Visible-Light-Controlled Histone Deacetylase Inhibitors for Targeted Cancer Therapy

Abstract

The lack of selectivity of anticancer drugs limits current chemotherapy. Light-activatable drugs, whose activity can be precisely controlled with external light, could provide a more localized action of the drugs in the tumor, thus decreasing side effects and increasing efficacy. Herein, we introduce a series of photoswitchable azobenzene histone deacetylase inhibitors (HDACis) whose activity can be controlled by external visible light. Initial HDACis isomerized under ultraviolet light and were up to >50-fold more active under illumination than in the dark in enzyme assays. These were then optimized toward compounds responding to more permeable and less harmful green light by introducing o-halogen atoms into the azobenzene. Selected compounds decreased cell viability only under illumination in four different cancer cell lines. Overall, we present photoswitchable HDACis with optimized activation wavelengths, which inhibit enzyme activity and cell viability only upon illumination with visible light, contributing to the still limited toolbox of photoswitchable anticancer drugs.

Introduction

A prevailing limitation of anticancer drugs is that they often fail to fully differentiate between cancer and healthy cells. This lack of selectivity limits their therapeutic window, which both decreases treatment’s efficacy and leads to undesired side effects. In parallel to the investigation of new targeted antitumor drugs, it is important to identify novel approaches that enable a more selective and localized effect of such drugs. For this purpose, several drug delivery systems have been developed, which direct the drug to the site of interest using internal (pH,¹ reactive oxygen species,² or overexpression of enzymes³ and receptors⁴) or external (temperature,⁵ ultrasound,⁶ or light^7,8) triggers. Among these, light is particularly attractive given its noninvasive and remote action, together with a precise modulation by adjusting the wavelength, intensity, and time of exposure.

Photopharmacology uses light as an external trigger to control drug activity with high spatiotemporal precision. It is based on molecules that change their structure upon illumination under particular light conditions, which is used to exercise an external control over its target receptor and finally in their biological function. The design of light-activatable molecules relies on two main approaches: caging and photoswitching. In the caging approach, a bioactive molecule is chemically modified with a photoremovable group that renders it inactive, and the active molecule is released at the target tissue upon illumination.⁹ In the case of photoswitches, a photochromic moiety is introduced within the structure of bioactive molecules; this enables reversible on/off switching between biologically active and inactive forms when illuminated.¹⁰ While the design of cages is simpler as it can employ well-validated drugs, the reversibility of photoswitches allows for greater control of biological function, as both the initiation and termination of drug effects and its dose can be locally controlled by light. Among the known photochromic moieties, azobenzenes are the most popular.¹¹⁻¹⁴ They can isomerize between the more stable trans configuration to cis under illumination with a particular wavelength, which is dependent on the type and substitution of the aryl groups. This results in a large change in polarity, geometry, and end-to-end distance, which can lead to a difference in target engagement.

Photopharmacology has huge potential in the field of cancer, where a more localized effect of drugs could be achieved via illumination of the tumor area. To date, some examples of azobenzene-based molecules have been described, which show more potent anticancer activities under illumination conditions than in the dark in vitro(15−19) and in nematodes.²⁰ However, there has been a lack of further progression to more advanced in vivo rodent models. On top of the standard medicinal chemistry parameters that need to be considered for the progression of small molecules to in vivo studies (potency, physicochemical and pharmacokinetic properties, etc.), for photoswitching molecules, photochemical properties also need to be optimized. These include the wavelength of isomerization, photostationary state (PSS) distribution, photochemical conversion efficiency, and thermal half-life of the metastable isomer.²¹ Arguably, the optimal photochemical profile of anticancer azobenzenes still needs to be determined, and for this purpose, molecular tools with a range of properties would be invaluable. As localized and dynamic control of drug action is pursued, photoisomerization parameters and kinetics must be optimized to correlate with the target receptor activity, timings and biological rythms, and physiologiocal conditions to obtain an improved therapeutic effect.

Histone deacetylases (HDACs) are promising targets in oncology, as they can reverse cancer-associated epigenetic states.^22,23 These enzymes remove acetyl groups from the amino-terminal lysine residues on histone tails and nonhistone proteins. This leads to condensed chromatin, which limits the binding of transcription factors to promoter sequences and represses gene expression. However, the broad pharmacology and lack of specificity of HDACis limit their clinical use.²⁴ Therefore, they are an ideal target for the development of photoswitchable inhibitors.

Most classical HDAC isozymes have a zinc-dependent active site, for which many examples of HDAC inhibitors have been described. These are divided into different structural classes, namely, hydroxamates, benzamides, cyclic peptides, and short chain fatty acids.^23,25 The typical pharmacophore of zinc-dependent HDACis consists of three structural elements: (1) a zinc binding domain (ZBD), which chelates the zinc atom at the active site pocket of the enzyme; (2) a spacer/linker, which is usually hydrophobic and lies in the channel; and (3) a cap group, which sits on the surface of the channel and can be exploited to confer isoform selectivity.^26,27 The most common zinc chelators are hydroxamic acids (HA) and o-aminoanilide (OAA), such as SAHA and mocetinostat, respectively (Figure 1A). HAs are often less selective but more potent than OAAs.^28,29 The reported photoswitchable HDACis utilize an azobenzene as the photoswitchable moiety, which is incorporated onto the linker region (Figure 1B). The top compounds from these studies are HA 1(18,30) and OAA 2,^19,31 both of which are more active in the cis form than in the stable trans form. While HA 1 isomerizes under a low wavelength of 365 nm and relaxes at a relatively slow rate (t_1/2 = 4.2 h at room temperature in buffer), OAA 2 has a stronger push–pull character that increases the activation wavelength to 470 nm and decreases its half-life to 60 μs at room temperature in PBS but exerts a surprising long lasting inhibitory activity in the presence of the target HDAC protein.

Figure 1.

Reported (A) conventional and (B) photoswitchable HDAC inhibitors.

In this work, we aimed to understand the SAR of the reported azobenzenes to design optimized photoswitchable HDAC inhibitors. First, we identified analogues with larger differences in enzyme activity between isomers. Second, we increased the wavelength of isomerization from the ultraviolet (UV) to the visible region. Third, we obtained compounds with robust differences in activity in cell viability assays.

Results and Discussion

The reported photoswitchable HDACis differ in their ZBG, linker, and cap group. We first wanted to understand the influence of these elements on the photochemical properties and biological activity of the analogues, which would guide further optimization. For this purpose, we prepared a library of azobenzene-containing HDACis (Table 1). Analogues containing the alkene linker were accessed from acrylate 3,¹⁸ which generated the azobenzene moiety via formation of the diazonium salt (Scheme 1). Further ester hydrolysis and amide coupling gave the desired compounds 11 and 12. For HAs, we obtained higher and more consistent yields by preparing the O-THP-protected hydroxamate before a final deprotection to the final compounds, rather than reacting ester 7/8 directly with hydroxylamine. Synthesis of analogues 17 and 18 without the alkene proceeded via Mills reaction. With the small library of photoswitchable HDACis in hand, we proceeded to evaluate their photochemical properties. We determined (1) the optimal wavelength to isomerize the thermostable trans to the cis configuration, (2) the rate of back-isomerization to the trans configuration after illumination, and (3) the proportion of each isomer under different light conditions. These data are summarized in Table 1, and the full data set can be found in Figures S1–S8.

Table 1. Photochemical Properties of a First Library of Azobenzene HDACis.

¹Determined by measuring UV–vis spectra at 25–50 μM in DMSO.

²Determined by measuring UV–vis spectra at 25–50 μM in DMSO over time at room temperature after illumination with λ_cis.

³Determined by HPLC at the isosbestic point of each isomeric pair (310–338 nm) after illumination of a 100 μM solution in DMSO with either λ_cis or λ_trans.

⁴In DMEM with 0.1% DMSO at 37 °C, measured by HPLC.

⁵Using CoolLed at 217 mW/cm² instead of Teleopto plates.

Scheme 1. Synthesis of a First Library of Azobenzene HDACis.

Reagents and conditions: (a) (i) NaNO₂, HCl, MeOH/H₂O, −5 °C to rt, 10 min; (ii) 4 or 5, KOH, −5 °C to rt, 18 h, 44% (quant); (b) MeI, K₂CO₃, acetone, 50 °C, 2 h, 85%; (c) NaOH, EtOH, rt, 8–18 h, 20–87%; (d) o-phenylenediamine, DIPEA, EDC, HOBt, DMF, rt, 18 h, 31–55%; (e) oxone, DCM/H₂O, rt, 1 h, 95%; (f) DCM/AcOH, 35 °C, 18 h, 53%; (g) (i) O-THP-hydroxylamine, DIPEA, EDC, HOBt, DMF, rt, 18 h, 55%; (ii) HCl, 1,4-dioxane, rt, 30 min, 46%; (h) (i) tert-butyl (2-aminophenyl)carbamate, DIPEA, EDC, HOBt, DMF, rt, 18 h, 37%; (ii) TFA, DCM, rt, 2 h, 66%.

The λ_max of the trans isomer, and consequently the optimal wavelength of isomerization, was mainly influenced by the para substituent of the azobenzene. The dimethylamine analogues had λ_max values of 446–462 nm, while the methoxy analogues had λ_max values of 360–378 nm; hence, the former isomerized under blue-light irradiation and the latter under violet-light irradiation. As expected, the dimethylamine analogues have a greater push–pull character and relaxed much quicker than the corresponding methoxy, and in some cases, the relaxation was too fast to observe the absorbance spectrum of the cis isomer. For instance, 18 had a t_1/2 of 20.8 h, while its close pair 2 had a t_1/2 of 4.3 min.

The strongest effect of the alkene linker was on the back-isomerization rate, decreasing the half-life 6–7-fold as compared to that of the nonlinker or saturated analogues (17 vs 1, and 18 vs 11 and 20). The alkene also induced a 14–18 nm bathochromic shift.

Figure 2 shows a summary of the photochemical properties of compound 11, which later was found to give among the most promising biological performances. The optimal conditions of isomerization of 11 from trans to cis and vice versa were with near-ultraviolet (380 nm) and green light (550 nm), respectively (Figure 2B). The conversion ratio from cis to trans was high (80%), and back-isomerization occurred with moderate efficiency, recovering 67% of the trans configuration (Figure 2C). Thermal relaxation occurred with a t_1/2 of 3.1 h in DMSO at room temperature, and under conditions for cellular assays (37 °C in DMEM with 0.1% DMSO), it decreased to 40 min (Figure 2D). 11 was stable after multiple cis/trans isomerization cycles (Figure 2E).

Figure 2.

Photochemical properties of OAA 11. (A) Chemical structures of photoisomers of 11. (B) UV–vis spectra of 11 (25 μM) in DMSO under different light conditions; the extracted Z was calculated from the irradiated spectra (380 nm) and its known E/Z composition. (C) Quantification of the E/Z composition of 11 (100 μM) in DMSO after different temperature and light conditions, measured by HPLC at the isosbestic point (321 nm). (D) Half-life estimation of cis-11 after irradiation (380 nm) following its absorbance at 380 nm, in DMSO at room temperature (25 μM) or DMEM at 37 °C (10 μM). (E) Absorbance of 11 (25 μM) in DMSO at 380 nm after multiple illumination cycles (380–550 nm, 2 min each). In all cases, illumination at 380 nm was performed at 11 mW/cm² and that at 550 nm at 13 mW/cm².

To study the inhibition of HDAC enzymes by the compounds, we used a fluorogenic assay with human recombinant HDAC1.³² The enzyme was incubated with an acetylated substrate and each inhibitor for 1 h at room temperature, under either dark or light conditions. We opted for illumination throughout the incubation period to maintain the maximum proportion of cis under light conditions. To obtain the maximum proportion of trans under the dark conditions, the stock solutions were heated to 60 °C for 20 min before the assay, which was shown to be sufficient to achieve 93.3–99.9% of trans (Figures S1–S8). Then, the deacetylated substrate generated a fluorescent coumarin after incubation of the mixture with trypsin at 37 °C for 20 min.

We identified appropriate illumination intensities for each wavelength, which were high enough to reach PSS but not damaging to the enzyme. For this purpose, we first determined the minimum light intensity required to isomerize the compounds (results for compound 11 are shown in Figure S19 as an example). Then, we compared the activity of the nontreated enzyme under dark and illumination conditions and determined the dose–response curves of SAHA under both conditions. In general, the chosen conditions did not affect the enzyme activity (Figure S20). In some isolated replicates, we observed small differences in absolute fluorescence values between light conditions, but in these cases, the IC₅₀ values of SAHA remained unchanged (Figure S21).

Table 2 shows the activity of the tested compounds under dark and light conditions. The cis isomer of HA 1 gave the highest potency with an IC₅₀ of 52 nm, consistent with previous reports showing that HAs are more potent than the corresponding OAAs.²⁸ However, alkene-containing OAA 11 (Figure 3A) and thiophene-OAA 20 gave the largest difference between both isomers, being 50- and 29-fold more active under light than dark conditions, respectively. Saturated OAA 21 was also active only in the cis form, albeit with a potency lower than that of the corresponding alkene 11. Interestingly, only HA 17 was more active in the trans form than in the cis form.

Table 2. Inhibition of Recombinant HDAC1 by the First Library of Azobenzenes^a.

	IC50 (μM)
compound	dark	light	IC50(dark)/IC50(light)
17	0.42	2.9	0.14
1	0.96	0.052	18
18	1.2	0.20	6
11	>10	0.20	>50
20	>10	0.35	>29
21	>10	3.4	>2.9

^aIC₅₀ values under either dark or illumination (365 or 380 nm) conditions, measured by a fluorescence assay.

Figure 3.

Inhibition of recombinant HDAC1 by the first library of azobenzenes. (A) Dose–response curve of 11 against HDAC1 under either dark or illumination conditions; IC₅₀ (dark) > 10 μM, and IC₅₀ (365 nm) = 200 nM. (B) IC₅₀ values of 1 against HDAC1 following incubation with the enzyme for different times after being either kept in the dark or illuminated for 30 min. Illumination at 365 or 380 nm, 2 mW/cm²; stocks kept at 37 °C in the dark overnight before use.

With an IC₅₀ of 200 nM in the cis form, OAA 11 was chosen as the most promising photoswitchable HDACi from this small library, and it was used for further optimization.

We also wanted to assess whether enzyme binding would stabilize the more active cis form and extend its half-life. For this purpose, the enzyme was incubated with 1 for 6 h, either with or without preillumination of the compound, and enzyme activity was then assessed every hour. We found that the difference in activity between dark and illuminated conditions was maintained over time (Figure 3B). Considering that the t_1/2 of this compound in DMSO and a buffer is 2.7 h, these results suggest that enzyme binding stabilizes the azobenzene in its less stable cis form.

To further optimize the compounds for future in vivo use, it was necessary to find inhibitors that were activated under visible light. Among the strategies that have been described to increase the isomerization wavelength of azobenzenes, we focused on the introduction of halogen atoms ortho to the azo moiety.³³⁻³⁶ This modification is known to separate the n → π* absorption bands of the two isomers, which often overlap around 400–500 nm, allowing their use to trigger isomerization under green light.

