Discovery, Synthetic Methodology, and Biological Evaluation for

Jun 13, 2013 - 11d plus UVA can induce a decrease in reactive oxygen species ... UVA-induced senescence-like characteristics in FB cells, which may ...
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Discovery, Synthetic Methodology, and Biological Evaluation for Antiphotoaging Activity of Bicyclic[1,2,3]triazoles: In Vitro and in Vivo Studies Hsin-Yu Hsieh,‡,∥ Wen-Chun Lee,†,∥ Gopal Chandru Senadi,† Wan-Ping Hu,*,‡ Jium-Jia Liang,† Tong-Rong Tsai,§ Yu-Wei Chou,‡ Kung-Kai Kuo,⊥ Chung-Yu Chen,§ and Jeh-Jeng Wang*,† †

Department of Medicinal and Applied Chemistry, ‡Department of Biotechnology, §School of Pharmacy, and ⊥Department of Surgery, School of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan S Supporting Information *

ABSTRACT: Novel bicyclic[1,2,3]triazoles (4, 7, 11, 15) have been synthesized using a one-pot metal free strategy with high structural diversity as photoprotective agents, and their effect on UVA-induced senescence in human dermal fibroblast cells (FB) and the associated mechanism are delineated. 11d plus UVA can induce a decrease in reactive oxygen species (ROS) production and senescence-associated β-galactosidase (SA-β-gal) activity but an increase in adenosine triphosphate (ATP) synthesis and mitochondrial membrane potential (ΔΨmt). The mRNA levels of six senescence-associated genes, matrix metalloproteinase-1 (MMP-1), was decreased, while elastin, procollagen I type I, fibronectin, COL1α1, and tissue inhibitor of metalloproteinase-1 (TIMP-1) were increased. 11d plus UVA also decreased MMP-1 and increased TIMP-1 protein levels. Additionally, the thickness of the murine dorsal skin and epidermis, by UVA, was decreased by topical 11d treatment. Our results indicate that bicyclic[1,2,3]triazoles protect UVA-induced senescence-like characteristics in FB cells, which may provide potential prevention against photoaging.

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mitochondrial function.10 Mitochondria are well-known to participate in the production of ROS which might be harmful if produced excessively.11 Numerous investigations have shown that collagen gets degraded in photoaged skin because of the inhibition of collagen synthesis mediated by matrix metalloproteinases (MMPs), which are a family of secreted or transmembrane zinc endopeptidases that are capable of digesting ECM.12 MMPs are divided into subclasses of collagenases, gelatinases, stromelysins, matrilysins, and membrane-type MMPs (MTMMPs) according to their substrate specificity and domain structure.13 It is reported that ROS affects the MMP gene expression through a signal transduction pathway.14 Photoprotection is a group of mechanisms that nature has developed to minimize the damages that an organism suffers when exposed to UV radiation. These mechanisms can be controlled or organized by certain organic and inorganic compounds or substances (e.g., melanin) produced by different terrestrial and aquatic sources. A number of photoprotective compounds such as scytonemins, mycosporines, mycosporinelike amino acids, phenylpropanoids and flavonoids (higher plants), melanins (humans and other animals and even some

INTRODUCTION Human skin is the potential anatomical barrier for pathogens and damage, which acts as a pivotal fence between internal and external environment in bodily defense.1 The basic biological processes involved in aging lead to reductions in function and ability to tolerate injury. UV irradiation induces photodamage of the skin, which is characterized by distinct alterations in the composition of the dermal extracellular matrix (ECM), resulting in wrinkles, laxity, coarseness, mottled pigmentation, and histological changes that include increased epidermal thickness and connective tissue alteration.2−4 Breakdown of a balance of combination between the connective tissue components (biomolecules including collagens, proteoglycans, and glycoproteins) leads to the detrimental effects, e.g., photoaging in dermal fibroblasts. Skin contains antioxidant defenses that nullify reactive oxygen species (ROS) including free radicals, but these defenses will be overwhelmed if the dose of UV light is high enough, and this would result in free radical damage to cellular components such as proteins, lipids, and DNA.5,6 ROS induced by oxidative stress can ultimately lead to apoptotic cell death.7 Especially, the accumulated ROS plays a critical role in the intrinsic aging and photoaging of human skin in vivo, thus suggesting it to be responsible for various skin cancers and other cutaneous inflammatory disorders.8,9 Recent evidence has indicated that photoaging is linked to the © 2013 American Chemical Society

Received: March 18, 2013 Published: June 13, 2013 5422

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Scheme 1. Synthetic Strategy for the Construction of Bicyclic[1,2,3]triazoles

approach to construct bicyclic[1,2,3]triazole structural motifs by one-pot reaction via [2 + 3] alkyne−azide cycloaddition, from yne-ols under microwave condition (Scheme 1). We were able to access diverse analogues of bicyclic[1,2,3]triazoles 4, 7, 11, and 15 (Scheme 1) with ease using this methodology.

bacteria), and several other UV-absorbing substances of unknown chemical structures from different organisms have been developed to counter the photodamage.15,16 1,2,3Triazoles have gained special attention because of their potent applications ranging from medicinal,17−19 materials,20−23 and biological24−28 research areas. More importantly, fused triazoles are of interest because of their various biological activities29−34 and clinical applications.35,36 1,2,3-Triazoles such as biscotrizole37 and drometrizole,38 which absorb UVA and UVB radiation, are used as active ingredients for the application of skin photoprotection. On the basis of this background, our research group has synthesized a novel series of bicyclic[1,2,3]triazoles as photoprotective agents that absorb light in the UVA region, and we have delineated their effect on UVA-induced senescence in human dermal fibroblast cells (FB) and the associated mechanism. Hence, we felt it would be attractive to develop a straightforward and general method for the synthesis of bicyclic [1,2,3]triazoles, which we feel would pave the way for the preparation of a wide variety of different bioactive compounds. Syntheses of these compounds through novel methodologies involving multiple bond formation in one pot are a major challenge. One particular area that has received substantial interest over the past few years is azide−alkyne cycloaddition. This type of reaction has become more important especially after the advent of the click reaction.39−43 While there are numerous methods for the synthesis of individual 1,2,3triazoles,44−47 only a few methods are reported in the literature for the synthesis of fused [1,2,3]triazole analogues.48−56 In recent years, extensive research has been done on intramolecular alkyne−azide click chemistry and in all reported methodologies the reactions were carried from azide predecessors.57−62 Therefore, we feel there remains a need for a scalable, efficient, and universal method for synthesis of this pivotal class of compounds in an environmentally benign manner. Herein, we report, for the first time, a novel metal free

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RESULTS AND DISCUSSION Chemistry. The complete optimization studies for the synthesis of bicyclic [1,2,3]triazoles are described in Supporting Information (Table S1 and Table S2). Under the optimized reaction conditions, various yne-ols with a variety of substituents were investigated for the formation of bicyclic [1,2,3]triazoles. As shown in Scheme 2, a series of substituents on the 5-phenylpent-4-yn-1-ol including ortho, para, and meta positions were well tolerated and the corresponding 5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole derivatives were obtained in good yields (Scheme 2, 4a−g). The yne-ol with an electron-donating group at the ortho, para, and meta positions gave slightly higher yields (4b, 4d, and 4f) than those of electron-withdrawing groups on the corresponding positions (4c, 4e, and 4g). The feasibility of the reaction with alkyl substituted yne-ol was also shown to produce the desired compound in 70% yield (4h). The structure of compound 4d was confirmed by ORTEP (Figure 1). In a similar approach reaction of 6-phenylhex-5-yn-1-ol with various substituents on the phenyl ring resulted in the desired 4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine derivatives. The yne-ol with an electron-donating group at ortho, para, and meta positions gave slightly higher yields (Scheme 2, 7b, 7d, and 7f) than those of electron-withdrawing groups on the corresponding positions (7c, 7e, and 7g). The reaction of allyl derivative also gave the target compound in good yield (7h). We further extended our studies by using diyne-ol as substrate under standard optimized conditions and observed smooth conversion to produce ethynyl bicyclic [1,2,3]triazoles. As shown in Scheme 3, a series of substituents on the 75423

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Scheme 2. Substrate Scope of Metal Free Cyclization for the Synthesis of 5,6- Dihydro-4H-pyrrolo[1,2-c][1,2,3]triazoles and 4,5,6,7-Tetrahydro[1,2,3]triazolo[1,5-a]pyridinesa

