Histone Deacetylase Inhibitors through Click Chemistry

Abstract

Histone deacetylase inhibitors (HDACi) are a relatively new class of chemotherapy agents. Herein, we report a click-chemistry based approach to the synthesis of HDACi. Fourteen agents were synthesized from the combination of two alkyne and seven azido precursors. The inhibition of HDAC1 and HDAC8 was then determined by in vitro enzymatic assays, after which the cytotoxicity was evaluated in the NCI human cancer cell line screen. A lead compound 5g (NSC746457) was discovered that inhibited HDAC1 at an IC₅₀ value of 104 ± 30 nM and proved quite potent in the cancer cell line screen with GI₅₀ values ranging from 3.92 μM to 10 nM. Thus, this click HDACi design has provided a new chemical scaffold that has not only revealed a lead compound, but one which is easily amendable to further structural modifications given the modular nature of this approach.

Introduction

Posttranscriptional modifications of histones are important processes in epigenetics.^1–3 The reversible acetylation of Lys residues,^4–6 for example, can help control the remodeling status of chromatin via charge-charge interactions between negatively charged DNA and neutral or positively charged histones. The removal and addition of these acetyl groups is catalyzed by the enzymes histone deacetylase (HDAC) and histone acetyltransferase (HAT), respectively. In the past ten years, studies on this process have attracted an increasing amount of attention, due in part to the observations that aberrant hypoacetylation of histones frequently occurs in tumor cells, resulting in the silencing of specific (tumor suppressor) genes.⁷ Accordingly, clinical and biological studies have shown that inhibition of HDACs can selectively inhibit cancer cell growth.⁸ It is has also been revealed that HDACs have a panel of non-histone targets, many of which are involved in tumorigenesis.^9–17 All of these features thus establish HDAC as an attractive target in cancer chemotherapy.^18–21

Thus far, eighteen human HDAC subtypes have been identified and accordingly divided into four classes based upon their homology to yeast HDACs.²⁰ Class I (yeast transcriptional regulator RPD3) and II (yeast Hda 1) HDACs require a zinc ion to mediate deacetylation and are therefore mechanistically different from the Class III (SIR2 yeast family) NAD⁺ dependent HDACs. While HDAC 11 possesses similar catalytic activity to Class I and II HDACs in terms of requiring a zinc ion, it actually serves as the sole member of Class IV due to low overall sequence similarity with Classes I and II.

In the case of the aforementioned Zn(II) dependant HDAC isozymes, the active site structure was first revealed by a homolog of the hyperthermophilic bacterium Aquifex aeolicus in 1999.²² In general, the active site is a tube-like pocket that accommodates the side chain of client Lys residues. Located at the bottom of this pocket is the Zn(II) cofactor, which acts as a Lewis acid to catalyze hydrolysis of the acetylated Lys side chain. The opening region (also called the cap region) of the active site consists of multiple loops and is thus highly malleable.

From this elucidation of the structure of the HDAC active site, progress on HDACi design has been greatly facilitated (Figure 1). In most cases, the reported inhibitor is composed of the following three moieties: a Zn(II) binding moiety, a spacer moiety and a “cap” moiety. Of the demonstrated Zn(II) binding moieties, the hydroxamic acid (HA) functionality is the most common. For example, the HA moiety exists in the naturally occurring compound TSA as well as in the clinically approved drug SAHA. In spite of the strong affinity of HA with Zn(II), however, the in vivo efficacy of HA-containing HDACi is impaired by the short clearance time of these agents.^23–25 Therefore, non-HA HDACi design has been explored.^26,27 These non-HA moieties include electrophilic ketones (e.g. 1 in Figure 1), O-aminoanilides (e.g. MS-275), thiols (or the pro-drug forms) (e.g. FK-228 and 2), and a variety of others. However, in terms of binding affinity, none are as strong as HA. As for the spacer moiety, proper length is the key factor in successful HDAC inhibition. For example, for SAHA and its analogs, six CH₂ units are preferred, whereas chiral-center-&-HA-containing HDACi (e.g. CHAPs and 3) prefer five CH₂ units between the HA and the chiral center.^28–30 Similarly, chiral-center-&-thiol-containing HDACi (e.g. 2) prefer five CH₂ units in the spacer moiety. In addition to these length requirements, introduction of an unsaturated moiety within the spacer region has also proved to be a valuable feature in enhancing potency (e.g. TSA, MS275, (S)-HDAC-42 and 4). Such value appears to arise from π-π stacking interactions between the unsaturated moiety and two conserved Phe residues that occur near the midpoint of the tube-like pocket (for HDAC8, they are Phe152 and Phe208) in the eleven Zn(II) dependant human HDACs.³¹ In regards to the “cap” moiety, a hydrophobic substituent, especially an aromatic substituent, is frequently incorporated. It should be noted that structure derivatization at the “cap” region is a key strategy in current HDACi design, since topological differences are observed in the corresponding “cap” regions of HDAC isozymes.

Figure 1.

Current HDACi.

Herein, we report a HDACi scaffold that is built upon a Huisgen 1,3-dipolar cycloaddition of alkyne and azido precursors (Figure 2). This reaction has been widely applied in chemical synthesis in recent years in efforts to develop new drugs.^32–35 It is now recognized as the premier example of the click reaction due to the convenient availability and high stability of the alkyne and azide precursors, as well as the mild reaction conditions and nearly quantitative yields. In our design, the precursors corresponding to the “cap” moiety of the HDACi contain an azido group, whereas the zinc chelating functionality precursors contain an alkyne group. The “clicked” products have a triazole ring in the spacer moiety, which may enhance the HDAC binding affinity via π-π stacking interactions. We have accordingly named these HDACi as click HDACi. Currently, fourteen click HDACi (5a–f and 6a–f) have been prepared from two alkyne precursors (7 and 8) and seven azido-substituted precursors (9a–g) via this combinatorial approach. From these fourteen HDACi, a lead compound, 5g, was discovered which exhibited an in vitro potency comparable to that of SAHA which was clinically approved in 2007 as a chemotherapy agent.

Figure 2.

Combinatorial Design of HDACi viaClick Chemistry.

Results and Discussion

The seven azido precursors 9a–g (Scheme 1) were synthesized from the corresponding halide substrates 10a-g via treatment with 0.5 M sodium azide in DMSO (Scheme 1).³⁶ All products, with the exceptions of 9c and 9d, were obtained after overnight stirring. In the case of 9e, however, the reaction had to be carried out at 0°C to provide satisfactory yields. For 9c and 9d, tetraethylammonium bromide was added to catalyze the reactions, which were subsequently complete after two days. Excluding 9e, all products could be used directly in the following steps without column purification.

Scheme 1.

Preparation of the Azido Precursors 9a–g.

The preparation of the alkyne precursor 8 began with the synthesis of the PMB-protected hydroxylamine 13 via established procedures (Scheme 2).³⁷ Subsequent amidation of 13 with the commercially available acid 14 provided intermediate 8 in 61% yield.

Scheme 2.

Preparation of Alkyne Precursor 8.

In contrast, synthesis of the alkyne precursor 7 (Scheme 3) was initiated through conversion of the propiolic acid 15 to the trans-iodopropenoic acid 16 in 75% yield according to reported procedures.³⁸ The synthesis of 18 from 16 utilized a known route, which was employed to prepare the corresponding (Z)-isomer.³⁹ This route entailed initial protection of the acid 16 with MEMCl to furnish the ester 17 (86% yield). 18 was subsequently obtained in 87% yield via a Sonogashira reaction. Deprotection of 18 with HCl to afford 19 (quantitative) followed by treatment with oxalyl chloride and a catalytic amount of DMF yielded an acid chloride intermediate. This acid chloride was used directly in the next step in which DIPEA & 13 (2:1) were added within 1 min at −10 °C to give 7 in 84% yield. Such rapid addition of the amine 13 was necessary as slow addition dramatically increased the yield of the by-product 20.

Scheme 3.

Preparation of Alkyne Precursor 7.

