Synthesis and biological activity of NOSH-naproxen (AVT-219) and NOSH-sulindac (AVT-18A) as potent anti-inflammatory agents with chemotherapeutic potential

Abstract

Nitric oxide- (NO) and hydrogen sulfide- (H₂S) releasing naproxen (NOSH-naproxen) and NO and H₂S-releasing sulindac (NOSH-sulindac) were synthesized and their cell growth inhibitory properties were evaluated in four different human cancer cell lines. These cell lines are of adenomatous (colon, pancreas), epithelial (breast), and lymphocytic (leukemia) origin. Using HT-29 human colon cancer cells, NOSH-naproxen and NOSH-sulindac increased apoptosis, and inhibited proliferation. NOSH-naproxen caused a G₀/G₁ whereas NOSH-sulindac caused a G₂/M block in the cell cycle. Both compounds exhibited significant anti-inflammatory properties, using the carrageenan rat paw edema model. Reconstitution and structure-activity studies representing a fairly close approximation to the intact molecule showed that NOSH-naproxen was approximately 8000-fold more potent than the sum of its parts in inhibiting cell growth. Our data suggest that these compounds merit further investigation as potential anti-cancer agents.

Figure 1. Structural components of NOSH-naproxen and NOSH-sulindac

The parent compounds, naproxen and sulindac are shown in the shaded boxes. ADT-OH releases H₂S and ONO₂ releases NO, both shown in dotted ellipses.

Figure 2. Effect of NOSH-NAP and NOSH-SUL on HT-29 colon cancer cell kinetics

NOSH-NAP and NOSH-SUL inhibited proliferation by altering cell cycle progression and inducing apoptosis. Cells were treated with vehicle, or NOSH-NAP (panels A-C) or NOSH-SUL (panels D-F) at their respective IC₅₀ for cell growth inhibition (80 and 90 nM) and analyzed at the indicated times. Panels A and D, proliferation by PCNA antigen expression; Panels B and E), cell cycle phases by PI staining and flow cytometry; