Design, synthesis, and structure–activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates

Design, synthesis, and structure–activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates

Accepted Manuscript Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activ...

857KB Sizes 8 Downloads 46 Views

Accepted Manuscript Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates Xufeng Cao, Yuanyuan Xu, Yongbing Cao, Ruilian Wang, Ran Zhou, Wenjing Chu, Yushe Yang PII:

S0223-5234(15)30207-5

DOI:

10.1016/j.ejmech.2015.08.023

Reference:

EJMECH 8059

To appear in:

European Journal of Medicinal Chemistry

Received Date: 19 May 2015 Revised Date:

6 August 2015

Accepted Date: 9 August 2015

Please cite this article as: X. Cao, Y. Xu, Y. Cao, R. Wang, R. Zhou, W. Chu, Y. Yang, Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates, European Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.08.023. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates

M AN U

SC

RI PT

Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and Yushe Yang a,* a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China b School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China ∗ Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail: [email protected] † These authors contributed equally to this work.

Abstract

EP

TE D

In order to further enhance the anti-Aspergillus efficacy of our previously discovered antifungal lead compounds (I) , two series of novel azoles featuring thieno[2,3-c]pyrrolidone and thieno[3,2-c]pyrrolidone nuclei were designed and evaluated for their in vitro activity on the basis of the binding mode of albaconazole using molecular docking, along with SARs of antifungal triazoles. Most of target compounds exhibited excellent activity against Candida and Cryptococcus spp., with MIC values in the range of 0.0625 µg/ml to 0.0156 µg/ml. The thieno[3,2-c]pyrrolidone unit was more suited for improving activity against Aspergillus spp. The most potent compound, 18a, was selected for further development due to its significant in vitro activity against Aspergillus spp. (MIC = 0.25 µg/ml), as well as its high metabolic stability in human liver microsomes.

AC C

Keywords: Azoles, Synthesis, Antifungal activity, CYP51, Molecular docking During the past two decades, there is no denying the fact that the incidence of systemic fungal infections, with an enormous growth, has led to high morbidity and mortality in immunocompromised patients, such as oncology patients undergoing chemotherapy, HIV-infected individuals, patients given potent pharmacologic immunomodulators and broad-spectrum antibiotics [1-4]. Clinically, the three major pathogens, including the Candida spp. (mortality rate: 20%~40%), Cryptococcus neoformans (mortality rate: 20%~70%), and Aspergillus spp. (mortality rate: 20%~70%) account for most of systemic fungal infections [5]. Currently, 4 different classes of antifungal agents, which include the polyenes (such as amphotericin B and nystatin), azoles, echinocandins (such as caspofungin and micafungin), and antimetabolites (such as 5-fluorocytosine), are commonly used in clinic. Among these antifungal drugs, azoles exert activity by selective inhibition of the lanosterol 14α-demethylase (P45014DM, CYP51), the key enzyme in sterol biosynthesis, which would cause the growth inhibition of fungal cells [6]. Based on their broader antifungal spectrum, higher 1

ACCEPTED MANUSCRIPT

O

N N

N OH N N

N N

N

O

F

Cl

Fluconazole

N N

N

N

F OH

N

N

O OH

F

Voriconazole

N

F

N N

O

Cl

Albaconazole

O

N

N

N

OH N N

F

F

Posaconazole

TE D

F

N

O

N

N F

N N

N

Itraconazole

Cl

N

N

M AN U

F

N

O

O

SC

N

RI PT

efficacy, and lower toxicity, azoles, including fluconazole, itraconazole, voriconazole, and posaconazole (Fig. 1) are most widely used in antifungal chemotherapy [7]. Besides that, albaconazole (Fig. 1), one of the new generation azoles, has been investigated in clinical trials due to its strong therapeutic efficacy for systemic aspergillosis [8, 9]. In recent years, the incidence of Aspergillus infections among immunocompromised patients has increased dramatically [10]. Unfortunately, this pathogen is intrinsically resistant to fluconazole. The broader-spectrum azoles, such as itraconazole, voriconazole, and posaconazole are active against Aspergillus, however, the mortality rate related to aspergillosis still remains unacceptably high [10]. Therefore, there is still a pressing need for novel antifungal azoles with excellent activity against a variety of clinical pathogens, particularly, Aspergillus spp.

Figure 1. Antifungal azoles (names refer to single enantiomers, except itraconazole, which is racemic).

AC C

EP

In this line, we have recently reported the discovery and development of several series of novel fused heterocycles-linked azoles (depicted by general formula I, the most potent compound from each classes of azoles, 19, 20, 21 and 22 were taken as examples) with high potency against Candida and Cryptococcus spp. Unfortunately, almost all of the target compounds discussed above showed only moderate or low potency (MIC = 2.0 µg/ml to 32.0 µg/ml) against Aspergillus fumigatus compared with albaconazole (MIC = 0.25 µg/ml), the highly potent lead compound [11]. Thus, this has prompted us to continue to undertake structural modification of potent original compounds in search of novel azoles with improved anti-Aspergillus efficacy. To further explore the potential mechanism of azoles and provide guidance for new scaffold design, albaconazole was docked into the active site of Candida albicans CYP51 (CACYP51) [12]. As shown in Fig. 2, the triazole ring and difluorophenyl group of albaconazole formed a coordination bond and hydrophobic interactions with heme group (Fe atom) and hydrophobic pocket (F126, I104 and I131), respectively. The C2 hydroxyl group of albaconazole might form hydrogen-bonding interaction with Y132 mediated by crystal waters [13, 14]. Most importantly, the quinazolinone ring of albaconazole formed hydrophobic interactions with Y118, L376 and F380.

2

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

Figure 2. The predicted binding mode of albaconazole and 18a complex (A), albaconazole (B) and compound 18a (C) in the active site of CACYP51.

Figure 3. Previous selected compounds and new structures. Based on the structural features of albaconazole and fused heterocycles-linked azoles (I), along with the binding mode of albaconazole (Fig. 3) [15-17], we aimed to develop a new scaffold (II) derived from azoles (I), in which the piperazine or piperidine ring (A ring of I) will be replaced by 3

ACCEPTED MANUSCRIPT

TE D

M AN U

SC

RI PT

a pyrrolidone ring (A ring of II) (Fig. 3). It was our hope that, introduction of a five-membered heterocyclic ring (B ring of II) fused to the pyrrolidone ring (A ring of II) as a linker, which was purposely designed to mimic the hydrophobic interactions of quinazolinone ring of albaconazole, might result in development of more potent compounds with higher anti-Aspergillus efficacy. Then, during the comprehensive surveys of the B ring of II (such as thiophene ring, thiazole ring, etc.), as well as the R1 and R2 groups attached to the B ring of II,two more potent classes of novel azoles, 10a–i and 18a–u, which contained thieno[2,3-c]pyrrolidone and thieno[3,2-c]pyrrolidone units respectively, caught our interest. This paper describes the synthesis and SAR studies of the novel antifungal azoles and the selection of 18a for further development. As shown in Scheme 1, side chain 5 was obtained by slight modifications of published procedures [18]. Thiophene-3-carboxylic acid 1 was methylated with methyl iodide and n-butyllithium in THF to yield compound 2, which was converted to bromide-substituted compound 4 through methyl esterification and bromination reactions. Treatment of 4 with N-bromosuccinimide (NBS) and benzoyl peroxide (BPO) in CH3CN afforded compound 5 as side chain.

AC C

EP

Scheme 1. Synthetic route of intermediate 5. Reagents and conditions: (a) THF, -78 °C, n-butyl lithium, 1h, CH3I, -78 °C then room temp, 2h; (b) MeOH, Conc. H2SO4, 0 °C then reflux, 4h; (c) DMF/AcOH (3:2, v/v), NBS, room temp, overnight; (d) CCl4, BPO, NBS, 80 °C, 12h. Synthesis of the title compounds 10a–i was carried out according to Scheme 2. The ring-opening reaction of oxirane 6 [19] with NaN3 followed by catalytic hydrogenation gave amino 7 [20, 21], which was subsequently treated with compound 5 to afford intermediate 8 [22]. Thus, compound 8 was subjected to hydrolysis reaction and cyclization [23] to obtain bromine-substituted compound 10a, which was considered for use as a pivotal precursor in Suzuki coupling to efficiently prepare R2-substituted title compounds 10b–i.

4

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

Scheme 2. Synthetic route of the title compounds 10a-i. Reagents and conditions: (a) NaN3, NH4Cl, DMF, 100 °C, 6 h; (b) H2, Pd/C, room temp, overnight; (c) K2CO3, CH3CN, 0 °C then room temp, overnight; (d) NaOH, MeOH, THF, 0 degree then room temp, 8h; (e) CH2Cl2, EDCI, HOBt, Et3N, 0 °C then room temp, 8h; (f) R2B(OH)2 or R2B(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10h. To further investigate the effect of different types of linkers (fused heterocycles of II) on their antifungal activity, many novel azoles 18a–u featuring R1 and R2-substituted thieno[3,2-c]pyrrolidone side chains were synthesized. As outlined in Scheme 3, intermediate 15 was synthesized via four steps [24]. Bromidation of the starting material 11 with Br2 in CHCl3, followed by oxidation with NaH2PO4/H2O2 and methyl esterification afforded methyl ester 14. In the presence of BPO, compound 14 was subsequently reacted with NBS in CCl4 to yield side chain 15.

Scheme 3. Synthetic route of intermediate 15. Reagents and conditions: (a) Br2, CHCl3, 0 °C then reflux, 4h; (b) CH3CN, NaH2PO4, H2O2, 0 °C then room temp, 8h; (c) MeOH, Conc. H2SO4, 0 °C then reflux, 4h; (d) CCl4, BPO, NBS, 80 °C, 12h. As shown in Scheme 4, another pivotal precursor, 18a was prepared from amino 7 by using procedures similar to those described above. Afterward, the title compounds 18b–u were prepared conveniently by Suzuki coupling of compound 18a with various boric acid esters or boric acid. All the new compounds described above were characterized by NMR and ESI-MS spectroscopic analysis. The in vitro minimum inhibitory concentration (MIC) of all title compounds were determined by the method recommended by the National Committee for Clinical Laboratory Standards 5

ACCEPTED MANUSCRIPT

M AN U

SC

RI PT

(NCCLS) using the serial dilution method in 96-well microtest plates [25, 26]. All of target compounds were evaluated for their antifungal activity against a panel of pathogenic fungi obtained from the American Type Culture Collection (ATCC) or clinical isolates. Fluconazole, voriconazole, and albaconazole purchased from respective manufacturers, as well as previously prepared azoles 19–22 [11], were served as the positive control drugs. The results of in vitro antifungal activities are summarized in Tables 1–2.

