Potent Synergy between Spirocyclic Pyrrolidinoindolinones and Fluconazole against Candida albicans

Abstract

A spiroindolinone (1S,3R,3aR,6aS)-1-benzyl-6′-chloro-5-(4-fluorophenyl)-7′-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3′-1H-indole]-2′,4,6-trione was previously reported to enhance the antifungal effect of fluconazole against C. albicans. A diastereomer of that compound was synthesized, along with various analogues. Many of the compounds were shown to enhance the antifungal effect of fluconazole against C. albicans, some with exquisite potency. One spirocyclic piperazine derivative, which we have named synazo-1, enhanced the effect of fluconazole with EC₅₀ of 300 pM against a susceptible strain of C. albicans and as low as 2 nM against some resistant strains. Synazo-1 exhibits true synergy with fluconazole with an FIC index below 0.5 in the strains tested. Synazo-1 exhibited low toxicity in mammalian cells relative to the concentrations required for the antifungal synergy.

Introduction

Candida sp. account for 80% of major systemic fungal infections.^[1] The mortality rate for invasive candidiasis has been estimated around 40%.^[2] Fluconazole has high bioavailability and is well tolerated;^[3–7] it is the first line antifungal treatment for candidiasis and is now available in generic form. However, there are reports of side effects, including hepatitis, leukopenia, thrombocytopenia, gastrointestinal distress, headache, anaphylaxis, and rash.^[8–13] There is a need for drugs that can potently enhance the efficacy of azoles. Moreover, resistance to fluconazole and other azole drugs remains a major problem^[14] particularly among immunocompromised patients.^[15]

Compounds that synergize with fluconazole at low concentrations (e.g., MIC₉₀ < 0.1 μg/mL) are unusual. The antifungal agents flucytosine,^[16–19] and fenpropimorph^[20] have been shown to potently synergize with fluconazole against various strains of C. albicans. Micafungin^[21–23] and caspofungin^[20] are highly potent, but not synergistic with fluconazole, with FIC indices above 1.0. A number of drugs commonly used against non-fungal human diseases have also been shown to synergize with azoles against C. albicans. The calcineurin inhibiting drug tacrolimus^[24,25] potently enhances the activity of fluconazole against C. albicans. Quite a few other compounds have been reported to inhibit the growth of Candida in synergy with fluconazole, with MIC₉₀s below 1 μg/mL, but not below 0.1 μg/mL: e.g., T. broussonetii extract,^[26] terbinafine,^[27] amlodarone,^[28] catechin, quercetin, epigallocatechin,^[29] simvastatin,^[30] tunicamycin,^[20] cationic peptides IJ3, IJ4^[31] and VS3,^[32] ketorolac,^[33] cyclosporin A,^[24,34] nystatin,^[35] sanguinarine,^[36] allicin,^[37] declofenac,^[33] leaf extracts of Lippia alba,^[38] diphenyldiselenide,^[39] balcalein,^[40] geldanamycin,^[36] pseudolaric acid B,^[41] and doxycycline.^[42] Hundreds of other compounds have been reported to exhibit antifungal activity in concert with azoles but not below 1 μM. Chemical synthesis can be used to improve the potency of lead molecules; in a recent study, several analogues of the azole synergizer berberine (MIC₈₀ 1.0 μg/mL) were identified with up to 8 times higher potency.^[43,44]

Several groups have screened large libraries of compounds in search of small molecules that synergize with azoles. In 2011, Spitzer and coworkers screened the Prestwick library of off-patent drugs^[45] and demonstrated that sertraline and thiethylperazine synergize with fluconazole. The MIC for sertraline was as low as 0.5 μg/mL in the presence of fluconazole. Also in 2011, LaFleur and coworkers screened a library of 120,000 compounds in search of molecules that could act in synergy with clotrimazole against C. albicans biofilms, but none were active below 1 μM.^[46] Starting in 2010 Lindquist, Schreiber, and others at the Broad Institute reported a massive screening campaign to identify small molecules capable of inhibiting growth of C. albicans in synergy with fluconazole with a particular interest in Hsp90 and calcineurin pathways.^[47,48] After an initial screen of over 300,000 compounds and a subsequent rescreening, 296 compounds were found to have fluconazole dependent potency against a partially resistant clinical strain CaCi-8, and lack of cytotoxicity against mammalian fibroblasts. Three of those compounds^[48–50] were selected for further optimization but none of the resulting compounds (ML189, ML212, and ML229) were active below 0.7 μM against CaCi-8. We speculated that some of the other 296 compounds identified in the Broad screen might merit further study as potentiators of fluconazole activity in C. albicans. We set out to identify promising candidates from the Broad assay and design analogs capable of potently inhibiting the growth of C. albicans in the presence of fluconazole. We were intrigued by the structurally interesting spirocyclic compound (1S,3R,3aR,6aS)-1-benzyl-6′-chloro-5-(4-fluorophenyl)-7′-methylspiro[1,2,3a,6a-tetrahydropyrrolo[3,4-c]pyrrole-3,3′-1H-indole]-2′,4,6-trione (PubChem CID 6584729) and the potential activity of synthetically accessible diastereomer 1 and related analogues (Figure 1).

Figure 1.

Fluconazole synergizer CID 6584729 and diastereomer 1.

Results and Discussion

Chemistry

As shown in Scheme 1 N-phenylmaleimides 4a and 4b were synthesized through a two-step condensation of substituted anilines with maleic anhydride.⁵¹ Anilines 2a and 2b were condensed with maleic anhydride to form the corresponding N-phenylmaleamic acids 3a and 3b that were cyclized using acetic anhydride in the presence of sodium acetate to afford the corresponding maleimides.

Scheme 1.

Synthesis of Substituted N-Phenylmaleimides. Reagents and conditions: (a) 1.0 equiv maleic anhydride, Et₂O, 23 °C, 15 min; (b) 0.7 equiv sodium acetate, (CH₃CO)₂O, 70 °C, 30 min.

Various substituted isatins were prepared from the corresponding anilines using the two-step Sandmeyer synthesis (Scheme 2).^[52] Anilines were reacted with the oxime of chloral, generated in situ, to afford isonitrosoacetanilides, which were pure by TLC. The isonitrosoacetanilides were cyclized, without purification, through an intramolecular Friedel-Crafts reaction to afford the corresponding isatins 6a–6d in good yield. N-Benzylisatins 7d and 7e were prepared by alkylation with benzyl bromide using sodium hydride as a base.^[53]

Scheme 2.

Synthesis of Substituted Isatins. Reagents and conditions: (a) 1.1 equiv chloral hydrate, 3.0 equiv NH₂OH·HCl, 9.0 equiv Na₂SO₄, 1.1 equiv HCl, H₂O, 70 °C, 1 h; (b) H₂SO₄, 23 °C, 5 min, (c) 1.1 equiv benzyl bromide, 1.1 equiv NaH, DMF, 23 °C, 2 h.

Spiroindolinones are readily accessible through one-pot three-component coupling reactions of isatins, amino acids, and maleimides.^[54–56] The reaction of isatin 6d, L-phenylalanine and maleimide 4a generated compound 1 as a single diastereomer in 74% yield (Scheme 3). The optically pure amino acid undergoes decarboxylation during the reaction; unless otherwise stated all spiroindolinones were isolated and tested as racemates. The relative stereochemistry of compound 1 was secured through a NOESY experiment (Scheme 3) and shown to match that of related spiroindolinones prepared from isatins and maleimides under the same reaction conditions.^[56] In particular, the strong nOe between protons on C3′ and C6a′ of the pyrrolidine ring indicate that they are on the same face and conversely that the benzyl group and succinimide ring are both on the opposing face. Furthermore the strong nOe between the fluorophenyl proton and the proton on C4 of the indolone ring is consistent with the stereochemistry of compound 1. Interestingly, the ¹H NMR spectra, ¹³C NMR spectra and nOes for the compound sold as CID 6584729 (by Vitas-M) were indistinguishable from those of compound 1. Thus commercial STK580951 is in fact the same as our synthetic compound 1 and does not match PubChem CID 6584729.

Scheme 3.

Synthesis of Compound 1 and nOes Used in the Assignment of Relative Configuration. Reagents and conditions: 3:1 MeOH/H₂O, 65 °C, 16 h.

Other analogues of spirocycle 1 were synthesized (Scheme 4) using phenylalanine, tryptophan, and N_ε-Boc-Lysine. In all cases, the limiting reagent, isatin 6 or 7, was completely consumed and the reaction gave the desired cycloadduct as a single diastereomer. The reactions of these amino acids were highly stereoselective, affording products with a relative configuration analogous to spiroindolinone 1. We did not observe or isolate other diastereomers of the spiroindolinones 8–13.

Scheme 4.

Synthesis of Spirocyclic Pyrrolidines Through a Three-Component, 1-Pot [1,3]-Dipolar Cycloaddition with Amino Acids. Reagents and conditions: 3:1 MeOH/H₂O, 65 °C, 4 h – 16 h

The formation of diastereomer 1, and 8–13 was anticipated based on the work of Pavlovskaya and coworkers,^[55] but is best explained by examining a larger body of work involving reactions of amino acids, enones, and either isatins or phenylglyoxalate derivatives, which are essentially acyclic analogues of isatins. All known reactions of amino acids, isatins, and enones react to give products consistent with syn-anti azomethine ylides (Figure 2, configuration A).^[57–60] However, in the corresponding reactions with phenylglyoxylate, the reactive configuration of the azomethine ylide seems to depend on the type of amino acid: proline gives products consistent with syn-anti azomethine ylides (Figure 2, configuration A),^[61] whereas acyclic amino acids give products consistent with anti-anti azomethine ylides (Figure 2, configuration B).^[62–64]

Figure 2.

Stereochemical configurations of azomethine ylides.

The 1,3-dipolar cycloaddition can proceed through either an endo or exo transition state. The products derived from all known reactions of amino acids, acyclic enones, and either isatins or phenylglyoxalates can be rationalized to arise through endo transition states (Scheme 5, path A).^[58–62,65] In contrast, reactions of amino acids, maleimides and either isatins or phenylglyoxalates can proceed through endo or exo transition states depending on the structure of the amino acid. Acyclic amino acids give products consistent with endo transition states (Scheme 5, paths B and C).^{[55,58,62–65]} The only known reaction of a cyclic six-membered ring amino acid, pipecolic acid, with isatin and an acyclic dipolarophile also gives products consistent with an endo transition state (Scheme 5, path B);^[59] however, the stereochemical outcome in the reaction of azomethine ylides with acyclic dipolarophiles cannot be extrapolated to reactions with maleimides. ^{[56, 60a]} In contrast, the cyclic five-membered ring amino acid proline gives products consistent with an exo transition state (Scheme 5, path D).^[56]

Scheme 5.

Endo/Exo Selectivity in Dipolar Cycloadditions of Azomethine Ylides.

