The first examples of chemical modification of oligomycin A

Abstract

The first examples of chemical modification of antibiotic oligomycin A are described. The interaction of oligomycin A with hydroxylamine yielded six-membered nitrone annelated with the antibiotic at the positions 3,4,5,6,7. The reaction with 1-aminopyridinium iodide in pyridine led to pyrazolo[1,5-a]pyridine conjugated with the antibiotic at the positions 2 and 3 (product of addition to the C₂–C₃ double bond followed by spontaneous oxidation). The structures of the compounds obtained were supported by NMR and mass spectrometry methods including the ¹⁵N-labeling of compounds.

Introduction

Oligomycins are cytotoxic macrolides that contain a 26-membered α,β-unsaturated lactone with a conjugated diene fused to a bicyclic spiroketal ring system. These compounds inhibit oxidative phosphorylation in mitochondria by preventing ATP synthesis. Their mode of action involves the decoupling of the F₀ and F₁ factors of mitochondrial ATPase responsible for facilitating proton transfer through the inner mitochondrial membrane.^{1, 2} The enzymatic complex F₀F₁ ATP synthase can be considered as a target for antitumor or anti-infection therapy.^{3, 4} Oligomycins display a variety of significant biological activities; in addition to the specific inhibition of mitochondrial ATPase, strong anti-actinobacterial,⁴ antifungal effects⁵ and antitumor^{1, 6} actions have been recorded. Oligomycins are among the topmost cell line selective agents; they block P-glycoprotein activity and trigger apoptosis in doxorubicin-resistant HepG2 cells.⁷

The oligomycin antibiotic complex was initially isolated in 1954 from a strain of Streptomyces diastatochromogenes.² The complex consists of variable proportions of three major components A, B and C-oligomycins depending on the strain and culture conditions.⁵ Later, from various Streptomyces strains, oligomycins D, T, F and others were isolated and identified.^{6, 8, 9, 10} The structures and absolute configuration of oligomycins A and C were established by chemical correlation of their individual degradation products with those derived from rutamycin;^{11, 12} however, no data on chemically modified derivatives of oligomycins have been published until now.

Results and discussion

Interaction of oligomycin A with hydroxylamine and 1-aminopyridine

Availability of oligomycin A in substantial quantities by fermentation provides the basis for the expedient investigation of its mode of action and improvement of its therapeutic potential through direct chemical modification. To this end, we report the first examples of chemical modification of oligomycin A. The strain producer of the antibiotic S. avermitilis NICB62 was used for biosynthesis and isolation of oligomycin A.

With the aim of exploring the reactivity of functional groups of oligomycin A that might open the way to the preparation of more selective antitumor or anti-infective oligomycin A derivatives, we first studied the interaction of the antibiotic with carbonyl-specific reagents. By the interaction of the antibiotic with hydroxylamine hydrochloride or 1-aminopyridinium iodide in pyridine, we managed to isolate novel derivatives 2 and 3a, respectively. The structures of novel compounds 2 and 3a were determined on the basis of HR-electrospray ionization (ESI)-MS data and with the use of 1D (¹H and ¹³C) and 2D NMR correlation spectra (¹H-¹H ¹COSY and ¹H-¹³C HETCOR). Spectral parameters of these compounds were compared with those of oligomycin A obtained in this study and with literature data (Table 1).^{8, 13, 14}

Table 1 ¹³C and ¹H NMR spectra of compounds 1, 2 and 3a (δ_C, δ_H p.p.m., J Hz) in CDCl₃^a

To elucidate the structures of novel derivatives, their ¹⁵N-labeled analogs ¹⁵N-2 and ¹⁵N-3 were synthesized with the use of ¹⁵N-hydroxyl amine or 1-¹⁵N-aminopyridine, respectively. For the ¹⁵N-labeled compounds, 1-D (¹H and ¹³C) spectra were measured and the coupling constants J_15N,1H and J_15N,13C were determined (Table 2). We showed in this study that compound 2 was a six-membered nitrone (2,3,4,5-tetrahydropyridine N-oxide) annelated with the macrolide cycle (Scheme 1), and that compound 3a was pyrazolo[1,5-a]pyridine annelated with the oligomycin A framework (Scheme 3). Similarly, the interaction of oligomycin A with 1-amino-4-methylpyridine yielded the analogous structure (3b).

