for highly functionalized molecules, and the increased length allowed for solubility. Trityl deprotection using DCM–TFA and iPr₃SiH provided largazole–biotin derivatives 5 and 6 in 41 % and 36 % yield, respectively, as shown in Scheme 7. This convergent route can be utilized to access additional amide-linked biological conjugates.

Scheme 7.

Synthesis of depsipeptide–biotin conjugate (5) and peptide isostere–biotin conjugate (6). Reagents and conditions: (a) thiophenol, 2 M potassium hydroxide, MeCN; (b) sodium bicarbonate, MeCN; (c) DCM–TFA (20 : 1), triisopropylsilane, 41 % (depsipeptide derivative) over three steps, 36 % (peptide isostere) over three steps.

2.3 Biochemical Evaluation

Compounds 1, 2, 5, and 43 were tested across an HDAC isoform panel of HDACs 1–9 to determine the biochemical inhibitory potency of these species relative to the parent natural product. The IC₅₀ values, reported in nM in Table 1, represent a summary of HDAC inhibitory activity. The peptide isostere thiol of parent largazole, 2, displayed inhibitory activity below 4 nM for HDACs 1–3, proving more potent than the parent natural product 1. The newly derivatized compound at the thiazole cap group of parent largazole, 43 (Figure 4), displayed sub-nanomolar inhibitory activity for HDACs 1 and 3 and below 3 nM for HDAC 2. A decrease in potency was seen across all derivatives for HDACs 6 and 8, as expected.

Table 1.

IC₅₀ values (nM) of compounds 1, 2, 5, and 43 across 92 HDAC isoforms 1–3, 6, and 8.

Compound	HDAC isoform IC50 (nM)
	1	2	3	6	8
Largazole (1)	10.09	18.65	9.09	165.6	1068
2	1.95	3.38	2.59	102	255.3
43	0.166	2.61	0.734	14.9	94
5	0.2388	2.84	1.064	36.51	129.3

Figure 4.

Structure of depsipeptide analogue with a hydroxymethyl handle off of the thiazole cap group.

Biotin–largazole conjugate 5 exhibited very little change in IC₅₀ activity when compared to compound 43. IC₅₀ values below 3 nM were maintained across HDACs 1–3 for 5 and 43. A full representation of the full HDAC panel for 43 (Figure 5A) and 5 (Figure 5B) is depicted in Figure 5.

Figure 5.

Inhibition of HDAC activity by largazole thiol and biotinylated largazole thiol. (A,B) Inhibition of HDAC 1–9 activity by (A) 43 and (B) 5 is presented in dose–response format. Data represent mean values of quadruplicate data ± s.d. IC₅₀ curves were fit by logistic regression.

Affinity chromatography assays were performed with the largazole–biotin derivative 5 to detect HDACs 1–3 binding in cell lysate. Streptavidin beads were added after a 16 h incubation with compound 5 or vehicle in 200 mg of HeLa nuclear extract. Figure 6 shows immunoblot analysis of the resulting eluates, confirming the enrichment of HDACs 1–3 from streptavidin beads conjugated to compound 5 over mock pull down (vehicle only) or input samples.

Figure 6.

1 mM largazole biotin (5) in 200 mL of HeLa nuclear extract (~ 200 mg); 1 mg of streptavidin beads added after 16 h incubation with 5.

3. Conclusion

We have developed a versatile, successful synthesis of new cap group derivatives of the potent natural product largazole with functional handles for conjugating probe molecules, reporters, and potential cancer-cell-specific targeting moieties. The potent biochemical activity observed for the thiazole-derivatized analogues reported herein demonstrates that the unencumbered cap group chemical space is a fertile area for future investigations into increasing the selectivity and therapeutic potential of largazole analogues. We are intensively investigating the chemical conjugation of other molecules to the largazole scaffold via the approach described herein and will report on this in due course.

4. Experimental Section

4.1 General Experimental Methods

All reactions were run under argon in oven-or flame-dried glassware. Thin-layer silica gel chromatography (TLC) was used to monitor reactions on 0.25 mm silica gel 60F plated with fluorescent indicator from Merck. Various stains were used to visualize TLC plates, including but not limited to anisaldehyde and Seebach’s with gentle heating. Column chromatography and preparatory TLC were used to purify all compounds. The solvent systems described below were run on silica gel 60 (230–400 mesh) purchased from Sorbent Technologies. Reagents were purchased from commercial sources and used either directly or after drying. ¹HNMR and ¹³C NMR were recorded using Varian 300, 400, or 500 MHz NMR spectrometers. Chemical shifts are reported in ppm relative to CHCl₃ at δ = 7.27 (¹HNMR) and δ = 77.23 (¹³C NMR) or tetramethylsilane (TMS) at δ = 0.00. Mass spectra were obtained on a Fisions VG Autospec. Optical rotations were collected at 589 nm on a Rudolph Research Automatic Polarimeter Autopol III. FT-IR spectra were collected on a Thermo Scientific Nicolet iS50.

