**3-Carboxylic Acid and Formyl-Derived Coumarins as Photoinitiators in Photo-Oxidation or Photo-Reduction Processes for Photopolymerization upon Visible Light: Photocomposite Synthesis and 3D Printing Applications**

**Mahmoud Rahal 1,2,3, Bernadette Graff 1,2, Joumana Toufaily <sup>3</sup> , Tayssir Hamieh 3,4 , Guillaume Noirbent <sup>5</sup> , Didier Gigmes <sup>5</sup> , Frédéric Dumur 5,\* and Jacques Lalevée 1,2,\***

	- <sup>2</sup> Université de Strasbourg, 67081 Strasbourg, France
	- <sup>3</sup> Laboratory of Materials, Catalysis, Environment and Analytical Methods (MCEMA) and LEADDER Laboratory, Faculty of Sciences, Doctoral School of Sciences and Technology (EDST), Lebanese University, Beirut 6573-14, Lebanon; joumana.toufaily@ul.edu.lb (J.T.); tayssir.hamieh@ul.edu.lb (T.H.)
	- <sup>4</sup> SATIE-IFSTTAR, Université Gustave Eiffel, Campus de Marne-La-Vallée, 25, allée des Marronniers, F-78000 Versailles, France
	- <sup>5</sup> Aix Marseille Univ, CNRS, ICR UMR 7273, F-13397 Marseille, France; guillaume.noirbent@outlook.fr (G.N.); didier.gigmes@univ-amu.fr (D.G.)

**Abstract:** In this paper, nine organic compounds based on the coumarin scaffold and different substituents were synthesized and used as high-performance photoinitiators for free radical photopolymerization (FRP) of meth(acrylate) functions under visible light irradiation using LED at 405 nm. In fact, these compounds showed a very high initiation capacity and very good polymerization profiles (both high rate of polymerization (Rp) and final conversion (FC)) using two and three-component photoinitiating systems based on coum/iodonium salt (0.1%/1% *w*/*w*) and coum/iodonium salt/amine (0.1%/1%/1% *w*/*w*/*w*), respectively. To demonstrate the efficiency of the initiation of photopolymerization, several techniques were used to study the photophysical and photochemical properties of coumarins, such as: UV-visible absorption spectroscopy, steady-state photolysis, real-time FTIR, and cyclic voltammetry. On the other hand, these compounds were also tested in direct laser write experiments (3D printing). The synthesis of photocomposites based on glass fiber or carbon fiber using an LED conveyor at 385 nm (0.7 W/cm<sup>2</sup> ) was also examined.

**Keywords:** coumarin; free radical polymerization; LED; photocomposites; direct laser write

#### **1. Introduction**

The development of new low-cost, environmentally friendly, and energy-efficient polymer synthesis remains more than ever at the heart of academic and industrial concerns and the subject of many new research strategies. In fact, thanks to technological development, light sources which are at the same time inexpensive, efficient, and with low energy consumption have been developed recently to induce photopolymerization reactions [1–4]. Nowadays, photopolymers are present in several fields such as coatings [5], dentistry [6], automotive [7], cosmetics [8], 3D printing, and holography [9], etc. For most of these industrial fields, photochemical polymerization uses ultraviolet radiation, a technique widely known as UV curing. However, this pathway based on UV lamps (Hg lamps) remains energy-consuming. Moreover, the ultraviolet light is harmful to human health (carcinogenic) and characterized by particularly low light penetration, which is a challenge for the photopolymerization of thick and filled samples [10]. Therefore, alternatives to UV lamps and the use of longer wavelengths (near UV or visible) can be advantageous.

**Citation:** Rahal, M.; Graff, B.; Toufaily, J.; Hamieh, T.; Noirbent, G.; Gigmes, D.; Dumur, F.; Lalevée, J. 3-Carboxylic Acid and Formyl-Derived Coumarins as Photoinitiators in Photo-Oxidation or Photo-Reduction Processes for Photopolymerization upon Visible Light: Photocomposite Synthesis and 3D Printing Applications. *Molecules* **2021**, *26*, 1753. https://doi.org/ 10.3390/molecules26061753

Academic Editor: Maria João Matos

Received: 22 February 2021 Accepted: 18 March 2021 Published: 21 March 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The use of light-emitting diodes (LEDs) perfectly fit this requirement for safer/cheaper, and more efficient irradiation devices than UV lamps or UV lasers [11–14]. In parallel, it is important to develop new photoinitiating systems able to absorb in the near UV or the visible range where their absorption spectrum overlaps that of the LED emission. To obtain this type of system, it is necessary to develop new organic molecules carrying chromophore groups capable of shifting their absorption spectrum towards the near-UV-visible range. These molecules will be called photoinitiator (PI), which can absorb the light and generate reactive species (in combination with additives) able to initiate the photopolymerization process.

In this paper, nine coumarin derivatives (noted Coum in Scheme 1) varying by the substitution pattern at the 3- and 7-positions of the coumarin core were synthesized and evaluated as photoinitiators for the FRP of acrylate and methacrylate monomers. In fact, coumarin derivatives have already been tested as photoinitiators of FRP and they have shown good polymerization profile (Rp and FC) as well as good photochemical and photophysical properties [15–19].

**Scheme 1.** The new series of coumarins (CoumA–CoumI) examined as photoinitiators of polymerization.

However, in the present work, coumarin-3-carboxylic acids, coumarin-3-aldehydes varying by the substitution pattern of the coumarin core and a coumarin of extended aromaticity have been studied as photoinitiators. Comparisons of the three families of coumarins have revealed that the substitution of the 3-position by electron-withdrawing groups such as a formyl group could improve the reactivity. The presence of a strong electron-donating group at the 7-position, such as diethylamine or a naphthalene group, could reinforce the electronic delocalization and the photoinitiating ability of the different systems. An optimum situation was found when electron-donating and electron-accepting groups were attached at both extremities of the coumarin core. Considering that the nitro group is among the most electron-withdrawing group, a coumarin bearing this electron acceptor was also designed and synthesized.

In fact, coumarin derivatives are usually characterized by very high fluorescence emission and can be used as fluorescent chromophores for several applications [20]. They are also characterized by high molar extinction coefficients in the near-UV and the visible range [19]. These novel coumarin-based photoinitiators were tested in photopolymerization of acrylate functions (TMPTA or TA) in both Thick (1.4 mm) and Thin sample (25 µm) using two and three-component photoinitiating systems PISs based on Coum/Iodonium salt (0.1%/1% *w*/*w*) and Coum/Iodonium salt/amine (NPG) (0.1%/1%/1% *w*/*w*/*w*). These systems were also used in 3D printing and photocomposite synthesis. These dyes are characterized by very high extinction coefficients with a broad absorption extending over the near UV/visible and high quantum yields were determined by fluorescence quenching. It is important to note that coumarin shows a dual photo-oxidation and photo-reduction character.

#### **2. Results**

Photoinitiation ability, the performance of photopolymerization, photophysical and photochemical properties as well as chemical mechanisms associated with the photopolymerization processes will be discussed in detail.

#### *2.1. Synthesis of the Different Dyes*

As mentioned in the introduction section, three families of coumarins have been examined as photoinitiators of polymerization. The first family concerned coumarin-3-carboxylic acids. The five dyes were prepared in solution by condensation of diethyl malonate with *ortho*-hydroxyarylaldehydes [21]. After hydrolysis of esters in acidic conditions (a mixture of hydrochloric acid and acetic acid), the solution was neutralized to provide the different dyes with reaction yields ranging from 75% for CoumA to 86% yield for CoumE. A similar procedure was used for CoumC except that the hydrolysis of the intermediate ester coumarin resulted in a decarboxylation reaction, providing CoumC in 72% yield. The presence of the dimethylamino group in CoumC is essential to activate the decarboxylation reaction since this reaction was not observed for the other coumarins, maintaining the acidic function on the coumarins (See Scheme 2) [22]. Using the Vilsmeier Haack reaction, CoumC could be converted as CoumG in a 77% yield.

Finally, CoumH and CoumI could be prepared starting from 2-thiopheneacetic acid and 4-diethylamino-2-hydroxybenzaldehyde. By Knoevenagel reaction, 7-(diethylamino)- 3-(thiophen-2-yl)-2*H*-chromen-2-one could be obtained in 58% yield and by means of a Vilsmeier Haack reaction, CoumH was isolated in pure form in 88% yield. Conversely, CoumI was prepared in two steps, first by bromination of 7-(diethylamino)-3-(thiophen-2-yl)-2*H*-chromen-2-one in 86% yield, followed by a Suzuki cross-coupling reaction with 3-nitrophenylboronic acid. Using this procedure, CoumI was obtained in 67% yield (See Scheme 3).

**Scheme 3.** Synthetic routes to CoumH and CoumI.

#### *2.2. Light Absorption Properties*

**ε** − − <sup>−</sup> **ε** − − − − UV-visible absorption spectra of the different coumarins in acetonitrile are depicted in Figure 1 (See also Table 1). These organic compounds are characterized by a high molar extinction coefficient in both near-UV and visible range (e.g., CoumC ε ~ 18000 M−<sup>1</sup> cm−<sup>1</sup> at 374 nm and 3500 M-1cm−<sup>1</sup> at 405 nm, and CoumF ε ~ 10200 M−<sup>1</sup> cm−<sup>1</sup> at 376 nm and 4800 M−<sup>1</sup> cm−<sup>1</sup> at 405 nm). So, these absorption properties afford a good overlap with the emission spectrum of the LEDs used in this work (LED at 405 nm for FRP, LED at 375 nm for the photolysis experiments and LED at 385 nm for the photocomposites synthesis).

λ **Figure 1.** UV-visible absorption spectra of the investigated compounds based on coumarin derivatives in ACN: (**1**) CoumA, (**2**) CoumB, (**3**) CoumC, (**4**) CoumD, (**5**) CoumE, (**6**) CoumF, (**7**) CoumG, (**8**) CoumH, and (**9**) CoumI.

**λ − − − −** In fact, the presence of different substituents on the coumarin scaffold can affect the absorption properties (Figure 2) of these compounds and their molar extinction coefficients can be affected. For example, taking CoumA as a standard structure among these 10 compounds, we observed a shift towards higher absorption range (e.g., CoumB, CoumD, and CoumF are strongly shifted), and towards lower absorption range (e.g., CoumE), so a bathochromic effect is observed by introduction of electron donor group (such as OH, OMe, and NR2) and a hypsochromic effect is observed by introduction of electron acceptor group (e.g., NO<sup>2</sup> in case of CoumE).

**Table 1.** Light absorption properties of coumarins at 405 nm and at λmax; singlet state energy (ES1) determined from the crossing point of absorption and fluorescence spectra.



**Figure 2.** HOMO and LUMO frontier orbitals and their respective calculated UV-visible absorption spectra for the different investigated compounds at the UB3LYP/6-31G\* level.

The electron-donating effect of these substituents is presented by ascending order: CoumH > CoumI > CoumG > CoumF > CoumC > CoumD > CoumB.

#### *2.3. Free Radical Photopolymerization*

#### 2.3.1. Photopolymerization of Methacrylate Function of Mix-MA

The FRP profiles of methacrylate functions using Mix-MA as the benchmark monomer was performed in thick sample and in the presence of two or three-component PISs based on Coum/Iod (or NPG) (0.1%/1% *w*/*w*) or Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*) respectively, upon visible light irradiation with a LED at 405nm are given in Figure 3 (See also Table 2).

**Figure 3.** Photopolymerization profiles of methacrylate functions (conversion vs. irradiation time) using MIX-MA in thick sample (1.4 mm) under visible light irradiation using a LED at 405 nm: (**A**) Coum/Iod (0.1%/1% *w*/*w*), (**B**) Coum/NPG (0.1%/1% *w*/*w*) and **(C)** Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*): (1) CoumA, (2) CoumB, (3) CoumC, (4) CoumD, (5) CoumE, (6) CoumF, (7) CoumG, (8) CoumH, (9) CoumI and (10) Iod/NPG (1%/1% *w*/*w*). Irradiation starts at t = 10 s.

The obtained results show that Coum/Iod (or NPG) is less reactive than threecomponent PISs (Coum/Iod/NPG), this result can be explained by a higher yield of reactive species (radicals) in the presence of Iod/NPG which is not able, alone, to initiate the FRP (e.g., FC = 24% for CoumI/Iod vs. 76% for CoumI/Iod/NPG, and FC = 0% for CoumA/Iod vs. 76% for CoumA/Iod/NPG; show Figure 3A,C curve 1). In fact, CoumB, CoumD, CoumF and CoumG showed a photoreduction process rather than a photo-oxidation process (e.g., FC = 0% for CoumB/Iod vs. 67% for CoumB/NPG, and FC = 0% for CoumD/Iod vs. 75% for CoumD/NPG), but CoumC and Coum8 show an opposite behavior with a photo-oxidation process probably more favorable than the photoreduction (FC = 70% for CoumC/Iod vs. 28% for CoumC/NPG; Figure 3A,B curve 3). The FRP profiles also show a low rate of polymerization, this can be due to the high oxygen inhibition effect.


**Table 2.** Final reactive functions conversion (FC%) for different monomers and different PISs upon visible light irradiation using a LED at 405 nm (400 s of irradiation and thickness = 1.4 mm).

<sup>a</sup> Coum/Iod (0.1%/1% *w*/*w*); <sup>b</sup> Coum/NPG (0.1%/1% *w*/*w*).

#### 2.3.2. Photopolymerization of Acrylates (TMPTA or TA)

In fact, iodonium salt or NPG alone cannot initiate the FRP of acrylate at 405 nm due to their absorption in the UV range [17,23]. Therefore, the coumarins derivatives are introduced in order to improve the absorption properties of photosensitive formulations.

Firstly, the most of Coumarin derivatives show high extinction coefficients at 405 nm. The photopolymerization profiles of acrylate functions in thick (1.4 mm) or thin (25 µm) samples (conversion vs. irradiation time) using TMPTA (or TA) as benchmark monomers are depicted in Figure 4 (see also Tables 2 and 3). The obtained results show that the twocomponent PISs based on Coum/Iod (0.1%/1% *w*/*w*) (or Coum/NPG) are able to strongly initiate the FRP, but a very higher performance [Final conversion (FC) and polymerization rate (Rp)] was acquired using the three-component PISs based on Coum/Iod/NPG which is quite efficient in the FRP of acrylate functions upon LED at 405 nm (e.g., FC = 60% for CoumC/Iod (0.1%/1% *w*/*w*) vs. 80% for CoumC/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*), Figure 4A and B curve 3).

Moreover, the Iod/NPG (1%/1% *w*/*w*) couple weakly initiates the FRP (FC = 47%). This is ascribed to the formation of a charge transfer complex (CTC) between Iod and NPG [24] which is able to generate reactive species when it absorbs light. Clearly, the presence of Coumarin as photoinitiator is improving the performance of the photopolymerization processes.

Some of the coumarins can show both photoreduction (electron transfer from NPG to Coumarin) and photoxidation (electron transfer from Coumarin to Iod) processes, while other derivatives show only photoreduction process, such as CoumA, CoumB and CoumE (e.g., FC = 0% for CoumB/Iod (0.1%/1% *w*/*w*) vs. FC = 78% for CoumB/NPG (0.1%/1% *w*/*w*)).

**Figure 4.** Photopolymerization profiles of acrylate functions (conversion vs irradiation time) using TMPTA in thick sample (1.4 mm) under visible light irradiation using a LED at 405 nm: (**A**) Coum/Iod (0.1%/1% *w*/*w*), (**B**) Coum/NPG (0.1%/1% *w*/*w*) and (**C**) Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*): (1) CoumA, (2) CoumB, (3) CoumC, (4) CoumD, (5) CoumE, (6) CoumF, (7) CoumG, (8) CoumH, (9) CoumI and (10) Iod/NPG (1%/1% *w*/*w*). The molar concentrations for 0.1 % *w*/*w* are 0.0055, 0.0051, 0.0049, 0.0048, 0.0045, 0.0044, 0.0043, 0.0032, and 0.0025 M for CoumA, CoumB, CoumC, CoumD, CoumE, CoumF, CoumG, CoumH, CoumI, respectelievly. The irradiation starts at t = 10 s.

**Table 3.** Final reactive functions conversion (FC%) for different monomers and different PISs upon visible light irradiation using a LED at 405 nm (150 s of irradiation and thickness = 25 µm).


<sup>a</sup> Coum/Iod (0.1%/1% *w*/*w*); <sup>b</sup> Coum/NPG (0.1%/1% *w*/*w*).

#### *2.4. D Printing Experiments Using Coum/Iod/amine PISs and Optical Microscopy Characterization*

New 3D patterns were obtained by direct laser write experiments of Coum/Iod/amine PISs using a laser diode at 405 nm and characterized by optical microscopy. These 3D patterns were obtained under air using different PISs based on Coum/Iod/TMA (0.05%/0.5%/0.235% *w*/*w*/*w*) in TA or TMPTA (See Figure 5). In fact, the high photosensitivity of this resin allowed an efficient polymerization process in the irradiated area so a high spatial resolution is observed. Markedly, a great thickness is obtained (~2090 µm) and these patterns were carried out in a very short irradiation time (~2–3 min). Using a wellestablished Type I photoinitiator (diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide—TPO) in similar direct laser write conditions; similar performances can be reached but requiring a higher content (0.5% *w*/*w*). This latter result demonstrates the interest in using Coum derivatives. It is important to note that the 3D patterns based on CoumC exhibit a blue fluorescence when these structures are characterized by the light of the microscope.

**Figure 5.** Characterization of 3D patterns by numerical optical microscopy obtained by free radical photopolymerization experiment (using TA or TMPTA as benchmark monomer) using a diode laser at 405 nm: (**A**) CoumC/Iod/4,N-N-TMA (0.05%/0.5%/0.235% *w*/*w*/*w*) in TA, (**B**) CoumH/Iod/TMA (0.05%/0.5%/0.19% *w*/*w*/*w*) in TMPTA and **(C)** CoumD/Iod/4,N-N-TMA (0.05%/0.5%/0.275% *w*/*w*/*w*) in TMPTA.

#### *2.5. Near-UV Conveyor Experiments for the Synthesis of Photocomposites Using Coum/Iod/NPG (0.1%/1%/1% w/w/w)*

Generally, photocomposites are materials composed of at least two components: matrix and reinforcement. The mixture of these two components leads to new interesting properties that the two components separately do not have. The production of composites in the last decades and until today represents a very dynamic market in different fields such as aeronautics, automotive, wind power, and buildings. So, due to their very high mechanical resistance and chemical resistance, the glass fibers are used in this work as a matrix for the photocomposite synthesis.

In this work, the proposed coumarin derivatives were tested for access to photocomposites upon near-UV light using a LED conveyor at 385 nm (0.7 W/cm<sup>2</sup> ). The curing results obtained are summarized in Figure 6. Firstly, photocomposites were prepared by impregnation of glass fibers with an acrylic resin (TMPTA) (50% glass fibers/50% acrylic resin) and irradiated upon a LED at 385 nm. Remarkably, a very fast polymerization was observed using Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*), where both the surface and the bottom are tack-free after some passes.

**Figure 6.** Photocomposites manufactured upon near-UV irradiation at 385 nm (0.7 W/cm<sup>2</sup> ) using glass fiber/resin (50%/50% *w*/*w*) in the presence of three-component PISs based on Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*): (**1**) CoumC, (**2**) CoumD, (**3**) CoumF, (**4**) CoumG, and (**5**) CoumH.

#### **3. Discussion**

For a better understanding of the photoinitiation ability, the photochemical properties of the studied coumarins were investigated. More particularly, their photolysis behaviors, fluorescence quenchings, and redox properties were investigated in the presence of additives (amine/iodonium salt), allowing to establish the photochemical mechanisms (see Scheme 4 below).


**Scheme 4.** Proposed chemical mechanisms.

#### *3.1. Steady-State Photolysis of Coumarins*

Steady-state photolysis of coumarins derivatives in ACN and under irradiation light using a LED at 375 nm have been performed to explain the obtained results in FRP. So, the photolysis of one of these compounds (CoumC) is presented in Figure 7. First of all, the photolysis of CoumC alone upon irradiation at 375 nm is very slow compared to that obtained with Iod, which is very fast. In fact, the appearance of a weak peak

between 425 and 500 nm and the evolution of the absorption peak of CoumC shows that a high interaction between CoumC and Iod took place by an electron transfer process, this process induced, during the irradiation, a photolysis of the CoumC and generation of new photoproducts. On the other hand, the photolysis of CoumC with Iod/NPG couple was very slow (Figure 7D curve 3) and poor consumption was obtained; these results can be explained by a high regeneration of CoumC in three-component PISs.

− − − **Figure 7.** (**A**) Photolysis of CoumC alone in ACN, (**B**) photolysis of CoumC with Iod (10−<sup>2</sup> M) in ACN, (**C**) photolysis of CoumC with Iod/NPG (10−2M) couple, (**D**) percentage of consumption of CoumC (1) without Iod, and with (2) with Iod (10−<sup>2</sup> M), and (3) with Iod/NPG vs. irradiation time—upon exposure to the LED@375 nm in ACN.

#### *3.2. Fluorescence Quenching and Cyclic Voltammetry Experiments for the Coumarins*

− − ϕ Fluorescence quenching and emission spectra of the different coumarins (e.g., CoumC) have been carried out in ACN and reported in Figure 8. Firstly, where the emission intensity of CoumC decreases when we added Iod or NPG, so an interaction between <sup>1</sup>Coum-C and Iod (or NPG) occurs, this result is in full agreement with FRP and photolysis experiments shown above. To compare the reactivity of different coumarin with Iod or NPG, the Stern-Volmer coefficient (Ksv) have been calculated according to Equation (1). For example, a very high quenching of CoumF with NPG and poor quenching of CoumC with NPG were observed, so Ksv for CoumF is higher than that of CoumC (Ksv = 44 M−<sup>1</sup> for CoumC and 400 M−<sup>1</sup> for CoumF), therefore a high electron transfer quantum yield is obtained for CoumF φ = 0.9) compared to that obtained for CoumC (φ = 0.6) (Table 4)

$$\phi\_{\rm S1} = \mathcal{K}\_{\rm SV} \text{[Iod]} / (1 + \mathcal{K}\_{\rm SV} \text{[Iod]}) \tag{1}$$

Δ − The free energy change (∆G) for the electron transfer between coumarins and Iod or NPG is an important parameter to evaluate the feasibility of this process. ∆G can be extracted from the ES1 and the electrochemical properties (Eox and Ered) (using Equation (1)) e.g., ∆G = −2.39 eV for CoumF/Iod which is more reactive in FRP of acrylate functions (TA monomer) (FC = 86%). All these data are gathered in Table 4.

