The occurrence and development of tumors rely on the nutritional supply from blood vessels, which also serve as the main pathway for tumor metastasis. Inhibiting angiogenesis is one of the main strategies for cancer treatments. Chiral drugs, encouraged and inspired by chiral natural products, make up a major portion of marketed drugs. However, as an important source for synthetic chemistry and drug discovery, the counterpart of chiral natural products, the enantiomers, has received little attention. Here, we constructed a compound library containing 100 racemates (±)-1–100 and 4 pairs of enantiomers (33, 48, 59, 68) of 3,4-dioxygenated-4-aryl-quinolin-2(1H)-one alkaloids. Through extensive activity screening, we found that the compounds with 3R, 4R configuration, opposite to the natural products, exhibited potent angiogenesis inhibitory activity in zebrafish, while the 3S, 4S-configured natural derivatives have no effects. More importantly, compound (+)-48, named as (3R, 4R)-CHNQD-00728, significantly inhibited hepatic tumor growth in doxycin hydrochloride-induced liver-specific enlargement zebrafish. Examining the phenomenon and unlocking dark matter of enantiomeric natural products in innovative drugs discovery can provide a new perspective on organic synthesis and medicinal chemistry, thus enabling a broader exploration.

MS/MS-based molecular networking is an effective strategy to rapidly dereplicate known compounds and to guide the discovery process for new and novel natural products. In the present study, the chemical diversity of indole diterpenoids from the marine-derived fungus Penicillium sp. N4-3 was investigated using molecular networking techniques. Guided by this information, targeted isolation resulted in two new indole diterpenoids shearinines R and S (1, 2) and an oxidative artifact shearinine T (3), together with the verification of two known analogs (4, 5). Furthermore, five indole diterpenoids (6−10), including three putatively new ones, shearinines U−W (6, 9, 10), were predicted from the molecular ion cluster by the combination of GNPS molecular networking and manual analysis of MS/MS fragmentation clusters. Shearinines T (3) and W (10) are characterized by an oxidative cleavage of the C-2–C-18 double bond. Feature fragment ions of these shearinines revealed two type of dominant ions related to the indole moiety and the breaking of C-9 side chain or Ring Ⅰ. Compound 1 showed antibacterial activities against a panel of pathogenic bacteria with IC₅₀ values ranging from 6.34 to 47.96 μg/mL and inhibited the growth of the human hepatic (HepG2) and gastric (SGC-7901) cancer cells lines with IC₅₀ values of 6.27 and 19.16 μg/mL, respectively.