4.2 tert-Butyl ((4-Cyano-5-iodothiazol-2-yl)methyl)carbamate (28)

To a solution of thiazole 27 (85 mg, 0.36 mmol) in 7.2 mL of THF at −78 °C, 1.1 mL of LDA (0.88 mmol, 0.8 M in THF) was added dropwise (it should be noted that the addition – of more than three equivalents of LDA would have resulted in decomposition of SM). The mixture was stirred at −78 °C for 20 min, then a THF solution (1.44 mL) of I₂ (365 mg, 1.44 mmol) was added. After stirring at the same temperature for 5 min, the solution was treated with saturated aq. NH₄Cl, diluted with ethyl acetate, separated, washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0 to 9 % ethyl acetate in DCM) to afford 97.4 mg (74 % yield) of thiazole iodide 28 as a brown solid.

¹HNMR (400 MHz, CDCl₃) δ 5.29 (s, 1H), 4.57 (d, J = 6.4 Hz, 2H), 1.46 (s, 9H); ¹³CNMR (100 MHz, CDCl₃) δ176.5, 155.6, 134.6, 113.8, 85.0, 81.0, 42.6, 28.3; IR (neat) 3364, 2927, 1678, 1516; HRMS (ESI): m/z calcd. for C₁₀H₁₂ IN₃NaO₂S⁺ (M + Na)⁺ 387.9587, found 387.9579.

4.3 tert-Butyl ((4-Cyano-5-(4-(hydroxymethyl)phenyl) thiazol-2-yl)methyl)carbamate (30)

Thiazole iodide 28 (550 mg, 1.5 mmol), 4-(hydroxymethyl) phenylboronic acid 29, (343 mg, 2.3 mmol), NaHCO₃ (380 mg, 4.5 mmol), and Pd(PPh₃)₄ were combined, and 16 mL of solvent (DME/H₂O= 3/1, v/v) was added. The mixture was heated to 120 °C and refluxed at that temperature for 14 h, then cooled to room temperature and diluted with water. DME was evaporated, and the aqueous layer was extracted with ethyl acetate and separated. The organic layer was washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0 to 11 % ethyl acetate in DCM) to afford 67 mg (19% yield) of protonated compound 27 and 354 mg (68% yield) of desired coupling product 30 as a white solid.

¹HNMR (400 MHz, CDCl₃) δ 7.61 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 5.64 (br s, 1H), 4.70 (s, 2H), 4.52 (d, J = 4.6 Hz, 2H), 2.81 (br s, 1H), 1.45 (s, 9H); ¹³CNMR (100 MHz, CDCl₃) δ 169.1, 143.7, 128.7, 128.1, 127.5, 127.0, 120.4, 115.3, 114.6, 80.8, 64.2, 42.3, 28.3 (33); IR (neat) 3332, 2978, 2227, 1689, 1513, 1279, 1248, 910, 728; HRMS (ESI): m/z calcd. for C₁₇H₁₉N₃O₂S⁺ (M+ Na)⁺ 368.1039, found 368.1030.

4.4 tert-Butyl ((4-Bromophenyl)sulfonyl)(4-(2-(((tert- butoxycarbonyl)amino)methyl)-4-cyanothiazol-5-yl)benzyl)-carbamate (31)

Benzyl alcohol 30 (35 mg, 0.1 mmol), NsNHBoc (45 mg, 0.15 mmol), and PPh₃ (39 mg, 0.15 mmol) were combined and 1 mL of THF was added. To the mixture was added DEAD (40% in toluene, w/w, 75 mL, 0.15 mmol) at 0 °C. After stirring at 0 °C for 30 min, the solution was washed with 1 N HCl, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0% to 4% ethyl acetate in DCM) to afford 57 mg (90% yield) of 31 as a yellow solid.