AC C

EP

TE D

Scheme 4. Synthetic route of the title compounds 18a-u. Reagents and conditions: (a) K2CO3, CH3CN, 0 °C then room temp, overnight; (b) NaOH, MeOH, THF, 0 degree then room temp, 6h; (c) CH2Cl2, EDCI, HOBt, Et3N, 0 °C then room temp, 8h; (d) R2B(OH)2 or R2B(pin), Cs2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 80 °C, 10h. As summarized in Table 1, almost all of thieno[2,3-c]pyrrolidone derivatives 10a–I exhibited remarkable activity against all of the tested strains, with the exception of Aspergillus fumigatus, which are superior or comparable to those of the reference drugs, including compounds 19–22, voriconazole and albaconazole. Generally, the aryl-substituted analogs 10b–I were more potent than bromide-substituted compound 10a. In addition, these analogs substituted with phenyl ring (10b), as well as some different types of nitrogen aromatic heterocycles (10c–i), showed similar activity. Table 1 In vitro antifungal activity of thieno[2,3-c]pyrrolidone derivatives. (MIC, µg/ml)a

6

ACCEPTED MANUSCRIPT N

S

OH N

N

F

N

Br

N

N F

N

O

R2

O

F

F

10b-i

10a C. alb compd

S

OH N

C. par

C.neo

C.gla

A.f um

a

22019

0.03125 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0078 0.0156 0.0156 0.03125 0.25 0.25 0.25 0.0156 0.0078

0.0625 0.03125 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0625 0.125 0.5 0.25 0.0156 0.0156

0.0625 0.03125 0.03125 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0156 0.0625 0.125 0.5 1 0.0156 0.0156

32609

537

0.125 0.0625 0.0156 0.0156 0.0078 0.0156 0.0156 0.0078 0.0156 0.0625 0.0625 0.125 0.5 4 0.0156 0.0078

0.125 0.0625 0.0625 0.0156 0.0156 0.0625 0.0156 0.0625 0.0156 0.0156 0.0625 1 4 1 0.0156 0.0156

SC

phenyl4-pyridyl3-pyridyl5-pyrimidinyl2-CN-5-pyridyl2-methoxy-5-pyridyl3-F-5-pyridyl2-methoxy-5-pyrimidinyl-

Y0109

M AN U

10a 10b 10c 10d 10e 10f 10g 10h 10i 19 20 21 22 fluconazole voriconazole albaconazole

SC5314

RI PT

R2 7544

32 32 >64 16 32 32 64 >64 16 2 2 32 >32 >64 0.25 0.25

AC C

EP

TE D

Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus fumigatus. Apparently, as all of the target compounds 10a–i discussed above (Table 1) were ineffective against Aspergillus fumigatus, a series of novel azole 18a–u were prepared through the bioisosteric approach reported previously [11]. Similar to compounds 10a–i, most of thieno[3,2-c]pyrrolidone derivatives showed excellent in vitro activity (Table 2) against Candida spp. and Cryptococcus neoforman. Most importantly, some azoles also had high or moderate potency against Aspergillus fumigatus. Compared with the most potent compound 18a (R2 = Br ), introduction of several types of phenyl groups (18b–l) on position 2 of the thiophene ring of thieno[3,2-c]pyrrolidone derivatives resulted in a slight decrease in activity. Substitution within this phenyl ring was also investigated. We found that the in vitro antifungal activity of para-substituted derivatives were stronger than those of meta-substituted compounds. (18c and 18d, 18e and18f, 18g and 18h, 18i and 18j, 18k and 18l ). Compounds (18g, 18i, 18k or 18h, 18j, 18l ) with a phenyl ring substituted with an electron-withdrawing group (–F, –Cl, –CN) exhibited activity similar to those observed in electron-donating groups (–CH3, –CH3O) substituted phenyl analogs (18c, 18e or 18d, 18f). In addition, most of the pyridine and pyrimidine ring analogs (18m–u) were tested and proved to be comparable to those of the phenyl analogs, except for 18p and18s, which featured an electron-withdrawing group (–CN, –F) at the ortho-position of the meta-pyridyl ring. Compared with the pyridine ring analogs 18p–t (–CN, –CH3O, –F) and pyrimidine ring analog 18u (–CH3O), unsubstituted aryl analogs 18m–o showed relatively stronger activity. Table 2 In vitro antifungal activity of thieno[3,2-c]pyrrolidone derivatives. (MIC, µg/ml)a

7

a

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

Abbreviations: C.alb., Candida albicans; C.par., Candida parapsilosis; C.neo., Cryptococcus neoformans; C.gla., Candida glabrata; A.fum., Aspergillus fumigatus. In order to further investigate the binding mode and the SARs of thieno[3,2-c]pyrrolidone derivatives 18a–u, a representative compound, 18a, was docked into the active site of Candida albicans CYP51 (CACYP51) [12]. Compound 18a and albaconazole exhibited similar conformations as their triazole ring and difluorophenyl moiety superimposed well (Fig. 2). A conserved water molecule in CYP51 [27] formed three hydrogen bonds with C2 hydroxyl group of 18a, propionate of porphyrin ring D and Y132. The water-mediated hydrogen bond network among 18a, heme group and CYP51 plays important roles in strengthening their binding affinity. The variable part, bromine-substituted thieno[3,2-c]pyrrolidone nuclei formed hydrophobic interactions with Y118, L376 and F380, similar to the quinazolinone ring of albaconazole. Moreover, the bromine atom of compound 18a formed additional hydrophobic interactions with M508. These results may provide a good explanation for the excellent in vitro activity of compound 18a. Because of its significant activity and broad antifungal spectrum, compound 18a was evaluated for prediction of its metabolic stability using human liver microsomes (Table 3). With high metabolic bioavailability (MF =80% ), a long half-life (t1/2 = 420.1 min) and low in vitro liver 8

ACCEPTED MANUSCRIPT microsomal clearance (Clint = 5.0 µL/min/mg) in human liver microsomes , compound 18a is regarded metabolically sufficiently stable [28-30].

RI PT

Table 3 Half-lives and intrinsic clearances of compounds 18a in human liver microsomesa

0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 µM, incubation at 37 °C, samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS. bt1/2: elimination half-life in human liver microsomes. cClint: intrinsic body clearance. dMF: metabolic bioavailability In conclusion, we identified two series of novel azoles (II) as potent antifungal agents containing a thienopyrrolidone nucleus. Summarizing the results of Tables 1–2, we can conclude that both potent in vitro antifungal activity and broad spectrum were well attained when different types of fused heterocycles linkers of I (19–22) were replaced with thienopyrrolidone nucleus. In addition, the linker of II, thieno[3,2-c]pyrrolidone nucleus has been identified for anti-Aspergillus activity. Among these compounds, bromide-substituted analog 18a displayed the most remarkable in vitro activity against Candida spp., Cryptococcus neoformans and Aspergillus fumigatus, which is superior or comparable to those of the reference drugs voriconazole and albaconazole. The observation described above was also clarified by a computational molecular docking of azoles with CYP51. Additionally, 18a was found to be stable metabolically in human liver microsomes. Further developments of compound 18a are ongoing in our laboratory.

Acknowledgment

TE D

M AN U

SC

a

AC C

EP

The authors thank Prof. Cheng Luo and Ran Zhou from Drug Discovery and Design Center, Shanghai Institute of Materia Medica, for molecular docking experiments; We also thank Ran Zhou for writing the script for the molecular docking analysis presented in the paper. We also thank Dr. Jia Liu for performing the metabolic stability assay. We are grateful to the National Science and Technology Major Project for the support of this research. The project described was supported by Key New Drug Creation and Manufacturing Program, China (No. 2014ZX09101004-003). This work was also supported by the National Natural ScienceFoundation of China (No. 21402223).

Supplementary data

Experimental details and spectroscopic characterization of compounds involved in the synthesis are available online.

References and notes [1] M.K. Kathiravan, A.B. Salake, A.S. Chothe, P.B. Dudhe, R.P. Watode, M.S. Mukta, S. Gadhwe, The biology and chemistry of antifungal agents: A review, Bioorg. Med. Chem. 20 (2012) 5678-5698. 9

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[2] A.H. Groll, J. Lumb, New developments in invasive fungal disease, Future Microbiol. 7 (2012) 179-184. [3] O. Turel, Newer antifungal agents, Expert Rev. Anti-Infect. Ther. 9 (2011) 325-338. [4] L.A. Sorbera, J. Aravamudan, E. Rosa, Therapeutic target for candidiasis, Drugs Future 36 (2011) 627-630. [5] J.-P. Latgé, Aspergillus fumigatus and Aspergillosis, Clin. Microbiol. Rev. 12 (1999) 310-350. [6] L. Meerpoel, P. Lewi, Conazoles, Molecules 15 (2010) 4129-4188. [7] S. Wang, G. Jin, W. Wang, L. Zhu, Y. Zhang, G. Dong, Y. Liu, C. Zhuang, Z. Miao, J. Yao, W. Zhang, C. Sheng, Design, synthesis and structure–activity relationships of new triazole derivatives containing N-substituted phenoxypropylamino side chains, Eur. J. Med. Chem. 53 (2012) 292-299. [8] C. Girmenia, New generation azole antifungals in clinical investigation, Expert Opin. Invest. Drugs 18 (2009) 1279-1295. [9] R. Guillon, F. Pagniez, C. Picot, D. Hedou, A. Tonnerre, E. Chosson, M. Duflos, T. Besson, C. Loge, P. Le Pape, Discovery of a novel broad-spectrum antifungal agent derived from albaconazole, ACS Med. Chem. Lett. 4 (2013) 288-292. [10] I. Hadrich, F. Makni, S. Neji, S. Abbes, F. Cheikhrouhou, H. Trabelsi, H. Sellami, A. Ayadi, Invasive Aspergillosis: Resistance to Antifungal Drugs, Mycopathologia 174 (2012) 131-141. [11] X. Cao, Z. Sun, Y. Cao, R. Wang, T. Cai, W. Chu, W. Hu, Y. Yang, Design, Synthesis, and Structure–Activity Relationship Studies of Novel Fused Heterocycles-Linked Triazoles with Good Activity and Water Solubility, J. Med. Chem. 57 (2014) 3687-3706. [12] B.C. Monk, T.M. Tomasiak, M.V. Keniya, F.U. Huschmann, J.D. Tyndall, J.D. O'Connell, 3rd, R.D. Cannon, J.G. McDonald, A. Rodriguez, J.S. Finer-Moore, R.M. Stroud, Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer, Proc. Natl. Acad. Sci. USA 111 (2014) 3865-3870. [13] C. Sheng, W. Zhang, H. Ji, M. Zhang, Y. Song, H. Xu, J. Zhu, Z. Miao, Q. Jiang, J. Yao, Y. Zhou, J. Zhu, J. Lü, Structure-Based Optimization of Azole Antifungal Agents by CoMFA, CoMSIA, and Molecular Docking, J. Med. Chem. 49 (2006) 2512-2525. [14] H. Ji, W. Zhang, Y. Zhou, M. Zhang, J. Zhu, Y. Song, J. Lu, J. Zhu, A Three-Dimensional Model of Lanosterol 14α-Demethylase of Candida albicans and Its Interaction with Azole Antifungals, J. Med. Chem. 43 (2000) 2493-2505. [15] X. Chai, J. Zhang, Y. Cao, Y. Zou, Q. Wu, D. Zhang, Y. Jiang, Q. Sun, New azoles with antifungal activity: Design, synthesis, and molecular docking, Bioorg. Med. Chem. Lett. 21 (2011) 686-689. [16] Y. Zou, Q. Zhao, J. Liao, H. Hu, S. Yu, X. Chai, M. Xu, Q. Wu, New triazole derivatives as antifungal agents: Synthesis via click reaction, in vitro evaluation and molecular docking studies, Bioorg. Med. Chem. Lett. 22 (2012) 2959-2962. [17] Y. Jiang, J. Zhang, Y. Cao, X. Chai, Y. Zou, Q. Wu, D. Zhang, Y. Jiang, Q. Sun, Synthesis, in vitro evaluation and molecular docking studies of new triazole derivatives as antifungal agents, Bioorg. Med. Chem. Lett. 21 (2011) 4471-4475. [18] M. Lang, M.H.J. Seifert, K.K. Wolf, A. Aschenbrenner, R. Baumgartner, T. Wieber, V. Trentinaglia, M. Blisse, N. Tajima, T. Yamashita, D. Vitt, H. Noda, Discovery and hit-to-lead optimization of novel allosteric glucokinase activators, Bioorg. Med. Chem. Lett. 21 (2011) 5417-5422. 10