Following preparation of these two libraries, a series of click reactions between alkyne precursors 7 and 8 and the seven azido precursors 9a–g were performed to afford intermediates 21a–g and 22a–g (Scheme 4). In this combinatorial chemistry step, the alkyne precursors were first desilylated with CsF (1 equiv.). Following workup, without purification, the crude desilylation intermediates were treated with 9a–g in the presence of a Cu(I) catalyst to give 21a–g and 22a–g. The purified products were then treated with TFA to yield the click HDACi 5a–g and 6a–g. These final target compounds were purified by C-18 reverse phase column chromatography due to the presence of an unidentified reddish impurity which appeared upon addition to normal phase silica gel.

Scheme 4.

Combinatorial Syntheses of Click HDACi.

In order to assess the biological activity of these fourteen compounds, they were first tested for inhibition of HDAC. Specifically, HDAC1 and HDAC8 were chosen for the preliminary screen. Selection of these two isozymes stemmed from the previously reported correlation between HDAC1 inhibition and cancer cell growth inhibition,^40–44 as well as the existence of preferences among several HDAC inhibitors for HDAC1 or HDAC8.^29,31,45,46 Accordingly, preliminary screening at a concentration of 0.50 μM (Table 1) was conducted. This screening revealed that one of the compounds, 5g, possessed an inhibitory potency towards HDAC1 that was quite similar to that of SAHA. In addition, several other inhibitors (5a-5f, 6a, 6b, 6g) possessed activity against HDAC1, albeit to a lesser extent than either SAHA or 5g. Of particular importance in this regard is 6g, given that it showed only 10 ± 1% inhibition at the concentration tested. This is notable due to the fact that it and 5g, which demonstrated 75 ± 5% inhibition, only differ in the length of the linker region. This suggests that a longer linker length considerably aids HDAC1 inhibition in this case.

Table 1.

Activity of all HDACi in the human HDAC1 and HDAC8 inhibition tests

	SAHA	5a	5b	5c	5d	5e	5f	5g	6a	6b	6c	6d	6e	6f	6g
HDAC 1	72 ± 2	31 ± 4	10 ± 3	8 ± 2	13 ± 4	8 ± 2	15 ± 3	75 ± 5	18 ± 3	9 ± 1	NS	NS	NS	NS	10 ± 1
HDAC 8	NS	12 ± 6	NS	NS	NS	NS	NS	NS	12 ± 2	12 ± 2	NS	26 ± 3	NS	NS	13 ± 2

The values given represent the percentage by which the HDAC activity was inhibited by a 0.50 μM HDACi treatment.

All of the assays were repeated at least three times.

When the inhibition capacity was less than 5%, NS (meaning no significant inhibition) was assigned.

While the aforementioned ten compounds showed inhibition against HDAC1, only five of the fourteen (5a, 6a, 6b, 6d, 6g) demonstrated any significant inhibition of HDAC8. Of these five, four also possessed activity against HDAC1 with 6d being the exception. Nonetheless, none of the inhibitors demonstrated as high of a degree of inhibition of HDAC8 as that seen with the inhibition of HDAC1 by SAHA and 5g.

Before better characterizing the activity of these compounds against HDAC, a list of the fourteen inhibitors was submitted to the National Cancer Institute (NCI) for acceptance into the human cancer cell line screening program which is provided at no cost. Of the fourteen, only one, 5g, ultimately advanced to the final five-dose screen. This selection for advancement stemmed from the results of the single-dose preliminary screen in which the percent inhibition of cell line growth was determined through comparison to untreated samples as described in detail in the Supplementary Information. Furthermore, in the five-dose screen (Figure 4), in which data for SAHA is included to serve as a standard, 5g proved to be active against each of the cancer cell lines in the screen, albeit to varying extents. The differences among the cell lines, however, were generally within or close to one order of magnitude. For example, the GI₅₀ values obtained against the various leukemia cell lines all fell within the range of 0.249 – 2.99 μM. This same trend appeared among the other cancer types for which the ranges were as follows: non-small cell lung 0.199 – 2.26 μM, colon 0.347 – 2.07 μM, CNS 0.318 – 1.53 μM, melanoma 0.271 – 1.08 μM, ovarian 0.187 – 3.21 μM, renal 0.0100 – 2.38 μM, prostate 0.580 – 1.30 μM, and breast 0.180 – 3.92 μM. As can be seen, the lower end of the range for the renal cancer cell lines is noticeably lower than those of the other cancer types. Specifically, the RXF 393 cell line possessed a GI₅₀ < 10 nM. This represents the lowest GI₅₀ obtained across the 60 cell lines; however, this low value does not appear to be specific to this type of cancer, but rather cell line specific as the next most sensitive renal cell line (TK-10) possesses a GI₅₀ value of 495 nM.

Figure 4.

Comparison of GI₅₀ values from the NCI human cancer cell line screen for 5g and SAHA. Tested doses included 1×10⁻⁴, 1×10⁻⁵, 1×10⁻⁶, 1×10⁻⁷ and 1×10⁻⁸ M. a) Activity against leukemia and non-small cell lung cancer cell lines; b) Activity against colon, central nervous system (CNS) and prostate cancer cell lines; c) Activity against melanoma and ovarian cancer cell lines; d) Activity against renal and breast cancer cell lines

Given the results of the NCI screen and the fact that 5g did not show any significant inhibition of HDAC8 at the concentration used in the preliminary screen, a more detailed analysis of HDAC1 inhibition was performed. It was found that 5g possesses an IC₅₀ value against HDAC1 (104 ± 30 nM) that is comparable to that found with SAHA (140 ± 65 nM). Thus, taken together, the results of the enzyme and cell-based assays provide support to the proposed correlation between the cytotoxicity of HDAC inhibitors and the ability of these agents to inhibit HDAC1. This notion is further supported by the NCI’s preliminary cell inhibition studies on the six candidates which were originally selected (Supplementary Information). Those compounds which demonstrated a high degree of HDAC1 inhibition (SAHA and 5g) also exhibited high levels of potency in the cell-based assay. In contrast, the weaker inhibitors of HDAC1 also generally exhibited reduced potency against cancer cells.

Conclusion

This paper highlights the utility of click chemistry in the synthesis of HDACi. Through this approach, a combinatorial strategy was employed which enabled generation of multiple HDACi. Such combinatorial approaches greatly enhance the efficiency of drug discovery as libraries of candidate molecules can be obtained rapidly. This fact was highlighted by the discovery of lead compound 5g, which was found to inhibit HDAC1 with an IC₅₀ = 104 ± 30 nM while demonstrating no significant inhibition of HDAC8 in preliminary testing. This level of inhibition was found to be comparable to that exhibited by the clinically approved drug SAHA (140 ± 65 nM). Furthermore, 5g was also found to be active across all cell lines in the NCI human cancer cell line screen with GI₅₀ values ranging from 3.92 μM to 10 nM.

In summary, we have developed a click chemistry approach to HDACi design which has not only led to a potent lead compound, but more importantly, one which is readily amendable to further derivatization. For example, functionalization of the aromatic ring, replacement of the aromatic ring with a heteroaromatic species, or introduction of a chiral center in the “cap” moiety can all occur quite readily by incorporating these features into the azide-containing fragment. This will allow modifications to occur prior to the combinatorial step, thus maintaining the modular nature of this approach. A large number of new candidate molecules can therefore be rapidly prepared via modification of the azide library.