Similar ylides can be accessed from three-component reactions with amines instead amino acids, but there are cases where the trends in ylide configuration^[66] and endo/exo selectivity^[67] no longer hold. Notably, Ardil and coworkers showed that 1,3-dipoles derived from N-methylpiperazine aminals and related compounds favor exo adducts over endo adducts — sometimes exclusively exo — in refluxing toluene.^[67] The assumption that isatins would react through path C (Scheme 5) may have led to the misassignment of the compound CID 6584729 by the commercial supplier along with over 100 spiroindolinones in the PubChem database. We cannot be sure of the stereochemistry of the compound CID 6584729 that was tested by Lindquist and coworkers since the experimental data for compounds in PubChem assay IDs 1979, 2467, and 2423 were never reported. One cannot rule out the possibility that the compound CID 6584729 tested in those assays was correctly assigned and generated through a more lengthy synthetic route than the one-pot reaction used in this and related work.

To provide access to diastereomeric spiroindolinones with defined absolute stereochemistries we carried out a three-component coupling with isatin 6d, N-phenylmaleimide and (2S, 4R)-4-hydroxyproline (Scheme 6). After 16 h the reaction generated an inseperable mixture of two spiroindolinones 24a and 24b in 30% yield along with unreacted isatin. The two optically pure stereoisomers were readily separated by silica gel chromatography after benzoylation of the hydroxy groups to afford esters 25a and 25b. The relative stereochemistry was assigned on the basis of diagnostic nOes. In particular, in both spiroindolinones 25a and 25b, there is an nOe between the bridgehead proton H^d and the arene proton H^g on the indolone ring. In spiroindolinone 25a, protons H^f′, H^a, and H^e′ on the β-face of the proline ring exhibit vicinal nOes between each other. Protons H^e′ and H^f′ on the β-face of the proline ring exhibit long-range nOes with protons H^c and H^d on the succinimide ring, respectively; and proton H^f′ exhibits highly diagnostic long-range nOes to the indolone aryl proton H^g. In spiroindolinone 25b, protons H^f′, H^a, H^e′ and H^b on the β-face of the proline ring exhibit vicinal nOes between each other. Protons H^e and H^f on the α-face of the proline ring exhibit long-range nOes with protons H^c and H^d on the succinimide ring, respectively; and proton H^f exhibits a highly diagnostic long-range nOe to the indolone aryl proton H^g. Thus, the [3+2] cycloaddition of trans-hydroxyproline assembles proceeds via exo addition of maleimide to an azomethine ylide with a syn-anti configuration (Scheme 5, path D).

Scheme 6.

Stereoselectivity in the Three-Component, 1-Pot [1,3]-Dipolar Cycloaddition with (2S, 4R)-4-Hydroxyproline. Stereochemistry was established by nOes (grey lines). Reagents and conditions: a) 3:1 MeOH/H₂O, 90 °C, 16 h, b) 1.1 equiv BzCl, 1.2 equiv Et₃N, DMF, 23 °C, 20 h.

The reaction of the six-membered ring amino acid N_ε-Boc-piperazine-2-carboxylic acid proceeds in manner analogous with pipecolic acid (Scheme 5, path B) to afford spirocyclic indolinone 14 as a single diastereomer. The relative stereochemistry of spirocyclic piperazine 14 was secured with nOes after removal of the Boc and shown to match that of compound 1. Related spiroindolinones 15–23 were also prepared stereoselctively from N_ε-Boc-piperazine-2-carboxylic acid (Scheme 4).

The Boc group was removed from the spirocyclic piperazine 14 using trifluoroacetic acid to give a 90% yield of the piperazine 26 (Scheme 7). Piperazine 26 served as the precursor for various N-acyl derivatives 27–31 in the following reactions (Scheme 7). Compound 27 was synthesized by acylating 26 with methyl 10-chloro-10-oxodecanoate in the presence of sodium carbonate. Carbodiimide mediated coupling of (S)-2-methoxy-2-phenylacetic acid with the racemic piperazine 26 in the presence of triethylamine resulted in a mixture of diastereomers; only one diastereomer 28 was readily purified but the relative stereochemistry was not assigned. Acylation of 26 with hydrocinnamoyl chloride in the presence of triethylamine afforded amide 29. The reaction of piperazine 26 with benzyl isocyanate generated the urea 30. Carbamate 31 was synthesized by acylation of piperazine 26 with benzyl chloroformate.

Scheme 7.

Synthesis of Pentacyclic Pyrrolidines Through Further Substitutions to Compound 26. Reagents and conditions: (a) 1:1 TFA/CH₂Cl₂ 23 °C, 15 min, 90%, (b) 1.04 equiv methyl 10-chloro-10-oxodecanoate, 1.0 equiv Na₂CO₃, CH₂Cl₂, 23 °C, 30 min, 53%, (c) 1.3 equiv (S)-2-methoxy-2-phenylacetic acid, 2.1 equiv EDC, 3.5 equiv Et₃N, CH₂Cl₂, 23 °C, 20 min, 45%, (d)1.05 equiv 3-phenylpropanoyl chloride, 1.1 equiv Et₃N, CH₂Cl₂, 23 °C, 3 h, 64%, (e) 1.1 equiv (isocyanatomethyl)benzene, CH₂Cl₂, 23 °C, 4 h, 75%, (f) 1.1 equiv benzyl chloroformate, 2.1 equiv DIPEA, CH₂Cl₂, 0 – 23 °C, 2.5 h, 54

Structure-Activity Relationships

CID 6584729 was reported to enhance the effect of fluconazole against the partially resistant clinical isolate of C. albicans CaCi-8 at EC₅₀ 0.12 μM.^[68] We determined the antifungal potency of the new spiroindolinones in combination with fluconazole against a susceptible strain (HLY4123) derived from a commonly used laboratory strain of C. albicans. The activity of compound 1 was promising, with an EC₅₀ of 0.011 μM. We then compared the activity of compound 1, derived from phenylalanine with the activity of spiroindolinones derived from other amino acids (Table 1, compounds 1, 8, 9, and 10). Neither tryptophan nor N_ε-Boc-lysine derivatives were better than the parent compound 1 derived from phenylalanine. Regardless of the maleimide substituent, N-benzylisatin derivatives exhibited relatively low activity (compounds 11 and 12). Compound 13 derived from 6-chloro-7-methylisatin but lacking the 4-fluoro substituent was exceedingly potent with an EC₅₀ of 1 nM.

Table 1.

Structure-Activity Relationships for Polycyclic Pyrrolidines Against the Susceptible Strain HLY4123 of C. albicans in the Presence of Fluconazole (0.25 μg/mL).

Compound	R5′	R2′	R3′	R5	R6	R7	R1	EC50 (μM)[a]
1	4-fluorophenyl	H	Ph	H	Cl	CH3	H	0.011 ± 0.004
8	Bn	H	Ph	H	H	H	H	~ 10 ± 1.3
9	Bn	H	3-indolyl	H	H	H	H	~ 10 ± 1.8
10	Bn	H	(CH2)3NHBoc	H	H	H	H	>100
11	Bn	H	Ph	H	H	H	Bn	>10
12	Ph	H	Ph	H	H	H	Bn	>100
13	Ph	H	Ph	H	Cl	CH3	H	0.001 ± 0.0005
14	Ph	CH2CH2N(Boc)		H	Cl	CH3	H	0.0056 ± 0.003
15	4-fluorophenyl	CH2CH2N(Boc)		H	Cl	CH3	H	0.037 ± 0.001
16	3,5-bis(F3C)phenyl	CH2CH2N(Boc)		H	Cl	CH3	H	0.0237 ± 0.01
17	Ph	CH2CH2N(Boc)		H	Cl	CH3	Bn	>100
18	Bn	CH2CH2N(Boc)		H	H	H	Bn	0.0318 ± 0.4
19	Bn	CH2CH2N(Boc)		H	H	H	H	>100
20	Ph	CH2CH2N(Boc)		H	H	H	Bn	>100
21	Bn	CH2CH2N(Boc)		MeO	H	H	H	230 ± 5.7
22	Ph	CH2CH2N(Boc)		H	H	CH3	H	0.213 ± 0.08
23	Ph	CH2CH2N(Boc)		H	CH3	CH3	H	0.0057 ± 0.006
26	Ph	CH2CH2NH		H	Cl	CH3	H	~10 ± 1.5
27	Ph	CH2CH2N[CO(CH2)8CO2Me		H	Cl	CH3	H	0.0379 ± 0.009
28	Ph	CH2CH2N[COCH(OCH3)C6H5]		H	Cl	CH3	H	0.035 ± 0.007
29	Ph	CH2CH2N[CO(CH2)2Ph]		H	Cl	CH3	H	0.0181 ± 0.004
30	Ph	CH2CH2N[CONHCH2Ph]		H	Cl	CH3	H	0.256 ± 0.1
31	Ph	CH2CH2N(Cbz)		H	Cl	CH3	H	0.0003 ± 0.00001

^[a]Each value is the arithmetic mean ± SD of three independent experiment.

When we employed the non-natural amino acid N_ε-Boc-piperazine-2-carboxylic acid, the resulting spirocyclic piperazine 14 was still highly active with an EC₅₀ of 5.6 nM. N-Benzylsuccinimide derivatives 18, 19, and 21 were not highly active. The substituents on the indolone ring were still important, even with the pentacyclic piperazine core (compounds 17, 20, 22, 23). We removed the Boc group from the piperazine ring of compound 14, leading to a loss of potency (compound 26). Surprisingly, the carboxyl oxygen of the carbamate moiety appears to be essential for high potency; because carbamates 14 and 31 were more potent than amides 27 and 28. Even more revealing, the isosteric amide 29 and urea 30 were two and three orders of magnitude less active, respectively, than benzyloxycarbamate 31. Ultimately, benzyloxycarbamate 31 proved to be exquisitely active in improving the efficacy of fluconazole with an EC₅₀ of 300 pM. The two hydroxyproline adducts 25a and 25b, exhibited almost no activity under the conditions of the assay; those results are not surprising given that the relative stereochemistry of those compounds was different from all the other compounds that were tested.

In general, substitution of small, hydrophobic groups on 6 and 7 positions of the indolone ring and benzyl substitution on the 3′ position of the central pyrrolidine improves the antifungal activity. Also, a phenyl moiety not benzyl in the succinamide ring enhances the antifungal activity. When the piperazine is present in the polycyclic pyrrolidine, carbamoyl moiety but not the acyl groups on the piperazine ring significantly improves the antifungal activity. On the other hand, fluorinated substituents on the para and meta positions of the phenyl group in the succinamide ring as well as N-substitution of the indolone moiety diminish the activity of the spiroindolinones against C. albicans.

Activity Against Resistant Cell Lines

A variety of resistant clinical isolates of C. albicans were screened with 64 μg/ml fluconazole and compound 31 at a single dose (3 μM) in a broth microdilution assay (Table 2).^[69] The strains grow at dramatically different rates. The published fluconazole minimum inhibitory concentrations (MICs) for these isolates convey the level of resistance. Strains for which growth in the presence of compound 31 and fluconazole was less than 25% of growth in the presence of fluconazole alone (Isolates 17, 23, 26, 33, 36, and 45) were selected for determination of EC₅₀s. Compound 31 was particularly active against clinical isolates 17, 26 and 36 and exhibited good activity against the highly resistant isolate 45.