Scheme 3. Substrate Scope of Metal Free Cyclization for the Synthesis of Ethynyl-5,6-dihydro-4H-pyrrolo[1,2e][1,2,3]triazoles and Ethynyl-4,5,6,7tetrahydro[1,2,3]triazolo[1,5-a]pyridinesa

a

Reaction conditions: 2, 5 (6.3 mmol), [(PhO2)PON3] (6.9 mmol), DBU (7.6 mmol), DMF (10 mL) at 150 °C, 50 W, 1 atm, 20 min.

a

Reaction conditions: 8, 12 (5.4 mmol), [(PhO2)PON3] (5.9 mmol), DBU (6.5 mmol), DMF (10 mL) at 150 °C, 50 W, 1 atm, 10 min.

effect of UVA on normal fibroblast (FB) cell viability was evaluated by the MTT assay. FB cells were exposed to different doses of UVA irradiation. Twenty-four hours after exposure cell viability was determined. As shown in Figure 2A, there was a decrease in the viability of FB with increasing doses of UVA irradiation. The cell viability was significantly inhibited at doses of 8 and 12 J/cm2 UVA irradiation. Thus, the subcytotoxic dose of UVA used throughout this study was 6 J/cm2. A previous report by Civatte et al.63 showed 24 J/cm2 as an environmental relevant dose of UVA. 24 J/cm2 of UVA equates to about 1 h and 12 min in the midday sun during the summer at latitude 48° N, and 6 J/cm2 is about 18 min at 12 a.m. in summer. In our research laboratory, it required about 21 min to obtain 6 J/ cm2 of UVA. Hence, the dosage used in this work is equal to the UVA dose that occurs for humans. In addition, data from morphology observation also showed that no significant inhibitory effect was seen when UVA was at a dose lower than 6 J/cm2 (Figure 2B). To examine the most effective concentration and time period of bicyclic [1,2,3]triazole treatment before UVA irradiation, cells were pretreated with 0, 1, 2.5, and 5 μM 11d for 2 and 4 h before 6 J/cm2 UVA irradiation. Twenty-four hours after exposure, cell proliferations were determined (Figure 2C). The proliferative effect of 11d plus UVA on FB cells is dose- and time-dependent. It is obvious that cells treated with dosage at 5 μM 11d for 4 h exhibited a marked cell proliferation. We selected a concentration of 5 μM and a time period of 4 h of treatment for further mechanistic studies.

Figure 1. ORTEP diagram for compounds 4d and 11d.

phenylhepta-4,6-diyn-1-ol including ortho, para, and meta positions were tolerated and the corresponding ethynyl-5,6dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole derivatives were obtained in good yields. The presence of electron-donating group or electronwithdrawing group on the ortho, para, and meta positions does not affect the product formation and reaction yield (Scheme 3, 11a−f,h) except in the case of p-NO2 with a yield of 40% (11g). On the other hand, alkyl diyne-ol also produced the corresponding target compound in good yield (11i). The structure of compound 11d was confirmed by ORTEP (Figure 1). To attain the ethynyl-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5a]pyridine derivatives as shown in Scheme 3, a series of substituents on the 8-phenylocta-5,7-diyn-1-ol with different electronic properties in ortho, para, and meta positions were tested, and the results show that the reaction underwent smooth conversion in good yields (Scheme 3, 15a−f,h) irrespective of electronic properties except in the case of pNO2 with a yield of 42% (15g). The corresponding alkyl diyneol also gave the desired 4,5,6,7-tetrahydro[1,2,3]triazolo[1,5a]pyridine derivative in good yield (15i). Biology. Effect of UVA Irradiation and Bicyclic[1,2,3]triazole on Human Dermal Fibroblast Cell Viability. The 5424

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Figure 2. (A) Dose-dependent effect of UVA irradiation on FB cell viability was determined by the MTT assay. (B) Morphological observation was used for the detection of FB cell viability. (C) Dose-response and time-response curves for 11d plus UVA tested for FB cell proliferation: (∗) p < 0.05 vs control group (0 μM and 0 J/cm2); (#) p < 0.05, (##) p < 0.01 vs UVA group. Similar results were obtained in three independent experiments.

Bicyclic[1,2,3]triazoles (4, 7, 11, 15) and UVA Stimulated FB Cell Proliferation vs UVA Group. Cell proliferation activity induced by compound plus UVA was examined using the MTT assay. FB cells were treated with 5 μM agents for 4 h before 6 J/cm2 UVA irradiation. Twenty-four hours after irradiation, more than 50% compounds plus UVA treatment exhibited a higher proliferation than the UVA group, and compound 11 plus UVA exhibited the most proliferation activity on FB cells as shown in Table 1. Intracellular ATP Content. ATP is the central parameter of cellular energetics, metabolic regulation, and cellular signaling; therefore, determination of intracellular ATP is worthwhile in the characterization of cellular physiology. FB cells were treated with 11 at 5 μM for 4 h followed by 6 J/cm2 UVA irradiation, and the intracellular ATP content was obtained immediately.64,65 When compared with that of UVA group, ATP formation significantly increased in FB cells exposed to most compounds 11 plus UVA treatment (Figure 3). Moreover compound 11d exhibited a higher ATP compared to other agents after UVA irradiation. Therefore, we chose compound 11d as a model for further studies. Mitochondrial Function. Photoaging is largely caused by the generation of ROS in the skin by UV. Mitochondrial

processes, especially those involving free radical production, damage, and propagation, are deeply implicated in aging. The mitochondrial membrane potential (ΔΨmt) is an important parameter not only for mitochondria but also for cellular status. Therefore, decreased formation of ROS and increased level of ΔΨmt were often used as an indication of photoprotection. In this experiment, FB cells were treated with and without 11d at 5 μM (Figure 4A and Figure 4C) or treated with various concentrations of 11d (Figure 4B and Figure 4D) for 4 h before receiving UVA irradiation. Compared with that of the untreated control, both ROS and ΔΨmt had no significant change after 11d treatment. In contrast, 11d plus UVA reduced intracellular ROS and increased ΔΨmt, compared with UVA irradiation alone (Figure 4A and Figure 4C). In addition, the decreased ROS and increased ΔΨmt were dose-dependent, as compared with that of UVA irradiation (Figure 4B and Figure 4D). Real-Time Quantitative PCR for Senescence-Associated mRNA Expression. Chronic UV irradiation accelerates the degradation of ECM components (collagen, elastin, and fibronectin) in dermal tissue, and an increase in MMP-1 level, which is associated with aging.66 Therefore, we used the qPCR measurements for elastin, procollagen I type I, fibronectin, 5425

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Protein Levels. In order to confirm our data obtained by real time qPCR, we performed immunoblot analysis of MMP-1, which plays an important role in aging processes.67 As expected from the mRNA data, the MMP-1 protein level had a significant decrease, while the TIMP-1 (MMP-1 inhibitor) had a significant increase after 11d plus UVA treatment, compared with the control group (Figure 6). SA-β-gal Activity. Cellular senescence is a program activated in response to various stress types, such as DNA damage, oxidative stress, and UV radiation. SA-β-gal is a biomarker for senescent and aging cells. The morphology of SA-β-gal-stained senescence cells was examined by histochemical staining. Our study revealed that the rates of senescence cells in the control group and 11d group were both low. However, the number of SA-β-gal-positive cells (blue point formation) of the UVA group was increased in comparison with the control group. The 11d plus UVA group showed a decrease in the number of SA-β-gal-positive cells compared with the UVA group, which implied some promotive effects of 11d on the cell senescence while combined with UVA irradiation (Figure 7). Epidermal Hypertrophy Skin Sections. Epidermal thickness is used as a parameter to reflect skin photoaging quantitatively, since epidermal hypertrophy is thought to cause wrinkle formation. In our study, the epidermal thickness of the dorsal skin of the mice was significantly increased by chronic UVA exposure. The epidermal hypertrophy was significantly reduced by topical application of 11d plus UVA (Figure 8).