Table 2 Spin coupling values J_{15N, 13C} and J_{15N, 1H} (Hz) in ¹³C and ¹H NMR spectra of ¹⁵N-2 and ¹⁵N-3a in CDCl₃

Scheme 1

Interaction of oligomycin A with hydroxylamine.

Elucidation of the structure of nitrone annelated with oligomycin A

Cyclic nitrones result from the intramolecular 1,3-azaprotio cyclotransfer reaction of the oxime group with the olefinic bond. The reaction requires the presence of the terminal olefinic electron-withdrawing ester group CO₂R. Also, the product(s) of reaction were shown to depend on the space-filling capacity of substituents.^{15, 16} For certain oximes, the two modes of reaction illustrated in Scheme 2 are in competition, and the preferred reaction path in any given case will be that of lowest energy.¹⁷

Scheme 2

Dipole formation from oximes; (a) intramolecular oxime–olefin cycloaddition and (b) intramolecular 1,3-azaprotio cyclotransfer reaction (APT).

In our case paths, (a) and (b) would lead to structures 4 or 2, respectively, with the same molecular formula C₄₄H₇₅NO₁₁ determined by HR-ESI-MS.

The detailed NMR investigation of compound 2 and its ¹⁵N-labeled derivative was performed to elucidate the structure of this compound and to select between nitrone 2 and the otherwise possible 1,2-oxazepin 4. In our NMR study of compound 2, we followed the approach that was described by Laatsch et al.⁸ and consisted in the study of the structural fragments of the antibiotic. There are four structural fragments in oligomycin A and its novel derivatives 2 and 3, which are characterized by relatively strong spin interactions between the geminal and vicinal hydrogen atoms F1 (C2–C6), F2 (C8–C10), F3 (C13–C26) and F4 (C28–C34).

The comparison of chemical shifts of ¹H and ¹³C atoms in segments F3 (C13–C26) and F4 (C28–C34) in compound 2 and oligomycin A shows that the differences in these segments are connected only with the differences in mutual dispositions of the atoms but not with the changes in atom hybridization or changes in the number of atoms. This conclusion is supported by rather close values of δC for the majority of carbon atoms of the structural segments F3 and F4 of 1 and 2 (differences ⩽1.5 p.p.m.). The exclusions are presented by C₂₀ (Δδ=2.4 p.p.m.), C₂₁ (Δδ=5.4 p.p.m.) and C₂₂ (Δδ=5.0 p.p.m.); however, even in these cases, the type of hybridization and the number of C–H and C–C bonds in compounds 1 and 2 are similar. It is also true of parameters of the ¹H NMR spectra of compounds 1 and 2. Contrary to the segments F3 and F4, the parameters of the ¹H and ¹³C NMR spectra of segments F1 and F2 in compounds 1 and 2 are substantially different. First of all, these differences show the presence of the C=N bond and the singular C2–C3 bond in compound 2 instead of C=O and double C2=C3 bond in compound 1. The elucidation of the structure of 2,3,4,5-tetrahydropyridine N-oxide annelated with the macrolide cycle at the positions 3,4,5,6,7 was also based on the comparison of the ¹H and ¹³C NMR spectra of compounds 2 and ¹⁵N-2 (Table 2). The value of ³J_{15N, 1H-6}=4.3 Hz supports the bond C₇=N and permitted us to exclude any structure with the C₁₁=N bond. The annelated 1,2-oxazepine structure that might be formed at the interaction of an unsaturated carbonyl compound with hydroxylamine is in contradiction with the values of constants of spin interactions ³J_{15N, 1HA-2}=4.0 Hz and ³J_{15N, 1HB-2}=2.3 Hz. For the same reason, we excluded from our consideration other cyclic structures with the N–O bond or acyclic structures with N–OH moiety; the latter must be excluded because of the presence of spin interaction between ¹⁵N=C-7 and C-3 (¹J_15N, C-3=7.0 Hz). Structure 2 is also well supported by the high value of the constant ¹J_15N, C–7=19.8 Hz¹⁸ and the value of the constant ⁵J_H-3, H-6=2.0 Hz, which can be considered as an analog of homoallylic interaction.