Table 2.

Effect of Compound 31 on the Growth of Resistant Clinical Strains in the Presence of Fluconazole.

		Growth (OD600) [b]		Potency
Isolate [a]	MICFlc[a]	+Fluc [c]	+Cpd 31 [d]+Fluc[c]	+Cpd 31 EC50 (nM)[c,e]
17	32	3.96	0.76	5 ± 0.011
23	32	6.77	0.60	53 ± 0.005
26	32	6.44	0.83	2 ± 0.002
33	64	7.38	0.88	11 ± 0.008
36	64	5.08	1.07	5 ± 0.251
45	128	9.17	1.03	16 ± 0.002

^[a]From reference 69.

^[b]growth measured after 16 h in SC at 30°C.

^[c][fluconazole]=64 μg/mL.

^[d][compound 31]=3 μM.

^[e]Each value is the arithmetic mean ± SD of three independent experiments.

A checkerboard assay was used to determine the fractional inhibitory concentrations for compound 31 and fluconazole against the fluconazole-susceptible strain (HLY4123) and two fluconazole-resistant clinical isolates 26 and 45 (Table 3).^[69]

Table 3.

FIC Indices for Compound 31 and Fluconazole in Different Strains of C. albicans (MIC₉₀ measured in μM).^[a]

Strain	MICcpd	MICcpd(+Flc)	MICFlc	MICFlc(+cpd)	FICI
HLY4123	>300	0.03	0.5	0.125	0.25
26	>30	0.3	836	105	0.14
45	>30	0.03	836	105	0.13

^[a]MIC data are derived from single replicates of 2 independent experiments; values were consistent between experiments.

In all of the strains tested, the FIC index was below 0.5, fitting the classical definition of synergy.^[70] Compound 31 alone did not have measurable toxicity against any of the strains at the solubility limit (between 30 and 300 μM). In the strains tested, fluconazole dramatically enhances the activity of compound 31 against C. albicans. Conversely compound 31 makes fluconazole more potent against those same strains but the effect is less dramatic. We have named compound 31 as synazo-1 (Figure 3).

Figure 3.

The structure of compound 31, renamed synazo-1.

Greater than 90% inhibition of C. albicans sterol α-demethylase (a.k.a. Erg11 or CYP51) would be expected at ten times the K_i for fluconazole, which has been determined to be 0.03 μM.^[71,72] When synazo-1 is present at 300 nM in the susceptible strain, the MIC₉₀ for fluconazole is reduced from 0.5 μM to 0.125 μM, consistent with the theoretical limit for fluconazole potency of around 0.3 μM.

Cytotoxicity of Synazo-1 Against Mammalian Cells

We compared the cytotoxicity of spiroindoline 1 and synazo-1 against NIH 3T3 cells at higher concentrations up to 1.5 mM (Figure 4). Both compounds exhibited only weak cytotoxicity. Synazo-1 was slightly more cytotoxic, but not at the concentrations required for antifungal synergy. According to PubChem, CID 6584729 was previously tested for cytotoxicity against NIH 3T3 cells (PubChem AID 2387) but the EC₅₀ was ≥160 μM, the limit of the assay. It is unclear how the biological activities reported for CID 6584729 should relate to that of the diastereomer 1 or whether they were even distinct compounds.

Figure 4.

Cytotoxicity of synazo-1 and compounds 1 against NIH 3T3 cells. Data represent arithmetic means ± SD of three independent experiments.

Drug-Like Parameters for Synazo-1

A wide range of readily calculated properties are often used as indicators of oral bioavailability. The calculated physicochemical properties of synazo-1 were compared with typical ranges for lead-like molecules.^[73–75] Synazo-1 flags just one of the common warnings for drug lead-like properties (Table 4) — molecular weight. For comparison, the orally available azole posoconazole is outside the range on four of the parameters. It is widely recognized that the average molecular weight and complexity of newly approved oral drugs has been increasing with each year.^[76–77]

Table 4.

Calculated Physicochemical Properties of Synazo-1.^[a]

Property	Range	Synazo-1
milogP	−4.0 to 4.2	4.173
TPSA	≤120 Å2	99.3 Å2
molecular weight	≤460	571
N+O	≥1	9
H-bond donors	≤5	1
rotatable bonds	≤10	4
halogens	≤7	1
fraction sp3	0.15–0.80	0.20
H-bond acceptors	≤9	5

^[a]Calculated with Molinspiration property calculation service. http://www.molinspiration.com/cgi-bin/properties.

Both the carbamate and imide moieties are potential liabilities for metabolism, yet when the stability of synazo-1 was tested in 10% FBS/phosphate buffered saline at 37 °C no decomposition was observed over 16 h.

Conclusions

In conclusion, we have designed, synthesized and studied spiroindolinones inspired by CID 6584729 that was previously reported to exhibit activity against C. albicans in combination with fluconazole. The relative stereochemistry of compound 1 and analogues was secured through 2D NMR experiments. The three-component, one-pot [3+2] dipolar cycloaddition of isatins, amino acids, and maleimides was found to proceed through endo addition of maleimides to a syn-anti azomethine ylide in all cases except for a proline derivative. A number of the new spiroindolinones were substantially more potent against C. albicans than the original lead compound 1 when used in combination with fluconazole. In particular, synazo-1 was exquisitely potent with an EC₅₀ of 300 pM against a susceptible strain. Synazo-1 also exhibited low nanomolar activity against a number of resistant isolates of Candida. When tested in both susceptible and resistant strains of C. albicans, synazo-1 was a true synergizer with an FIC index below 0.5. Synazo-1 has many of the calculable parameters associated with orally available drug molecules and represents a promising candidate for development as an antifungal synergizer.

Experimental Section

Chemistry

Analytical High-Performance Liquid Chromatography (HPLC)

The HPLC instrument consisted of an Agilent Technologies series 1200 autosampler, series 1200 UV/Vis detector, series 1100 pump, using ChemStation software (Agilent Technologies, Santa Clara, CA, USA). The analytical column was a reverse-phase Waters Nova-pak^® C₁₈ 150 mm × 3.9 mm column. A gradient elution was used (flow rate 0.2 mL/min), starting with 80% water and progressing to 100% acetonitrile over a period of 1 h, with both solvents containing 0.1% formic acid. All compounds have purity ≥95% (254 nm) by HPLC.

General Experimental Procedures

NMR spectral data were recorded at room temperature using a Bruker 500 or 600 MHz spectrometer unless stated otherwise. The NMR data are reported as follows: chemical shifts in ppm from an internal tetramethylsilane standard on the δ scale, multiplicity (br=broad, app=apparent, s=singlet, d=doublet, t=triplet, q=quartet, and m=multiplet), coupling constants (Hz), and integration. Analytical thin layer chromatography (TLC) was performed using EMD Reagents 0.25 mm silica gel 60-F plates. “Flash” chromatography on silica gel was performed using Silicycle silica gel (40–63 μm). All reactions were carried out under an atmosphere of nitrogen in glassware that was evacuated and back-filled with nitrogen three times. Reactions were carried out at room temperature unless otherwise indicated. Unless otherwise noted, all reagents were commercially obtained and, where appropriate, purified prior to use. THF, Et₂O, DMF and CH₂Cl₂ were dried by filtration through alumina according to the procedure of Grubbs and coworkers.^[78] For final compounds the purity was determined by HPLC (Agilent Technologies series 1200).

General procedure for the synthesis of pyrrolidines through a three-component dipolar cycloaddition (1, 8–23, 24a and 24b):^[79]

A 100 mL round bottom flask was charged with substituted isatin (1.0 equiv), N-substituted maleimide (1.1 equiv), the amino acid (1.1 equiv) and a stir bar. A 3:1 (v/v) mixture of water and methanol was added to the reaction flask such that the concentration of isatin was 0.25 M. The reaction was heated at reflux by immersing the reaction flask in a hot oil bath at 90 °C up to the level of the flask’s contents. Initially a clear solution was obtained and CO₂ evolution was observed. However, after a few hours the reaction mixture became cloudy. The reaction was monitored for consumption of the substituted isatin by TLC (EtOAc/hex). Upon consumption of the substituted isatin, the reaction was cooled to room temperature. Next, the reaction mixture was quenched by pouring it into a mixture of ice and sat. aq. NaHCO₃. The resulting solid was washed thoroughly with water in Büchner funnel to afford a grey solid. The solid was then dissolved in minimum amount of CH₂Cl₂ and purified by flash chromatography with EtOAc/hex (1:1) to afford the racemic substituted pyrrolidine.

(±)-(3R,3′R,3a′R,6a′S)-3′-benzyl-6-chloro-5′-(4-fluorophenyl)-7-methyl -2′,3′,3a′,6a′-tetrahydro-4′H-spiro[indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′(5′H)-trione (1)

A 100 mL round bottom flask was charged with 1-(4-fluorophenyl)-1H-pyrrole-2,5-dione (0.50 g, 2.55 mmol, 1.0 equiv), p-fluoro-N-phenylmaleimide (0.53 g, 2.8 mmol, 1.1 equiv), L-phenylalanine (0.46 g, 2.8 mmol, 1.1 equiv) and a stir bar. A 3:1 mixture of water and methanol (11 mL) was added to the reaction flask. The content of the reaction flask was heated at reflux by immersing the reaction flask in a hot oil bath up to the level of the flask’s contents. Initially a clear solution was obtained and CO₂ was expelled. After few hours a cloudy solution was observed. Upon consumption of the substituted isatin (16 h), the reaction was allowed to cool to room temperature. Next, the reaction mixture was quenched by pouring it into a mixture of ice and sat. aq. NaHCO₃. The resulting solid was washed thoroughly with water in Büchner funnel to afford a grey solid. The solid was then dissolved in minimum amount of CH₂Cl₂ and purified by flash chromatography with different combinations of EtOAc/hex to afford the pyrrolidine 1 as a white solid (0.93 mg, 1.9 mmol, 74%): R_f=0.35 (1:1 EtOAc/hex); mp 212–214. The ¹H NMR chemical shifts were concentration-dependent in CDCl₃, particularly within the range 0.5–2 mM. ¹H NMR (600 MHz, CDCl₃): δ 8.00 (s, 1H), 7.41-7.38 (m, 2H), 7.27-7.16 (m, 6H), 7.02 (d, J=8.0 Hz, 2H), 6.81 (d, J=8.5 Hz, 1H), 4.72-4.71 (m, 1H), 3.75-3.69 (m, 1H), 3.45 (dd, J=14.0, 4.0 Hz, 1H), 2.73 (dd, J=13.8, 10.5 Hz, 1H), 2.16 (s, 1H), 1.99 (s, 3H); ¹³C NMR (125 MHz, CDCl₃): δ 180.4, 175.1, 174.4, 163.4, 161.4, 140.5, 139.1, 136.2, 128.9, 128.8, 128.3, 127.6, 126.7, 124.6, 124.5, 123.5, 118.2, 116.6, 116.4, 68.2, 58.9, 51.6, 47.6, 37.9, 13.5; IR (thin film) 3201, 3065, 1710, 1696, 1623, 1601, 1510; HRMS (ESI): m/z calculated for C₂₈H₂₉ClN₄O₅Na [M+Na]⁺ 559.1724, found 559.1743. HPLC purity: 95.76%, t_R=21.73 min.