Experimental Section

All solvents were dried with a solvent-purification system from Innovative Technology, Inc. All reagents were obtained from commercial sources and used without further purification. Analytical TLC was carried out on silica gel 60 F254 aluminum-backed plates (E. Merck). The 230–400 mesh size of the same absorbent was utilized for all chromatographic purifications. The 200–400 mesh silica gel 60 RP-18 (from EMD^™) was utilized to purify the target click HDACi. ¹H and ¹³C NMR spectra were recorded at the indicated field strengths. The high-resolution mass spectra were collected at The Ohio State University Campus Chemical Instrumentation Center. The purities of click HDACi were analyzed by HPLC (refer to the supporting document). The purity of the key target compound 5g is 98.3%, with the purities of the other thirteen target compounds all being higher than 96%. As for the HDAC1 and HDAC8 inhibition studies, the assay kits were obtained from Biomol International LP. All assays were repeated at least three times. Briefly, the assay was performed in two stages. In the first stage, HDAC8 solution (15 μL, 1 U totally), HDACi solution (10 μL) and HDAC8 substrate solution (25 μL) were added to the 96-well microplate. The reaction proceeded at 30 °C for 45 min. The second stage was initiated by the addition of 50 μL of Developer which stopped HDAC activity and produced the fluorescent signal (λ_ex = 350 nm; λ_em = 450 nm). The cell line assays for the selected six target compounds, 5b (NSC746454), 5e (NSC746455), 5f (NSC746456), 5g (NSC746457), 6e (NSC746458) and 6g (NSC746459), were performed by the NIH.⁴⁷ Cell lines tested consisted of the following: CCRF-CEM, HL-60(TB), K-562, MOLT-4, RPMI-8226 and SR (Leukemia); A549/ATCC, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M and NCI-H460 (Non-Small Cell Lung Cancer); COLO 205, HCT-116, HCT-15, HT29, KM12 and SW-620 (Colon Cancer); SF-628, SF-295, SF-539, SNB-19, SNB-75 and U251 (CNS Cancer); LOX IMVI, MALME-3M, M14, SK-MEL-28, SK-MEL-5, UACC-257 and UACC-62 (Melanoma); IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8 and SK-OV-3 (Ovarian Cancer); 786-0, A498, ACHN, CAKI-1, RXF 393, SN12C, TK-10 and UO-31 (Renal Cancer); PC-3 and DU-145 (Prostate Cancer); MCF7, NCI/ADR-RES, MDA-MB-231/ATCC, HS578T, MDA-MB-435, BT-549 and T-47D (Breast Cancer). Full experimental details are available free of charge via the Internet at http://dtp.nci.nih.gov/branches/btb/ivclsp.html.^{48, 49}

General procedure for the preparation of Click HDACi 5a-g and 6a-g from 21a-g and 22a-g

Triisopropylsilane (3 mmol, 0.6 mL, 3 equiv), PMB protected substrate (21a–g or 22a–g, 1 mmol, 1.0 equiv) and TFA (2 mL) were added in sequence to a reaction vessel equipped containing DCM (40 mL). The mixture was stirred at room temperature and monitored by TLC. Upon completion, the reaction mixture was diluted with acetonitrile (100 mL) and then neutralized with DOWEX^® MARATHON^® WBA anion exchange resin (from Aldrich). The resin was washed additionally with acetonitrile (50 mL × 2). The combined organic solution was evaporated to provide a solid, which was purified to afford the final product.

(E)-3-(1-benzyl-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (5a)

The crude product was first dissolved with a minimal amount of 5% methanol in DCM, after which the pure compound was precipitated with hexanes (4× the original volume) as a slightly pink solid (28 mg 5a from 100 mg 21a, 42% yield). ¹H NMR (500 MHz, CD₃OD):δ 8.15 (s, 1H), 7.50 (d, J = 15.8 Hz, 1H), 7.42-7.28 (m, 5H), 6.62 (d, J = 15.8 Hz, 1H), 5.60 (s, 2H); ¹³C NMR (125 MHz, CD₃OD):δ 165.8, 145.2, 136.7, 130.2, 129.8, 129.3, 125.7, 120.4, 55.1 (one peak less due to overlap ~130); HRMS (ESI) calcd for C₁₂H₁₂N₄O₂Na [M+Na]⁺ 267.0858, found 267.0860.

(E)-N-hydroxy-3-(1-phenethyl-1H-1,2,3-triazol-4-yl)acrylamide (5b)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 5: 1) to provide 5b as a white solid (65 mg 5b from 174 mg 21b, 55% yield). ¹H NMR (500 MHz, DMF-d₇):δ 8.32 (s, 1H), 7.48 (d, J = 15.7 Hz, 1H), 7.36-7.20 (m, 5H), 6.74 (d, J = 15.7 Hz, 1H), 4.73 (t, J = 7.3 Hz, 2H), 3.27 (t, J = 7.3 Hz, 2H); ¹³C NMR (125 MHz, CD₃OD): δ 163.9, 144.4, 139.0, 129.9, 129.6, 128.5, 127.8, 125.5,120.6, 52.0, 37.1; HRMS (ESI) calcd for C₁₃H₁₄N₄O₂Na [M+Na]⁺ 281.1014, found 281.1017.

(E)-N-hydroxy-3-(1-(3-phenylpropyl)-1H-1,2,3-triazol-4-yl)acrylamide (5c)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 5: 1) to provide a pale colored solid (35 mg product 5c from 100 mg 21c, 50% yield). ¹H NMR (500 MHz, CD₃OD):δ 8.15 (s, 1H), 7.51 (d, J = 15.8 Hz, 1H), 7.30-7.24 (m, 2H), 7.21-7.15 (m, 3H), 6.62 (d, J = 15.8 Hz, 1H), 4.42 (t, J = 7.2 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.41 (tt, J₁ = J₂ = 7.4 Hz, 2H); ¹³C NMR (125 MHz, CD₃OD):δ 165.9, 144.9, 142.0, 129.7, 129.6, 129.4, 127.4, 125.8,120.2, 51.0, 33.6, 33.0; HRMS (ESI) calcd for C₁₄H₁₆N₄O₂Na [M+Na]⁺ 295.1171, found 295.1168.

(E)-N-hydroxy-3-(1-(2-phenoxyethyl)-1H-1,2,3-triazol-4-yl)acrylamide (5d)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 6: 1) to provide a white solid (69 mg 5d from 163 mg 21d, 61% yield). ¹H NMR (500 MHz, DMF-d₇):δ 8.52 (s, 1H), 7.52 (d, J = 15.8 Hz, 1H), 7.38-7.24 (m, 2H), 7.07-6.92 (m, 3H), 6.79 (d, J = 15.7 Hz, 1H), 4.91 (t, J = 5.0 Hz, 2H), 4.51 (t, J = 5.0 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 163.8, 159.3, 144.7, 130.6, 128.3, 126.0, 122,2, 120.8,115.7, 67.3 50.6; HRMS (ESI) calcd for C₁₃H₁₄N₄O₃Na [M+Na]⁺ 297.0964, found 297.0966.

(E)-N-hydroxy-3-(1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)acrylamide (5e)

The crude product was purified via C-18 reverse phase column chromatography (water acetonitirile = 6: 1) to provide a pale colored solid (22mg product 5e from 90 mg 21e, 35%). ¹H NMR (500 MHz, DMF-d₇):δ 11.19 (s, 1H), 8.48 (s, 1H), 8.17 (d, J = 7.4 Hz, 2H), 7.80 (t, J = 7.9 Hz, 1H), 7.67 (dd, J₁ = J₂ =7.8 Hz, 2H), 7.62 (d, J = 15.2, 1H), 6.85 (d, J = 15.7 Hz, 1H), 6.34 (s, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 192.2, 143.6, 134.4, 134.4, 129.2, 128.3, 127.7, 126.4, 119.8, 56.1 (one peak less due to the overlap with solvent peak ~ δ 162); HRMS (ESI) calcd for C₁₃H₁₂N₄O₃Na [M+Na]₊ 295.0807, found 295.0811.

(E)-N-hydroxy-3-(1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)acrylamide (5f)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitrile: TFA = 6: 1: 0.03) to provide a pink solid (67 mg 5f from 187 mg 21f, 71% yield). ¹H NMR (500 MHz, DMF-d₇):δ 10.60 (s, 1H), 8.49 (s, 1H), 7.69 (d, J = 7.5 Hz, 2H), 7.57 (d, J = 15.7 Hz, 1H), 7.36 (apparent t, J = 7.4 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 6.81 (d, J = 15.7 Hz, 1H), 5.51 (s, 2H); ¹³C NMR (125 MHz, CD₃OD):δ δ 165.4, 144.5, 140.0, 129.9, 128.5, 127.2, 124.9, 120.8, 120.4, 53.6; HRMS (ESI) calcd for C₁₃H₁₃N₅O₃Na [M+Na]⁺ 310.0916, found 310.0923.

(E)-3-(1-cinnamyl-1H-1,2,3-triazol-4-yl)-N-hydroxyacrylamide (5g)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitrile = 5: 1) to provide a white solid (96 mg 5g from 199 mg 21g, 70% yield). ¹H NMR (500 MHz, DMF-d₇):δ 10.2 (br s, 1H), 8.48 (s, 1H), 7.58-7.48 (m, 3H), 7.37 (apparent t, J = 7.4 Hz, 2H), 7.30 (t, J = 7.1 Hz, 1H), 6.82 (d, J = 16.0 Hz, 1H), 6.77 (d, J = 15.8 Hz, 1H), 6.60 (dt, J₁ = 15.5 Hz, J₂ = 6.2 Hz,1H), 5.29 (d, J = 6.1Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 163.9, 144.9, 137.2, , 129.7, 135.3, 129.2, 128.3, 127.8, 125.3, 124.5, 120.9, 52.8; HRMS (ESI) calcd for C₁₄H₁₄N₄O₂Na [M+Na]⁺ 293.1014, found 293.1020.

1-Benzyl-1H-[1,2,3]triazole-4-carboxylic acid hydroxamide (6a)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 6: 1) to provide a white solid (16 mg 6a from 53 mg 22a, 47% yield). ¹H NMR (500 MHz, DMF-d₇): δ 11.40-9.20 (bd s, 1H), 8.64 (s, 1H), 7.58-7.28 (m, 5H), 5.76 (s, 2H); ¹³C NMR (125 MHz, DMF-d₇): δ 158.9, 143.1, 137.0, 129.9, 129.3, 129.1, 127.1, 54.4; HRMS (ESI) calcd for C₁₀H₁₀N₄O₂Na [M+Na]⁺ 241.0701, found 241.0697.

1-Phenethyl-1H-[1,2,3]triazole-4-carboxylic acid hydroxamide (6b)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 6: 1) to provide a white solid (21 mg 6b from 63 mg 22b, 51% yield). ¹H NMR (500 MHz, DMF-d₇): δ 11.21 (s, 1H), 9.30 (s, 1H), 8.48 (s, 1H), 7.40-7.20 (m, 5H), 4.78 (t, J = 7.3 Hz, 2H), 3.29 (t, J = 7.3 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇): δ 158.3, 141.9, 138.0, 129.0, 128.7, 126.9, 126.2, 51.3, 36.2; HRMS (ESI) calcd for C₁₁H₁₂N₄O₂Na [M+Na]⁺ 255.0858, found 255.0862.

1-(3-Phenyl-propyl)-1H-[1,2,3]triazole-4-carboxylic acid hydroxamide (6c)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 6: 1) to provide a white solid (31 mg 6c from 63 mg 22c, 74% yield). ¹H NMR (500 MHz, DMF-d₇):δ 11.27 (s, 1H), 9.38 (s, 1H), 8.61 (s, 1H), 7.36-7.20 (m, 5H), 4.54 (t, J = 7.1 Hz, 2H), 2.66 (t, J = 7.7 Hz, 2H), 2.26 (tt, J₁ = J₂ = 7.1 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 159.1, 142.8, 142.0, 129.4, 127.0, 126.9, 55.7, 50.5, 33.2, 32.7; HRMS (ESI) calcd for C₁₂H₁₄N₄O₂Na [M+Na]⁺ 269.1014, found 269.1014.

1-(2-Phenoxy-ethyl)-1H-[1,2,3]triazole-4-carboxylic acid hydroxamide (6d)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 6: 1) to provide a white solid (15 mg 6d from 40 mg 22d, 56% yield). ¹H NMR (500 MHz, DMF-d₇):δ 10.60-9.90 (bd s, 1H), 8.65 (s, 1H), 7.34-7.28 (m, 2H), 7.00-6.94 (m, 2H), 4.95 (t, J = 5.1 Hz, 2H), 4.53 (t, J = 5.1, 2H); ¹³C NMR (125 MHz, DMF-d₇): δ 159.4, 159.1, 143.0, 130.6, 127.6, 122.2, 115.7, 67.3, 50.7; HRMS (ESI) calcd for C₁₁H₁₂N₄O₂Na [M+Na]⁺ 271.0807, found 271.0807.

1-(2-Oxo-2-phenyl-ethyl)-1H-[1,2,3]triazole-4-carboxylic acid hydroxyamide (6e)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 7: 1) to provide a white solid (56 mg 6e from 116 mg 22e, 72% yield). ¹H NMR (500 MHz, DMF-d7):δ 11.40-9.20 (bd s, 2H), 8.62 (s, 1H), 8.16 (d, J = 7.3 Hz, 2H), 7.78 (t, J = 7.4 Hz, 1H), 7.65 (dd, J₁ = J₂ = 7.8 Hz, 2H), 6.38 (s, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 192.9, 159.1, 143.0, 135.5, 135.3, 130.1, 129.3, 128.9, 57.3; HRMS (ESI) calcd for C₁₁H₁₀N₄O₃Na [M+Na]⁺ 269.0651, found 269.0629.

1-Phenylcarbamoylmethyl-1H-[1,2,3]triazole-4-carboxylic acid hydroxyamide (6f)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 7: 1) to provide a white solid (45 mg 6f from 120 mg 22f, 55% yield). ¹H NMR (500 MHz, DMF-d₇):δ 10.77 (s, 1H), 10.50-10.00 (bd s, 1H), 8.65 (s, 1H), 7.70 (d, J = 7.4 Hz, 2H), 7.35 (dd, J₁ = J₂ = 7.9 Hz, 2H), 7.11 (t, J = 7.4 Hz, 1H), 5.57 (s, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 165.3, 159.1, 142.8, 140.1, 129.9, 128.8, 124.8, 120.4, 53.6; HRMS (ESI) calcd for C₁₁H₁₁N₅O₃Na [M+Na]⁺ 284.0760, found 284.0743.

1-(3-Phenyl-allyl)-1H-[1,2,3]triazole-4-carboxylic acid hydroxyamide (6g)

The crude product was purified via C-18 reverse phase column chromatography (water: acetonitirile = 5: 1) to provide a white solid (21 mg 6 from 62 mg 22 50% yield). ¹H NMR (500 MHz, DMF-d₇): δ 10.60-9.80 (bd s, 2H), 8.61 (s, 1H), 7.53 (d, J = 7.6 Hz, 2H), 7.38 (dd, J₁ = J₂ = 7.5 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 6.79 (d, J = 15.9, 1H), 6.62 (td, J₁ = 15.7 Hz, J₂ = 6.6 Hz, 1H), 5.33 (d, J = 6.5, 2H); ¹³C NMR (125 MHz, DMF-d₇): δ 159.4, 159.1, 143.0, 130.6, 127.6, 122.2, 115.7, 67.3, 50.7; HRMS (ESI) calcd for C₁₂H₁₂N₄O₂Na [M+Na]⁺ 267.0858, found 267.0855.

(E)-N-(4-methoxybenzyloxy)-5-(trimethylsilyl)pent-2-en-4-ynamide (7)

The acid 19 (168 mg, 1.0 mmol, 1.0 equiv) was added to a reaction vessel containing DCM (3 mL). To this solution, oxalyl chloride (130 μL, 1.6 mmol, 1.6 equiv) and a trace amount of DMF were added. The mixture was stirred at room temperature for 1 hour. The solvent was then removed under vacuum to afford a yellow-reddish syrup. DCM (4 mL) was added, and the flask was cooled to 0°C (solution A). The amine 13 (285 mg 1.5 mmol, 1.5 equiv) and N,N′-diisopropyethylamine (DIPEA) (0.52 mL, 3.0 mmol, 3.0 equiv) were added to a second reaction vessel containing DCM (10 mL) (solution B). Solution B was cooled to 0°C, after which solution A was added within 1 min. The mixture was stirred for one half hour at 0°C. The reaction mixture was then poured onto ice cold 0.1 N HCl (20 mL), after which DCM (20 mL) was added. The organic layer was additionally washed with ice cold 0.1 N HCl (2 × 20 mL). The DCM solution was dried with Na₂SO₄, and the solvent was evaporated. The crude product was purified via flash column chromatography (hexanes: ethyl acetate, 87:13 to 83:17) to provide pure 7 as a white oil (253 mg, 84%). ¹H NMR (500 MHz, CD₃CN): δ 9.44 (bd s, 1H), 7.33 (d, J = 8.6 Hz, 2H), 6.92 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 15.5 Hz, 1H), 6.17 (d, J = 14.2 Hz, 1H), 4.79 (s, 2H), 3.79 (s, 3H), 0.20 (s, 9H); ¹³C NMR (125 MHz, CD₃CN):δ 163.0, 161.0, 132.3, 132.0, 128.7, 121.4, 114.8, 103.2, 102.8, 78.4, 55.9, – 0.33; HRMS (ESI) calcd for C₁₆H₂₁NO₃SiNa [M+Na]⁺ 326.1188, found 326.1193.