The formation of a tetrahydropyridine cycle in compound 2 results in the formation of a new asymmetric atom at C-3. The high value of the constant ³J_{H-3, H-4}=10 Hz suggests that the atoms H-3 and H-4 are trans-oriented in six-membered nitrone and that the C3 atom has an S configuration (Figure 1).

Figure 1

Three-dimensional structure of compound 2.

Elucidation of the structure of pyrazolo[1,5-a]pyridine conjugated with oligomycin A

An unexpected product of the intermolecular cycloaddition to the C₂–C₃ double bond followed by simultaneous dehydrogenation (3a) was formed by the interaction of 1-aminopyridinium iodide with 1 in pyridine. The carbonyl groups did not participate in this reaction (Scheme 3).

Scheme 3

Interaction of oligomycin A with 1-aminopyridinium iodide in pyridine.

It has been shown previously that the 1,3-dipolar cycloaddition of polymer-bound alkynes to azomethine imines produced substituted pyrazolo[1,5-a]pyridine derivatives.¹⁹ However, we could not find the examples of the interaction of 1-aminopyridine with the double bond-containing compounds followed by spontaneous dehydrogenation. Still, there are examples of the interaction of 1-aminopyridine with double bond-containing compounds substituted at the double bond with groups that are split off in the reaction producing pyrazolo[1,5-a]pyridine derivatives.²⁰ Formation of pyrazolo[1,5-a]pyridine derivatives involves a regioselective [3+2] cycloaddition of 1-aminopyridine to alkenes or alkynes.

To help elucidate the structure of 3a, 1-¹⁵NH₂-pyridine was prepared from ¹⁵NH₂OH and pyridine, producing ¹⁵N-3a with 1 in pyridine. The NMR data for 1-¹⁵N-aminopyridine are presented in Table 3. 1-Amino-4-methylpyridine gave 5-methylpyrazolo[1,5-a]pyridine annelated with the macrolide (3b) by the interaction with 1 in similar conditions. The structures of 3a and 3b were supported by mass spectrometry and NMR spectroscopy. We failed to prepare a product of the interaction of 1-amino-2-methylpyridine with oligomycin A, supposedly because of steric hindrances.

Table 3 Chemical shifts δ_C and δ_H (p.p.m.) and spin coupling constants J_{15N, 13C} and J_{15N, 1H} (Hz) in ¹³C and ¹H NMR spectra of 1-¹⁵N-aminopyridinium iodide in DMSO-d₆