Finally, the FRP results of acrylate functions can be explained by a global mechanism based on the different results obtained by the characterization techniques (steady-state photolysis, Fluorescence quenching and cyclic voltammetry). First of all, the photoinitiator (Coumarin) goes to its excited state once it absorbs suitable light energy, and as it is not able to give reactive species alone, the Iod salt (or NPG), therefore, interacts with its excited state and will be able to dissociate and give reactive species responsible to initiate the FRP (r1–r2). The addition of NPG to the photosensitive formulation is very important

ϕ

because of the formation of a charge-transfer complex between Iod salt and NPG [Iod-NPG]CTC able to generate reactive species (r3–r4). Moreover, a hydrogen transfer process from NPG to Coumarins can occur which generates two types of radicals (Coum-H• , NPG(-H) • ) (r5). In fact, a decarboxylation of NPG(-H) • can take place and leads to the radical formation (NPG(-H, -CO2) • ), which react with Iod salt to produce reactive species (Ar• and NPG(-H, CO2) + ) (r6–r7). Ar• and NPG(-H,-CO2) • ) (r1–r9) radicals are assumed as the reactive species responsible to the FRP of the (meth)acrylate functions. The coumarins consumption is reduced in three-component PIS (Figure 6); this can be explained by a regeneration of the photoinitiator, which is in agreement on r8-r9 (See Scheme 4).

**Figure 8.** (**A**) Fluorescence quenching of CoumC by Iod, (**B**) ES1 determination, (**C**) determination of KSV (Stern–Volmer coefficient), and (**D**) oxidation potential (Eox) determination of CoumC.

Δ **Δ Δ <sup>−</sup> <sup>−</sup> Table 4.** Parameters characterizing the chemical mechanisms associated with <sup>1</sup>Coum/Iod or <sup>1</sup>Coum/NPG interaction in acetonitrile. For Iod and NPG, a reduction and oxidation potential of −0.2 and 1.03 eV were used respectively for the ∆Get calculation.


− −

The photoinitiation ability is a strong interplay between these different reactions (r1-r9), but their light absorption properties and intersystem crossing behavior (singlet vs. triplet state pathways, lifetimes) must also be taken into account. Therefore, a deeper characterization of their structure/reactivity/efficiency relationship is beyond the scope of the present work.

● ●

● ● ●

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#### **4. Materials and Methods**

#### *4.1. Synthesis of the Coumarins*

All reagents and solvents were purchased from Aldrich, Alfa Aesar, or TCI Europe and used as received without further purification. Mass spectroscopy was performed by the Spectropole of Aix-Marseille University. ESI mass spectral analyses were recorded with a 3200 QTRAP (Applied Biosystems SCIEX) mass spectrometer. The HRMS mass spectral analysis was performed with a QStar Elite (Applied Biosystems SCIEX) mass spectrometer. Elemental analyses were recorded with a Thermo Finnigan EA 1112 elemental analysis apparatus driven by the Eager 300 software. <sup>1</sup>H and <sup>13</sup>C NMR spectra were determined at room temperature in 5 mm o.d. tubes on a Bruker Avance 400 spectrometer and on a Bruker Avance 300 spectrometer of the Spectropole: The <sup>1</sup>H chemical shifts were referenced to the solvent peak CDCl<sup>3</sup> (7.26 ppm), and the <sup>13</sup>C chemical shifts were referenced to the solvent peak CDCl<sup>3</sup> (77 ppm). 7-(Diethylamino)-3-(thiophen-2-yl)-2*H*-chromen-2-one and 3-(5-bromothiophen-2-yl)-7-(diethylamino)-2*H*-chromen-2-one used as intermediates of reaction have been synthesized according to procedures previously reported in the literature, without modifications and in similar yields [18].

Synthesis of 2-oxo-2*H*-chromene-3-carboxylic acid (CoumA)

δ Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to a solution of salicylaldehyde (1.22 g, 10 mmol, M = 122.12 g/mol) dissolved in absolute ethanol (30 mL). After stirring and heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (1.43 g, 75% yield). <sup>1</sup>H NMR (400 MHz, DMSO-d6) δ 8.75 (s, 1H), 7.91 (dd, *J* = 7.7, 1.2 Hz, 1H), 7.80–7.68 (m, 1H), 7.42 (dd, *J* = 15.4, 7.9 Hz, 2H). Analyses were consistent with those previously reported in the literature [25]. δ

Synthesis of 7-hydroxy-2-oxo-2*H*-chromene-3-carboxylic acid (CoumB),

Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to the solution of 2,4-dihydroxybenzaldehyde (1.38 g, 10 mmol, M = 138.12 g/mol) dissolved in absolute ethanol (30 mL). After stirring and heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis, and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (1.61 g, 78% yield). <sup>1</sup>H NMR (400 MHz, DMSO-*d6*) δ 8.68 (s, 1H), 7.75 (d, *J* = 8.6 Hz, 1H), 6.85 (dd, *J* = 8.6, 2.3 Hz, 1H), 6.74 (d, *J* = 2.1 Hz, 1H). Analyses were consistent with those previously reported in the literature [25]. δ

Synthesis of 7-(diethylamino)-2*H*-chromen-2-one (CoumC)

δ

δ

δ

Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to the solution of 4-(Diethylamino)-salicylaldehyde (1.93 g, 10 mmol) dissolved in absolute ethanol (30 mL). After stirring and heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (1.56 g, 72% yield). <sup>1</sup>H NMR (400 MHz, CDCl3) δ 7.53 (d, *J* = 9.3 Hz, 1H), 7.24 (d, *J* = 8.8 Hz, 1H), 6.60–6.44 (m, 2H), 6.03 (d, *J* = 9.3 Hz, 1H), 3.41 (q, *J* = 7.1 Hz, 4H), 1.21 (t, *J* = 7.1 Hz, 6H). Analyses were consistent with those previously reported in the literature [26]. δ δ

δ

δ

Synthesis of 8-methoxy-2-oxo-2*H*-chromene-3-carboxylic acid (CoumD)

δ Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to the solution of 3-methoxysalicylaldehyde (1.52 g, 10 mmol, M = 152.15 g/mol) dissolved in absolute ethanol (30 mL). After stirring and heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (1.80 g, 82% yield). <sup>1</sup>H NMR (400 MHz, DMSO-*d6*) δ 8.50 (s, 1H), 7.42–7.26 (m, 3H), 3.91 (s, 3H). Analyses were consistent with those previously reported in the literature [27]. δ

Synthesis of 6-nitro-2-oxo-2*H*-chromene-3-carboxylic acid (CoumE)

δ δ Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to the solution of 2-hydroxy-5-nitrobenzaldehyde (1.67 g, 10 mmol, M = 167.12 g/mol) dissolved in absolute ethanol (30 mL). After stirring and heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (2.02 g, 86% yield). <sup>1</sup>H NMR (400 MHz, DMSO-d6) δ 8.81 (d, *J* = 2.8 Hz, 1H), 8.51–8.33 (m, 2H), 7.59 (d, *J* = 9.1 Hz, 1H) Analyses were consistent with those previously reported in the literature [24].

Synthesis of 3-oxo-3*H*-benzo[f]chromene-2-carboxylic acid (CoumF)

Dimethyl malonate (2.64 g, 20 mmol, M = 132.11 g/mol) and piperidine (1 mL, 10 mmol) were mixed and added to the solution of 2-hydroxy-1-naphthaldehyde (1.72 g, 10 mmol, M = 172.18 g/mol) dissolved in absolute ethanol (30 mL). After stirring and

δ

δ

δ

heating to reflux for 6 h, the solvent was removed under reduced pressure. Then, concentrated HCl (20 mL) and acetic acid (20 mL) were added for hydrolysis and the solution was refluxed overnight. After cooling, the solution was poured onto ice and aq. 40% NaOH was added until pH = 5. A pale precipitate formed. After stirring for another 30 min, the mixture was filtered, washed with water, pentane, and dried under vacuum (1.82 g, 76% yield). <sup>1</sup>H NMR (400 MHz, DMSO-*d6*) δ 9.37 (s, 1H), 8.60 (d, *J* = 8.3 Hz, 1H), 8.31 (d, *J* = 9.0 Hz, 1H), 8.09 (d, *J* = 7.8 Hz, 1H), 7.77 (ddd, *J* = 8.3, 7.0, 1.3 Hz, 1H), 7.65 (dt, *J* = 12.9, 2.9 Hz, 1H), 7.60 (d, *J* = 9.1 Hz, 1H). Analyses were consistent with those previously reported in the literature [28]. δ δ δ

Synthesis of 7-(diethylamino)-2-oxo-2*H*-chromene-3-carbaldehyde (CoumG)

δ 20 mL of POCl<sup>3</sup> was added dropwise to 20mL of dry DMF at 0 ◦C under Argon and stirred during 30 minutes at 50 ◦C. Then 7-(diethylamino)-2H-chromen-2-one (15.0 g, 69.1 mmol, M = 217.27g/mol) in 100 mL of DMF was added to the mixture and the mixture was heated to 60 ◦C overnight. Afterward, the mixture was poured into 500 mL of ice water and a solution of NaOH 20% was added. The precipitate was filtered and washed with water. (13.12 g, 77% yield). <sup>1</sup>H NMR (300 MHz, CDCl3) δ 10.08 (s, 1H), 8.21 (s, 1H), 7.46–7.32 (m, 1H), 6.61 (dd, *J* = 9.0, 2.5 Hz, 1H), 6.45 (d, *J* = 2.3 Hz, 1H), 3.46 (q, *J* = 7.1 Hz, 4H), 1.23 (t, *J* = 7.1 Hz, 6H). Analyses were consistent with those previously reported in the literature [26]. δ δ

Synthesis of 5-(7-(diethylamino)-2-oxo-2*H*-chromen-3-yl)thiophene-2-carbaldehyde (CoumH)

δ δ 7-(Diethylamino)-3-(thiophen-2-yl)-2*H*-chromen-2-one (3.00 g, 10 mmol, M = 299.39 g/mol) was dissolved in DMF (7 mL) and POCl<sup>3</sup> (1.8 mL, 20 mmol) was slowly added at 0 ◦C. The mixture was heated up to 80 ◦C overnight. After cooling, the solution was quenched with water. The mixture was extracted with DCM several times. The organic phases were combined, dried over magnesium sulfate and the solvent removed under reduced pressure. It was used without any further purification (2.88 g, 88% yield). <sup>1</sup>H NMR (400 MHz, CDCl3) δ 9.90 (s, 1H), 8.02 (s, 1H), 7.77 (d, *J* = 4.1 Hz, 1H), 7.73 (d, *J* = 4.1 Hz, 1H), 7.38 (d, *J* = 8.9 Hz, 1H), 6.69 (dd, *J* = 8.9, 2.5 Hz, 1H), 6.56 (d, *J* = 2.4 Hz, 1H), 3.46 (q, *J* = 7.1 Hz, 4H), 1.25 (t, *J* = 7.1 Hz, 6H). Analyses were consistent with those previously reported in the literature [29]. δ

Synthesis of 7-(diethylamino)-3-(5-(3-nitrophenyl)thiophen-2-yl)-2*H*-chromen-2-one (CoumI)

Tetra*kis*(triphenylphosphine)palladium (0) (0.46 g, 0.744 mmol, M = 1155.56 g.mol−<sup>1</sup> ) was added to a mixture of 3-(5-bromothiophen-2-yl)-7-(diethylamino)-2*H*-chromen-2-one (2.31 g, 6.11 mmol, M = 378.28 g.mol−<sup>1</sup> ), 3-nitrophenylboronic acid (1.53 g, 9.16 mmol, M = 166.93 g.mol−<sup>1</sup> ), toluene (54 mL), ethanol (26 mL) and an aqueous potassium carbonate solution (2 M, 6.91 g in 25 mL water, 26 mL) under vigorous stirring. The mixture was

stirred at 80 ◦C for 48 h under a nitrogen atmosphere. After cooling to room temperature, the reaction mixture was poured into water and extracted with ethyl acetate. The organic layer was washed with brine several times, and the solvent was then evaporated. The residue was purified by filtration on a plug of silicagel using a mixture of DCM/ethanol as the eluent (67% yield, 1.72 g). <sup>1</sup>H NMR (400 MHz, CDCl3) δ 8.49 (t, *J* = 1.9 Hz, 1H), 8.10 (ddd, *J* = 8.2, 2.2, 1.0 Hz, 1H), 7.98–7.92 (m, 2H), 7.64 (d, *J* = 4.0 Hz, 1H), 7.56 (d, *J* = 8.0 Hz, 1H), 7.43 (d, *J* = 4.0 Hz, 1H), 7.35 (d, *J* = 8.9 Hz, 1H), 6.64 (dd, *J* = 8.8, 2.5 Hz, 1H), 6.55 (d, *J* = 2.4 Hz, 1H), 3.45 (q, *J* = 7.1 Hz, 4H), 1.24 (t, *J* = 7.1 Hz, 6H); HRMS (ESI MS) *m*/*z*: theor: 420.1144 found: 420.1147 (M+. detected); Anal. calc. for C23H20N2O4S: C, 65.7, H, 4.8, O, 15.2; found: C 65.5, H 4.7, O 15.4. δ

−

−

#### *4.2. Other Chemical Compounds*

−

All the other chemicals (Figure 9) were selected with the highest purity available and used as received. Di-*tert*-butyl-diphenyliodonium hexafluorophosphate (Iod) and TMA (4,*N*,*N*-Trimethylaniline) were obtained from Lambson Ltd. (Wetherby, UK). Trimethylolpropane triacrylate (TMPTA), di(trimethylolpropane) tetraacrylate (TA), Mix-MA, *N*-Phenylglycine (NPG) were obtained from Allnex or Sigma Aldrich (Darmstadt, Germany). TMPTA, TA, and Mix-MA were selected as benchmark monomers for the radical polymerizations.

**Figure 9.** Other chemical compounds used in this paper.

**Mix-MA**

#### *4.3. Irradiation Light Sources*

We used differentlight-emitting diodes (LEDs) aslight sources: (1) at 405 nm(I = 110 mW/cm<sup>2</sup> ) for the photopolymerization experiments, (2) at 375 nm (I = 40 mW/cm<sup>2</sup> ) for the photolysis of Coumarins and (3) at 385 nm (I = 0.7 W/cm<sup>2</sup> ) for the photocomposite synthesis.

#### *4.4. Real-Time Fourier Transform Infrared Spectroscopy (RT-FTIR): Kinetic Followed and Final Conversion (FC) Determination for the Photopolymerisation*

In this research, the ability of coumarins to initiate the photopolymerization of (meth)acrylate functions (FRP) was studied using two and three-component photoini-

tiating systems based on Coum/Iod salt (or NPG) (0.1%/1% *w*/*w*) and Coum/Iod/NPG (0.1%/1%/1% *w*/*w*/*w*). The percentage of the different chemical compounds is calculated according to the monomer weight. Kinetic study, as well as the reactive function conversion, were monitored by the evolution of the double bond vs. time. In fact, the polymerization experiments were performed in both thick (1.4 mm) and thin (25 µm) samples, they were obtained by deposition of the formulation into the mold (1.4 mm) or between two propylene films in order to reduce O<sup>2</sup> inhibition, respectively. In addition, excellent solubility of all coumarin derivatives (excluding the CoumE) were observed. For the thick and thin samples, the evolution of the (meth)acrylate functions for TMPTA or Mix-MA were followed by RT-FTIR spectroscopy (JASCO FTIR 6600) at about 6150 cm−<sup>1</sup> and 1630 cm−<sup>1</sup> , respectively. The procedure used to monitor the photopolymerization profile was described in detail in [30,31].

#### *4.5. Redox Potentials*

The reduction (Ered) or oxidation (Eox) potentials for the different coumarin derivatives were determined by cyclic voltammetry in ACN using tetrabutylammonium hexafluorophosphate as the supporting electrolyte (potential vs. saturated calomel electrode–SCE). The free energy change (∆Get) for an electron transfer reaction was calculated using equation (2) [27], where Eox, Ered, E\*, and C represent the oxidation potential of the electron donor, the reduction potential of the electron acceptor, the excited state energy level (determined from luminescence experiments) and the coulombic term for the initially formed ion pair, respectively. Here, C was neglectedm as usually done for polar solvents.

$$
\Delta \mathbf{G}\_{\text{el}} = \mathbf{E}\_{\text{ox}} \text{ - } \mathbf{E}\_{\text{red}} \text{ - } \mathbf{E}^\* + \mathbf{C} \tag{2}
$$

#### *4.6. UV-Vis Absorption and Photolysis Experiments*

The absorption properties (UV-visible absorption spectrum and molar extinction coefficient) as well as the steady state photolysis of the investigated Coumarin derivatives (CoumA–CoumI) in acetonitrile have been investigated using a JASCO V730 spectrometer.

#### *4.7. Fluorescence Experiments*

The fluorescence properties of these organic compounds in ACN were studied using a JASCO FP-6200 spectrofluorimeter. The fluorescence quenching of <sup>1</sup> coumarin by Iod or NPG were examinated from the classical Stern-Volmer treatment [32] (I0/I = 1 + kq τ0[Q], where I<sup>0</sup> and I stand for the fluorescent intensity of coumarin in the absence and the presence of Iod or NPG, respectively; τ<sup>0</sup> stands for the lifetime of coumarin in the absence of Iod and [Q] stand for the concentration of quencher, in our study Iod or NPG).

#### *4.8. Computational Procedure*

Molecular orbital calculations were carried out with the Gaussian 03 suite of programs [33,34]. Electronic absorption spectra for the different compounds were calculated with the time-dependent density functional theory at the MPW1PW91-FC/6-31G\* level of theory on the relaxed geometries calculated at the UB3LYP/6-31G\* level of theory.

#### *4.9. Near-UV Conveyor for Photocomposite Synthesis*

Photocomposites have been achieved using glass fibers for the reinforcement and an organic resin based on acrylates (50%/50% *w*/*w*). The photosensitive resin has been deposited on glass fibers, then the mixture was irradiated using a LED conveyor at 385 nm (0.7 W/cm<sup>2</sup> ). A Dymax-UV conveyor was used, the distance between the belt and the LED was fixed to 15 mm, and the belt speed was fixed to 2 m/min.

#### *4.10. Laser Writing and 3D patterns Characterization*

For the direct laser write experiments, a computer-controlled laser diode at 405 nm (spot size = 50 µm) was used, and the 3D patterns obtained were characterized by a numerical optical microscope (DSX-HRSU from OLYMPUS Corporation) [35].

#### **5. Conclusions**

Nine coumarins varying by the substitution pattern at the 3- and 7-positions of the coumarin core have been tested and proposed as highly efficient photoinitiators for the FRP of meth(acrylates) functions under visible light irradiation using a LED at 405 nm. Remarkably, these photoinitiators can be used in 3D printing experiments and these dyes showed a very high efficiency in the photocomposite synthesis (significant curing of the surface and the bottom) using a LED conveyor at 385 nm. The challenge remains, therefore, to develop new coumarins absorbing at longer wavelengths e.g., in the near-infrared range for a better penetration of light into thick/filled samples.

**Author Contributions:** Conceptualization, J.L. and F.D.; methodology, J.L. and F.D.; software, B.G.; validation, all authors; formal analysis, J.L.; investigation, M.R. and G.N.; resources, J.L., F.D., J.T., T.H., and D.G.; data curation, J.L.; writing—original draft preparation, M.R., B.G., F.D., and J.L.; writing—review and editing, all authors; visualization, J.L.; supervision, J.L.; project administration, J.L.; funding acquisition, J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by The Association of Specialization and Scientific Guidance, the Centre National de la Recherche Scientifique, Aix Marseille Université and the Université de Haute Alsace. This research was also funded by the Agence Nationale de la Recherche (ANR agency) through the PhD grant of Guillaume Noirbent (ANR-17-CE08-0054 VISICAT project).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The Lebanese group would like to thank "The Association of Specialization and Scientific Guidance" (Beirut, Lebanon) for funding and supporting this scientific work.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


### *Article* **Synthesis and Biological Activity Evaluation of Coumarin-3- Carboxamide Derivatives**

**Weerachai Phutdhawong <sup>1</sup> , Apiwat Chuenchid <sup>2</sup> , Thongchai Taechowisan <sup>3</sup> , Jitnapa Sirirak <sup>2</sup> and Waya S. Phutdhawong 2,\***


**Abstract:** A series of novel coumarin-3-carboxamide derivatives were designed and synthesized to evaluate their biological activities. The compounds showed little to no activity against gram-positive and gram-negative bacteria but specifically showed potential to inhibit the growth of cancer cells. In particular, among the tested compounds, 4-fluoro and 2,5-difluoro benzamide derivatives (**14b** and **14e**, respectively) were found to be the most potent derivatives against HepG2 cancer cell lines (IC<sup>50</sup> = 2.62–4.85 µM) and HeLa cancer cell lines (IC<sup>50</sup> = 0.39–0.75 µM). The activities of these two compounds were comparable to that of the positive control doxorubicin; especially, 4-flurobenzamide derivative (**14b**) exhibited low cytotoxic activity against LLC-MK2 normal cell lines, with IC<sup>50</sup> more than 100 µM. The molecular docking study of the synthesized compounds revealed the binding to the active site of the CK2 enzyme, indicating that the presence of the benzamide functionality is an important feature for anticancer activity.

**Keywords:** coumarin3-carboxamides; coumarins; pyranocoumarins; anticancer activity; antibacterial activity

#### **1. Introduction**

Coumarin is one of the potent secondary metabolites in plants [1,2] and fungi [3], and it is characterized by several pharmacological properties [4]. Like decursin **1** and decursinol **2**, these coumarins have pyranocoumarin moiety (Figure 1), having been isolated from the medicinal plant Angelica genus and shown potential for treating inflammatory diseases such as cancer, hepatic fibrosis, diabetic retinopathy, and neurological disorders [5]. The dehydrated derivative of decursinol, xanthyletin **3**, has also been shown to possess several biological properties, such as anti-tumor and antibacterial activities [6]. With a benzopyrone skeleton, coumarin is versatile and can be easily transformed into a large variety of functionalized skeletons. As a result, many coumarin derivatives have been designed, synthesized, and evaluated to address broad biological activities [7] such as antibacterial [8], antifungal [9], antioxidant [10], anti-inflammatory [11], anticancer [12], anticoagulant [13], and antiviral activities [14]. The synthetic *N*-phenyl coumarin carboxamide **4a** has been designed and shown to possess high antibacterial activity against *Helicobacter pylori* (*H. pylori)*, with the minimum inhibitory concentration (MIC) = 1 µg/mL [15], while the benzyl substitution of coumarin carboxamides **4b–d** has been shown to arrest breast cancer cell (BT474 and SKBR-3) growth by inhibiting ErbB-2 and ERK1 MAP kinase. Moreover, compounds **4b**–**d** are specific to cancer cell lines, with no cytotoxicity against normal human fibroblasts [16]. In our ongoing search for novel compounds to overcome drug resistance, the diverse biological activities of coumarins have been interesting. In the current study,

**Citation:** Phutdhawong, W.; Chuenchid, A.; Taechowisan, T.; Sirirak, J.; Phutdhawong, W.S. Synthesis and Biological Activity Evaluation of Coumarin-3- Carboxamide Derivatives. *Molecules* **2021**, *26*, 1653. https://doi.org/ 10.3390/molecules26061653

Academic Editor: Maria João Matos

Received: 26 February 2021 Accepted: 12 March 2021 Published: 16 March 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

we designed novel pyranocoumarin-3-carboxamide derivatives with the expectation that the carboxamide part could possess active pharmacological properties **4a**–**d** and that the pyran ring moiety could also show specific biological proteins, as in the case of xanthyletin **3**. The synthesized compounds were examined to evaluate their antibacterial activity and cytotoxicity against HepG2 and HeLa cell lines. As the coumarins were attractive casein kinase 2 (CK2) inhibitors [17], molecular docking was used to study the possible interactions of novel coumarin-3-carboxamides with the CK2 enzymes.