(E)-N-(4-methoxybenzyloxy)-5-(trimethylsilyl)-N-((E)-5-(trimethylsilyl)pent-2-en-4-ynoyl)pent-2-en-4-ynamide (20)

Compound 20 was a by-product obtained during the preparation of 7. ¹H NMR (500 MHz, CD₃CN):δ 7.25 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 15.9 Hz, 1H), 6.48 (d, J = 16.0 Hz, 1H), 6.34 (d, J = 15.9 Hz, 1H), 6.12 (d, J = 16.0 Hz, 1H), 5.00 (s, 1H), 3.78 (s, 1H), 0.22 (s, 9H), 0.18 (s, 9H); ¹³C NMR (125 MHz, CD₃CN):δ 161.3, 160.8, 149.0, 131.1, 131.0, 129.7, 129.6, 128.8, 116.4, 114.8, 108.2, 103.1, 103.1, 101.6, 77.8, 55.9, −0.30, −0.51; HRMS (ESI) calcd for C₂₄H₃₁NO₄Si₂Na [M+Na]⁺ 476.1689, found 476.1683.

N-(4-methoxybenzyloxy)-3-(trimethylsilyl)propiolamide (8)

Commercially available acid 14 (710 mg, 5.0 mmol, 1.0 equiv) was added to a reaction vessel containing DCM (10 mL). To this solution, oxalyl chloride (430 μL, 5.5 mmol, 1.1 equiv) and a trace amount of DMF were added. The mixture was stirred at room temperature for 2 hours. The solution was then placed in an ice bath, and most of the acidic gases (HCl & SO₂) were removed under vacuum (~20 bar) to give a yellow solution. Freshly dried DCM (10 mL) was then added (solution A). The amine 13 (950 mg, 5.0 mmol, 1.0 equiv) and DIPEA (2.8 mL, 16.1 mmol, 3.2 equiv) were added to a second reaction vessel containing DCM (50 mL) (solution B). Solution B was cooled to 0 °C, after which solution A was added within 1 min. The mixture was then stirred for 30 min at 0 °C. The reaction mixture was poured onto ice cold 0.1 N HCl (200 mL), before DCM (200 mL) was also added. The organic layer was additionally washed with ice cold 0.1 N HCl twice (2 × 100 mL). The DCM solution was dried with Na₂SO₄, and the solvent was evaporated. The crude product was purified via flash column chromatography (hexanes: ethyl acetate, 85:15 to 80:20) to provide 8 as pale color oil (0.85 g, 61%). ¹H NMR (500 MHz, CDCl₃):δ 8.43 (bd s, 1H), 7.32 (d, J = 8.5 Hz, 2H), 6.89 (dd, J = 8.5 Hz, 2H), 4.85 (s, 2H), 3.80 (s, 3H), 0.20 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): δ 160.1, 151.0, 131.0, 126.9, 114.0, 95.0, 94.4, 78.2, 55.3, 0.8; HRMS (ESI) calcd for C₁₄H₁₉NO₃SiNa [M+Na]⁺ 300.1032, found 300.1033.

General procedure to prepare the azido precursors 9a-g

An alkyl halide 10a–g (1.5 g, 1.0 equiv) was added to a reaction vessel containing a solution of NaN₃ in DMSO (1.1 equiv, 0.5 M). The reaction was monitored by NMR analysis. Upon completion of the reaction, water (50 mL) was added, and the product was extracted with ether (3 × 50 mL). The combined organic layers were washed with water (2 × 50 mL) and brine (50 mL), and dried with magnesium sulfate. The organic solvent was removed to provide the azido compound. For 9c and 9d, a catalytic amount of tetraethylammonium bromide (0.05 mol%, 0.005 equiv) was added. The spectral data were in agreement with those reported.^{36, 50–53}

(Azidomethyl)benzene (9a)

From 10a (1.5 g, 11.3 mmol, 1.0 equiv), 9a was obtained in 95% yield as a colorless liquid. ¹H NMR (500 MHz, CDCl₃):δ 7.75-7.40 (m, 5H), 4.35 (s, 2H).³⁶

(2-Azidoethyl)benzene (9b)

From 10b (1.5 g, 10.2 mmol, 1.0 equiv), 9b was obtained in 93% yield as a colorless liquid. ¹H NMR (500 MHz, CDCl₃)δ 7.33 (t, J = 7.2 Hz, 2H), 7.26 (d, J = 7.1 Hz, 1H), 7.23 (d, J = 7.2 Hz, 2H), 3.51 (t, J = 7.3 Hz, 2H), 2.91 (t, J = 7.3 Hz, 2H).^50,51

(3-Azidopropyl)benzene (9c)

From 10c (1.5 g, 9.30 mmol, 1.0 equiv), 9c was obtained in 90% yield as a colorless liquid. ¹H NMR (500 MHz, CDCl₃): δ 7.30 (t, J = 7.3 Hz, 2H), 7.24-7.16 (m, 3H), 3.29(t, J = 7.3 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 1.92 (tt, J₁ = J₂ = 7.2 Hz, 2H).^{50, 51}

(2-Azidoethoxy)benzene (9d)

From 10d (1.5 g, 9.2 mmol, 1.0 equiv), 9d was obtained in 95% yield as a colorless liquid. ¹H NMR (500 MHz, CDCl₃)δ 7.34-7.28 (m, 2H), 7.02-6.97 (m, 1H), 6.95-6.89 (m, 2H), 4.16 (d, J = 5.0 Hz, 2H), 3.60 (d, J = 5.0 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃):δ 158.4, 129.8, 121.6, 114.8, 67.1, 50.4.

2-Azido-1-phenylethanone (9e)

From 10e (1.5 g, 8.0 mmol, 1.0 equiv), 9e was obtained in 75% yield after flash column chromatography (2% to 5% ether in hexanes) as a colorless liquid. ¹H NMR (500 MHz, CDCl₃): δ 7.91-7.88 (m, 2H), 7.65-7.60 (m, 1H), 7.53-7.48 (m, 2H), 4.56 (s, 2H).⁵²

2-Azido-N-phenylacetamide (9f)

From 10f (1.5 g, 8.5 mmol, 1.0 equiv), 9f was obtained in 90% yield as a colorless liquid. ¹H NMR (500 MHz, CDCl₃):δ 8.00 (bd s, 1H), 7.54 (d, J = 8.6 Hz, 2H), 7.35 (dd, J₁ = 8.4 Hz, J₂ = 7.5 Hz, 2H), 7.16 (d, J = 8.6 Hz, 1H), 4.15 (s, 2H).⁵³

2-Azido-N-phenylacetamide (9g)

From 10g (1.5 g, 9.4 mmol, 1.0 equiv), 9g was obtained in 87% yield as a colorless liquid. ¹H NMR (500 MHz, DMSO-d₆)δ 7.51-7.47 (m, 2H), 7.38-7.31 (m, 2H), 7.31-7.26 (m, 1H), 6.71 (d, J = 15.8 Hz, 1H), 6.39 (dt, J₁ = 15.8 Hz, J₂ = 6.6 Hz, 1H), 4.04 (d, J = 6.6 Hz, 2H).³⁶

O-(4-methoxybenzyl)hydroxylammonium chloride (13)

N-hydroxyphthalimide 11 (4.9 g, 30 mmol, 1.0 equiv) & potassium carbonate (5.0 g, 36 mmol, 1.2 equiv) were added to a reaction vessel containing DMF (100 mL). To this solution, 1-(chloromethyl)-4-methoxybenzene (PMBCl) (4.7 mL, 33 mmol, 1.1 equiv) was added. The reaction continued overnight at room temperature. Water (500 mL) and benzene (500 mL) were then added. The aqueous layer was additionally extracted with benzene (2 × 100 mL). The combined organic layers were washed with sat. NaHCO₃ solution (3 × 200 mL), and dried with magnesium sulfate. Crude 12 was obtained after removal of the solvent, and was directly employed in the next step. To this crude product in methanol (500 mL), hydrazine monohydrate was added (4.5 mL, 92.5 mmol, 3.1 equiv). The mixture was stirred overnight at room temperature. TLC analysis indicated the presence of a new compound. Upon completion of the reaction, the solution pH was adjusted to 2 via the addition of aqueous 2M HCl. The mixture was cooled in an ice bath for 1 hour, after which the white precipitate (by product) was removed. The solvent was removed, and the residue/syrup was partitioned between DCM (200 mL) & ammonium bicarbonate solution (pH = 8, 200 mL). The organic layer was dried with Na₂SO₄. Addition of 1M HCl in ether then caused the product 13 to precipitate as a white solid (4.3 g, 75.6% yield for two steps). Spectral data were in agreement with those reported.^{37 1}H NMR (500 MHz, DMSO-d₆): δ 11.10 (bd s, 3H), 7.35 (d, J = 8.6 Hz, 2H), 6.96 (d, J = 8.6 Hz, 2H), 4.97 (s, 2H), 3.76 (s, 3H).

