A platform for the discovery of new macrolide antibiotics

Abstract

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

Extended Data Figure 1 |. Synthesis of a C2-fluoro 14-membered azaketolide by a late-stage fluorination reaction.

Subjection of β-keto lactone 25 to potassium tert-butoxide (1.0 equiv) at –78 °C followed by N-fluorobenzenesulfonimide (1.0 equiv) afforded FSM-22391 in 43% yield.

Extended Data Figure 2 |. Synthesis of a 15-membered azaketolide with a modified C2-substituent.

Thermolysis of a β-keto tert-butyl ester substrate (55) proceeded at a lower temperature (80 °C) and afforded a 15-membered macrocycle without substitution at C2 (56). This macrocycle served as a nearly ideal intermediate for preparation of macrolides with diverse C2-substitutions. For example, an allyl group was introduced at C2 by treatment of 56 with sodium tert-butoxide (1.1 equiv) and allyl iodide (1.1 equiv) at –40 °C followed by warming the reaction solution to 23 °C. The product 57 (obtained in 62% yield) was then transformed to FSM-56156 in two steps (72% yield).

Extended Data Figure 3 |. Synthesis of a 15-membered azacethromycin hybrid.

Macrolactone 63 was prepared from amine 60 and aldehyde 61 in two steps by a reductive amination–macrocyclization sequence. Treatment of 63 with paraformaldehyde (6.0 equiv) and acetic acid (10.0 equiv) furnished adduct 64 as a crystalline solid (84% yield). The imidazolidine group within 64 served to protect both the secondary amine and the cyclic carbamate functions, and permitted the introduction of a quinoline heterocycle via a Heck reaction. Methanolysis (TFA, CH₃OH) cleaved the imidazolidine group, affording FSM-20919 (29%, two-step yield).

Extended Data Figure 4 |. Synthesis of a 15-membered azaketolide with a modified C10-substituent.

N-tert-butylsulfinyl imine 68 (prepared in five steps from amide 10) allowed for the stereocontrolled introduction of various C10-substituents. For example, addition of allylmagnesium bromide proceeded with >20:1 stereoselectively to furnish the adduct depicted; subsequent cleavage of the sulfinyl (HCl, CH₃OH) and tert-butyldiphenylsilyl (Bu₄NF) groups within the adduct then furnished left-hand intermediate 69 (81% yield). Amine 69 and aldehyde 35 were coupled by a reductive amination reaction (NaBH₃CN, 60–75% yield). The product (70) was then transformed to FSM-11044 in a three-step sequence that consisted of a macrocyclization reaction (72% yield), a methanolysis reaction (quantitative yield) and lastly a [3+2] dipolar cycloaddition reaction (92% yield).

Extended Data Figure 5 |. Synthesis of a 15-membered azaketolide with a modified C13-substituent.

Modification of position C13 was achieved by modification of a single component, in this case the ketone building block 74 depicted above. Reductive coupling of 78 and 35 united the left- and right-halves; subsequent thermal macrocyclization provided macrolactone 80. The allyl group within intermediate 80 was cleaved upon ozonolysis (O₃, trifluoroacetic acid); reductive workup with sodium cyanoborohydride afforded alcohol 81. Subjection of 81 to bis(2-methoxyethyl)aminosulfur trifluoride afforded the fluoroethyl-substituted macrocycle 82 (30%, two-step yield), which was transformed to FSM-11453 by a [3+2] dipolar cycloaddition reaction.

Extended Data Figure 6 |. Synthesis of a 15-membered azaketolide with a modified desosamine sugar residue.

The 15-membered macrolactone 90 was synthesized using thioglycoside 84 and alkyne 89 as building blocks (in lieu of building blocks 28 and 8 used for the synthesis of 15-membered azaketolide FSM-20707). Treatment of 90 with tributyltin hydride (2.0 equiv), acetic acid (5.0 equiv), and tetrakis(triphenylphosphine)palladium (2 mol%) led to cleavage of the allyloxycarbonyl protective group, giving rise to amine 91 (92% yield). The latter intermediate has been transformed into a number of fully synthetic macrolides with modified desosamine sugar residues. For example, acylation of the primary amino group of intermediate 91 with pyridine-2-carbonyl chloride (2.0 equiv) in the presence of trimethylamine (3.0 equiv) followed by removal of the tert-butoxycarbonyl group afforded FSM-21887 (86%, two-step yield).