**Figure 1.** Synthetic and natural-occurring coumarins with biological activities.

#### **2. Results and Discussion**

#### *2.1. Chemistry*

The preparation of pyranocoumarin-3-carboxamide was applied from the previous synthetic strategies reported by Faulgues and colleagues [18] and was described in Scheme 1. Commercially available 2,4-dihydroxy benzyldehyde **5** and 3-hydroxy-3-methyl-1-butene **6** were used as the starting materials and were subjected to Lewis acid–promoted Friedel-Crafts alkylation reaction in dioxane with BF3-diethylate to obtain the aldehyde **7** in 53% yield as a major product together with many unidentified products [19]. The aldehyde **7** was cyclized to form a pyrano ring using 2,3-dichloro-5,6-dicyano-l,4-benzoquinone (DDQ), and the benzopyran **8** was obtained in a very good yield. The cyclization of benzopyran **8** with the malonic anhydride **9** in pyridine and aniline at room temperature for 24 h according to a previous report [20] gave a poor yield of the pyranocoumarin-3-carboxylic acid **10,** due to the difficulty of purification. The amidation of the acid **10** with aniline using *N,N'*-dicyclohexyl carbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) gave the amide **12** in 11% yield after recrystallization.

**Scheme 1.** Synthesis of pyranocoumarin-3-carboxamide **12.**

To improve the yield of pyranocoumarin-3-carboxamide **12**, the coumarin-3-carboxylic acid **13** was prepared in good yield prior to amidation with appropriate anilines using

hexafluorophosphate azabenzotriazole tetramethyl Uronium (HATU) and Et3N to obtain amide **14a**–**g** in 43%–51% yields (Scheme 2). Then, the cyclization of **14a** by refluxing with DDQ in benzene gave pyranocoumarin-3-carboxamide **12** in 66% yield (Scheme 3). To study the effect of the substituent at C3 of coumarin ring, the carboxyl group was decarboxylated using Cu powder to give coumarin **15** in 53% yield (Scheme 4). Then, the synthesized coumarins were further evaluated for their antibacterial and anticancer activities.

**Scheme 2.** Synthesis of coumarin-3-carboxamides **14a**–**g.**

**Scheme 3.** Cyclization of pyranocoumarin-3-carboxamide **12.**

**Scheme 4.** Decarboxylation of coumarin-3-carboxylic acid.

#### *2.2. Antibacterial Activity*

Coumarin derivatives **10**, **12**, **13**, **14a**–**g**, and **15** were evaluated for their antibacterial activity against *Bacillus cereus*, *Bacillus subtilis*, *Staphylococcus aureus*, *Escherichia coli*, *Salmonella typhimuriumthrough* using the microbroth dilution method. Penicillin G and solvent were used as positive and negative controls, respectively, and the MIC (µg/mL) values were obtained (Table 1). The results show that only compounds **10** and **13** exhibited moderate antibacterial activities against gram-positive bacteria, while the other tested compounds displayed MIC values of more than 128 µg/mL. This may be because the carboxylic acid at the C3 position played an essential role in the antibacterial activity. Compound **15**, without the carboxyl group, showed no antibacterial activity, and the carboxamides **14a**–**g**, displayed no activity. Meanwhile, coumarin-3-carboxylic acid **13** was the most active among the tested compounds, with an MIC value of 32 µg/mL against *B. cereus*; however, it was less active than the reference drug penicillin G. Moreover, all the tested compounds showed no activity against any gram-negative bacteria.


**Table 1.** MIC of coumarin derivatives **10**, **12a**, **13**, **14a**–**g**, **15** and penicillin G against *B. cereus*, *B. subtilis*, *S. aureus*, *E. coli*, *S. typhimurium*.

#### *2.3. Anticancer Activity*

All synthesized compounds were evaluated for in vitro cytotoxic activity against two cancer cell lines (HepG2 and HeLa cell lines) and normal cell lines (LLC-MK2) through an MTT assay, and the results are presented in Table 2. Most of the tested compounds displayed potent anticancer activity. The *N*-phenyl coumarin-3-carboxamides **12** and **14a** showed significantly more potency than the parent acids **10** and **13**, respectively, against HepG2 cell lines. Moreover, compound **15**, with no substituent at C3, showed better activity than the parent acid **13**. The effect of substituents on the phenyl ring was compared with the effect of substituents on the carboxamide **14a** and it was found that the phenyl bearing fluorine atoms **14b** and **14e** possessed similar effects on the potency, while, the phenyl bearing 4-chlorine and 4-bromine atoms showed less activity against both cancer cell lines. Moreover, the phenyl bearing 4-methyl and 4-methoxyl groups displayed weak activity against the test cancer cell lines. From these results, the size and electron-donating group of the *para*-substituted benzene ring may affect anticancer properties. From these tested compounds, the amide **14e** displayed the most potent anticancer activity; however, it exhibited high cytotoxic activity against the normal cell line, with IC<sup>50</sup> = 1.33 µM. Interestingly, amide **14b** displayed slightly lower activity than **14e**, but it showed low cytotoxicity against the normal cell line. This compound possessed anticancer activity comparable to those of the tested anticancer drugs doxorubicin and acridine orange.

#### *2.4. Molecular Docking*

Casein Kinase 2 enzyme is a key player in the pathophysiology of cancer [21,22]. Using the iGEMDOCK v2.1 software [23], molecular docking was performed to investigate binding positions and intermolecular interactions between coumarin **10**, **12, 13**, **14a–f**, and **15** and the binding site of CK2. Coumarins **10**, **12**, **13**, **14a**–**f**, and **15** were docked to the active site of CK2 co-crystallized with **G12** (PDB ID: 2QC6). Moreover, **G12** was also redocked to CK2, and its total energy and hydrogen bond length were compared with those of coumarins **10**, **12, 13**, **14a**–**f**, and **15**. The molecular docking results show that the binding position of redocked **G12** was roughly the same as that of co-crystallized **G12** in CK2 (Figure 2a). Moreover, all synthesized compounds were bound to the active site of CK2, and the binding positions were similar to that of **G12** (Figure 2b,c), and their binding energies (−118.99 to −89.19 kcal/mol) were lower than that of **G12** (−79.10 kcal/mol) (Table 3). Figure 3 also illustrates the hydrogen bond interactions in the binding of coumarin **12**, **14a**, **14b**, **14d**, and **14e** in the cavity of CK-2 compared with that of **G12**. Coumarins **14a–f** had much lower binding energy (−118.99 to −105.53 kcal/mol) than **G12**, and their *N*-phenyl ring was also bound to similar positions of the **G12** phenolic ring (Figure 2c). Key

amino acid residues, including LYS68, ASN118, ASN117, and ASP175, formed a hydrogen bond with **14a**–**f**, where ASN117 and ASN118 interacted with the hydroxy group of **14a–f**, while *N*-phenyl ring interacted with LYS68 and ASP175. The substitution group on the *N*-phenyl ring of **14a**–**f** also influenced the number of hydrogen bonds in the CK2 active site. Moreover, a comparison of the halogen substitution groups on the *N*-phenyl ring showed that the Cl and Br substitution groups on the *N*-phenyl ring formed no hydrogen bond, while the F substitution group **14b** and **14e** could form hydrogen bonds with LYS68 and ASP175 (Figure 3d,f), which may be because Cl and Br are larger than F in size. Additionally, the experimental results show that the coumarins **14b** and **14e** had good anticancer activities.


**Table 2.** In vitro anticancer activities of synthesized coumarins compared with doxorubicin and acridine orange.

**Figure 2.** (**a**) Redocked **G12** (brown) and **G12** (green) in the cavity of CK-2 (PDB ID: 2QC6). (**b**) Comparison of the bindings of **10** (pink), **12** (sky blue), **13** (orange), 15 (violet), and **G12** (green) in the CK-2 cavity. (**c**) Comparison of the bindings of **14a** (yellow), **14e** (blue), **14f** (purple), **14g** (red), and **G12** (green) in the CK-2 cavity (PDB ID: 2QC6).


**Table 3.** Summary of binding energy, amino acid interactions, and hydrogen bond length of coumarin derivatives in CK2 binding site.

**Figure 3.** Hydrogen bond interactions in the bindings of (**a**) **G12**,(**b**) **12**,(**c**) **14a**, (**d**) **14b**,(**e**) **14d**, and (**f**) **14e** in the CK-2 cavity.

#### **3. Materials and Methods**

#### *3.1. Chemistry*

General information: Solvents and reagents were purchased from commercial suppliers TCI Chemicals (Tokyo, Japan), Sigma-Aldrich (Bangalore, India), and Fluka (Dorset, UK). Structure determination was conducted by analyzing the <sup>1</sup>H, <sup>13</sup>C, and <sup>19</sup>F NMR spectra (Bruker 300 apparatus) and the infrared (IR) spectrum was determined using PerkinElmer Frontier Fourier-transform infrared spectrometer. Melting point was conducted using Stuart SMP2 melting point apparatus and high-resolution mass spectroscopy was analyzed by Thermo scientific, Orbitrap Q Exactive Focus.

#### 3.1.1. Synthesis of 2,4-Dihydroxy-5-(3-methylbut-2-en-1-yl)-benzaldehyde **7**

First, 2,4-dihydroxy benzaldehyde **5** (1.5 g, 10.8 mmol) in dioxane (5 mL) was added to a stirred solution of 3-hydroxy-3-methyl-1-butene **6** (1.5 mL, 14.3 mmol) and boron trifluoride diethyl etherate (BF3-OEt2, 1.5 mL) in dioxane (3 mL), and stirring was continued for 2.5 h at room temperature. Dichloromethane (50 mL) was added, and the resulting solution was extracted with water (3 × 50 mL). The combined organic layer was dried over Na2SO<sup>4</sup> before evaporation to dryness and then purified via column chromatography (silica gel, 4:1 hexane:EtOAc) to obtain a white solid of 2,4-dihydroxy-5-(3-methylbut-2-en-1-yl) benzaldehyde **7** (0.48 g, 53% yield): m.p. 124–125 ◦C (lit. [19] 121–123 ◦C), <sup>1</sup>H-NMR (300 MHz, CDCl3): δ 11.27 (s, 1H), 9.69 (s, 1H, OH), 7.26 (s, 1H), 6.37 (s, 1H), 6.10 (s, 1H, OH), 5.30 (tt, *J* = 4.53, 1.35 Hz, 1H), 3.30 (d, *J* = 7.2 Hz, 2H), 1.77 (s, 3H), 1.61 (s, 3H) ppm.

#### 3.1.2. Synthesis of 7-Hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde **8**

A mixture of compound **7** (1.0 g, 4.85 mmol) and DDQ (1.2 g, 5.28 mmol) in benzene (10 mL) was refluxed for 6 h, and the precipitate was filtered off. The filtrate was evaporated to dryness to afford the crude product, which was purified via column chromatography (silica gel, 10:1 hexane:EtOAc) to obtain a white solid of 7-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde **8** (0.91 g, 92% yield): m.p. 82–83 ◦C (lit. [20] 95–96 ◦C), <sup>1</sup>H NMR (300 MHz, CDCl3): δ 11.41 (br, OH), 9.68 (s, 1H), 7.16 (s, 1H), 6.35 (s, 1H), 6.30 (d, *J* = 7.86 Hz, 1H), 5.59 (d, *J* = 9.93 Hz, 1H), 1.59 (s, 3H) 1.48 (s, 3H) ppm.

#### 3.1.3. Synthesis of 8,8-Dimethyl-2-oxo-2H,8H-pyrano[3,2-g]chromene-3-carboxylic acid **10**

Compound **8** (1.0 g, 4.90 mmol) and malonic acid **9** (1.0 g, 9.60 mmol) were dissolved in pyridine (5.5 mL) containing aniline (0.5 mL) and stirred for 24 h at room temperature. Afterward, the rection mixture was poured into ice-cold 10% HCl (80 mL). The yellow precipitate was washed with cold water to remove mineral acid and then air-dried to yield a yellow solid (recrystallization by 2:1:2; EtOAc:EtOH:hexane) of 8,8-dimethyl-2-oxo-2H,8H-pyrano[3,2-g]chromene-3-carboxylic acid **10** (0.23 g, 17% yield): m.p. 187–188 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3): δ 8.80 (s, 1H), 7.29 (s, 1H), 6.85 (s, 1H), 6.40 (d, *J* = 10.02 Hz, 1H), 5.81 (d, *J* = 10.02 Hz, 1H), 1.52 (s, 6H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3): δ 164.54 (C), 163.28 (C), 160.87 (C), 156.60 (C), 150.99 (CH), 132.34 (C), 127.13 (CH), 120.17 (CH), 120.06 (CH), 112.50 (C), 110.59 (C), 104.38 (CH), 79.33 (C), 28.75 (2CH3) ppm. IR: 3051.36 (C-H, aromatic), 2922.46 (C-H, aliphatic), 1743.12 (C=O, acid) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C15H13O<sup>5</sup> (M)<sup>+</sup> 273.0758, found 273.0757.

3.1.4. Synthesis of 8,8-Dimethyl-2-oxo-*N*-phenyl-2H,8H-pyrano[3,2-g]chromene-3 carboxamide **12**

A mixture of compound **10** (0.10 g, 0.36 mmol), aniline **11** (0.040 mL, 0.43 mmol), DCC (0.10 g, 0.44 mmol), and DMAP (8 mg, 0.065 mmol) in dry CH2Cl<sup>2</sup> (5 mL) was stirred at room temperature for 18 h. Afterward, the reaction mixture was filtered, and the filtrate was evaporated under vacuum. The residue was recrystallized using EtOH to obtain a yellow solid of 8,8-dimethyl-2-oxo-*N*-phenyl-2H,8H-pyrano[3,2-g]chromene-3-carboxamide **12 (**14 mg, 11% yield): m.p. 187–188 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3): δ 10.81(s, 1H), 8.89 (s, 1H), 7.75 (d, *J* = 1.14 Hz, 2H), 7.39 (t, *J* = 6.54 Hz, 2H), 7.30 (s, 1H), 7.18 (t, *J* = 1.14 Hz, 1H), 6.80 (s, 1H), 6.40 (d, *J* = 9.90 Hz, 1H), 5.78 (d, *J* = 9.96 Hz, 1H), 1.55 (s, 6H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3): δ 162.20 (C), 160.03 (C), 159.55 (C), 156.35 (C), 148.67 (CH), 137.30 (C), 131.82 (CH), 129.02 (2CH), 126.72 (CH), 124.57 (CH), 120.53 (2CH), 120.43 (CH), 119.58 (C), 114.71 (C), 112.73 (C), 75.72 (C), 28.64 (2CH3) ppm. IR: 3198.94 (N-H), 3059.35 (C-H, aromatic), 2969.10 (C-H, aliphatic), 1699.85 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H18O4N (M)<sup>+</sup> 348.1230, found 348.1230.

#### 3.1.5. Synthesis of 3-Carboxy-6-(3-methyl-2-butenyl)-7-hydroxy-coumarin **13**

Compound **7** (1.0 g, 4.85 mmol) and malonic acid **9** (1.0 g, 9.60 mmol) were dissolved in pyridine (5.5 mL) containing aniline (0.5 mL), and stirred for 24 h at room temperature. Afterward, the reaction mixture was poured into ice-cold 10% HCl (80 mL), and the yellow precipitate obtained was washed with cold water to remove mineral acid and then air-dried to yield a yellow solid of 3-carboxy-6-(3-methyl-2-butenyl)-7-hydroxy-coumarin **13** (1.10g, 88% yield): m.p. 237–238 ◦C (lit. [20] 218–224 ◦C), <sup>1</sup>H NMR (300 MHz, CDCl3+methanol-d4) δ 8.79 (s. 1H), 7.40 (s, 1H), 6.85 (s, 1H), 5.32 (tt, *J* = 7.4, 1.3 Hz, 1H), 3.34 (d, *J* = 7.29 Hz, 2H), 1.79 (s, 3H), 1.70 (s, 3H) ppm., <sup>13</sup>C-NMR (75 MHz, CDCl3+methanol-d4): δ 163.58 (C), 162.79 (C), 162.37 (C), 154.78 (C), 150.43 (CH), 133.49 (C), 129.32 (CH), 127.92 (C), 119.16 (CH), 110.24 (C), 107.85 (C), 100.76 (CH), 26.34 (CH2), 24.42 (CH3), 16.50 (CH3) ppm., IR: 3303.61 (O-H), 3049.81 (C-H, aromatic), 2911.92 (C-H, aliphatic), 1733.10 (C=O, acid), 1718.83 (C=O, lactone) cm−<sup>1</sup> .

3.1.6. General Procedure of Coumarin-3-carboxamides Preparation (**14a**–**g**)

Triethanolamine (TEA) (0.1 mL, 1.36 mmol) was added to a solution of compound **13** (70 mg, 0.26 mmol) and HATU (0.12 g, 0.36 mmol) in dry THF (5 mL), and the mixture was stirred at room temperature for 30 min. The obtained dark clear mixture was treated with aniline derivatives (**11a**–**g**) (1.2 eq.). The resulting mixture was stirred at room temperature for 18 h. Dichloromethane (50 mL) was added, and the resulting solution was extracted with sat. NaCl (3 × 30 mL). The remaining organic layer was dried over Na2SO<sup>4</sup> before evaporation to dryness. After evaporation of the solvent in vacuo, the crude product was purified via preparative thin-layer chromatography (silica gel, 4:1 hexane:EtOAc) to give yellow solids of coumarin-3-carboxamide (**14a**–**g**)

7-Hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-*N*-phenyl-2H-chromene-3-carboxamide **14a** (47 mg, 51% yield): m.p. 258–259 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridine-d5): δ 10.86 (s, NH), 8.90 (s, 1H), 7.73 (d, *J* = 7.62 Hz, 2H), 7.42 (s, 1H), 7.35 (t, *J* = 7.62 Hz, 2H), 7.12 (t, *J* = 7.38 Hz, 1H), 6.84 (s, 1H), 5.40 (tt, *J* = 7.26, 1.35 Hz, 1H), 3.43 (d, *J* = 7.26 Hz, 2H), 1.81 (s, 3H), 1.74 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.38 (C), 162.81 (C), 160.56 (C), 155.49 (C), 149.36 (CH), 138.08 (CH), 134.13(C), 130.04 (CH), 128.97 (2CH), 128.68 (C), 124.35 (CH), 121.14 (CH), 120.49 (2CH), 112.94 (C), 111.33 (C), 101.68 (CH), 27.85 (CH2), 25.84 (CH3), 17.85 (CH3) ppm., IR: 3195.27 (O-H), 2917.31 (C-H, aliphatic), 1695.54 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H20O4N (M)<sup>+</sup> 350.1387, found 350.1386.

*N*-(4-Fluorophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-carboxamide **14b** (45 mg, 47% yield): m.p. 259–261 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3): δ 10.56 (br, NH), 8.89 (s, 1H), 7.68 (dd, *J* = 8.97, 4.92 Hz, 2H), 7.42 (s, 1H), 7.07 (dd, *J* = 9.12, 8.79 Hz 2H), 6.83 (s, 1H), 5.34 (t, *J* = 7.23 Hz, 1H), 3.35 (d, *J* = 7.29 Hz, 2H), 1.80 (s, 3H), 1.72 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3): δ 163.04 (C), 162.57 (C), 160.72 (C), 159.64 (d, *J* = 242.5 Hz, C), 155.46 (C), 149.71 (CH), 134.43 (C), 133.84 (d, *J* = 3.0 Hz, C), 130.23 (CH), 128.80 (C), 120.91 (CH), 115.72 (d, *J* = 22.5 Hz, 2CH), 112.86 (C), 112.31 (d, *J* = 7.5 Hz, 2CH), 111.68 (C), 101.70 (CH), 27.74 (CH2), 25.87 (CH3), 17.82 (CH3) ppm., <sup>19</sup>F NMR (282 MHz, CDCl3, std. TFA): −118.08 (s, 1F) ppm., IR: 3208.66 (O-H), 3155.21 (N-H), 3048.54 (C-H, aromatic), 2913.86 (C-H, aliphatic), 1698.12 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H19O4NF (M)<sup>+</sup> 368.1293, found 368.1293.

*N*-(4-Chlorophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-carboxamide **14c** (44 mg, 44% yield): m.p. 282–283 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridined5): δ 10.92 (s, NH), 8.89 (s, 1H), 7.69 (d, *J* = 8.88 Hz, 2H), 7.37 (s, 1H), 7.30 (dd, *J* = 8.89, 2.01 Hz, 2H), 6.83 (s, 1H), 5.41 (t, *J* = 7.23 Hz, 1H), 3.43 (d, *J* = 7.23 Hz, 2H), 1.81 (s, 3H), 1.74 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.65 (C), 162.81 (C), 160.64 (C), 155.57 (C), 149.50 (CH), 136.76 (C), 134.06 (C), 130.09 (CH), 129.15 (C), 128.95 (2CH), 128.83 (C), 121.64 (2CH), 121.17 (CH), 112.56 (C), 111.27 (C), 101.68 (CH), 27.85 (CH2), 25.83 (CH3), 17.84 (CH3) ppm. IR: 3196.03 (O-H), 3122.34 (N-H), 3073.18 (C-H, aromatic), 2911.85 (C-H, aliphatic), 1698.47 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H19O4N35Cl (M)<sup>+</sup> 384.0997, found 384.0996.

*N*-(4-Bromophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3-carboxamide **14d** (52 mg, 47% yield)): m.p. 276–277 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridined5): δ 10.92 (s, NH), 8.89 (s, 1H), 7.69 (d, *J* = 8.88 Hz, 2H), 7.44 (d, *J* = 9.66 Hz, 2H), 7.39 (s, 1H), 6.83 (s, 1H), 5.40 (t, *J* = 7.14 Hz, 1H), 3.43 (d, *J* = 7.26 Hz, 2H), 1.81 (s, 3H), 1.73 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.67 (C), 162.80 (C), 160.65 (C), 155.57 (C), 149.51 (CH), 137.25 (C), 134.04 (C), 131.89 (2CH), 130.09 (CH), 128.83 (C), 121.96 (2CH), 121.16 (CH), 116.80 (C), 112.52 (C), 111.25 (C), 101.67 (CH), 27.85 (CH2), 25.83 (CH3), 17.84 (CH3) ppm. IR: 3192.94 (O-H), 3070.45 (C-H, aromatic), 2915.14 (C-H, aliphatic), 1697.23 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H19O4N79Br (M)<sup>+</sup> 428.0492, found 428.0492.