¹HNMR (400 MHz, CDCl₃) δ 8.32 (dd, J = 8.3, 1.7 Hz, 1H), 7.79–7.69 (m, 5H), 7.54 (d, J = 8.2 Hz, 1H), 5.54 (br s, 1H), 4.99 (s, 2H), 4.57 (d, J = 5.9 Hz, 2H), 1.46 (s, 9H), 1.28 (s, 9H); ¹³CNMR (100 MHz, CDCl₃) δ 176.2, 169.2, 150.2, 147.6, 140.0, 134.5, 133.8, 133.1, 132.4, 132.0, 128.5, 128.4, 127.5, 124.6, 120.7, 114.7, 85.7, 80.8, 50.6, 42.5, 28.3 (33), 27.8 (33); IR(neat) 3353, 2980, 1730, 1543, 1367, 1280, 1249, 1151, 1123, 1062, 918, 852, 760; HRMS (ESI): m/z calcd. for C₂₈H₃₁N₅NaO₈S₂⁺ (M+ Na)⁺ 652.1506, found 652.1498.

4.5 (R)-2′-(((tert-Butoxycarbonyl)amino)methyl)-4-me-thyl-5′-(4-(((4-nitrophenyl)sulfonamido)methyl)phenyl)-4,5-di-hydro-[2,4′-bithiazole]-4-carboxylic Acid (33)

Thiazole nitrile 31 (377 mg, 0.6 mmol), a-methyl-cysteine-HCl (124 mg, 0.72 mmol), and NaHCO₃ (76 mg, 0.9 mmol) were combined and 10 mL of methanol was added. To the mixture was added Et₃N (0.16 mL, 1.2 mmol). After refluxing at 70 °C for 36 h, the solvent was removed under reduced pressure. The residue was purified by flash column chromatography on silica gel (9% to 30% ethyl acetate in DCM) to afford 230 mg (32 : 33 = 1 : 1, 59%) of 32 and 33 as a yellow foam. 32 and 33 were used as a mixture for the next reaction without further purification. An analytically pure sample could be obtained by esterification, separation, then hydrolysis.

(R=H, 33), [α]_D²⁸ = −8° (c = 1.126 in CH₃OH); ¹HNMR (400 MHz, CD₃OD) δ 7.81 (dd, J = 7.8, 1.2 Hz, 1H), 7.74 (dd, J = 7.6, 1.4 Hz, 1H), 7.66 (ddd, J = 7.8, 7.6, 1.4 Hz, 1H), 7.59 (ddd, J = 7.8, 7.6, 1.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 4.46 (s, 2H), 4.29 (s, 2H), 3.76 (d, J = 11.3 Hz, 1H), 3.22 (d, J = 11.3 Hz, 1H), 1.45 (s, 3H), 1.45 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 176.0, 169.5, 162.6, 156.8, 147.7, 141.6, 140.0, 138.2, 133.8, 133.2, 132.1, 130.2, 129.7, 128.9, 127.5, 124.3, 84.8, 79.6, 46.3, 41.7, 41.5, 27.3, 22.6; IR (neat) 3120, 2974, 2938, 1693, 1665, 1392, 1365, 1347, 1172, 1154, 1079, 863; HRMS (ESI): m/z calcd. for C₂₇ H₃₀N₅O₈S₃⁺ (M + H)⁺ 648.1251, found 648.1231

4.6 2-(Trimethylsilyl)ethyl (S,E)-3-((((R)-2′-(((tert-Butoxycarbonyl)amino)methyl)-4-methyl-5′-(4-(((4-nitrophe-nyl)sulfonamido)methyl)phenyl)-4,5-dihydro-[2,4′-bithiazole]-4-carbonyl)-L-valyl)oxy)-7-(tritylthio)hept-4-enoate (35)

19 (336 mg, 0.4 mmol) was dissolved in 40 mL of CH₃CN. 4 mL of diethylamine was added at 0 °C. The bath was removed and the resulting solution was stirred at room temperature for 2 h, then evaporated, azeotroped with toluene (2X2 mL), and dried under vacuum. In another round flask, acid 32/33 (230 mg, 0.35 mmol, mixture of 32 and 33, 1 : 1) was dissolved in 55 mL of DCM. PyBOP (416 mg, 0.8 mmol) and DIPEA (210 mL, 1.2 mmol) were added and the mixture was allowed to stir at room temperature for 20 minutes. To the resulting solution was added a DCM solution (total 25 mL) of crude amine. After 3 h, the reaction was concentrated and submitted immediately to column chromatography (0 % to 25 % ethyl acetate in DCM) to afford the macrocyclization precursor (236 mg, 58 %, 34 : 35 = 1 : 1) as a yellow foam. 34 and 35 were used as a mixture for the next reaction without further purification.