(±)-(3R,3′R,3a′R,6a′S)-3′,5′-Dibenzyl-2′,3′,3a′,6a′-tetrahydro-4′H-spiro [indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′(5′H)-trione (8)

Using the general procedure for the synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 8 as a white solid (0.65 mg, 1.5 mmol, 60%): R_f=0.50 (1:1 EtOAc/hex); mp 138–140; ¹H NMR (600 MHz, CDCl₃): δ 7.50-7.49 (m, 2H), 7.40-7.38 (m, 3H), 7.29 (br s, 1H), 7.26-7.25 (m, 1H), 7.24-7.22 (m, 3H), 7.18-7.16 (m, 2H), 6.80 (t, J=7.5 Hz, 1H), 6.69 (d, J=7.8 Hz, 1H), 6.50 (d, J=7.5 Hz, 1H), 4.83 (d, J=14.0 Hz, 1H), 4.69 (d, J=14.0 Hz, 1H), 4.68-4.67 (m, 1H), 3.58-3.56 (m, 1H), 3.42 (d, J=7.6 Hz, 2H), 2.60 (dd, J=13.8, 10.4 Hz, 1H), 2.01 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 180.1, 175.8, 174.5, 140.3, 139.3, 135.8, 129.8, 129.0, 128.9, 128.8, 128.7, 128.1, 126.9, 126.5, 126.4, 122.6, 109.7, 67.6, 58.5, 51.5, 47.7, 42.7, 38.2; IR (thin film) 3850, 3646, 2971, 2843, 1697, 1619, 1054, 1032; HRMS (ESI): m/z calculated for C₂₇H₂₃N₃O₃Na [M+Na]⁺ 460.1637, found 460.1620. HPLC purity: 100%, t_R=20.04 min.

(±)-(3R,3′R,3a′R,6a′S)-3′-((1H-Indol-3-yl)methyl)-5′-benzyl-2′,3′,3a′,6a′-tetrahydro-4′H-spiro[indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′(5′H)-trione (9)

Using the general procedure for the synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 9 as a yellow solid (0.83 mg, 1.7 mmol, 70%): R_f=0.35 (1:1 EtOAc/hex); mp 130–132; ¹H NMR (600 MHz, CDCl₃): δ 7.96 (br s, 1H), 7.60 (d, J=7.9 Hz, 1H), 7.52 (d, J=7.3 Hz, 2H), 7.42-7.39 (m, 2H), 7.36-7.33 (m, 1H), 7.30-7.29 (m, 1H), 7.17-7.11 (m, 2H), 7.09-7.06 (m, 2H), 6.80-6.77 (m, 1H), 6.62 (d, J=7.7 Hz, 1H), 6.49 (d, J=7.5 Hz, 1H), 4.86 (d, J=14.0 Hz, 1H), 4.80-4.76 (m, 1H), 4.70 (d, J=14.0 Hz, 1H), 3.60 (t, J=7.7 Hz, 1H), 3.49 (dd, J=14.6, 3.7 Hz, 1H), 3.41 (d, J=7.7 Hz, 1H), 2.82 (dd, J=14.6, 10.3 Hz, 1H), 2.13 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 180.3, 176.0, 174.8, 140.4, 136.2, 135.9, 129.6, 129.0, 128.8, 128.2, 127.5, 126.8, 126.5, 122.5, 122.4, 122.1, 119.5, 119.1, 113.6, 111.1, 109.7, 67.7, 58.1, 51.6, 47.7, 42.6, 27.7; IR (thin film) 3679, 2971, 2864, 2843, 1695, 1619, 1054, 1032; HRMS (ESI): m/z calculated for C₂₉H₂₄N₄O₃Na [M+Na]⁺ 499.1746, found 499.1739. HPLC purity: 98.81%, t_R=21.98 min.

(±)-tert-Butyl (4-((3R,3′R,3a′R,6a′S)-5′-benzyl-2,4′,6′-trioxo-3′,3a′,4′,5′, 6′,6a′-hexahydro-2′H-spiro[indoline-3,1′-pyrrolo[3,4-c]pyrrol]-3′-yl) butyl)carbamate (10)

Using the general procedure for the synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 10 as a white solid (0.42 mg, 0.82 mmol, 33%): R_f=0.35 (4:6 EtOAc/hex); mp 109–111; ¹H NMR (600 MHz, CDCl₃): δ 7.45-7.44 (m, 2H), 7.38-7.33 (m, 3H), 7.29 (br s, 1H), 7.20-7.17 (m, 1H), 6.79-6.76 (m, 2H), 6.36 (d, J=7.4 Hz, 1H), 4.78 (d, J=14.0 Hz, 1H), 4.70-4.63 (m, 1H), 4.61 (d, J=14.0 Hz, 1H), 4.34-4.32 (m, 1H), 3.50 (t, J=7.7 Hz, 1H), 3.42 (d, J=7.7 Hz, 1H), 3.18-3.08 (m, 2H), 2.06-1.91 (m, 2H), 1.57-1.53 (m, 2H), 1.43 (s, 9H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 180.15, 175.8, 174.5, 140.3, 135.8, 129.8, 129.2, 128.8, 128.2, 126.5, 126.4, 122.7, 109.8, 68.1, 58.3, 51.7, 48.1, 42.7, 40.1, 31.2, 30.0, 28.5, 24.6, 14.3; IR (thin film) 3707, 2971, 2843, 1345, 1054, 1032; HRMS (ESI): m/z calculated for C₂₉H₃₄N₄O₅Na [M+Na]⁺ 541.2427, found 541.2411. HPLC purity: 97.81%, t_R=20.74 min.

(±)-(3R,3′R,3a′R,6a′S)-1,3′,5′-Tribenzyl-2′,3′,3a′,6a′-tetrahydro-4′H-spi ro[indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′(5′H)-trione (11)

Using the general procedure for the synthesis of pyrrolidines outlined above, 1-benzylindoline-2,3-dione (0.07 g, 0.32 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 11 as a white solid (0.08 mg, 0.15 mmol, 46%): R_f=0.60 (1:1 EtOAc/hex); mp 173–175; ¹H NMR (600 MHz, CDCl₃): δ 7.50-7.49 (m, 2H), 7.39-7.12 (m, 14H), 6.82-6.79 (m, 1H), 6.64 (d, J=9.3 Hz, 1H), 6.54 (d, J=8.7 Hz, 1H), 4.88-4.81 (m, 2H), 4.80-4.72 (m, 1H), 4.71-4.65 (m, 2H), 3.64-3.60 (m, 1H), 3.44-3.39 (m, 2H), 2.61 (dd, J=16.6, 12.3 Hz, 1H), 2.01 (br s, 1H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 178.7, 175.9, 174.4, 142.6, 139.3, 135.8, 135.4, 129.8, 129.0, 128.9, 128.9, 128.8, 128.6, 128.1, 127.8, 127.3, 126.5, 126.4, 125.8, 122.6, 109.1, 67.5, 58.5, 51.8, 47.7, 43.6, 42.7, 38.2; IR (thin film) 3850, 3708, 2971, 2864, 1453, 1054, 1032; HRMS (ESI): m/z calculated for C₃₄H₂₉N₃O₃Na [M+Na]⁺ 550.2106, found 550.2108. HPLC purity: 98.45%, t_R=23.19 min.

(±)-(3R,3′R,3a′R,6a′S)-1,3′-Dibenzyl-5′-phenyl-2′,3′,3a′,6a′-tetrahydro-4′H-spiro[indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′(5′H)-trione (12)

Using the general procedure for the synthesis of pyrrolidines outlined above, 1-benzylindoline-2,3-dione (0.30 g, 1.3 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 12 as a yellow solid (0.38 mg, 0.74 mmol, 57%): R_f=0.60 (1:1 EtOAc/hex); mp 183–185; ¹H NMR (600 MHz, [D₆]DMSO): δ 7.57-7.52 (m, 2H), 7.46-7.44 (m, 1H), 7.40-7.37 (m, 4H), 7.34-7.33 (m, 3H), 7.31-7.25 (m, 3H), 7.21-7.17 (m, 2H), 7.14 (dd, J=7.4, 1.5 Hz, 1H), 6.97 (t, J=7.5 Hz, 1H), 6.84 (d, J=7.8 Hz, 1H), 4.90 (d, J=15.6 Hz, 1H), 4.76 (d, J=15.6 Hz, 1H), 4.52-4.50 (m, 1H), 3.73 (dd, J=7.5, 1.8 Hz, 1H), 3.63 (dd, J=7.8, 2.7 Hz, 1H), 2.61 (dd, J=16.6, 12.3 Hz, 1H), 3.32 (br s, 1H), 2.82-2.78 (m, 1H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 178.7, 175.9, 174.4, 142.6, 139.3, 135.8, 135.4, 129.8, 129.0, 128.9, 128.9, 128.8, 128.6, 128.1, 127.8, 127.3, 126.5, 126.4, 125.8, 122.6, 109.1, 67.5, 58.5, 51.8, 47.7, 43.6, 42.7, 38.2; IR (thin film) 3680, 2966, 2865, 1706, 1054, 1032; HRMS (ESI): m/z calculated for C₃₄H₂₉N₃O₃Na [M+Na]⁺ 550.2106, found 550.2108. HPLC purity: 95.07%, t_R=23.09 min.