*N*-(2,5-Difluorophenyl)-7-hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3 carboxamide **14e** (46 mg, 46% yield): m.p. 248–250 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridined5): δ 12.27 (s, NH), 8.90 (s, 1H), 8.40 (ddd, *J* = 10.46, 6.66, 3.15 Hz, 1H), 7.43 (s, 1H), 7.04 (ddd, *J* = 9.57, 9.18, 4.89 Hz, 1H), 6.86 (s, 1H), 6.65–6.75 (m,1H), 5.41 (t, *J* = 7.35 Hz, 1H), 3.43

(d, *J* = 7.23 Hz, 2H), 1.81 (s, 3H), 1.74 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.90 (C), 162.63 (C), 160.90 (C), 158.57, (d, *J* = 238.5 Hz, C), 158.55, (d, *J* = 239.3 Hz, C), 155.75 (C), 149.73 (C), 134.03 (C), 130.18 (CH), 128.88 (C), 127.65 (d, *J* = 11.3 Hz, CH), 121.19 (CH), 115.20 (dd, *J* = 27.4, 9 Hz, CH), 112.28 (C), 111.18 (C), 110.03 (dd, *J* = 24.0, 7.5 Hz, CH), 109.09 (d, *J* = 30 Hz, CH), 101.74 (CH), 27.83 (CH2), 25.82 (CH3), 17.83 (CH3) ppm. <sup>19</sup>F NMR (282 MHz, CDCl3+pyridine-d5, std. TFA): −117.72 (d, *J* = 14.10 Hz, 1F), -136.03 (d, *J* = 14.10 Hz, 1F) ppm., IR: 3252.42 (O-H), 3130.54 (N-H), 2073.1 (C-H, aromatic), 2915.45 (C-H, aliphatic), 1701.64 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C21H17O4NF<sup>2</sup> (M+Na)<sup>+</sup> 408.1018, found 408.1013.

7-Hydroxy-6-(3-methylbut-2-en-1-yl)-2-oxo-N-(p-tolyl)-2H-chromene-3-carboxamide **14f** (41 mg, 43% yield): m.p. 276–277 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridine-d5): δ 10.80 (s, NH), 8.90 (s, 1H), 7.65 (d, *J* = 7.44 Hz, 2H), 7.41 (s, 1H), 7.15 (d, *J* = 8.31 Hz, 2H), 6.85 (s, 1H), 5.41 (tt, *J* = 7.32, 1.35 Hz, 1H), 3.43 (d, *J* = 7.20 Hz, 2H), 2.31 (s, 3H), 1.81 (s, 3H), 1.74 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.33 (C), 162.79 (C), 160.40 (C), 155.45 (C), 149.19 (CH), 135.56 (C), 134.01 (C), 133.91 (C), 130.00 (CH), 129.47 (2CH), 121.22 (CH), 120.45 (2CH), 113.03 (C), 111.31 (C), 101.66 (CH), 27.86 (CH2), 25.83 (CH3), 20.91 (CH3), 17.84 (CH3) ppm. IR: 3187.07 (O-H), 3130.54 (N-H), 3073.13 (C-H, aromatic), 2913.70 (C-H, aliphatic), 1698.29 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C22H22O4N (M)<sup>+</sup> 364.1543, found 364.1540.

7-Hydroxy-*N*-(4-methoxyphenyl)-6-(3-methylbut-2-en-1-yl)-2-oxo-2H-chromene-3 carboxamide **14g** (43 mg, 44% yield): m.p. 259–261 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3+pyridined5): δ 10.74 (s, NH), 8.88 (s, 1H), 7.63 (d, *J* = 9.03 Hz, 2H), 7.41 (s, 1H), 6.91 (d, *J* = 9.06 Hz, 2H), 6.82 (s, 1H), 5.34 (tt, *J* = 7.35, 1.47 Hz, 1H), 3.82 (s, 3H), 3.36 (d, *J* = 7.20 Hz, 2H), 1.80 (s, 3H), 1.72 (s, 1H) ppm., <sup>13</sup>C NMR (75 MHz, CDCl3+pyridine-d5): δ 163.00 (C), 162.28 (C), 160.44 (C), 156.65 (C), 156.65 (C), 155.34 (C), 149.37 (CH), 134.40 (C), 131.01 (C), 130.16 (CH), 128.33 (C), 122.29 (2CH), 120.94 (CH), 113.20 (C), 111.74 (C), 101.71 (CH), 55.56 (CH3), 27.76 (CH2), 25.81 (CH3), 17.82 (CH3) ppm. IR: 3182.53 (O-H), 3111.40 (N-H), 3073.14 (C-H, aromatic), 2911.91 (C-H, aliphatic), 1695.83 (C=O, amide) cm−<sup>1</sup> ; HREI-MS (*m*/*z*) calculated for C22H22O5N (M)<sup>+</sup> 380.1493, found 380.1490.

3.1.7. Synthesis of 8,8-Dimethyl-2-oxo-*N*-phenyl-2H,8H-pyrano[3,2-g]chromene-3 carboxamide **12**

A mixture of compound **14a** (1.7 g, 4.85 mmol) and DDQ (1.2 g, 5.28 mmol) in benzene (10 mL) was refluxed for 6 h, and the precipitate was filtered off. The filtrate was evaporated to dryness to afford the crude product, which was purified via column chromatography (silica gel, 10:1 hexane:EtOAc) to obtain a white solid of 8,8-dimethyl-2-oxo-N-phenyl-2H,8H-pyrano[3,2-g]chromene-3-carboxamide **12** (1.11 g, 66% yield): m.p. 187–188 ◦C, <sup>1</sup>H NMR (300 MHz, CDCl3): δ 10.81(s, 1H), 8.89 (s, 1H), 7.75 (d, *J* = 1.14 Hz, 2H), 7.39 (t, *J* = 6.54 Hz, 2H), 7.30 (s, 1H), 7.18 (t, *J* = 1.14 Hz, 1H), 6.80 (s, 1H), 6.40 (d, *J* = 9.90 Hz, 1H), 5.78 (d, *J* = 9.96 Hz, 1H), 1.55 (s, 6H) ppm.

#### 3.1.8. Synthesis of 6-(3-Methyl-2-buteny1)-7-hydroxycoumarin **15**

Compound **13** (0.20 g, 0.73 mmol) in 2 mL quinoline containing 0.3 g Cu powder was heated for 3 min at 215–220 ◦C in an oil bath. The mixture was cooled to room temperature and diluted with CH2Cl<sup>2</sup> (30 mL) prior to extraction with 10% HCI (2 × 30 mL) and then with water (30 mL). The solvent was evaporated, leaving a tacky orange solid, which was purified via column chromatography (silica gel, 1:1 hexane:EtOAc) to yield a cream solid of 6-(3-Methyl-2-buteny1)-7-hydroxycoumarin **15** (0.09 g, 53% yield), m.p. 134–136 ◦C (lit. 133 ◦C [24]), <sup>1</sup>H NMR (300 MHz, CDCl3): δ 7.67 (d, *J* = 9.42 Hz, 1H), 7.20 (s, 1H), 7.06 (s, 1H), 6.24 (d, *J* = 9.42 Hz, 1H), 5.33 (tt, *J* = 8.73, 1.41 Hz, 1H), 3.38 (d, *J* = 7.23 Hz, 2H), 1.78 (s, 3H), 1.75 (s, 3H).

#### *3.2. Determination of Antibacterial Activity*

The antibacterial activities of coumarin derivatives **10**, **12**, **13**, **14a–g**, and **15** were evaluated against five reference standard bacteria, both gram-positive and gram-negative: *B. cereus* TISTR 2372, *B. subtilis* TISTR 001, *S. aureus* TISTR 2392, *E. coli* TISTR 073, and *S. typhimurium* TISTR 2519, using a standard microbroth dilution method [25].

The MIC values of coumarin derivatives **10**, **12**, **13**, **14a–g**, and **15** were determined through the microbroth dilution method in 96-well microtitre plates. The bacterial cultures were prepared from overnight cultures on nutrient broth (NB) at 37 ◦C for 24 h by diluting in NB compared with 0.5 McFarland. Coumarin derivatives **10**, **12**, **13**, **14a–g**, and **15** (5000 µg/mL) were prepared in EtOH, and 128 µg/mL of these were added to the first wells. Two-fold serial dilutions were prepared, and final concentrations of 128 to 1 µg/mL were achieved. The positive controls for penicillin G were determined, with the final concentrations from 128 to 1 µg/mL. In addition, an extra row of EtOH was used as a vehicle control to determine its possible inhibitory activity. Finally, 10 µL of bacterial suspension was added to each well. After the bacteria were incubated at 37 ◦C for 24 h, the microtitre plates were visually examined for bacterial growth; the growth rate was monitored at the optical density at 600 nm with a microplate reader. In each row, the well containing the lowest concentration that showed no visible growth was considered the MIC.

#### *3.3. Cell Viability Assay*

The cell viability assays of coumarin derivatives **10**, **12**, **13**, **14a**–**g**, and **15** were conducted against three cancer cell lines (HepG2, HeLa, and MDA-MB-231) and one normal cell line (LLC-MK2) using an MTT assay [26].

Stock solutions of coumarin derivatives **10**, **12a**, **13**, **14a**–**g**, and **15** were prepared in EtOH at a concentration of 5000 µg/mL. Prior to use, the stock solutions were further diluted to 128 µg/mL and added to the first wells. Two-fold serial dilutions were prepared, and final concentrations of 128 to 1 µg/mL in culture medium were achieved. Cells were seeded at a density of 5 × 104 cells/well in a 96-well plate and incubated for 16 h, followed by treatment with the test compounds. The control culture contained the carrier solvent of 2.5% dimethyl sulfoxide (DMSO). After 24 h, HepG2, HeLa, MDA-MB-231, and LLC-MK2 cells were then incubated with MTT (500 µg/mL) for 4 h. Then, DMSO was added to dissolve the blue formazan crystals formed, which were formed as a result of the action of cellular oxidoreductase enzymes on the MTT dye. Finally, the optical density at 450 nm was determined using a microplate reader.

#### **4. Conclusions**

We designed and synthesized a series of coumarin-3-carboxamides and evaluated their antibacterial and anticancer activities. The carboxylic acid at the C3 position of coumarins was necessary for the antibacterial activity, as seen for compounds 10 and 13, which showed moderate antibacterial activities against the tested gram-positive bacteria. Meanwhile, most of the tested compounds showed potent anticancer activity, and the 4-fluorophenyl coumarin-3′ -carboxazine 4b was by far the most active anticancer, with activity comparable to that of the anticancer drug doxorubicin, and it had low cytotoxicity against a normal cell line. The molecular docking study revealed the binding to the active site of the CK2 enzyme, indicating that the presence of the phenyl carboxamide is important for anticancer activity.

**Author Contributions:** Conceptualization, project administration, supervision, W.P.; Methodology, investigation, validation, writing—original draft, A.C.; conceptualization, methodology, microbiology testing supervision, T.T.; molecular modeling supervision, J.S.; methodology, conceptualization, project administration, writing—review and editing, supervision, W.S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** We gratefully acknowledge the Department of Chemistry and the Department of Microbiology, Faculty of Science, Silpakorn University, for the financial support and antibacterial and anticancer assay. We also thank the Chulabhorn Research Institute for the measurements of the HR-ESI mass spectroscopy investigation and the Rice Department.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Studies of Coumarin Derivatives for Constitutive Androstane Receptor (CAR) Activation**

**Shin-Hun Juang <sup>1</sup> , Min-Tsang Hsieh 1,2 , Pei-Ling Hsu 1,†, Ju-Ling Chen 1,3,†, Hui-Kang Liu <sup>4</sup> , Fong-Pin Liang <sup>1</sup> , Sheng-Chu Kuo <sup>1</sup> , Chen-Yuan Chiu <sup>5</sup> , Shing-Hwa Liu <sup>5</sup> , Chen-Hsi Chou <sup>3</sup> , Tian-Shung Wu <sup>3</sup> and Hsin-Yi Hung 3,\***


**Abstract:** Constitutive androstane receptor (CAR) activation has found to ameliorate diabetes in animal models. However, no CAR agonists are available clinically. Therefore, a safe and effective CAR activator would be an alternative option. In this study, sixty courmarin derivatives either synthesized or purified from *Artemisia capillaris* were screened for CAR activation activity. Chemical modifications were on position 5,6,7,8 with mono-, di-, tri-, or tetra-substitutions. Among all the compounds subjected for in vitro CAR activation screening, 6,7-diprenoxycoumarin was the most effective and was selected for further preclinical studies. Chemical modification on the 6 position and unsaturated chains were generally beneficial. Electron-withdrawn groups as well as long unsaturated chains were hazardous to the activity. Mechanism of action studies showed that CAR activation of 6,7-diprenoxycoumarin might be through the inhibition of EGFR signaling and upregulating PP2Ac methylation. To sum up, modification mimicking natural occurring coumarins shed light on CAR studies and the established screening system provides a rapid method for the discovery and development of CAR activators. In addition, one CAR activator, scoparone, did showed anti-diabetes effect in *db*/*db* mice without elevation of insulin levels.

**Keywords:** Yin Chen Hao; constitutive androstane receptor; coumarin; scoparone

#### **1. Introduction**

Constitutive androstane receptor (CAR, NR1I3), a member of the superfamily of nuclear receptors, is a xenobiotic receptor responsible for the regulation of drug metabolism as well as the pathological involvement of various diseases such as cancer, diabetes, inflammatory disease, metabolic disease and liver disease, suggesting a potential target for drug discovery [1]. Predominantly expressed in the liver, once CAR becomes activated, it disassociates from the cytoplasmic protein complex and with retinoid X receptor (RXR) binds to specific DNA region and influence target gene expression [2].

CAR has been proven to involve in various energy pathways as well as metabolic pathways [3]. The well-established function of CAR is to regulate bile acid detoxification and bilirubin clearance via the induction of metabolizing enzymes and transporters, such as Mrp2–4, Oatp 2, Cyp3a11, Sult2a1 and Ugt1a1 [4–6]. Moreover, CAR knock out (KO) mice

**Citation:** Juang, S.-H.; Hsieh, M.-T.; Hsu, P.-L.; Chen, J.-L.; Liu, H.-K.; Liang, F.-P.; Kuo, S.-C.; Chiu, C.-Y.; Liu, S.-H.; Chou, C.-H.; et al. Studies of Coumarin Derivatives for Constitutive Androstane Receptor (CAR) Activation. *Molecules* **2021**, *26*, 164. https://doi.org/10.3390/ molecules26010164

Received: 16 November 2020 Accepted: 28 December 2020 Published: 31 December 2020

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**Copyright:** © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

led to high incidence of hepatic necrosis and high level of alanine aminotransferase (ALT) and bilirubin, suggesting an important function of CAR activation for hepatoprotective effect and a potential role in treating cholestasis [7]. A direct CAR agonist, TCPOBOP, was found to decrease liver injury and hepatocyte apoptosis in the treated mice as well as to improve insulin sensitivity in ob/ob mice model [8]. In addition, CAR activation ameliorates hyperglycemia as well as fatty liver by suppressing glucose production, stimulating glucose uptake and usage in the liver and inhibiting hepatic lipogenesis and induction of β–oxidation [9]. Recent metabolomic research revealed CAR activation significantly lower mRNA expression that involved in gluconeogenic pathway, up-regulate glucose utilization pathways, enhance specific fatty acid synthesis and impair β–oxidation [10]. β–

Since CAR is a potential target for various diseases, neither CAR agonists nor antagonists are seen clinically. 6-(4-Chlorophenyl) imidazo [2,1-b] [1,3] thiazole-5-carbaldehyde *O*-(3,4-dichlorobenzyl) oxime (CITCO), a human CAR agonist, is used extensively in biological studies, but failed to be further developed due to toxicity. Another commonly used agonist, TCPOBOP (Figure 1), is a mouse CAR agonist, which also points out the difficulty of developing CAR activators—species specificity [11]. Luckily, Yin Chen Hao (*Artemisia capillaris*) in the Asteraceae family has long been used to treat jaundice in Traditional Chinese Medicine (TCM). The extract of Yin Chen Hao Tang (YCHT) was found to activate CAR and then accelerated bilirubin clearance in animal models [12,13]. In 2015, a meta-analysis studied 15 randomized controlled trials (RCT) including 1405 cases ranging from 2000 to 2014 and found YCHT reduced the elevated levels of cholestasis serum markers significantly either in short or long curative time periods and also improved the clinical efficiency of other anti-cholestasis medications [14]. Further, 6,7-dimethylesculetin (scoparone), a major active principal isolated from Yin Chin Hao, also exerted CAR activation activity both in vitro and in vivo [12]. Scoparone exerted plenty of pharmacological activites, such as hepatoprotective effect, antioxidation, anti-inflammation, and anti-cholestasis [15]. In addition, scoparone (10 µM) potentiated chenodeoxycholic acid (10 µM, a potent FXR agonist) effect to increase the expression of bile salt export pump. However, scoparone alone (up to 100 µM) had no effect on FXR activation [16]. β– μ μ μ

**Figure 1.** Structures of CAR direct activators. TCPOBOP is a mouse CAR direct activator. CITCO is a human CAR direct activator.

Inspired by the findings above, this study was designed to mimic coumarins from Yin Chen Hao, to systemically synthesize coumarin derivatives to study structure-activity relationship of coumarin derivatives on CAR activation and to establish in vitro CAR activation screening method for discovering and developing therapeutic CAR activation agents in the future. Further, the mechanism of action of the most active compound will be discussed and a possible indication, anti-diabetes, of a CAR activator will be evaluated in vivo.

#### **2. Results and Discussion**

#### *Chemistry*

Although coumarin derivatives have been studied extensively, their effects on CAR activation are largely underinvestigated. In coumarins isolated from Yin Chen Hao, all the substitutions are on C5, C6, C7, C8 positions [17]. Since the exact binding site of scoparone is unclear, ligand-based drug design is applied. The first strategy is to evaluate

the substitution effect on C5, C6, C7, C8 positions alone and together to establish SAR (Tables 1–3). Various alkoxy groups including different chain length, branch, and unsaturation were synthesized and studied in these four positions. These modifications included the structures of natural coumarins, such as 5-hydroxycoumarin, 5-methoxycoumarin, umbeliferone, methylumbeliferone, aurapten, 8-hydroxycoumarin, 8-methoxycoumarin, esculetin, isoscopoletin, scopoletin, scoparone, ayapin, isoscopolin, scopolin, fraxinol, and leptodactylone. Their natural occurrence is provided in the reference column of the tables. From the references, we can find these coumarins can be found in many clinically used TCMs or herbs. Moreover, we also synthesized eight new derivatives (compounds **11**, **20**, **44**, **46–49**, **50**) for comparison of SAR. The syntheses of the coumarin derivatives were mainly via Perkin reaction [18] or Knoevenagel reaction [19] or Pechmann reaction [20] or Wittig reactions [21,22] or others [23,24] and the spectral data for the known compounds were identical with the literature values. – **–**

**Table 1.** Mono-substituted coumarin structures and their CAR activation.



**Table 1.** *Cont.*

<sup>a</sup> CAR activation fold was calculated: (the luminescence value of the tested compound- the luminescence value of scoparone)/the luminescence value of scoparone (as positive control) × 100%. 

**Table 2.** Di-substituted coumarins and their CAR activation.


<sup>a</sup> CAR activation fold was calculated: (the luminescence value of the tested compound- the luminescence value of scoparone)/the luminescence value of scoparone (as positive control) × 100%.


**Table 3.** Tri- and tetra-substituted coumarins and their CAR activation.

<sup>a</sup> CAR activation fold was calculated: (the luminescence value of the tested compound- the luminescence value of scoparone)/the luminescence value of scoparone (as positive control) × 100%.

Moreover, syntheses of coumarins disubstituted mainly on the C6 and C7 positions were carried out (Table 2) The starting material, 6,7-dihydroxycoumarin, was commercially available. Various alkoxyl substitutions were synthesized by treating 6,7-dihydroxycoumarin in dimethylformamide with the corresponding alkyl bromide. Tri-and tetrasubstituted coumarins were also synthesized and are summarized in Table 3. Scopolin (**53**), isoscopolin (**52**), compound **59** and **60** were isolated from Yin Chen Hao [17].

#### **3. Establishment of In Vitro CAR Activation Screening Assay**

μ Until now, there is no commercially available high throughput screening method for CAR activation. Traditionally, CAR activation can be detected in western blots by its downstream signals such as UGT1A1, MRP2 or CYP2B6. In Figure 2, we confirmed that the protein expressions of downstream genes of CAR, UGT1A and MRP2, were upregulated after scoparone and CITCO treatment. However, this method was slow and pricy. In order to rapidly screen all the compounds for CAR activation at less cost, an in vitro CAR activation screening assay was developed: DNA was extracted from Hep3B cells and UGT1A1 promoter was obtained using PCR amplification. Ligation between UGT1A1 promoter and pGL4 vector was performed and the plasmid was amplified via *E. coli* replication. The desired plasmid was extracted from E. coli and transfected into Hep3B. The desired colony bearing UGT1A1 promoter-pGL4 plasmid was selected by neomycin. CAR activation was measured by adding tested compounds to Hep3B for several hours and luminescence was measured. Higher activation will get higher luminescence readout. This assay system was validated with two different CAR agonists: CITCO (direct agonist) and scoparone (an indirect agonist). CITCO could induce about 50% elevation of luciferase activity while scoparone as a positive control could enhance luciferase activity too. Different concentration of scoparone at 1, 5, 10 and 20 µM had been tested either for luciferase assay or downstream UGT1A1 and MRP2 expression (data not shown) and 10 µM was selected to be the best condition for the following work.

μ

After successfully establishing the in vitro assay, all of the coumarin compounds were subjected to the screening (at 10 µM) and their SAR was discussed. Although the reporter system can successfully identify CAR-activating compounds under 8 h and display better efficiency and sensitivity compared with western blots, it only presented a medium luciferase response. According to previous studies, CITCO was reported to only moderately

enhance human CAR (less than 2-fold), compared with the enhance provided by TCPOBOP for murine CAR (5- to 10-fold) in luciferase reporter assays [11,54]. These results suggested that human CAR might have a weaker response to agonists compared to murine CAR, which might be the same situation as in our reporter system. Another possibility for the medium response in our reporter system might be the contribution of the whole gtPBREM element which might include response-suppressive elements that was used and the single nucleotide variation of DR3 element which was reported to slightly reduce the original UGT1A1 activity [55].