Table 1. Effect of Bicyclic[1,2,3]triazoles plus UVA on FB cell proliferation vs UVA Groupa compd + UVA

survival (%)

control UVA 4a 4d 4e 4f 4g 7a 7b 7d 7e 7f 7g 11a 11b 11c 11d 11e 11f 11g 11h 15a 15c 15d 15e 15f 15h

100 ± 0.02 63.95 ± 0.01 61.06 ± 0.02 58.02 ± 0.06 52.84 ± 0.04 70.29 ± 0.03 69.98 ± 0.02 57.02 ± 0.02 61.04 ± 0.03 63.67 ± 0.03 61.97 ± 0.06 54.05 ± 0.02 51.99 ± 0.03 62.74 ± 0.01 71.22 ± 0.02 75.21 ± 0.02 95.32 ± 0.01 88.18 ± 0.03 73.16 ± 0.04 84.06 ± 0.05 80.67 ± 0.03 68.68 ± 0.02 78.89 ± 0.02 61.92 ± 0.02 71.26 ± 0.03 73.01 ± 0.03 55.74 ± 0.05

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CONCLUSIONS The biological results show that compound 11d can suppress UVA-induced damage in human fibroblasts, including a decrease in ROS production and SA-β-gal activity, but an increase in ATP synthesis and mitochondrial membrane potential (ΔΨmt). Besides ROS being involved in the UVAdependent induction of MMP-1,68 the major collagenolytic enzyme responsible for collagen destruction in the dermis, ROS also result in structural and functional alterations in the ECM. Thus, it was of interest to study the effect of the 11d plus UVA on the aging-related gene expression levels. In our study, the mRNA levels of MMP-1 were decreased, while elastin, procollagen I type I, fibronectin, COL1α1, and TIMP-1 were increased. Data from Western blot analysis also confirmed that 11d plus UVA decreased MMP-1 and increased TIMP-1 protein levels (Figure 9). Moreover, UVA irradiation resulted in increased thickness of the epidermis, and 11d plus UVA treatment decreased this effect by HE staining in animal model. In addition, we have developed an efficient approach for the synthesis of bicyclic [1,2,3]triazoles by a one-pot metal free intramoleculer [2 + 3] alkyne−azide cycloaddition directly from yne-ol under microwave conditions. The feasibility of the reaction was proven to work in conventional heating, and this makes it a practical methodology for both industry and academia. The key advantages of this method are metal free, one-pot, much shorter reaction time of 10−20 min, broad functional group tolerance, and good to excellent yields. By use of this methodology, various fused triazoles can be accessed by altering the length of alkynols. Taken together, bicyclic[1,2,3]triazole treatment could protect skin fibroblasts against UVAinduced photoaging. These compounds could be developed as helpful skin photoprotective agents.

Cells were cultured with agents at 5 μM for 4 h before 6 J/cm2 UVA irradiation. Twenty-four hours after irradiation, cell growth and viability were assessed using the MTT assay. The data are expressed as the mean ± SD.

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Figure 3. Effect of 11 plus UVA treatment on the intracellular ATP levels of the FB cells. Cells were treated with 5 μM 11a−h for 4 h followed by 6 J/cm2 UVA. Relative ATP levels were calculated as the percentage of the control group (0 μM and 0 J/cm2) level: (∗∗) p < 0.01 vs control group; (#) p < 0.05, (##) p < 0.01 vs UVA group. Similar results were obtained in three independent experiments.

Col1α1, MMP-1, and TIMP-1 (MMP-1 inhibitor) expressions with or without 11d, UVA, or 11d plus UVA treatment. Compared with the control group, the mRNA expression had no significant change after 11d treatment. However, the mRNA expression of elastin, procollagen I type I, fibronectin, Col1α1, and TIMP-1 had a significant increase after 11d plus UVA treatment, while the MMP-1 decreased, compared with the UVA irradiated group (Figure 5). 5426

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Figure 4. FB cells were treated with or without 11d at 5 μM or treated with various concentrations of 11d for 4 h before receiving sham irradiation or irradiated with UVA (6 J/cm2) and stained with (A, B) DCFH-DA and (C, D) DiOC6 and analyzed immediately by flow cytometry: (∗) p < 0.05, (∗∗) p < 0.01 vs control group; (##) p < 0.01 vs UVA group. Similar results were obtained in three independent experiments.

Figure 5. Expression of elastin, procollagen I type I, fibronectin, COL1α1,TIMP-1, MMP-1 mRNA in different groups. The mRNA expression analyzed by quantitative real-time PCR and the expressional levels were normalized to the level of GAPDH mRNA: (∗) p < 0.05, (∗∗) p < 0.01 vs control group; (##) p < 0.01 vs UVA control. Similar results were obtained in three independent experiments.

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using CDCl3 or DMSO-d6 as a solvent. 1H NMR chemical shifts are referenced to tetramethylsilane (TMS) (0 ppm) or CDCl3 (7.26 ppm). 13C NMR was referenced to CDCl3 (77.0 ppm). The abbreviations used are as follows: s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet. Mass spectra and high resolution mass spectra were measured using electron impact (EI,

EXPERIMENTAL SECTION

Chemistry. Materials and Methods. 1H NMR and 13C NMR spectra were recorded on a 400 MHz Varian Unity Plus and Varian Mercury plus spectrometer or 200 MHz Oxford spectrometer. The chemical shift (δ) values are reported in parts per million (ppm), and the coupling constants (J) are given in Hz. The spectra were recorded 5427

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Figure 6. Immunoblot analysis of MMP-1 and TIMP-1 protein in different groups. After exposure to UVA (6 J/cm2) with or without 11d (5 μM), cell lysates were collected and immunoblotted with specific antibodies as indicated. For the internal control, the same amounts of protein extract were also probed with antibody against actin. Shown is the pattern of the MMP-1 and TIMP-1 by FB cells treated with or without 11d before receiving UVA irradiation: (∗∗) p < 0.01 vs control group; (#) p < 0.05, (##) p < 0.01 vs UVA group. Similar results were obtained in three independent experiments.

Figure 7. UVA irradiation induced SA-β-gal expression in FB cells and protective effect of compound 11d on it. The expression of SA-β-gal was detected using cytochemical staining methods as described in Materials and Methods, and cell in blue was considered as positive SA-β-gal staining. Similar results were obtained in three independent experiments: (∗∗) p < 0.01 vs control group; (##) p < 0.01 vs UVA group. Similar results were obtained in three independent experiments. the crude compound obtained was purified by column chromatography using hexane/ethyl acetate gradient to obtain pure product. 3-Phenyl-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4a). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 80%; mp 102−104 °C. 1H NMR (CDCl3, 400 MHz) δ 7.76 (dd, J = 6.8, 1.6 Hz, 2H), 7.41 (t, J = 8.0 Hz, 2H), 7.29 (t, J = 7.4 Hz, 1H), 4.32 (t, J = 7.4 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H), 2.85 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 139.35, 138.37, 131.21, 128.71 (2C), 127.37, 125.07 (2C), 46.16, 28.33, 21.74. HRMS (ESI, m/z) for C11H12N3 [(M + H)+], calcd 186.1031; found 186.1032. Anal. Calcd for C11H11N3: C, 71.33; H, 5.99; N, 22.69. Found: C, 70.85; H, 6.15; N, 21.72. 2-(5,6-Dihydro-4H-pyrrolo[1,2-e][1,2,3]triazol-3-yl)benzenamine (4b). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 75%; mp 215−217 °C. 1H NMR (CDCl3, 400 MHz) δ 7.22 (dd, J = 7.6 Hz, 1.2 Hz, 1H), 7.09 (td, J = 8.4 Hz, 1.6 Hz, 1H), 6.78−6.70 (m, 2H), 4.38 (t, J = 7.2 Hz, 2H), 3.13 (t, J = 6.8 Hz, 2H), 2.88 (quintet, J = 8.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 145.42 (2C), 138.73 (2C), 128.60, 127.42, 117.25, 116.80, 46.51, 28.59, 22.73. HRMS (ESI, m/z) for C11H13N4 [(M + H)+], calcd 201.1135; found 201.1144. Anal. Calcd for C11H12N4: C, 65.98; H, 6.04; N, 27.98. Found: C, 65.38; H, 6.13; N, 27.98. 3-(2-Trifluorophenyl)-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4c). The title compound was prepared according to the