(±)-(3R,3′R,3a′R,6a′S)-3′-Benzyl-6-chloro-7-methyl-5′-phenyl-2′,3′,3a′, 6a′-tetrahydro-4′H-spiro[indoline-3,1′-pyrrolo[3,4-c]pyrrole]-2,4′,6′ (5′H)-trione (13)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.30 g, 1.7 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 13 as a white solid (0.21 g, 0.44 mmol, 26%): R_f=0.60 (1:1 EtOAc/hex); mp 168–170; ¹H NMR (600 MHz, [D₆]DMSO): δ 10.12 (s, 1H), 7.54-7.51 (m, 2H), 7.45-7.43 (m, 1H), 7.39-7.35 (m, 4H), 7.29-7.27 (m, 2H), 7.19-7.17 (m, 1H), 6.97 (d, J=8.0 Hz, 1H), 6.90 (d, J=8.0 Hz, 1H), 4.55 (br s, 1H), 3.72 (t, J=7.5 Hz, 1H), 3.53 (d, J=7.8 Hz, 1H), 3.38-3.35 (m, 2H), 2.27 (s, 3H); ¹³C NMR (125 MHz, [D₆]DMSO): δ 142.8, 140.6,135.1, 134.5, 132.9, 129.4, 129.3, 128.7, 127.6, 127.3, 126.4, 125.0, 121.9, 117.3; IR (thin film) 3850, 3626, 2971, 2864, 1710, 1693, 1014, 1032; HRMS (ESI): m/z calculated for C₂₇H₂₂ClN₃O₃Na [M+Na]⁺ 494.1247, found 494.1255. HPLC purity: 95.00%, t_R=23.41 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′, 4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (14)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.81 g, 4.1 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 14 as a light pink solid (1.3 g, 2.5 mmol, 60%): R_f=0.35 (4:6 EtOAc/hexanes); mp 201–203; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.90 (s, 1H), 7.54-7.51 (m, 2H), 7.47-7.46 (m, 1H), 7.31 (d, J=7.8 Hz, 2H), 7.06 (d, J=7.8 Hz, 1H), 6.73 (d, J=7.8 Hz, 1H), 4.31 (br s, 1H), 3.85 (t, J=7.5 Hz, 2H), 3.64-3.65 (m, 1H), 3.55 (d, J=7.8 Hz, 1H), 2.74-2.58 (m, 2H) 2.25 (app s, 4H), 2.13-2.08 (m, 1H), 1.41 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 313 K): δ 177.9, 174.7, 173.4, 153.6, 142.8, 134.6, 132.1, 128.9, 128.5, 126.9, 124.6, 123.2, 122.8, 117.3, 79.1, 71.7, 62.3, 58.7, 57.6, 50.4, 45.9, 44.9, 27.9, 13.7; IR (thin film) 3840, 3708, 3626, 2971, 2843, 1713, 1695, 1032; HRMS (ESI): m/z calculated for C₂₈H₂₉ClN₄O₅Na [M+Na]⁺ 559.1724, found 559.1743. HPLC purity: 96.96%, t_R=22.18 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-6-chloro-2′-(4-fluorophenyl)-7-meth yl-1′,2,3′-trioxo-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (15)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.12 g, 0.63 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 15 as a white solid (0.17 mg, 0.31 mmol, 49%): R_f=0.35 (4:6 EtOAc/hex); mp 246–248; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.42 (s, 1H), 7.39-7.35 (m, 2H), 7.32-7.29 (m, 2H), 7.02 (d, J=7.8 Hz, 2H), 6.75 (d, J=7.8 Hz, 1H), 4.35 (dd, J=12.6, 1.8 Hz, 1H), 3.89-3.83 (m, 2H), 3.74-3.71 (m, 1H), 3.56 (d, J=7.8 Hz, 1H), 2.74-2.58 (m, 2H) 2.77-2.73 (m, 1H), 2.74-2.54 (m, 1H), 2.50 (s, 3H), 2.29-2.09 (M, 1H), 1.44 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 313 K): δ 178.5, 175.3, 173.9, 161.0, 154.2, 143.3, 135.1, 129.7, 128.8, 125.1, 123.7, 122.6, 117.9, 116.6, 116.4, 79.6, 72.2, 58.1, 51.0, 46.6, 45.6, 45.9, 28.5, 14.3; IR (thin film) 3850, 3708, 2965, 2866, 1706, 1689, 1032; HRMS (ESI): m/z calculated for C₂₈H₂₈ClFN₄O₅Na [M+Na]⁺ 577.1630, found 577.1644. HPLC purity: 100%, t_R=22.20 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-2′-(3,5-bis(trifluoromethyl)phenyl)-6-chloro-7-methyl-1′,2,3′-trioxo-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro [indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxy late (16)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.50 g, 2.6 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 16 as a white solid (0.13 mg, 0.30 mmol, 12%): R_f=0.5 (2:3 EtOAc/hex); mp 199–201; ¹H NMR (600 MHz, CDCl₃, 320 K): δ 8.51 (s, 1H), 7.93-7.92 (m, 3H), 7.12 (d, J=8.1 Hz, 1H), 6.70 (d, J=8.0 Hz, 1H), 4.59 (br s, 1H), 4.11 (br s, 1H), 3.80–3.82 (m, 1H), 3.81-3.78 (m, 1H), 3.74-3.72 (m, 1H), 2.82–2.89 (m, 1H), 2.65–2.71 (m, 1H), 2.33–2.34 (m, 2H), 2.17 (s, 3H), 1.47 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO): δ 177.6, 174.2, 173.0, 154.4, 141.2, 136.8, 133.0, 132.7, 126.2, 124.5, 123.8, 123.7, 122.5, 118.4, 81.0, 72.9, 62.3, 58.3, 57.6, 50.5, 46.17, 45.6, 28.4, 13.8; IR (thin film) 3187, 1724, 1706, 1680, 1599, 1276, 1132; HRMS (ESI): m/z calculated for C₃₀H₂₇ClF₆N₄O₅Na [M+Na]⁺ 695.1472, found 695.1450. HPLC purity: 98.46%, t_R=24.08 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-1,2′-dibenzyl-1′,2,3′-trioxo-2′,3′,3a′, 3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo [1,2-a]pyrazine]-5′(1′H)-carboxylate (18)

Using the general procedure for the synthesis of pyrrolidines outlined above, 1-benzylindoline-2,3-dione (0.30 g, 1.3 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 18 as a white solid (0.39 mg, 0.66 mmol, 56%): R_f=0.8 (1:1 EtOAc/hex); mp 191–192; ¹H NMR (600 MHz, [D₆]DMSO): δ 7.38-7.37 (m, 4H), 7.36-7.32 (m, 5H), 7.31-7.27 (m, 1H), 7.22-7.20 (m, 1H), 6.87 (d, J=7.8 Hz, 1H), 6.82-6.79 (m, 1H), 6.50 (d, J=7.2 Hz, 1H), 4.87 (s, 2H), 4.65 (AB q, J=14.7 Hz, 2H), 4.36-4.34 (m, 1H), 3.85-3.83 (m, 1H), 3.79-3.76 (m, 1H), 3.72-3.71 (m, 1H), 3.49 (d, J=7.8 Hz, 1H), 2.68-2.64 (m, 1H), 2.51–2.53 (m, 1H), 2.12-2.09 (m, 1H), 1.44 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 313 K): δ 176.1, 175.8, 174.5, 155.2, 143.7, 136.6, 136.4, 130.1, 129.2, 129.0, 128.2, 128.1, 127.9, 127.7, 126.7, 124.2, 122.7, 109.7, 79.7, 71.7, 58.2, 50.8, 46.3, 45.5, 43.1, 42.2, 28.5; IR (thin film) 3850, 3626, 2971, 2862, 1707, 1693, 1678, 1032; HRMS (ESI): m/z calculated for C₃₅H₃₆N₄O₅Na [M+Na]⁺ 615.2584, found 615.2572. HPLC purity: 100%, t_R=24.32 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-2′-benzyl-1′,2,3′-trioxo-2′,3′,3a′,3b′, 4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (19)

Using the general procedure for the synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.37 g, 2.5 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 19 as a white solid (0.66 mg, 1.3 mmol, 53%): R_f=0.45 (1:1 EtOAc/hex); mp 207–208; ¹H NMR (600 MHz, CDCl₃): δ 7.46-7.43 (m, 3H), 7.36-7.35 (m, 3H), 7.23-7.20 (m, 1H), 6.83-6.81 (m, 1H), 6.77 (d, J=7.7 Hz, 1H), 6.32 (d, J=6.8 Hz, 1H), 4.80 (d, J=14.1 Hz, 1H), 4.63 (d, J=14.1 Hz, 1H), 4.22-4.12 (m, 1H), 3.80-3.78 (m, 1H), 3.60-3.58 (m, 1H), 3.41 (d, J=7.9 Hz, 1H), 2.79-2.45 (m, 2H), 2.27-2.18 (m, 2H), 1.46 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): δ 180.4, 175.8, 174.7, 156.1, 140.5, 135.8, 129.7, 129.2, 128.9, 128.8, 128.2, 126.5, 122.6, 109.9, 68.1, 58.3, 51.6, 48.1, 42.6, 40.1, 31.1, 30.0, 28.5, 24.6; IR (thin film) 3679, 2971, 2864, 1706, 1642, 1054, 1032, 1012; HRMS (ESI): m/z calculated for C₂₈H₃₀N₄O₅Na [M+Na]⁺ 525.2114, found 525.2106. HPLC purity: 100%, t_R=22.09 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-2′-benzyl-1′,2,3′-trioxo-2′,3′,3a′,3b′, 4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (20)

Using the general procedure for the synthesis of pyrrolidines outlined above, 1-benzylindoline-2,3-dione (0.30 g, 1.3 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 20 as a yellow solid (0.43 mg, 0.75 mmol, 71%): R_f=0.75 (1:1 EtOAc/hex); mp 210–212; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 7.54-7.51 (m, 2H), 7.46-7.44 (m, 1H), 7.35-7.34 (m, 6H), 7.29-7.26 (m, 2H), 7.03-7.02 (m, 2H), 6.92 (d, J=8.4 Hz, 1H), 4.91 (s, 2H), 4.40-4.38 (m, 1H), 3.92-3.89 (m, 2H), 3.81-3.77 (m, 1H), 3.61 (d, J=7.8 Hz, 1H), 2.84-2.77 (m, 1H), 2.68-2.63 (m, 1H), 2.24-2.16 (m, 2H), 1.45 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): δ 176.7, 175.7, 174.3, 155.2, 144.2, 137.1, 133.1, 130.7, 130.0, 129.7, 129.5, 128.4, 128.1, 127.9, 126.9, 124.8, 123.4, 110.2, 80.1, 72.4, 58.8, 51.7, 47.0, 46.8, 43.6, 28.9; IR (thin film) 3850, 3671, 2972, 2843, 1712, 1690, 1135, 1032; HRMS (ESI): m/z calculated for C₃₄H₃₄N₄O₅Na [M+Na]⁺ 601.2427, found 601.2415. HPLC purity: 100%, t_R=23.46 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-2′-benzyl-5-methoxy-1′,2,3′-trioxo-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4] pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (21)