We thus introduced di- and tetra-ortho fluorine and chlorine atoms to the scaffold of azobenzene 11, affording 32a–e. These compounds were prepared via formation of the diazonium salt of premade 22a–e, reacting with phenol 23a–e, respectively, to form the azobenzene moiety, followed by phenol methylation (Scheme 2A). The alkene was then introduced through a Heck reaction with tert-butyl acrylate, which after deprotection and amide coupling with o-phenylenediamine furnished the desired products.

Scheme 2. Synthesis of Halogenated Azobenzene HDACis.

Reagents and conditions: (a) for R = OH, (i) NaNO₂, HCl, H₂O, 0 °C, 20 min; (ii) 23a–e, NaOH, 0 °C, 2 h, 25–95%; (b) for R = NMe₂, NaNO₂, H₂SO₄, AcOH, DMF, 0 °C, 2 h; (ii) 24a or 24b, NaOH, 0 °C to rt, 3 days, 40–74%; (c) MeI, K₂CO₃, acetone, 50 °C, 2–3 h, 86–95%; (d) tert-butyl acrylate, P(o-tol)₃, Pd(OAc)₂, Et₃N, DMF, 100 °C, 18 h, 45–75%; (e) TFA, DCM, rt, 1 h, quant; (f) o-phenylenediamine, DIPEA, EDC, HOBt, DMF, rt, 18 h, 28–84%; (g) (i) NaNO₂, HCl, H₂O, 0 °C, 1 h; (ii) 23a or 23b, NaOH, K₂CO₃, 0 °C, 1 h; (h) NaOH, THF, MeOH, H₂O, rt, 18 h, 40% quant.

For the sake of comparison, tetrafluoro and tetrachloro substitutions were also introduced into non-alkene OAA 18. These compounds were also prepared via formation of the diazonium salt to give azobenzenes 35a and 35b (Scheme 2B). We thereafter obtained better yields by methylating both the phenol and acid groups, followed by ester hydrolysis and amide coupling to 38a and 38b.

It was also of interest to prepare the tetrafluoro and tetrachloro analogues with the p-dimethylamine. While push–pull azobenzenes with the p-dimethylamine have among the fastest thermal relaxation rates, tetrahalogenated azobenzenes are among the most stable, and both substitution patterns provide bathochromic shifts; thus, it was of interest to combine both features and determine the photochemical properties of the resulting compounds 33a and 33b. These were prepared in a manner similar to that of the corresponding p-methoxy 32a and 32b. In this case, reaction of the diazonium salts with dimethylamine 24a and 24b was much slower than with phenols 23a–e.

All p-methoxy tetrahalogenated compounds gave maximum amounts of the cis isomer upon illumination with green light (550 nm), and back-isomerization to trans proceeded with blue light (455 nm) (Figure 4). Conversely, difluoro 32d and dichloro 32e gave an absorption profile similar to that of the corresponding nonsubstituted 11 (Figures S12 and S13) but had slightly longer half-lives (Table 3).

Figure 4.

UV–vis spectra of halogenated compounds under different light conditions: (A) 32a, (B) 32b, (C) 33a, (D) 33b, and (E) 38e. trans and cis were calculated from the compound’s irradiated spectra at two wavelengths and their known E/Z composition. (F) Half-life estimation of the cis isomers after irradiation (420 or 550 nm) following their absorbance at selected wavelengths, in DMSO at room temperature.

Table 3. Photochemical Properties of ortho-Halogenated Compounds.

compound	R	X	Y	n	λmaxtrans (nm)a	λmaxcis (nm)a	λcis (nm)a	λtrans (nm)a	t1/2b	% cis at λcisc	% trans at λtransc
32a	OMe	F	F	1	358	434	550	455	9.3 days	77	74
32b	OMe	Cl	Cl	1	338	452	550	455	13.8 h	45	88
32c	OMe	F	Cl	1	358	442	550	455	7.4 days	68	85
32d	OMe	F	H	1	388	448	380	550	9.9 h	67	59
32e	OMe	Cl	H	1	396	464	380	455	4.3 h	64	62
33a	NMe2	F	F	1	430		420	550	5.4 h	–	–
33b	NMe2	Cl	Cl	1	412		420	550	8.1 min	–	–
38a	OMe	F	F	0	348	428	550	455	42 daysd/4.7 dayse	84	67
38b	OMe	Cl	Cl	0	336	452	550	455	8.3 h	46	84
39f	OMe	F	F	1	358	438	550	455	9.1 days	72	83

^aDetermined by measuring UV–vis spectra at 25–100 μM in DMSO.

^bDetermined by measuring UV–vis spectra at 25–100 μM in DMSO over time at room temperature after illumination with λ_cis.

^cDetermined by HPLC at the isosbestic point of each isomeric pair (290–336 nm) after illumination of a 100 μM solution in DMSO with either λ_cis or λ_trans.

^dEstimation, full back-isomerization not reached.

^eIn DMEM with 0.1% DMSO at 37 °C, measured by HPLC.

^fWith HA instead of OAA.

As expected, tetrafluorination had a pronounced effect on the rate of back-isomerization, with an increase in the half-life of up to 72-fold. The fluorine atoms lower the n orbital of the Z isomer, hence hugely increasing its thermal stability.³⁷ The incorporation of difluoro–dichloro also led to a large 57-fold increase, while tetrachlorination had a small effect. Also, the presence of the alkene generally increased t_1/2 as evidenced by comparing 32a to 38a (Figure 4F), which is consistent with the previous results. Thus, the slowest compound herein is non-alkene tetrafluoro 38a, with a t_1/2 in DMSO at room temperature of 42 days. When measured under the conditions used for cellular assays (37 °C in DMEM with 0.1% DMSO), the t_1/2 was reduced to 4.7 days.

In terms of the proportion of each isomer at the PSS, the ortho-halogenated compounds gave moderate to high percentages of the cis isomer (64–84%) under illumination with λ_cis except for the tetrachloro analogues (32b and 38b), where only 45–46% of the cis configuration was generated upon illumination with green light.

Another difference in the halogenated compounds was found in the conditions required to obtain maximum amounts of the more stable trans isomer. While for the nonhalogenated inhibitors heating to 60 °C for 20 min was sufficient, the tetrahalogenated compounds required 80 °C for 1–2 h.

We then tested the inhibition of recombinant HDAC1 by these compounds under continuous illumination. Pleasingly, most compounds were more active in the cis than in the trans form (Table 4). In particular, tetrafluoro 32a and 33a (Figure 5B) and difluoro–dichloro 32c (Figure 5A) had no or little activity at the highest tested concentration (10 μM) in the dark but were active under illumination with IC₅₀ values in the low micromolar range. Although these compounds had potencies lower than that of the parent 11, they have the advantage of being activated under visible light.

Table 4. Inhibition of Recombinant HDAC1 by the Halogenated Azobenzenes under Dark or Illumination Conditions, Measured by a Fluorescence Assay.

	IC50 (μM)
compound	dark	light	IC50(dark)/IC50(light)
32a	>10	2.0 (550 nm)	>5.0
32b	naa	≈10 (550 nm)
32c	>10	3.9 (550 nm)	>2.6
32d	≈10	0.46 (380 nm)	≈22
32e	>10	>10 (380 nm)
33a	naa	1.2 (420 nm)	>8.3
33b	>10	≈5 (420 nm)	>2
38a	≈10	1.7 (550 nm)	≈5.9
38b	naa	naa(550 nm)
39	0.14	0.054 (550 nm)	2.6

^aNot active.

Figure 5.

Inhibition of recombinant HDAC1 by the halogenated azobenzenes. Dose–response curves against HDAC1 of (A) 32c (dark vs 550 nm, 6 mW/cm²) and (B) 33a (dark vs 420 nm, 7 mW/cm²). (C) Distribution of all compounds in terms of their relative activity between isomers, half-life of cis, and optimal wavelength of isomerization.

The potency of HA 39 was higher than that of OAA 32a, but 39 exhibited a smaller difference between the two isomers, which agrees with the trends observed above for activity of HAs versus OAAs.

At this point, we had a range of photoswitchable inhibitors of recombinant HDAC1 with higher potencies under illumination and with a range of distinct photochemical properties in terms of their activation wavelengths and half-times (Figure 5C). Given the promise of HDAC inhibitors to induce apoptosis to cancer cells, we proceeded to study the effects of the best analogues in HeLa cells. We first confirmed that representative compounds were able to inhibit HDACs in cells in a whole-cell HDAC assay, which accounts for activity against class 1 and 2b HDAC isoforms (Figure S22). We then proceeded to evaluate the effect of the compounds in a viability assay against HeLa cells. The compounds were either kept in the dark or illuminated before addition onto preseeded cells and then incubated for 24–48 h before the proportion of live cells was measured with an MTS assay.

While the positive control SAHA induced cell death after treatment for 24 h, we found small effects with our compounds at this time point, and clear dose–response curves were obtained only 48 h after compound addition.

From the UV-activatable compounds, OAAs 11 and 20 showed again the best results, with no activity in the dark up to 100 μM, and IC₅₀ values under illumination of 12 and 7.4 μM, respectively, with the viability decreasing to 30% (Table 5 and Figure 6A). The observed large effect is remarkable, considering that 11 back-isomerizes under these conditions with a t_1/2 of 40 min. Given that continuous exposure to HDAC inhibitors is required to achieve a full response,^29,38 these results support the previous findings (Figure 3B) that enzyme engagement in the cis form is maintained for longer than its half-life in an enzyme-free solution.

Table 5. Effect of Selected Compounds on the Viability of HeLa Cells under Dark or Illumination Conditions, Measured by an MTS Assay.

	IC50a (μM)
compound	dark	light	IC50(dark)/IC50(light)
17	≈25b	≈25b(380 nm)	≈1
1	≈12b	≈12b(380 nm)	≈1
18	nac	≈19b(380 nm)	>5.3
11	nac	12 (380 nm)	>8.3
20	nac	7.4 (380 nm)	>14
21	nac	nac(380 nm)
32a	nac	37 (550 nm)	>2.7
32c	nac	37 (550 nm)	>2.7
32d	nac	nac(380 nm)
32e	nac	nac(380 nm)
38a	>100	29 (550 nm)	>3.4
39	6.7	13 (550 nm)	0.52

^aIC₅₀ corresponds to the concentration of the compound that reduces cell viability by half.

^bEstimation, full dose–response not obtained.

^cNot active.

Figure 6.

Cellular activity of 20 and 32c, measured as the percentage of live cells relative to the 0.5% DMSO control. (A) Dose–response curves of 20 on the viability of HeLa cells (MTS assay) under either dark or preillumination (380 nm) conditions for 24 and 48 h; dark n.a., IC₅₀ (365 nm, 24 h) = 13 μM, IC₅₀ (365 nm, 48 h) = 7.4 μM. (B) Dose–response curve of 32c on the viability of HeLa cells (MTS assay) under either dark or preillumination (550 nm) conditions for 48 h; dark n.a., IC₅₀ (550 nm, 24 h) = 37 μM. Effect of 50 μM (C) 20 and (D) 32c on HeLa, HT29, MCF7, and KG1 cells under either dark or preillumination (380 or 550 nm) conditions, measured by MTS (HeLa, HT29, and MCF7) and CellTiterGlo (KG1) assays.

Among the halogenated compounds, the findings were again consistent with the results for recombinant HDAC1, with tetrafluoro 32a and 38a, and difluoro–dichloro 32c, inducing cell death only under light conditions.

HAs showed worse results in these cell assays, with no or little difference in activity between dark and illumination conditions.

The faster 2, 33a, and 33b were inactive under both dark and illumination conditions. It is likely that in this case back-isomerization occurred too fast for the cis isomer to have enough time to engage with the target, and in this case, constant illumination would be necessary.

HDACis have been proposed as a potential therapeutic strategy against cancer stem cells (CSCs).^39,40 We selected three cell lines that have a strong CSC phenotype (HT29,^41,42 MCF7⁴³⁻⁴⁵ and KG1⁴⁶) and tested the best compounds at 50 μM under dark and illumination conditions. Compounds 11, 20, 32a, 32c, and 38a had little or no effect in the dark but significantly reduced the viability of these cells after 48 h when they were preilluminated (Figure 6C,D and Figure S23).

Conclusions

The ability to localize the effect of anticancer drugs with high spatiotemporal precision could overcome the limitations of current chemotherapy. Photoswitching molecules are particularly attractive, as their therapeutic effect could be modulated with external light. For this purpose, photoswitches with a range of properties will be necessary to shed light on the optimal profile required for clinical progression. One of the properties that will likely be important is the wavelength of activation, as longer wavelengths in the visible spectrum are less harmful and more permeable. In this work, we aimed to contribute to building this toolbox of molecules.

Herein, we chose a well-established target for anticancer drugs, for which photoswitching molecules were already known to facilitate further optimization. Initial SAR studies identified alkene 11, which had remarkable differences in activity in the recombinant enzyme assays, with the metastable cis form being >50-fold more active than the stable trans form, with an IC₅₀ of 200 nM against HDAC1. It had a half-life of 40 min under cell medium conditions, and it isomerized under near-UV light.

To increase the wavelength of isomerization, the next round of SAR was based on adding halogen atoms ortho to the azobenzene group. Tetrafloro 32a and 33a and difluoro–dichloro 32c were particularly attractive. Despite a drop in potency, these were still more active in the cis form, with >3–8-fold differences with the trans form. Notably, they isomerized under green and blue illumination, respectively, and had half-lives different from that of alkene 11.

Importantly, we found a good translation of the enzyme activities onto effects in cells, particularly of the benzamide analogues. In four different cancer cell lines, our top compounds caused a significant reduction in viability upon treatment for 48 h after illumination, but no or less activity in the dark.

All in all, we have developed a small library of photoswitchable HDAC inhibitors with a range of biological and photochemical properties. These vary in terms of their activation wavelength, from near-ultraviolet to green wavelengths, and half-lives, from minutes to days. Most examples are more active in the cis form than in the trans form, with >50-fold differences in recombinant enzyme inhibition assays and >14-fold differences in cell viability assays. We expect that not only the disclosed molecules but also the findings and the explored SAR will be highly valuable in paving the way for further progression of azobenzene-based drugs as cancer therapeutics.

Experimental Section

General Synthesis Methods

All reagents were obtained from commercial sources and used without further purification. Reactions were conducted under an inert atmosphere of nitrogen or argon unless not using anhydrous solvents. Anhydrous solvents were obtained from a solvent purification system (PureSolv-EN) and kept under a nitrogen atmosphere. Temperatures of <25 °C were obtained using the following cooling baths: 0 °C ice/water and −5 °C ice/water with NaCl. Concentrations (c) in the general procedures refer to the limiting reagent and are given in millimoles per milliliter.

Analytical thin layer chromatography (TLC) was carried out on aluminum sheets coated with silica gel (Macherey-Nagel, 60F, 0.2 mm, ALUGRAM Sil G/UV254). The spots were visualized by UV irradiation (254 nm) and by staining with a KMnO₄ solution followed by heating.

Flash column chromatography was performed on 60 silica gel (Panreac, 40–63 μm particle size) or with a Biotage Isolera One automated system with Biotage KP-C18-SH cartridges. The eluents are specified in each case.

NMR spectra were recorded on a Varian-Mercury 400 MHz spectrometer. Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant, and integration. Chemical shifts (δ) are reported in parts per million downfield from TMS and are referenced to the residual solvent peak. Coupling constants (J) are reported in hertz.

Low-resolution mass spectra (m/z) were recorded on a Quattro Micro MS detector (Waters). Selected peaks are reported in daltons, and their intensities given as percentages of the base peak. High-resolution mass spectra (HRMS) were recorded on a FIA with an ultra-high-performance liquid chromatography (UPLC) Aquity instrument (Waters) coupled to an LCT Premier Orthogonal Accelerated TOF instrument (Waters). Data from mass spectra were analyzed by electrospray ionization in positive and negative modes using MassLynx version 4.1 (Waters).

Analytical high-performance liquid chromatography (HPLC) was performed on a Thermo Ultimate 3000SD instrument (Thermo Scientific Dionex) coupled to a PDA detector and an LTQ XL ESI-ion trap mass spectrometer (Thermo Scientific) with a Sunfire C18 2.5 μm, 4.6 mm × 50 mm (Waters) column or on an ESI Quattro Micro MS detector (Waters) with a ZORBAX Extend-C18 3.5 μm, 2.1 mm × 50 mm (Agilent) column. The purity of the final compounds was determined to be >95%.

Hydroxamic acid 1(30) and o-aminoanilide 2(19) were prepared as previously reported.

Syntheses of compounds 19–21 and 39 are shown in Scheme S1.

Ethyl (E)-3-(4-((E)-(4-Hydroxyphenyl)diazenyl)phenyl)acrylate¹⁸ (6)

To a solution of (E)-ethyl 3-(4-aminophenyl)acrylate 3 (400 mg, 2.09 mmol) in MeOH (3.0 mL) and HCl (1 M, 5.7 mL, 5,7 mmol) at −5 °C was added a solution of sodium nitrite (171 mg, 2.47 mmol) in water (1.5 mL) dropwise, keeping the temperature below 0 °C. The mixture was then stirred at room temperature for 10 min and cooled to −5 °C. A solution of phenol (179 mg, 1.90 mmol) and KOH (235 mg, 4.18 mmol) in MeOH (7.0 mL) was added dropwise, keeping the temperature below 0 °C. The resulting suspension was stirred at room temperature overnight, diluted with EtOAc, washed with 1 × 1 M HCl and 1× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give azobenzene 6 (621 mg, 2.10 mmol, quant) as a brown solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.40 (s, 1H), 7.92 (d, J = 8.6 Hz, 2H), 7.86–7.78 (m, 4H), 7.72 (d, J = 16.0 Hz, 1H), 7.01–6.91 (m, 2H), 6.74 (d, J = 16.0 Hz, 1H), 4.21 (q, J = 7.1 Hz, 2H), 1.27 (t, J = 7.1 Hz, 3H); m/z (ESI+) 297.3 (MH⁺, 100%).

Ethyl (E)-3-(4-((E)-(4-Methoxyphenyl)diazenyl)phenyl)acrylate¹⁸ (8)

To a solution of phenol 6 (563 mg, 1.90 mmol) in acetone (28.4 mL) were added K₂CO₃ (2.63 g, 19.0 mmol) and methyl iodide (950 μL, 15.2 mmol), and the mixture was heated to 50 °C. After 2 h, the mixture was concentrated in vacuo, diluted with water, and filtered to give 8 (503 mg, 1.62 mmol, 85%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.98–7.90 (m, 2H), 7.93–7.86 (m, 2H), 7.73 (d, J = 16.1 Hz, 1H), 7.69–7.62 (m, 2H), 7.07–6.97 (m, 2H), 6.51 (d, J = 16.0 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 3.91 (s, 3H), 1.35 (t, J = 7.1 Hz, 3H); m/z (ESI+) 311.3 (MH⁺, 100%).

(E)-3-(4-((E)-(4-Methoxyphenyl)diazenyl)phenyl)acrylic Acid (9)

To a solution of ethyl ester 8 (503 mg, 1.62 mmol) in ethanol (3.2 mL) was added NaOH (10 wt %, 1.3 mL, 3.2 mmol). The mixture was stirred at room temperature for 8 h, acidified with 1 M HCl, filtered, washed with water, and dried to give acid 9 (398 mg, 1.41 mmol, 87%) as a brown solid: ¹H NMR (400 MHz, DMSO-d₆) δ 7.95–7.82 (m, 6H), 7.66 (d, J = 16.0 Hz, 1H), 7.18–7.09 (m, 2H), 6.64 (d, J = 15.9 Hz, 1H), 3.87 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 167.4, 162.3, 152.7, 146.3, 142.9, 136.5, 129.4, 124.8, 122.8, 120.6, 114.7, 55.7; m/z (ESI+) 283.3 (MH⁺, 100%); HRMS (m/z) [M – H]⁻ calcd for C₁₆H₁₃N₂O₃^– 281.0923, found 281.0932.

(E)-N-(2-Aminophenyl)-3-(4-((E)-(4-methoxyphenyl)diazenyl)phenyl)acrylamide (11)

To a solution of acid 9 (100 mg, 0.354 mmol) in anhydrous DMF (1.8 mL) were added benzene-1,2-diamine (38 mg, 0.35 mmol), DIPEA (93 μL, 0,53 mmol), EDC (102 mg, 0.531 mmol), and HOBt (81 mg, 0.53 mmol), and the mixture was stirred at room temperature overnight, diluted with water, extracted with 3× DCM, dried over anhydrous Na₂SO₄, washed with 2× brine/water, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 90% EtOAc in hexane) to give benzamide 11 (72 mg, 0.19 mmol, 55%) as an orange solid: mp 200.1–200.3 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.45 (s, 1H), 7.96–7.88 (m, 4H), 7.82 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 15.7 Hz, 1H), 7.37 (d, J = 7.0 Hz, 1H), 7.19–7.11 (m, 2H), 7.02 (d, J = 15.7 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.6, 1.5 Hz, 1H), 4.98 (s, 2H), 3.88 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.3, 162.3, 152.4, 146.3, 141.6, 138.5, 137.2, 128.8, 125.9, 124.8, 124.7, 123.8, 123.4, 123.0, 116.3, 116.0, 114.7, 55.7; m/z (ESI+) 373.3 (MH⁺, 15%); HRMS (m/z) [M – H]⁻ calcd for C₂₂H₁₉N₄O₂^– 371.1503, found 371.1513.

(E)-Ethyl 3-(4-((E)-(4-(Dimethylamino)phenyl)diazenyl)phenyl)acrylate (7)

To a solution of (E)-ethyl 3-(4-aminophenyl)acrylate 3 (400 mg, 2.09 mmol) in MeOH (3.0 mL) and HCl (1 M, 5.7 mL, 5.7 mmol) at −5 °C was added a solution of sodium nitrite (171 mg, 2.47 mmol) in water (1.5 mL) dropwise, keeping the temperature below 0 °C. The mixture was then stirred at room temperature for 10 min and cooled to −5 °C. A solution of N,N-dimethylaniline (240 μL, 1.90 mmol) and potassium hydroxide (235 mg, 4.18 mmol) in MeOH (7.0 mL) was added dropwise, keeping the temperature below 0 °C. The resulting suspension was stirred at room temperature overnight, diluted with EtOAc, washed with 1 × 1 M HCl and 1× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 15% EtOAc in hexane) to give azobenzene 7 (270 mg, 0.835 mmol, 44%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.91 (d, J = 8.6 Hz, 2H), 7.86 (d, J = 8.5 Hz, 2H), 7.72 (d, J = 16.0 Hz, 1H), 7.67–7.58 (m, 2H), 6.77 (d, J = 9.4 Hz, 2H), 6.48 (d, J = 16.0 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 3.11 (s, 6H), 1.35 (t, J = 7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 167.0, 154.0, 152.8, 143.9, 143.7, 135.2, 128.9, 125.5, 122.7, 118.6, 111.7, 60.6, 40.4, 14.4; m/z (ESI+) 324.3 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₉H₂₂N₃O₂⁺ 324.1704, found 324.1707.

(E)-3-(4-((E)-(4-(Dimethylamino)phenyl)diazenyl)phenyl)acrylic Acid (10)

To a suspension of ethyl ester 7 (489 mg, 1.51 mmol) in ethanol (3.0 mL) was added NaOH (10 wt %, 1.2 mL, 3.0 mmol). The mixture was stirred at room temperature overnight, acidified with 1 M HCl, filtered, and washed with water to give a brown solid, which shows 36% remaining starting material. The solid was diluted with ethanol (3.0 mL) and treated with NaOH (10 wt %, 1.2 mL, 3.0 mmol). The mixture was stirred at room temperature overnight, acidified with 1 M HCl, and filtered, and the solid rediluted in MeOH and concentrated in vacuo to give acid 10 (88 mg, 0.30 mmol, 20%) as a brown solid: ¹H NMR (400 MHz, DMSO-d₆) δ 7.88–7.72 (m, 6H), 7.64 (d, J = 16.0 Hz, 1H), 6.90–6.78 (m, 2H), 6.60 (d, J = 16.0 Hz, 1H), 3.07 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 167.5, 153.2, 152.8, 143.1, 142.7, 135.2, 129.3, 125.1, 122.2, 119.8, 111.6, 39.9; m/z (ESI+) 296.3 (MH⁺, 100%); HRMS (ESI+) [M – H]⁻ calcd for C₁₇H₁₆N₃O₂^– 294.1247, found 294.1248.

(E)-N-(2-Aminophenyl)-3-(4-((E)-(4-(dimethylamino)phenyl)diazenyl)phenyl)acrylamide (12)

To a solution of acid 10 (62 mg, 0.21 mmol) in anhydrous DMF (1.0 mL) were added benzene-1,2-diamine (23 mg, 0.21 mmol), DIPEA (55 μL, 0.32 mmol), EDC (60 mg, 0.32 mmol), and HOBT (48 mg, 0.32 mmol). The mixture was stirred at room temperature overnight, diluted with water, extracted with 3× DCM, dried over anhydrous Na₂SO₄, washed with 2× brine/water, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 100% EtOAc in hexane) to give benzamide 12 (25 mg, 0.065 mmol, 31%) as an orange solid: mp 218.1–218.9 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 7.87–7.73 (m, 6H), 7.61 (d, J = 15.7 Hz, 1H), 7.37 (dd, J = 8.0, 1.5 Hz, 1H), 6.98 (d, J = 15.7 Hz, 1H), 6.95–6.89 (m, 1H), 6.88–6.81 (m, 2H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.5, 1.5 Hz, 1H), 4.97 (s, 2H), 3.08 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.4, 152.9, 152.7, 142.7, 141.6, 138.8, 135.9, 128.7, 125.8, 125.0, 124.7, 123.5, 123.1, 122.5, 116.3, 116.0, 111.6, 39.9; m/z (ESI⁺) 386.3 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₃H₂₄N₅O⁺ 386.1985, found 386.1975.

4-Nitrosobenzoic Acid⁴⁷ (14)

To a suspension of 4-aminobenzoic acid 13 (1.00 g, 7.29 mmol) in DCM (11.2 mL) was added a solution of Oxone (8.97 g, 14.6 mmol) in water (44.9 mL). The resulting suspension was stirred at room temperature for 1 h, filtered, and dried to give 4-nitrosobenzoic acid (1.05 g, 6.94 mmol, 95%) as a yellow solid: ¹H NMR (400 MHz, DMSO-d₆) δ 8.30–8.23 (m, 1H), 8.07–7.99 (m, 1H); m/z (ESI+) 152.2 (MH⁺, 100%).

(E)-4-((4-Methoxyphenyl)diazenyl)benzoic Acid⁴⁸ (16)

A solution of 4-methoxyaniline (204 mg, 1.65 mmol) and nitroso 14 (250 mg, 1.65 mmol) in DCM (10.3 mL) and acetic acid (10.3 mL) was stirred at 35 °C overnight. Then it was cooled o 0 °C and filtered to give azobenzene 16 (226 mg, 0.882 mmol, 53%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 8.15–8.08 (m, 2H), 7.97–7.93 (m, 2H), 7.93–7.89 (m, 2H), 7.24–7.05 (m, 2H), 3.89 (s, 3H); m/z (ESI+) 257.2 (MH⁺, 100%).

(E)-4-((4-Methoxyphenyl)diazenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (S1)

To a solution of acid 16 (80 mg, 0.31 mmol) in anhydrous DMF (1.6 mL) were added O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (40 mg, 0.34 mmol), DIPEA (82 μL, 0.47 mmol), EDC (90 mg, 0.47 mmol), and HOBt (72 mg, 0,47 mmol). The mixture was stirred at room temperature overnight, diluted with water, extracted with 3× DCM, washed with a 1:1 brine/water mixture, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (20% to 30% EtOAc in hexane) to give S1 (61 mg, 0.17 mmol, 55%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 11.81 (s, 1H), 7.99–7.92 (m, 4H), 7.91 (d, J = 8.6 Hz, 2H), 7.20–7.12 (m, 2H), 5.03 (s, 1H), 4.08 (d, J = 10.1 Hz, 1H), 3.88 (s, 3H), 3.62–3.48 (m, 1H), 1.81–1.67 (app s, 3H), 1.61–1.48 (m, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6, 162.5, 153.7, 146.2, 133.8, 128.6, 125.0, 122.2, 114.8, 101.0, 61.4, 55.8, 27.9, 24.7, 18.3; m/z (ESI+) 356.2 (MH⁺, 30%); HRMS (ESI-) [M – H]⁻ calcd for C₁₉H₂₀N₃O₄^– 354.1459, found 354.1443.