**Figure 2.** CAR downstream signal protein expression after treatment of CITCO or scoparone.

#### **4. Structure-Activity Relationships**

μM) – μ μ All the compounds were compared to scoparone (**39**) and the resulting CAR activation fold results (%) are shown in Tables 1–3. This is the readout of the tested compound minus the readout of scoparone and then divided by the scoparone readout. Among all sixty compounds, twenty compounds exhibited better or similar CAR activation ability than scoparone and 6,7-diprenoxycoumarin (**50**) was the best compound with a 50% increased CAR activation fold. In addition, none of the compounds have any cytotoxicity at this concentration (IC<sup>50</sup> > 50 µM) except for **47** (IC<sup>50</sup> = 31 µM). As for a detailed SAR discussion of the mono- and disubstituted coumarins, hydroxyl groups except on position 8 exhibited similar or less activity than scoparone. Methoxy substitution is better on position 6 than positions 5, 7 or 8. Electron withdrawing groups, such as an acetyl group or a trifluoromethyl group decrease the activity, suggesting the importance of enough electrons. A medium carbon chain length such as a *n*-butyl group on the 6 position is good. Prenyl groups are general good, except in the 7 position. Longer unsaturated chains such as geranyl or farnesyl reduce the activity. To sum up, electron withdrawing groups would be detrimental to the activity. Modification on the 6 position generally increases the activity. Longer unsaturated chains are not good for the activity. Among tri- and tetra-substituted coumarins, 5,7-dimethoxy-8-hydroxycoumarin (**57**) and 6,7,8-trimethoxycoumarin (**58**) showed comparable CAR activation effects to scoparone. 6-Methoxy-7,8-methylenedioxycoumarin (**59**) exhibited a good activation effect (36% increase) and 5,6-dimethoxy-7,8- methylenedioxycoumarin (**60**) also had good activity.

Moreover, if we examined coumarins reported to exist in Yin Chen Hao, such as esculetin (**35**), isoscopoletin (**37**), scopoletin (**38**), scoparone (**39**), scopolin (**53**), isoscopolin (**52**), 5,6,7-trimethoxycoumarin (**54**), leptodactylone (**57**), compound **59** and **60**, we can find that compounds **52**, **53**, **57**, **59** and **60** had superior CAR activating activity than scoparone, implying that the anti-cholestasis effect of YCHT might be an added effect of all the coumarins inside.

#### **5. Mechanistic Studies of 6,7-Diprenoxycoumarin (50)**

First, the translocation of CAR protein was confirmed after treatment with **50** (Figure 3A), and the translocation activity could be inhibited by okadaic acid (OA), the phosphatase

inhibitor which inhibits CAR from translocating into the nucleus. In addition, the protein expression of nuclear CAR increased after **50** treatment as shown in Figure 3B.

**Figure 3.** Image of immunofluorescence staining for CAR translocation in Hep3B cells after 6,7-diprenoxycoumarin (**50**) (10 μM) for 2 h. The negative control group **Figure 3.** Image of immunofluorescence staining for CAR translocation in Hep3B cells after 6,7-diprenoxycoumarin (**50**) treatment (**A**) and scoparone treatment (**C**). Hep3B cells were treated with **50** (10 µM) for 2 h. The negative control group was pre-treated with 10 nM okadaic acid (OA). The blue and green fluorescence stands for signals of nuclei and CAR protein, respectively. The protein expression of nuclear CAR after 6,7-diprenoxycoumarin (**50**) treatment for 2 h (**B**).

Like phenobarbital-mediated CAR activation, **50** activated CAR through inhibiting EGFR phosphorylation [56]. Western blot results indicated that **50** could reduce EGFinduced phosphorylation of EGFR in Hep3B (Figure 4). The up-stream protein, PP2Ac, which is responsible for CAR dephosphorylation was increased with its active form, methyl-PP2Ac (Figure 5).

) of 10 μM **Figure 4.** 6,7-Diprenoxycoumarin (**50**) antagonized EGF-induced phosphorylation EGFR in Hep3B. The Hep3B cells (1 × 10 6 ) were treated without (Ctrl) or with EGF in the absent **50** or present (EGF + **50**) of 10 µM **50** for 15 min and phosphorylation status of EGFR was measured. ) of 10 μM

(5, 10, 20 and 40 μM) (5, 10, 20 and 40 μM) **Figure 5.** The concentration-dependent study of PP2Ac methylation status under 6,7-diprenoxycoumarin (**50**) treatment. Hep3B cells (1 × 10 6 ) were treated with **50** (5, 10, 20 and 40 µM) for 24 h. Treated cell lysates were prepared as described in the Methods section and methyl-PP2Ac expression was analyzed by western blot.

The mRNA and protein expression level of CAR-related down-stream genes, MRP2 and UGT1A1, were also enhanced by **50** (UGT1A1 protein level was not significantly increased in our study) (Figures 6 and 7). These data strongly supported that the CAR activation of coumarin derivatives might through the inhibiting the EGFR signaling and upregulating PP2Ac methylation.

cells were treated with 10 μM

cells were treated with 10 μM

of MRP2 and UGT1A1 after 10 μM 6,7

of MRP2 and UGT1A1 after 10 μM 6,7

) of 10 μM

(5, 10, 20 and 40 μM)

of MRP2 and UGT1A1 after 10 μM 6,7 cells were treated with 10 μM **Figure 6.** The time course study of mRNA expression of MRP2 and UGT1A1 after 10 µM 6,7 diprenoxycoumarin treatment. Hep3B cells were treated with 10 µM **50** for 2, 4, 6, 8 and 10 h. The total mRNA was obtained and the expression of downstream genes were analyzed with PCR. The ratio of MRP2 to GAPDH was calculated using Image J and normalization.

**Figure 7.** The protein expressions of MRP2 after 6,7-diprenoxycoumarin (**50**) treatment. Hep3B cell (1 × 10 6 ) were treated with **50** (5, 10, 20 and 40 µM) for 24 h. Treated cell lysates were prepared as described in the Methods section and the expression of MRP2 was analyzed by western blot.

(5, 10, 20 and 40 μM) for 24 h. Treated cell lysates were prepared as

didn't show

μ μ

#### **6. Further In Vivo Hypoglycemic Effect and Pharmacokinetic Studies**

From previous literature, CAR activator has been found to regulate glucose metabolizing gene expression [9]. Therefore, in vivo hypoglycemic effect was evaluated using scoparone and compound **50** (Figure 8). Db/db mice were treated with scoparone (100 mg/kg, P.O.) or vehicle (CMC) for two weeks. The scoparone-treated group showed a significant improvement in oral glucose tolerance test (OGTT), which was not observed in vehicle group (A). Further analysis showed the level of fructosamine, a glycation end product, also decreased after treatment, indicating blood glucose levels became lower after treatment (B). However, the insulin level (C) or HOMA-IR (D) did not change, indicating that glucose-lowering effect was not due to an insulinotropic effect. Compound **50** didn't show any in vivo efficacy even by intraperitoneal injection, which led us to perform a

μ μ

pharmacokinetic study of scoparone and **50** (Supplementary Figure S21). After analytical method validation, C57BL/6 mice were given scoparone (i.p.) or **50** and the resulting plasma concentration-time are presented in Tables S1 and S2. In the pharmacological assay, the desired scoparone concentration is ~2 µg/mL (10 µM), which can be achieved when the dose given is 0.5 mg/mouse (Table S1). However, the desired concentration of **50** (~3 µg/mL, 10 µM) cannot be obtained even if the amount of drug is increased to 0.5 mg/mice. Thus, the CAR activating effect of **50** may not be seen in vivo, which can account for the fact no blood sugar lowering effects of **50** were observed in vivo.

μU/ **Figure 8.** Treatment of scoparone on db/db mice for two weeks improves glucose tolerance and insulin sensitivity. Treatment of scoparone on db/db mice for two weeks improves glucose tolerance. (**A**) db/db mice were treated with scoparone or vehicle (P.O.) for 2 weeks. After 12 h of fasting, glucose tolerance tests were performed and blood glucose levels were assessed (*n* = 6) (**B**) Area under curve of blood glucose level were calculated. Fructosamine (**C**) and insulin (**D**) levels were measured. (**E**) Homeostasis Model Assessment (HOMA) was calculated as HOMA-IR index = insulin(µU/mL) × glucose(mmol/L)/22.5. (\* statistically significant compared with Control group; # statistically significant compared with db/db group; Probability of < 0.05 was considered to be significant)

#### **7. Conclusions**

CAR activation has already been associated with amelioration of various diseases such as diabetes. However, clinical CAR agonists are still lacking, hterefore, a safe and effective CAR activator would be an interesting alternative option for these diseases. In this study, sixty coumarin derivatives either synthesized or purified from Yin Chen were screened for CAR activation activity. Among all the compounds, **50** is the most effective for CAR

activation and was selected for further studies. Modification on the 6 position is generally more beneficial than at other positions. Electron-withdrawn groups are detrimental to the activity. Mechanism of action studies showed that CAR activation of **50** might be through the inhibition of EGFR signaling and upregulated PP2Ac methylation. In vivo OGTT, scoparone can lower blood sugar and fructosamine levels without insulinotropic effects. In addition, a preliminary pharmacokinetic study also indicated the desired scoparone blood concentration can be achieved. This study established also a screening system providing a rapid method for CAR activator discovery and development and provided scientific evidence for the effects of coumarins on CAR activation.

#### **8. Materials and Methods**

#### *8.1. General Information*

All of the solvents and reagents were obtained commercially and used without further purification. The progress of all the reactions was monitored by TLC on 2 × 6 cm pre-coated silica gel 60 F<sup>254</sup> plates of thickness 0.25 mm (Merck). The chromatograms were visualized under 254 or 366 nm UV light. Silica gel 60 (Merck, particle size 0.063–0.200 mm) was used as the adsorbant for column chromatography.

NMR spectra were obtained on an AV400 NMR spectrometer (Bruker). MS spectra were measured either at the instruments center of National Chung Hsing University (JMS-700 spectrometer, JEOL, Japan) or National Tsing Hua University (MAT-95XL HRMS, Finnigan, San Jose, CA). CITCO was purchased from Sigma-Aldrich (Oakville, ON, Canada).

#### *8.2. Chemistry*

*6-Trifluoromethoxycoumarin* (**11**). 2-Hydroxy-4-trifluoromethoxylbenzaldehyde (0.5 g, 2.4 mmol) and methoxycarbonylmethylene-triphenylphosphorane (0.9 g, 2.8 mmol) were dissolved in *N*, *N*-diethylaniline (5.0 mL) and refluxed for 4 h. The reaction mixture was purified by silica gel column chromatography (*n*-hexane/ethyl acetate (8:2)) to give the intermediate. Then the intermediate was dissolved in diphenyl ether (5.0 mL), heated to 200 ◦C for 4 h. The mixture was partitioned with water and dichloromethane and dried over MgSO4. The desired compound was obtained by silica gel column chromatography (*n*-hexane/ethyl acetate (8:2)) to give 6-trifluoromethoxycoumarin (**11**, 0.3 g, 1.3 mmol). Yellow needle-like crystals. Yield: 55.0%. M.P.: 83–85 ◦C. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.71 (1H, d, *J* = 9.6 Hz), 7.43–7.38 (2H, m), 7.28 (1H, s), 6.53 (1H, d, *J* = 9.6 Hz). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 159.8, 152.1, 145.0, 142.3, 124.7, 120.3 (q, *J* = 258 Hz), 119.7, 119.4, 118.3, 117.9. <sup>19</sup>F-NMR (470 MHz, CDCl3): δ = −58.31. HRMS [ESI]<sup>+</sup> calculated for C10H5F3O3: 230.0191; found [M]<sup>+</sup> 230.0192.

*7-Trifluoromethoxycoumarin* (**20**). 3-Trifluoromethoxylphenol (1.0 g, 5.6 mmol), palladium (II) acetate (5.0 mol%) and trifluoroacetic acid (8.4 mL) were mixed in dichloromethane (5.0 mL) under nitrogen and stirred at room temperature for 7 days. Brine solution was added and the misture extracted with ethyl acetate. The ethyl acetate layer was washed with brine and dried over anhydrous MgSO4. The solvent was removed in vacuo. The residue was purified by column chromatography (*n*-hexane/ethyl acetate (7:3)) to give 7-trifluoromethoxycoumarin (**20**, 0.06 mg, 0.3 mmol) and recovered starting material. White crystals. Yield: 4.0%. M.P.: 69–71 ◦C. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.68 (1H, d, *J* = 9.6 Hz), 7.50 (1H, d, *J* = 8.4 Hz), 7.15 (1H, s), 7.11 (1H, d, *J* = 8.4 Hz), 6.24 (1H, d, *J* = 9.6 Hz). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 159.7, 154.6, 151.2, 142.4, 129.0, 120.1 (q, *J* = 258 Hz), 117.2, 116.7, 116.6, 109.1. <sup>19</sup>F-NMR (470 MHz, CDCl3): δ = −57.48. HRMS [ESI]<sup>+</sup> calculated for C10H5F3O3: 230.0191; found [M]<sup>+</sup> 230.0190.

#### *8.3. General Procedure for the Synthesis of Various Alkoxyl Courmarin Derivatives*

Varied alkoxy-substituted coumarins were prepared by using the corresponding alkyl bromide. The 8-hydroxycoumarin or 6,7-dihydroxycoumarin, alkyl bromide and potassium carbonate were mixed in DMF. The reaction mixture was heated to 200 ◦C for 1–2 h and then cooled to room temperature. The reaction mixture was diluted with H2O and extracted with dichloromethane. The organic layers were combined, dried over anhydrous MgSO<sup>4</sup> and concentrated under vacuum. The residue was purified by silica gel column chromatography (*n*-hexane/rthyl acetate (7:3)) to give the various alkoxysubstituted coumarins.

*8-n-Butoxycoumarin* (**31**). Yield: 22.2%. White needle-like crystals. M.P.: 79–81◦C. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.65 (1H, d, *J* = 9.6), 7.17–7.13 (1H, m), 7.06–7.00 (2H, m), 6.40 (1H, d, *J* = 9.6 Hz), 4.09–4.06 (2H, m), 1.86–1.79 (2H, m), 1.54–1.52 (2H, m), 0.98–0.94 (3H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 160.4, 146.8, 143.9, 143.6, 124.2, 119.5, 119.1, 116.7, 115.0, 69.1, 31.1, 19.1, 13.7; HRMS [ESI]<sup>+</sup> calculated for C13H15O<sup>3</sup> [M + H]<sup>+</sup> 218.0943; found 218.0944.

*6,7-Dialloxycoumarin* (**44**). Yield: 74.6%. White needle-like crystals. M.P.: 84–85 ◦C. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.56 (1H, d, *J* = 9.6 Hz), 6.86 (1H, s), 6.82 (1H, s), 6.24 (1H, d, *J* = 9.6 Hz), 6.10–5.99 (2H, m), 5.46–5.28 (4H, m), 4.65–4.59 (4H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 161.3, 152.3, 149.9, 145.3, 143.2, 132.7, 131.9, 118.5, 118.0, 113.4, 111.5, 110.9, 101.5, 70.4, 69.8; HRMS [ESI]<sup>+</sup> calculated for C15H14O4: 258.0892; found [M + H]<sup>+</sup> 259.0893.

*6,7-Di-n-butoxycoumarin* (**46**). Yield: 89.9%. Yellow needle-like crystals. M.P.: 77–79 ◦C. <sup>1</sup>H- NMR (400 MHz, CDCl3): δ 7.56 (1H, d, *J* = 9.6 Hz), 6.84 (1H, s), 6.78 (1H, s), 6.22 (1H, d, *J* = 9.6 Hz), 4.04–3.96 (4H, m), 1.85–1.75 (4H, m), 1.51–1.46 (4H, m), 0.98–0.96 (6H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 161.5, 153.1, 150.0, 146.6, 143.3, 113.1, 111.2, 110.5, 110.9, 69.6, 68.9, 31.1, 30.7, 19.1, 19.1, 13.7, 13.7; HRMS [ESI]<sup>+</sup> calculated for C17H22O4: 290.1518; found [M]<sup>+</sup> 290.1519.

*6,7-Dipentoxycoumarin* (**47**). Yield: 48.6%. Yellow solid. M.P.: 57–59 ◦C. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.56 (1H, d, *J* = 9.6 Hz), 6.83 (1H, s), 6.78 (1H, s), 6.23 (1H, d, *J* = 9.6 Hz), 4.03–3.96 (4H, m), 1.88–1.78 (4H, m), 1.46–1.35 (4H, m), 0.93–0.89 (6H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 161.5, 153.1, 150.0, 146.0, 143.3, 113.1, 111.2, 110.4, 100.9, 69.8, 69.2, 28.8, 28.4, 28.1, 28.0, 22.3, 22.3, 13.9, 13.9; HRMS [ESI]<sup>+</sup> calculated for C19H26O4: 318.1831; found [M]<sup>+</sup> 318.1834.

*6,7-Di-isopentoxycoumarin* (**48**). Yield: 84.2%. White solid. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.57 (1H, d, *J* = 9.6 Hz), 6.84 (1H, s), 6.79 (1H, s), 6.23(1H, d, *J* = 9.2 Hz), 4.07–3.99 (4H, m), 1.87–1.80 (2H, m), 1.76–1.55 (4H, m), 0.96–0.94 (12H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 161.5, 153.1, 150.0, 146.0, 143.3, 113.1, 111.2, 110.4, 100.9, 68.3, 67.7, 37.8, 37.4, 25.1, 22.5, 22.5; HRMS [ESI]<sup>+</sup> calculated for C19H26O4: 318.1831; found [M]<sup>+</sup> 318.1832.

*6,7-Dihexoxycoumarin* (**49**). Yield: 51.0%. Yellow solid. <sup>1</sup>H-NMR (400 MHz, CDCl3): δ 7.56 (1H, d, *J* = 9.5 Hz), 6.83 (1H, s), 6.78 (1H, s), 6.22 (1H, d, *J* = 9.5 Hz), 4.03–3.96 (4H, m), 1.86–1.77 (4H, m), 1.46–1.32 (12H, m), 0.89–0.87 (6H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 161.5, 153.1, 150.0, 146.0, 143.3, 113.0, 111.2, 110.4, 100.8, 69.8, 69.2, 31.4, 31.4, 29.8, 28.7, 25.6, 25.5, 22.5 (2C), 13.9 (2C); HRMS [ESI]<sup>+</sup> calculated for C21H31O<sup>4</sup> [M + H]+347.2215; found [M]<sup>+</sup> 346.4605.

*6,7-Digeranoxycoumarin* (**51**). Yield: 55.2%. Yellow solid. <sup>1</sup>H-NMR (400MHz, CDCl3): δ 7.54 (1H, d, *J* = 9.6 Hz), 6.82 (1H, s), 6.75 (1H, s), 6.19 (1H, d, *J* = 9.2 Hz), 5.46–5.40 (2H, m), 5.01 (2H, s), 4.46–4.51 (4H, m), 2.89 (2H, s), 2.82 (2H, s), 2.06–1.91 (8H, m), 1.73–1.53 (18H, m). <sup>13</sup>C-NMR (100 MHz, CDCl3): δ = 161.5, 152.2, 150.0, 145.6, 143.3, 141.5, 141.2, 131.8, 131.8, 123.6, 123.5 119.3, 118.8, 113.1, 111.2, 110.8, 101.3, 66.6, 66.3, 39.4, 39.4, 26.2, 26.1, 25.6, 25.5, 17.4 (2C), 16.7, 16.6; HRMS [ESI]<sup>+</sup> calculated for C29H38O4: 450.2770; found 450.2772.

#### *8.4. Cell Lines*

Human hepatocellular carcinoma cell line (Hep 3B) was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan).

#### *8.5. Cell Culture*

Cells were maintained in minimum essential medium (MEM, Invitrogen) containing 10% fetal bovine serum (HyClone®, Tianjin, China), 100 units/mL penicillin, 100 µg/mL streptomycin, 2 mM L-glutamine (penicillin-streptomycin-glutamine 100X from GIBCO, Invitrogen) and 1 mM sodium pyruvate at 37 ◦C humidified incubator with 5% CO2.

#### *8.6. Cell Cytotoxicity Assay of Coumarin Derivatives*

The colorimetric assay for cellular growth and survivals described by Hansen et al. with modifications [57]. The MTT (3-(4,5-cimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide, Sigma-Aldrich Inc., Germany) assay was utilized to determine the IC<sup>50</sup> value of each compound. Cells (5000 cells/well) were seeded into 96-well plates for 24 h and subsequently the cells were treated with vehicle or tested compounds at pre-determined concentration. After 72 h treatment, MTT solution was added to the final concentration of 0.5 mg/mL and continue cultured for 2 h at 37 ◦C. Afterward, the cells were lysed with lysis buffer (40% DMF and 20% SDS in H2O) overnight at 37 ◦C to lysate the cells. The absorbance at 570 nm was then detected by a microplate reader and the IC<sup>50</sup> value was calculated.

#### *8.7. Reverse Transcription Polymerase Chain Reaction (RT-PCR)*

Total RNA was isolated from control and treated cells using a TRIzol reagent (Invitrogen) according to the manufacturer's instruction. The RNA concentration was measured by a spectrophotometer and equal amounts of total RNA (2 µg) were reverse—transcribed using a RevertAid First Strand cDNA Synthesis Kit (K1622, Fermentas, Hanover, MD, USA). Amplification of cDNA was performed in the PCR reaction buffer (0.2 mM dNTP, 1.5 mM MgCl2, and 0.5 µM of each primer) containing 2.5 units of *Taq* DNA polymerase. The PCR products were resolved by agarose electrophoresis, visualized by ethidium bromide straining and quantified by Image J®. The PCR cycling procedures and sequences of primers are listed below. PCR conditions: (1) UGT1A1: 94 ◦C, 30 s; 62 ◦C, 45 s; 72 ◦C, 30 s; 27 cycles; (2) MRP2: 94 ◦C, 30 s; 62 ◦C, 45 s; 72 ◦C, 30 s; 27 cycles; (3) CAR: 94 ◦C, 30 s; 59 ◦C, 45 s; 72 ◦C, 30 s; 35 cycles; (4) GADPH: 94 ◦C, 30 s; 62 ◦C, 45 s; 72 ◦C, 30 s; 27 cycles; (4) gtPBREM: 94 ◦C, 30 s; 63 ◦C, 30 s; 68 ◦C, 60 s; 30 cycles.


*8.8. Primer Sequences*

#### *8.9. Cloning and Transfection*

The genomic DNA was isolated from Hep3B cells using Wizard® Genomic DNA Purification Kit (Promega, Fitchburg, WI, USA). The promoter region of *UGT1A1* gene, gtPBREM element, was amplified by AccuPrimeTM Taq DNA Polymerase High Fidelity Kit (Invitrogen, MA, USA). The sequence of the amplified element was confirmed by DNA sequencing company and cloned into pGL4 luciferase reporter vector. The pGL4-gtPBREM plasmid was transfected into Hep3B cells. The stable pGL4-gtPBREM transfected Hep3B cells, Hep3B-gtPBREM cells, were established after G418 selection. The luciferase activity represented of CAR activation ability was measured using individual established clone. Further, CAR-shRNA was obtained from the Genomic Research Center and transfected into Hep3B-gtPBREM cells. The transient transfected shCAR-Hep3B-gtPBREM cells were used for further luciferase assays.