Extended Data Figure 7 |. Synthesis of a 16-membered azaketolide.

Homologation of aldehyde 35 was achieved by a Wittig olefination reaction (CH₃OCH₃PPh₃⁺Cl^–, NaHMDS) followed by hydrolysis of the resulting enol ether to afford aldehyde 93 in 65% yield. Reductive coupling of amine 15 and aldehyde 93 furnished macrocyclization precursor 94 (73% yield). The 16-membered macrolactone 95 was obtained in 78% yield upon thermolysis of 94 (1mM, 132 °C). Two additional steps transformed 95 to the 16-membered azaketolide FSM-21397.

Extended Data Figure 8 |. Synthesis of a 14-membered macrolide with a trans-olefin linkage.

Mesylate 98 was prepared in quantitative yield by treatment of alcohol 34 with methanesulfonyl chloride (1.50 equiv) and triethylamine (2.0 equiv). Displacement of mesylate 98 with sodium 1-phenyl-1H-tetrazole-5-thiolate (2.0 equiv) followed by oxidation of the resulting thioether with ammonium molybdate (0.20 equiv)–hydrogen peroxide (100 equiv) afforded sulfone 100 in 70% yield. Aldehyde 101 and sulfone 100 were coupled in a Julia–Kocienski olefination reaction (NaHMDS, –78 → 23 °C) to provide a 4.8:1 mixture of E- and Z-olefin isomers. The E-isomer 102 was isolated and desilylated (Bu₄NF, 79%). Thermolysis of the product 103 (1 mM, 132 °C) furnished the 14-membered macrocycle 104 in 83% yield. 104 was then transformed to FSM-21079 in two additional steps (methanolysis and [3+2] dipolar cycloaddition).

Extended Data Figure 9 |. Synthesis of a 15-membered macrolide with an amide linkage (C9-N9a).

Oxidation of aldehyde 35 with sodium chlorite (10.0 equiv) in the presence of sodium dihydrogen phosphate (10.0 equiv) and 2-methyl-2-butene (100 equiv) afforded carboxylic acid 107 in 70% yield. Acid 107 and amine 15 were coupled in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2.00 equiv) to provide amide 108. Macrocyclization of 108 (1 mM, 132 °C) proceeded in 81% yield to afford macrolactam 109. Methanolysis and [3+2] dipolar cycloaddition then transformed 109 to FSM-21344 in two steps.

Extended Data Figure 10 |. Synthesis of a 15-membered macrolide with an amide linkage (C10-N9a).

Amine 112 was prepared in 70% yield by displacement of mesylate 98 with sodium azide followed by reduction of the resulting alkyl azide (H₂, Pd). The coupling of amine 112 and acid 113 proceeded in 54% yield. The product, amide 114, was desilylated (Bu₄NF, 80%) to afford the macrocyclization precursor 115. Thermal macrocyclization of 115 followed by cleavage of the methoxycarbonyl protective group afforded lactam 116 in 80% yield. Copper-catalyzed [3+2] dipolar cycloaddition then provided FSM-21473 in 64% yield.

Figure 1 |. Summary of macrolide antibiotic development by semisynthesis.

To date, all macrolide antibiotics are produced by chemical modification (semisynthesis) of erythromycin, a natural product produced on ton-scale by fermentation. Depicted are the approved semisynthetic macrolide antibiotics clarithromycin, azithromycin, and telithromycin along with the dates of their FDA approval and the number of steps for their synthesis from erythromycin. The previous ketolide clinical candidate cethromycin and the current clinical candidate solithromycin are also depicted. It is evident that increasingly lengthy sequences are being employed in macrolide discovery efforts.

