Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline

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Design, Synthesis, and Preliminary Studies of Spiro-Isoxazoline-Peroxides against Human-Cytomegalovirus (HCMV) and Glioblastoma (GBM6)# Prasanta Das, Mohammad H Hasan, Dipanwita Mitra, Ratna Bollavarapu, Edward J. Valente, Ritesh Tandon, Drazen Raucher, and Ashton T. Hamme J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b00746 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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The Journal of Organic Chemistry

Design, Synthesis, and Preliminary Studies of SpiroIsoxazoline-Peroxides against Human-Cytomegalovirus (HCMV) and Glioblastoma (GBM6)║ Prasanta Das,*,† Mohammad H. Hasan,‡ Dipanwita Mitra,‡ Ratna Bollavarapu,‡ Edward J. Valente,§ Ritesh Tandon,‡ Drazen Raucher,Δ and Ashton T. Hamme II*,† ║Dedicated

to the memory of Professor Leo A. Paquette

†Department

‡Department

of Chemistry and Biochemistry, Jackson State University, Jackson, MS 39217, USA. of Microbiology and Immunology, University of Mississippi Medical Center, Jackson,

Mississippi 39216, USA §Department

ΔDepartment

of Chemistry, University of Portland, Portland, OR 97203, USA of Cellular and Molecular Biology, University of Mississippi Medical Center, Jackson,

MS, 39216, USA Email: [email protected], and [email protected]

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ABSTRACT: The association between glioblastoma (GBM) and human cytomegalovirus (HCMV) infection has been the intensely debated topic over decades for developing new therapeutic option. In this regard, the peroxides from natural and synthetic sources served as potential antiviral and anticancer agents in the past. Herein a concise and efficient strategy has been demonstrated to access a novel class of peroxides containing a spiro-isoxazoline to primarily investigate the biological activities. The synthetic compounds were evaluated for in vitro antiviral and antiproliferative activity against human cytomegalovirus (HCMV) and glioblastoma cell line (GBM6), respectively. While compound 13m showed moderate anti-CMV activity (IC50 = 19 µM), surprisingly, an independent biological assay for compound 13m revealed its antiproliferative activity against human glioblastoma cell line (GBM6) with IC50 = 10 µM. Hence, the unification of an isoxazoline and peroxide heterocycles could be a potential direction to initiate HCMV-GBM drug discovery program.

INTRODUCTION

Glioblastoma (GBM) is considered as grade IV (most aggressive) astrocytoma, whose malignancy often becomes the primary cause of brain cancer in adults.1 The prognosis of GBM is poor with patients surviving for only a year or less. Moreover, the diagnosis of GBM is often delayed due to non-specific signs and symptoms. Despite the multimodality treatment (surgery with chemotherapy and radiotherapy),2 recurrences of the disease,2b chemotherapy resistance, and scarcity in therapeutic drugs1,3 have lowered the survival rate in patients (9% in 5-years).2c Moreover, viruses are known to be causative agents in certain human tumors.4 Interestingly, several studies showed a significant connection between human cytomegalovirus (HCMV) infection and glioblastoma (GBM).5 Though the oncomodulatory role of HCMV is still controversial,6 the antiviral drug valganciclovir was found to be beneficial in combination with the therapy for GBM.6a,7 As herpesviruses are ubiquitous human pathogens, carried by 70–100% of the world’s population and several herpesviruses are known to be oncogenic8 (e.g., Kaposi Sarcoma-Associated Virus and Epstein Barr Virus), it would be useful to find novel drug agents that are ACS Paragon Plus Environment

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effective against both herpesviruses and cancers. Since the etiology of GBM is unknown and HCMV infection has a putative role in GBM progression, some investigators have postulated that HCMV genes might have significant roles in promoting GBM pathogenesis and disease progression. Moreover, the emergence of drug-resistance, often caused by the long-term uses of existing frontline drugs (e.g., ganciclovir (GCV), cidofovir (CDV), and foscarnet (FOS)), has lessened the available treatment option.9 To find novel drugs, natural and synthetic peroxides containing an endo- or spiro-linkage have attracted significant attention due to their diverse biological activities.10 For example, artemisinin (Qinghaosu, 1) is widely known for its promising antimalarial activities;11 however, its derivatives (2-4), hybrids (5 and 6), and dimer (7) have attracted further interest as anticancer and antiviral prophylactic agents (Figure 1).12 In this regard, artesunate (ART, 4) has been found to be an antiviral agent against human cytomegalovirus (HCMV).12d,13 Figure 1. Natural and Synthetic Peroxides

Despite the significant success, artemisinin’s mode of action is a debated topic till to date; however, investigators have hypothesized that peroxide linkage could play an analogous role10c,11a,14 for its anti-


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CMV and -GBM activity. Over the past decades, synthetic peroxides, containing less structurally intriguing scaffolds, have extensively been evaluated for antimalarial activities, while their anti-CMV and anti-GBM activity scarcely studied. Therefore, along with the tentative progression in understanding the connection of genetic and molecular pathogenesis of HCMV and GBM, substantial movement in developing new drugs is highly warranted. Furthermore, spiro-linked isoxazolines are widely known architectures in several natural and synthetic compounds to exhibit significant biological activity.15 In this context, we previously accomplished the total synthesis of spiroisoxazoline containing complex natural product (11deoxyfistularin-3) as well as the library of small spiro-heterocycles to study their anticancer activity.16 Continuing our investigation into novel pharmacophores, we now envisage incorporating both an isoxazoline and a peroxide within a common framework (Scheme 1) as a source to initiate a drugdiscovery program. We also hypothesized that the isoxazoline ring could combine essential drug properties like polarity, hydrophilicity, and solubility into the molecule while the peroxo linkage might be responsible for its antiviral (HCMV) and antiproliferative (GBM) activity. Scheme 1. Previous and Current Methods for Spiro-Isoxazolines

Envisaging a novel spiro-peroxide pharmacophore, we found that isoxazole bearing a hydroperoxide is essential for the halo-peroxidation to construct the desired spiro-isoxazoline-peroxide core (Scheme


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1). Therefore, to determine the biological relevance of peroxo linkage, we were primarily interested in synthesizing an array of racemic spiro-isoxazoline-peroxides for biological assays (Scheme 1). RESULTS AND DISCUSSION Chemistry As depicted in Scheme 2, the strategy for the synthesis of spiro-isoxazoline-peroxides is mainly relied on three key steps: 1,3-dipolar cycloaddition,16 hydroperoxidation,17 and halo-peroxidation.18 The most crucial halo-peroxidation involves a dearomatization in which the isoxazole ring serves as a masked alkene or diene. Based on our previous experience in spiro-cyclization, we imagined establishing a feasible method for halo-peroxidation that would enable us to access a library of spiroperoxides. The synthesis of spiro-isoxazoline-peroxide 13a primarily began with an isoxazole precursor 12a bearing a hydroperoxide (Scheme 2). In doing so, the required isoxazole moiety 11a was first synthesized (96% yield) following a 1,3-dipolar cycloaddition reaction between commercially available tert-alkynol 10 and phenylnitrile oxide (generated in situ from its corresponding oximoyl chloride).16c Based on our previous experience, regioselective selection of 1,3-dipolar cycloaddition predominantly produced 3,5-disubstituted isoxazole 11a over 3,4-disubstituted product. Moreover, the synthesis of five and six-membered cyclic peroxides such as 1,2-dioxolanes, 1,2,4-trioxolanes (ozonides), 1,2-dioxanes, 1,2-dioxenes, 1,2,4-trioxanes, and 1,2,4,5-tetraoxanes are prevalent in literature.10d Among the various synthetic methods for oxolanes, we imagined constructing a peroxo bridge between a spiro and tertiary center from its corresponding pendent tert-hydroperoxide. To get the information about terthydroperoxide, tert-alcohol 11a was tentatively treated with 30% H2O2/H2SO417 to afford 12a in 50% yield. Afterward, we have also realized an improvement in reactivity using 50% H2O2 to provide 12a in 75% yield (Scheme 2). Under this reaction condition, we also anticipated the formation of a minor dimerized product through a feasible dialkylation of H2O2; however, to our surprise, no trace of evidence for the dimerized product was detected, presumably due to steric congestion adjacent to the