Using the general procedure for the synthesis of pyrrolidines outlined above, indoline-2,3-dione (0.45 g, 2.5 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 21 as a white solid (0.66 mg, 1.3 mmol, 53%): R_f=0.35 (1:1 EtOAc/hex); mp 218–220; ¹H NMR (600 MHz, [D₆]DMSO): δ 10.43 (s, 1H), 7.38-7.36 (m, 2H), 7.33-7.32 (m, 2H), 7.29-7.27 (m, 1H), 6.77 (dd, J=8.7, 2.1 Hz, 1H), 6.73-6.71 (m, 1H), 6.07 (s, 1H), 4.64 (AB q, J=15 Hz, 2H), 4.31 (br s, 1H), 3.87-3.83 (m, 1H), 3.75-3.73 (m, 1H), 3.68-3.64 (m, 1H), 3.45 (d, J=7.8 Hz, 1H), 2.61-2.59 (m, 2H), 2.18-2.16 (m, 1H), 2.04 (td, J=11.2, 3.0 Hz, 1H), 1.40 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): δ 177.7, 176.1, 174.7, 154.9, 153.8, 136.4, 129.1, 127.9, 127.5, 126.0, 115.4, 113.4, 110.5, 79.7, 72.3, 61.2, 57.9, 55.5, 50.6, 46.3, 45.2, 42.0, 28.5; IR (thin film) 3850, 3648, 2967, 1710, 1641, 1130, 1032; HRMS (ESI): m/z calculated for C₂₈H₃₀N₄O₅Na [M+Na]⁺ 555.2222, found 555.2239. HPLC purity: 96.22%, t_R=23.24 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′, 3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4] pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (22)

Using the general procedure for the synthesis of pyrrolidines outlined above, 7-methylindoline-2,3-dione (1.0 g, 6.2 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 22 as a white solid (2.0 g, 4.0 mmol, 60%): R_f=0.3 (1:1 EtOAc/hex); mp 212–214; ¹H NMR (600 MHz, [D₆]DMSO, 398 K): δ 10.1 (s, 1H), 7.53-7.51 (m, 2H), 7.45-7.42 (m, 1H), 7.34-7.33 (m, 2H), 7.05 (d, J=7.8 Hz, 1H), 6.88-6.86 (m, 1H), 6.76 (d, J=7.2 Hz, 1H), 4.37-4.35 (m, 1H), 3.87-3.82 (m, 2H), 3.75-3.73 (m, 1H), 3.55-3.54 (m, 1H), 2.79-2.75 (m, 1H), 2.65-2.61 (m, 1H), 2.31-2.22 (m, 4H), 2.21-2.19 (m, 1H), 1.44 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO): δ 178.6, 175.5, 174.1, 154.5, 141.8, 132.7, 131.5, 129.6, 129.0, 127.5, 124.8, 123.9, 122.2, 119.5, 79.6, 72.3, 58.1, 50.9, 49.1, 46.7, 28.5, 16.7, 14.6; IR (thin film) 3229, 2973, 2360, 2340, 1714, 1696, 1391; HRMS (ESI): m/z calculated for C₂₈H₃₀N₄O₅Na [M+Na]⁺ 525.2114, found 525.2108. HPLC purity: 99.18%, t_R=20.85 min.

(±)-tert-Butyl (3R,3a′R,3b′S,9a′S)-6,7-dimethyl-1′,2,3′-trioxo-2′-phenyl -2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4] pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (23)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6,7-dimethylindoline-2,3-dione (0.38 g, 2.2 mmol, 1.0 equiv) was used and the product was purified by flash chromatography with EtOAc/hex to afford the pyrrolidine 23 as a white solid (0.60 g, 1.20 mmol, 61%): R_f=0.3 (1:1 EtOAc/hex); mp 215–217; ¹H NMR (500 MHz, [D₆]DMSO): δ 10.6 (s, 1H), 7.53-7.52 (m, 2H), 7.50-7.44 (m, 1H), 7.29-7.28 (m, 2H), 6.77 (d, J=7.5 Hz, 1H), 6.60 (d, J=7.5 Hz, 1H), 4.29 (br s, 1H), 3.82-3.79 (m, 2H), 3.64–3.65 (m, 1H), 3.47-3.46 (m, 1H), 2.62 (br s, 2H), 2.19-2.17 (m, 4H), 2.09-2.07 (m, 4H), 1.39 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 320 K): δ 178.8, 175.5, 174.1, 141.8, 138.7, 132.7, 129.6, 129.0, 127.4, 123.6, 123.5, 122.4, 118.2, 79.6, 72.4, 58.1, 50.9, 46.7, 28.5, 20.1, 13.5; IR (thin film) 3739, 3244, 2976, 1712, 1696, 1417, 1390; HRMS (ESI): m/z calculated for C₂₉H₃₂N₄O₅Na [M+Na]⁺ 539.2271, found 539.2268. HPLC purity: 98.24%, t_R=21.47 min.

Synthesis of (+)-(3S,3a′S,7′R,8a′S,8b′R)-6-chloro-7-methyl-1′,2,3′-tri oxo-2′-phenyl-2′,3′,3a′,6′,7′,8′,8a′,8b′-octahydro-1′H-spiro[indoline-3,4′-pyrrolo[3,4-a]pyrrolizin]-7′-yl benzoate (25a) and (−)-(3R,3a′R,7′R, 8a′R,8b′S)-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,6′,7′,8′, 8a′,8b′-octahydro-1′H-spiro[indoline-3,4′-pyrrolo[3,4-a]pyrrolizin]-7′-yl benzoate (25b)

Using the general procedure for the synthesis of pyrrolidines outlined above, 6-chloro-7-methylindoline-2,3-dione (0.50 g, 2.6 mmol, 1.0 equiv) was used. The resulting crude reaction mixture was dissolved in cold DCM and the products precipitated as a yellow solid. Then the solid was collected by vacuum filtration and washed with cold DCM until the solid became white. The pyrrolidine products were collected as a mixture of two diastereomers. The resulting products were highly insoluble in most of the organic solvents and this made it very hard to separate the two diastereomers by flash column chromatography.

The mixture of two stereoisomers (0.030 g, 0.07 mmol, 1 equiv) from the above procedure was added to a 5 mL oven-dried round-bottom flask. The flask was fitted with a septum and purged with nitrogen. Next, 1.4 mL of DMF was added to the reaction flask such that the concentration of the mixture of two diastereomers was 0.05 M. Next, benzoyl chloride (9.0 μL, 0.075 mmol, 1.1 equiv) and triethylamine (0.01 mL, 0.08 mmol, 1.2 equiv) was added to the reaction flask and the reaction was allowed to stir at room temperature until the consumption of the alcohols confirmed by TLC (9:1 DCM/MeOH).Upon consumption of the alcohols, the reaction mixture was concentrated under vacuum to give a yellow solid. The solid was then purified by flash chromatography with DCM/Et₂O (20:1) to afford esters 25a and 25b.

The first stereoisomer 25a (0.012 g, 0.02 mmol, 65%) was isolated as a white solid. R_f=0.55 (20:1 DCM/Et₂O); mp 236–238; [α]_D²⁰=54.5 (c=0.13 in MeOH); ¹H NMR (600 MHz, [D₆]Acetone): δ 9.67 (s, 1H), 7.97 (dd, J=8.4, 1.2 Hz, 2H), 7.61-7.58 (m, 1H), 7.51 (d, J=7.8 Hz, 1H), 7.48-7.37 (m, 5H), 7.28 (d, J=7.8 Hz, 2H), 7.16 (d, J=7.8 Hz, 1H), 5.63-5.61 (m, 1H), 4.71-4.69 (m, 1H), 4.35 (d, J=9.6 Hz, 1H), 3.82 (dd, J=10.2, 6.6 Hz, 1H), 3.52 (dd, J=11.1, 5.1 Hz, 1H), 3.01 (d, J=10.8 Hz, 1H), 2.76-2.72 (m, 1H), 2.56-2.52 (m, 1H), 2.33 (s, 3H); ¹³C NMR (125 MHz, [D₆]Acetone): δ 177.4, 176.6, 174.9, 165.6, 135.6, 133.0, 130.2, 129.3, 128.6, 128.4, 128.1, 127.2, 125.6, 124.4, 122.4, 118.1, 74.7, 65.1, 55.3, 53.4, 53.1, 37.7, 13.3; IR (thin film) 3685, 2978, 2851, 1721, 1054, 1032, 1012; HRMS (ESI): m/z calculated for C₃₀H₂₄ClN₃O₅Na [M+Na]⁺ 564.1302, found 564.1302. HPLC purity: 96.63%, t_R=21.95 min.

The second stereoisomer 25b (0.015 g, 0.03 mmol, 81%) was also isolated as a white solid. R_f=0.48 (20:1 DCM/Et₂O); mp 221–223; [α]_D²⁰= −80.2 (c=0.23 in MeOH); ¹H NMR (600 MHz, [D₆]Acetone): δ 9.69 (s, 1H), 8.09 (dd, J=8.4, 1.2 Hz, 2H), 7.67 (t, J=7.5 Hz, 1H), 7.54 (t, J=7.8 Hz, 1H), 7.48-7.37 (m, 3H), 7.28 (d, J=7.8 Hz, 2H), 7.13 (d, J=8.4 Hz, 1H), 5.44-5.42 (m, 1H), 4.55-4.52 (m, 1H), 4.39 (d, J=10.2 Hz, 1H), 4.00 (dd, J=9.9, 6.9 Hz, 1H), 3.38 (dd, J=10.2, 6.0 Hz, 1H), 3.28 (dd, J=9.9, 6.3 Hz, 1H), 2.83-2.78 (m, 1H), 2.39-2.36 (m, 1H), 2.35 (s, 3H); ¹³C NMR (125 MHz, [D₆]Acetone): δ 177.4, 176.5, 174.8, 165.6, 135.6, 133.3, 133.0, 130.1, 129.5, 128.6, 128.2, 127.3, 125.5, 125.2, 124.6, 122.4, 74.7, 64.9, 54.4, 53.5, 53.3, 36.6, 13.3; IR (thin film) 3686, 2980, 2481, 1720, 1050, 1030, 1014; HRMS (ESI): m/z calculated for C₃₀H₂₄ClN₃O₅Na [M+Na]⁺ 564.1302, found 564.1305. HPLC purity: 95.36%, t_R=21.68 min.

Synthesis of (±)-tert-butyl (3R,3a′R,3b′S,9a′S)-1-benzyl-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro [indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxy late (17):^[53]

A 5 mL oven-dried round-bottom flask was charged with sodium hydride (60 wt % in mineral oil, 0.01 g, 0.37 mmol, 1.1 equiv) and the mineral oil dispersed in sodium hydride was removed by washing with hexanes (3 × 2 mL). The resulting white solid of sodium hydride was suspended in 0.2 mL of DMF. The suspension was cooled to 0 °C in an ice bath, after which pyrrolidines 14 (0.18 g, 0.34 mmol, 1.0 equiv) was added as a solid over the course of 15 min. After the addition was complete the ice bath was removed and the solution was stirred for 1 h at room temperature. Next, benzyl bromide (48 μL, 0.40 mmol, 1.2 equiv) was added to the reaction flask and the reaction was allowed to stir at room temperature until the consumption of 15 confirmed by TLC (2:3 EtOAc/hex). Upon consumption of the 14, the reaction mixture was concentrated under vacuum to give a yellow solid. The solid was then purified by flash chromatography with EtOAc/hex (4:1) to afford the pyrrolidine 17, a white solid (0.17 g, 0.27 mmol, 82%): R_f=0.65 (2:3 EtOAc/hex); mp 145–146; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 7.51-7.48 (m, 2H), 7.42-7.41 (m, 1H), 7.34-7.31 (m, 4H), 7.26-7.24 (m, 1H), 7.18-7.17 (m, 2H), 7.13 (d, J=8.1 Hz, 1H), 6.86 (d, J=8.1 Hz, 1H), 5.21-5.16 (m, 2H), 4.37-4.35 (m, 1H), 3.87-3.86 (m, 2H), 3.74-3.72 (m, 1H), 3.60 (d, J=8.1 Hz, 1H), 2.80-2.78 (m, 1H), 2.67-2.65 (m, 1H), 2.34-2.32 (m, 1H), 2.27 (s, 3H), 2.19-2.17 (m, 1H), 1.42 (s, 9H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): δ 177.6, 175.2, 173.8, 154.2, 143.4, 137.8, 136.6, 132.6, 129.6, 129.5, 129.0, 127.7, 127.5, 125.8, 125.1, 124.5, 124.0, 118.6, 79.7, 71.0, 63.9, 58.6, 51.9, 46.5, 45.8, 45.1, 30.7, 28.5, 14.7; IR (thin film) 3680, 2971, 2843, 1713, 1054, 1032, 1012; HRMS (ESI): m/z calculated for C₃₅H₃₅ClN₄O₅Na [M+Na]⁺ 649.2194, found 649.2194. HPLC purity: 100%, t_R=23.13 min.