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

[19] J. Pesti, C.-K. Chen, L. Spangler, A.J. DelMonte, S. Benoit, D. Berglund, J. Bien, P. Brodfuehrer, Y. Chan, E. Corbett, C. Costello, P. DeMena, R.P. Discordia, W. Doubleday, Z. Gao, S. Gingras, J. Grosso, O. Haas, D. Kacsur, C. Lai, S. Leung, M. Miller, J. Muslehiddinoglu, N. Nguyen, J. Qiu, M. Olzog, E. Reiff, D. Thoraval, M. Totleben, D. Vanyo, P. Vemishetti, J. Wasylak, C. Wei, The Process Development of Ravuconazole: An Efficient Multikilogram Scale Preparation of an Antifungal Agent (1), Org. Process Res. Dev. 13 (2009) 716-728. [20] T. Konosu, T. Miyaoka, Y. Tajima, S. Oida, Triazole antifungals. III. Stereocontrolled synthesis of an optically active triazolylmethyloxirane precursor to antifungal oxazolidine derivatives, Chem. Pharm. Bull. 39 (1991) 2241-2246. [21] T. Konosu, Y. Tajima, N. Takeda, T. Miyaoka, M. Kasahara, H. Yasuda, S. Oida, Triazole antifungals. IV. Synthesis and antifungal activities of 3-acylamino-2-aryl-2-butanol derivatives, Chem. Pharm. Bull. 39 (1991) 2581-2589. [22] B.S. Freeze, M. Hirose, L. Hong Myung, T.B. Sells, Z. Shi, L.R. Takaoka, S. Vyskocil, T. Xu, Preparation of heteroaryls as PI3K and mTor kinases inhibitors for treating proliferative, inflammatory or cardiovascular disorders, in, Millennium Pharmaceuticals, Inc., USA . 2012, pp. 268pp. [23] M. Prat, M.A. Buil, M.D. Fernandez, J. Castro, J.M. Monleon, L. Tort, G. Casals, M. Ferrer, J.M. Huerta, S. Espinosa, M. Lopez, V. Segarra, A. Gavalda, M. Miralpeix, I. Ramos, D. Vilella, M. Gonzalez, M. Cordoba, A. Cardenas, F. Anton, J. Beleta, H. Ryder, Discovery of novel quaternary ammonium derivatives of (3R)-quinuclidinyl carbamates as potent and long acting muscarinic antagonists, Bioorg. Med. Chem. Lett. 21 (2011) 3457-3461. [24] A. Pasternak, R.K. Dejesus, Y. Zhu, L. Yang, S. Walsh, B. Pio, A. Shahripour, H. Tang, K. Belyk, D. Kim, Inhibitors of the renal outer medullary potassium channel, in, Merck Sharp & Dohme Corp., USA . 2012, pp. 229pp. [25] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved Standard, edn 3; Document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2009. [26] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved Standard, edn 3; Document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. [27] N. Strushkevich, S.A. Usanov, H.W. Park, Structural basis of human CYP51 inhibition by antifungal azoles, J. Mol. Biol. 397 (2010) 1067-1078. [28] R.S. Obach, Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes, Drug Metab. Dispos. 27 (1999) 1350-1359. [29] Y.Y. Lau, G. Krishna, N.P. Yumibe, D.E. Grotz, E. Sapidou, L. Norton, I. Chu, C. Chen, A.D. Soares, C.C. Lin, The use of in vitro metabolic stability for rapid selection of compounds in early discovery based on their expected hepatic extraction ratios, Pharm. Res. 19 (2002) 1606-1610. [30] A. Fritsche, A.S. Elfringhoff, J. Fabian, M. Lehr, 1-(2-Carboxyindol-5-yloxy)propan-2-ones as inhibitors of human cytosolic phospholipase A2α: Synthesis, biological activity, metabolic stability, and solubility, Bioorg. Med. Chem. 16 (2008) 3489-3500.

11

ACCEPTED MANUSCRIPT Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates

M AN U

SC

RI PT

Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and Yushe Yang a,* a State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China b School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433, China c Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China ∗ Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail: [email protected] † These authors contributed equally to this work.

Highlights

AC C

EP

TE D

• Two series of novel azoles featuring thienopyrrolidone nuclei were designed and synthesized. • A molecular docking of azoles with CYP51 was conducted to understand the mechanism of action and provide guidance for new scaffold design. • The target compounds exhibited excellent activity with a broad spectrum. • Compound 18a exhibit significant anti-Aspergillus efficacy (MIC = 0.25 µg/ml). • Compound 18a was metabolically stable in human liver microsomes (MF =80% ).

1

ACCEPTED MANUSCRIPT

Design, synthesis, and structure-activity relationship studies of novel thienopyrrolidone derivatives with strong antifungal activity against Aspergillus fumigates Xufeng Caoa,†, Yuanyuan Xua,†, Yongbing Caob, Ruilian Wangb, Ran Zhouc, Wenjing Chua, and a

RI PT

Yushe Yang a,* State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Shanghai

Institute for Biological Sciences, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China b

School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,

c

SC

China

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute

M AN U

of Materia Medica, Chinese Academy of Sciences, 555 Zuchong Zhi Road, Shanghai 201203, China

Corresponding author: Yushe Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:



[email protected]

These authors contributed equally to this work.

I. Biological test

TE D

Experimental details

Minimum Inhibitory Concentration Testing. The in vitro minimum inhibitory concentration (MIC) of all title compounds were determined by the method recommended by the National

EP

Committee for Clinical Laboratory Standards (NCCLS) using the serial dilution method in 96well microtest plates [1, 2]. The tested fungi species included six pathogenic fungi. Candida albicans, Candida parapsilosis 22019 and Cryptococcus neoformans 32609 were purchased

AC C

from the American Type Culture Collection (ATTC), and other strains were provided by Shanghai Changhai Hospital. Candida parapsilosis 22019 was used as the quality-controlled strain, and tested in each assay. Fluconazole, voriconazole, and albaconazole purchased from respective manufacturers, as well as previously prepared azoles 19–21, were served as the positive control drugs. All of test compounds were dissolved in dimethyl sulfoxide (DMSO), serially diluted in growth medium. The strains were incubated at 35 °C. The MIC values was determined at 24 h for Candida spp., at 48 h for Aspergillus fumigatus, and 72 h for Cryptococcus neoformans 32609.

S1

ACCEPTED MANUSCRIPT

Metabolic Stability Assay. The assay was performed with liver microsomes pooled from human (adult male & female, catalogue no. 99268, BD Gentest, USA). The incubation was performed as follows: microsomes in 0.1 M trishydroxymethyl aminomethane/hydrochloric acid buffer pH 7.4 (0.33 mg/mL microsomal protein), cofactor MgCl2 (5 mM), tested compound

RI PT

(final concentration 0.1 µM, cosolvent (0.01% DMSO), 0.005% bovine serum albumin), and then NADPH (1 mM) at 37 °C for 60 min. The reaction can be started by the addition of liver microsomes or the tested compound or NADPH. Aliquots were sampled at 0, 7, 17, 30, and 60 min incubation, and enzymatic reaction was stopped by protein precipitation in methanol. After

SC

centrifugation, samples were then analyzed by LC/MS/MS. The assay evaluated the metabolic stability of compounds by measuring the in vitro half-life (t1/2) and human liver microsomal

M AN U

clearance (Clint) as previously described [3-5]

Molecular Docking. Structure of Candia albicans CYP51 was built based on crystal structure of S. cerevisiae CYP51 [6] (PDB code: 4K0F) using SWISS-MODEL [7]. GOLD Suite v5.0 was utilized to perform docking analysis. Iron atom of heme group was treated as metal ion to predict the coordination geometry. The position of C1 atom of the ligand in template CYP51 was

II. Chemistry

TE D

selected as the centroid of the binding site. GoldScore was employed to evaluate the fitness.

General Chemical Methods. Compounds not described below were purchased from commercial vendors. Provided samples were of greater than 95% purity, as determined by the

EP

suppliers, via NMR. Melting points (uncorrected) were determined on an X-4 melting point apparatus. Optical rotations were determined with a Perkin-Elmer 241 polarimeter at 22 °C and at 589 nm using a sodium lamp and a 1 ml cell. Data are reported as follows: [α]22D

AC C

(concentration g/100ml, solvent). 1H NMR spectral data and

13

C NMR spectral data were

recorded on a Bruker 300 NMR or a Bruker 400 NMR or a Bruker 500 NMR spectrometer using TMS as an internal standard, chemical shifts are given in parts per million (d) values and coupling constants (J) in Hertz. EI-MS spectra were obtained on a Finnigan MAT 95 mass spectrometer and ESI-MS spectra were obtained on a Krats MS 80 mass spectrometer. Elemental analysis was obtained via a vario EL spectrometer. HPLC analysis was conducted for all assayed compounds on an Agilent 1100 series LC system (PLATISILTM ODS 5µm 250 × 4.6 mm) with two solvent systems (acetonitrile/water or methanol/buffer (0.1% CF3COOH in water)). All the

S2

ACCEPTED MANUSCRIPT

assayed compounds possess ≥ 95% purity. Silica gel thin-layer chromatography was performed on precoated plates GF254 (Qindao Haiyang Chemical, China). Column chromatography was performed on silica gel H (200-300 mesh) , and the solvent proportions were expressed on a volume:volume basis. Chemicals and solvents used were commercially available without any

RI PT

pretreatment.

2-methylthiophene-3-carboxylic acid (2). 1 (15.00 g, 0.12 mol) was dissolved in THF (50 ml). Under argon protection, n-butyl lithium (2.0 M THF solution, 120 ml) was added dropwise at -78 °C. The reaction mixture was stirred for 1 h at -78 °C. To this mixture was added CH3I

SC

(8.7 ml, 0.14 mol), then the reaction mixture was allowed to warm to room temperature and stirred for 2 hours. Slowly quenched the reaction with hydrogen chloride (2.0 M ethyl acetate

M AN U

solution), then stirred the mixture at room temperature for 30 min before concentrating in vacuo. The residue was diluted with H2O (100 ml) and extracted with ethyl acetate (200 ml). The combined organic layers were washed with H2O (50 ml ×2) and brine (50 ml ×2), dried over anhydrous Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure to give a yellow crude solid, which was purified by recrystallization from acetic acid/H2O to afford 2 (14.00 g, 82.1% ) as a white solid: mp 108-110 °C. 1H NMR (400 MHz, CDCl3):δ 7.45 (d, J =

TE D

5.5 Hz, 1H), 7.01 (d, J = 5.5, 1H), 2.73 (s, 3H). MS (EI) m/z: 142 (M+).

methyl 2-methylthiophene-3-carboxylate (3). 2 (14.00 g, 98.60 mmol) was dissolved in methanol (60 ml). Concentrated sulfuric acid (30 ml) was added dropwise at 0 °C. The reaction

EP

mixture was stirred for 4 h at reflux, then was cooled to room temperature, and the solvent was evaporated under reduced pressure. The residue was partitioned between CH2Cl2 (200 ml) and

AC C

H2O (60 ml). The organic phase was washed with H2O (50 ml), saturated aqueous sodium bicarbonate solution (50 ml) and saturated brine solution (50 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure to give 3 (12.00 g, 78.0%) as a colorless viscous oil. 1H NMR (400 MHz, CDCl3):δ 7.38 (d, J = 5.5 Hz, 1H), 6.98 (d, J = 5.1, 1H), 3.85 (s, 3H), 2.73 (s, 3H). MS (EI) m/z: 156 (M+).

methyl 5-bromo-2-methylthiophene-3-carboxylate (4). 3 (12.00 g, 76.82 mmol) was dissolved in 100 ml DMF/acetic acid (3:2). N-bromosuccinimide (13.70 g, 77.00 mmol) was added at room temperature and stirred overnight. The reaction mixture was diluted with H2O (30 S3

ACCEPTED MANUSCRIPT

ml) and extracted with ethyl acetate (60 ml ×2). The organic phase was washed with saturated aqueous sodium bisulfate solution (30 ml) and saturated brine solution (30 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure to give 4 (16.10 g, 789.1%) as a pale yellow viscous oil. 1H NMR (400 MHz, CDCl3):δ 7.33 (s, 1H), 3.84 (s, 3H),

RI PT

2.67 (s, 3H). MS (EI) m/z: 235 (M+).

methyl 5-bromo-2-(bromomethyl)thiophene-3-carboxylate (5). 4 (10.00 g, 42.53 mmol) and benzoyl peroxide (1.10 g, 4.53 mmol) was dissolved in 150 ml CCl4. N-bromosuccinimide

SC

(7.96 g, 44.72 mmol) was added in batches at room temperature. The reaction mixture was allowed to warm to 80 °C and stirred for 12 hours. After cooling, the solvent was evaporated

M AN U

under reduced pressure. The residue was partitioned between ethyl acetate (200 ml) and H2O (80 ml). The organic phase was washed with H2O (60 ml) and saturated brine solution (60 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum: ethyl acetate 80:1~60:1) to give 5 (8.23 g, 61.6%) as a white solid: mp 95-96 °C. 1H NMR (400 MHz, CDCl3):δ 7.35 (s, 1H),

TE D

5.00 (s, 2H), 3.86 (s, 3H). MS (EI) m/z: 312 (M+).

(2R,3R)-3-amino-2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)butan-2-ol (7). A mixture of epoxide 6 (10.00 g, 40.00 mmol), sodium azide (3.90 g, 60.00 mmol), ammonium chloride (2.73 g, 51.00 mmol) and DMF(60.0 ml) was stirred at 80 °C for 10h. After cooling, the mixture

EP

was partitioned between ethyl acetate (200 ml ×2) and brine (80 ml). The organic phase was washed with H2O (60 ml ×2) and saturated brine solution (60 ml ×2), dried over Na2SO4, and

AC C

filtrated, then the solvent was evaporated under reduced pressure to afford dark brown oil, which was used for the following reaction without further purification. The crude solid was dissolved in methanol (40 ml), to the solution was added 10% palladium on carbon (1.60 g) at room temperature. The mixture was stirred under hydrogen at 1 atm overnight and then filtered, concentrated under reduced pressure to give 7 (7.35 g, 68.8%) as a white solid: mp 146-148°C. [α]22D –71.0°(c 0.5, CHCl3). 1H NMR (300 MHz, DMSO) δ 8.25 (s, 1H), 7.63 (s, 1H), 7.25 (dd, J = 16.0, 8.9 Hz, 1H), 7.17-7.06 (m, 1H), 6.86 (td, J = 8.5, 2.3 Hz, 1H), 5.48 (s, 1H), 4.65 (q, J = 14.4 Hz, 2H), 3.41 (dd, J = 6.5, 2.7 Hz, 1H), 0.69 (d, J = 6.5 Hz, 3H). MS (EI) m/z: 268(M+).

S4

ACCEPTED MANUSCRIPT

methyl

5-bromo-2-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-

butan-2-ylamino}methyl]thiophene-3-carboxylate (8). Under nitrogen flow, K2CO3 (0.84g, 6.05 mmol) was added to a stirred solution of 7 (1.35 g, 5.03 mmol) and 5 (1.74 g, 5.56 mmol) in

RI PT

CH3CN (50 ml) at 0 °C. After addition, the reaction mixture was was allowed to warm to room temperature and stirred overnight. The solvent was evaporated under reduced pressure. The residue was partitioned between ethyl acetate (60 ml) and H2O (30 ml). The organic phase was washed with saturated brine solution (30 ml), dried over Na2SO4, and filtrated, then the solvent

SC

was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum: ethyl acetate 6:1~4:1) to give 8 (1.68 g, 66.6%) as a colorless oil. H NMR (400 MHz, CDCl3):δ 7.83 (s, 1H), 7.77 (s, 1H), 7.38-7.31 (m, 2H), 6.75-6.63 (m, 2H),

M AN U

1

5.08 (d, J = 14.3, 1H), 4.89-4.84 (m, 2H), 4.53 (d, J = 17.0, 1H), 4.09 (d, J = 17.0, 1H), 3.80 (s, 3H), 3.26 (q, J = 6.9, 1H), 0.91 (d, J = 6.6 Hz, 3H). MS (EI) m/z: 500 (M+).

5-bromo-2-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-butan-2ylamino}methyl]thiophene-3-carboxylic acid (9). NaOH (1.0 M aqueous solution, 7.85 ml)

TE D

was added to a stirred mixture of 8 (786.50 mg,1.57 mmol) and MeOH/THF (10 ml/10 ml) at 0°C. The mixture was stirred for 8 h at room temperature, then the solvent was evaporated under reduced pressure. The residue was added to ice and acided (PH = 2) by addition of hydrogen chloride (1.0 M aqueous solution). The aqueous solution was extracted with ethyl

EP

acetate/isopropanol (45 ml/5 ml), the combined organic phase was washed with saturated brine solution, dried over Na2SO4, and filtrated, the solution was concentrated in vacuo to give 9

AC C

(650.00 mg, 85.0%) as a colorless viscous oil. 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.74 (s, 1H), 7.46-7.36 (m, 2H), 6.83-6.71 (m, 2H), 5.23-5.06 (m, 3H), 4.59 (d, J = 19.1 Hz, 1H), 4.26 (d, J = 14.6 Hz, 1H), 1.13 (d, J = 7.2 Hz, 3H). MS (EI) m/z: 486 (M+).

2-bromo-5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2yl]-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10a). HOBt (66.00 mg, 0.49 mmol) and triethylamine (125.00 mg, 1.23 mmol) were added to a stirred mixture of 9 (195.00 mg, 0.40 mmol) and CH2Cl2 (10 ml) at 0°C. The mixture was stirred for 0.5 h at room temperature, then the solvent was evaporated under reduced pressure. To this mixture was added EDCI (118.00 mg,

S5

ACCEPTED MANUSCRIPT

0.61 mmol), then the reaction mixture was allowed to warm to room temperature and stirred for 8 hours. The organic layers were washed with H2O (20 ml×2) and hydrogen chloride (1.0 M aqueous solution), then neutralized by addition of saturated aqueous sodium bicarbonate solution. The organic phase was washed with saturated brine solution (20 ml), dried over Na2SO4, and

RI PT

filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2: MeOH 200:1~100:1) to give 10a (131.00 mg, 69.3%) as a pale yellow solid: mp 136-138 °C. 1H NMR (400 MHz, CDCl3) δ 7.81 (s, 1H), 7.67 (s, 1H), 7.38 (dd, J = 14.8, 8.0 Hz, 1H), 7.23 (s, 1H), 6.78-6.68 (m, 2H), 5.11 (d, J = 14.2 Hz, 1H), 4.98

3H).

13

SC

(d, J = 17.9 Hz, 2H), 4.55 (d, J = 18.6 Hz, 1H), 4.21 (d, J = 14.2 Hz, 1H), 1.08 (d, J = 7.1 Hz, C NMR (101 MHz, CDCl3): δ 165.44, 158.00, 153.62, 151.52, 140.85, 130.31, 128.30,

123.21, 123.14 (2C, overlap), 118.14, 111.86, 104.35, 79.85, 55.43 (2C, overlap), 24.87, 13.52.

M AN U

MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for C18H15BrF2N4O2S: 468.0165, Found: 468.0116.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-phenyl-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10b). A mixture of 10a (100.00 mg, 0.21 mmol),

TE D

cesium carbonate (139.20 mg, 0.43 mmol), phenylboronic acid (34.00 mg, 0.28 mmol) and tetrakis(triphenylphosphine)palladium (0) (25.00 mg, 0.02 mmol) in dioxane (15 ml) and H2O (5 ml) was degassed and flushed with argon. The mixture was hearted at 80 °C for 10 h. The solvent was evaporated under reduced pressure. The residue was diluted with H2O (30 ml) and

EP

extracted with ethyl acetate (40 ml ×2). The combined organic layers were washed with H2O (20 ml ×2) and brine (20 ml ×2), dried over anhydrous Na2SO4, and filtrated, then the solvent was

AC C

evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2: MeOH 200:1~50:1) to give 10b (58.00 mg, 59.2%) as a white solid: mp 122-124 °C. 1H NMR (500 MHz, DMSO) δ8.27 (s, 1H),7.81-7.26 (m, 7H), 7.25(d, J = 15.1 Hz, 2H),6.93 (s, 1H), 5.01 (d, J = 16.1 Hz, 2H), 4.86 (d, J = 13.5 Hz, 1H), 4.71 (d, J = 12.6 Hz, 1H), 4.39 (d, J = 12.9 Hz, 1H), 1.16 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.12, 150.68, 150.40, 148.51, 144.68, 139.78, 132.00, 131.41, 129.22, 125.55, 124.43, 124.31 (2C, overlap), 110.81, 103.96, 78.65, 55.09 (2C, overlap), 39.75, 13.37. MS (EI) m/z: 466 (M+). HRMS (EI): Anal. Calcd for C24H20F2N4O2S: 466.1318, Found: 466.1329.

S6

ACCEPTED MANUSCRIPT

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyridin-4-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10c). 10c (63.00 mg, 63.3%) was prepared from 10a (100.00 mg, 0.21 mmol) and 4-pyridinylboronic acid (37.82 mg, 0.31 mmol) in the same manner as described for 10b. white solid: mp 136-139 °C. 1H NMR (500 MHz,

RI PT

CDCl3) δ 8.78 (d, J = 6.6 Hz, 2H), 7.86 (s, 1H), 7.78 (s, 1H), 7.38 (dd, J = 14.8, 8.0 Hz, 1H), 7.32 (d, J = 6.6 Hz, 2H), 7.18 (s, 1H), 6.83-6.76 (m, 2H), 5.31-5.06 (m, 3H), 4.68 (d, J = 18.5 Hz, 1H), 4.37 (d, J = 14.3 Hz, 1H), 1.06 (d, J = 6.8 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ

161.32, 151.42, 150.97, 149.96, 145.91, 143.64, 140.57, 140.08, 129.91, 122.84, 122.70 (2C,

SC

overlap), 119.43, 117.65, 111.36, 103.83, 79.43, 54.90 (2C, overlap), 47.49, 13.00. MS (EI) m/z:

M AN U

467 (M+). HRMS (EI): Anal. Calcd for C23H19F2N5O2S: 467.1218, Found: 467.1229.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyridin-3-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10d). 10d (71.12 mg, 71.2%) was prepared from 10a (100.00 mg, 0.21 mmol) and 3-pyridinylboronic acid pinacol ester (62.78 mg, 0.31 mmol) in the same manner as described for 10b. white solid: mp 125-127 °C. 1H NMR (500 MHz, CDCl3) δ 8.89 (s, 1H), 8.60 (s, 1H), 7.90 (d, J = 8.1 Hz, 2H), 7.74 (s, 1H), 7.52 (s, 1H),

TE D

7.45 (d, J = 7.1 Hz, 1H), 7.38 (dd, J = 7.6, 4.8 Hz, 1H), 6.88-6.70 (m, 2H), 5.20 (d, J = 12.3 Hz, 1H), 5.17-5.08 (m, 2H), 4.71 (d, J = 18.5 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.16 (d, J = 7.0 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 165.82, 163.87, 161.79, 159.15, 157.09, 151.89, 150.56,

149.30, 146.95, 140.48, 133.23, 133.08, 123.32, 123.21 (2C, overlap), 116.96, 111.43, 104.30,

EP

79.74, 55.39 (2C, overlap), 29.70, 13.48. MS (EI) m/z: 467 (M+). HRMS (EI): Anal. Calcd for

AC C

C23H19F2N5O2S: 467.1236, Found: 467.1218.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyrimidin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10e). 10e (56.00 mg, 56.6%) was prepared from 10a (100.00 mg, 0.21 mmol) and 5-pyrimidinylboronic acid pinacol ester (52.82 mg, 0.26 mmol) in the same manner as described for 10b. white solid: mp 138-140 °C. 1H NMR (500 MHz, CDCl3) δ 9.18 (s, 1H), 8.96 (s, 2H), 7.89 (s, 1H), 7.74 (s, 1H), 7.55 (s, 1H), 7.43-7.36 (m, 1H), 6.86-6.73 (m, 2H), 5.21-5.12 (m, 3H), 4.72 (d, J = 18.7 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.44, 161.92, 158.00, 153.62, 151.52, 144.18, 141.46, 130.38, 128.30, 123.24, 123.14 (2C, overlap), 118.14, 111.72, 104.35,

S7

ACCEPTED MANUSCRIPT

79.85, 55.41 (2C, overlap), 24.56, 13.52. MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for C22H18F2N6O2S: 468.1216, Found: 468.1228.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-cy-

RI PT

anopyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10f). 10f (61.00 mg, 58.0%) was prepared from 10a (100.00 mg, 0.21 mmol) and 2-cyanopyridine-5-boronic acid pinacol ester (64.40 mg, 0.28 mmol) in the same manner as described for 10b. white solid: mp 101-103 °C. 1H NMR (500 MHz, CDCl3) δ 8.50 (d, J = 1.9 Hz, 1H), 7.89 (s, 1H), 7.78 (d, J = 2.3 Hz, 1H), 7.72

SC

(s, 1H), 7.56 (dd, J = 8.1, 2.2 Hz, 1H), 7.56-7.43 (m, 1H), 7.18 (s, 1H), 6.82-6.73 (m, 2H), 5.375.29 (m, 3H), 4.72 (d, J = 18.8 Hz, 1H), 4.26 (d, J = 14.3 Hz, 1H), 1.03 (d, J = 7.1 Hz, 3H). 13C

M AN U

NMR (101 MHz, CDCl3): δ 165.44, 161.92, 158.00, 153.62, 151.52, 144.18, 141.46, 137.73, 136.58, 132.19, 130.66, 130.38, 128.30, 123.24, 123.14 (2C, overlap), 118.14, 111.72, 104.35, 79.88, 55.43 (2C, overlap), 24.38, 13.32. MS (EI) m/z: 492(M+). HRMS (EI): Anal. Calcd for C24H18F2N6O2S: 492.1236, Found: 492.1218.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-me-

TE D

thoxypyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10g). 10g (67.00 mg, 63.1%) was prepared from 10a (100.00 mg, 0.21 mmol) and 2-methoxy-5-pyridinylboronic acid (62.50 mg, 0.28 mmol) in the same manner as described for 10b. white solid: mp 114-116 °C. 1H NMR (500 MHz, CDCl3) δ 8.39 (d, J = 2.3 Hz, 1H), 7.86 (s, 1H), 7.75 (dd, J = 8.6, 2.5 Hz, 1H), 7.71

EP

(s, 1H), 7.43 (d, J = 7.1 Hz, 1H), 7.33 (s, 1H), 6.82-6.71 (m, 3H), 5.17 (d, J = 13.7 Hz, 1H), 5.06 (d, J = 14.3 Hz, 2H), 4.66 (d, J = 18.4 Hz, 1H), 4.29 (d, J = 14.3 Hz, 1H), 3.95 (s, 3H), 1.13 (d, J

AC C

= 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 166.09, 164.10, 151.83, 149.44, 146.22, 144.19, 140.25, 136.56, 130.43, 130.20, 123.48, 123.37, 123.26 (2C, overlap), 115.48, 111.79, 111.31, 104.28, 79.68, 55.46 (2C, overlap), 53.75, 29.70, 13.44. MS (EI) m/z: 497(M+). HRMS (EI): Anal. Calcd for C24H21F2N5O3S: 497.1316, Found: 497.1328.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-fluoropyridin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10h). 10h (66.18 mg, 63.8%) was prepared from 10a (100.00 mg, 0.21 mmol) and 3-fluoropyridine-5-boronic acid pinacol ester (63.68 mg, 0.29 mmol) in the same manner as described for 10b. white solid: mp 103-106 °C. 1H

S8

ACCEPTED MANUSCRIPT

NMR (500 MHz, CDCl3) δ 8.69 (s, 1H), 8.43 (d, J = 18.2 Hz, 1H), 7.86 (s, 1H), 7.73 (s, 1H), 7.59 (d, J = 9.1 Hz, 1H), 7.53 (s, 1H), 7.49-7.38 (m, 1H), 6.85-6.74 (m, 2H), 5.32-5.11 (m, 3H), 4.70 (d, J = 18.6 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H).

13

C NMR (101

MHz, CDCl3): δ 165.64, 163.81, 161.82, 151.91, 151.12, 143.99, 142.71, 140.56, 137.51, 131.32,

RI PT

130.33, 123.28, 123.14 (2C, overlap), 119.76, 117.91, 111.69, 104.34, 79.86, 55.39 (2C, overlap), 29.71, 13.51. MS (EI) m/z: 485(M+). HRMS (EI): Anal. Calcd for C23H18F3N5O2S: 485.1136, Found: 485.1128.

SC

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-methoxypyrimidin-5-yl)-5,6-dihydro-4H-thieno[2,3-c]pyrrol-4-one (10i). 10i (57.15 mg, 53.8%)

M AN U

was prepared from 10a (100.00 mg, 0.21 mmol) and 2-methoxypyrimidine-5-boronic acid pinacol ester (68.80 mg, 0.29 mmol) in the same manner as described for 10b. white solid: mp 120-122 °C. 1H NMR (500 MHz, CDCl3) δ 8.72 (s, 2H), 7.87 (s, 1H), 7.72 (s, 1H), 7.46-7.38 (m, 2H), 6.82-6.72 (m, 2H), 5.18 (d, J = 14.1 Hz, 1H), 5.14-5.03 (m, 2H), 4.68 (d, J = 18.5 Hz, 1H), 4.29 (d, J = 14.3 Hz, 1H), 4.05 (s, 3H), 1.14 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.33, 163.79, 161.79, 156.49, 151.95, 150.34, 141.94, 140.60, 130.38, 123.31, 123.18, 122.32

TE D

(2C, overlap), 116.71, 111.82, 104.31, 79.81, 55.34 (2C, overlap), 55.16, 24.85, 13.49. MS (EI) m/z: 498 (M+). HRMS (EI): Anal. Calcd for C23H20F2N6O3S: 498.1226, Found: 498.1312. 5-bromo-3-methylthiophene-2-carbaldehyde (12). 11 (50.00 g, 0.40 mol) was dissolved in

EP

chloroform (300 ml). Bromine (66.50 g, 0.42 mol) was slowly added dropwise at 0 °C. The resulting mixture was heated under reflux for 4 hours. The reaction mixture was diluted with

AC C

CH2Cl2 (60 ml). The organic phase was washed with H2O (50 ml) and saturated aqueous sodium bicarbonate solution (50 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum: ethyl acetate 30:1~20:1) to give 12 (51.80 g, 63.1%) as a brown solid: mp 55-57 °C. 1

H NMR (400 MHz, CDCl3) δ 9.89 (s, 1H), 6.95 (s, 1H), 2.52 (s, 3H). MS (EI) m/z: 204 (M+). 5-bromo-3-methylthiophene-2-carboxylic acid (13). 12 (10.00 g, 48.80 mmol) was dissolved

in CH3CN (150 ml). Saturated aqueous monosodium phosphate solution (7.80 g, 50.20 mmol) was slowly added dropwise at 0 °C, followed by 10% aqueous hydrogen peroxide solution (5.10

S9

ACCEPTED MANUSCRIPT

ml, 48.86 mmol), and stirred for 0.5 h. Then, saturated aqueous sodium chlorite solution (4.41 g, 48.80 mmol) was slowly added, and stirred for 8 h at room temperature. The mixture was diluted with aqueous sodium hydrogen carbonate (50 ml) and stirred for 0.5 h at 0 °C. The residue was acided (PH = 2) by addition of hydrogen chloride (1.0 M aqueous solution).The resultant

RI PT

precipitate was collected by filtration, washed with diethyl ether then dried under vacuum to afford 13 (7.00 g, 65.0%) as a white solid: mp 121-122 °C. 1H NMR (400 MHz, CD3OD) δ 13.99 (s, 1H), 8.28-7.54 (m, 1H), 3.26 (m, 3H). MS (EI) m/z: 220 (M+).