(E)-3-Iodopropenoic acid (16)

Propiolic acid 15 (10 mL, 162.0 mmol, 1.0 equiv) was added over 1 min to a reaction vessel containing a solution of CuI (200 mg, 1.1 mmol, 0.007 equiv) and HI (40 mL, initially 57%, 303.2 mmol, 1.9 equiv). The reaction mixture was immediately immersed in a pre-heated oil bath (130°C) for 30 min. The reaction was then cooled to room temperature over a 15 min period. A white precipitate formed, after which the solution was kept at room temperature for another 15 min. The liquid was removed by filtration, and the solid was washed water (3 × 70 mL). Following a period of drying, the pure iodoacid 16 was obtained as white needles (23.9 g, 75%). ¹H-NMR (500 MHz, CDCl₃): δ 10.54-8.66 (bd s, 1H), 8.07 (d, J = 14.9 Hz, 1H), 6.88 (d, J = 14.9 Hz, 1H). Spectral data were in agreement with those reported.³⁸

(2-Methoxyethoxy)methyl-(2E)-3-iodoprop-2-enoate (17)

K₂CO₃ (3.42 g, 24.8 mmol, 1.2 equiv) and 2-methoxyethoxymethyl chloride (MEMCl) (3.2 mL, 27.6 mmol, 1.3 equiv) were added to a reaction vessel containing a solution of E-iodopropenoic acid 16 (4.2 g, 21.2 mmol, 1.0 equiv) in DMF (20 mL). The mixture was stirred at ambient temperature for 1 hour, poured onto water (20 mL), and then extracted with ether (3 × 20 mL). The combined organic layers were washed with water, brine, dried over MgSO₄, and concentrated. Flash column chromatograhphy (hexanes: ethyl acetate, 20:1 to 10:1) provided 5.2 g (86%) of 17 as a colorless oil: ¹H NMR (500 MHz, C₆D₆): δ 7.62 (d, J = 14.8 Hz, 1H) 6.62 (d, J = 14.8 Hz, 1H), 5.13 (s, 2H), 3.49 (t, J = 4.7 Hz, 2H), 3.18 (t, J = 4.7 Hz, 2H), 3.04 (s, 3H); ¹³C NMR (125 MHz, C₆D₆):δ 163.1, 136.6, 100.2, 89.9, 71.8, 69.9, 58.7; HRMS (ESI) calcd for C₇H₁₁IO₄Na [M+Na]⁺ 308.9600, found 308.9605.

(2-Methoxyethoxy)methyl-(2E)-5-(trimethylsilyl)pent-2-en-4-ynoate (18)

Pd(PPh₃)₂Cl₂ (0.56 g, 0.80 mmol, 0.8 equiv) and CuI (0.31 g, 1.63 mmol, 0.1 equiv) were added to a reaction vessel containing a solution of the iodide 17 (4.65 g, 16.3 mmol, 1.0 equiv) in Et₃N (60 mL). The solution was degassed with argon. Trimethylsilylacetylene (3.8 mL, 29.3 mmol, 1.8 equiv) was then added and the solution was stirred for 1 h. The mixture was poured onto sat. NH₄Cl (200 mL) and extracted with ether (3 × 100 mL). The combined organic layers were washed with brine, dried over MgSO₄, and concentrated. The thick oily residue was subjected to flash column chromatography (hexanes: ethyl acetate, 20:1 to 13:1) to yield 3.6 g (87%) of 18 as a pale oil: ¹H NMR (500 MHz, CDCl₃)δ 6.77 (d, J = 16.0 Hz, 1H), 6.23 (d, J = 16.0 Hz, 1H), 5.38 (s, 2H), 3.77(m, 2H), 3.53(m, 2H), 3.36 (s, 3H), 0.20 (s, 9H); ¹³C NMR (125 MHz, CDCl₃)δ 165.1, 130.6, 126.0, 105.9, 101.1, 89.8, 71.5, 69.6, 59.1, −0.43; HRMS (ESI) calcd for C₁₂H₂₀O₄SiNa [M+Na]⁺ 279.1029, found 279.1032.

(E)-5-(trimethylsilyl)pent-2-en-4-ynoic acid (19)

MEM-protected 18 (3.07 g, 12.0 mmol, 1.0 equiv) and 3N HCl (12 mL) were added to a reaction vessel containing THF (150 mL). The reaction was stirred at room temperature for 3 days, during which time it was monitored by TLC (5% MeOH in dichloromethane, R_f = 0.10). The solution was then concentrated and the residue partitioned between water (250 mL) and CH₂Cl₂ (100 mL). The water was further extracted with CH₂Cl₂ (2 × 100 mL). The combined organic layers were washed with brine, dried over MgSO₄, and concentrate give 2.01 g (~100%) of the crude acid 19 as pale yellow solid. The crude product is pure enough for NMR analysis. ¹H NMR (500 MHz, CDCl₃):δ 0.20 (s, 9H), 6.22 (d, J = 15.9 Hz, 1H), 6.80 (d, J = 15.9 Hz, 1H), 12.5 ~ 11.0 (bd s, 1H); ¹³C NMR (125 MHz, CDCl₃):δ −0.34, 101.1, 107.1, 127.6, 130.4, 171.4; HRMS (ESI) calcd for C₈H₁₂O₂SiNa [M+Na]⁺ 191.0502, found 191.0504.

General procedure for the preparation of 21a-g

The alkyne precursor 7 (152 mg, 0.5 mmol, 1.0 equiv) was added to a reaction vessel containing THF (5 mL) and MeOH (5 mL). Cesium fluoride (91 mg, 0.6 mmol, 1.2 equiv) was then added, after which the reaction was monitored by TLC analysis. The desilylation intermediate was slightly more polar than the reactant. Upon disappearance of the reactant, the solvent was removed, followed by partitioning between DCM (50 mL) and brine (50 mL). A trace amount of acetic acid was added to neutralize the aqueous solution. The brine phase was additionally extracted with DCM (2 × 50 mL). The combined DCM solution was dried with Na₂SO₄, and the solvent was removed. The obtained white solid was dissolved with THF (10 mL). To this solution, the azido compound 9a–g (0.75 mmol, 1.5 equiv), catalyst CuIP(OEt)₃ (7 mg, 0.02 mmol, 0.04 equiv) and one drop of DIPEA were added sequentially. The reaction continued overnight at room temperature, and was monitored by TLC analysis. Frequently, more and more white precipitate (product) formed as the reaction progressed. The solvent was removed, and the residue was partitioned between DCM (50 mL) and 1% CuSO₄ aqueous solution (50 mL). The aqueous layer was additionally extracted with DCM (2 × 50 mL). The combined DCM layers were dried with Na₂SO₄. After removal of the solvent, the crude product was purified via flash column chromatography to afford the final product 21a–g.