70 eV). Melting points were determined on a Mel-Temp apparatus and are uncorrected. All products reported showed 1H NMR spectra in agreement with the assigned structures. The purity of the tested compounds was determined by combustion elemental analyses conducted by the instrument center of National Cheng Kung University by Elementar Vario EL III CHN recorder elemental analyzer. All tested compounds yielded data consistent with a purity of at least 95% compared with the theoretical values. Microwave reactions were performed on a CEM Discover instrument. Reaction progress and product mixtures were routinely monitored by TLC using Merck TLC aluminum sheets (silica gel 60 F254), and compounds were visualized with aqueous KMnO 4 . Column chromatography was carried out with 230−400 mesh silica gel 60 (Merck) and a mixture of hexane/ethyl acetate as eluent. All chemicals and reagents were purchased from commercial suppliers (Sigma Aldrich, Alfa Aesar, and Merck) and were used without further purification. General Procedure for Synthesis of Compounds 4a−h, 7a− h, 11a−i, and 15a−i. To an ice cooled solution of the corresponding yne-ol 2, 5, 8, and 12 (1.0 g, 1.0 equiv) in DMF (10 mL) were added the corresponding [(PhO2)PON3] (1.1 equiv) and DBU (1.2 equiv), and the reaction mixture was stirred at 150 °C, 50 W, 1 atm by microwave, under an argon atmosphere for 10−20 min. The reaction was monitored by TLC analysis. The crude product was extracted with ethyl acetate by washing with water followed by drying over anhydrous Na2SO4. Organic layer was concentrated under reduced pressure and 5428

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white solid. Yield 76%; mp 138−140 °C. 1H NMR (CDCl3, 400 MHz) δ 8.52 (s, 1H), 8.19 (td, J = 7.6, 1.2 Hz, 1H), 8.14 (dd, J = 8.0, 2.0 Hz, 1H), 7.60 (t, J = 8.4 Hz, 1H), 4.43 (t, J = 7.4 Hz, 2H), 3.21 (t, J = 7.4 Hz, 2H), 2.96 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 148.66, 139.35, 137.51, 133.07, 130.86, 129.85, 122.05, 119.75, 46.44, 28.49, 21.82. HRMS (ESI, m/z) for C11H11N4O2 [(M + H)+], calcd 231.0882; found 231.0883. Anal. Calcd for C11H10N4 O2: C, 57.39; H, 4.38; N, 24.34. Found: C, 57.58; H, 4.52; N, 24.10. 3-(4-Methoxyphenyl)-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4f). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 98−100 °C. 1H NMR (CDCl3, 400 MHz) δ 7.71 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 8.8 Hz, 2H), 4.38 (t, J = 7.4 Hz, 2H), 3.85 (s, 3H), 3.10 (t, J = 7.2 Hz, 2H), 2.88 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 159.07, 137.46, 130.78, 128.40, 126.51(2C), 114.24(2C), 55.31, 46.25, 28.50, 21.76. HRMS (ESI, m/ z) for C12H14N3O [(M + H)+], calcd 216.1137; found 216.1135. Anal. Calcd for C12H13N3O: C, 66.96; H, 6.09; N, 19.52. Found: C, 67.07; H, 6.10; N, 18.81. 3-(4-Nitrophenyl)-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4g). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 40%; mp 148−150 °C. 1H NMR (CDCl3, 400 MHz) δ 8.26 (d, J = 8.8 Hz, 2H), 7.93 (d, J = 8.8 Hz, 2H), 4.43 (t, J = 7.4 Hz, 2H), 3.19 (t, J = 7.4 Hz, 2H), 2.69 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 146.70, 140.18, 137.60, 132.57, 125.42 (2C), 124.29 (2C), 46.44, 28.43, 21.99. HRMS (ESI, m/z) for C11H11N4O2 [(M + H)+], calcd 231.0882; found 231.0880. Anal. Calcd for C11H10N4O2: C, 57.39; H, 4.38; N, 24.34. Found: C, 57.35; H, 4.47; N, 23.12. 3-Allyl-5,6-dihydro-4H-pyrrolo[1,2-e][1,2,3]triazole (4h). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a colorless oil. Yield 70%. 1H NMR (CDCl3, 400 MHz) δ 6.01−5.91 (m, 1H), 5.16−5.08 (m, 2H), 4.30 (t, J = 7.6 Hz, 2H), 3.46 (d, J = 6.4 Hz, 2H), 2.85−2.73 (m, 4H); 13C NMR (CDCl3, 100 MHz) δ 137.92, 134.66, 116.40, 46.262, 30.30, 29.68, 28.36, 20.53. HRMS (ESI, m/z) for C8H12N3 [(M + H)+], calcd 150.1026; found 150.1029. 3-Phenyl-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (7a). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 83%; mp 62−64 °C. 1H NMR (CDCl3, 400 MHz) δ 7.75 (dd, J = 8.4, 1.2 Hz, 2H), 7.42 (t, J = 8.0 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1H), 4.40 (t, J = 6.4 Hz, 2H), 3.00 (t, J = 6.4 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.94 (quintet, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 142.37, 131.68, 129.86, 128.59 (2C), 127.27, 126.11 (2C), 46.37, 22.26, 21.78, 20.17. HRMS (ESI, m/z) for C12H14N3 [(M + H)+], calcd 200.1188; found 200.1186. Anal. Calcd for C12H13N3: C, 72.23; H, 6.58; N, 21.09. Found: C, 72.15; H, 6.56; N, 21.07. 2-(4,5,6,7-Tetrahydro[1,2,3]triazolo[1,5-a]pyridin-3-yl)phenylamine (7b). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 75%; mp 135−137 °C. 1H NMR (CDCl3, 400 MHz) δ 7.19 (dd, J = 7.6, 1.6 Hz, 1H), 7.11 (td, J = 8.0, 1.6 Hz, 1H), 6.78 (dd, J = 8.0, 1.2 Hz, 1H), 6.74 (td, J = 7.6, 1.2 Hz, 1H), 5.10 (bs, 2H), 4.42 (t, J = 6.0 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H), 2.10 (quintet, J = 6.0 Hz, 2H), 1.93 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 145.45, 142.35, 130.31, 128.48, 128.07, 117.21, 116.37, 115.25, 46.45, 22.34, 22.14, 20.27. HRMS (ESI, m/z) for C12H15N4 [(M + H)+], calcd 215.1297; found 215.1296. Anal. Calcd for C12H14N4: C, 67.27; H, 6.59; N, 26.15. Found: C, 66.70; H, 6.54; N, 24.91. 3-(2-Trifluorophenyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5a]pyridine (7c). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a colorless oil. Yield 68%. 1H NMR (CDCl3, 400 MHz) δ 7.78 (d, J = 8.0 Hz, 1H), 7.59 (td, J = 7.6, 1.2 Hz, 1H), 7.53 (td, J = 8.4, 1.2 Hz, 1H), 7.38 (d, J = 7.2 Hz, 1H), 4.45 (t, J = 6.0 Hz, 2H), 2.68 (t, J = 6.0 Hz, 2H), 2.11 (quintet, J = 6.0 Hz, 2H), 1.90 (quintet, J = 5.6 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 140.89, 132.64, 131.62, 131.46,

Figure 8. Photomicrographs of HE-stained dorsal mouse skin. In the UVA-irradiated group, there was an increase in epidermal thickness; however, 11d plus UVA treatment decreased epidermal thickness.