Synthesis of (±)-(3R,3a′R,3b′S,9a′S)-6-chloro-7-methyl-2′-phenyl-3a′,4′,5′,6′,7′,9a′-hexahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyro lo[1,2-a]pyrazine]-1′,2,3′(2′H,3b′H)-trione (26):^[80]

A 10 mL round bottom flask was charged with pyrrolidine 14 (0.57 g, 1.1 mmol, 1 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Next, 3.1 mL CH₂Cl₂ was added to the reaction flask such that the concentration of 14 was 0.34 M and stirred for 10 min. A pink color solution resulted. Then 3.1 mL trifluoroacetic acid was slowly added to the reaction mixture by syringe. A dark brown color solution resulted. The deprotection reaction was stirred until TLC indicated complete consumption of the pyrrolidine 14, 15 min. Upon consumption of the 14, the reaction mixture was concentrated under vacuum to give a brown oil. 5 mL of saturated aqueous NaHCO₃ was added to the oil and the aqueous layer was then extracted with (3 × 5 mL) CH₂Cl₂. The combined organic layers were then washed with brine and dried over Na₂SO₄. The resulting organic solution was concentrated under vacuum to afford 26, a pink solid, (0.42 g, 0.96 mmol, 87%): R_f=0.81 (20:80:5 EtOAc/hex/Et₃N); mp 196–199; ¹H NMR (600 MHz, [D₆]DMSO): δ 10.89 (s, 1H), 7.58-7.53 (m, 2H), 7.47-7.45 (m, 1H), 7.30 (d, J=7.4 Hz, 2H), 7.06 (d, J=8.0 Hz, 1H), 6.71 (d, J=8.0 Hz, 1H), 3.77-3.76 (m, 2H), 3.51-3.50 (m, 1H), 3.42 (br s, 1H), 3.30 (d, J=11.9 Hz, 1H), 2.79 (app d, J=12.1 Hz, 1H), 2.43-2.41 (m, 1H), 2.25 (s, 3H), 2.24-2.21 (m, 2H); ¹³C NMR (125 MHz, CDCl₃, 310 K): δ 178.8, 175.5, 174.2, 143.4, 134.9, 132.7, 129.6, 128.9, 127.4, 125.2, 124.1, 122.5, 117.7, 72.6, 58.7, 50.7, 48.7, 47.1, 46.7, 44.9, 14.3; IR (thin film) 3850, 3671, 2971, 2864, 1709, 1054, 1032, 1013; HRMS (ESI): m/z calculated for C₂₃H₂₁ClN₄O₃Na [M+Na]⁺ 459.1200, found 459.1190. HPLC purity: 100%, t_R=17.25 min.

Synthesis of (±)-methyl 10-((3R,3a′R,3b′S,9a′S)-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro [indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazin]-5′(1′H)-yl)-10-oxodecanoate (27):^[81]

A 15 mL round bottom flask was charged with pyrrolidine 26 (0.13 g, 0.30 mmol, 1 equiv), Na₂CO₃ (0.03 g, 0.30 mmol, 1.0 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Then 6 mL of CH₂Cl₂ was added to the reaction flask such that the concentration of 26 was 0.05 M. Next, methyl 10-chloro-10-oxodecanoate (0.07 mL, 0.31 mmol, 1.04 equiv) was added drop-wise to the reaction mixture by syringe. The reaction was allowed to stir at room temperature until the consumption of 26 confirmed by TLC (20:80:5 EtOAc/hex/Et₃N). Upon consumption of 26, the reaction mixture was concentrated under vacuum to give a yellow oil. The oil was then purified by flash chromatography with EtOAc/hex/Et₃N (80:20:5) to afford the pyrrolidine 27, a yellow solid, (0.10 g, 0.16 mmol, 53%): R_f=0.75 (20:80:5 EtOAc/hex/Et₃N); mp 110–111; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.42 (s, 1H), 7.53-7.51 (m, 2H), 7.45-7.43 (m, 1H), 7.33 (d, J=7.8 Hz, 2H), 7.03 (d, J=8.4 Hz, 1H), 6.78 (d, J=7.8 Hz, 1H), 4.58 (br s, 1H), 4.08 (br s, 1H), 3.86 (t, J=7.8 Hz, 1H), 3.73-3.72 (m, 1H), 3.61-3.58 (m, 4H), 2.61-2.82 (m, 4H), 2.34-2.27 (m, 5H), 2.22-2.18 (m, 1H), 1.59-1.53 (m, 4H), 1.22-1.42 (m, 9H); ¹³C NMR (125 MHz, CDCl₃, 313 K): δ 178.2, 174.6, 174.3, 172.2, 171.7, 141.8, 136.6, 131.7, 129.4, 128.9, 126.3, 124.4, 123.5, 122.3, 118.7, 72.7, 58.7, 51.4, 50.4, 48.7, 46.2, 45.3, 44.5, 41.1, 34.1, 33.4, 29.7, 29.3, 29.2, 25.3, 24.9, 13.6; IR (thin film) 3817, 3671, 2921, 2863, 1710, 1617, 1054, 1032; HRMS (ESI): m/z calculated for C₃₄H₃₉ClN₄O₆Na [M+Na]⁺ 657.2456, found 657.2435. HPLC purity: 97.45%, t_R=23.69 min.

Synthesis of (±)-(3R,3a′R,3b′S,9a′S)-6-chloro-5′-((S)-2-methoxy-2-phenylacetyl)-7-methyl-2′-phenyl-3a′,4′,5′,6′,7′,9a′-hexahydrospiro [indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-1′,2,3′(2′H, 3b′H)-trione (28):^[82]

A 15 mL round bottom flask was charged with pyrrolidine 26 (0.20 g, 0.46 mmol, 1.0 equiv), (S)-2-methoxy-2-phenylacetic acid (0.10 g, 0.60 mmol, 1.3 equiv), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (0.18 g, 0.96 mmol, 2.1 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Then 6 mL of CH₂Cl₂ was added to the reaction flask such that the concentration of 26 was 0.07 M. Next triethylamine (0.22 mL, 1.6 mmol, 3.5 equiv) was added to the reaction mixture and the reaction was allowed to stir at room temperature until the consumption of 26 confirmed by TLC (20:80:5 EtOAc/hex/Et₃N). Upon consumption of the 26, the reaction mixture was quenched with 5 mL of 1 N aq. HCl and the aqueous layer was then extracted with (3 × 5 mL) CH₂Cl₂. The combined organic layers were then washed with brine and dried over Na₂SO₄. The resulting organic solution was concentrated under vacuum to give a yellow oil. The oil was then purified by flash chromatography with EtOAc/hex/Et₃N (80:20:5) to afford the pyrrolidine 28, a white solid, (0.12 g, 0.21 mmol, 45%): R_f=0.65 (1:1 EtOAc/hex); mp 127–129; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.42 (s, 1H), 7.51-7.49 (m, 2H), 7.44-7.41 (m, 1H), 7.39-7.35 (m, 4H), 7.32-7.31 (m, 3H), 7.01 (d, J=8.0 Hz, 1H), 6.74 (d, J=8.0 Hz, 1H), 5.17-5.16 (m, 1H), 4.71-4.70 (m, 1H), 4.19-4.17 (m, 1H), 3.79-3.75 (m, 1H), 3.64-3.60 (m, 1H), 3.54 (app dd, J=8.0, 2.9 Hz, 1H), 3.39-3.37 (m, 3H), 2.73-2.64 (m, 2H), 2.27-2.22 (m, 4H), 2.07-2.05 (m, 1H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): (2 rotamers observed) δ 178.5, 175.3, 173.9, 154.9, 137.4, 135.2, 132.6, 129.5, 129.0, 128.9, 128.6, 127.6, 127.5, 127.3, 125.2, 123.6, 122.6, 117.8, 81.8, 81.0, 72.1, 68.9, 63.9, 58.6, 58.2, 57.3, 56.3, 50.9, 46.5, 45.3, 44.5, 41.7, 32.5, 30.6, 30.1, 21.8, 19.0, 17.1; IR (thin film) 3850, 3708, 2971, 2921, 1712, 1643, 1054, 1032, 1012; HRMS (ESI): m/z calculated for C₃₂H₂₉ClN₄O₅Na [M+Na]⁺ 607.1724, found 607.1736. HPLC purity: 97.04%, t_R=20.02 min.