#### *8.10. Luciferase Assays*

The Hep3B-gtPBREM cells and shCAR-Hep3B-gtPBREM cells (1.5 × 10<sup>5</sup> cells/well) were seeded into 12-well plates for overnight then pre-determined concentration compounds were added for desired time. After treatment, transfected cells were washed twice in PBS, lysated with 1× passive lysis buffer and luciferase intensity of equal amount of cell lysate was measured by a Synergy HT Multi-Mode Microplate Reader (BioTek, Winooski, VT, USA).

#### *8.11. Nuclear and Cytoplasmic Extraction*

Cells were harvest by centrifugation after trypsin-EDTA treatment. The ice-cold CER I (10 µL/1 × 10<sup>6</sup> cells) was added to the cell pellet following the manufacturer's instruction. The tube was vigorously vortexed on the highest setting for 15 s to fully suspend the cell pellet, incubated on ice for 10 min, then ice-cold CER II was added to the tube and vortexed for 5 s on the highest setting. After incubating in ice for 1 min, tube was vortexed for 5 s on the highest setting then subjected to centrifuge for 5 min at 16,000× *g*. The supernatant (cytoplasmic fraction) was transfected to a clean pre-chilled tube and the nuclear fraction (pellet) was re-suspended in ice-cold NER. After 40 min incubation on ice, the nuclear fraction was subjected to centrifuge for 10 min at 16,000× *g* and the supernatant was transferred to a pre-chilled tube for further investigation.

#### *8.12. Protein Extraction*

1 × 10<sup>6</sup> cells were seeded in a 10 cm plate, incubated at 37 ◦C for overnight and treated with various concentrations of compounds for the indicated times. After incubation, the culture medium was removed and washed twice with 2 mL ice-cold 1× PBS. Three hundred µL of protein lysis buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% SDS, 1% Triton X-100, 1 mM PMSF) was added into each plate, scraped with a rubber policeman, collected into a premarked microtube and the incubated in ice for 1 h, sonicated by Ultrasonic Cell Disruptor for three time at 5 s each. The cell lysated was cleared by centrifugation at 12,000× *g* for 15 min at 4 ◦C. Supernatants were transferred into a fresh marked microtube and stored at −20 ◦C for future use.

#### *8.13. Western Blots*

Protein concentration of each sample was determined using a BCA Protein Assay Kit (Pirece®, Thermo Scientific). Different percentages of SDS-PAGE gels were prepared according to the molecular size of proteins of interest. After complete polymerization of stacking gels, each sample with equal amount of total protein was separated in polyacrylamide gels (stacking gel: 50 v, 60 min; separating gel: 100 v, 60 min). After separating, wet-transfer method was utilized to transfer proteins onto the PVDF membrane. The gels were assembled in the transfer sandwich and put in a transfer tank filled with transfer buffer (25 mM glycine, 0.15% ethanolamine, 20% methanol). The electro-transfer was performed at the voltage of 100 volt for 80 min at 4 ◦C. After transfer, PVDF-membrane was blocked with 5% BSA in 1× TBS-Tween at room temperature for 1 h. Membrane was washed with 10 mL 1× TBS-Tween three times and incubated with primary antibodies overnight at 4 ◦C with gentle agitation. After primary antibody was removed, the membrane was washed three times with 10 mL 1× TBS-Tween for 10 min at room temperature with constant shaking. The horseradish peroxide (HRP)-conjugated secondary antibody was added to react with the membrane for 1 h at room temperature with gentle agitation. After incubation, membrane was washed with 1× TBS-Tween 3 times for 10 min. For detection, the membrane was immersed completely in the mixture of 0.5 mL luminosol reagent and 0.5 mL peroxidase for 1 min. The emitted fluorescence from the membrane was caught on a LAS-4000 biomolecule imager (FUJI.).

#### *8.14. Immunofluorescence*

Hep3B cells (4 × 10<sup>5</sup> ) were grown on coverslips for overnight then washed twice with 1× PBS and fixed with 4% paraformaldehyde in 1× PBS for 30 min at room temperature. Cells were then permeabilized with 1× PBS containing 0.1% (*v/v*) Triton X-100 for 10–15 min and blocked in 1× PBS containing 5% BSA for 1 h at room temperature. CAR antibody (1:200 dilutions) was added and reacted at room temperature for 2 h in 1× PBS containing 5% BSA. FITC-conjugated goat anti-rabbit IgG antibody was added for another 1 h at room temperature. Coverslips were mounted and images were acquired under a fluorescence microscope.

#### *8.15. Animals*

Six-week old male C57/BL6 mice (*n* = 6) were acquired from the Animal Center of the College of Medicine, National Taiwan University (Taipei, Taiwan) and 6-week-old male *db/db* mice (BKS.Cg-Dock7m +/+ Leprdb/JNarl) (*n* = 12) were acquired from the National Laboratory Animal Center (Tainan, Taiwan). Experimental animals were allowed to acclimate to the controlled photoperiod (a cycle of 12-h light/12-h dark), humidity (40–60% relative humidity) and temperature (22 ± 2 ◦C) with ad libitum supply of standard chow diets and drinking water for one week prior to the experimental treatment in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources Commission on Life Sciences, 2011) [58]. Acclimated animals were randomly separated into three cohorts: control group (C57/BL6 mice treated with vehicle for two weeks, *n* = 6), db/db group (*db/db* mice treated with vehicle for two weeks, *n* = 6), and db/db+SP group (*db/db* mice treated with 100 mg/kg scoparone for two weeks, *n* = 6). (IACUC Approval NO.: 20140532; Duration: 2015.01.01–2015.12.31; Ethical Committee: National Taiwan University College of Medicine and College of Public Health Institutional Animal Care and Use Committee (IACUC).)

#### *8.16. Oral Glucose Tolerance Test (OGTT)*

The control mice or diabetic mice with or without drugs treatment for 2 weeks received an oral glucose challenge (2 g/kg). Mice were under slight anesthesia by an intraperitoneal injection of pentobarbital (50 mg/kg) and blood samples (20–50 µL via orbital sinus at each time point) were collected following: 0, 15, 30, 60, 90, and 120 min after delivery of the glucose load. Blood glucose levels were determined using SURESTEP blood glucose meter (Lifescan). Glucose tolerance was determined and performed as the curve (AUC) and delta AUC (∆AUC) using Prism 5 software (GraphPad).

#### *8.17. Determination of Blood Insulin and Fructosamine*

To determine the amount of insulin and fructosamine after fasting overnight, blood samples (0.5–0.8 mL) of euthanasia animals were collected. After centrifugation, the serum was analyzed by insulin and fructosamine immunoassay kits according to the respective instructions of the manufacturer (Mercodia AB Inc., Uppsala, Sweden; Hospitex Diagnostics Lp, League City, TX, USA). The homeostasis model assessment of insulin resistance (HOMA-IR) index is calculated with the formula of the values of fasting glucose and insulin divided by 22.5 as previously described [59].

#### *8.18. Statistical Analysis*

The values in the graphs are given as mean ± S.E.M. The significance of difference was evaluated by the paired Student's t-test. When more than one group was compared with one control, significance was evaluated according to one-way analysis of variance (ANOVA). Probability values of <0.05 were considered to be significant.

**Supplementary Materials:** The online version of this article contains Supplementary Materials.

**Author Contributions:** H.-Y.H., S.-H.J. suggested the work protocol, interpreted the results, prepared and revised the manuscript; M.-T.H. and F.-P.L. curative data; P.-L.H., J.-L.C., and C.-Y.C. performed

the laboratory experiments; H.-Y.H., S.-H.J. and S.-H.L. for funding acquisition, resources and data analysis. H.-K.L. performed PEPCK assay; C.-H.C. provided support for PK study; S.-C.K. and T.-S.W. provided natural coumarins and interpretated data. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Ministry of Science and Technology, Taiwan, granted to H.-Y. Hung. And the APC was funded by Hsin-Yi Hung.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** This work was supported by Ministry of Science and Technology Taiwan granted to H.-Y. Hung.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors**

**Eva Shannon Schiffrer <sup>1</sup> , Matic Proj <sup>1</sup> , Martina Gobec <sup>1</sup> , Luka Rejc <sup>2</sup> , Andrej Šterman <sup>1</sup> , Janez Mravljak <sup>1</sup> , Stanislav Gobec <sup>1</sup> and Izidor Sosiˇc 1,\***


**Abstract:** The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7*H*-furo[3,2-*g*]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure–activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.

**Keywords:** immunoproteasome; psoralen core; non-peptidic; electrophilic compounds; warhead scan

#### **1. Introduction**

In mammals, most intracellular proteins are destined for degradation, which involves the proteasome, a multiprotease complex [1–3]. The 26S proteasome represents the heart of the ubiquitin-proteasome system that is responsible for the maintenance of protein homeostasis and the regulation of various cellular processes [4–6]. It is a nucleophilic hydrolase with *N*-terminal Thr1 acting as a nucleophile to cleave the peptide bond of proteins [7]. The 26S proteasome is comprised of a 20S core particle (CP) and 19S regulatory units. The 20S core is a 720 kDa large barrel-shaped structure assembled of four stacked rings, each consisting of seven subunits. The two outer α rings provide structural integrity and act like "gates" allowing the entry of unfolded proteins to the two inner β rings, which contain three catalytically active subunits responsible for proteolysis of substrates [8]. Subunit β1 shows caspase-like activity, subunit β2 trypsin-like activity, whereas subunit β5 exhibits chymotrypsin-like activity [9,10]. There are three individual CP types: the constitutive proteasome (cCP), which is expressed in all eukaryotic cells, the thymoproteasome (tCP) [11], which is exclusive to cortical thymic epithelial cells, and the immunoproteasome (iCP) [12], which is expressed in cells of hematopoietic origin, but can also be induced in other tissues. Namely, the induction of iCP in other cell types is possible during acute immune and inflammatory responses [13–15]. Exposure to inflammatory factors, such as tumor necrosis factor α and interferon-γ causes the expression of the iCP active subunits β (designated as β1i, β2i, β5i), which replace their constitutive counterparts [12,16].

**Citation:** Schiffrer, E.S.; Proj, M.; Gobec, M.; Rejc, L.; Šterman, A.; Mravljak, J.; Gobec, S.; Sosiˇc, I. Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors. *Molecules* **2021**, *26*, 356. https:// doi.org/10.3390/molecules26020356

Academic Editor: Maria João Matos Received: 13 December 2020 Accepted: 9 January 2021 Published: 12 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Increased expression of cCP and iCP can lead to a number of diseases. These include many types of cancer, infections, inflammatory and autoimmune diseases (Crohn's disease, ulcerative colitis, hepatitis, and rheumatoid arthritis), as well as neurological disorders [17–23]. The cCP and the iCP therefore represent validated targets for the design of new pharmacologically active compounds [24–27]. The druggability of both CPs is clearly represented by the clinically used covalent inhibitors bortezomib, carfilzomib, and ixazomib, which are used for the treatment of multiple myeloma and mantle-celllymphoma [27]. Selective inhibition of the iCP's β5i [28] subunit or simultaneously acting on β1i and β5i catalytic activities [29,30] are both approaches that are being investigated in the treatment of autoimmune and inflammatory diseases. In addition, such strategy should cause fewer adverse effects, as the expression of iCP is induced during the course of disease processes [31,32]. By avoiding cCP inhibition, the protein degradation would thus not be inhibited in most eukaryotic cells. β β β

α γ

β β β β

The most advanced iCP inhibitors that are frequently utilized in functional studies of iCP inhibition are represented in Figure 1. Please note that only a selected number of derivatives is depicted; namely, the most studied β5i-selective inhibitor PR-957 [28], β1i and β5i dual inhibitors KZR-616 [29] and 'compound 22′ [33], as well as the most selective β5i inhibitor DPLG-3 [34]. Structurally, these compounds all possess a peptidic backbone. Moreover, the former three compounds are all endowed with an electrophilic warhead, which reacts with the catalytic Thr1 of the proteasome subunits to form a covalent bond and to confer improved inhibition [24]. β β β ′ β

**Figure 1.** Structures of the most studied iCP-selective peptidic inhibitors. For a more thorough overview on subunit-selective iCP inhibitors, the reader is referred to recent reviews [32,35].

β Because peptidic compounds, such as bortezomib and carfilzomib, are prone to poor metabolic stability and low bioavailability due to the unfavorable physico-chemical characteristics [36–38], there is a need to develop inhibitors with non-peptidic scaffolds. Despite being significantly less represented, there were some recent reports on non-peptidic inhibitors of the iCP (mostly inhibiting the β5i subunit) and the representative compounds are shown in Figure 2 [39–44]. As with peptidic compounds, irreversible inhibitors of non-peptidic nature can be obtained through structure-guided optimization, whereby an electrophilic warhead is properly positioned onto the structure of the non-covalently binding scaffold [45]. An essential prerequisite for this strategy to work is that the position of the electrophilic moiety allows the formation of the covalent bond between the inhibitor and the catalytic Thr1.

Recently, we discovered non-peptidic and β5i-selective inhibitors with a central psoralen core [39]. The most potent non-covalent inhibitor obtained during structure-activity relationship (SAR) studies possessed a phenyl substituent at position 4′ (see Figure 3 for psoralen atom numbering). This compound was also transformed into two potent irreversible covalent inhibitors by adding electrophilic warheads at position 3, i.e., succinimidyl ester and oxathiazolone. Of these two compounds, the oxathiazolone-based inhibitor showed the most promising inhibitory characteristics (Figure 2, 'compound 42′ ) as it was a potent and selective iCP inhibitor [39]. It was demonstrated previously that oxathiazolones inhibit iCP via cyclocarbonylation of the β-OH and α-NH<sup>2</sup> of the active site Thr1 [41]. Nevertheless, this structural fragment is deemed hydrolytically unstable making it less

suitable for further development [41]. This fact prompted us to investigate other possible warheads that could be attached at the same position of the psoralen core. Previously, we already determined that acrylamides and nitrile-based warheads led to worse inhibition of the iCP [39]. However, to further map the warhead chemical space attached onto the psoralen core, we prepared a new focused set of compounds with different electrophilic fragments attached at position 3 (Figure 3), and evaluated their influence on the inhibition of all six catalytic subunits of both CPs. The selection of warheads in this study was based both on previously well described Thr targeting warheads (e.g., vinyl sulfones, α',β'-epoxyketones) [24], as well as on biologically less represented electrophilic moieties. In addition, to minimize the influence of non-covalently binding portion of the molecule on overall inhibitory potency, we used the same core compound with a phenyl substituent at position 4′ . β ′ ′ β α

β **Figure 2.** A selection of non-peptidic iCP inhibitors. 'Compound 42' [39] was the most selective irreversible β5i subunit inhibitor from the initial series of psoralen-based inhibitors. It represents the parent compound for studies in this manuscript. ′

**Figure 3.** Schematic representation of the work described in this study. The numbering system for the psoralen ring is shown for clarity, as well as general nomenclature for the warhead moieties used.

#### **2. Results and Discussion**

α β

*2.1. Syntheses of 3-Substituted Psoralens*

′ To prepare 3-allyl-substituted psoralen, ethyl acetoacetate was used as a starting material (Scheme 1). It was first alkylated using NaH as a base to obtain compound **1**, which was subjected to Pechmann reaction conditions to yield 7-hydroxycoumarin derivative **2**. After OH group alkylation with 2-bromoacetophenone, the final allyl-substituted compound **4** was obtained by base-catalyzed condensation of the coumarin derivative **3** into psoralen ring (Scheme 1). A compound with 3-vinyl-based warhead attached at position 3 (compound **7**) was obtained via a similar route. The crucial intermediate

′

β

7-hydroxy-4-methyl-4-vinyl-2*H*-chromen-2-one (**5**) was obtained in high yield by heating resorcinol derivative and crotonyl chloride at 60 ◦C in acetone. This was followed by a 2-bromoacetophenone-mediated alkylation and cyclization into psoralen yielding compounds **6** and **7**, respectively.

**Scheme 1.** Synthesis of compounds with allyl (**4**) and vinyl (**7**) warheads attached at position 3 of the psoralen ring. Reagents and conditions: (a) allyl bromide, NaH (60%), THF, 0 ◦C to rt, overnight; (b) resorcinol, 98% H2SO<sup>4</sup> , dioxane, 0 ◦C to rt, overnight; (c) 2-bromoacetophenone, K2CO<sup>3</sup> , KI, dioxane, 100 ◦C, 24 h; (d) 1 M NaOH, propan-2-ol, 80 ◦C, 40 min; (e) crotonyl chloride, K2CO<sup>3</sup> , acetone, 60 ◦C, 24 h; (f) 1 M KOH, EtOH, 85 ◦C, 2 h.

Compounds **4** and **7** were further used as synthons to prepare derivatives with other electrophilic moieties at position 3 (Scheme 2). The former was used in a Wacker-type oxidation of the terminal olefin by the combination of Pd(OAc)<sup>2</sup> and Dess-Martin periodinane to prepare the compound with ethyl methyl ketone moiety, i.e., compound **8**. The vinylsubstituted derivative **7** was a starting point for three different warhead-decorated psoralens, namely vinyl sulfone **9** (via NH4I-induced sulfonylation of vinyl at position 3 with DMSO), 3-bromo-4,5-dihydroisoxazole **10** [46] (via cycloaddition of the alkene with 1,1,-dibromoformaldoxime), and pinacolate ester **11** (via transition-metal-free synthesis of alkylboronate from vinyl and bis(pinacolato)diboron) (Scheme 2).

− **Scheme 2.** Synthesis of compounds **8**, **9**, **10**, and **11** with ketone, vinyl sulfone, 3-bromo-4,5-dihydroisoxazole, and pinacolate ester, respectively, as warheads. Reagents and conditions: (a) Dess–Martin periodinane, Pd(OAc)<sup>2</sup> , CH3CN, H2O, 50 ◦C, overnight; (b) DMSO, H2O, NH<sup>4</sup> I, 130 ◦C, 36 h; (c) 1,1-dibromoformaldoxime, DMF, NaHCO<sup>3</sup> , −15 ◦C to rt, 5 h; (d) bis(pinacolato)diboron, CsF, 1,4-dioxane, MeOH, 100 ◦C, 12 h.

α β

α

α β

′

′

α β

The synthesis of 3-propanal-substituted psoralen **15** was initiated by a coumarin derivative **12** possessing ethyl propionate moiety at position 3 (Scheme 3). The acidic hydrolysis yielded propanoic acid **13**, which was transformed into aldehyde derivative **14** by first forming an acid chloride, followed by in situ reduction with hydrogen gas using Pd/BaSO<sup>4</sup> as a catalyst. Interestingly, an attempt to prepare α-ketoaldehyde (which is a known Thr-targeting warhead [24]) from compound **15** by Riley oxidation with SeO<sup>2</sup> resulted in the formation of α,β-unsaturated aldehyde derivative **16** (Scheme 3, Figures 4 and 5). α α β

−

α β **Scheme 3.** Synthesis of compounds with aldehyde- (**15**) and α,β-unsaturated aldehyde-based (**16**) warheads attached at position 3 of the psoralen ring. Reagents and conditions: (a) 1 M HCl, dioxane, reflux, 2 h; (b) i. SOCl<sup>2</sup> , DMF, toluene, rt, 17 h; ii. H<sup>2</sup> , Pd/BaSO<sup>4</sup> , toluene, 100 ◦C, 2 h; (c) 1 M NaOH, propan-2-ol, 60 ◦C, 15 min; (d) SeO<sup>2</sup> , dioxane, H2O, MW, 150 ◦C, 1 h. Synthesis of compound **12** was described previously [39].

′ ′ ′ **Figure 4.** COSY experiment for **16**. Circled cross-peaks indicate coupling between aldehyde proton CHO and the adjacent C2′ -H, and between C2′ -H and C3′ -H.

′

α β

′ ′ ′

′ **Figure 5.** NOESY experiment for **16**. Only cross-peaks that indicate coupling between CH<sup>3</sup> protons and C4-H and C3′ -H are shown.

α β To further confirm the structure of α,β-unsaturated aldehyde **16**, two-dimensional NMR experiments correlation spectroscopy (COSY) and Nuclear Overhauser effect spectroscopy (NOESY) were recorded. In the COSY spectrum (Figure 4), a clear correlation between the aldehyde proton CHO and the adjacent C2′ -H was observed. In addition, the NOESY experiment showed a coupling between the CH<sup>3</sup> protons and C4-H and C3′ -H (Figure 5).

The fact that the most advanced selective iCP inhibitors and also carfilzomib, which is a marketed cCP and iCP inhibitor, possess an α',β'-epoxyketone fragment as the Thr-targeting warhead, encouraged us to prepare two such psoralen-based compounds (Scheme 4). Both **20** and **21** were synthesized from the corresponding precursors **17**, **18**, and **19** by a HATU-mediated amide bond formation (Scheme 4).

**Scheme 4.** Synthesis of epoxyketone-based compounds **20** and **21**. Reagents and conditions: (a) i. TFA, CH2Cl<sup>2</sup> , 0 ◦C, 30 min; ii. HATU, HOBt×H2O, DIPEA, DMF (**20**) or CH2Cl<sup>2</sup> (for **21**), rt, 24 h. Syntheses of compounds **17**, **18**, and **19** were based on previously described procedures [39], compound **17**; [47], compound **18**; [48], compound **19**. All spectral data ( <sup>1</sup>H-NMR, HRMS) corresponded well to the original reports.

β To prepare 3-azetidin-2-one-substituted psoralen **24**, a previously synthesized compound **22** was used as a crucial intermediate. It was first *N*-acylated with 3-bromopropanoyl chloride to yield 3-bromopropanamide **23**, and then cyclized into the β-lactam ring by using NaO*t*Bu as a base (Scheme 5).

β β β

′

**β β β β β β**

β β β β β β

β β β

**Scheme 5.** Synthesis of alkyl bromide-based psoralen **23** and psoralen **24** with azetidin-2-one as a warhead. Reagents and conditions: (a) 3-bromopropanoyl chloride, K2CO<sup>3</sup> , CH2Cl<sup>2</sup> , 0 ◦C to rt, 3 h; (b) NaO*t*Bu, DMF, 0 ◦C to rt, 24 h. Synthesis of compound **22** was described previously [39].

#### *2.2. Biochemical Evaluation*

β β β

′ The target compounds were evaluated for their inhibitory potencies on both CPs (Table 1) using subunit selective fluorogenic substrates (for details, see Materials and Methods Section). The data were calculated as residual activities (RAs) of individual subunits of CPs in the presence of 1 µM of each compound. This concentration was used due to poor solubility of all final compounds at higher concentrations, emphasizing the need for development of inhibitors with improved solubility. The previously described oxathiazolone derivative 'compound 42′ and carfilzomib were used as positive control using the same concentration (1 µM) to enable a better comparison between compounds.

β

β β β β β β β β β **β β β β β β Table 1.** Inhibitory potencies of compounds against all catalytically active subunits (β5i, β2i, and β1i) of the iCP and against all catalytically active subunits (β5, β2, β1) of the human cCP. In the assays, the following substrates were used: Suc-LLVY-AMC for β5i and β5; Boc-LRR-AMC for β2i and β2; Ac-PAL-AMC for β1i; Ac-nLPnLD-AMC for β1.