Figure 9. Proposed mechanism for 11d plus UVA reduced cell senescence in FB cells. general procedure and purified by column chromatography to obtain a colorless oil. Yield 65%. 1H NMR (CDCl3, 400 MHz) δ 7.74 (d, J = 8.0 Hz, 1H), 7.61−7.47 (m, 3H), 4.38 (t, J = 7.4 Hz, 2H), 2.92 (t, J = 7.2 Hz, 2H), 2.80 (quintet, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 140.36, 137.32, 132.29, 131.49, 130.04 (d, J = 1.5 Hz), 128.49, 128.13, 125.94 (q, J = 5.3 Hz), 123.77 (q, J = 272 Hz), 46.39, 28.07, 20.96. HRMS (ESI, m/z) for C12H11F3N3 [(M + H)+], calcd 254.0905; found 254.0904. 3-(3-Methoxyphenyl)-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4d). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 86−88 °C. 1H NMR (CDCl3, 400 MHz) δ 7.43 (s, 1H), 7.35−7.27 (m, 2H), 6.86 (dd, J = 8.4, 2.8 Hz, 1H), 4.38 (t, J = 7.4 Hz, 2H), 3.87 (s, 3H), 3.12 (t, J = 7.4 Hz, 2H), 2.88 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 160.00, 139.36, 138.54, 132.63, 129.78, 117.60, 132.52, 110.24, 55.32, 46.25, 28.40, 21.85. HRMS (ESI, m/z) for C12H14N3O [(M + H)+], calcd 216.1137; found 216.1138. Anal. Calcd for C12H13N3O: C, 66.96; H, 6.09; N, 19.52. Found: C, 66.85; H, 6.09; N, 19.52. 3-(3-Nitrophenyl)-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (4e). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a 5429

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Journal of Medicinal Chemistry

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obtain a white solid. Yield 75%; mp 140−142 °C. 1H NMR (CDCl3, 400 MHz) δ 7.33 (dd, J = 7.6, 1.6 Hz, 1H), 7.13 (td, J = 7.6, 1.6 Hz, 1H), 6.72−6.66 (m, 2H), 4.36 (bs, 2H), 4.33 (t, J = 7.2 Hz, 2H), 2.98 (t, J = 6.8 Hz, 2H), 2.80 (quintet, J = 7.6 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 147.99, 144.01, 131.99, 129.98, 123.97, 117.61, 114.27, 106.87, 89.35, 83.87, 46.85, 28.03, 20.71. HRMS (ESI, m/z) for C13H13N4 [(M + H)+], calcd 255.1135; found 225.1143. Anal. Calcd for C13H12N4: C, 69.62; H, 5.39; N, 24.98. Found: C, 68.92; H, 5.43; N, 24.78. 3-Ethynyl-2-fluorobenzene-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (11c). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 75%; mp 64−66 °C. 1H NMR (CDCl3, 400 MHz) δ 7.53 (dt, J = 7.2, 1.6 Hz, 1H), 7.36−7.29 (m, 1H), 7.15−7.07 (m, 2H), 4.36 (t, J = 7.4 Hz, 2H), 3.02 (t, J = 7.2 Hz, 2H), 2.85 (quintet, J = 7.2 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 162.40 (q, J = 250.1 Hz), 144.56, 133.32, 130.27 (d, J = 7.6 Hz), 123.64 (d, J = 3.1 Hz), 123.57, 115.41 (d, J = 20.5 Hz), 111.13 (d, J = 15.2 Hz), 86.04, 83.78 (d, J = 3.0 Hz), 46.87, 28.03, 20.72. HRMS (ESI, m/z) for C13H11FN3 (M + H)+], calcd 228.0937; found 228.0936. Anal. Calcd for C13H11FN3: C, 68.71; H, 4.44; N, 18.49. Found: C, 68.43; H, 4.43; N, 18.31. 3-Ethynyl-3-methoxybenzene-5,6-dihydro-4H-pyrrolo[1,2c][1,2,3]triazole (11d). The title compound was prepared according to the general procedure and purified by column chromatography to obtain light yellow solid. Yield 78%; mp 83−85 °C. 1H NMR (CDCl3, 400 MHz) δ 7.25 (t, J = 7.6 Hz, 1H), 7.13 (td, J = 7.6, 1.2 Hz, 1H), 7.07−7.06 (m, 1H), 6.90 (dd, J = 8.4, 2.8 Hz, 1H), 4.36 (t, J = 7.4 Hz, 2H), 3.81 (s, 3H), 3.02 (t, J = 7.4 Hz, 2H), 2.84 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 159.23, 144.15, 129.36, 124.01, 123.92, 123.50, 116.22, 115.21, 92.62, 78.67, 55.24, 46.86, 28.10, 20.82. HRMS (ESI, m/z) for C14H13N3ONa [(M + Na)+], calcd 262.0956; found 262.0954. Anal. Calcd for C14H13N3O: C, 70.28; H, 5.48; N, 17.56. Found: C, 70.39; H, 5.44; N, 17.57. 3-Ethynyl-3-nitrobenzene-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (11e). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 75%; mp 138−140 °C. 1H NMR δ 8.37− 8.36 (m, 1H), 8.19 (dd, J = 8.4, 2.4 Hz, 1H), 7.84 (td, J = 7.6, 1.2 Hz, 1H), 7.54 (t, J = 8.0 Hz, 1H), 4.41 (t, J = 7.4 Hz, 2H), 3.07 (t, J = 7.4 Hz, 2H), 2.89 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 148.05, 144.84, 137.14, 129.43, 126.23, 124.42, 123.17 (2C), 90.32, 81.59, 46.99, 28.15, 20.90. HRMS (ESI, m/z) for C13H11N4O2 [(M + H)+], calcd 255.0882; found 255.0883. Anal. Calcd for C13H10N4O2: C, 61.41; H, 3.96; N, 22.04. Found: C, 61.64; H, 4.27; N, 20.97. 3-Ethynyl-4-methoxybenzene-5,6-dihydro-4H-pyrrolo[1,2c][1,2,3]triazole (11f). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 80−82 °C. 1H NMR (CDCl3, 400 MHz) δ 7.47 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.36 (t, J = 7.4 Hz, 2H), 3.82 (s, 3H), 3.01 (t, J = 7.4 Hz, 2H), 2.83 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 159.80, 143.78, 133.06(2C), 124.32, 114.62, 113.95(2C), 92.68, 77.46, 55.27, 46.85, 28.14, 20.83. HRMS (ESI, m/z) for C14H13N3ONa [(M + Na)+], calcd 262.0956; found 262.0957. Anal. Calcd for C14H13N3O: C, 70.58; H, 5.48; N, 17.56. Found: C, 69.74; H, 5.48; N, 17.20. 3-((4-Nitrophenyl)ethynyl)-5,6-dihydro-4H-pyrrolo[1,2-e][1,2,3]triazole (11g). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 40%; mp 232−234 °C. 1H NMR (DMSO, 400 MHz) δ 8.33 (bs, 2H), 8.26 (d, J = 8.8 Hz, 2H), 4.75 (d, J = 9.2 Hz, 2H), 3.25 (t, J = 6.4 Hz, 2H), 2.39−2.37 (m, 2H); 13C NMR (DMSO, 100 MHz) δ 147.01, 138.10, 129.43 (2C), 126.89, 123.07 (2C), 78.02 (2C), 50.74, 29.50, 25.36, 23.66. HRMS (ESI, m/z) for C13H11O2N4 [(M + H)+], calcd 255.0877; found 255.2107. Anal. Calcd for C13H10N4O2: C, 61.41; H, 3.96; N, 22..04. Found: C, 60.32; H, 4.37; N, 21.42 3-Ethynyl-4-ethylbenzene-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (11h). The title compound was prepared according to