(E)-N-Hydroxy-4-((4-methoxyphenyl)diazenyl)benzamide (17)

To a solution of S1 (46 mg, 0.13 mmol) in MeOH (1.3 mL) was added HCl in 1,4-dioxane (4 M, 1.8 mL, 7.0 mmol). After 30 min, the mixture was concentrated in vacuo, and the resulting red solid was diluted with Et₂O and filtered. The solid was purified by flash column chromatography (0% to 20% MeOH in DCM) to give hydroxamic acid 17 (16 mg, 0,059 mmol, 46%) as an orange solid: mp 198.8–201.4 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 11.39 (s, 1H), 9.16 (s, 1H), 7.99–7.91 (m, 4H), 7.88 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.9 Hz, 2H), 3.88 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.4, 162.5, 153.4, 146.2, 134.4, 128.2, 124.9, 122.2, 114.8, 55.8; m/z (ESI+) 272.3 (MH⁺, 100%); HRMS (ESI-) [M – H]⁻ calcd for C₁₄H₁₂N₃O₃^– 270.0885, found 270.0884.

tert-Butyl (2-Aminophenyl)carbamate⁴⁹ (S2)

To a solution of benzene-1,2-diamine (1.00 g, 9.25 mmol) in methanol (18.5 mL) was added di-tert-butyl dicarbonate (2.02 g, 9.25 mmol). The solution was stirred at room temperature for 24 h, concentrated under reduced pressure, diluted with brine, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give protected diamine S2 (1.90 g, 9.14 mmol, 99%) as a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 7.27 (d, J = 6.9 Hz, 1H), 7.01 (ddd, J = 8.0, 7.2, 1.5 Hz, 1H), 6.87–6.78 (m, 2H), 6.27 (s, 1H), 1.51 (s, 9H); m/z (ESI+) 356.2 (MH⁺, 2%).

tert-Butyl (E)-(2-(4-((4-Methoxyphenyl)diazenyl)benzamido)phenyl)carbamate (S3)

To a solution of acid 16 (80 mg, 0.31 mmol) in anhydrous DMF (1.6 mL) were added tert-butyl (2-aminophenyl)carbamate S2 (65 mg, 0,31 mmol), DIPEA (82 μL, 0.47 mmol), EDC (90 mg, 0.47 mmol), and HOBt (72 mg, 0.47 mmol). The mixture was stirred at room temperature overnight, diluted with water, extracted with 3× DCM, dried over anhydrous Na₂SO₄, washed with a 1:1 brine/water mixture, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 20% EtOAc in hexane) to give S3 (51 mg, 0.11 mmol, 37%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 8.10 (d, J = 8.6 Hz, 2H), 8.01–7.90 (m, 4H), 7.82 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 7.3 Hz, 2H), 7.16 (dd, J = 6.8, 1.3 Hz, 1H), 7.07–6.99 (m, 2H), 6.88 (s, 1H), 3.91 (s, 3H), 1.53 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.1, 162.7, 154.9, 154.8, 147.1, 135.5, 131.1, 129.9, 128.5, 126.3, 126.2, 126.0, 125.3, 124.6, 122.8, 114.5, 81.7, 55.8, 28.4; m/z (ESI+) 447.3 (MH⁺, 40%); HRMS (ESI-) [M – H]⁻ calcd for C₂₅H₂₅N₄O₄^– 445.1857, found 445.1881.

(E)-N-(2-Aminophenyl)-4-((4-methoxyphenyl)diazenyl)benzamide (18)

To a solution of Boc-protected S3 (47 mg, 0.10 mmol) in DCM (2.1 mL) was added TFA (405 μL, 5.26 mmol). The mixture was stirred at room temperature for 2 h and concentrated in vacuo. The resulting red solid was diluted with Et₂O and filtered. The solid was purified by flash column chromatography (EtOAc in hexane) to give benzamide 18 (24 mg, 0.069 mmol, 66%) as an orange solid: mp 271.2–271.3 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.82 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 8.01–7.85 (m, 4H), 7.18 (t, J = 7.9 Hz, 3H), 6.98 (q, J = 6.4, 5.1 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.61 (t, J = 7.6 Hz, 1H), 4.96 (s, 2H), 3.89 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.6, 162.5, 153.6, 146.2, 143.1, 136.2, 129.1, 126.8, 126.7, 125.0, 123.1, 122.0, 116.2, 116.1, 114.8, 55.8; m/z (ESI+) 347.3 (MH⁺, 20%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₁₉N₄O₂⁺ 347.1503, found 347.1503.

(E)-4-((4-(Dimethylamino)phenyl)diazenyl)-N-((tetrahydro-2H-pyran-2-yl)oxy)benzamide (S4)

To a solution of 4-dimethylaminoazobenzene-4′-carboxylic acid (200 mg, 0.743 mmol) in anhydrous DMF were added DIPEA (650 μL, 3.7 mmol), EDC (427 mg, 2.23 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (96 mg, 0.82 mmol), and HOBt (57 mg, 0.37 mmol). The mixture was stirred at room temperature overnight, diluted with water, extracted with 3× DCM, washed with 1× NaHCO₃ and 1× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (20% to 50% EtOAc in hexane) to give S4 (179 mg, 0.486 mmol, 65%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 11.76 (s, 1H), 7.94–7.88 (m, 2H), 7.86–7.79 (m, 4H), 6.89–6.81 (m, 2H), 5.02 (s, 1H), 4.14–4.01 (m, 1H), 3.61–3.47 (m, 1H), 3.08 (s, 6H), 1.74 (s, 3H), 1.66–1.45 (m, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.7, 154.2, 152.9, 142.6, 132.5, 128.4, 125.2, 121.6, 111.6, 101.0, 61.4, 39.9, 27.9, 24.8, 18.3; m/z (ESI+) 369.4 (MH+, 100%); HRMS (ESI+) [M – H]⁻ calcd for C₂₀H₂₃N₄O₃^– 367.1776, found 367.1776.

(E)-4-((4-(Dimethylamino)phenyl)diazenyl)-N-hydroxybenzamide (19)

To a solution of S4 (150 mg, 0.407 mmol) in MeOH (4.1 mL) was added HCl in 1,4-dioxane (4 M, 1.75 mL, 7.00 mmol). After 30 min, the mixture was concentrated in vacuo. The crude product was diluted with Et₂O, filtered, washed with more Et₂O, and dried to give hydroxamic acid 19 (76 mg, 0.27 mmol, 66%) as a dark purple solid: mp 164.0–164.2 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 7.93–7.85 (m, 2H), 7.85–7.77 (m, 4H), 6.90–6.82 (m, 2H), 3.08 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.5, 153.7, 152.9, 142.6, 133.0, 128.1, 125.4, 121.5, 112.0, 40.0; m/z (ESI+) 285.3 (MH⁺, 100%); HRMS (ESI+) [M – H]⁻ calcd for C₁₅H₁₅N₄O₂^– 283.1190, found 283.1200.

tert-Butyl (2-((E)-3-(4-((E)-(4-Methoxyphenyl)diazenyl)phenyl)acrylamido)-4-(thiophen-2-yl)phenyl)carbamate (S5)

To a solution of acid 9 (40 mg, 0.14 mmol) in dry DMF (720 μL) were added tert-butyl (2-amino-4-(thiophen-2-yl)phenyl)carbamate³⁸ (45 mg, 0.16 mmol), DIPEA (37 μL, 0.21 mmol), EDC (41 mg, 0.21 mmol), and HOBt (32 mg, 0.21 mmol). The mixture was stirred at room temperature overnight, diluted with DCM, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 30% EtOAc in hexane) to give amide S5 (30 mg, 0.054 mmol, 38%) as an orange solid: ¹H NMR (400 MHz, CDCl₃) δ 8.60–8.46 (m, 1H), 7.93 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.1 Hz, 2H), 7.84–7.75 (m, 2H), 7.63 (d, J = 8.2 Hz, 2H), 7.39 (q, J = 8.9 Hz, 2H), 7.23 (d, J = 3.6 Hz, 1H), 7.20 (d, J = 5.0 Hz, 1H), 7.12 (s, 1H), 7.08–6.96 (m, 3H), 6.62 (d, J = 15.6 Hz, 1H), 3.90 (s, 3H), 1.53 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 164.8, 162.5, 154.4, 153.6, 147.2, 143.3, 141.8, 136.5, 130.1, 129.0, 128.2, 128.0, 125.1, 125.0, 124.6, 123.9, 123.4, 123.2, 123.0, 122.5, 121.6, 114.4, 81.3, 55.8, 28.5; m/z (ESI+) 555.0 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₃₁H₃₁N₄O₄S⁺ 555.2061, found 555.2086.

(E)-N-(2-Amino-5-(thiophen-2-yl)phenyl)-3-(4-((E)-(4-methoxyphenyl)diazenyl)phenyl)acrylamide (20)

To a solution of Boc-protected S5 (29 mg, 0.052 mmol) in DCM (1.0 mL) was added TFA (120 μL, 1.57 mmol). The mixture was stirred at room temperature for 2 h, basified with saturated aqueous NaHCO₃, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give an orange solid. The crude product was purified by C18 column chromatography to give 20 (15 mg, 0.033 mmol, 63%) as an orange solid: mp 199.5–200.5 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.53 (s, 1H), 7.98–7.90 (m, 4H), 7.83 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 2.1 Hz, 1H), 7.67 (d, J = 15.8 Hz, 1H), 7.37 (d, J = 5.0 Hz, 1H), 7.26 (dd, J = 8.4, 2.3 Hz, 1H), 7.23 (d, J = 3.7 Hz, 1H), 7.16 (d, J = 9.0 Hz, 2H), 7.09–6.99 (m, 2H), 6.80 (d, J = 8.3 Hz, 1H), 5.25 (s, 2H), 3.88 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.5, 162.3, 152.4, 146.3, 144.4, 141.4, 138.8, 137.2, 128.8, 128.2, 124.8, 123.7, 123.5, 123.3, 123.0, 123.0, 122.2, 121.9, 121.0, 116.2, 114.7, 55.7; m/z (ESI+) 455.4 (MH⁺, 45%); HRMS (ESI-) [M – H]⁻ calcd for C₂₆H₂₁N₄O₂S^– 453.1391, found 453.1406.

Methyl 3-(4-Nitrophenyl)propanoate⁵⁰ (S6)

To a suspension of 3-(4-nitrophenyl)propanoic acid (500 mg, 2.56 mmol) in MeOH (10.2 mL) was added sulfuric acid (960 μL, 17.9 mmol). The mixture was heated to 80 °C for 3 h and concentrated in vacuo. The residue was diluted with water, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methyl ester S6 (547 mg, 2.61 mmol, quant) as a light brown solid: ¹H NMR (400 MHz, DMSO-d₆) δ 8.17–8.11 (m, 2H), 7.56–7.49 (m, 2H), 3.58 (s, 3H), 2.99 (t, J = 7.5 Hz, 2H), 2.71 (t, J = 7.5 Hz, 2H). NMR data were in agreement with the reported values.⁵⁰

Methyl 3-(4-Aminophenyl)propanoate¹⁸ (S7)

A suspension of nitro S6 (547 mg, 2.61 mmol) and 10% palladium on carbon (28 mg, 0.026 mmol) in MeOH (52.3 mL) was stirred at room temperature under a hydrogen atmosphere for 5 h. The mixture was then filtered through Celite and concentrated in vacuo to give amine S7 (452 mg, 2.52 mmol, 96%) as a light orange solid: ¹H NMR (400 MHz, CDCl₃) δ 7.08–6.90 (m, 2H), 6.67–6.56 (m, 2H), 3.66 (s, 3H), 2.84 (t, J = 7.8 Hz, 2H), 2.57 (t, J = 7.8 Hz, 2H); m/z (ESI+) 180.3 (MH⁺, 100%). NMR data were in agreement with the reported values.⁵¹

Methyl (E)-3-(4-((4-Hydroxyphenyl)diazenyl)phenyl)propanoate¹⁸ (S8)

To a solution of S7 (136 mg, 0.760 mmol) in MeOH (1.5 mL) and HCl (2.1 mL, 2.1 mmol) at −5 °C was added a solution of sodium nitrite (62 mg, 0,90 mmol) in water (530 μL) dropwise, keeping the temperature below 0 °C. The mixture was then stirred at room temperature for 10 min and cooled to −5 °C. A solution of phenol (65 mg, 0.69 mmol) and potassium hydroxide (85 mg, 1.52 mmol) in MeOH (2.7 mL) was added dropwise, keeping the temperature below 0 °C. The resulting suspension was stirred at room temperature overnight, diluted with EtOAc, washed with 1 × 1 M HCl and 1× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give azobenzene S8 (210 mg, 0.74 mmol, quant.) as a dark brown solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.28 (s, 1H), 7.81–7.75 (m, 2H), 7.75–7.70 (m, 2H), 7.44–7.38 (m, 2H), 6.98–6.90 (m, 2H), 3.59 (s, 3H), 2.93 (t, J = 7.6 Hz, 2H), 2.69 (t, J = 7.4 Hz, 2H). NMR data were in agreement with the reported values.¹⁸

Methyl (E)-3-(4-((4-Methoxyphenyl)diazenyl)phenyl)propanoate (S9)

To a solution of phenol S8 (200 mg, 0.703 mmol) in acetone (10.0 mL) were added methyl iodide (350 μL, 5.63 mmol) and potassium carbonate (972 mg, 7.03 mmol). The mixture was heated to 50 °C for 1 h and concentrated under reduced pressure. The residue was dissolved in EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy S9 (176 mg, 0.590 mmol, 84%) as an orange solid: ¹H NMR (400 MHz, CDCl₃) δ 7.95–7.86 (m, 2H), 7.85–7.77 (m, 2H), 7.36–7.29 (m, 2H), 7.05–6.97 (m, 2H), 3.89 (s, 3H), 3.68 (s, 3H), 3.03 (t, J = 7.8 Hz, 2H), 2.68 (t, J = 7.8 Hz, 2H). NMR data were in agreement with the reported values.¹⁸

(E)-3-(4-((4-Methoxyphenyl)diazenyl)phenyl)propanoic Acid (S10)

Sodium hydroxide (456 mg, 1.140 mmol) was added to a solution of methyl ester S9 (170 mg, 0.570 mmol) in MeOH (1.1 mL). The mixture was heated to 50 °C. After 30 min, the mixture was concentrated, diltued with water, acidified with 1 M HCl, filtered, and dried to give acid S10 (152 mg, 0.535 mmol, 94%) as an orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ 12.20 (s, 1H), 7.91–7.83 (m, 2H), 7.77 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.5 Hz, 2H), 7.17–7.08 (m, 2H), 3.86 (s, 3H), 2.91 (t, J = 7.6 Hz, 2H), 2.60 (t, J = 7.6 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 173.7, 161.9, 150.5, 146.2, 144.2, 129.3, 124.5, 122.3, 114.6, 55.7, 34.9, 30.2; m/z (ESI+) 285.4 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₆H₁₇N₂O₃⁺ 285.1234, found 285.1206.

(E)-N-(2-Aminophenyl)-3-(4-((4-methoxyphenyl)diazenyl)phenyl)propanamide (21)

To a solution of acid S10 (50 mg, 0.18 mmol) in anhydrous DMF (880 μL) were added benzene-1,2-diamine (23 mg, 0.21 mmol), DIPEA (46 μL, 0.26 mmol), EDC (51 mg, 0.26 mmol), and HOBt (40 mg, 0.26 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by C18 column chromatography to give benzamide 21 (12 mg, 0.032 mmol, 18%) as an orange solid: mp 235.0–235.2 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.16 (s, 1H), 7.89 (d, J = 8.9 Hz, 2H), 7.80 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.2 Hz, 2H), 7.18–7.09 (m, 3H), 6.89 (td, J = 7.6, 1.6 Hz, 1H), 6.70 (dd, J = 8.1, 1.5 Hz, 1H), 6.53 (td, J = 7.5, 1.5 Hz, 1H), 3.87 (s, 3H), 3.02 (t, J = 7.6 Hz, 2H), 2.70 (t, J = 7.7 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 170.2, 161.9, 150.4, 146.2, 144.6, 142.0, 129.3, 125.9, 125.4, 124.5, 123.3, 122.3, 116.1, 115.8, 114.6, 55.7, 37.0, 30.9; m/z (ESI+) 375.4 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₂₃N₄O₂⁺ 375.1816, found 375.2101.