SC

methyl 5-bromo-3-methylthiophene-2-carboxylate (14). 14 (8.70 g, 75.8%) was prepared from 13 (10.80 g, 48.85 mmol) in the same manner as described for 3. As a colorless viscous oil. H NMR (400 MHz, CDCl3) δ 6.89 (s, 1H), 3.84 (s, 3H), 2.50 (s, 3H). MS (EI) m/z: 234 (M+).

M AN U

1

methyl 5-bromo-3-(bromomethyl)thiophene-2-carboxylate (15). 14 (5.00 g, 21.27 mmol) and benzoyl peroxide (0.50 g, 2.00 mmol) was dissolved in 200 ml CCl4. N-bromosuccinimide (3.90 g, 21.91 mmol) was added in batches at room temperature. The reaction mixture was allowed to warm to 80 °C and stirred for 12 hours. After cooling, the solvent was evaporated

TE D

under reduced pressure. The residue was partitioned between ethyl acetate (300 ml) and H2O (100 ml). The organic phase was washed with H2O (80 ml) and saturated brine solution (80 ml), dried over Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum: ethyl acetate 80:1~60:1)

EP

to give 15 (4.00 g, 60.0%) as a white solid: mp 81-82°C. 1H NMR (400 MHz, CDCl3) δ 7.16 (s,

AC C

1H), 4.85 (s, 2H), 3.88 (s, 3H). MS (EI) m/z: 312 (M+).

methyl

5-bromo-3-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-

butan-2-ylamino}methyl]thiophene-2-carboxylate (16). 16 (4.86 g, 80.7%) was prepared from 7 (3.21 g, 12.00 mmol) and 15 (4.14 g, 13.18 mmol) in the same manner as described for 8. As a colorless viscous oil 1H NMR (400 MHz, CDCl3) δ 7.89 (s, 1H), 7.75 (s, 1H), 7.36 (dd, J = 15.6, 8.8 Hz, 1H), 7.17 (s, 1H), 6.73 (t, J = 8.4 Hz, 2H), 4.93 (d, J = 14.3 Hz, 1H), 4.77 (d, J = 14.5 Hz, 1H), 4.26 (d, J = 14.1 Hz, 1H), 3.97 (d, J = 14.1 Hz, 1H), 3.85 (s, 3H), 3.23-3.10 (m, 1H), 0.92 (d, J = 6.4 Hz, 3H). MS (EI) m/z: 500 (M+).

S10

ACCEPTED MANUSCRIPT

5-bromo-3-[{(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)-butan-2ylamino}methyl]thiophene-2-carboxylic acid (17). 17 (0.88 g, 78.6%) was prepared from 16 (1.16 g, 2.31 mmol) in the same manner as described for 9. As a pale yellow viscous oil 1H NMR (500 MHz, DMSO) δ 8.38 (s, 1H), 7.74 (s, 1H), 7.61 (s, 1H), 7.54-7.44 (m, 1H), 7.06 (m, 2H),

RI PT

5.10 (d, J = 14.5 Hz, 1H), 4.74 (d, J = 14.6 Hz, 1H), 4.56 (d, J = 13.6 Hz, 1H), 4.48 (d, J = 13.5 Hz, 1H), 3.60 (d, J = 6.4 Hz, 1H), 1.18 (d, J = 6.1 Hz, 3H). MS (EI) m/z: 486 (M+).

2-bromo-5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-

SC

yl]-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18a). 18a (0.18 g, 62.3%) was prepared from 17 (0.30 g, 0.62 mmol) in the same manner as described for 10a. white solid: mp 146-148 °C. 1H

M AN U

NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.71 (s, 1H), 7.43-7.38 (m, 1H), 7.11 (s, 1H), 6.82-6.73 (m, 2H), 5.15 (d, J = 14.2 Hz, 1H), 4.90 (d, J = 18.6 Hz, 2H), 4.48 (d, J = 18.5 Hz, 1H), 4.26 (d, J = 14.2 Hz, 1H), 1.11 (d, J = 7.1 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 164.04, 163.97,

161.47, 151.53, 144.19, 135.07, 130.37, 124.20, 123.32, 123.16 (2C, overlap), 111.70, 104.22, 79.62, 55.40 (2C, overlap), 29.78, 13.25. MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for

TE D

C18H15BrF2N4O2S: 468.0136, Found: 468.0128.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-phenyl-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18b). A mixture of 18a (100.00 mg, 0.21 mmol), cesium carbonate (139.20 mg, 0.43 mmol), phenylboronic acid (34.00 mg, 0.28 mmol) and

EP

tetrakis(triphenylphosphine)palladium (0) (25.00 mg, 0.02 mmol) in dioxane (15 ml) and H2O (5 ml) was degassed and flushed with argon. The mixture was hearted at 80 °C for 10 h. The

AC C

solvent was evaporated under reduced pressure. The residue was diluted with H2O (20 ml) and extracted with ethyl acetate (30 ml ×2). The combined organic layers were washed with H2O (20 ml ×2) and brine (20 ml ×2), dried over anhydrous Na2SO4, and filtrated, then the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (CH2Cl2: MeOH 200:1~50:1) to give 18b (61.00 mg, 62.1%) as a white solid: mp 142-144 °C. 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.68 (d, J = 15.1 Hz, 1H), 7.63 (d, J = 7.3 Hz, 2H), 7.52-7.32 (m, 4H), 7.27 (s, 1H), 6.84-6.63 (m, 2H), 5.18 (d, J = 13.8 Hz, 1H), 5.08-4.86 (m, 2H), 4.53 (d, J = 18.3 Hz,1H), 4.31 (d, J = 14.3 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H). 13

C NMR (101 MHz, CDCl3): δ 165.67, 155.06, 152.91, 151.76, 144.13, 133.67, 133.09, 129.06,

S11

ACCEPTED MANUSCRIPT

128.96, 126.31, 123.47, 123.33 (2C, overlap), 116.90, 111.58, 104.25, 79.70, 55.50 (2C, overlap), 20.98, 13.40. MS (EI) m/z: 466 (M+). HRMS (EI): Anal. Calcd for C24H20F2N4O2S: 466.1336, Found: 466.1318.

RI PT

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-methylphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18c). 18c (67.20 mg, 65.6%) was prepared from 18a (100.00 mg, 0.22 mmol) and 4-methylphenylboronic acid (36.03 mg, 0.26 mmol) in the same manner as described for 18b. white solid: mp 173-175 °C. 1H NMR (400

SC

MHz, CDCl3) δ 7.96 (d, J = 6.9 Hz, 1H), 7.75 (s, 1H), 7.54 (d, J = 8.1 Hz, 2H), 7.43-7.38 (m, 1H), 7.27-7.20 (m, 3H), 6.83-6.74 (m, 2H), 5.37-5.10 (m, 1H), 5.06-4.86 (m, 2H), 4.60 (d, J = 13

C NMR (101 MHz,

M AN U

17.5 Hz, 1H), 4.44-4.21 (m, 1H), 2.41 (s, 3H), 1.17 (d, J = 6.9 Hz, 3H).

CDCl3): δ 162.02, 155.44, 153.01, 151.35, 143.94, 139.17, 134.49, 132.60, 129.88, 128.46, 126.21, 123.37, 123.29 (2C, overlap), 116.37, 111.63, 104.25, 72.80, 55.69 (2C, overlap), 21.75, 21.30, 13.35. MS (EI) m/z: 480 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O2S: 480.1426, Found: 480.1435.

TE D

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-methylphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18d). 18d (58.20 mg, 55.1%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-methylphenylboronic acid (36.03 mg, 0.26 mmol) in the same manner as described for 18b. white solid: mp 170-171 °C. 1H NMR (500

EP

MHz, CDCl3) δ 7.88 (s, 1H), 7.70 (s, 1H), 7.48-7.36 (m, 4H), 7.32 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 7.5 Hz, 1H), 6.82-6.73 (m, 2H), 5.20 (d, J = 12.7 Hz, 1H), 4.91 (d, J = 15.2 Hz, 2H), 4.54 (d, J

AC C

= 18.1 Hz, 1H), 4.33 (d, J = 14.3 Hz, 1H), 2.43 (s, 3H), 1.16 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 161.80, 155.31, 152.88, 151.76, 144.12, 138.97, 133.69, 133.00, 129.77, 129.08, 126.98, 123.47, 123.39, 123.36 (2C, overlap), 116.76, 111.59, 104.25, 72.83, 55.50 (2C, overlap), 29.66, 21.44, 13.38. MS (EI) m/z: 480 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O2S: 480.1416, Found: 480.1412.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-methoxyphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18e). 18e (68.00 mg, 62.3%) was prepared from 18a (100.00 mg, 0.22 mmol) and 4-methoxyphenylboronic acid (38.00 mg, 0.25

S12

ACCEPTED MANUSCRIPT

mmol) in the same manner as described for 18b. white solid: mp 107-109 °C. 1H NMR (400 MHz, CDCl3) δ 7.57 (d, J = 8.6 Hz, 2H), 7.52-7.43 (m, 4H), 6.96 (d, J = 8.7 Hz, 2H), 6.83-6.71 (m, 2H), 5.19 (d, J = 13.8 Hz, 1H), 4.96-4.85 (m, 2H), 4.53 (d, J = 18.2 Hz, 1H), 4.33 (d, J = 14.3 Hz, 1H), 3.86 (s, 3H), 1.16 (d, J = 7.0 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 159.86,

RI PT

152.69, 151.19, 143.68, 135.45, 131.69, 129.97, 127.21, 126.03, 123.06, 122.89 (2C, overlap), 115.38, 114.12, 111.11, 103.79, 72.30, 54.99 (2C, overlap), 54.59, 29.21, 12.92. MS (EI) m/z: 496 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O3S: 496.1426, Found: 496.1418.