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tert-center. Moreover, a sharp singlet at ~8 ppm in 1H-NMR also confirms the presence of -OOH19 in 12a, which was further corroborated by HRMS. Having characterized the hydroperoxide 12a, we next planned to perform a halonium-ion mediated spiro-cyclization. The halo-peroxidation of an unsaturated hydroperoxide reported in the past;18 however, a similar electrophilic cyclization of pendent hydroperoxide on an aromatic isoxazoline system is relatively challenging. To the best of our knowledge, there is no report on spiro-isoxazoline-peroxide to date. Since spiro-peroxide is analogous to our other spiro-derivatives,16c we tentatively treated 12a following our previously developed reaction condition, pyridinium tribromide (PTB) in CH2Cl2,16c to see the feasibility of bromo-peroxidation. To our disappointment, we were scarcely able to isolate the desired spiro-isoxazoline-peroxide (±)-13a ( 20% yield); however, this setback prompted us to investigate a suitable reaction condition. Scheme 2. Synthetic Strategy for Spiro-Isoxazoline-Peroxide (±)-13a

To determine the optimized reaction condition, isoxazole-hydroperoxide 12a was used as a model substrate (Table 1). In our initial study, some commercially available brominating agents, such as NBS (N-bromo

succinimide),

DBDMH

(1,3-Dibromo-5,5-dimethylhydantoin),

Br2/CCl4,

and

PTT

(trimethylphenyl ammonium tribromide), were screened. As indicated in Table 1, we observed a smooth bromo-hydroperoxidation of 12a while using NBS and DBDMH, delivering the desired 4-bromo-spiroperoxide 13a in 65% and 80%. To further improve the yield, various tribromide sources such as PTB and PTT were investigated, wherein both the reagents were found sluggish for our system as the desired product 13a was barely isolable. However, a remarkable improvement in yields (50% and 70%, entry 5 and 6, Table 1) was observed using PTB/K2CO3 and PTT/K2CO3, presumably due to an enhanced nucleophilicity of hydroperoxide in the presence of a base. Further screening for an efficient bromoACS Paragon Plus Environment

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cyclization led us to identify molecular Br2/CCl4 to produce 13a in 88% yield. To our surprise, searching for minimizing the risk of hazardous reagent led us to explore an unusual iodine-mediated brominating agent. Although serendipitously discovered, the combination of phenyliodine(III) diacetate (PIDA) and ZnBr2 in CH2Cl2 was found to be efficient for bromo-hydroperoxidation as the overall reaction time dramatically reduced to 0.5 h with an excellent conversion (95% yield, entry 7, Table 1). We initially speculated the in-situ formation of PhIBr2 resembling PhICl2;20 however, to our knowledge, there is no report either on PhIBr2 or PIDA/ZnBr2 mediated bromination so far. Regardless of various uses of hypervalent iodine reagents in organic synthesis, aryliodine(III) dihalide such as PhICl220 has received growing interest in organic synthesis due to its ready availability, stability, mildness, selectivity, and safe handling over molecular chlorine (Cl2). In mark contrast to PhICl2, we found that PIDA/ZnBr2 is analogous to an earlier condition PIDA/KBr or PIDA/Bu4NBr that mediates an intramolecular oxidative bromocyclization for homoallylic sulfonamide.21a Surprisingly, PIDA/ZnBr2 is relatively a powerful brominating agent than PIDA/NaBr and PIDA/KBr as depicted in entry (7-9), Table 1. At this point, we have not realized any oxidative decomposition of peroxide 12a, which was further confirmed by performing a controlled experiment employing 12a with PIDA alone; instead, we recovered the peroxide 12a exclusively (entry 13, Table 1). Along the lines of the reported observation,21b we postulated that instant generation of yellow color upon mixing PIDA and ZnBr2 in CH2Cl2 might be due to the formation of acetylhypobromite (AcOBr) or bromine (Br2), which instantly prompted the bromonium ion mediated spirocyclization (Scheme 3). To extend scope toward its chloro- and iodo- derivatives, we next executed the similar reaction but using ZnCl2/PIDA and ZnI2/PIDA, in entry 11 and 12, Table 1. To our surprise, PIDA/ZnCl2 was found to be as reactive as PIDA/ZnBr2 to produce the corresponding chloro-derivative 13a (X = Cl) in 92% yield; however, no iodo-peroxidation reaction occurred for PIDA/ZnI2, apparently due to unreactive nature of ZnI2 to PIDA under the reaction condition. Though PIDA mediated bromination is known, to our knowledge, this is the first instance where we have discovered PIDA/ZnBr2 and PIDA/ZnCl2 as powerful agents for bromo- and chloro-peroxidation.


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However, herein we focus our interest toward a library of bromo-spiro-peroxides and the details of some of our findings. Table 1. Optimization of Halo-Peroxidationa

aGeneral

conditions: Hydroperoxide 12a (0.2 mmol, 1.0 equiv), X-source (0.3 mmol, 1.5 equiv),

additive (0.3 mmol, 1.5 equiv), CH2Cl2 (2 mL, 0.1 M) at rt. bTime required for the reaction. cBased on isolated product after purification by chromatography. dProduct obtained as chloro-spiro-peroxide. eNo iodo-derivative was isolated. fControlled reaction performed without halogen source. As depicted in Table 2, using a conventional 1,3-dipolar cycloaddition, a variety of substituted isoxazoles 11 (a-o) were efficiently synthesized from its corresponding 1,3-dipoles and alkyne 10. In doing so, various 1,3-dipole precursors A (hydroximoyl chloride), possessing aromatic, aliphatic, and ester substituents, were employed to access a wide range of substituted isoxazoles. To check the compatibility of aromatic substitutions, the presence of electron-donating (entry 2 and 3, Table 2) and electron withdrawing (entry 10 and 11, Table 2) groups found well tolerated; as corresponding substituted isoxazoles were isolated in high yield and no regioisomeric products were observed. Next, we investigated the tolerance of halogen functionality in 1,3-dipolar cycloaddition reaction with alkyne ACS Paragon Plus Environment

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10 (entry 4-9, Table 2). Halogens (F, Cl, Br, and I) were also found to be the suitable substrates, delivering the substituted isoxazoles (entry 4-9, Table 2) in excellent yields. We next realized that incorporating F and CF3 functionality might be advantageous because of their diverse and promising biological activities.22 Having succeeded with aromatic substitutions, we next directed our interest toward aliphatic functionality to tune the hydrophobicity of isoxazole ring for biomedical interest (Table 2). Therefore, few alkanes (Me, n-Pr, and i-Bu) substituted 1,3-dipole-precursors were employed to obtain desired compounds 11(l-n) in good yields (Table 2). We have also noticed an excellent 3,5regioselectivity for the entire list of compounds (Table 2), presumably due to steric congestion between 3- and 4-substitutions. Meanwhile, extending the scope of isoxazolines to include acid, ester, and amide functionalities was found significant in terms of planned biological studies. Hence, we decided to perform the DABCO-promoted 1,3-dipolar cycloaddition reaction between methyl-nitroacetate and tertalkynol under a sealed tube reflux condition;16c as a result, the desired product 11o was isolated in 94% yield (entry 15, Table 2) as a single isomer. Table 2. Synthesis of tert-Alcohol Containing Isoxazoles 11(a-o)a,d