The molecular formulas of compounds 3a and 3b were found by HR-ESI-MS to be C₅₀H₇₆N₂O₁₁ and C₅₁H₇₈N₂O₁₁, respectively. The determination of the structure of compound 3a was similarly based on the comparison of hybridization and the number of hydrogen atoms connected with similar atoms in compounds 1 and 3a (Table 1). This comparison shows that serious differences are present only for atoms C2 and C3, which are both sp² hybridized in 3a and 1 but deprotonated in 3a. C1 and other carbon atoms of the fragment F1, excluding C2 and C3, retain their type of hybridization (sp² for C1, sp³ for C4, C5, C6) and the number of hydrogen atoms connected with them (0 for C1, 1 for each of C4, C5, C6). However, the dramatic differences between the spin coupling constants for similar hydrogen atoms in 1 and 3 show big differences in the steric structures of fragment F1 in oligomycin A and 3a. NMR spectral parameters of fragments F3 and F4 are close in compound 3a and oligomycin A. All carbon atoms of the 1-aminopyridine moiety in 3a retain the starting sp² type of hybridization, but in 3a one of the carbon atoms around 1-N is deprotonated, and the symmetry presented in the starting 1-aminopyridine is absent (atoms in positions 2/6 and 3/5 are not equivalent). The presence of two carbonyl groups in compound 3a is supported by the presence of ¹³C NMR signals at δ 217.76 and 215.93 p.p.m. In accordance with Ms data, it is suggested that compound 3a has a structure of pyrazolo[1,5-a]pyridine annelated at the C₂–C₃ bond with the macrolide cycle. The selection between the possible structures 3a and 5 was based on the investigation of NMR spectra of ¹⁵N-3a (Table 3). The interaction between ¹⁵N of the pyrazolopyridine moiety and C4-H of the macrolide cycle (³J_{15N, 4-H}=4.0 Hz) supports the structure of 3a as this coupling constant value is incompatible with the interaction through four bonds present in structure 5.

The low value of the ¹J_15N,13C constant is in agreement with literature data. In aza-arenes, the direct spin coupling constant ¹J_15N,13C is close to 0 Hz, being both positive or negative.¹⁸ Using the published data, we can compare this coupling constant with the analogous constant ¹J_15N,13C in oximes (C=N-O), which has a rather small absolute value (0.5–5 Hz).¹⁸

In conclusion, the product of the interaction of oligomycin A with 1-aminopyridinium iodide has the structure of pyrazolo[1,5-a]pyridine annelated with the macrolide at the C2–C3 bond. The product of the interaction of oligomycin A with 1-amino-4-methylpyridinium iodide is its C-methylated analog (3b). The significant differences in the NMR spectra of 3a and 3b can be registered only in the area of the signals of the pyridine nucleus (δ, p.p.m., J, Hz for hydrogen atoms of the pyridine cycle in compound 3b are the following: 3′-H: δ 7.73bs; 4′-CH₃: δ 2.43s; 5′-H: δ 6.73dd, J=7.0, 1.8; 6′-H: δ 8.29, J=7.0).

Cytotoxic, antibacterial and antifungal properties and structure-activity relationships for oligomycin A derivatives are under investigation. Preliminary studies have shown that compounds 2, 3a and 3b are less cytotoxic than the starting antibiotic. Although oligomycin A is highly active against Aspergillus niger (MIC=0.125 μg ml⁻¹) and has moderate activity against Cfndida albicans and Cryptococcus humicolus, compound 2 is practically devoid of antifungal and anti-yeast properties. Compound 3 is active against A. niger with MIC=2 μg ml⁻¹, and its anti-yeast properties are close to those of oligomycin A.

Methods

Experimental section

Oligomycin A (1), with a purity of 95%, was elaborated in the Scientific Research Centre for Biotechnology of Antibiotics BIOAN, Moscow, using selection strain-producer S. avermitilis NIC B62 with the level of oligomycin A production about 1 g l⁻¹. Fermentation was performed for 8 days at 28 °C in liquid medium. Isolation and purification were performed by extraction with acetone-hexane mixture followed by crystallization.