<sup>1</sup> RA values are means from at least three independent determinations. Ac-PAL-AMC, acetyl-Pro-Ala-Leu-7 amino-4-methylcoumarin; Ac-nLPnLD-AMC, acetyl-Nle-Pro-Nle-Asp-AMC; Boc-LRR-AMC, *t*-butyloxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin; Suc-LLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin. carf.: carfilzomib.

Given the fact that all assayed compounds possessed the same non-covalently binding portion, we were able to thoroughly assess the contributions of attached warheads to the inhibition of all catalytically active subunits of iCP and cCP. The assay results showed that all new psoralens were worse inhibitors of β5i subunit of iCP in comparison to the parent oxathiazolone-based 'compound 42′ (Table 1, Figure 6). This is most probably due to the mispositioning of the electrophilic carbons of all compounds and the catalytic Thr1O*<sup>γ</sup>* in the β5i active site. Interestingly, all compounds inhibited β5i activity with a similar potency at 1 µM with RA values ranging from 62 to 78%. Of the 12 prepared compounds, 3-bromo-4,5-dihydroisoxazole-substituted psoralen **10** and compound **16** with an α,β-unsaturated aldehyde as the warhead were the most promising. The former showed RA value of

α β

62 ± 5%, whereas for the latter RA was determined at 65 ± 3% (see also postulated binding modes for 10 and 16 in Figure 7). It was not surprising to see that all 12 compounds also exhibited worse inhibition of the β5 subunit of cCP, albeit these differences were much less pronounced as for the β5i subunit. Of note, compounds **9**, **11**, **23**, and **24** were slightly better inhibitors of β1i subunit of iCP in comparison to the 'compound 42′ . All psoralen-based compounds (with oxathiazolone included) did not inhibit other subunits of both CPs (i.e., β2i, β2, and β1), whereas carfilzomib completely abolished activity of all subunits at 1 µM (Table 1, Figure 6). β β β ′ β β β

β

′

*<sup>γ</sup>* β β

**Figure 6.** Inhibition results represented as bar charts of inhibition percentage. carf.: carfilzomib.

′ β ′ γ **Figure 7.** Molecular modelling. Binding site residues are presented as green sticks with labels for some of the key residues. (**A**) Covalent docking of '42′ (magenta) into the β5i subunit (PDB: 5M2B). Please note that only the initial intermediate formed after the nucleophilic attack of OH group of Thr1 onto the carbonyl group of the oxathiazolone is represented. Co-crystalized ligand Ro19 is presented with blue sticks and dashed yellow lines for hydrogen bonds. (**B**) Noncovalent docking of **10** (cyan) and **16** (yellow) reveals good alignment of the psoralen core with the proposed pose of '42′ (magenta). However, the distance from the electrophilic carbons of **10** and **16** to the catalytic Thr1O<sup>γ</sup> is too large to form a covalent bond.

δ

≥

#### **3. Materials and Methods**

#### *3.1. General Chemistry Methods*

Reagents and solvents were obtained from commercial sources (Acros Organics (Thermo Fisher Scientific, Waltham, MA, USA), Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), TCI Europe (Tokyo Chemical Industry, Tokyo, Japan), Alfa Aesar (Thermo Fisher Scientific, Waltham, MA, USA), Fluorochem (Fluorochem Ltd., Derbyshire, UK) and were used as received. Carfilzomib were purchased from MedChemExpress. For reactions involving air or moisture sensitive reagents, solvents were distilled before use and these reactions were carried out under nitrogen or argon atmosphere. Reactions using microwaves were performed on a standard monomode microwave reactor MONOWAVE 200 (Anton Paar, Graz, Austria). Reactions were monitored using analytical thin-layer chromatography plates (Merck 60 F254, 0.20 mm), and the components were visualized under UV light and/or through staining with the relevant reagent. Normal phase flash column chromatography was performed on Merck Silica Gel 60 (particle size 0.040–0.063 mm; Merck, Germany). <sup>1</sup>H and <sup>13</sup>C-NMR spectra were recorded at 295 K on a Bruker Avance III 400 MHz spectrometer (Bruker, Billerica, MA, USA) operating at frequencies for <sup>1</sup>H-NMR at 400 MHz and for <sup>13</sup>C-NMR at 101 MHz. The chemical shifts (δ) are reported in parts per million (ppm) and are referenced to the deuterated solvent used. The coupling constants (*J*) are given in Hz, and the splitting patterns are designated as follows: s, singlet; br s, broad singlet; d, doublet; app d, apparent doublet; dd, doublet of doublets; ddd, doublet of doublets of doublets; t, triplet; dt, doublet of triplets; td, triplet of doublets; m, multiplet. All <sup>13</sup>C-NMR spectra were proton decoupled. Mass spectra data and high-resolution mass measurements were performed on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). The purity of the compounds used in biochemical assays was determined with analytical normal-phase HPLC on an Agilent 1100 LC modular system (Agilent, Santa Clara, CA, USA) that was equipped with a photodiode array detector set to 254 nm. A Kromasil 3-CelluCoat column (150 mm × 4.6 mm; 3 µm particle size) was used, with a flow rate of 1.0 mL/min and a sample injection volume of 5–20 µL. An isocratic eluent system of A (hexane) and B (isopropanol) was used; the ratio used is described for each compound below. The purities of the test compounds used for the biological evaluations were ≥95%, unless stated otherwise.

#### *3.2. Syntheses*

Synthesis of ethyl 2-acetylpent-4-enoate (**1**):

To a solution of ethyl acetoacetate (7.28 mL, 7.50 g, 57.60 mmol, 1 equiv.) in 50 mL of anhydrous THF, NaH in mineral oil (60%, 2.30 g, 57.60 mmol, 1 equiv.) was added and the resulting suspension stirred under argon at 0 ◦C. After 20 min, a solution of allyl bromide (4.99 mL, 6.97 g, 57.60 mmol, 1 equiv.) in 25 mL of anhydrous THF was added dropwise. The reaction mixture was stirred at room temperature overnight. Next, cold H2O (25 mL) was added and THF was evaporated under reduced pressure. The resulting suspension was extracted with Et2O (3 × 25 mL), the organic layer separated, dried over anhydrous Na2SO4, and evaporated. The product was purified by column chromatography (Et2O/petroleum ether, 1/5, *v*/*v*). Yield: 71%, clear liquid. <sup>1</sup>H-NMR (400 MHz, DMSO-*d*6) δ 1.17 (t, *J* = 7.1 Hz, 3H, CH3CH2), 2.18 (s, 3H, COCH3), 2.42–2.47 (m, 2H, CH2CHCH2CH), 3.73 (dd, *J* = 7.8, 6.8 Hz, 1H, CH), 4.07–4.15 (qd, 2H, *J* = 7.1, 1.6 Hz, CH3CH2), 4.98–5.10 (m, 2H, CH2CHCH2CH), 5.67–5.77 (m, 1H, CH2CHCH2CH).

Synthesis of 3-allyl-7-hydroxy-4-methyl-2*H*-chromen-2-one (**2**):

This compound was prepared using Pechmann condensation as follows. A solution of resorcinol (4.06 g, 36.90 mmol, 1 equiv.) and ethyl 2-acetylpent-4-enoate (**1**) (6.90 g, 40.50 mmol, 1.1 equiv.) in dioxane (80 mL) was cooled to 0 ◦C, followed by drop-wise addition of concentrated H2SO<sup>4</sup> (98%, 19.60 mL, 405 mmol, 10 equiv.). The reaction mixture was stirred at room temperature overnight. Dioxane was then evaporated under reduced pressure and the semi-solid mixture was added portion-wise to an ice-cold solution of KOH

(40 g) in H2O (100 mL). The pH was adjusted to 13 with KOH and the resulting white solid was filtered off. The filtrate was extracted with EtOAc (3 × 25 mL) and the combined organic extracts were dried over anhydrous Na2SO<sup>4</sup> and evaporated under reduced pressure. The compound was purified by column chromatography (EtOAc/*n*-hexane, 1/1.5, *v*/*v*, dry loading) yielding a pale-yellow solid. Yield: 13%. <sup>1</sup>H-NMR (400 MHz, DMSO-*d*6) δ 2.34 (s, 3H, CH3), 3.30 (d, *J* = 6.0 Hz, 2H, Ar-CH2CHCH2), 4.98–5.06 (m, 2H, Ar-CH2CHCH2), 5.84 (ddt, *J* = 16.3, 10.3, 6.0 Hz, 1H, Ar-CH2CHCH2), 6.70 (d, *J* = 2.4 Hz, 1H, Ar-H), 6.80 (dd, *J* = 8.7, 2.4 Hz, 1H, Ar-H), 7.63 (d, *J* = 8.7 Hz, 1H, Ar-H), 10.44 (s, 1H, OH); HRMS (ESI) *m/z* calculated for C13H11O<sup>3</sup> [M − H]<sup>−</sup> 215.0714, found 215.0707.

Synthesis of 3-allyl-4-methyl-7-(2-oxo-2-phenylethoxy)-2*H*-chromen-2-one (**3**):

This compound was synthesized following a previously described procedure [39]. Briefly, to a solution of 3-allyl-7-hydroxy-4-methyl-2*H*-chromen-2-one (**2**) (0.99 g, 4.56 mmol, 1 equiv.) in dioxane (70 mL), K2CO<sup>3</sup> (2.52 g, 18.22 mmol, 4 equiv.) and KI (76 mg, 0.46 mmol, 0.1 equiv.) were added. After 10 min of stirring at 100 ◦C, 2-bromoacetophenone (1.36 g, 6.83 mmol, 1.5 equiv.) was added and the mixture was further stirred at 100 ◦C for 24 h. The solvent was then removed under reduced pressure, followed by addition of H2O (30 mL) to the residue. The aqueous phase was extracted with EtOAc (3 × 30 mL), the combined organic extracts were washed with brine (20 mL), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The compound was purified by crystallization from MeOH yielding pale-yellow crystalline solid. Yield: 67%. <sup>1</sup>H-NMR (400 MHz, DMSO-*d*6) δ 2.38 (s, 3H, CH3), 3.32 (d, *J* = 6.0 Hz, 2H, Ar-CH2CHCH2), 5.03 (ddd, *J* = 8.5, 3.0, 1.3 Hz, 2H, Ar-CH2CHCH2), 5.75 (s, 2H, CH2), 5.86 (ddt, *J* = 16.2, 10.2, 6.0 Hz, 1H, Ar-CH2CHCH2), 7.03 (dd, *J* = 8.9, 2.6 Hz, 1H, Ar-H), 7.07 (d, *J* = 2.5 Hz, 1H, Ar-H), 7.59 (app dd, *J* = 10.6, 4.8 Hz, 2H, 2 × Ar-H), 7.68–7.77 (m, 2H, 2 × Ar-H), 8.04 (app dd, *J* = 8.4, 1.2 Hz, 2H, 2 × Ar-H); HRMS (ESI) *m/z* calculated for C21H19O<sup>4</sup> [M + H]<sup>+</sup> 335.1280, found 335.1272.

Synthesis of 6-allyl-5-methyl-3-phenyl-7*H*-furo[3,2-*g*]chromen-7-one (**4**):

This compound was synthesized following a previously described procedure [39]. Namely, to a heated (80 ◦C) and stirred solution of **3** (0.25 g, 0.75 mmol, 1 equiv.) in propan-2-ol (25 mL), an aqueous solution of NaOH (7.5 mL, 1 M, 10 equiv.) was added. The reaction mixture was stirred at 80 ◦C for 40 min. After the reaction was complete (monitored by TLC), propan-2-ol was evaporated under reduced pressure. The aqueous residue was acidified with HCl (6 mL, 1 M) to pH 5, then H2O (20 mL) was added, the aqueous layer was extracted with CH2Cl<sup>2</sup> (3 × 25 mL), and the combined organic extracts were evaporated under reduced pressure. The compound was purified by column chromatography (Et2O/petroleum ether, 1/3, *v*/*v*). White solid, yield: 70%. <sup>1</sup>H-NMR (400 MHz, DMSO-*d6*) δ 2.54 (s, 3H, CH3), 3.40 (d, *J* = 6.0 Hz, 2H, Ar-CH2CHCH2), 5.01–5.11 (m, 2H, Ar-CH2CHCH2), 5.89 (ddt, *J* = 16.1, 10.2, 6.0 Hz, 1H, Ar-CH2CHCH2), 7.41–7.48 (m, 1H, Ar-H), 7.51–7.60 (m, 2H, 2 × Ar-H), 7.79 (s, 1H, Ar-H), 7.80–7.82 (m, 1H, Ar-H), 7.83 (t, *J* = 1.6 Hz, 1H, Ar-H), 8.18 (s, 1H, Ar-H), 8.48 (s, 1H, Ar-H); <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.49 (s, 3H, CH3), 3.49 (d, *J* = 6.0 Hz, 2H, Ar-CH2CHCH2), 5.01–5.16 (m, 2H, Ar-CH2CHCH2), 5.94 (ddt, *J* = 16.2, 10.1, 6.0 Hz, 1H, Ar-CH2CHCH2), 7.44 (ddd, *J* = 7.4, 4.0, 1.3 Hz, 1H, Ar-H), 7.49–7.51 (m, 1H, Ar-H), 7.51–7.57 (m, 2H, 2 × Ar-H), 7.61–7.64 (m, 1H, Ar-H), 7.65 (t, *J* = 1.7 Hz, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, DMSO-*d*6) δ 15.19, 30.94, 99.48, 115.69, 116.46, 116.99, 121.18, 121.29, 122.81, 127.23, 127.84, 129.22, 130.72, 134.50, 144.29, 148.39, 149.94, 155.92, 160.58; HRMS (ESI) *m/z* calculated for C21H17O<sup>3</sup> [M + H]<sup>+</sup> 317.1172, found 317.1166. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 99%.

Synthesis of 7-hydroxy-4-methyl-4-vinyl-2*H*-chromen-2-one (**5**):

A suspension of 1-(2,4-dihydroxyphenyl)ethan-1-one (502 mg, 3.3 mmol, 1 equiv.), crotonyl chloride (395 µL, 429 mg, 4.1 mmol, 1.25 equiv.) and K2CO<sup>3</sup> (1.47 g, 10.6 mmol, 3.2 equiv.) in acetone (25 mL) was heated at 60 ◦C for 24 h. The solvent was then evaporated under reduced pressure, followed by the addition of EtOAc (100 mL). The organic phase was extracted with H2O (100 mL), and the aqueous phase acidified with 2 M HCl and

further extracted with EtOAc (2 × 100 mL). The combined organic extracts were dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The compound was purified by column chromatography (EtOAc/*n*-hexane, 1/4, *v*/*v*). White solid, yield: 71%. <sup>1</sup>H-NMR (400 MHz, DMSO-*d*6) δ 2.45 (s, 3H, CH3), 5.51 (dd, *J* = 12.0 Hz, 2.4 Hz, 1H, Ar-CHCH2), 6.02 (dd, *J* = 17.4 Hz, 2.4 Hz, 1H, Ar-CHCH2), 6.67 (d, *J* = 2.4 Hz, 1H, Ar-H), 6.72 (dd, *J* = 17.4, 12.0 Hz, 1H, Ar-CHCH2), 6.79 (d, *J* = 8.9, 2.4 Hz, 1H, Ar-H), 7.66 (d, *J* = 8.9 Hz, 1H, Ar-H), 10.51 (br s, 1H, OH). HRMS (ESI) *m/z* calculated for C12H9O<sup>3</sup> [M − H]<sup>−</sup> 201.0557, found 201.0549.

Synthesis of 4-methyl-7-(2-oxo-2-phenylethoxy)-3-vinyl-2*H*-chromen-2-one (**6**):

This compound was synthesized following a previously described procedure [39]; using the procedure as for **5**. The compound was purified by crystallization from EtOH yielding off-white crystalline solid. Yield: 79%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.48 (s, 3H, CH3), 5.38 (s, 2H, OCH2), 5.63 (dd, *J* = 11.8, 1.9 Hz, 1H, Ar-CH=CH2), 6.04 (dd, *J* = 17.6, 1.9 Hz, 1H, Ar-CH=CH2), 6.71 (dd, *J* = 17.6, 11.8 Hz, 1H, Ar-CH=CH2), 6.77 (d, *J* = 2.6 Hz, 1H, Ar-H), 6.96 (dd, *J* = 9.0, 2.6 Hz, 1H, Ar-H), 7.49–7.57 (m, 2H, Ar-H), 7.59 (d, *J* = 8.9 Hz, 1H, Ar-H), 7.70–7.63 (m, 1H, Ar-H), 7.95–8.04 (m, 2H, 2 × Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 15.37, 70.55, 101.43, 112.79, 114.88, 120.10, 122.26, 126.34, 127.99, 128.99, 129.03, 134.14, 134.26, 146.69, 153.55, 160.18, 160.39, 193.18; HRMS (ESI) *m/z* calculated for C20H17O<sup>4</sup> [M + H]<sup>+</sup> 321.1121, found 321.1123.

Synthesis of 5-methyl-3-phenyl-6-vinyl-7*H*-furo[3,2-*g*]chromen-7-one (**7**):

To a solution of 4-methyl-7-(2-oxo-2-phenylethoxy)-3-vinyl-2*H*-chromen-2-one (**6**) (132 mg, 0.4 mmol, 1 equiv.) in EtOH (5 mL), KOH (1.2 mL, 1 M, 1.2 mmol, 3 equiv.) was added and the reaction mixture stirred at 85 ◦C for 2 h. The solvent was then evaporated, followed by the addition of H2O (20 mL). The suspension was acidified with concentrated HCl to pH = 1 and extracted with CH2Cl<sup>2</sup> (2 × 50 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The product was purified by column chromatography (EtOAc/*n*-hexane, 1/1, *v*/*v*). Yellow solid; yield: 78 %; <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.60 (s, 3H, CH3), 5.69 (dd, *J* = 11.8 Hz, 1.8 Hz, 1H, Ar-CH=CH2), 6.05 (dd, *J* = 17.7 Hz, 1.8 Hz, 1H, Ar-CH=CH2), 6.77 (dd, *J* = 17.7, 11.8, 1H, Ar-CH=CH2), 7.42–7.47 (m, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.51–7.56 (m, 2H, Ar-H), 7.62–7.66 (m, 2H, Ar-H), 7.82 (s, 1H, Ar-H), 8.04 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 15.92, 99.74, 116.23, 117.38, 121.23, 122.31, 122.82, 123.98, 127.59, 128.04, 129.19, 129.26, 131.11, 142.79, 146.88, 150.39, 156.67, 160.21; HRMS (ESI) *m/z* calculated for C20H15O<sup>3</sup> [M + H]<sup>+</sup> 303.1016, found 303.1019. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 98%.

Synthesis of 5-methyl-6-(2-oxopropyl)-3-phenyl-7*H*-furo[3,2-*g*]chromen-7-one (**8**):

This compound was synthesized following a previously described procedure [49]. Briefly, to a stirred solution of olefin **4** (158 mg, 0.5 mmol, 1 equiv.) in CH3CN (3.5 mL) and H2O (0.5 mL), Pd(OAc)<sup>2</sup> (5.6 mg, 0.025 mmol, 5 mol %) and Dess-Martin periodinane (254 mg, 0.6 mmol, 1.2 equiv.) were added. The reaction mixture was warmed to 50 ◦C and stirred under an argon atmosphere overnight. The reaction mixture was then filtered through a small pad of Celite and washed with EtOAc, and the filtrate was concentrated. The residue was purified by column chromatography (EtOAc/*n*-hexane = 1/2, *v*/*v*, dry loading). White solid, yield: 40%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.32 (s, 3H, CH2COCH3), 2.44 (s, 3H, CH3), 3.88 (s, 2H, CH2COCH3), 7.44 (t, *J* = 7.4 Hz, 1H, Ar-H), 7.48–7.57 (m, 3H, 3 × Ar-H), 7.63 (app dd, *J* = 8.0, 1.0 Hz, 2H, 2 × Ar-H), 7.83 (s, 1H, Ar-H), 8.01 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 16.19, 30.15, 42.42, 100.13, 116.08, 117.27, 118.41, 122.51, 124.19, 127.77, 128.19, 129.41, 131.26, 143.01, 149.65, 150.81, 156.85, 161.93, 204.58; HRMS (ESI) *m/z* calculated for C21H17O<sup>4</sup> [M + H]<sup>+</sup> 333.1121, found 333.1127. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 98%.

Synthesis of (*E*)-5-methyl-6-(2-(methylsulfonyl)vinyl)-3-phenyl-7*H*-furo[3,2-*g*]chromen-7-one (**9**):

To a solution of 5-methyl-3-phenyl-6-vinyl-7*H*-furo[3,2-*g*]chromen-7-one (**7**) (100 mg, 0.33 mmol, 1 equiv.) in DMSO (1 mL), H2O (0.5 mL) and NH4I (191 mg, 1.32 mmol, 4 equiv.) were added. The reaction mixture was stirred at 130 ◦C for 36 h. Then, it was

cooled to room temperature, followed by slow addition of Na2S2O<sup>3</sup> × 5H2O until the discoloration of mixture. Subsequently, H2O (20 mL) was added and the aqueous phase extracted with EtOAc (3 × 20 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The product was purified by column chromatography (EtOAc/*n*-hexane, 1/2, *v*/*v*). Yellow solid; yield: 64%; <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.63 (s, 3H, Ar-CH3), 3.14 (s, 3H, SO2CH3), 7.44–7.48 (m, 1H, Ar-CHCHSO2CH3), 7.52–7.57 (m, 4H, Ar-CHCHSO2CH<sup>3</sup> and 3 × Ar-H), 7.61–7.65 (m, 3H, Ar-H), 7.87 (s, 1H, Ar-H), 8.11 (s, 1H, Ar-H); <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 15.08, 42.96, 60.48, 65.58, 100.25, 116.60, 116.89, 121.68, 122.48, 124.96, 127.66 (2C), 128.31, 129.38 (2C), 130.69, 143.40, 149.56, 150.40, 157.12, 161.99; HRMS (ESI) *m/z* calculated for C21H17O5S [M + H]<sup>+</sup> 381.0791, found 381.0795.; Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 99%.