(E)-4-((2,6-Difluoro-4-iodophenyl)diazenyl)-3,5-difluorophenol (25a)

A solution of sodium nitrite (133 mg, 1.92 mmol) in water (5.0 mL) was added dropwise to a suspension of 2,6-difluoro-4-iodoaniline 22a (490 mg, 1.92 mmol) in water (8.0 mL) and concentrated HCl (2.0 mL) at 0 °C. The suspension was stirred at the same temperature for 20 min and then added to a solution of 3,5-difluorophenol 23a (250 mg, 1.92 mmol) and sodium hydroxide (154 mg, 3.84 mmol) in water (4.0 mL) dropwise, keeping the temperature below 0 °C and basic pH via addition of aqueous 2 M NaOH. The mixture was then stirred at the same temperature for an additional 2 h, acidified with 1 M HCl, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 30% EtOAc in hexane) to give azobenzene 25a (191 mg, 0.482 mmol, 25%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.47–7.38 (m, 2H), 6.55 (d, J = 10.7 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 160.1 (m), 157.7 (d, J = 269 Hz), 155.1 (d, J = 263 Hz), 131.9, 126.4, 122.4 (d, J = 27 Hz), 100.7 (d, J = 23 Hz), 92.8 (d, J = 10 Hz); m/z (ESI+) 397.0 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₂H₆N₂OF₄I⁺ 396.9455, found 396.9400.

(E)-1-(2,6-Difluoro-4-iodophenyl)-2-(2,6-difluoro-4-methoxyphenyl)diazene (27a)

To a solution of phenol 25a (191 mg, 0.482 mmol) in acetone (6.9 mL) were added potassium carbonate (666 mg, 4.82 mmol) and methyl iodide (240 μL, 3.86 mmol). The mixture was stirred at 50 °C for 1 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy 27a (184 mg, 0.449 mmol, 93%) as a brown solid: ¹H NMR (400 MHz, CDCl₃) δ 7.46–7.39 (m, 2H), 6.64–6.56 (m, 2H), 3.88 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.1 (t, J = 14 Hz), 157.7 (dd, J = 262, 7 Hz), 155.1 (dd, J = 265, 6 Hz), 131.9 (t, J = 10 Hz), 126.2 (t, J = 9 Hz), 122.4 (dd, J = 24, 3 Hz), 99.1 (dd, J = 24, 3 Hz), 92.8, 56.4; m/z (ESI+) 411.0 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₃H₈F₄IN₂O⁺ 410.9612, found 410.9604.

tert-Butyl (E)-3-(4-((E)-(2,6-Difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorophenyl)acrylate (28a)

To a solution of iodo 27a (180 mg, 0.439 mmol) in anhydrous DMF (1.8 mL) were added tri-o-tolylphosphine (13 mg, 0.044 mmol), triethylamine (133 mg, 1.32 mmol), tert-butyl acrylate (84 mg, 0.66 mmol), and palladium(II) acetate (4.9 mg, 0.022 mmol). The mixture was degassed with argon for 10 min and heated to 100 °C overnight. The mixture was diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 20% EtOAc in hexane) to give 28a (128 mg, 0.312 mmol, 71%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.40 (d, J = 15.9 Hz, 1H), 7.15–7.05 (m, 2H), 6.58–6.49 (m, 2H), 6.34 (d, J = 15.9 Hz, 1H), 3.81 (s, 3H), 1.47 (s, 9H); m/z (ESI+) 411.3 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₁₉F₄N₂O₃⁺ 411.1326, found 411.1328.

(E)-3-(4-((E)-(2,6-Difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorophenyl)acrylic Acid (30a)

A solution of tert-butoxy 28a (112 mg, 0.273 mmol) in DCM (1.4 mL) and TFA (1.4 mL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure to give acid 30a as a dark red solid, which was used in the following step without purification: ¹H NMR (400 MHz, DMSO-d₆) δ 7.77 (d, J = 10.5 Hz, 2H), 7.61 (d, J = 16.0 Hz, 1H), 7.08–6.99 (m, 2H), 6.80 (d, J = 16.0 Hz, 1H), 3.92 (s, 3H).

(E)-N-(2-Aminophenyl)-3-(4-((E)-(2,6-difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorophenyl)acrylamide (32a)

To a solution of acid 30a (97 mg, 0.27 mmol) in DMF (1.4 mL) were added benzene-1,2-diamine (35 mg, 0.33 mmol), DIPEA (72 μL, 0.41 mmol), EDC (79 mg, 0.41 mmol), and HOBt (63 mg, 0.41 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 50% EtOAc in hexane) to give benzamide 32a (52 mg, 0.12 mmol, 43%) as an orange solid: mp 199.3–200.3 °C. NMR data were collected after heating a DMSO-d₆ solution of the product at 80 °C for 1 h to obtain the trans form: ¹H NMR (400 MHz, DMSO-d₆) δ 9.46 (s, 1H), 7.65–7.53 (m, 3H), 7.37 (dd, J = 7.9, 1.5 Hz, 1H), 7.13–6.97 (m, 3H), 6.93 (td, J = 7.6, 1.6 Hz, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 4.97 (s, 2H), 3.91 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.4 (d, J = 14 Hz), 162.7, 156.7 (dd, J = 260, 7 Hz), 154.87 (d, J = 258 Hz), 141.6, 138.8 (d, J = 10 Hz), 136.5, 130.9 (d, J = 10 Hz), 126.6, 126.0, 125.0 (t, J = 9 Hz), 124.7, 123.2, 116.3, 116.0, 111.9 (d, J = 22 Hz), 99.7 (dd, J = 24, 3 Hz), 56.8; m/z (ESI+) 445.2 (MH⁺, 45%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₁₇F₄N₄O₂⁺ 445.1282, found 445.1270.

(E)-3,5-Dichloro-4-((2,6-dichloro-4-iodophenyl)diazenyl)phenol (25b)

A solution of sodium nitrite (34 mg, 0.49 mmol) in water (5.0 mL) was added dropwise to a suspension of 2,6-dichloro-4-iodoaniline 22b(52,53) (141 mg, 0.491 mmol) in water (8.0 mL) and concentrated HCl (520 μL) at 0 °C. The suspension was stirred at the same temperature for 20 min and then added to a solution of 3,5-dichlorophenol 23b (80 mg, 0.49 mmol) and sodium hydroxide (39 mg, 0.98 mmol) in water (4.0 mL) dropwise, keeping the temperature below 0 °C and basic pH via addition of 2 M NaOH. The mixture was then stirred at the same temperature for an additional 2 h, acidified with 1 M HCl, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give azobenzene 25b (111 mg, 0.240 mmol, 49%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 8.07 (s, 2H), 7.06 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ 160.0, 146.9, 137.8, 137.5, 129.8, 126.5, 117.0, 94.7; m/z (ESI+) 460.9 (MH⁺, 75%); HRMS (ESI+) [MH]⁻ calcd for C₁₂H₄Cl₄IN₂O^– 458.8128, found 458.8107.

(E)-1-(2,6-Dichloro-4-iodophenyl)-2-(2,6-dichloro-4-methoxyphenyl)diazene (27b)

To a solution of phenol 25b (107 mg, 0.232 mmol) in acetone (3.3 mL) were added potassium carbonate (320 mg, 2.32 mmol) and methyl iodide (120 μL, 1.85 mmol). The mixture was stirred at 50 °C for 1 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy 27b (105 mg, 0.221 mmol, 95%) as a brown solid. The crude product was taken to the following step without further purification: ¹H NMR (400 MHz, CDCl₃) δ 7.79 (s, 2H), 7.01 (s, 2H), 3.88 (s, 3H); m/z (ESI+) 475.0 (MH⁺, 90%).

tert-Butyl (E)-3-(3,5-Dichloro-4-((E)-(2,6-dichloro-4-methoxyphenyl)diazenyl)phenyl)acrylate (28b)

To a solution of iodo 27b (90 mg, 0.19 mmol) in anhydrous DMF (950 μL) were added tri-o-tolylphosphine (5.8 mg, 0.019 mmol), tert-butyl acrylate (41 μL, 0.28 mmol), triethylamine (79 μL, 0.57 mmol), and palladium(II) acetate (2.1 mg, 9.5 μmol). The mixture was degassed with argon for 10 min and then heated to 100 °C overnight. The mixture was allowed to cool to room temperature, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1% to 2% EtOAc in hexane) to give 28b (45 mg, 0.095 mmol, 50%) as a dark red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.56 (s, 2H), 7.47 (d, J = 16.0 Hz, 1H), 7.02 (s, 2H), 6.42 (d, J = 16.0 Hz, 1H), 3.88 (s, 3H), 1.54 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.5, 160.4, 148.5, 140.6, 140.0, 136.1, 130.5, 128.5, 127.8, 123.5, 115.6, 81.3, 56.2, 28.3; m/z (ESI+) 475.1 (MH⁺, 70%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₁₉Cl₄N₂O₃⁺ 475.0144, found 475.0136.

(E)-3-(3,5-Dichloro-4-((E)-(2,6-dichloro-4-methoxyphenyl)diazenyl)phenyl)acrylic Acid (30b)

A solution of tert-butyl ester 28b (58 mg, 0.12 mmol) in DCM (610 μL) and TFA (610 μL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure to give acid 30b, which was taken to the following step without purification: ¹H NMR (400 MHz, DMSO-d₆) δ 8.09 (s, 2H), 7.61 (d, J = 16.1 Hz, 1H), 7.34 (s, 2H), 6.81 (d, J = 16.0 Hz, 1H), 3.92 (s, 3H); m/z (ESI+) 418.9 (MH⁺, 45%).

(E)-N-(2-Aminophenyl)-3-(3,5-dichloro-4-((E)-(2,6-dichloro-4-methoxyphenyl)diazenyl)phenyl)acrylamide (32b)

To a solution of acid 30b (50 mg, 0.12 mmol) in anhydrous DMF (600 μL) were added benzene-1,2-diamine (16 mg, 0.14 mmol), DIPEA (31 μL, 0.18 mmol), EDC (34 mg, 0.18 mmol), and HOBt (28 mg, 0.18 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (20% to 30% EtOAc in hexane) to give benzamide 32b (42 mg, 0.082 mmol, 69%) as an orange solid: mp 234.0–234.5 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.40 (s, 1H), 7.94 (s, 2H), 7.58 (d, J = 15.7 Hz, 1H), 7.39 (dd, J = 7.9, 1.5 Hz, 1H), 7.34 (s, 2H), 7.10 (d, J = 15.8 Hz, 1H), 6.93 (ddd, J = 8.5, 7.4, 1.6 Hz, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.5, 1.5 Hz, 1H), 4.97 (br s, 2H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.8, 160.7, 146.8, 141.5, 139.3, 137.5, 135.9, 129.2, 128.4, 126.5, 126.4, 125.9, 124.6, 123.2, 116.3, 116.0, 115.9, 56.7; m/z (ESI+) 509.1 (MH⁺, 70%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₁₇Cl₄N₄O₂⁺ 509.0100, found 509.0098.

2-Chloro-6-fluoro-4-iodoaniline⁵⁴ (22c)

To a solution of 2-chloro-6-fluoroaniline (1.00 g, 6.87 mmol) in acetonitrile (13.8 mL) was added N-iodosuccinimide (1.55 g, 6.87 mmol) portionwise. The mixture was stirred at room temperature overnight, diluted with water, extracted with 3× hexane, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1% EtOAc in hexane) to give iodo 22c (540 mg, 1.99 mmol, 29%) together with 6% remaining starting material: ¹H NMR (400 MHz, CDCl₃) δ 7.30 (t, J = 1.8 Hz, 1H), 7.16 (dd, J = 9.7, 1.8 Hz, 1H), 4.04 (s, 2H).

(E)-3-Chloro-4-((2-chloro-6-fluoro-4-iodophenyl)diazenyl)-5-fluorophenol (25c)

A solution of sodium nitrite (118 mg, 1.71 mmol) in water (5.0 mL) was added dropwise to a suspension of iodo 22c (463 mg, 1.71 mmol) in water (8.0 mL) and concentrated HCl (1.8 mL) at 0 °C. The suspension was stirred at the same temperature for 30 min and then added to a solution of 3-chloro-5-fluorophenol 23c (250 mg, 1.71 mmol) and sodium hydroxide (136 mg, 3.41 mmol) in water (4.0 mL) dropwise, keeping the temperature below 0 °C and basic pH via addition of aqueous 2 M NaOH. The mixture was then stirred at the same temperature for an additional 2 h, acidified with 1 M HCl, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1% to 10% EtOAc in hexane) to give azobenzene 25c (268 mg, 0.625 mmol, 37%) as a red solid: ¹H NMR (400 MHz, methanol-d₄) δ 7.80 (t, J = 1.7 Hz, 1H), 7.65 (dd, J = 9.9, 1.7 Hz, 1H), 6.89 (dd, J = 2.6, 1.5 Hz, 1H), 6.63 (dd, J = 13.1, 2.6 Hz, 1H); ¹³C NMR (101 MHz, methanol-d₄) δ 162.9 (d, J = 14 Hz), 155.7 (d, J = 265 Hz), 153.3 (d, J = 264 Hz), 140.8 (d, J = 9 Hz), 137.6 (d, J = 6 Hz), 135.9 (d, J = 4 Hz), 133.4, 132.8 (d, J = 3 Hz), 126.4 (d, J = 23 Hz), 114.6 (d, J = 3 Hz), 104.3 (d, J = 23 Hz), 93.4 (d, J = 9 Hz); m/z (ESI+) 429.0 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₂H₆Cl₂F₂IN₂O⁺ 428.8864, found 428.8860.

