3.2. Synthesis of Coelenterazine Analogues

All the reactions were monitored by TLC with 0.25 mm silica gel plates (60GF-254). UV light, iodine stain, and ninhydrin were used to visualize the spots. Silica gel was utilized for column chromatography purification. ¹H NMR and ¹³C-NMR were recorded on a Bruker DRX spectrometer at 300 or 400 MHz, δ in parts per million, and J in hertz, using TMS as an internal standard. ESI-MS spectra were recorded on an API 4000 spectrometer. Melting points were determined uncorrected on an electrothermal melting point apparatus. HPLC tests were measured with Agilent Technologies 1260 liquid chromatography.

For most of CTZ analogues, substituents on the C-6 phenyl ring of coelenterazine were introduced by first preparing 2-amino-3-benzyl-5-bromopyrzine (1-1) via regioselective palladium-catalyzed Neigishi-type cross-coupling of benzylzinc bromide and 2-amino-3,5-dibromopyrazine. For the A-series CTZ analogues [15] (Figure 4), key precursors 2 were obtained with a good yield via Sonogashira coupling reactions with substituted phenylacetylene.

Synthesis of the A-series CTZ analogues.

For the B-series CTZ analogues [16] (Figure 5), the key intermediates 4 were synthesized with good to moderate yields by Suzuki coupling reaction with the appropriately substituted arylboronic acid. However, compounds 4-15 and 4-16 were generated from compound 1-1 and substituted arylboronic acid using two sequential Suzuki coupling reactions.

Synthesis of the B-series CTZ analogues.

For the T-series CTZ analogues (Figure 6) [15], the compound 1-1 underwent a Sonogashira coupling reaction with trimethylsilylacetylene and then became 2-amino-3-benzyl-5-ethynyl-pyrazine (6) by removing the trimethylsilyl group. Then, the key precursors 8 were synthesized using a click reaction of the compound 6 and azides.

Synthesis of the T-series CTZ analogues.

The benzylketoacetal (3), which was required for the final condensation step, was synthesized by the Grignard reagent benzylmagnesium chloride and ethyl diethoxyacetate. Then, the coelenteramines 2, 4, and 8 were condensed with the compound 3 respectively, yielding the coelenterazine analogues of A-series, B-series and T-series, respectively.

Synthesis details are available in the Supplementary Materials.