Synthesis of (±)-(3R,3a′R,3b′S,9a′S)-6-chloro-7-methyl-2′-phenyl-5′-(3-phenylpropanoyl)-3a′,4′,5′,6′,7′,9a′-hexahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-1′,2,3′(2′H,3b′H)-trione (29)

A 10 mL round bottom flask was charged with pyrrolidine 26 (0.05 g, 0.11 mmol, 1.0 equiv), triethylamine (18.0 μL, 0.13 mmol, 1.1 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Then 2.0 mL of CH₂Cl₂ was added to the round bottom flask by syringe. Then hydrocinnamoyl chloride (18.0 μL, 0.12 mmol, 1.05 equiv) was added to the reaction flask with a syringe. The reaction mixture was allowed to stir for 3 h at room temperature. The reaction was monitored for consumption of 26 by TLC (100:5 EtOAc/Et₃N). Upon consumption of 26, the reaction mixture was quenched with 5 mL of saturated aqueous NaHCO₃ and the aqueous layer was then extracted with (3 × 5 mL) CH₂Cl₂. The combined organic layers were then washed with brine and dried over Na₂SO₄. The resulting organic solution was concentrated under vacuum to give a yellow oil. The oil was then purified by flash chromatography with EtOAc/hex/Et₃N (60:40:5) to afford the pyrrolidine 29, a white solid, (0.04 g, 0.07 mmol, 64%): R_f=0.2 (60:40:5 EtOAc/hex/Et₃N); mp 236–239; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.40 (s, 1H), 7.53-7.51 (m, 2H), 7.45-7.42 (m, 1H), 7.34-7.33 (m, 2H), 7.27-7.23 (m, 4H), 7.17-7.15 (m, 1H), 7.03 (d, J=7.8 Hz, 1H), 6.78 (d, J=8.4 Hz, 1H), 4.59 (s, 1H), 7.09-7.06 (m, 1H), 3.87-3.84 (m, 1H), 3.73-3.69 (m, 1H), 3.59-3.57 (m, 1H), 2.90-2.88 (m, 2H), 2.84-2.82 (m, 2H), 2.78-2.74 (m, 2H), 2.31-2.28 (m, 4H), 2.19-2.15 (m, 1H); ¹³C NMR (125 MHz, [D₆]DMSO, 310 K): δ 178.6, 178.5, 175.4, 175.3, 174.0, 170.6, 143.4, 141.9, 135.2, 132.7, 129.6, 129.0, 128.8, 128.7, 127.5, 126.3, 125.2, 123.9, 122.6, 117.9, 72.2, 60.2, 58.9, 51.2, 48.5, 45.5, 44.9, 34.5, 31.2, 14.3; IR (thin film) 3423, 2917, 1708, 1633, 1620, 1384, 1204; HRMS (ESI): m/z calculated for C₃₂H₂₉ClN₄O₄Na [M+Na]⁺ 591.1775, found 591.1763. HPLC purity: 98.20%, t_R=21.15 min.

Synthesis of (3R,3a′R,3b′S,9a′S)-N-benzyl-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxamide (30)

A 10 mL round bottom flask was charged with pyrrolidine 26 (0.06 g, 0.15 mmol, 1.0 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Then 3.0 mL of CH₂Cl₂ was added to the round bottom flask by syringe. Next, (isocyanatomethyl)benzene (0.02 mL, 0.16 mmol, 1.1 equiv) was added to the reaction flask by syringe. Then the reaction mixture was allowed to stir for 4 h at room temperature. The reaction was monitored for consumption of 26 by TLC (50:50:5 EtOAc/Hex/Et₃N). Upon consumption of 26, the resulting organic solution was concentrated under vacuum to give a yellow oil. The oil was then purified by flash chromatography with EtOAc/hex/Et₃N (60:40:5) to afford the pyrrolidine 30, a white solid, (0.63 g, 0.11 mmol, 75%): R_f=0.24 (50:50:5 EtOAc/Hex/Et₃N); mp 258-259; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.44 (s, 1H), 7.53-7.51 (m, 2H), 7.45-7.44 (m, 1H), 7.33-7.32 (m, 2H), 7.29-7.28 (m, 4H), 7.20 (m, 1H), 7.03 (d, J=8.0 Hz, 1H), 6.76 (d, J=8.0 Hz, 1H), 4.47-4.45 (m, 1H), 4.29-4.27 (m, 2H), 3.95 (d, J=14.1 Hz, 1H), 3.82-3.77 (m, 2H), 3.56 (d, J=7.9 Hz, 1H), 2.75-2.71 (m, 1H), 2.64-2.58 (m, 1H), 2.28 (s, 3H), 2.25-2.23 (m, 2H); ¹³C NMR (125 MHz, [D₆]DMSO): δ 178.7, 175.4, 174.1, 157.6, 143.4, 141.4, 135.1, 132.7, 129.6, 129.1, 128.6, 127.5, 126.9, 125.1, 123.9, 122.6, 117.9, 72.3, 58.3, 51.1, 47.5, 46.7, 45.7, 44.1, 43.6 ; IR (thin film) 3618, 2922, 2360, 2340, 1712, 1616, 1536, 1387; HRMS (ESI): m/z calculated for C₃₁H₂₈ClN₅O₄Na [M+Na]⁺ 592.1727, found 592.1724. HPLC purity: 97.18%, t_R=19.94 min.

Synthesis of (±)-benzyl (3R,3a′R,3b′S,9a′S)-6-chloro-7-methyl-1′,2,3′-trioxo-2′-phenyl-2′,3′,3a′,3b′,4′,6′,7′,9a′-octahydrospiro[indoline-3,9′-pyrrolo[3′,4′:3,4]pyrrolo[1,2-a]pyrazine]-5′(1′H)-carboxylate (31):^[83]

A 5 mL round bottom flask was charged with pyrrolidine 26 (0.20 g, 0.45 mmol, 1.0 equiv), N,N-Diisopropylethylamine (0.17 mL, 0.96 mmol, 2.1 equiv) and a stir bar. The flask was fitted with a septum and purged with nitrogen. Then 0.92 mL of CH₂Cl₂ was added to the round bottom flask by syringe. The reaction mixture was cooled to 0 °C with an ice bath. Meanwhile another 5 mL pear-shaped flask was charged with benzyl chloroformate (0.07 mL, 0.50 mmol, 1.1 equiv) and the flask was fitted with a septum and purged with nitrogen. Then 0.34 mL of CH₂Cl₂ was added to the pear shaped vial containing the benzyl chloroformate and stirred for 10 min. Next the contents of the 5 mL pear shaped vial was slowly added to the 5 mL round bottom flask containing 26. Ice bath was removed and the reaction mixture was allowed to stir for 2.5 h at room temperature. The reaction was monitored for consumption of 26 by TLC (50:50:5 EtOAc/Hex/Et₃N). Upon consumption of 26, the reaction mixture was quenched with 5 mL of saturated aqueous NaHCO₃ and the aqueous layer was then extracted with (3 × 5 mL) CH₂Cl₂. The combined organic layers were then washed with brine and dried over Na₂SO₄. The resulting organic solution was concentrated under vacuum to give a yellow oil. The oil was then purified by flash chromatography with EtOAc/hex/Et₃N (60:40:5) to afford the pyrrolidine 31, a white solid, (0.14 g, 0.25 mmol, 54%): R_f=0.81 (1:1 EtOAc/hex); mp 209–211; ¹H NMR (600 MHz, [D₆]DMSO, 400 K): δ 10.40 (s, 1H), 7.53-7.51 (m, 2H), 7.45-7.42 (m, 1H), 7.37-7.35 (m, 6H), 7.32-7.31 (m, 1H), 7.03 (d, J=8.0 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 5.16 (s, 2H), 4.48 (d, J=12.7 Hz, 1H), 3.99 (d, J=13.1 Hz, 1H), 3.89-3.87 (m, 1H), 3.82-3.79 (m, 1H), 3.61 (d, J=8.0 Hz, 1H), 2.91-2.86 (m, 2H), 2.78-2.75 (m, 1H), 2.34-2.25 (m, 5H); ¹³C NMR (125 MHz, [D₆]DMSO): δ 178.5, 175.3, 173.9, 154.9, 143.4, 137.3, 135.2, 132.6, 129.5, 129.0, 128.9, 128.3, 128.1, 127.4, 72.3, 66.9, 58.2, 51.0, 47.2, 43.5, 30.9, 21.1, 14.3 ; IR (thin film) 3850, 3678, 3262, 2966, 2864, 1715, 1694, 1032; HRMS (ESI): m/z calculated for C₃₁H₂₇ClN₄O₅Na [M+Na]⁺ 593.1567, found 593.1584. HPLC purity: 97.60%, t_R=21.96 min.

Biological Evaluation

Strains, media, and compounds

The C. albicans strains HLY4123 was used as the susceptible laboratory strain for the antifungal evaluation in this study. HLY4123 carries a GFP reporter for ERG3 expression and was constructed by plasmid transformation of the commonly used laboratory C. albicans strain CAI4. Selected resistant C. albicans strains with different mechanisms of becoming drug resistance were obtained from Dr. David Rogers.^{Error! Bookmark not defined.} The strains were cultured at 30 °C under constant shaking (200 rpm) in synthetic complete (SC) medium containing 2% glucose. The stock solution of fluconazole (Sigma-Aldrich, USA) was prepared in sterile water (0.1 mg/mL), whereas the other test compounds were prepared in DMSO. The commercial sample sold as CID 6584729 was obtained from Vitas-M (supplier number STK 580951).

Dose-Response Curves for Test Compounds Against C. albicans with and without Fluconazole: C. albicans was grown in SC medium overnight and then diluted to an effective OD600 of 0.0625. Serial ten-fold dilutions of the test compounds (0.15–1500 μM) were prepared in DMSO in 1.5 mL Eppendorf tubes. To each well in columns B-D (triplicate analysis) of a 24-well Palcon plate was added 2.5 μL of fluconazole solution. To each well in all four columns of the plate was added 1 mL of cells in SC medium such that the column A served as a control to assess the EC₅₀ of the compound in the absence of fluconazole. Then to each well in rows 2–5 was added a solution of the compound in DMSO (2 μL each) such that the final solution fluconazole in columns 2–4 was 0.25 μg/mL and the concentration of compound in each row varied from 0.003 μM to 30 μM. The plates were incubated in a rotary shaker/incubator at 30 °C for 16 h. The contents of each well were re-suspended with a micropipettor and a 20 μL aliquot was added to a polystyrene cuvette and diluted with 680 μL of deionized water. The suspension was triturated again immediately before measuring the absorbance at 600 nm (OD₆₀₀) for cell densities. EC₅₀ values were determined by fitting to a standard curve using the Excel-based tool ED50PLUS v1.0 (Mario H. Vargas).

Determination of FIC₉₀s with a Checkerboard Assay: Checkerboard assays were carried out using four 24-well plates. The results on each plate were normalized by duplication of one row and one column with a row and column on another plate. C. albicans was grown in SC medium overnight and then diluted to an effective OD₆₀₀ of 0.0625. Serial ten-fold dilutions of the test compounds (150 mM) were prepared in DMSO in 1.5 mL Eppendorf tubes. Serial two-fold dilutions of the fluconazole (6.53 μM) were prepared in sterile water in 1.5 mL Eppendorf tubes. To each well was added 2 μL of a stock solution of compound in DMSO and 2.5 μL of a stock solution of fluconazole in water. Concentrations of compound in rows 1–11 varied from 300 μM to 0.0003 nM; the last row 12 contained no compound. Concentrations of fluconazole in each column B-H varied from 0.0625 to 2 μM; the first column A contained no fluconazole. The plates were incubated in a rotary shaker/incubator at 30 °C for 16 h. The contents of each well was resuspended with a micropipettor and a 20 μL aliquot was added to a polystyrene cuvette and diluted with 680 μL of deionized water. The suspension was triturated again immediately before measuring the absorbance at 600 nm. The MIC₉₀ values were determined as the lowest concentrations of the drugs (alone or in combination) that inhibited fungal growth by 90% compared with that of the drug-free wells.

Molecular Properties

Physicochemical properties were calculated from the SMILES representation of synazo-1 using the Molinspiration Property Calculation Service at www.molinspiration.com.