(E)-3-(1-benzyl-1H-1,2,3-triazol-4-yl)-N-(4-methoxybenzyloxy) acrylamide (21a)

A white solid was obtained (87%, 158 mg 21a product from 0.5 mmol 7) via flash column chromatography (MeOH: DCM, from 1.2: 100 to 1.5: 100). ¹H NMR (500 MHz, CD₂Cl₂ & CD₃OD):δ 7.91 (s, 1H), 7.50 (d, J = 15.7 Hz, 1H), 7.42-7.28 (m, 7H), 6.90 (d, J = 8.4 Hz, 2H), 6.54 (d, J = 15.7 Hz, 1H), 5.56 (s, 2H), 4.84 (s, 2H), 3.79 (s, 3H); ¹³C NMR (125 MHz, CD₂Cl₂ & CD₃OD):δ 164.8, 160.8, 144.5, 135.4, 131.6, 129.7, 129.4, 129.2, 128.7, 128.3, 125.0, 119.6, 114.4, 78.5, 55.6, 54.8; HRMS (ESI) calcd for C₂₀H₂₀N₄O₃Na [M+Na]⁺ 387.1433, found 387.1431.

(E)-N-(4-methoxybenzyloxy)-3-(1-phenethyl-1H-1,2,3-triazol-4-yl) acrylamide (21b)

A white solid was obtained (97% yield, 184 mg 21b from 0.5 mmol 7) via flash column chromatography (MeOH: DCM, from 0.8: 100 to 1.5: 100). ¹H NMR (500 MHz, DMF-d₇)δ 11.28 (s, 1H), 8.36 (s, 1H), 7.56 (d, J = 15.7 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.35-7.20 (m, 5H), 6.98 (d, J = 8.6 Hz, 2H), 6.72 (d, J = 15.5 Hz, 1H), 4.89 (s, 2H), 4.74 (t, J = 7.3 Hz, 2H), 3.83 (s, 3H), 3.27 (t, J = 7.3 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 164.1, 161.0, 144.2, 138.9, 131.9, 129.9, 129.6, 129.6, 129.4, 127.7, 125.8, 120.2, 114.7, 78.1, 56.0, 52.0, 37.0; HRMS (ESI) calcd for C₂₁H₂₂N₄O₃Na [M+Na]⁺ 401.1590, found 401.1585.

(E)-N-(4-methoxybenzyloxy)-3-(1-(3-phenylpropyl)-1H-1,2,3-triazol-4-yl)acrylamide (21c)

A white solid was obtained (95% yield, 186 mg 21c from 0.5 mmol 7) via flash column chromatography (MeOH: DCM, from 0.8: 100 to 1.5: 100). ¹H NMR (500 MHz, DMF-d₇)δ 11.29 (s, 1H), 8.49 (s, 1H), 7.60 (d, J = 15.7 Hz, 1H), 7.40 (d, J = 8.5 Hz, 2H), 7.35-7.30 (m, 2H), 7.30-7.25 (m, 2H), 7.25-7.20 (m, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.75 (d, J = 15.5 Hz, 1H), 4.90 (s, 2H), 4.50 (t, J = 7.3 Hz, 2H), 3.83 (s, 3H), 2.67 (t, J = 7.3 Hz, 2H), 2.25 (t, J = 7.3 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇): δ164.1, 161.0, 144.5, 142.2, 131.9, 129.7, 129.6, 129.5, 127.2, 125.8, 120.3, 114.8, 78.1, 56.0, 50.4, 33.3, 32.8 (one peak less due to overlap); HRMS (ESI) calcd for C₂₂H₂₄N₄O₃Na [M+Na]⁺ 415.1746, found 415.1745.

(E)-N-(4-methoxybenzyloxy)-3-(1-(2-phenoxyethyl)-1H-1,2,3-triazol-4-yl)acrylamide (21d)

A white solid was obtained (89 % yield, 176 mg 21d from 0.5 mmol 7) via flash column chromatography (MeOH: DCM, from 1.0: 100 to 2.0: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.26 (s, 1H), 8.55 (s, 1H), 7.61 (d, J = 15.7 Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.36-7.27 (m, 2H), 7.06-6.90 (m, 5H), 6.77 (d, J = 14.8 Hz, 1H), 4.91 (t, J = 5.0 Hz, 2H), 4.89 (s, 2H), 4.51 (t, J = 5.1 Hz, 2H), 3.83 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 164.2, 161.0, 159.4, 144.5, 131.9, 130.6, 129.6, 129.5, 126.5, 122.3, 120.4, 115.7, 114.8, 78.1, 67.4, 56.0, 50.6; HRMS (ESI) calcd for C₂₁H₂₂N₄O₄Na [M+Na]⁺ 417.1539, found 417.1543.

(E)-N-(4-methoxybenzyloxy)-3-(1-(2-oxo-2-phenylethyl)-1H-1,2,3-triazol-4-yl)acrylamide (21e)

A pale yellow solid was obtained (38 % yield, 110 mg 21e from 0.73 mmol 7) via flash column chromatography (MeOH: DCM, from 1.5: 100 to 2: 100). ¹H NMR (500 MHz, DMF-d₇)δ 11.30 (s, 1H), 8.48 (s, 1H), 8.17 (d, J = 7.4 Hz, 2H), 7.78 (t, J = 7.4 Hz, 1H), 7.72-7.62 (m, 3H), 7.41 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 15.6 Hz, 1H), 6.35 (s, 2H), 4.91 (s, 2H), 3.83 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 192.3, 163.4, 160.2, 143.8, 134.8, 134.6, 131.1, 129.3, 128.9, 128.7, 128.6, 126.9, 119.7, 114.0, 77.3, 56.4, 55.3; HRMS (ESI) calcd for C₂₁H₂₀N₄O₄Na [M+Na]⁺ 415.1382, found 387.1378.

(E)-N-(4-methoxybenzyloxy)-3-(1-(2-oxo-2-(phenylamino)ethyl)-1H-1,2,3-triazol-4-yl)acrylamide (21f)

A white solid was obtained (92%, 187 mg product 21f from 0.5 mmol 7) via flash column chromatography (MeOH: DCM, from 2.5: 100 to 3.5: 100). ¹H NMR (500 MHz, DMF-d₇) δ 11.29 (s, 1H), 10.60 (s, 1H), 8.53 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 15.7 Hz, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.36 (dd, J₁ = 7.6 Hz, J₂ = 8.4 Hz, 2H), 7.12 (t, J = 7.4 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.78 (d, J = 15.7 Hz, 1H), 5.52 (s, 2H), 4.90 (s, 2H), 3.83 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇) δ 164.6, 163.4, 160.2, 143.6, 139.3, 131.1, 129.2, 128.9, 128.7, 126.8, 124.1, 119.6, 114.0, 77.3, 55.2, 52.8 (one peak less due to the overlap); HRMS (ESI) calcd for C₂₁H₂₁N₅O₄Na [M+Na]⁺ 430.1491, found 430.1492.

(E)-3-(1-cinnamyl-1H-1,2,3-triazol-4-yl)-N-(4-methoxybenzyloxy)acrylamide (21g)

A white solid was obtained (86% yield, 216 mg product 21g from 150 mg 7) via flash column chromatography (MeOH: DCM, from 1.0: 100 to 2.0: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.29 (s, 1H), 8.52 (s, 1H), 7.62 (d, J = 15.7 Hz, 1H), 7.53 (d, J = 7.3 Hz, 2H), 7.42-7.36 (m, 4H), 7.31 (tt, J₁ = 7.3 Hz, J₂ = 2.0 Hz, 1H), 6.98 (d, J = 8.6 Hz, 2H), 6.78 (m, 2H), 6.61 (td, J₁ = 15.8 Hz, J₂ = 6.3 Hz, 1H), 5.30 (d, J = 6.1 Hz, 2H), 4.89 (s, 2H), 3.83 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 164.1, 161.0, 144.7, 137.3, 135.4, 131.9, 129.8, 129.7, 129.4, 129.3, 127.8, 125.7, 124.5, 120.4, 114.8, 78.1, 56.0, 52.8; HRMS (ESI) calcd for C₂₂H₂₂N₄O₃Na [M+Na]⁺ 413.1590, found 413.1581.