130.06, 129.80, 128.61, 126.32 (q, J = 5.3 Hz), 123.80 (q, J = 272.7 Hz), 46.27, 22.47, 20.06, 19.97. HRMS (ESI, m/z) for C13H13F3N3 [(M + H)+], calcd 268.1062; found 268.1063. 3-(3-Methoxyphenyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5a]pyridine (7d). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 94−96 °C. 1H NMR (CDCl3, 400 MHz) δ 7.40−7.27 (m, 3H), 6.87 (dd, J = 8.0, 2.8 Hz, 1H), 4.42 (t, J = 6.4 Hz, 2H), 3.87 (s, 3H), 3.01 (t, J = 6.4 Hz, 2H), 2.09 (quintet, J = 6.0 Hz, 2H), 1.96 (quintet, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 159.85, 142.27, 133.03, 130.00, 129.61, 118.46, 113.36, 111.37, 55.28, 46.42, 22.31, 21.88, 20.20. HRMS (ESI, m/z) for C13H16N3O [(M + H)+], calcd 230.1293; found 230.1294. Anal. Calcd for C13H15N3O: C, 68.10; H, 6.59; N, 18.33. Found: C, 68.11; H, 6.59; N, 18.33. 3-(3-Nitrophenyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (7e). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 77%; mp 155−157 °C. 1H NMR (CDCl3, 400 MHz) δ 8.54 (s, 1H), 8.18 (td, J = 8.0, 1.2 Hz, 1H), 8.12 (dd, J = 8.4, 2.4 Hz, 1H), 7.60 (t, J = 8.0 Hz, 1H), 4.54 (t, J = 5.6 Hz, 2H), 3.09 (t, J = 5.6 Hz, 2H), 2.15 (quintet, J = 6.0 Hz, 2H), 2.04 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 148.44, 140.24, 133.43, 131.77, 130.88, 129.65, 121.84, 120.34, 46.50, 22.13, 21.74, 19.97. HRMS (ESI, m/z) for C12H13N4O2 [(M + H)+], calcd 245.1038; found 245.1037. Anal. Calcd for C12H12N4 O2: C, 59.01; H, 4.95; N, 22.94. Found: C, 58.97; H, 4.97; N, 22.95. 3-(4-Methoxyphenyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5a]pyridine (7f). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 76%; mp 78−80 °C. 1H NMR (CDCl3, 400 MHz) δ 7.68 (d, J = 8.8 Hz, 2H), 6.97 (d, J = 9.2 Hz, 2H), 4.40 (t, J = 6.0 Hz, 2H), 3.84 (s, 3H), 2.98 (t, J = 6.4 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.93 (quintet, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 158.93, 142.30, 129.04, 127.42 (2C), 124.42, 114.07 (2C), 55.25, 46.38, 22.35, 21.73, 20.27. HRMS (ESI, m/z) for C13H16N3O [(M + H)+], calcd 230.1293; found 230.1292. Anal. Calcd for C13H15N3O: C, 68.10; H, 6.59; N, 18.33. Found: C, 68.05; H, 6.56; N, 18.30. 3-(4-Nitrophenyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (7g). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 42%; mp 218−220 °C. 1H NMR (CDCl3, 400 MHz) δ 8.28 (d, J = 8.8 Hz, 2H), 7.96 (d, J = 8.8 Hz, 2H), 4.46 (t, J = 6.0 Hz, 2H), 3.08 (t, J = 6.0 Hz, 2H), 2.14 (quintet, J = 6.0 Hz, 2H), 2.03 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 146.60, 140.46, 138.16, 131.63, 126.26 (2C), 124.10 (2C), 46.57, 22.15, 22.03, 20.03. HRMS (ESI, m/z) for C12H13N4O2 [(M + H)+], calcd 245.1038; found 245.1037. Anal. Calcd for C12H12N4 O2: C, 59.01; H, 4.95; N, 22.94. Found: C, 59.14; H, 4.99; N, 21.89. 3-Allyl-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (7h). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a colorless oil. Yield 75%. 1H NMR (CDCl3, 400 MHz) δ 5.99−5.89 (m, 1H), 5.11−5.05 (m, 2H), 4.34 (t, J = 6.0 Hz, 2H), 3.44−3.42 (m, 2H), 2.71 (t, J = 6.4 Hz, 2H), 2.04 (quintet, J = 6.0 Hz, 2H),1.90 (quintet, J = 6.4 Hz, 2H); 13 C NMR (CDCl3, 100 MHz) δ 140.95, 135.08, 130.59, 116.23, 46.44, 30.05, 22.82, 20.29, 20.19. 3-Phenylethynyl-5,6-dihydro-4H-pyrrolo[1,2-c][1,2,3]triazole (11a). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 81%; mp 67−69 °C. 1H NMR (CDCl3, 400 MHz) δ 7.56−7.51 (m, 2H), 7.54−7.32 (m, 3H), 4.35 (t, J = 7.6 Hz, 2H), 3.01 (t, J = 7.4 Hz, 2H), 2.83 (quintet, J = 7.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 144.12, 131.50 (2C), 128.52, 128.28 (2C), 123.98, 122.54, 92.67, 78.84, 46.85, 28.09, 20.79. HRMS (ESI, m/z) for C13H11N3Na [(M + Na)+], calcd 232.0851; found 232.0852. Anal. Calcd for C13H11N3: C, 74.62; H, 5.30; N, 20.08. Found: C, 74.72; H, 5.30; N, 20.06. 2-((5,6-Dihydro-4H-pyrrolo[1,2-e][1,2,3]triazol-3-yl)ethynyl)benzenamine (11b). The title compound was prepared according to the general procedure and purified by column chromatography to 5430

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Journal of Medicinal Chemistry

Article

3-Ethynyl-3-nitrobenzene-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15e). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 133−135 °C. 1H NMR (CDCl3, 400 MHz) δ 8.36 (s, 1H), 8.19 (dd, J = 8.0, 2.4 Hz, 1H), 7.84 (td, J = 8.0, 1.6 Hz, 1H), 7.55 (t, J = 8.0 Hz, 1H), 4.41 (t, J = 6.4 Hz, 2H), 2.96 (t, J = 6.4 Hz, 2H), 2.12 (quintet, J = 6.0 Hz, 2H), 1.98 (quintet, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 148.10, 137.90, 129.43, 126.92, 126.19 (2C), 124.55, 123.11, 91.29, 81.43, 46.38, 22.40, 20.16, 19.59. HRMS (ESI, m/z) for C14H13N4O2 [(M + H)+], calcd 269.1038; found 269.1037. Anal. Calcd for C14H12N4O2: C, 62.68; H, 4.51; N, 20.88. Found: C, 62.78; H, 4.77; N, 20.06. 3-Ethynyl-4-methoxybenzene-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15f). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 80%; mp 64−66 °C. 1 H NMR (CDCl3, 400 MHz) δ 7.47 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 4.37 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H), 2.91 (t, J = 6.4 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.94 (quintet, J = 6.0 Hz, 2H); 13 C NMR (CDCl3, 100 MHz) δ 159.78, 135.94, 133.05(2C), 128.08, 114.78, 113.96 (2C), 93.63, 77.32, 55.29, 46.31, 22.48, 20.16, 19.71. HRMS (ESI, m/z) for C15H16N3O [(M + H)+], calcd 254.1293; found 254.1292. Anal. Calcd for C15H15N3O: C, 71.13; H, 5.97; N, 16.59. Found: C, 71.00; H, 6.11; N, 16.46. 3-((4-Nitrophenyl)ethynyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15g). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 42%; mp 155−157 °C. 1H NMR (CDCl3, 400 MHz) δ 8.39 (d, J = 8.8 Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 4.44 (t, J = 6.0 Hz, 2H), 3.21 (t, J = 6.4 Hz, 2H), 2.11 (quintet, J = 5.6 Hz, 2H), 1.95 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 184.93, 151.03 (2C), 133.17 (2C), 127.70, 113.72 (2C), 46.32 (2C), 29.69, 22.28, 22.20, 19.52. HRMS (ESI, m/z) for C14H12O2N4Na [(M + Na)+], calcd 291.0852; found 291.0824. 3-Ethynyl-4-ethylbenzene-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15h). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 62−64 °C. 1H NMR (CDCl3, 400 MHz) δ 7.45 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 4.37 (t, J = 6.4 Hz, 2H), 2.91 (t, J = 6.4 Hz, 2H), 2.66 (q, J = 8.0 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.94 (quintet, J = 6.4 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 144.96, 136.12, 131.51 (2C), 127.96, 127.88 (2C), 119.83, 93.86, 77.92, 46.30, 28.80, 22.46, 20.13, 19.68, 15.30. HRMS (ESI, m/z) for C16H18N3 [(M + H)+], calcd 252.1501; found 252.1500. Anal. Calcd for C16H17N3: C, 76.46; H, 6.82; N, 16.72. Found: C, 75.93; H, 6.88; N, 16.32. 3-(Hex-1-ynyl)-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15i). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a colorless oil. Yield 75%; 1H NMR (CDCl3, 400 MHz) δ 4.32 (t, J = 6.0 Hz, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.08−2.02 (m, 2H), 1.94−1.88 (m, 2H), 1.60 (quintet, J = 6.0 Hz, 2H), 1.50 (quintet, J = 7.2 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 135.44, 128.11, 94.83, 69.70, 46.11, 30.44, 22.35, 21.78, 19.90, 19.59, 19.00, 13.45. HRMS (ESI, m/z) for C12H18N3 [(M + H)+], calcd 204.1495; found 204.1503. Experimental Protocol for Biological Studies. Cell Culture. Fibroblasts were obtained from adult foreskin specimens as previously described69 and were maintained in DMEM medium supplemented with10% FBS, 100 U/mL penicillin G, and 100 μg/mL streptomycin sulfate (Gibco, BRL) at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed every 2−3 days. Fibroblasts were passaged at confluence by trypsinization with a solution of 0.25% trypsin−EDTA and were used between passages 5 and 10, grown to >75% confluency. UVA Irradiation. The method for UVA irradiation was described in our previous study.70 For UVA irradiation, a specific UVA lamp emitting a peak wavelength of 365 nm (UVP, Upland, CA, U.S.) was used. The cultured cells were pretreated with different agents at 5 μM for 4 h before UVA irradiation. The cultured cells were rinsed with

