Provided is a preparation method of composition rich in compound Sp-1. The method performs isomer separation on a reaction product from a first step, and then performs a two-step chemical synthesis, so as to prepare a composition comprising high-purity compound Sp-1, wherein an HPLC purity of the single isomer Sp-1 is greater than 90%.Provided is a composition rich in compound Sp-1. The composition has an activity inhibition on tumor cell proliferation in-vitro significantly higher than that of NUC-1031 and compound Rp-1. Also provided are a use of the compound and a pharmaceutical composition comprising the compound and at least one pharmaceutically acceptable excipient.