General procedure for the preparation of 22a-g

The alkyne precursor 8 (589 mg, 2.12 mmol, 1.0 equiv) was added to a reaction vessel containing THF (50 mL) and MeOH (50 mL). Cesium fluoride (322 mg, 2.12 mmol, 1.0 equiv) was then added, after which the reaction was monitored by TLC analysis. The desilylation intermediate was slightly more polar than 8. Upon the disappearance of 8, the solution was split into ten equal fractions. These fractions were used directly in the ensuing reactions as work-up led to degradation of the alkyne. To each fraction of the alkyne solution, the azido compound 9a–g (0.32 mmol, 1.5 equiv), catalyst CuIP(OEt)₃ (10 mg, 0.028 mmol, 0.1 equiv) and one drop of DIPEA were added sequentially. The reaction was allowed to proceed to completion by stirring overnight at room temperature. The solvent was removed, and the residue was partitioned between DCM (50 mL) and 1% CuSO₄ aqueous solution (50 mL). The aqueous phase was additionally extracted with DCM (2 × 50 mL). The combined organic fractions were then dried (Na₂SO₄). After removal of the solvent, the crude product was purified via flash column chromatography.

1-Benzyl-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22a)

A white solid was obtained (83% yield, 60 mg 22a from 0.212 mmol 8) via flash column chromatography (MeOH: DCM, from 0.5: 100 to 0.8: 100). ¹H NMR (500 MHz, DMF-d₇): δ 11.76 (bd s, 1H), 8.72 (s, 1H), 7.60-7.25 (m, 7H), 6.96 (d, J = 7.3 Hz, 2H), 5.77 (s, 2H), 4.95 (s, 2H), 3.82 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇): δ 161.0, 159.1, 142.8, 137.1, 131.8, 130.0, 129.5, 129.3, 127.7, 114.7, 78.5, 56.0, 54.5; HRMS (ESI) calcd for C₁₈H₁₈N₄O₃Na [M+Na]⁺ 361.1277, found 361.1263.

1-Phenethyl-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22b)

A white solid was obtained (95% yield, 63 mg 22b from 0.189 mmol 8) via flash column chromatography (MeOH: DCM, from 0.5: 100 to 0.75: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.69 (bd s, 1H), 8.56 (s, 1H), 7.42 (d, J = 8.6 Hz, 2H), 7.32-7.20 (m, 5H), 6.96 (d, J = 8.7 Hz, 2H), 4.93 (s, 2H), 4.77 (t, J = 7.3 Hz, 2H), 3.81 (s, 3H), 3.28 (t, J = 7.4 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 161.0,142.4, 138.8, 131.8, 129.9, 129.6, 129.3, 127.8, 127.7, 114.8, 78.5, 56.0, 52.2, 37.0; HRMS (ESI) calcd for C₁₉H₂₀N₄O₃Na [M+Na]⁺ 375.1433, found 375.1425.

1-(3-Phenyl-propyl)-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22c)

A white solid was obtained (67% yield, 53 mg 22c from 0.212 mmol 8) via flash column chromatography (MeOH: DCM, from 0.5: 100 to 0.6: 100). ¹H NMR (500 MHz, DMF-d₇): δ 11.74 (bd s, 1H), 8.69 (s, 1H), 7.45 (d, J = 8.4 Hz, 2H), 7.36-7.20 (m, 5H), 6.98 (d, J = 8.5 Hz, 2H), 4.97 (s, 2H), 4.54 (t, J = 7.0 Hz, 2H), 3.83 (s, 3H), 2.67 (t, J = 7.7 Hz, 2H), 2.27 (tt, J₁ = J₂ = 7.4 Hz, 2H); ¹³C NMR (125 MHz, DMF-d₇):δ 161.0, 159.2, 142.6, 142.1, 131.8, 129.5, 129.3, 127.6, 127.2, 114.8, 78.5, 56.0, 50.7, 33.3, 32.8 (One less peak due to overlap); HRMS (ESI) calcd for C₂₀H₂₂N₄O₃Na [M+Na]⁺ 389.1590, found 389.1585.

1-(2-Phenoxy-ethyl)-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22d)

A white solid was obtained (56 % yield, 44 mg 22d from 0.212 mmol 8) via flash column chromatography (MeOH: DCM, from 0.5: 100 to 1.0: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.81-11.72 (bd s, 1H), 8.73 (s, 1H), 7.44 (d, J = 8.6 Hz, 2H), 7.31 (t, J = 8.0 Hz, 2H), 7.01-6.95 (m, 5H), 4.98-4.94 (m, 4H), 4.54 (t, J = 5.2 Hz, 2H), 3.82 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 161.0, 159.4, 159.1, 142.6, 131.8, 130.7, 129.3, 128.2, 122.3, 115.7, 114.7, 78.5, 63.7, 56.0, 50.8; HRMS (ESI) calcd for C₁₉H₂₀N₄O₄Na [M+Na]⁺ 391.1382, found 391.1379.

1-(2-Oxo-2-phenyl-ethyl-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22e)

A pale yellow solid was obtained (76 % yield, 74 mg 22e from 0.265 mmol 8) via flash column chromatography (MeOH: DCM, from 0.25: 100 to 1: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.83 (s, 1H), 8.69 (s, 1H), 8.17 (d, J = 7.6 Hz, 2H), 7.78 (t, J = 7.3 Hz, 1H), 7.65 (dd, J₁ = J₂ = 7.5 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 6.99 (d, J = 8.2 Hz, 2H), 6.39 (s, 2H), 4.99 (s, 2H), 3.84 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 192.9, 161.0, 159.2, 142.6, 135.6, 135.4, 131.9, 130.1, 129.5, 129.3, 114.8, 78.5, 57.4, 56.0 (One less peak due to overlap); HRMS (ESI) calcd for C₁₉H₁₈N₄O₄Na [M+Na]⁺ 389.1266, found 389.1218.

1-Phenylcarbamoylmethyl-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22f)

The crude product was dissolved in a minimal amount of 5% MeOH in DCM. The pure compound was then precipitated via addition of hexane (2:1 = Hexane: Solution). Following centrifugation (5000 rpm, 5 min), the supernatant was removed, and the solid was sonicated in the presence of a 1% EDTA aqueous solution. The solution was then filtered to afford the pure compound (81% yield, 82 mg 22f from 0.265 mmol 8). ¹H NMR (500 MHz, DMF-d₇):δ 11.90-11.70 (bd s, 1H), 10.65 (s, 1H), 8.72 (s, 1H), 7.69 (d, J = 8.0 Hz, 2H), 7.45 (d, J = 8.5 Hz, 2H), 7.36 (dd, J₁ = J₂ = 7.8 Hz, 2H), 7.12 (t, J = 7.3 Hz, 1H), 6.98 (d, J = 8.5 Hz, 2H), 5.57 (s, 2H), 4.98 (s, 2H), 3.83 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 165.2, 161.0, 159.2, 142.4, 140.0, 131.8, 129.4, 129.3, 124.9, 120.4, 114.8, 78.5, 56.0, 53.7 (One less peak due to overlap); HRMS (ESI) calcd for C₁₉H₁₉N₅O₄Na [M+Na]⁺ 404.1335, found 404.1319.

1-(3-Phenyl-allyl)-1H-[1,2,3]triazole-4-carboxylic acid (4-methoxy-benzyloxy)-amide (22g)

A white solid was obtained (80% yield, 62 mg of 22g from 0.212 mmol 8) via flash column chromatography (MeOH: DCM, from 0.25: 100 to 0.5: 100). ¹H NMR (500 MHz, DMF-d₇):δ 11.76 (s, 1H), 8.69 (s, 1H), 7.54 (d, J = 7.4 Hz, 2H), 7.44 (d, J = 8.6 Hz, 2H), 7.38 (dd, J₁ = J₂ = 7.5 Hz, 2H), 7.31 (t, J = 7.3 Hz, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.80 (d, J = 15.9 Hz, 1H), 6.63 (td, J₁ = 15.8 Hz, J₂ = 6.6 Hz, 1H), 5.34 (d, J = 6.5, 2H), 4.96 (s, 2H), 3.82 (s, 3H); ¹³C NMR (125 MHz, DMF-d₇):δ 161.0, 159.1, 142.7, 137.3, 135.6, 131.8, 129.8, 129.4, 129.3, 127.8, 127.5, 124.4, 114.7, 78.5, 56.0, 53.0; HRMS (ESI) calcd for C₂₀H₂₀N₄O₃Na [M+Na]⁺ 387.1433, found 387.1430.

Figure 3.

Comparison of HDAC1 inhibition by SAHA and 5g.

Abbreviations

HDAC

histone deacetylase

HDACi

histone deacetylase inhibitor

SAHA

Suberoylanilide hydroxamic acid

TSA

trichostatin A

HA

hydroxamic acid