(E)-1-(2-Chloro-6-fluoro-4-iodophenyl)-2-(2-chloro-6-fluoro-4-methoxyphenyl)diazene (27c)

To a solution of phenol 25c (268 mg, 0.625 mmol) in acetone (8.9 mL) were added potassium carbonate (863 mg, 6.25 mmol) and iodomethane (310 μL, 5.00 mmol). The mixture was stirred at 50 °C for 1 h, concentrated under reduced pressure, diluted with water, extracted with 2× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy 27c (238 mg, 0.537 mmol, 86%) as a brown solid: ¹H NMR (400 MHz, CDCl₃) δ 7.71 (t, J = 1.6 Hz, 1H), 7.50 (dd, J = 9.6, 1.7 Hz, 1H), 6.97–6.91 (m, 1H), 6.67 (dd, J = 12.9, 2.8 Hz, 1H), 3.88 (s, 3H); m/z (ESI+) 443.0 (MH⁺, 20%); HRMS (ESI+) [MH]⁺ calcd for C₁₃H₈Cl₂F₂IN₂O⁺ 442.9021, found 442.9034.

tert-Butyl (E)-3-(3-Chloro-4-((E)-(2-chloro-6-fluoro-4-methoxyphenyl)diazenyl)-5-fluorophenyl)acrylate (28c)

DMF (2.1 mL) degassed with argon was added to iodo 27c (238 mg, 0.537 mmol), tri-o-tolylphosphine (16 mg, 0.054 mmol), and palladium(II) acetate (6,0 mg, 0.027 mmol). Then, triethylamine (220 μL, 1.61 mmol) and tert-butyl acrylate (120 μL, 0.806 mmol) were added, and the mixture was degassed with argon for 10 min. The mixture was heated to 100 °C overnight, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1% to 5% EtOAc in hexane) to give 28c (108 mg, 0.244 mmol, 45%) as a brown oil: ¹H NMR (400 MHz, CDCl₃) δ 7.52–7.42 (m, 2H), 7.25 (dd, J = 11.2, 1.7 Hz, 1H), 6.95 (dd, J = 2.7, 1.5 Hz, 1H), 6.68 (dd, J = 12.8, 2.6 Hz, 1H), 6.41 (d, J = 15.9 Hz, 1H), 3.89 (s, 3H), 1.54 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.5, 162.0 (d, J = 13 Hz), 154.3 (d, J = 266 Hz), 153.0 (d, J = 262 Hz), 140.3 (d, J = 21 Hz), 140.2 (d, J = 2 Hz), 137.0 (d, J = 9 Hz), 136.5 (d, J = 6 Hz), 133.3 (d, J = 8 Hz), 132.5 (d, J = 4 Hz), 125.6 (d, J = 3 Hz), 123.7, 114.7 (d, J = 22 Hz), 112.2 (d, J = 3 Hz), 102.4 (d, J = 24 Hz), 81.4, 56.3, 28.3; m/z (ESI+) 443.4 (MH⁺, 25%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₁₉Cl₂F₂N₂O₃ 443.0735, found 443.0709.

(E)-N-(2-Aminophenyl)-3-(3-chloro-4-((E)-(2-chloro-6-fluoro-4-methoxyphenyl)diazenyl)-5-fluorophenyl)acrylamide (32c)

A solution of tert-butoxy 28c (108 mg, 0.244 mmol) in DCM (1.2 mL) and TFA (1.2 mL) was stirred at room temperature for 1 h and then concentrated in vacuo.

To a solution of the crude acid mentioned above in DMF (1.2 mL) were added benzene-1,2-diamine (32 mg, 0.29 mmol), DIPEA (64 μL, 0.37 mmol), EDC (70 mg, 0,37 mmol), and HOBt (56 mg, 0.37 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (30% to 35% EtOAc in hexane) to give benzamide 32c (32 mg, 0.067 mmol, 28%) as an orange solid. NMR data were collected after heating a DMSO-d₆ solution of the product at 80 °C for 1 h to obtain the trans form: ¹H NMR (400 MHz, DMSO-d₆) δ 9.43 (s, 1H), 7.84 (s, 1H), 7.71 (dd, J = 11.8, 1.7 Hz, 1H), 7.58 (d, J = 15.7 Hz, 1H), 7.37 (dd, J = 7.9, 1.5 Hz, 1H), 7.26 (dd, J = 2.7, 1.4 Hz, 1H), 7.13 (dd, J = 13.4, 2.7 Hz, 1H), 7.08 (d, J = 15.8 Hz, 1H), 6.93 (td, J = 7.6, 1.6 Hz, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.6, 1.5 Hz, 1H), 4.98 (s, 2H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.8, 162.3 (d, J = 14 Hz), 153.4 (d, J = 263 Hz), 152.0 (d, J = 259 Hz), 141.6, 138.6 (d, J = 10 Hz), 138.4 (d, J = 10 Hz), 136.2, 135.1 (d, J = 6 Hz), 132.0 (d, J = 8 Hz), 131.1, 126.6, 126.0, 125.4 (d, J = 3 Hz), 124.6, 123.2, 116.3, 116.0, 115.0 (d, J = 21 Hz), 112.6 (d, J = 3 Hz), 102.9 (d, J = 24 Hz), 56.8; m/z (ESI+) 477.2 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₁₇Cl₂F₂N₄O₂⁺ 477.0691, found 477.0769.

(E)-3-Fluoro-4-((2-fluoro-4-iodophenyl)diazenyl)phenol (25d)

A solution of sodium nitrite (62 mg, 0.89 mmol) in water (2.3 mL) was added dropwise to a suspension of 2-fluoro-4-iodoaniline (211 mg, 0.892 mmol) in water (3.8 mL) and concentrated HCl (940 μL) at 0 °C. The suspension was stirred at the same temperature for 20 min and then added to a solution of 3-fluorophenol (100 mg, 0.892 mmol) and sodium hydroxide (71 mg, 1.8 mmol) in water (1.9 mL) dropwise, keeping the temperature below 0 °C and basic pH via addition of aqueous 2 M NaOH. The mixture was then stirred at the same temperature for an additional 2 h, acidified with 1 M HCl, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give azobenzene 25d (198 mg, 0.550 mmol, 62%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.99 (s, 1H), 7.94 (dd, J = 10.2, 1.8 Hz, 1H), 7.74–7.69 (m, 1H), 7.67 (d, J = 8.9 Hz, 1H), 7.41 (t, J = 8.3 Hz, 1H), 6.81 (dd, J = 12.7, 2.5 Hz, 1H), 6.78–6.61 (m, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.6 (d, J = 12 Hz), 161.5 (d, J = 257 Hz), 158.5 (d, J = 260 Hz), 139.7 (d, J = 7 Hz), 134.2 (d, J = 4 Hz), 133.5 (d, J = 7 Hz), 126.0 (d, J = 22 Hz), 118.8, 118.7, 112.8 (d, J = 2 Hz), 103.5 (d, J = 22 Hz), 98.2 (d, J = 8 Hz); m/z (ESI+) 361.1 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₂H₈N₂OF₂I⁺ 360.9644, found 360.9619.

(E)-1-(2-Fluoro-4-iodophenyl)-2-(2-fluoro-4-methoxyphenyl)diazene (27d)

To a solution of phenol 25d (175 mg, 0.486 mmol) in acetone (6.9 mL) were added potassium carbonate (672 mg, 4.86 mmol) and methyl iodide (240 μL, 3.89 mmol). The mixture was stirred at 50 °C for 1 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy 27d (170 mg, 0.454 mmol, 94%) as a red solid, which was used in the following step without further purification.: ¹H NMR (400 MHz, DMSO-d₆) δ 7.94 (dd, J = 10.2, 1.7 Hz, 1H), 7.88–7.55 (m, 2H), 7.42 (t, J = 8.3 Hz, 1H), 7.13 (dd, J = 12.9, 2.6 Hz, 1H), 6.92 (dd, J = 9.1, 2.7 Hz, 1H), 3.88 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.3 (d, J = 11 Hz), 161.4 (d, J = 258 Hz), 158.6 (d, J = 261 Hz), 139.6 (d, J = 7 Hz), 134.3–134.2 (m), 134.2 (m), 126.1 (d, J = 22 Hz), 118.8, 118.4, 112.0 (d, J = 3 Hz), 102.3 (d, J = 23 Hz), 98.8 (d, J = 8 Hz), 56.4; m/z (ESI+) 375.1 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₃H₁₀N₂OF₂I⁺ 374.9800, found 374.9804.

tert-Butyl (E)-3-(3-Fluoro-4-((E)-(2-fluoro-4-methoxyphenyl)diazenyl)phenyl)acrylate (28d)

To a solution of iodo 27d (125 mg, 0.334 mmol) in anhydrous DMF (1.3 mL) were added tri-o-tolylphosphine (10 mg, 0.033 mmol), tert-butyl acrylate (73 μL, 0,50 mmol), triethylamine (140 μL, 1.0 mmol), and palladium(II) acetate (3.8 mg, 0.017 mmol). The mixture was degassed with argon for 10 min and then heated to 100 °C overnight. The mixture was allowed to cool to room temperature, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (2% to 5% EtOAc in hexane) to give 28d (94 mg, 0.25 mmol, 75%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.84 (dd, J = 9.4, 8.5 Hz, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.55 (d, J = 16.0 Hz, 1H), 7.38 (dd, J = 11.3, 1.8 Hz, 1H), 7.34 (dd, J = 8.4, 1.8 Hz, 1H), 6.84–6.73 (m, 2H), 6.42 (d, J = 16.0 Hz, 1H), 3.89 (s, 3H), 1.54 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.8, 164.3 (d, J = 11 Hz), 162.2 (d, J = 259 Hz), 160.1 (d, J = 258 Hz), 141.6 (d, J = 7 Hz), 141.5 (d, J = 2 Hz), 138.8 (d, J = 8 Hz), 135.6 (d, J = 7 Hz), 124.2 (d, J = 3 Hz), 122.7, 118.9 (d, J = 2 Hz), 118.4, 116.0 (d, J = 21 Hz), 111.2 (d, J = 3 Hz), 102.0 (d, J = 23 Hz), 81.1, 56.1, 28.3; m/z (ESI+) 375.2 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₂₁F₂N₂O₃⁺ 375.1515, found 375.1550.

(E)-N-(2-Aminophenyl)-3-(3-fluoro-4-((E)-(2-fluoro-4-methoxyphenyl)diazenyl)phenyl)acrylamide (32d)

A solution of tert-butoxy 28d (36 mg, 0.096 mmol) in DCM (480 μL) and TFA (480 μL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure.

To a solution of the crude acid mentioned above in anhydrous DMF (480 μL) were added benzene-1,2-diamine (12 mg, 0.11 mmol), DIPEA (25 μL, 0.14 mmol), EDC (28 mg, 0.14 mmol), and HOBt (22 mg, 0.14 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by C18 column chromatography to give benzamide 32d (13 mg, 0.032 mmol, 33%) as an orange solid: mp 196.1–200.6 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 9.45 (s, 1H), 7.74–7.64 (m, 3H), 7.57–7.51 (m, 2H), 7.30 (dd, J = 7.9, 1.5 Hz, 1H), 7.09 (dd, J = 12.9, 2.7 Hz, 1H), 6.99 (d, J = 15.8 Hz, 1H), 6.91–6.81 (m, 2H), 6.68 (dd, J = 8.0, 1.5 Hz, 1H), 6.51 (td, J = 7.5, 1.5 Hz, 1H), 4.92 (s, 2H), 3.82 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 164.3 (d, J = 11 Hz), 163.0, 161.4 (d, J = 258 Hz), 159.3 (d, J = 256.0 Hz), 141.6, 140.2 (d, J = 7 Hz), 139.8 (d, J = 8 Hz), 137.4 (d, J = 2 Hz), 134.5 (d, J = 7 Hz), 125.9, 125.4, 124.7, 123.8 (d, J = 3 Hz), 123.3, 118.4, 118.1, 116.4 (d, J = 20 Hz), 116.2, 116.0, 112.0 (d, J = 2 Hz), 102.3 (d, J = 23 Hz), 56.4; m/z (ESI+) 409.3 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₁₉F₂N₄O₂⁺ 409.1471, found 409.1445.

(E)-3-Chloro-4-((2-chloro-4-iodophenyl)diazenyl)phenol (25e)

A solution of sodium nitrite (134 mg, 1.94 mmol) in water (5.0 mL) was added dropwise to a suspension of 2-chloro-4-iodoaniline 22e (493 mg, 1.94 mmol) in water (8.0 mL) and concentrated HCl (2.0 mL) at 0 °C. The suspension was stirred at the same temperature for 20 min and then added to a solution of 3-chlorophenol 23e (250 mg, 1.94 mmol) and sodium hydroxide (156 mg, 3.89 mmol) in water (4.0 mL) dropwise, keeping the temperature below 0 °C and basic pH via addition of aqueous 2 M NaOH. The mixture was then stirred at the same temperature for an additional 2 h, acidified with 1 M HCl, extracted with 3× DCM, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give azobenzene 25e (540 mg, 1.37 mmol, 71%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.97 (s, 1H), 8.10 (d, J = 1.8 Hz, 1H), 7.85 (dd, J = 8.5, 1.8 Hz, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 6.90 (dd, J = 9.0, 2.5 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.5, 147.6, 141.1, 138.5, 137.5, 137.1, 134.6, 119.1, 119.0, 116.7, 115.8, 98.6; m/z (ESI+) 393.0 (MH⁺, 100%); HRMS (ESI+) [MH]⁻ calcd for C₁₂H₆Cl₂IN₂O^– 390.8907, found 390.8926.

(E)-1-(2-Chloro-4-iodophenyl)-2-(2-chloro-4-methoxyphenyl)diazene (27e)

To a solution of phenol 25e (466 mg, 1.19 mmol) in acetone (16.9 mL) were added potassium carbonate (1.64 g, 11.9 mmol) and methyl iodide (593 μL, 9.49 mmol). The mixture was stirred at 50 °C for 1 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give methoxy 27e (455 mg, 1.12 mmol, 94%) as a brown solid, which was used in the following step without further purification: ¹H NMR (400 MHz, CDCl₃) δ 7.92 (d, J = 1.8 Hz, 1H), 7.85 (d, J = 9.1 Hz, 1H), 7.67 (dd, J = 8.5, 1.8 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 2.7 Hz, 1H), 6.88 (dd, J = 9.1, 2.7 Hz, 1H), 3.90 (s, 3H); m/z (ESI+) 407.0 (MH⁺, 100%).

tert-Butyl (E)-3-(3-Chloro-4-((E)-(2-chloro-4-methoxyphenyl)diazenyl)phenyl)acrylate (28e)

To a solution of iodo 27e (452 mg, 1.11 mmol) in anhydrous DMF (5.6 mL) were added tri-o-tolylphosphine (34 mg, 0.11 mmol), tert-butyl acrylate (240 μL, 1.67 mmol), triethylamine (460 μL, 3.33 mmol), and palladium(II) acetate (12 mg, 0.056 mmol). The mixture was degassed with argon for 10 min and then heated to 100 °C overnight. The mixture was allowed to cool to room temperature, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (1% to 2% EtOAc in hexane) to give 28e (286 mg, 0.702 mmol, 63%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.86 (d, J = 9.1 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.68 (d, J = 1.8 Hz, 1H), 7.54 (d, J = 15.9 Hz, 1H), 7.46 (dd, J = 8.5, 1.9 Hz, 1H), 7.08 (d, J = 2.6 Hz, 1H), 6.89 (dd, J = 9.1, 2.7 Hz, 1H), 6.43 (d, J = 16.0 Hz, 1H), 3.89 (s, 3H), 1.54 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.8, 163.1, 149.4, 143.3, 141.3, 138.6, 138.1, 135.9, 130.0, 126.9, 122.7, 119.3, 118.6, 115.0, 114.3, 81.1, 56.1, 28.3; m/z (ESI+) 407.2 (MH⁺, 25%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₂₁Cl₂N₂O₃⁺ 407.0924, found 407.0925.

(E)-N-(2-Aminophenyl)-3-(3-chloro-4-((E)-(2-chloro-4-methoxyphenyl)diazenyl)phenyl)acrylamide (32e)

A solution of tert-butoxy 28e (122 mg, 0.300 mmol) in DCM (1.5 mL) and TFA (1.5 mL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure.