4.7 N-(4-((1²Z,2²Z,2⁴R,5S,8S)-5-Isopropyl-2⁴-methyl-3,6,10-tri- oxo-8-((E)-4-(tritylthio)but-1-en-1-yl)-2⁴,2⁵-dihydro-7-oxa-4,11- diaza-1(4,2),2(2,4)-dithiazolacyclododecaphane-1⁵-yl) benzyl)-4-nitrobenzenesulfonamide (36)

Acyclic precursor (236 mg, 0.2 mmol, mixture of 34 and 35, 1 : 1) was dissolved in 30 mL of DCM, and 6 mL of TFA was added to the solution at 0 °C. The reaction was allowed to warm to room temperature and stirred for 16 h. Solvents were evaporated and the crude amino acid was azeotroped with toluene (10 X 3 mL) to remove residual TFA. The crude amino acid was then dissolved in 300 mL of CH₃CN (to ~ 0.001 M) and DIPEA (0.32 mL, 1.8 mmol) was added. The resulting moderately opaque solution was allowed to stir for 10 min, before an MeCN (10 mL) solution of HATU (230 mg, 0.6 mmol) and HOBt (81 mg, 0.6 mmol) was added dropwise. The reaction was allowed to stir for 26 h, then concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0% to 11% AcOEt in DCM for the first column, 25% to 75 % ethyl acetate in hexane for the second column, and 0% to 9% AcOEt in DCM for the third column) to afford 40 mg (20% yield for two steps) of 36 as a yellow foam.

[α] ²⁶ = + 19° (c = 0.147 in CHCl₃); ¹HNMR (500 MHz, CD₃OD) δ 7.91 (dd, J = 7.8, 1.3 Hz, 1H), 7.80 (dd, J = 7.8, 1.3 Hz, 1H), 7.71 (ddd, J = 7.9, 7.5, 1.4 Hz, 1H), 7.64 (dddd, J = 7.7, 7.6, 5.1, 1.2 Hz, 1H), 7.57–7.18 (m, 15H), 5.71 (ddd, J = 16.1, 8.4, 6.7 Hz, 1H), 5.63 (m, 1H), 5.48 (dd, J = 15.4, 6.3 Hz, 1H), 5.11 (d, J = 17.5 Hz, 1H), 4.55 (d, J = 3.8 Hz, 1H), 4.37 (s, 1H), 4.34 (d, J = 17.5 Hz, 1H), 3.77 (d, J = 11.6 Hz, 1H), 3.24 (d, J = 11.6 Hz, 1H), 3.20 (m, 1H), 2.89 (dd, J = 15.9, 10.6 Hz, 1H), 2.67 (dd, J = 15.9, 3.1 Hz, 1H), 2.20 (dd, J = 17.3, 17.0 Hz, 1H), 2.12–2.02 (m, 2H), 1.92–1.88 (m, 2H), 1.77 (s, 3H), 1.59 (m, 1H), 0.73 (d, J = 6.9 Hz, 3H), 0.57 (d, J = 6.9 Hz, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 175.8, 171.8, 170.3, 168.3, 167.2, 149.3, 146.3, 144.6, 142.9, 140.8, 135.4, 134.8, 133.8, 133.5, 133.0, 131.6, 131.3, 130.8, 129.3, 129.2, 128.9, 127.7, 125.8, 83.9, 73.6, 58.9, 47.6, 47.4, 44.4, 41.3, 41.0, 35.2, 32.5, 32.4, 30.8, 30.5, 27.4, 24.5, 23.8, 19.8, 17.3, 14.4; IR (neat) 3362, 2960, 2926, 2853, 1734, 1668, 1540, 1443, 1344, 1259, 1165, 1071, 972, 853; HRMS (ESI): m/z calcd. for C₅₃H₅₃N₆O₈S₄⁺ (M + H)⁺ 1029.2802, found 1029.2778.

4.8 Largazole-Biotin Conjugate (5)

Thiophenol (10.4 mL, 0.1 mmol), KOH (3.6 mg, 64 mmol), water (30 mL), and acetonitrile (1.2 mL) were combined to give a colorless solution. To 8.2 mg (9.1 mmol) of Ns amine 36 was added 0.4 mL of the above solution at room temperature. The reaction was allowed to stir for 2 h then diluted with 4 mL of hexane and submitted directly to column chromatography (100% AcOEt then 25% MeOH in DCM) to afford 6.0 mg (78% yield) of free amine as a yellow oil.

Free amine (6.0 mg, 9 mmol), biotin derivative 40 (8.1 mg, 18 mmol), and NaHCO₃ (1.5 mg, 18 mmol) were combined. DMF (0.3 mL) was added and the reaction was allowed to stir for 14 h then concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (100% AcOEt then 5% to 11% MeOH in DCM) then by preparative TLC (9% MeOH in DCM three times) to afford the pure coupling product 41 as a yellow oil.