the general procedure and purified by column chromatography to obtain a white solid. Yield 77%; mp 76−78 °C. 1H NMR (CDCl3, 400 MHz) δ 7.45 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz, 2H), 4.34 (t, J = 7.4 Hz, 2H), 3.00 (t, J = 7.4 Hz, 2H), 2.82 (quintet, J = 7.4 Hz, 2H), 2.66 (q, J = 7.4 Hz, 2H), 1.23 (t, J = 6.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 144.97, 143.96, 131.46(2C), 127.83 (2C), 124.09, 119.63, 92.83, 78.11, 46.80, 28.72, 28.05, 20.74, 15.20. HRMS (ESI, m/z) for C15H16N3 [(M + H)+], calcd 238.1344; found 238.1343. Anal. Calcd for C15H15N3: C, 75.92; H, 6.37; N, 17.71. Found: C, 74.82; H, 6.33; N, 17.77. 3-(Hex-1-ynyl)-5,6-dihydro-4H-pyrrolo[1,2-e][1,2,3]triazole (11i). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a colorless oil. Yield 70%. 1H NMR (CDCl3, 400 MHz) δ 4.30 (t, J = 7.2 Hz, 2H), 2.93 (t, J = 6.4 Hz, 2H), 2.80 (quintet, J = 7.6 Hz, 2H), 2.41 (t, J = 7.2 Hz, 2H), 1.60 (quintet, J = 6.8 Hz, 2H), 1.48 (quintet, J = 6.8 Hz, 2H), 0.94 (t, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 141.80, 122.04, 93.82, 69.76, 46.57, 30.26, 27.86, 21.70, 20.39, 18.85, 13.34. 3-Phenylethynyl-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15a). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 85%; mp 88−90 °C. 1H NMR (CDCl3, 400 MHz) δ 7.55−7.51 (m, 2H), 7.36−7.32 (m, 3H), 4.37 (t, J = 6.0 Hz, 2H), 2.91 (t, J = 6.4 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.94 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 136.30, 131.48 (2C), 128.46, 128.28 (2C), 127.74, 122.66, 93.65, 78.64, 46.28, 22.41, 20.10, 19.63. HRMS (ESI, m/z) for C14H14N3 [(M + H)+], calcd 224.1188; found 224.1189. Anal. Calcd for C14H13N3: C, 75.31; H, 5.87; N, 18.82. Found: C, 75.11; H, 5.97; N, 18.48. 2-((4,5,6,7-Tetrahydro[1,2,3]triazolo[1,5-a]pyridin-3-yl)ethynyl)benzenamine (15b). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 77%; mp 142−144 °C. 1H NMR (CDCl3, 400 MHz) δ 7.34 (dd, J = 7.6, 1.6 Hz, 1H), 7.14 (td, J = 7.2, 1.2 Hz, 1H), 6.72−6.67 (m, 2H), 4.37 (t, J = 6.0 Hz, 2H), 2.90 (t, J = 6.4 Hz, 2H), 2.07 (quintet, J = 6.0 Hz, 2H), 1.94 (quintet, J = 6.0 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 148.17 (2C), 136.41, 132.37, 128.15, 118.04, 114.62, 107.43, 90.66, 83.98, 46.61, 22.70, 20.41, 19.91. HRMS (ESI, m/z) for C14H15N4 [(M + H)+], calcd 239.1291; found 239.1300. Anal. Calcd for C14H14N4: C, 70.57; H, 5.92; N, 23.51. Found: C, 69.78; H, 6.15; N, 22.48. 3-Ethynyl-2-fluorobenzene-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15c). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 77%; mp 86−88 °C. 1 H NMR (CDCl3, 400 MHz) δ 7.53 (dt, J = 7.4, 2.0 Hz, 1H), 7.36− 7.30 (m, 1H), 7.15−7.08 (m, 2H), 4.39 (t, J = 6.0 Hz, 2H), 2.94 (t, J = 6.4 Hz, 2H), 2.09 (quintet, J = 6.0 Hz, 2H), 1.96 (quintet, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 162.48 (q, J = 250.1 Hz), 136.80, 133.33, 130.22 (d, J = 8.4 Hz), 127.55, 123.98 (d, J = 3.8 Hz), 115.49 (d, J = 20.4 Hz), 111.41 (d, J = 15.9 Hz), 87.15, 83.72 (d, J = 3.1 Hz), 46.34, 22.47, 20.10, 19.64. HRMS (ESI, m/z) for C14H13FN3 [(M + H)+], calcd 242.1093; found 242.1094. Anal. Calcd for C14H12FN3: C, 69.70; H, 5.01; N, 17.42. Found: C, 69.68; H, 5.21; N, 16.89. 3-Ethynyl-3-methoxybenzene-4,5,6,7-tetrahydro[1,2,3]triazolo[1,5-a]pyridine (15d). The title compound was prepared according to the general procedure and purified by column chromatography to obtain a white solid. Yield 78%; mp 58−60 °C. 1 H NMR (CDCl3, 400 MHz) δ 7.25 (t, J = 6.4 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H), 7.07 (s, 1H), 6.90 (dd, J = 8.4, 2.8 Hz, 1H), 4.38 (t, J = 6.0 Hz, 2H), 3.82 (s, 3H), 2.92 (t, J = 6.4 Hz, 2H), 2.08 (quintet, J = 6.0 Hz, 2H), 1.98 (quintet, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) δ 159.28, 136.24, 129.39, 127.74, 124.04, 123.67, 116.27, 115.17, 93.65, 78.47, 55.28, 46.32, 22.46, 20.15, 19.67. HRMS (ESI, m/z) for C15H16N3O [(M + H)+], calcd 254.1293; found 254.1292. Anal. Calcd for C15H15N3O: C, 71.13; H, 5.97; N, 16.59. Found: C, 71.08; H, 6.09; N, 16.22. 5431

dx.doi.org/10.1021/jm400394s | J. Med. Chem. 2013, 56, 5422−5435

Journal of Medicinal Chemistry

Article

Table 2. Primers for Amplification primer name

primer sequence

MMP-1 (collagenase I)

forward: 5′-GCTGGGAGCAAACACATCTGAGGT-3′ reverse: 5′-TGAGCCGCAACACGATGTAAGTTG-3′ forward: 5′-CACCCACAGACGGCCTTCT-3′ reverse: 5′-CTTCTGGTGTCCGCACGAA-3′ forward: 5′-GGACTAGTCCCCCCAAGAACCTGACAACTT-3′ reverse: 5′-CGACGCGTCGTCCCCTCCCAAAGTCTTCTT-3′ forward: 5′-AAGCAGCAGCAAAGTTCGGT-3′ reverse: 5′-ACTAAGCCTGCAGCAGCTCCATA-3′ forward: 5′-ACCTACGGATGACTCGTGCTTTGA-3′ reverse: 5′-CAAAGCCTAAGCACTGGCACAACA-3′ forward: 5′-AATGGTGCTCCTGGTATTGCTGGT-3′ reverse: 5′- ACCAGTGTCTCCTTTGCTGCCA-3′ forward: 5′-GAAGGTGAAGGTCGGAGTCAACG-3′ reverse: 5′-AGTCCTTCCACGATAACCAAAGTTG-3′