To a solution of the crude acid mentioned above in anhydrous DMF (1.5 mL) were added benzene-1,2-diamine (39 mg, 0.36 mmol), DIPEA (79 μL, 0.45 mmol), EDC (86 mg, 0.45 mmol), and HOBt (69 mg, 0,45 mmol). The mixture was stirred at room temperature overnight, diluted with water, filtered, and dried to give benzamide 32e (81 mg, 0.18 mmol, 61%) as a red solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.47 (s, 1H), 8.02–7.92 (m, 1H), 7.82–7.70 (m, 3H), 7.61 (d, J = 15.7 Hz, 1H), 7.37 (dd, J = 7.9, 1.6 Hz, 1H), 7.35 (d, J = 2.7 Hz, 1H), 7.11 (dd, J = 9.1, 2.7 Hz, 1H), 7.06 (d, J = 15.8 Hz, 1H), 6.93 (td, J = 7.6, 1.6 Hz, 1H), 6.76 (dd, J = 8.0, 1.5 Hz, 1H), 6.59 (td, J = 7.5, 1.4 Hz, 1H), 4.99 (br s, 2H), 3.91 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.2, 163.0, 148.0, 142.3, 141.6, 139.2, 137.4, 137.1, 134.6, 130.0, 126.8, 125.9, 125.5, 124.7, 123.3, 118.8, 118.2, 116.3, 116.0, 115.1, 115.0, 56.3; m/z (ESI+) 441.3 (MH⁺, 90%); HRMS (ESI+) [MH]⁺ calcd for C₂₂H₁₉Cl₂N₄O₂⁺ 441.0880, found 441.0881.

3,5-Difluoro-N,N-dimethylaniline (25a)

To a solution of 3,5-difluoroaniline (500 mg, 3.87 mmol) in acetonitrile (7.7 mL) were added potassium carbonate (1.34 g, 9.68 mmol) and iodomethane (1.2 mL, 19 mmol). The mixture was heated to 60 °C overnight, cooled to room temperature, diluted with water, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give dimethylaniline 25a (642 mg, 4.08 mmol, quant) as a yellow solid: ¹H NMR (400 MHz, CDCl₃) δ 6.47 (d, J = 9.1 Hz, 2H), 6.34 (t, J = 9.0 Hz, 1H), 3.00 (s, 6H); m/z (ESI+) 158.2 (MH⁺, 100%).

(E)-4-((2,6-Difluoro-4-iodophenyl)diazenyl)-3,5-difluoro-N,N-dimethylaniline (26a)

A solution of sodium nitrite (88 mg, 1.3 mmol) in sulfuric acid (880 μL, 16.5 mmol) was heated to 70 °C and then cooled to 0 °C. To this mixture was added a solution of 2,6-difluoro-4-iodoaniline 22a (325 mg, 1.27 mmol) in DMF (4.0 mL) and acetic acid (1.5 mL). After the mixture had been stirred for 2 h at 0 °C, a solution of 3,5-difluoro-N,N-dimethylaniline 24a (100 mg, 0.636 mmol) was added dropwise. The mixture was stirred for an additional 1 h at 0 °C and at room temperature for 3 days. The mixture was then diluted with DCM, washed with 2× saturated aqueous NaHCO₃ and 1× brine, dried over anhydrous Na₂SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give azobenzene 26a (108 mg, 0.255 mmol, 40%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.38 (d, J = 8.0 Hz, 2H), 6.25 (d, J = 13.5 Hz, 2H), 3.08 (s, 6H); ¹³C NMR (100 MHz, CDCl₃) δ 158.6 (dd, J = 260, 8 Hz), 155.1 (dd, J = 260, 4 Hz), 153.2 (t, J = 14 Hz), 153.6 (m), 132.4 (m), 122.2 (m), 95.2 (m), 90.7 (t, J = 10 Hz), 40.4; m/z (ESI+) 424.1 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₁₄H₁₁F₄IN₃⁺ 423.9928, found 423.9988.

tert-Butyl (E)-3-(4-((E)-(4-(Dimethylamino)-2,6-difluorophenyl)diazenyl)-3,5-difluorophenyl)acrylate (29a)

To a mixture of iodo 26a (251 mg, 0.593 mmol), tri-o-tolylphosphine (18 mg, 0.059 mmol) and palladium(II) acetate (6,7 mg, 0.030 mmol) under argon were added degassed anhydrous DMF (2.4 mL), triethylamine (250 μL, 1.78 mmol), and tert-butyl acrylate (130 μL, 0.890 mmol), and the mixture was degassed with argon for an additional 10 min and then heated to 100 °C overnight. The mixture was diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 15% EtOAc in hexane) to give 29a (155 mg, 0.366 mmol, 62%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.46 (d, J = 15.9 Hz, 1H), 7.14 (d, J = 9.4 Hz, 2H), 6.37 (d, J = 15.9 Hz, 1H), 6.25 (d, J = 13.4 Hz, 2H), 3.08 (s, 6H), 1.53 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.61, 158.6 (dd, J = 260, 8 Hz), 155.7 (dd, J = 258, 5 Hz), 153.2 (t, J = 14 Hz), 140.8 (t, J = 3 Hz), 135.9 (t, J = 10 Hz), 133.2 (t, J = 11 Hz), 123.1, 122.6 (t, J = 9 Hz), 111.8 (d, J = 24 Hz), 95.2 (dd, J = 26, 2 Hz), 81.2, 40.3, 28.3; m/z (ESI+) 424.3 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₁H₂₂F₄N₃O₂⁺ 424.1648, found 424.1685.

(E)-N-(2-Aminophenyl)-3-(4-((E)-(4-(dimethylamino)-2,6-difluorophenyl)diazenyl)-3,5-difluorophenyl)acrylamide (33a)

A solution of tert-butoxy 29a (150 mg, 0.354 mmol) in DCM (1.8 mL) and TFA (1.8 mL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure to give a dark red solid.

To a solution of the crude acid mentioned above in DMF (1.8 mL) were added benzene-1,2-diamine (45 mg, 0.42 mmol), DIPEA (92 μL, 0.52 mmol), EDC (101 mg, 0.525 mmol), and HOBt (80 mg, 0.52 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (30% to 100% EtOAc in hexane) to give benzamide 33a (127 mg, 0.278 mmol, 79%) as an orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.42 (s, 1H), 7.62–7.46 (m, 3H), 7.37 (d, J = 7.8 Hz, 1H), 7.02 (d, J = 15.7 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 6.76 (d, J = 7.9 Hz, 1H), 6.61–6.56 (m, 3H), 4.97 (s, 2H), 3.09 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.9, 157.7 (dd, J = 258, 9 Hz), 156.3–153.5 (m), 153.5, 141.6, 137.0–136.5 (m, 2 × C), 131.8 (t, J = 10 Hz), 125.9, 125.7, 124.7, 123.3, 120.9 (t, J = 9 Hz), 116.3, 116.0, 111.7 (d, J = 24 Hz), 95.2 (d, J = 25 Hz), 40.2; m/z (ESI+) 458.2 (MH⁺, 100%); HRMS (ESI+) [MH]⁺ calcd for C₂₃H₂₀F₄N₅O⁺ 458.1598, found 458.1562.

(E)-3,5-Dichloro-4-((2,6-dichloro-4-iodophenyl)diazenyl)-N,N-dimethylaniline (26b)

A solution of sodium nitrite (73 mg, 1.05 mmol) in sulfuric acid (730 μL, 13.7 mmol) was heated to 70 °C and then cooled to 0 °C. To this mixture was added a solution of 2,6-dichloro-4-iodoaniline 22b(52,53) (303 mg, 1.05 mmol) in DMF (4.0 mL) and acetic acid (1.5 mL). After the mixture had been stirred for 2 h at 0 °C, a solution of 3,5-dichloro-N,N-dimethylaniline 24b(55,56) (100 mg, 0.526 mmol) was added dropwise. The mixture was stirred for an additional 1 h at 0 °C and at room temperature over the weekend. The mixture was then diluted with DCM, washed with 2× NaHCO₃ and 1× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give a dark orange oil. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give azobenzene 26b (191 mg, 0.391 mmol, 74%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.74 (s, 2H), 6.71 (s, 2H), 3.08 (s, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 151.3, 148.6, 137.5, 135.3, 132.6, 128.0, 112.4, 90.6, 40.3; m/z (ESI+) 488.0 (MH⁺, 70%); HRMS (ESI+) [MH]⁺ calcd for C₁₄H₁₁ClIN₃⁺ 487.8746, found 487.8721.

tert-Butyl (E)-3-(3,5-Dichloro-4-((E)-(2,6-dichloro-4-(dimethylamino)phenyl)diazenyl)phenyl)acrylate (29b)

To a mixture of iodo 26b (332 mg, 0.679 mmol), tri-o-tolylphosphine (21 mg, 0.068 mmol) and palladium(II) acetate (7,6 mg, 0.034 mmol) under argon were added degassed anhydrous DMF (2.7 mL), triethylamine (280 μL, 2.04 mmol), and tert-butyl acrylate (150 μL, 1.02 mmol), and the mixture was degassed with argon for 10 min and then heated to 100 °C overnight. The mixture was diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give 29b (201 mg, 0.411 mmol, 60%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.53 (s, 2H), 7.46 (d, J = 16.0 Hz, 1H), 6.73 (s, 2H), 6.39 (d, J = 15.9 Hz, 1H), 3.08 (s, 6H), 1.54 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 165.6, 151.2, 149.2, 140.3, 135.5, 135.2, 132.6, 128.4, 127.8, 122.9, 112.6, 81.2, 40.4, 28.3; m/z (ESI+) 488.2 (MH⁺, 65%); HRMS (ESI+) [MH]⁺ calcd for C₂₁H₂₂Cl₄N₃O₂⁺ 488.0455, found 488.0456.

(E)-N-(2-Aminophenyl)-3-(3,5-dichloro-4-((E)-(2,6-dichloro-4-(dimethylamino)phenyl)diazenyl)phenyl)acrylamide (33b)

A solution of tert-butoxy 29b (120 mg, 0.245 mmol) in DCM (1.2 mL) and TFA (1.2 mL) was stirred at room temperature. After 1 h, the mixture was concentrated under reduced pressure to give a dark red solid.

To a solution of the crude acid mentioned above in DMF (1.2 mL) were added benzene-1,2-diamine (32 mg, 0.29 mmol), DIPEA (64 μL, 0.37 mmol), EDC (70 mg, 0.37 mmol), and HOBt (56 mg, 0.37 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (20% to 50% EtOAc in hexane) to give benzamide 33b (108 mg, 0.206 mmol, 84%) as a dark orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ 9.37 (s, 1H), 7.88 (s, 2H), 7.56 (d, J = 15.7 Hz, 1H), 7.39 (d, J = 7.8 Hz, 1H), 7.06 (d, J = 15.8 Hz, 1H), 6.97–6.91 (m, 1H), 6.91 (s, 2H), 6.76 (dd, J = 8.0, 1.4 Hz, 1H), 6.69–6.51 (m, 1H), 4.98 (s, 2H), 3.10 (s, 6H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.9, 151.6, 147.7, 141.5, 136.2, 136.1, 133.3, 131.7, 128.2, 126.4, 125.9, 125.6, 124.6, 123.3, 116.3, 116.0, 112.2, 39.9; m/z (ESI+) 522.0 (MH⁺, 90%); HRMS (ESI+) [MH]⁺ calcd for C₂₃H₂₀Cl₄N₅O⁺ 522.0416, found 522.0427.

4-Bromo-2,6-difluoroaniline⁵⁷ (S11)

To a solution of 2,6-difluoroaniline (2.00 g, 15,5 mmol) in acetonitrile (31.0 mL) was added N-bromosuccinimide (2.76 g, 15.5 mmol) portionwise. The mixture was stirred at room temperature overnight, diluted with water, extracted with 8× hexane, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give brominated product S11 (1.90 g, 9.13 mmol, 59%) as a light brown solid: ¹H NMR (400 MHz, CDCl₃) δ 7.02–6.97 (m, 2H), 3.65 (br s, 2H); m/z (ESI+) 208.2 (MH⁺, 88%). NMR data were in agreement with the reported values.⁵⁷

4-Amino-3,5-difluorobenzonitrile⁵⁸ (S12)

To a solution of bromo S11 (1.68 g, 8.08 mmol) in anhydrous DMF (16.2 mL) was added copper(I) cyanide (2.17 g, 24.2 mmol). The mixture was stirred at 160 °C overnight, poured onto 30% aqueous ammonia, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (2% to 15% EtOAc in hexane) to give nitrile S12 (397 mg, 2.58 mmol, 32%) as a light purple solid: ¹H NMR (400 MHz, CDCl₃) δ 7.08 (dd, J = 6.0, 2.2 Hz, 1H), 4.20 (s, 1H); m/z (ESI+) 155.2 (MH⁺, 100%). NMR data were in agreement with the reported values.⁵⁸

4-Amino-3,5-difluorobenzoic Acid⁵⁸ (34a)

A solution of nitrile S12 (397 mg, 2.58 mmol) in aqueous sodium hydroxide (13.4 mL, 13.4 mmol) was heated to 110 °C overnight and washed with hexane, and the aqueous layer acidified with 1 M HCl, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give acid 34a (444 mg, 2.56 mmol, quant) as a beige solid: ¹H NMR (400 MHz, CDCl₃) δ 7.64–7.53 (m, 2H). NMR data were in agreement with the reported values.⁵⁸

Methyl (E)-4-((2,6-Difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorobenzoate (36a)

A solution of sodium nitrite (128 mg, 1.85 mmol) in water (1.4 mL) was added dropwise to a suspension of acid 34a (385 mg, 2.22 mmol) in water (8.0 mL) and concentrated HCl (370 μL) at 0 °C. The suspension was stirred at the same temperature for 1 h, and then a solution of 3,5-difluorophenol 23a (241 mg, 1.85 mmol), sodium hydroxide (79 mg, 2.0 mmol), and potassium carbonate (410 mg, 2.97 mmol) in water (2.8 mL) was added dropwise, keeping the temperature below 0 °C. The mixture was then stirred at the same temperature for an additional 1 h, acidified with 1 M HCl, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure.

To a solution of the crude product mentioned above (581 mg, 1.85 mmol) in acetone (26.4 mL) were added potassium carbonate (2.56 g, 18.5 mmol) and methyl iodide (925 μL, 14.8 mmol). The mixture was stirred at 50 °C for 3 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (5% to 10% EtOAc in hexane) to give 36a (95 mg, 0.28 mmol, 15%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 7.74–7.66 (m, 2H), 6.65–6.56 (m, 2H), 3.96 (s, 3H), 3.89 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 164.6 (t, J = 3 Hz), 163.6 (t, J = 14 Hz), 157.9 (dd, J = 263, 7 Hz), 154.9 (dd, J = 256, 4 Hz), 135.2 (t, J = 11 Hz), 131.7 (t, J = 9 Hz), 126.2 (t, J = 9 Hz), 114.2–113.7 (m), 99.2 (dd, J = 24, 3 Hz), 56.4, 53.0; m/z (ESI+) 343.2 (MH⁺, 100%).