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aGeneral

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conditions 1: Alkynol 10 (1 equiv), hydroximoyl chloride A (1.5 equiv), triethylamine (2

equiv), CH2Cl2 (10 mL) at rt. bTime required for completion of the reaction. cBased on isolated product after purification by chromatography. dGeneral condition 2: Alkynol 10 (1 equiv), methylnitroacetate (2.5 equiv), DABCO (0.2 equiv), methanol (10 mL) at 80 °C in sealed vessel for 3-days. Having a variety of isoxazoles in hands, tert-alcohols 11(a-o) were subsequently treated with 50% H2O2/H2SO4 to obtain the corresponding tert-hydroperoxides (Table 3). The results demonstrate that the isoxazoles bearing electro-donating (4-Me, 4-OMe), electron-withdrawing (4-NO2, 3-NO2), and halogen (4-F, 4-Cl, 4-Br, 4-I, 2,6-Cl2, and 4-CF3) substituents attached to the benzene ring all react smoothly under optimized reaction condition to aﬀord substituted hydroperoxides in moderate to good yields (5080%; Table 3). On the contrary, the nitro compounds (11j and 11k) provided 12j and 12k in 45% and 48% respectively, albeit in lower yield in comparison to other substitutions (entry 10 and 11, Table 3). Furthermore, hydroperoxidation smoothly responded to aliphatic (Me, n-Pr, and i-Bu) substituents, providing hydroperoxides 12(l-n) in good yields (entry 12-14, Table 3). Table 3. Synthesis of tert-Hydroperoxide Containing Isoxazoles 12 (a-o)a,(d-i)


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aGeneral

condition: Tert-alcohol 11 (1 equiv), a mixture of 50% hydrogen peroxide (6 equiv) and

sulfuric acid (1.5 mL) heated at 45-50 °C. bTime required for completion of the reaction. cBased on isolated product after purification by chromatography. d-iPhosphoric acid (1.5 mL) was added instead of H2SO4. Much to our satisfaction, an ester functionality on isoxazole also afforded corresponding terthydroperoxide 12o in moderate yield (58%; entry 15). The scope of hydroperoxidation protocol was further evaluated by exploring H3PO4 as an alternative acid source,23 which confers an improved transformation as evident in entry 5-7, 10, and 11 in Table 3. Importantly, hydroperoxidation for the entire list (11 (a-o) was also consistent with our previous observation as we have not observed any dialkylated product for hydroperoxide in Table 3. We finally employed tert-hydroperoxides 12 (a-o) for halo-peroxidation using our optimized reaction condition; to our delight, we have isolated a list of desired racemic spiro-peroxides in excellent yields (83-95%, Table 4) following a variable reaction times (0.5-2 h) in Table 4. Along with the desired peroxides, we have also observed an over-bromination for 13b and 13c that possess OMe and Me functionality. Accordingly, it suggests PIDA/ZnBr2 a stronger brominating agent that engendered a feasible α-bromination of Ar–OMe24 and a benzylic bromination25 of Ar-Me to provide 13b and 13c in 85% and 87% yields, respectively (Table 4). As the over brominated products contains a peroxo linkage, we realized to evaluate their biological activity, accordingly.