TIMP-1 COL1α1 elastin fibronectin procollagen Iα1 GAPDH

phosphate-buffered saline (PBS) and then irradiated with 6 J/cm2 UVA in PBS to avoid the formation of medium-derived toxic photoproducts induced by UV exposure. The doses of irradiation were measured by a UVX digital radiometer (UVP, Upland, CA, U.S.), and the incident irradiance at the surface of the cells was found to be 4.762 mW/cm2 at the target distance of 17 cm. The calculation formula for designated time for UVA treatment is [energy (J/cm2)] = [power (W/ cm2)][exposure time (s)]. Immediately after phototreatment, PBS was removed and medium was added to the cells. All the following experiments were performed three times in triplicate. Morphology Observation. FB cells (5 × 105 cells/well) were seeded on six-well plates. Cells were irradiated with 0, 2, 4, and 6 J/ cm2 UVA radiation. Twenty-four hours after exposure, photos were taken by using a microscope at 200× phase. Cell Viability. Cell viability was assessed by the MTT assay, a mitochondrial function assay based on the ability of viable cells to reduce the redox indicator MTT to insoluble formazan crystals by mitochondrial dehydrogenase. Briefly, cells were seeded in a 96-well plate at a cell density of 10 000 cells/well. After an overnight incubation, the cells were treated with compounds at 5 μM for 4 h followed by 6 J/cm2 UVA irradiation and incubation for 24 h. The medium was then discarded and replaced with 10 μL of MTT dye. Plates were incubated at 37 °C for 2 h. The resulting formazan crystals were solubilized in 100 μL of DMSO, and the optical density was read at 540 nm with a microplate reader (MRX-II, Dynex Technology, Chantilly, VA). ATP Content Bioluminescence Assay. The amount of intracellular ATP was determined by bioluminescent assay based on the measurement of the light output of the luciferin−luciferase reaction. After treatment with compounds at 5 μM for 4 h followed by 6 J/cm2 UVA irradiation, total cell extracts from cultured FB cells were obtained immediately by lysing solution. After centrifugation to remove cell debris, we collected supernatants for ATP measurement. The total amount of intracellular ATP was determined according to the protocol provided with the ATPLite assay kit (Perkin-Elmer, Boston, MA). Determination of Intracellular ROS Level. To evaluate intracellular reactive oxygen species (ROS) levels, 2′,7′-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes) fluorescent dye was used to clarify this issue. The nonpolar DCFH-DA is converted to the polar derivative DCFH by esterases when it is taken up by the cell. DCFH is nonfluorescent but is rapidly oxidized to the highly fluorescent DCF by intracellular H2O2 or nitric oxide. Cells were treated with or without 11d at 5 μM or treated with various concentrations of 11d for 4 h before receiving UVA irradiation. Then DCFH-DA (10 μM) was added into cultured cells for 30 min at 37 °C. The fluorescence of the samples was measured with a flow cytometer. The 2′,7′-dichlorofluorescein (DCF) data were recorded using FL-1 photomultiplier.

Assessment of Mitochondrial Membrane Potential (ΔΨmt). FB cells were cultured in 35 mm dishes and allowed to reach exponential growth for 24 h before treatment. Cells were treated with or without 11d at 5 μM or treated with various concentrations of 11d for 4 h before receiving UVA irradiation. The medium was removed, and the adherent cells were trypsinized. The cells were pelleted by centrifugation at 400g for 5 min and stained in a 100 nM/mL DiOC6 dye (Molecular Probes, Eugene, OR) for 30 min at room temperature and washed with PBS twice and resuspended in PBS. The samples were analyzed immediately for fluorescence (FL-1 detector, filter 530/ 30 nm band-pass) on a FACScan flow cytometer (Elite ESP, Beckman Coulter, Brea, CA). Histograms were analyzed using Windows Multiple Document interface software (WinMDI). Senescence-Associated β-Galactosidase (SA β-gal) Staining. The SA-β-gal staining used to detect the senescence has been previously reported.71 Briefly, cells were treated with or without 11d or UVA irradiation. Six days after exposure, cells were washed twice with phosphate buffered saline (PBS) and fixed with 2% formaldehyde, 0.2% glutaraldehyde for 5 min and stained with 1 mg/mL 5-bromo-4chloro-3-indoyl-β-galactoside (X-gal) solution [the solvent was composed of dimethylformamide, 5 mM potassium ferrocyanide, 150 mM NaCl, 40 mM citric acid and sodium phosphate, and 2 mM MgCl2, pH 6.0] for at least 12 h. At the end of incubation, cells were washed with PBS and the β-gal positive cells were determined. Twohundred cells were counted for each treatment under an inverted microscope using a 20× magnification objective lens. Quantitative RT-PCR. Total RNA was extracted from FB cells with 11d and UVA and with or without 11d plus UVA treatment using the Trizol reagent (Invitrogen). An amount of 2 μg of total RNA was used for reverse transcription using RevertAid H Minus first strand cDNA synthesis kit (Fermentas, MA, U.S.) following the manufacturer’s instructions. For real-time qPCR, the ABI PRISM 7900 sequence detection system (ABI) was used. Nine microliters of master mix (2× Maxima SYBR Green/ROXqPCR master mix, 0.3 μM forward primer, 0.3 μM reverse primer) and 1 μL of 100 ng cDNA were added to the 96-well plates and amplified using a suitable program. At the completion of cycling, melting curve analysis was performed to establish the specificity of the amplicon production. Data were analyzed according to the comparative Ct method and were normalized by GAPDH expression. The primers used for amplification are indicated in Table 2. Protein Extraction and Western Blot Analysis. Total cell extracts from cultured fibroblast cells were obtained by lysing the cells in ice-cold RIPA buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, and 0.25% sodium deoxycholate) containing 2 mM PMSF, 2 μg/mL aprotinin, 2 μg/mL leupeptin, 2 mM Na3VO4, and 2 mM NaF. After centrifugation at 14 000 rpm for 10 min, the protein in the supernatants was quantified by the Bradford method (Bio-Rad). An amount of 20 μg of protein per lane was applied in 10% SDS−polyacrylamide gel. After electrophoresis, protein 5432

dx.doi.org/10.1021/jm400394s | J. Med. Chem. 2013, 56, 5422−5435

Journal of Medicinal Chemistry

Article

was transferred from the gel to the polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA). The membranes were blocked at room temperature for 1 h in PBS + 0.1% Tween 20 (PBS-T) containing 5% skim milk. After a brief rinse with PBS-T, the membrane was incubated with primary antibody at room temperature for 2 h or at 4 °C overnight. Rabbit monoclonal antibodies against MMP-1 were purchased from Millipore (Bedford, MA). TIMP-1 was purchased from Cell Signaling Technology (Beverly, MA, U.S.). Mouse monoclonal antibody against actin was purchased from Chemicon International Inc. (Temecula, CA). The membrane was incubated with the corresponding horseradish peroxidase labeled secondary antibody (Santa Cruz Biotechnology) at room temperature for 1 h. Membranes were washed with PBS-T four times for 15 min, and the protein blots were visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer Life Sciences, Boston, MA). The relative amounts of specific proteins were quantified by densitometry scanning of X-ray films and analyzed by AlphaView Image software (Alpha Innotech Corporation, CA). Animal Model. Six-week-old female ICR strain mice were obtained from BioLASCO Taiwan. The animals were housed under controlled temperature (24 ± 2 °C), humidity (50 ± 10%), and light (12 h light/ 12 h dark cycles, without any UV emission). The animals were acclimatized for at least 1 week prior to the start of the experiments. Six-week-old mice were separated into three groups (n = 4 for each group): the UVA control, the 11d plus UVA, and negative control (without treatment) groups. UVA Irradiation of Mice. Before irradiation, the dorsal hair of the mice was shaved off with an electric clipper. UVA irradiation was administered through a UVA lamp with intensity of 4.762 mW/cm2. The doses of irradiation were measured by a UVX digital radiometer (UVP, Upland, CA, U.S.). A single dose of 3 J/cm2 UVA was delivered to the dorsal skin of each mouse in the experimental group . Then a thin layer of 0.1% 11d (0.5 g per square) was applied to the dorsal skin immediately after UVA irradiation three times a week for up to 12 weeks (total UVA irradiation dose of 108 J/cm2). The mice were sacrificed after the final irradiation, and the skin samples were collected for histology analysis. Topical Application of 11d. 11d Emulsion. Compound 11d and Tefose 63 were added to oily phase, and the mixture was allowed to stir until it became homogeneous (approximately 30 min). Both the aqueous and oily phases were heated to 70−80 °C, and then the oily phase was slowly transferred into the aqueous phase with moderate stirring (approx 30 min). Finally the emulsion was cooled to 25 °C while stirring (Table 3 and Figure 10).

Figure 10. Manufacturing process for 11d emulsion.

were analyzed by Student’s t-test. P values of