(E)-4-((2,6-Difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorobenzoic Acid (37a)

To a solution of methyl ester 36a (95 mg, 0.28 mmol) in THF (6.2 mL) and MeOH (3.1 mL) was added an aqueous solution of sodium hydroxide (0.8 M, 4.3 mL, 3.5 mmol). The mixture was stirred at room temperature overnight, concentrated, acidified with 1 M HCl, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give acid 37a (95 mg, 0.29 mmol, quant) as a red solid. The product was taken to the following step without further purification: ¹H NMR (400 MHz, methanol-d₄) δ 7.78–7.73 (m, 2H), 6.90–6.76 (m, 2H), 3.95 (s, 3H); m/z (ESI+) 329.2 (MH⁺, 100%).

(E)-N-(2-Aminophenyl)-4-((2,6-difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorobenzamide (38a)

To a solution of acid 37a (95 mg, 0.29 mmol) in DMF (1.4 mL) were added benzene-1,2-diamine (38 mg, 0.35 mmol), DIPEA (76 μL, 0.43 mmol), EDC (83 mg, 0.43 mmol), and HOBt (66 mg, 0.43 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EtOAc in hexane) to give benzamide 38a (50 mg, 0.12 mmol, 41%) as an orange solid. NMR data were collected after heating a DMSO-d₆ solution of the product at 80 °C for 2 h to obtain the trans form (15% of cis remained): ¹H NMR (400 MHz, DMSO-d₆) 1:1.6 mixture of isomers, major isomer reported δ 9.88 (s, 1H), 7.92 (d, J = 9.8 Hz, 2H), 7.16 (dd, J = 7.8, 1.5 Hz, 1H), 7.11–7.03 (m, 2H), 7.02–6.97 (m, 1H), 6.78 (dd, J = 8.0, 1.4 Hz, 1H), 6.60 (td, J = 7.5, 1.4 Hz, 1H), 5.03 (s, 2H), 3.92 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.7, 162.3, 156.9 (dd, J = 260, 7 Hz), 154.0 (dd, J = 257, 4 Hz), 143.6, 137.1 (t, J = 8 Hz), 132.6 (t, J = 6 Hz), 127.1, 127.1, 125.1–124.7 (m), 122.1, 116.0, 115.9, 112.6 (d, J = 23 Hz), 99.8 (dd, J = 24, 3 Hz), 56.9; m/z (ESI+) 419.2 (MH⁺, 55%); HRMS (ESI+) [MH]⁺ calcd for C₂₀H₁₅F₄N₄O₂⁺ 419.1126, found 419.1127.

Methyl (E)-3,5-Dichloro-4-((2,6-dichloro-4-methoxyphenyl)diazenyl)benzoate (36b)

A solution of sodium nitrite (106 mg, 1.53 mmol) in water (1.4 mL) was added dropwise to a suspension of 4-amino-3,5-dichlorobenzoic acid 34b (537 mg, 2.61 mmol) in water (8 mL) and concentrated HCl (300 μL) at 0 °C. The suspension was stirred at the same temperature for 1 h, and then a solution of 3,5-dichlorophenol 23b (250 mg, 1.53 mmol), sodium hydroxide (66 mg, 1.6 mmol), and potassium carbonate (339 mg, 2.45 mmol) in water (2.8 mL) was added dropwise, keeping the temperature below 0 °C. The mixture was then stirred at the same temperature for an additional 1 h, acidified with 1 M HCl, extracted with 3× EtOAc, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% to 70% EtOAc in hexane) to give a 1:4 mixture of the product and starting material.

To a solution of the mixture mentioned above (76 mg) in acetone (2.9 mL) were added potassium carbonate (276 mg, 2.00 mmol) and methyl iodide (100 μL, 1.60 mmol). The mixture was stirred at 50 °C for 3 h, concentrated under reduced pressure, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EtOAc in hexane) to give 36b (77 mg, 0.19 mmol, 94%) as a red solid: ¹H NMR (400 MHz, CDCl₃) δ 8.09 (s, 2H), 7.03 (s, 2H), 3.97 (s, 3H), 3.89 (s, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 164.6, 160.7, 151.6, 140.3, 130.9, 130.7, 130.4, 126.9, 115.7, 56.3, 53.0; m/z (ESI+) 406.9 (MH⁺, 45%).

(E)-3,5-Dichloro-4-((2,6-dichloro-4-methoxyphenyl)diazenyl)benzoic Acid (37b)

To a solution of methyl ester 36b (77 mg, 0.19 mmol) in THF (4.2 mL) and MeOH (2.1 mL) was added an aqueous solution of sodium hydroxide (2.9 mL, 2.4 mmol). The mixture was stirred at room temperature overnight, concentrated, acidified with 1 M HCl, and filtered to give acid 37b (30 mg, 0.076 mmol, 40%) as a red solid: ¹H NMR (400 MHz, methanol-d₄) δ 8.10 (s, 2H), 7.20 (s, 2H), 3.94 (s, 3H); m/z (ESI+) 393.0 (MH⁺, 70%).

(E)-N-(2-Aminophenyl)-3,5-dichloro-4-((2,6-dichloro-4-methoxyphenyl)diazenyl)benzamide (38b)

To a solution of acid 37b (95 mg, 0.24 mmol) in DMF (1.2 mL) were added benzene-1,2-diamine (31 mg, 0.29 mmol), DIPEA (63 μL, 0.36 mmol), EDC (69 mg, 0.36 mmol), and HOBt (55 mg, 0.36 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 3× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (EtOAc in hexane) to give benzamide 38b (44 mg, 0.091 mmol, 38%) as an orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.05 (s, 1H), 8.25 (s, 2H), 7.36 (s, 2H), 7.22 (d, J = 7.6 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 6.89 (d, J = 7.9 Hz, 1H), 6.73 (t, J = 7.6 Hz, 1H), 3.93 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 162.4, 160.9, 149.0, 141.3, 139.1, 135.9, 129.5, 129.0, 127.2, 127.1, 125.4, 123.5, 117.9, 117.1, 116.0, 56.7; m/z (ESI+) 483.1 (MH+, 70%); HRMS (m/z) [MH]⁺ calcd for C₂₀H₁₅Cl₄N₄O₂⁺ 482.9944, found 482.9887.

(E)-3-(4-((E)-(2,6-Difluoro-4-methoxyphenyl)diazenyl)-3,5-difluorophenyl)-N-hydroxyacrylamide (39)

To a solution of acid 30a (50 mg, 0.11 mmol) in DMF (550 μL) were added O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (15 mg, 0.13 mmol), EDC (32 mg, 0.16 mmol), HOBt (25 mg, 0.16 mmol), and DIPEA (30 μL, 0.16 mmol). The mixture was stirred at room temperature overnight, diluted with EtOAc, washed with 2× brine, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure to give an orange solid.

A solution of the crude protected hydroxamide mentioned above (50 mg, 0.11 mmol) in DCM (740 μL) and TFA (370 μL) was stirred at room temperature for 2 h and then concentrated under reduced pressure. The crude product was purified by C18 column chromatography to give hydroxamide 39 (10 mg, 0.027 mmol, 25%) as an orange solid: ¹H NMR (400 MHz, DMSO-d₆) δ 10.90 (s, 1H), 9.18 (s, 1H), 7.57 (d, J = 10.9 Hz, 2H), 7.49 (d, J = 16.2 Hz, 1H), 7.02 (d, J = 11.9 Hz, 2H), 6.65 (d, J = 15.6 Hz, 1H), 3.91 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 163.5–163.1 (m), 161.9, 156.7 (dd, J = 260, 7 Hz), 154.8 (dd, J = 258, 6 Hz), 138.8, 135.5, 130.8, 125.1 (d, J = 9 Hz), 123.4, 111.8 (d, J = 21 Hz), 99.7 (d, J = 23 Hz), 56.8; m/z (ESI+) 370.2 (MH⁺, 100%); HRMS (ESI-) [M – H]⁻ calcd for C₁₆H₁₀F₄N₃O₃^– 368.0664, found 368.0641.

Photochemistry

Ultraviolet–visible (UV–vis) spectra were recorded using a Tecan Spark 20M Multimode Microplate reader. Samples (200 μL of compound solution/well) were prepared at 25–100 μM in DMSO. Samples were measured between 300 and 800 nm with 2 nm fixed intervals in 96-well transparent plates. Illumination at different wavelengths was performed for 2 min using 96-well LED array plates (LEDA Teleopto) placed below the samples. Illumination was done at the highest potency for each plate, which corresponded to 7.0 mW/cm² for 365 nm, 11 mW/cm² for 380 nm, 19 mW/cm² for 405 nm, 13 mW/cm² for 420 nm, 14 mW/cm² for 455 nm, 14 mW/cm² for 470 nm, 19 mW/cm² for 500 nm, and 13 mW/cm² for 550 nm. Potencies were measured using a Thorlabs PM100D power energy meter connected with a standard photodiode power sensor (S120VC).

The E/Z composition under different temperature and light conditions was determined by HPLC and quantified by integration at 254 nm.

The spectrum of the pure Z isomer was estimated by subtracting the spectrum of the pure E isomer from the spectrum of the PSS upon illumination at a wavelength at which the proportion of each isomer had been previously determined by HPLC, using the equation

where A_λ is the absorbance in the PSS, A_E is the absorbance in the dark, and %E is the fraction of the E isomer at the PSS.

In the cases in which there was a mixture of E and Z isomers in the dark, the spectrum of each isomer was determined by measuring the absorbance of the PSS upon illumination at two different wavelengths where the proportion of each isomer had been previously determined by HPLC, using the equation

To determine the thermal relaxation rates of the cis isomers at room temperature, a 25–100 μM solution of the compounds in DMSO was illuminated for 2 min, and then the absorbance was measured at the wavelength with the largest difference in absorbance between both isomers, at fixed intervals in the dark. Thermal half-lives were calculated by plotting the absorbance readings versus time and fitting the obtained curve to an exponential decay function, with GraphPad Prism 6. For the rates at 37 °C, the 10 mM stock in DMSO was illuminated for 2 min and then diluted to a 10 μM solution in DMEM with 0.1% DMSO. The solution was kept in an incubator at 37 °C, and samples were taken at fixed intervals and analyzed by HPLC.

HDAC1 Assays

Assay Reagents

Recombinant HDAC1 (BPS Bioscience, snap frozen in 2 μg aliquots upon receipt, diluted to 200 ng/mL with HDAC buffer), HDAC buffer (25 mM Tris-HCl, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, set to pH 8 with concentrated HCl; BSA at 0.1 mg/mL added before the assay), substrate (Boc-Lys(Ac)-AMC, Enzo, 10 mM stock, diluted to 30 μM with HDAC buffer), stop solution (6 mg/mL trypsin and 300 μM SAHA (from 40 mM DMSO stock) in HDAC buffer).

Stock solutions of the compounds were diluted to 1 mM in DMSO and then heated to 60 or 80 °C to obtain solutions with the maximum amount of the trans isomer (dark). For the “light” solutions, the diluted solutions were illuminated with 96-well LED array plates for 3 min at the maximum potency. The 1 mM solutions were then serially diluted with HDAC buffer to 10 times the desired concentration, maintaining DMSO at 10%.

To initiate the enzyme reaction, reagents were added to 96-well low-binding black flat plates with a clear bottom in this order: compounds (10 μL, 10 μM to 3 nM), enzyme (40 μL, 8 ng/well), and substrate (50 μL, 15 μM). In control wells without the compound, inhibitor, or enzyme, HDAC buffer was added instead. Plates were kept at room temperature for 1 h either in the dark or under illumination with 96-well LED array plates at preoptimized potencies (2 mW/cm² for 365 and 380 nm, 7 mW/cm² for 420 nm, and 6 mW/cm² for 550 nm). Then, 50 μL of the stop solution was added and the plates were incubated at 37 °C for 20 min in the dark. Fluorescence was measured at a λ_em of 460 nm and a λ_ex of 380 nm. Enzyme activity is expressed as a percentage with respect to the nontreated cells, after subtracting the blank (well with no enzyme and no compound).

Cell Assays

HeLa, MCF7, and HT29 cells were maintained in adherent cultures in DMEM supplemented with 10% FBS. KG1 cells were maintained in a suspension culture in IMDM supplemented with 20% FBS.

Stock solutions of the compounds were heated to 60 or 80 °C to obtain solutions with the maximum amount of the trans isomer (dark). For the “light” solutions, the stock solutions were illuminated with 96-well LED array plates for 3 min at the maximum potency. The solutions were then serially diluted with DMEM to give 20 times the desired final concentration, maintaining DMSO at 10%.

Whole-Cell Inhibition Assay

See the HDAC1 Assays for details about the reagents, buffers, and assay solutions.

HeLa cells were seeded in transparent flat bottom plates at a density of 15 × 10³ cells/well in a volume of 45 μL. After 24 h, 2.5 μL of the compound solution and 2.5 μL of the HDAC substrate solution in DMEM were added to final concentrations of 50 and 100 μM, respectively. The plates were incubated at 37 °C for 3 h, and 50 μL of the stop solution was added. The plates were incubated for an additional 1 h at 37 °C. The fluorescence was measured at a λ_em of 460 nm and a λ_ex of 380 nm. Enzyme activity is expressed as a percentage with respect to the nontreated cells, after subtracting the blank (nontreated cells with no substrate).

Cell Viability Assay

Cells were seeded in 96-well black flat bottom plates with a clear bottom at a density of 5 × 10³ cells/well (HeLa) or 1 × 10⁴ cells/well (MCF7, HT29, and KG1), in a volume of 95 μL. After 24 h, 5 μL of compound solutions (20×) were added. Cells were incubated for 48 h.

Viability of HeLa, MCF7, and HT20 cells was determined with a CellTiter 96 assay (Promega), according to the manufacturer’s instructions. Briefly, 20 μL of CellTiter 96 reagent was added to each well, the plates were incubated at 37 °C for 2 h, and the absorbance at 490 nm was recorded. Viability is expressed as a percentage with respect to the nontreated cells.

Viability of KG1 cells was determined with a CellTiter-Glo (Promega) assay, according to the manufacturer’s instructions. Briefly, 100 μL of CellTiter-Glo reagent was added to each well, the plates were kept at room temperature for 10 min, and the luminescence was recorded. Viability is expressed as a percentage with respect to the nontreated cells.

Glossary

Abbreviations

HDAC

histone deacetylase

HA

hydroxamic acid

LED

light-emitting diode

OAA

o-aminoanilide

PSS

photostationary state

SAR

structure–activity relationship

THP

tetrahydropyran

TLC

thin layer chromatography

ZBD

zinc binding domain

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c01713.

• ¹H and ¹³C NMR spectra, HPLC traces, and full photochemical characterization of the final compounds (PDF)

• Molecular formula strings (CSV)

The authors declare no competing financial interest.

Special Issue

Published as part of the Journal of Medicinal Chemistry virtual special issue “New Drug Modalities in Medicinal Chemistry, Pharmacology, and Translational Science”.