Coupling product 41 was dissolved in 0.6 mL of DCM and cooled to 0 °C. TFA (30 mL) and iPr₃SiH (1.0 mL, 6 mmol) were added to the solution at 0 °C. The bath was removed and the reaction was allowed to stir at room temperature for 1 h. The solvent was removed by argon flow and the residue was purified by flash column chromatography on silica gel (0 to 9% MeOH in CHCl₃) to afford 4.0 mg of free thiol 5 (53% for two steps). This was further purified by preparative TLC (9% MeOH in DCM two times) to afford the pure sample for biological evaluation.

[α]_D²⁵ = + 52.0° (c = 0.050 in CH₃OH); ¹HNMR (600 MHz, CD₃OD) δ 7.48 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 5.92–5.82 (m, 2H), 5.72–5.63 (m, 2H), 5.13 (d, J = 8.0 Hz, 1H), 4.59–4.55 (m, 2H), 4.49 (dd, J = 7.7, 4.7 Hz, 1H), 4.44–4.40 (m, 3H), 4.30 (dd, J = 7.7, 4.7 Hz, 1H), 3.77 (d, J = 11.6 Hz, 1H), 3.60 (s, 1H), 3.27 (d, J = 11.6 Hz, 1H), 3.21–3.15 (m, 5H), 2.98–2.91 (m, 2H), 2.76–2.69 (m, 3H), 2.56 (dd, J = 6.9, 6.9 Hz, 1H), 2.45 (dd, J = 13.5, 6.5 Hz, 1H), 2.36 (m, 1H), 2.28 (dd, J = 7.4, 7.4 Hz, 2H), 2.23–2.09 (m, 6H), 1.81 (s, 3H), 1.76–1.29 (m, 23H), 0.74 (d, J = 6.9 Hz, 3H), 0.59 (d, J = 6.9 Hz, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 176.1, 176.0, 175.9, 175.8, 171.8, 170.4, 168,3, 167.2, 166.0, 144.7, 143.0, 142.5, 133.7, 131.4, 129.7, 129.1, 128.8, 84.0, 73.8, 64.3, 63.3, 61.6, 58.9, 57.0, 44.4, 43.6, 41.2, 41.0, 40.2, 38.7, 37.0, 36.9, 36.8, 35.2, 32.7, 30.1, 29.8, 29.5, 27.5, 26.9, 26.7, 26.6, 24.5, 19.8, 17.3; IR (neat) 3308, 2930, 2859, 1656, 1551, 1462, 1262, 667; HRMS (ESI): m/z calcd. for C₅₀H₇₂N₉O₈S₄⁺ (M + H)⁺ 1054.4381, found 1054.4346.

4.9 Methyl (S,E)-3-((S)-2-((R)-2′-(((tert-Butoxycarbonyl) amino)methyl)-4-methyl-5′-(4-(((4-nitrophenyl)sulfonamido)- methyl)phenyl)-4,5-dihydro-[2,4′-bithiazole]-4-carboxami- do)-3-methylbutanamido)-7-(tritylthio)hept-4-enoate (38)

Boc-amine 26 (420 mg, 0.66 mmol) was dissolved in 10 mL of DCM and 1 mL of TFA was added to the solution at 0 °C. The reaction was allowed to warm to room temperature and stirred for 2 h. Solvents were evaporated and the crude amino acid was azeotroped with toluene (2 X 2 mL) to remove residual TFA. Acid 32/33 (470 mg, ca. 0.72 mmol, 32 : 33 = 1 : 1) was dissolved in 50 mL of DCM. PyBOP (730 mg, 1.4 mmol) and DIPEA (630 mL, 3.6 mmol) were added and the mixture was allowed to stir at room temperature for 20 minutes. To the resulting solution was added a DCM solution (total 10 mL) of crude amine. After 4 h, the reaction was concentrated and submitted immediately to column chromatography (16% to 33% ethyl acetate in DCM) to afford macrocyclization precursor 37 : 38 (1 : 1) as a yellow foam.