SC

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-methoxyphenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18f). 18f (75.00 mg, 72.1%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-methoxyphenylboronic acid (38.00 mg, 0.25

M AN U

mmol) in the same manner as described for 18b. white solid: mp 106-108 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H), 7.71-7.61 (m, 2H), 7.56-7.51 (m, 1H), 7.33 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 6.9 Hz, 1H), 7.21 (d, J = 7.7 Hz, 1H), 7.14 (s, 1H), 6.82-6.72 (m, 2H), 5.18 (d, J = 13.0 Hz, 1H), 4.91 (d, J = 15.2 Hz, 2H), 4.52 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.2 Hz, 1H), 3.83 (s, 3H), 1.14 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 160.07, 152.83, 151.72, 144.12,

TE D

135.04, 131.99, 130.26, 128.47, 123.50, 123.40 (2C, overlap), 118.83, 117.12, 114.20, 112.10, 111.54, 104.22, 79.65, 55.55 (2C, overlap), 55.39, 29.69, 13.40. MS (EI) m/z: 496 (M+). HRMS (EI): Anal. Calcd for C25H22F2N4O3S: 496.1416, Found: 496.1423.

EP

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-chlorophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18g). 18g (72.00 mg, 68.1%) was prepared from 18a (100.00 mg, 0.22 mmol) and 4-chlorophenylboronic acid (44.00 mg, 0.28

AC C

mmol) in the same manner as described for 18b. white solid: mp 133-135 °C. 1H NMR (400 MHz, CDCl3) δ 7.87-7.79 (m, 1H), 7.76 (s, 1H), 7.70-7.65 (m, 2H), 7.44-7.39 (m, 2H), 7.37-7.33 (m, 1H), 7.27 (s, 1H), 6.89-6.68 (m, 2H), 5.28-4.91 (m, 3H), 4.55 (d, J = 18.0 Hz, 1H), 4.36 (d, J = 13.7 Hz, 1H), 1.17 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 162.02, 152.85, 143.84, 135.01, 132.12, 129.41, 128.57, 127.78, 127.49, 123.25, 123.17 (2C, overlap), 117.22, 111.67, 104.29, 72.81, 55.79 (2C, overlap), 29.72, 13.38. MS (EI) m/z: 500 (M+). HRMS (EI): Anal. Calcd for C24H19ClF2N4O2S: 500.1026, Found: 500.1018.

S13

ACCEPTED MANUSCRIPT

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-chlorophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18h). 18h (72.00 mg, 68.3%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-chlorophenylboronic acid (44.00 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 130-132 °C. 1H NMR (500

RI PT

MHz, CDCl3) δ 7.71 (s, 1H), 7.67 (dd, J = 6.4, 5.1 Hz, 2H), 7.56-7.48 (m, 3H), 7.33 (d, J = 9.7 Hz, 1H), 7.29 (s, 1H), , 6.83-6.73 (m, 2H), 5.20 (d, J = 14.0 Hz, 1H), 4.94 (d, J = 16.3 Hz, 2H), 4.54 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H).

13

C NMR (101

MHz, CDCl3): δ 162.16, 152.82, 151.80, 144.11, 132.89, 132.05, 131.95, 130.79, 130.46, 128.47,

SC

123.44, 123.32 (2C, overlap), 122.05, 117.61, 115.65, 113.10, 111.60, 104.26, 72.84, 55.49 (2C, overlap), 29.70, 13.43. MS (EI) m/z: 500 (M+). HRMS (EI): Anal. Calcd for C24H19ClF2N4O2S:

M AN U

500.1216, Found: 500.1118.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-fluorophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18i). 18i (74.00 mg, 62.6%) was prepared from 18a (100.00 mg, 0.22 mmol) and 4-fluorophenylboronic acid (39.00 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 119-120 °C. 1H NMR (500

TE D

MHz, CDCl3) δ 7.88 (s, 1H), 7.72 (s, 1H), 7.70-7.64 (m, 2H), 7.51-7.43 (m, 1H), 7.21 (s, 1H), 7.13 (t, J = 8.6 Hz, 2H), 6.83-6.74 (m, 2H), 5.20 (d, J = 13.2 Hz, 1H), 4.92 (d, J = 16.1 Hz, 2H), 4.53 (d, J = 18.1 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H).

13

C NMR (101

MHz, CDCl3): δ 162.48, 160.55, 150.90, 149.74, 147.17, 143.08, 142.09, 136.91, 132.41, 131.78,

EP

121.29, 121.19 (2C, overlap), 116.13, 109.60, 107.93, 102.24, 77.77, 53.40 (2C, overlap), 27.63, 16.37. MS (EI) m/z: 484 (M+). HRMS (EI): Anal. Calcd for C24H19F3N4O2S: 484.1216, Found:

AC C

484.1218.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-fluorophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18j). 18j (64.70 mg, 63.6%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-fluorophenylboronic acid (39.00 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 114-116 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H), 7.70 (s, 1H), 7.64 (dd, J = 11.5, 7.7 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J = 6.9 Hz, 1H), 7.44 (t, J = 7.5 Hz, 2H), 7.37-7.29 (m, 1H), 6.88-6.76 (m 2H), 5.18 (d, J = 13.5 Hz, 1H), 4.92 (d, J = 15.5 Hz, 2H), 4.52 (d, J = 18.2 Hz, 1H), 4.31 (d, J = 14.2 Hz, 1H), 1.14 (d,

S14

ACCEPTED MANUSCRIPT

J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.43, 152.81, 151.76, 144.14, 135.45, 135.10, 133.88, 132.02, 130.44, 128.48, 126.18, 124.44, 123.44, 123.31 (2C, overlap), 117.66, 111.60, 104.25,72.56, 55.45 (2C, overlap), 29.35, 13.43. MS (EI) m/z: 484 (M+). HRMS (EI): Anal.

RI PT

Calcd for C24H19F3N4O2S: 484.1126, Found: 484.1118.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(4-cyanophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18k). 18k (75.00 mg, 72.3%) was prepared from 18a (100.00 mg, 0.22 mmol) and 4-cyanophenylboronic acid pinacol ester (60.69

SC

mg, 0.26 mmol) in the same manner as described for 18b. white solid: mp 150-152 °C. 1H NMR (500 MHz, CDCl3) δ 7.87 (s, 1H), 7.77-7.71 (m, 2H), 7.55 (dd, J = 10.6, 4.3 Hz, 2H), 7.47 (td, J = 7.5, 2.6 Hz, 2H), 7.40 (s, 1H), 6.78-6.66 (m 2H), 5.21 (d, J = 13.9 Hz, 1H), 4.98 (d, J = 18.0

M AN U

Hz, 2H), 4.57 (d, J = 18.3 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.16 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.16, 152.77, 151.88, 144.13, 138.01, 135.25, 132.13, 131.94, 128.46, 126.61, 123.37, 123.27 (2C, overlap), 118.74, 118.39, 111.66, 104.30, 79.77, 55.41 (2C, overlap), 29.69, 13.47. MS (EI) m/z: 491 (M+). HRMS (EI): Anal. Calcd for C25H19F2N5O2S: 491.1216,

TE D

Found: 491.1226.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-cyanophenyl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18l). 18l (67.63 mg, 65.2%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-cyanophenylboronic acid (33.11 mg, 0.23

EP

mmol) in the same manner as described for 18b. white solid: mp 148-150 °C. 1H NMR (500 MHz, CDCl3) δ 7.90-7.85 (m, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.69 -7.59 (m, 2H), 7.56-7.51 (m, 2H), 7.33 (s, 1H), 6.85-6.72 (m, 2H), 5.18 (d, J = 13.7 Hz, 1H), 4.95 (d, J = 17.7

AC C

Hz, 2H), 4.54 (d, J = 18.3 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.14 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 163.74, 152.85, 151.75, 144.12, 135.03, 134.60, 132.08, 130.13, 129.50, 128.61, 123.33, 123.22 (2C, overlap), 120.81, 118.29, 118.14, 113.52, 111.61, 104.26, 79.83, 55.41 (2C, overlap), 29.62, 13.45. MS (EI) m/z: 491 (M+). HRMS (EI): Anal. Calcd for C25H19F2N5O2S: 491.1226, Found: 491.1221.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyridin-4-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18m). 18m (64.70 mg, 65.5%) was

S15

ACCEPTED MANUSCRIPT

prepared from 18a (100.00 mg, 0.22 mmol) and 4-pyridinylboronic acid (35.28 mg, 0.29 mmol) in the same manner as described for 18b. white solid: mp 178-180 °C. 1H NMR (400 MHz, CDCl3) δ 8.67 (d, J = 5.5 Hz, 2H), 7.85 (s, 1H), 7.72 (s, 1H), 7.52 (d, J = 6.1 Hz, 2H), 7.48 (s, 1H), 7.43 (m, 1H), 6.83-6.74 (m, 2H), 5.20 (d, J = 13.6 Hz, 1H), 4.98 (d, J = 14.3 Hz, 2H), 4.56

RI PT

(d, J = 14.3 Hz, 1H), 4.30 (d, J = 14.0 Hz, 1H), 1.15 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 165.03, 152.62, 151.90, 151.12, 150.69, 144.13, 140.88, 135.44, 130.39, 123.35, 123.26 (2C, overlap), 120.14, 118.96, 111.68, 104.31, 79.92, 55.39 (2C, overlap), 29.70, 13.48. MS (EI) m/z: 467 (M+). HRMS (EI): Anal. Calcd for C23H19F2N5O2S: 467.1216, Found:

SC

467.1228.

M AN U

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyridin-3-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18n). 18n (70.00 mg, 71.2%) was prepared from 18a (100.00 mg, 0.22 mmol) and 3-pyridinylboronic acid pinacol ester (58.85 mg, 0.29 mmol) in the same manner as described for 18b. white solid: mp 156-158 °C. 1H NMR (500 MHz, CDCl3) δ 8.91 (s, 1H), 8.61 (d, J = 4.2 Hz, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.87 (s, 1H), 7.72 (s, 1H), 7.44 (dd, J = 15.1, 8.0 Hz, 1H), 7.42-7.36 (m, 1H), 7.35 (s, 1H), 6.86-6.78 (m, 2H), 5.20

TE D

(d, J = 13.7 Hz, 1H), 4.97 (d, J = 17.6 Hz, 2H), 4.56 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H), 1.15 (d, J = 7.0 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 165.23, 152.87, 151.84, 15059,

149.65, 147.03, 144.12, 134.49, 133.57, 130.40, 129.94, 123.92, 123.41, 123.30 (2C, overlap), 118.05, 111.60, 104.27, 79.75, 55.58 (2C, overlap), 29.68, 13.44. MS (EI) m/z: 467 (M+). HRMS

EP

(EI): Anal. Calcd for C23H19F2N5O2S: 467.1232, Found: 467.1226.