All reagents and solvents were purchased from Aldrich (St Louis, MO, USA), Fluka (St Louis, MO, USA) and Merck (Darmstsdt, Germany). All solutions were dried over sodium sulfate and evaporated under reduced pressure on a Buchi rotary evaporator at <35 °C (Buchi, Dietikon, Switzerland). The progress reaction products, column eluates and all final samples were analyzed by TLC. ¹⁵NH₂OH.HCl was obtained from Aldrich. ¹⁵N-1-aminopyridinium iodide was obtained by the method described from ¹⁵NH₂OSO₃H and pyridine.²¹ TLC was performed on Merck G60F₂₅₄ precoated plates. Reaction products were purified by column chromatography on Merck silica gel 60 (0.04–0.063 mm). The NMR spectra were recorded on a Unity+400 (Varian, Palo Alto, CA, USA) spectrometer at 400.0 MHz for nuclei ¹H, and at 100.6 MHz for nuclei ¹³C. The NMR elucidation was made with the use of homo ¹H, ¹H (2D COSY) and hetero ¹H, ¹³C (2D HETCOR) correlation as well as ¹H and ¹³C 1D spectra of the antibiotic, its derivatives and their ¹⁵N-labeled compounds. ESI mass spectra were recorded on a Finnigan MAT 900S spectrometer (Finnigan, Bremen, Germany). UV spectra were obtained on a UV/VIS double beam spectrometer UNICO in methanol. IR spectra were recorded on Thermo Nicolet iS10 (Thermo Scientific, Waltham, MA, USA). Optical rotation was measured on AA55 Polarimeter (Optical Activity, Huntingdon, UK).

Synthesis of oligomycin A annelated with 2,3,4,5-tetrahydropyridine-1-oxide (2)

To a solution of oligomycin A (100 mg, 0.13 mmol) in pyridine (4 ml) was added NH₂OH.HCl (80 mg, 1.1 mmol) and KOAc (10 mg, 0.1 mmol) and the reaction mixture was stirred at room temperature for 48 h. Then it was poured on ice, acidified by 1N HCl till pH 3, and the reaction product was extracted with EtOAc. The extract was washed with water and dried over Na₂SO₄. The reaction product was purified by column chromatography in CHCl₃-MeOH (25:1) to give 50 mg (53%) of compound 2 as a colorless amorphous solid with m.p. 142–148 °C. R_F 0.42 (CHCl₃/MeOH, 10:1). MW calculated for C₄₅H₇₅NO₁₁ 805.5340, observed in ESI mass spectrum 806.5330 (M+H)⁺. UV spectrum, λ_max (MeOH): 232 (ɛ 12.193). IR (KBr) (cm⁻¹): 1730, 1700, 1458, 1384, 988. [α]_D²⁰=+160° (C1, MeOH). ¹⁵N-2 was obtained similarly with the use of ¹⁵NH₂OH.HCl.

Synthesis of oligomycin A annelated with pyrazolo[1,5-a]pyridine (3a)

To a stirred solution of oligomycin A (100 mg, 0.13 mmol) in pyridine (4 ml) was added 1-aminopyridinium iodide (80 mg, 0.36 mmol) and K₂CO₃ (60 mg, 0.43 mmol) and the reaction mixture was heated at 40 °C for 48 h. Then it was poured on ice, acidified by 1 N HCl till pH 3, and the reaction product was extracted with CHCl₃. The extract was washed with water to pH 7, dried over Na₂SO₄ and purified by column chromatography in hexane/aceton (4:1) to obtain colorless amorphous solid (38 mg, 42%). M.p. 92–94 °C, R_F 0.54 (hexane/aceton, 2:1). MW calculated for C₅₀H₇₆N₂O₁₁ 880.5449, observed in ESI mass spectrum 881.5461 (M+H)⁺, 903.5160 (M+Na)⁺. UV spectrum, λ_max (MeOH): 226 (ɛ 9.03), 301 (1.3). IR (KBr) (cm⁻¹): 1699, 1516, 1456, 988. [α]_D²⁰=+140° (C1, MeOH). ¹⁵N-3a was obtained similarly with the use of 1-¹⁵NH₂-C₅H₅N.

Synthesis of oligomycin A annelated with 5′-methyl-pyrazolo[1,5-a]pyridine (3b)

Compound 3b was obtained from oligomycin A and 1-amino-4-methylpyridine by a similar method with 24% yield. M.p. 110–112 °C. MW calculated for C₅₁H₇₈N₂O₁₁ 894.5606, observed in ESI mass spectrum 895.573 (M+H)⁺. UV spectrum, λ_max (MeOH): 297 (ɛ 6.9).

[α]_D²⁰=+136° (C1, MeOH)⌋