Figure 2 |. A convergent, fully synthetic route to the 14-membered azaketolide 25.

a, Graphical representation of the convergent synthesis of azaketolide 25 from eight variable building blocks (represented by colored spheres). Downward, Y-shaped arrows signify convergent coupling reactions. b, Synthesis of azaketolide 25, reagents and conditions (subscripts _L and _R indicate left and right halves, respectively): (a_L) LiHMDS, LiCl, 98%; (b_L) EtⁱPr₂N, COCl₂; (c_L) ⁱPrMgCl, CH₃Li, 76% over 2 steps, 92% of recovered (R,R)-pseudoephenamine; (d_L) NaHMDS; (e_L) NaN₃, 88% over 2 steps; (f_L) NH₃, Ti(OⁱPr)₄, NaBH₄, 95%; (g_L) Bu₄NF, 92%; (a_R) Pd[(S)-SegPhos]Cl₂, AgSbF₆, 93%, 92% ee; (b_R) ethylene glycol, PPTS, 92%; (c_R) KH, MeI, 97%; (d_R) (ⁱBu)₂AlH, 96%; (e_R) Et₃N, MgBr₂•OEt₂, 91%; (f_R) AgOTf, 81%, 16:1 β:α; (g_R) HCl, 100%; (h) NaCNBH₃, 82%; (i) 132 °C, 1 mM, PhCl, 78%; (j) CuSO₄, sodium L-ascorbate, 86%.

Figure 3 |. A convergent, fully synthetic route to the 15-membered azaketolide 38.

a, Graphical representation of the convergent synthesis of azaketolide 38 from eight variable building blocks (represented by colored spheres). Downward, Y-shaped arrows signify convergent coupling reactions. b, Synthesis of azaketolide 38, reagents and conditions (subscripts _L and _R indicate left and right halves, respectively): (a_L) LiHMDS, LiCl, 98%; (b_L) EtⁱPr₂N, COCl₂; (c_L) ⁱPrMgCl, CH₃Li, 76% over 2 steps, 92% of recovered (R,R)-pseudoephenamine; (d_L) NaHMDS; (e_L) NaN₃, 88% over 2 steps; (f_L) NH₃, Ti(OⁱPr)₄, NaBH₄, 95%; (g_L) Bu₄NF, 92%; (a_R) ^tBuLi, MgBr₂, 81%; (b_R) KH, MeI, 99%; (c_R) H₅IO₆, 99%; (d_R) ZnCl₂, 91%; (e_R) AgOTf, 70%; (f_R) HF (aq); (g_R) Dess–Martin periodinane, 87% over 2 steps; (h) NaCNBH₃, 86%; (i) 132 °C, 1 mM, PhCl, 94%; (j) CuSO₄, sodium L-ascorbate, 93%.

Figure 4 |. A convergent, fully synthetic route to solithromycin.

a, Graphical representation of the convergent synthesis of solithromycin, which was previously only accessible by semisynthesis. This route has been adapted for the synthesis of >30 novel ketolide antibiotic candidates, as well as the FDA-approved ketolide telithromycin. Downward, Y-shaped arrows signify convergent coupling reactions. b, Synthesis of solithromycin, reagents and conditions (subscripts _L and _R indicate left and right portions, respectively): (a_L) lithium (1S,2R)-1-phenyl-2-(pyrrolidin-1-yl)-1-propanolate, 85%; (b_L) CuSO₄, sodium L-ascorbate, 95%; (a_R) ^tBuLi, MgBr₂, 81%; (b_R) KH, MeI, 99%; (c_R) H₅IO₆, 99%; (d_R) ZnCl₂, 91%; (e_R) AgOTf, 70%; (f_R) HF (aq), 95%; (g_R) Dess–Martin periodinane, 92%; (h) Cp₂TiCl₂, cyclopentylmagnesium bromide, 80%; (i) Dess–Martin periodinane, 97%; (j) Bu₄NF, 95%; (k) 132 °C, 0.5 mM, PhCl, 66%; (l) KO^tBu, FN(SO₂Ph)₂, 85%; (m) Im₂CO, DBU; (n) imidazole hydrochloride, 60 °C, 87% over 2 steps.

Figure 5 |. Minimum inhibitory concentrations (μg/mL) for selected analogs against 9 Gram-positive and 5 Gram-negative microorganisms.

iErmA – inducible erythromycin ribosome methyltransferase A; cErmA – constitutive erythromycin ribosome methyltransferase A; MsrA – macrolide streptogramin resistance efflux pump A; MefA – macrolide efflux protein A; ErmB – erythromycin ribosome methyltransferase B; Erythro – erythromycin; Azithro – azithromycin; Telithro – telithromycin; Solithro – solithromycin.