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Table 4. Synthesis of Spiro-Isoxazoline-Peroxides (±)-13 (a-o)a

aReaction

conditions: Hydroperoxide 12 (a-o) (1 equiv), PIDA (1.5 equiv), ZnBr2 (1.5 equiv), CH2Cl2

(2 mL) at rt for 0.5-2 h, product isolated after purification by chromatography. bα-mono-brominated product was isolated. cMono bromination at benzylic position was observed. Having

investigated

the

compatibility

of

various

aromatic

and

aliphatic

substituted

spiroisoxazolines, we next targeted a few amides containing spiro-peroxide monomers and dimers to facilitate the biological study. To probe the effect of amide substituents on peroxides’ bioactivity, we ACS Paragon Plus Environment

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tentatively chose few commercially available amines. Now that an artemisinin peroxide containing dimers are known for their anticancer activity, we also envisaged synthesizing amide containing peroxodimer using hydroxymoloka‘i-amine, previously used for the total synthesis of cytotoxic natural product 11-deoxyfistularin-3. As depicted in Table 5, compound 13o was employed instantly under LiOH/MeOH:H2O (3:1)16b to afford corresponding acid 14 in 90% yield (Table 5). Subsequently, following a conventional amide coupling method26,16b involving EDC/DMAP, the corresponding acid 14 and various amines and diamines generated a range of amide monomers 15(a-d) and 15f, respectively in good yields (Table 5). Table 5. Synthesis of Spiro-Isoxazoline-Peroxo-Amides (±)-15 (a-g)

Interestingly, while 4-hydroxybenzylamine used for amide-coupling reaction, we isolated an amide 15b in 38% yield along with its amide-ester 15e in 35% yield and the later was also evaluated for the biological study. Finally, using hydroxymoloka‘i-amine, we were able to synthesize 15g as the desired peroxo dimer in 70% yield (Table 5). ACS Paragon Plus Environment

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A plausible mechanism has been proposed in Scheme 3. The in situ generated acetylhypobromite (AcOBr) or bromine (Br2) could facilitate a dearomatization of the isoxazole though a reactive bromonium ion intermediate (Scheme 3). Momentarily, the pendent hydroperoxide reorients in a sixmember transition to facilitate the opening of bromonium ion that leads to the formation of the desired 4-bromo-spiro-peroxide. Moreover, the stereoelectronic effect of the isoxazoline O-atom could further assist an oxonium ion mediated opening of bromonium ion to accelerate the spirocyclization (Scheme 3). Eventually, a crystal structure of 13i (Scheme 3) further substantiated the presence of spiro-linkage in peroxide; it also evident the anti-relationship between O- and Br-atoms. Scheme 3. X-ray Structure for 13i and Mechanistic Insight

Biology. The Synthesized compounds were investigated for the antiviral and anticancer activity. Antiviral Activity against HCMV: Initial screening for the test compounds (13a-13o and 15a-15g) having a potential HCMV inhibitory effect was performed using quantitative fluorescence microscopy. HFF-1 cells were pretreated with the test compounds (10 µM) or DMSO (control) for 1 hour followed by infection with HCMV (Towne-BAC-GFP strain) at a multiplicity of infection (MOI) of 3.0. This engineered HCMV strain contains a green fluorescent protein (GFP) and thus the infection is associated with the expression of GFP.27 If a compound inhibits HCMV replication, the expression of GFP, quantified as mean fluorescence intensity (MFI), is reduced. As shown in Figure 2, significant reduction in MFI is evident upon the treatment with compounds 13f, 13k, 13m, and 15g. However, other tested ACS Paragon Plus Environment

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compounds show MFI that is not significantly different from the control. This data demonstrates that these four compounds may have potential anti-HCMV properties.

Figure 2. Screening of Compounds for Anti-HCMV Activity. Confluent HFF-1 cells were pretreated with the compounds (10 µM) or DMSO (Control) for 1 hour followed by infection with HCMV (Towne-BAC-GFP) strain at MOI 3.0. At 11 days post infection, cells were fixed in 3.7% formaldehyde and MFI was quantified using a microwell image cytometer (Celigo, Nexcelcom Bioscience LLC, Lawerence, MA. Error bars represent standard error of mean (SEM) from three independent experiments. A two-tailed unpaired t-test with Welch’s correction (unequal variance assumption) was used for statistical analysis of differences. P values

Design, Synthesis, and Preliminary Studies of Spiro-isoxazoline

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