For R=H 38, [α]_D²⁶ = 55.8° (c = 0.163 in CHCl₃); ¹HNMR (400 MHz, CDCl₃) δ 8.04 (m, 1H), 7.67 (m, 1H), 7.61–7.58 (m, 2H), 7.36–7.34 (m, 10H), 7.27–7.14 (m, 13H), 6.63 (d, J = 8.9 Hz, 1H), 6.46 (d, J = 9.4 Hz, 1H), 5.87 (br s, 1H), 5.46 (ddd, J = 15.4, 6.8, 6.3 Hz, 1H), 5.37–5.31 (m, 2H), 4.76 (m, 1H), 4.57 (d, J = 6.0 Hz, 2H), 4.31 (m, 2H), 3.93 (dd, J = 8.9, 8.6 Hz, 2H), 3.67 (m, 2H), 3.56 (m, 1H), 3.53 (s, 3H), 3.45 (d, J = 11.6 Hz, 1H), 3.19 (d, J = 11.6 Hz, 1H), 2.52 (m, 2H), 2.13–1.98 (m, 2H), 1.73 (m, 1H), 1.44 (s, 12H), 1.26–1.20 (m, 4H), 0.77 (d, J = 6.7 Hz, 3H), 0.62 (d, J = 6.7 Hz, 3H); ¹³CNMR (100 MHz, CDCl₃) δ 174.4, 171.7,169.8, 167.4, 162.3, 148.0, 144.8, 141.6, 137.8, 134.2, 132.3, 130.6, 130.5, 130.3, 129.6, 129.5, 129.4, 127.8, 127.6, 126.5, 124.5, 85.5, 80.4, 66.5, 58.6, 51.7, 47.2, 47.1, 43.7, 42.3, 40.6, 38.6, 31.4, 31.3, 30.5, 29.6, 24.1, 19.2, 18.4, 18.1, 17.0, 14.1, 12.6; IR (neat) 3367, 2964, 2927, 1717, 1652, 1541, 1511, 1366, 1164, 972, 752; HRMS (ESI): m/z calcd. for C₅₉H₆₅N₇NaO₁₀ S₄⁺ (M+ Na)⁺ 1182.3568, found 1182.3583.

4.10 N-(4-((1²Z,2²Z,2⁴R,5S,8S)-5-Isopropyl-2⁴-methyl-3,6,10-trioxo-8-((E)-4-(tritylthio)but-1-en-1-yl)-2⁴,2⁵-dihydro-4,7,11- triaza-1(4,2),2(2,4)-dithiazolacyclododecaphane-1⁵-yl)benzyl)- 4-nitrobenzenesulfonamide (39)

Acyclic precursor 37/38 obtained above was azeotroped with toluene (232 mL) to remove residual AcOEt, then combined with LiOH·H₂O (139 mg, 3.3 mmol). Solvent (THF-MeOH - H₂O, 2 + 2 + 1 mL) was added and the reaction mixture was allowed to stir for 2.5 h. The reaction mixture was diluted with 10 mL of water, then the pH was adjusted to 2 by using 1 N HCl. Organic solvents were evaporated and the remaining aqueous layer was extracted with DCM (3X10 mL) then dried over Na₂SO₄, filtered, and evaporated. The crude acid was dissolved in 15 mL of DCM and 3 mL of TFA was added to the solution at 0 °C. The reaction was allowed to warm to room temperature and stirred for 3 h. Solvents were evaporated and the crude amino acid was azeotroped with toluene (2 X 2 mL) to remove residual TFA. The crude amino acid was then taken up in 700 mL MeCN and DIPEA (1 mL, 5.7 mmol) was added. The resulting moderately opaque solution was allowed to stir for 10 min, before HATU (500 mg, 1.32 mmol) and HOBt (178 mg, 1.32 mmol) were added. The reaction was allowed to stir for 14 h, then concentrated and redissolved in AcOEt. The solution was washed with saturated aqueous NH₄Cl, NaHCO₃, and brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (0% to 33% AcOEt in DCM for the first column, 16 % to 33% AcOEt in DCM for the second column, then 33% to 66 % AcOEt in DCM for PTLC) to afford 80 mg (12% yield for five steps) of 39 as white foam. (The product does dissolve in pure DCM and mixtures of DCM/AcOEt, DCM/MeOH and CHCl₃/MeOH but does not dissolve in pure CHCl₃, AcOEt, MeCN, or MeOH. Use 66% AcOEt in DCM for developing TLC.)