AC C

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(pyrimidin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18o). 18o (63.00 mg, 63.8%) was prepared from 18a (100.00 mg, 0.21 mmol) and 5-pyrimidinylboronic acid pinacol ester (59.13 mg, 0.29 mmol) in the same manner as described for 18b. white solid: mp 126-128 °C. 1H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H), 9.00 (s, 2H), 7.89 (s, 1H), 7.74 (s, 1H), 7.49-7.38 (m, 2H), 6.85- 6.71 (m, 2H), 5.21 (d, J = 14.1 Hz, 1H), 5.01 (d, J = 18.5 Hz, 2H), 4.59 (d, J = 18.4 Hz, 1H), 4.32 (d, J = 14.2 Hz, 1H), 1.17 (d, J = 7.0 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 164.87, 158.37, 153.82, 152.85, 146.16, 144.10, 135.81, 130.39, 128.29, 123.30, 123.17 (2C, overlap),

S16

ACCEPTED MANUSCRIPT

119.04, 111.68, 104.32, 79.91, 55.39 (2C, overlap), 29.69, 13.50. MS (EI) m/z: 468 (M+). HRMS (EI): Anal. Calcd for C22H18F2N6O2S: 468.1216, Found: 468.1228.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-cy-

RI PT

anopyridin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18p). 18p (65.00 mg, 61.6%) was prepared from 18a (100.00 mg, 0.21 mmol) and 2-cyanopyridine-5-boronic acid pinacol ester (64.40 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 239240 °C. 1H NMR (500 MHz, CDCl3) δ 9.01 (d, J = 1.8 Hz, 1H), 8.04 (dd, J = 8.1, 2.3 Hz, 1H),

SC

7.88 (s, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.74 (s, 1H), 7.49 (s, 1H), 7.44 (dd, J = 15.4, 8.3 Hz, 1H), 6.87-6.71 (m, 2H), 5.20 (d, J = 14.1 Hz, 1H), 5.02 (d, J = 18.0 Hz, 2H), 4.59 (d, J = 18.4 Hz,

M AN U

1H), 4.31 (d, J = 14.2 Hz, 1H), 1.16 (d, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 164.75, 152.86, 151.81,148.09, 147.96, 144.11, 136.45, 133.69, 132.97, 130.31, 128.69, 123.23, 123.16 (2C, overlap), 119.81, 116.95, 111.87, 104.33, 74.99, 55.41 (2C, overlap), 24.85, 13.51. MS (EI) m/z: 492 (M+). HRMS (EI): Anal. Calcd for C24H18F2N6O2S: 492.1218, Found: 492.1223.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-cy-

TE D

anopyridin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18q). 18q (62.80 mg, 60.5%) was prepared from 18a (100.00 mg, 0.21 mmol) and 3-cyanopyridine-5-boronic acid pinacol ester (65.76 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 218220 °C. 1H NMR (500 MHz, CDCl3) δ 9.07 (s, 1H), 8.84 (s, 1H), 8.15 (s, 1H), 7.86 (s, 1H), 7.72

EP

(s, 1H), 7.43-7.38 (m, 2H), 6.83-6.71 (m, 2H), 5.19 (d, J = 13.7 Hz, 1H), 5.00 (d, J = 17.7 Hz, 2H), 4.57 (d, J = 18.4 Hz, 1H), 4.30 (d, J = 14.1 Hz, 1H), 1.14 (d, J = 6.9 Hz, 3H).

13

C NMR

AC C

(101 MHz, CDCl3): δ 162.71, 157.00, 155.03, 150.78, 149.82, 149.55, 148.04, 145.31, 142.10, 134.02, 128.29, 121.22 121.14 (2C, overlap), 117.44, 113.91, 108.92, 108.52, 102.29, 77.72, 53.35 (2C, overlap), 27.64, 11.48. MS (EI) m/z: 492 (M+). HRMS (EI): Anal. Calcd for C24H18F2N6O2S: 492.1228, Found: 492.1216.

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-methoxypyridin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18r). 18r (75.60 mg, 72.1%) was prepared from 18a (100.00 mg, 0.21 mmol) and 2-methoxy-5-pyridinylboronic acid (62.50 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 130-132 °C. 1H NMR

S17

ACCEPTED MANUSCRIPT

(500 MHz, CDCl3) δ 8.47 (d, J = 2.3 Hz, 1H), 7.88 (s, 1H), 7.80 (dd, J = 8.6, 2.5 Hz, 1H), 7.72 (s, 1H), 7.49-7.42 (m, 1H), 7.20 (s, 1H), 6.88-7.66 (m, 3H), 5.20 (d, J = 13.4 Hz, 1H), 4.94 (d, J = 10.7 Hz, 2H), 4.54 (d, J = 18.2 Hz, 1H), 4.32 (d, J = 14.2 Hz, 1H), 3.99 (s, 3H), 1.15 (d, J = 6.9 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 164.44, 161.77, 153.00, 151.77, 151.43, 144.49,

RI PT

144.09, 136.78, 133.12, 130.45, 123.46, 123.34, 123.31 (2C, overlap), 116.66, 115.59, 111.38, 104.27, 79.68, 55.46 (2C, overlap), 53.80, 29.70, 13.40. MS (EI) m/z: 497(M+). HRMS (EI): Anal. Calcd for C24H21F2N5O3S: 497.1323, Found: 497.1318.

SC

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-fluoropyridin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18s). 18s (71.80 mg, 70.2%) was prepared from 18a (100.00 mg, 0.21 mmol) and 2-fluoropyridine-5-boronic acid (40.50 mg, 0.29

M AN U

mmol) in the same manner as described for 18b. white solid: mp 233-235 °C. 1H NMR (500 MHz, CDCl3) δ 8.47 (d, J = 1.9 Hz, 1H), 7.98 (td, J = 8.4, 2.5 Hz, 1H), 7.86 (s, 1H), 7.73-7.66 (m, 1H), 7.47-7.38 (m, 1H), 7.27 (s, 1H), 7.00 (dd, J = 8.5, 2.8 Hz, 1H), 6.82- 6.70 (m, 2H), 5.17 (d, J = 13.6 Hz, 1H), 4.95 (d, J = 17.6 Hz, 2H), 4.54 (d, J = 18.3 Hz, 1H), 4.29 (d, J = 14.2 Hz, 1H), 1.13 (d, J = 6.9 Hz, 3H). 13C NMR (101 MHz, CDCl3): δ 163.20, 150.90, 149.74, 147.17,

TE D

143.08, 142.09, 136.91, 132.41, 131.66, 128.35, 126.11, 121.32, 121.21 (2C, overlap), 116.13, 109.76, 108.23, 102.24, 77.77, 56.21 (2C, overlap), 27.59, 16.41. MS (EI) m/z: 485 (M+). HRMS (EI): Anal. Calcd for C23H18F3N5O2S: 485.1136, Found: 485.1118.

EP

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(3-fluoropyridin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18t). 18t (70.00 mg, 68.1%) was prepared from 18a (100.00 mg, 0.21 mmol) and 3-fluoropyridine-5-boronic acid pinacol ester

AC C

(63.68 mg, 0.29 mmol) in the same manner as described for 18b. white solid: mp 205-207 °C. 1H NMR (500 MHz, CDCl3) 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 8.46 (d, J = 2.3 Hz, 1H), 7.87 (s, 1H), 7.71 (s, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.46-7.38 (m, 1H), 7.37 (s, 1H), 6.83-6.71 (m, 2H), 5.19 (d, J = 14.1 Hz, 1H), 4.98 (d, J = 17.9 Hz, 2H), 4.55 (d, J = 18.4 Hz, 1H), 4.30 (d, J = 14.3 Hz, 1H), 1.14 (d, J = 7.0 Hz, 3H).

13

C NMR (101 MHz, CDCl3): δ 164.86, 160.70,

152.85, 151.94, 148.67,144.11, 142.91, 137.92, 135.14, 131.31, 130.34, 123.28, 123.20 (2C, overlap), 120.05, 118.90, 111.65, 104.23, 79.74, 55.41 (2C, overlap), 29.51, 13.40. MS (EI) m/z: 485 (M+). HRMS (EI): Anal. Calcd for C23H18F3N5O2S: 485.1136, Found: 485.1118.

S18

ACCEPTED MANUSCRIPT

5-[(2R,3R)-3-(2,4-difluorophenyl)-3-hydroxy-4-(1H-1,2,4-triazol-1-yl)butan-2-yl]-2-(2-methoxypyrimidin-5-yl)-5,6-dihydro-4H-thieno[3,2-c]pyrrol-4-one (18u). 18u (66.50 mg, 63.1%) was prepared from 18a (100.00 mg, 0.21 mmol) and 2-methoxypyrimidine-5-boronic acid

RI PT

pinacol ester (66.10 mg, 0.28 mmol) in the same manner as described for 18b. white solid: mp 191-193 °C. 1H NMR (400 MHz, CDCl3) δ 8.69 (s, 2H), 7.82 (s, 1H), 7.65 (s, 1H), 7.41-7.36 (m, 1H), 7.20 (s, 1H), 6.78-6.66 (m, 2H), 5.13 (d, J = 14.1 Hz, 1H), 4.90 (d, J = 18.1 Hz, 2H), 4.49 (d, J = 18.4 Hz, 1H), 4.24 (d, J = 14.2 Hz, 1H), 3.98 (s, 3H), 1.08 (d, J = 7.0 Hz, 3H). 13C NMR

SC

(101 MHz, CDCl3): δ 165.54, 161.55, 156.59, 152.98, 151.76, 146.85, 144.13, 134.29, 130.38, 123.30, 123.13, 122.33 (2C, overlap), 117.74, 111.71, 104.32, 75.11, 55.38 (2C, overlap), 54.96,

M AN U

25.82, 13.45. MS (EI) m/z: 498 (M+). HRMS (EI): Anal. Calcd for C23H20F2N6O3S: 498.1218, Found: 498.1226.

References and notes

TE D

[1] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved Standard, edn 3; Document M38-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2008. [2] Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards: Reference method for broth dilution antifungal susceptibility testing of Yeast. Approved Standard, edn 3; Document M27-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2009.

EP

[3] R.S. Obach, Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: An examination of in vitro half-life approach and nonspecific binding to microsomes, Drug Metab Dispos 27 (1999) 1350-1359.

AC C

[4] Y.Y. Lau, G. Krishna, N.P. Yumibe, D.E. Grotz, E. Sapidou, L. Norton, I. Chu, C. Chen, A.D. Soares, C.C. Lin, The use of in vitro metabolic stability for rapid selection of compounds in early discovery based on their expected hepatic extraction ratios, Pharm Res 19 (2002) 16061610. [5] A. Fritsche, A.S. Elfringhoff, J. Fabian, M. Lehr, 1-(2-Carboxyindol-5-yloxy)propan-2-ones as inhibitors of human cytosolic phospholipase A2α: Synthesis, biological activity, metabolic stability, and solubility, Bioorganic & Medicinal Chemistry 16 (2008) 3489-3500. [6] B.C. Monk, T.M. Tomasiak, M.V. Keniya, F.U. Huschmann, J.D. Tyndall, J.D. O'Connell, 3rd, R.D. Cannon, J.G. McDonald, A. Rodriguez, J.S. Finer-Moore, R.M. Stroud, Architecture of

S19

ACCEPTED MANUSCRIPT

a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer, Proc Natl Acad Sci U S A 111 (2014) 3865-3870.

AC C

EP

TE D

M AN U

SC

RI PT

[7] K. Arnold, L. Bordoli, J. Kopp, T. Schwede, The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling, Bioinformatics 22 (2006) 195-201.

S20