Alkyl shikimates (SAEs) bearing linear and branched alkyl groups (R) ranging from CH3 to C5H11 were synthesized and assayed for proliferative activity toward human dermal fibroblasts; proliferation increase (PRA), measured using a CCK-8 assay kit, was used as the primary endpoint. Several SAEs exhibited significant PRA, depending on the concentration added to the culture medium and alkyl group structure. However, shikimic acid (SA) added to the culture medium did not induce fibroblast proliferation. SAEs with CH3, C2H5, and C3H7 alkyl groups (SAE-1, SAE-2, and SAE-3a,b) induced a 30–50% increase in cell proliferation at a concentration of 5.5 mM, while SAEs with a C4H9 groups (SAE-4a,b,c) showed low PRA and those with a C5H11 groups (SAE-5a,b,c) exhibited strong cytotoxicity (CYT: decrease in cell viability) at this concentration. The proliferation rates of SAEs at 5.5 mM correlated significantly with their calculated log P (ClogP) values, which reflect hydrophobicity and cell membrane permeability (quadratic regression; coefficient of determination, r2 = 0.977–0.953. Based on these results, we proposed a mechanism in which membrane-permeable SAEs could form complexes with carboxylesterase (CES) present in dermal fibroblasts and be subsequently hydrolyzed into SA and alcohols (ALs) intracellularly, with the resulting SA potentially inducing PRA. In contrast, SA added extracellularly cannot enter cells due to its high hydrophilicity and therefore cannot induce PRA. In order to evaluate the stability of the proposed complexes, we estimated binding energies between CES and SAEs by quantum mechanical methods; however, the results showed no significant differences in binding energy attributable to alkyl chain length or steric effects of the SAE alkyl groups. The most plausible explanation for the differences in PRA among SAEs is that intracellularly regenerated SA promotes fibroblast proliferation, whereas the regenerated ALs may cause CYT. Alkyl group-dependent differences in AL toxicity may account for the concentration-dependent variation in SAE-induced PRA. In fact, when the CYT of linear alkyl alcohols (ALs-1, 2, 3a, 4a, and 5a) was measured by CCK-8 at a concentration of 100 mM, the results showed that differences in AL alkyl chain length significantly influenced cell viability, which was consistent with their ClogP values (CYT order; R = C5H11 > C4H9 > C3H7 > C2H5 ≥ CH3).

Alkyl shikimates promote proliferation of human dermal fibroblasts and exhibit structure–activity relationships

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2026年6月2日

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Hidetada Morishita,
Koji Umezawa,
Masanobu Kojima

摘要

Alkyl shikimates (SAEs) bearing linear and branched alkyl groups (R) ranging from CH₃ to C₅H₁₁ were synthesized and assayed for proliferative activity toward human dermal fibroblasts; proliferation increase (PRA), measured using a CCK-8 assay kit, was used as the primary endpoint. Several SAEs exhibited significant PRA, depending on the concentration added to the culture medium and alkyl group structure. However, shikimic acid (SA) added to the culture medium did not induce fibroblast proliferation. SAEs with CH₃, C₂H₅, and C₃H₇ alkyl groups (SAE-1, SAE-2, and SAE-3a,b) induced a 30–50% increase in cell proliferation at a concentration of 5.5 mM, while SAEs with a C₄H₉ groups (SAE-4a,b,c) showed low PRA and those with a C₅H₁₁ groups (SAE-5a,b,c) exhibited strong cytotoxicity (CYT: decrease in cell viability) at this concentration. The proliferation rates of SAEs at 5.5 mM correlated significantly with their calculated log P (ClogP) values, which reflect hydrophobicity and cell membrane permeability (quadratic regression; coefficient of determination, r² = 0.977–0.953. Based on these results, we proposed a mechanism in which membrane-permeable SAEs could form complexes with carboxylesterase (CES) present in dermal fibroblasts and be subsequently hydrolyzed into SA and alcohols (ALs) intracellularly, with the resulting SA potentially inducing PRA. In contrast, SA added extracellularly cannot enter cells due to its high hydrophilicity and therefore cannot induce PRA. In order to evaluate the stability of the proposed complexes, we estimated binding energies between CES and SAEs by quantum mechanical methods; however, the results showed no significant differences in binding energy attributable to alkyl chain length or steric effects of the SAE alkyl groups. The most plausible explanation for the differences in PRA among SAEs is that intracellularly regenerated SA promotes fibroblast proliferation, whereas the regenerated ALs may cause CYT. Alkyl group-dependent differences in AL toxicity may account for the concentration-dependent variation in SAE-induced PRA. In fact, when the CYT of linear alkyl alcohols (ALs-1, 2, 3a, 4a, and 5a) was measured by CCK-8 at a concentration of 100 mM, the results showed that differences in AL alkyl chain length significantly influenced cell viability, which was consistent with their ClogP values (CYT order; R = C₅H₁₁ > C₄H₉ > C₃H₇ > C₂H₅ ≥ CH₃).