[α]_D ³⁰ = + 17.5° (c = 0.257 in CH₂Cl₂); ¹HNMR (400 MHz, CD₂Cl₂) δ 8.03 (dd, J = 7.5, 2.2 Hz, 1H), 7.82 (dd, J = 7.8, 1.5 Hz, 1H), 7.72–7.68 (m, 2H), 7.31–7.18 (m, 23H), 6.92 (d, J = 8.1 Hz, 1H), 6.72 (d, J = 9.5 Hz, 1H), 6.63 (d, J = 10.5 Hz, 1H), 6.21 (dd, J = 6.4, 6.4 Hz, 1H), 5.52–5.40 (m, 2H), 5.07 (dd, J = 17.5, 8.4 Hz, 1H), 4.86 (m, 2H), 4.47 (dd, J = 10.4, 3.3 Hz, 1H), 4.35 (dd, J = 6.4, 1.8 Hz, 1H), 4.24 (dd, J = 17.5, 3.1 Hz, 1H), 3.71 (d, J = 11.7 Hz, 1H), 3.24 (d, J = 11.7 Hz, 1H), 2.67 (dd, J = 14.6, 4.2 Hz, 1H), 2.52 (dd, J = 14.8, 9.1 Hz, 1H), 2.37 (m, 1H), 2.20 (dd, J = 7.7, 7.1 Hz, 2H), 2.02–1.98 (m, 2H), 1.77 s, 3H), 0.87 (dd, J = 6.9, 6.6 Hz, 1H), 0.71 (d, J = 6.8 Hz, 3H), 0.40 (d, J = 6.8 Hz, 3H); ¹³CNMR (100 MHz, CD₂Cl₂) δ 173.7, 170.1, 170.0, 166.0, 147.7, 144.8, 142.1, 141.9, 138.4, 133.7, 133.5, 132.9, 130.8, 130.5, 130.2, 129.6, 129.5, 128.3, 127.9, 127.8, 126.6, 126.5, 125.2, 83.8, 66.9, 58.5, 48.2, 47.4, 44.6, 41.3, 40.5, 38.7, 32.3, 31.9, 31.7, 31.6, 30.0, 24.0, 19.7, 16.0, 14.3; IR (neat) 3351, 2960, 2927, 1661, 1539, 1499, 1364, 1265, 1163, 1070, 844, 733, 700, 583; HRMS (ESI): m/z calcd. for C₅₃H₅₃N₇NaO₇S₄ (M+ Na)⁺ 1050.2781, found 1050.2801.

4.11 Largazole Peptide Isostere-Biotin Conjugate (6)

Thiophenol (10.4 mL, 0.1 mmol), KOH (3.6 mg, 64 mmol), water (30 mL), and DMF (1.2 mL; acetonitrile was used as solvent to deprotect the Ns group of the depsipeptide macro- cyclic substrate 36 mentioned above, however this peptide macrocycle 39 is not soluble in acetonitrile therefore DMF was used here) were combined to give a colorless solution. To 8.5 mg (8.3 mmol) of Ns amine 39 was added 1.0 mL of above solution at room temperature. The reaction was allowed to stir for 3 h then evaporated and purified by column chromatography (50% AcOEt in DCM then 50% MeOH in DCM) to afford 6.1 mg (87% yield) of free amine as a yellow solid. Free amine (6.1 mg, 7.2 mmol), biotin derivative 40 (8.2 mg, 14 mmol), and NaHCO₃ (1.2 mg, 14 mmol) were combined. DMF (0.5 mL) was added and the reaction was allowed to stir for 15 h then concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (100% AcOEt then 5 % to 50% MeOH in DCM) then by preparative TLC (11% MeOH in DCM two times) to afford the pure coupling product 42 as a yellow solid.

Coupling product 42 was combined with 0.6 mL of DCM to give a suspension. TFA (30 mL) was added at 0°C and the suspension soon became a yellow solution. iPr₃SiH (1.0 mL, 6 mmol) was added to the solution at 0 °C. The bath was removed and the reaction was allowed to stir at room temperature for 1 h. Solvent was removed by argon flow and the residue was purified by flash column chromatography on silica gel (4 to 50 % MeOH in DCM) to afford 3.1 mg of free thiol 6 (41 % for two steps). This was further purified by preparative TLC (11% MeOH in DCM two times) to afford the pure sample for biological evaluation.