Synthesis of 6-(3-bromo-4,5-dihydroisoxazol-5-yl)-5-methyl-3-phenyl-7*H*-furo[3,2-*g*]chromen-7-one (**10**)

To a cooled (−15 ◦C) solution of 5-methyl-3-phenyl-6-vinyl-7*H*-furo[3,2-*g*]chromen-7 one (**7**) (145 mg, 0.48 mmol, 1 equiv.) and 1,1-dibromoformaldoxime (148 mg, 0.73 mmol, 1.5 equiv.) in DMF (10 mL), an aqueous solution of NaHCO<sup>3</sup> (1 mL, 112 mg, 1.3 mmol, 2.7 equiv.) was added. The reaction was then warmed to room temperature and stirred for 5 h. Then, the solution was diluted with CH2Cl<sup>2</sup> (20 mL) and washed with brine (20 mL). The organic extract was dried over Na2SO4, filtered, and the solvents removed under reduced pressure. The product was purified by column chromatography (EtOAc/*n*hexane, 1/4, *v*/*v*) to yield pale yellow solid. Yield: 76%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.60 (s, 3H, Ar-CH3), 3.47 (dd, *J* = 17.1, 11.8 Hz, 1H, one H of CH2), 3.62 (dd, *J* = 17.1, 10.4 Hz, 1H, one H of CH2), 6.09 (dd, *J* = 11.8, 10.4 Hz, 1H, CH2CHO), 7.42–7.48 (m, 2H, Ar-H), 7.51–7.57 (m, 2H, Ar-H), 7.60–7.64 (m, 2H, Ar-H), 7.83 (s, 1H, Ar-H), 8.06 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 15.27, 46.05, 77.81, 100.01, 116.61, 116.75, 119.73, 122.36, 124.29, 127.60, 128.19, 129.33, 130.84, 137.27, 143.12, 150.88, 151.48, 157.17, 159.50; HRMS (ESI) *m/z* calculated for C21H15O4NBr [M + H]<sup>+</sup> 424.0179, found 424.0179. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 99%.

Synthesis of 5-methyl-3-phenyl-6-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)- 7*H*-furo[3,2-*g*]chromen-7-one (**11**):

5-Methyl-3-phenyl-6-vinyl-7*H*-furo[3,2-*g*]chromen-7-one (**7**) (0.09 mmol, 1 equiv.) was dissolved in 1,4-dioxane (2 mL) and then bis(pinacolato)diboron (1,5 equiv.), cesium fluoride (2,5 equiv.) and MeOH (5 equiv.) were added. The reaction proceeded at 100 ◦C for 12 h. The reaction mixture was then diluted with EtOAc (15 mL) and filtrated over silica. The filtrate was evaporated under reduced pressure and the product purified from the crude mixture by column chromatography (EtOAc/*n*-hexane, 1/9, gradient to 1/1, *v*/*v*) to yield yellow solid. Yield: 33%. <sup>1</sup>H-NMR (400 MHz, CD3OD): δ 0.94 (t, *J* = 8.5 Hz, 2H, CH2), 1.13 (s, 12H, C(CH3)2), 2.48 (s, 3H, Ar-CH3), 2.68 (t, *J* = 8.5 Hz, 2H, CH2), 7.30–7.35 (m, 1H, Ar-H), 7.41–7.47 (m, 3H, Ar-H), 7.62–7.62 (m, 2H, Ar-H), 7.99 (s, 1H, Ar-H), 8.05 (s, 1H, Ar-H); <sup>13</sup>C-NMR (100 MHz, CDCl3): δ 14.12, 17.77, 23.59 (4C), 29.40, 83.06, 85.22 (2C), 87.21, 102.00, 102.50, 105.99, 127.09 (2C), 127.58, 127.64, 128.75 (2C), 136.06, 141.54, 146.52, 152.51, 157.66, 161.81; HRMS (ESI) *m/z* calculated for C26H28O5B [M + H]<sup>+</sup> 431.2024, found 431.2023.; Purity by HPLC (0–18 min; 95% *n*-hexane/isopropanol): 97%.

Synthesis of 3-(4-methyl-2-oxo-7-(2-oxo-2-phenylethoxy)-2*H*-chromen-3-yl)propanoic acid (**13**):

To a stirred solution of **12** (788 mg, 2 mmol, 1 equiv.) in dioxane (20 mL), HCl (1 M, 20 mL, 10 equiv.) was added. The reaction mixture was heated at reflux temperature for 2 h. Dioxane was then evaporated under reduced pressure, the precipitate that formed filtered off and washed with H2O. Yield: 94%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.44 (s, 3H, CH3), 2.67 (t, *J* = 7.6 Hz, 2H, CH2CH2COOH), 2.96 (t, *J* = 7.6 Hz, 2H, CH2CH2COOH), 5.38 (s, 2H, CH2), 6.78 (d, *J* = 2.6 Hz, 1H, Ar-H), 6.95 (dd, *J* = 8.9, 2.6 Hz, 1H, Ar-H), 7.49–7.60 (m, 3H, 3 × Ar-H), 7.66 (t, *J* = 7.4 Hz, 1H, Ar-H), 7.82–8.07 (m, 2H, 2 × Ar-H); HRMS (ESI) *m/z* calculated for C21H17O<sup>6</sup> [M − H]<sup>−</sup> 365.1031, found 365.1030.

Synthesis of 3-(4-methyl-2-oxo-7-(2-oxo-2-phenylethoxy)-2*H*-chromen-3-yl)propanal (**14**):

To a suspension of **13** (366 mg, 1 mmol, 1 equiv.) in toluene (20 mL), dried over 3 Å molecular sieves, a catalytic amount of anhydrous DMF (5 drops) and SOCl<sup>2</sup> (218 µL, 357 mg, 3 mmol, 3 equiv.) were added under argon. The reaction mixture was stirred at room temperature for 17 h and then the volatiles were evaporated to obtain a white solid that was dried under vacuum for 15 min to remove SOCl2. Toluene (20 mL), dried over 3 Å molecular sieves, was added to the dried solid (under argon), followed by the addition of 10% Pd/BaSO<sup>4</sup> (72 mg, 30% [*w*/*w*]). The reaction mixture was heated to 100 ◦C and stirred under a stream of hydrogen (1 atm) for 2 h. The reaction mixture was then evaporated to dryness and the compound was purified by column chromatography (EtOAc/*n*-hexane, 1/1, *v*/*v*, dry loading). White solid, yield: 59%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.43 (s, 3H, CH3), 2.77 (t, *J* = 7.4 Hz, 2H, CH2CH2CHO), 2.94 (t, *J* = 7.4 Hz, 2H, CH2CH2CHO), 5.38 (s, 2H, CH2), 6.78 (d, *J* = 2.6 Hz, 1H, Ar-H), 6.95 (dd, *J* = 8.9, 2.6 Hz, 1H, Ar-H), 7.50–7.59 (m, 3H, 3 × Ar-H), 7.66 (t, *J* = 7.4 Hz, 1H, Ar-H), 7.04–8.06 (m, 2H, 2 × Ar-H), 9.83 (t, *J* = 1.0 Hz, 1H, CH2CH2CHO); HRMS (ESI) *m/z* calculated for C21H19O<sup>5</sup> [M + H]<sup>+</sup> 351.1227, found 351.1221.

Synthesis of 3-(5-methyl-7-oxo-3-phenyl-7*H*-furo[3,2-*g*]chromen-6-yl)propanal (**15**):

To a suspension of **14** (519 mg, 1.48 mmol, 1 equiv.) in propan-2-ol (35 mL), an aqueous solution of NaOH (14.8 mL, 1 M, 10 equiv.) was added. The reaction mixture was stirred at 60 ◦C for 15 min. After the reaction was complete (monitored by TLC), the resulting red solution was acidified with 1 M HCl (15 mL) to get a yellow precipitate. The reaction mixture was evaporated under reduced pressure. H2O (50 mL) was added to the dry residue, which was extracted with CH2Cl<sup>2</sup> (2 × 50 mL), the combined organic extracts were washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and evaporated under reduced pressure. The compound was purified by column chromatography (EtOAc/*n*hexane, 1/2, *v*/*v*, dry loading). White solid, yield: 61%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.55 (s, 3H, CH3), 2.82 (t, *J* = 7.4 Hz, 2H, CH2CH2CHO), 3.01 (t, *J* = 7.4 Hz, 2H, CH2CH2CHO), 7.47–7.41 (m, 1H, Ar-H), 7.49 (s, 1H, Ar-H), 7.50–7.57 (m, 2H, 2 × Ar-H), 7.60–7.67 (m, 2H, 2 × Ar-H), 7.83 (s, 1H, Ar-H), 8.00 (s, 1H, Ar-H), 9.86 (t, *J* = 1.0 Hz, 1H, CH2CH2CHO); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 15.67, 20.85, 42.53, 99.97, 115.99, 117.34, 122.46, 122.98, 124.09, 127.72, 128.18, 129.39, 131.24, 142.94, 147.70, 150.56, 156.58, 161.72, 201.40; HRMS (ESI) *m/z* calculated for C21H17O<sup>4</sup> [M + H]<sup>+</sup> 333.1121, found 333.1116. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 87%.

Synthesis of 3-(5-methyl-7-oxo-3-phenyl-7*H*-furo[3,2-*g*]chromen-6-yl)acrylaldehyde (**16**): To a solution of the aldehyde **15** (53 mg, 0.16 mmol, 1 equiv.) in a mixture of dioxane (1.5 mL) and H2O (20 µL), SeO<sup>2</sup> (35 mg, 0.32 mmol, 2 equiv.) was added and the reaction mixture was irradiated in a microwave reactor at 150 ◦C (250 W) for 1 h. The reaction mixture was then evaporated to dryness and the compound was purified by column chromatography (EtOAc/*n*-hexane, 1/2, *v/v*, dry loading). White solid, yield: 10%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.74 (s, 3H, CH3), 7.32 (dd, *J* = 15.8, 7.5 Hz, 1H, CHCHCHO), 7.46 (t, *J* = 7.4 Hz, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.52–7.59 (m, 2H, 2 × Ar-H), 7.61–7.69 (m, 3H, 2 × Ar-H and CHCHCHO), 7.85 (s, 1H, Ar-H), 8.14 (s, 1H, Ar-H), 9.74 (d, *J* = 7.5 Hz, 1H, CHCHCHO); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 16.21, 100.29, 116.93, 117.39, 118.27, 122.59, 124.84, 127.79, 128.42, 129.49, 130.86, 134.87, 143.45, 143.55, 151.20, 152.79, 157.88, 158.95, 194.53; HRMS (ESI) *m/z* calculated for C21H15O<sup>4</sup> [M + H]<sup>+</sup> 331.0965, found 331.0979. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 97%.

Synthesis of 5-methyl-*N*-((*S*)-1-((*R*)-2-methyloxiran-2-yl)-L-oxopropan-2-yl)-7-oxo-3 phenyl-7*H*-furo[3,2-*g*]chromene-6-carboxamide (**20**):

To a cooled (0 ◦C) solution of compound **17** (160 mg, 0.50 mmol, 1 equiv.) in DMF (4 mL), HATU (285 mg, 0.75 mmol, 1.5 equiv.) and HOBt hydrate (115 mg, 0.75 mmol, 1.5 equiv.) were added. In a separate round-bottom flask, compound **18** (115 mg, 0.50 mmol, 1 equiv.) was dissolved in CH2Cl<sup>2</sup> (3 mL) at 0 ◦C, followed by the addition of TFA (3 mL). After 30 min of stirring at 0 ◦C, the volatiles were evaporated under reduced pressure thoroughly, the residue was dissolved in CH2Cl<sup>2</sup> and slowly added to the mixture containing compound **17** at 0 ◦C. After 5 min, DIPEA (348 µL, 285 mg, 2.0 mmol, 4 equiv.) was added and the reaction mixture stirred at room temperature for 24 h. Then, the solvent was evaporated and the product purified by column chromatography (EtOAc/*n*-hexane, 1/1, *v*/*v*, dry loading) without additional work-up. Off-white solid, yield: 16%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 1.44 (d, *J* = 7.0 Hz, 3H, CHCH3), 1.57 (s, 3H, CH3), 2.75 (s, 3H, Ar-CH3), 2.96 (d, *J* = 5.0 Hz, 1H, one H of oxirane CH2), 3.39 (app d, *J* = 5.0 Hz, 1H, one H of oxirane CH2), 4.72 (p, *J* = 6.7 Hz, 1H, CHCH3), 7.42–7.48 (m, 1H, Ar-H), 7.51 (s, 1H, Ar-H), 7.52–7.57 (m, 2H, Ar-H), 7.59–7.65 (m, 3H, CONH + Ar-H), 7.85 (s, 1H, Ar-H), 8.13 (s, 1H, Ar-H), <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 16.87, 16.92 (2C), 48.68, 52.70, 59.15, 100.04, 116.76, 117.32, 118.53, 122.43, 124.69, 127.63, 128.23, 129.33, 130.71, 143.27, 150.83, 155.64, 157.64, 160.02, 163.86, 208.01; HRMS (ESI) *m/z* calculated for C25H22O6N [M + H]<sup>+</sup> 432.1442, found 432.1438. Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 96%.

Synthesis of 5-methyl-*N*-((*S*)-1-((*R*)-2-methyloxiran-2-yl)-L-oxo-3-phenylpropan-2-yl)- 7-oxo-3-phenyl-7*H*-furo[3,2-*g*]chromene-6-carboxamide (**21**):

To a cooled (0 ◦C) solution of compound **17** (28 mg, 0.087 mmol, 1 equiv.) in CH2Cl<sup>2</sup> (4 mL), HATU (40 mg, 0.11 mmol, 1.2 equiv.) and HOBt hydrate (17 mg, 0.11 mmol, 1.2 equiv.) were added. In a separate round-bottom flask, compound **19** (27 mg, 0.087 mmol, 1 equiv.) was dissolved in CH2Cl<sup>2</sup> (2 mL) at 0 ◦C, followed by the addition of TFA (2 mL). After 30 min of stirring at 0 ◦C, the volatiles were evaporated under reduced pressure thoroughly, the residue was dissolved in CH2Cl<sup>2</sup> and slowly added to the mixture containing compound **17** at 0 ◦C. After 5 min, DIPEA (58 µL, 45 mg, 0.35 mmol, 4 equiv.) was added and the reaction mixture stirred at room temperature for 24 h. Then, the solvent was evaporated and the product purified by column chromatography (EtOAc/*n*-hexane, 1/2, *v*/*v*, dry loading) without additional work-up. Off-white solid, yield: 11%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 1.55 (s, 3H, CH3), 2.65 (s, 3H, Ar-CH3), 2.88 (dd, *J* = 13.7, 8.7 Hz, 1H, one H of oxirane CH2), 2.98 (d, *J* = 5.0 Hz, 1H, one H of oxirane CH2), 3.27 (dd, *J* = 13.7, 4.8 Hz, 1H, one H of CHCH2Ph), 3.47 (dd, *J* = 5.0, 0.5 Hz, 1H, one H of CHCH2Ph), 4.99 (symm m, 1H, CHCH3), 7.27–7.35 (m, 5H, Ar-H), 7.42–7.47 (m, 1H, Ar-H), 7.50 (d, *J* = 0.4 Hz, 1H, Ar-H), 7.52–7.56 (m, 2H, Ar-H), 7.59–7.63 (m, 2H, Ar-H), 7.84 (s, 1H, Ar-H), 7.85 (br d, *J* = 6.7 Hz, 1H, CONH), 8.11 (s, 1H, Ar-H); <sup>13</sup>C-NMR was not recorded due to insufficient amount of the final product; HRMS (ESI) *m/z* calculated for C31H26O6N [M + H]<sup>+</sup> 508.1755, found 508.1755. Purity by HPLC (0–18 min; 95% *n*-hexane/isopropanol): 97%.

Synthesis of 3-bromo-*N*-((5-methyl-7-oxo-3-phenyl-7*H*-furo[3,2-*g*]chromen-6-yl)methyl) propanamide (**23**):

To a cooled (0 ◦C) solution of compound **22** (92 mg, 0.3 mmol, 1 equiv.) in CH2Cl<sup>2</sup> (10 mL), K2CO<sup>3</sup> (50 mg, 0.36 mmol, 1.2 equiv.) was added. After 5 min, 3-bromopropanoyl chloride (36 µL, 62 mg, 0.36 mmol, 1.2 equiv.) was added drop-wise at 0 ◦C. The reaction mixture was then stirred at room temperature for 3 h. The reaction was quenched by the addition of H2O (20 mL) and the mixture was extracted with EtOAc (3 × 50 mL). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The product was purified by column chromatography (EtOAc/*n*-hexane, 1/2, *v*/*v*). White solid, yield: 90%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.73 (s, 3H, CH3), 2,74 (t, *J* = 6.7 Hz, 2H, COCH2), 3.61 (t, *J* = 6.7 Hz, 2H, CH2Br), 4.52 (d, *J* = 6.2 Hz, 2H, Ar-CH2NH), 7.42–7.47 (m, 1H, Ar-H), 7.51–7.56 (m, 3H, Ar-H), 7.61–7.64 (m, 2H, Ar-H), 7.83 (s, 1H, Ar-H), 8.07 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 15.59, 27.13, 36.69, 39.51, 100.01, 116.75, 117.07, 120.68, 122.43, 124.31, 127.61 (2C), 128.15, 129.32 (2C), 130.96, 142.99, 149.61, 150.66, 156.81, 162.37, 169.52; HRMS ( *m/z* ) (ESI): calculated for C22H19O4NBr [M + H]<sup>+</sup> 440.0492, found: 440.0490; Purity by HPLC (0–18 min; 70% *n*hexane/isopropanol): 96%.

Synthesis of 1-((5-methyl-7-oxo-3-phenyl-7*H*-furo[3,2-*g*]chromen-6-yl)methyl)azetidin-2-one (**24**):

To a cooled (0 ◦C) solution of compound **23** (66 mg, 0.15 mmol, 1 equiv.) in DMF (15 mL), NaO*t*Bu (16 mg, 0.17 mmol, 1.1 equiv.) was added. The reaction mixture was stirred at room temperature for 24 h. The reaction was quenched by the addition of H2O (20 mL) and the mixture was extracted with EtOAc (3 × 50 mL). The combined

organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The product was purified by column chromatography (EtOAc/*n*-hexane, 1/2, *v*/*v*). White solid, yield: 87%. <sup>1</sup>H-NMR (400 MHz, CDCl3) δ 2.73 (s, 3H, CH3), 2.74 (t, *J* = 6.4 Hz, 2H, azetidin-2-one-CH2), 3.61 (t, *J* = 6.4 Hz, 2H, azetidin-2-one-CH2), 4.52 (s, 2H, Ar-CH2N), 7.42–7.47 (m, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.53–7.56 (m, 2H, Ar-H), 7.61–7.65 (m, 2H, Ar-H), 7.84 (s, 1H, Ar-H), 8.07 (s, 1H, Ar-H); <sup>13</sup>C-NMR (101 MHz, CDCl3) δ 29.72, 31.95, 37.04, 38.55, 102.72, 112.28, 115.88, 116.54, 116.80, 122.43, 122.51, 127.62 (2C), 128.16, 128.74, 129.33 (2C), 132.78, 143.00, 156.80, 157.68, 178.20; HRMS ( *m/z* ) (ESI): calculated for C22H18O4N [M + H]<sup>+</sup> 360.1230, found: 360.1222; Purity by HPLC (0–18 min; 70% *n*-hexane/isopropanol): 87%.

#### *3.3. Residual Activity Measurements*

The screening of compounds was performed at 1 µM final concentrations in the assay buffer (0.01% SDS, 50 mM Tris-HCl, 0.5 mM EDTA, pH 7.4). Stock solutions of compounds were prepared in DMSO. To 50 µL of each compound, 25 µL of 0.8 nM human iCP or human cCP (both from Boston Biochem, Inc., Cambridge, MA, USA) was added. After 30 min incubation at 37 ◦C, the reaction was initiated by the addition of 25 µL of 100 µM relevant fluorogenic substrate: acetyl-Nle-Pro-Nle-Asp-AMC (Ac-nLPnLD-AMC, [Bachem, Bubendorf, Switzerland]) for β1, acetyl-Pro-Ala-Leu-7-amino-4-methylcoumarin (Ac-PAL-AMC, [Boston Biochem, Inc., Cambridge, MA, USA]) for β1i, *t*-butyloxycarbonyl-Leu-Arg-Arg-7-amino-4-methylcoumarin (Boc-LRR-AMC, [Bachem, Bubendorf, Switzerland]) for β2 and β2i, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC [Bachem, Bubendorf, Switzerland]) for β5 and β5i. The reaction progress was recorded on the BioTek Synergy HT microplate reader by monitoring fluorescence at 460 nm (λex = 360 nm) for 90 min at 37 ◦C. The initial linear ranges were used to calculate the velocity and to determine the residual activity.

In the case of the β1, β1i, β2, and β2i activity inhibition determination, the assay buffer was modified; SDS was replaced with the proteasomal activator PA28α (Boston Biochem, Inc., Cambridge, MA, USA).

#### *3.4. Molecular Modelling*

Compounds were prepared for docking using LigPrep (Schrödinger Suite 2020-2, Schrödinger, LLC, New York, NY, USA, 2020) to account for all possible tautomers and ionization states at pH 7.0 ± 2.0. The X-ray structure (PDB: 5M2B, [43]) of yeast 20S proteasome with human β5i and β1 subunits in complex with noncovalent inhibitor Ro19 was used for docking. The binding site is defined by the chain K (β5i) and neighbouring L (β1), so all other chains were removed. Protein Preparation Wizard [50] was used to add hydrogen atoms, protonate residues at pH 7, refine the H-bond network and to perform a restrained minimization. The receptor's grid box required for docking calculations was centred on the corresponding co-crystallized ligand. Noncovalent docking was performed using Glide [51], with the following parameters: XP (extra precision), flexible ligand sampling, perform postdocking minimization. Covalent docking was performed with CovDock program [52] using the pose prediction mode with default setup and Thr1 defined as the reactive residue. Nucleophilic addition to a double bond (oxathiazolone) was selected as the reaction.

#### **4. Conclusions**

Here, we showed that the introduction of 12 new electrophilic warheads at position 3 of the psoralen ring led to compounds with abrogated inhibition of the iCP (especially β5i subunit). As already described in the Introduction, it is imperative that the initial noncovalent binding of a given compound is followed by the positioning of the electrophilic 'warhead' near the desired nucleophilic amino-acid residue of the protein to achieve covalent interaction. Poor inhibition results were in our cases most probably due to the mispositioning of the electrophilic carbon and the catalytic Thr1O*<sup>γ</sup>* (Figure 7). The

oxathiazolone thus remains the optimal electrophilic moiety for this compound class. Despite somewhat disappointing results, the obtained data will help steer our future research in the field of psoralen-based iCP inhibitors, e.g., when designing inhibitors which simultaneously inhibit two iCP subunits as it was established that simultaneous inhibition of β1i and β5i is necessary to achieve significant anti-inflammatory effects.

**Author Contributions:** Conceptualization, J.M., S.G., and I.S.; formal analysis, M.G. and I.S.; funding acquisition, S.G.; investigation, E.S.S., M.P., L.R., and A.Š.; methodology, E.S.S., M.P., M.G., and I.S.; project administration, S.G.; supervision, J.M. and I.S.; writing—original draft, E.S.S.; writing—review & editing, S.G. and I.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Slovenian Research Agency, research core funding No. P1-0208, Grant number N1-0068 to S.G., and Grant number J3-1745 to M.G.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors acknowledge Maja Frelih for HRMS measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of all compounds, except compound **21** are available from the authors.

#### **References**


*Communication*