[α]_D²⁹ = +88.0 °C (c=0.10 in CH₃OH); ¹HNMR (600 MHz, CD₃OD,) δ 7.49 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 2H), 5.70–5.56 (m, 3H), 5.17 (d, J = 17.5 Hz, 1H), 4.93 (m, 1H), 4.91 (ddd, J = 12.2, 5.9, 4.1 Hz, 1H), 4.52 (d, J = 3.0 Hz, 1H), 4.48 (m, 1H), 4.44 (s, 2H), 4.37 (d, J = 17.5, 1H), 4.29 (dd, J = 7.9, 4.5 Hz, 1H), 3.71 (d, J = 11.9 Hz, 1H), 3.65 (s, 1H), 3.60 (s, 1H), 3.40 (d, J = 11.9 Hz, 1H), 3.21–3.14 (m, 4H), 2.92 (dd, J = 12.8, 5.0 Hz, 1H), 2.77–2.69 (m, 2H), 2.64 (dd, J = 14.9, 4.0 Hz, 1H), 2.55 (1H, dd, J = 6.9, 6.9 Hz), 2.47–2.40 (m, 2H), 2.35 (dd, J = 13.5, 6.8 Hz, 1H), 2.28 (dd, J = 7.4, 7.4 Hz, 1H), 2.18 (dd, J = 13.5, 7.2 Hz, 2H), 1.88 (3H, s), 1.75–1.36 (m, 23H), 0.73 (d, J = 6.9 Hz, 3H), 0.41 (d, J = 6.9 Hz, 3H); ¹³CNMR (125 MHz, CD₃OD) δ 176.2, 176.0, 175.9, 175.8 172.2, 172.1, 169.6, 167.7, 160.0, 144.8, 142.8, 142.7, 132.5, 131.3, 129.8, 129.0, 128.7, 84.2, 64.3, 63.4, 61.6, 59.2, 57.0, 44.7, 43.6, 41.1, 41.0, 40.1, 39.1, 37.5, 37.0, 36.9, 36.8, 33.3, 30.7, 30.1, 29.7, 29.5, 27.5, 26.9, 26.7, 26.6, 24.6, 24.3, 20.0, 15.8; IR (neat) 3300, 2927, 2854, 1677, 1548, 1435, 1205, 1132, 801, 724; HRMS (ESI): m/z calcd. for C₅₀H₇₂N₁₀NaO₇S₄⁺ (M+ Na)⁺ 1075.4360, found 1075.4379.

4.12 HDAC Biochemical Assay

Compounds 1, 2, 5, and 43 were tested against HDACs 1–9 and the activity was determined with an optimized homogenous assay performed in a 384-well plate. Reactions were performed in assay buffer (50 mM HEPES, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, pH 7.4; additional 200 mM TCEP was added for HDAC6) and followed by fluorogenic release of 7-amino-4-methylcoumarin from the substrate upon deacetylase and trypsin enzymatic activity. Fluorescence measurements were obtained every five minutes using a multilabel plate reader and plate-stacker (Envision; Perkin- Elmer). Each plate was analyzed by plate repeat, and the first derivative within the linear range was imported into analytical software (Spotfire DecisionSite). Replicate experimental data from incubations with inhibitor were normalized to DMSO controls ([DMSO] < 0.5%). The IC₅₀ was determined by logistic regression with unconstrained maximum and minimum values. The recombinant, full-length HDAC protein (BPS Biosciences) was incubated with fluorophore conjugates substrate, MAZ1600 and MAZ1675 at K_m=[substrate].

4.13 Biochemical Pull-Down Studies

HeLa cells (ATCC) were cultured in DMEM supplemented with 10 % FBS and Pen Strep (100 U/mL). Nuclear extracts were collected from ~ 250 mL of HeLa cell pellet using NE-PER extraction reagents (ThermoFisher Scientific) according to the manufacturer’s protocol. For pull-down studies, compound 5 or DMSO was added to 200 mL of HeLa nuclear extract (1 mg/mL total protein) at a final concentration of 1 mM and 0.1 %, respectively. After 16 h incubation at 4 °C, 200 mg streptavidin Dynabeads (MyOne Streptavidin T1 ThermoFisher Scientific), equilibrated in NER buffer at 1 mg/mL, was added to each protein-compound mixture and incubated for 1 h at 4 °C with gentle rotation. Beads were then collected, washed four times with wash buffer (10 mM HEPES pH 8.0, 300 mM NaCl, 10 mM KCl, 1.5 mM MgCl₂, and 0.2 % Triton X-100), and finally resuspended and boiled in 90 mL of 1X NuPAGE LDS sample buffer (ThermoFisher Scientific) to elute bound protein. Eluate and input samples were run on 4–12% Bis-Tris NuPAGE gels (ThermoFisher Scientific) and transferred to PVDF membranes. Membranes were blocked for 1h at RT in Odyssey blocking buffer (TBS; LI–COR Biosciences) and then incubated overnight at 4 °C in blocking buffer supplemented with 0.2 % Tween-20 and a primary antibody diluted 1 : 1000 (anti-HDAC1, Cell Signaling #2062; anti-HDAC2, Cell Signaling #2540; anti-HDAC3, Cell Signaling #2632). Next, membranes were washed three times with TBST at RT (5 min each) and incubated for 1 h at RT in blocking buffer supplemented with 0.2 % Tween-20, 0.01% SDS, and IRDye 800CW goat anti-rabbit IgG (LI COR Biosciences) diluted 1 : 7000. Following three 5 min washes in TBST, membranes were rinsed in TBS and imaged on the Odyssey CLx imaging system (auto intensity, 84 mm resolution, high quality; LI–COR Biosciences).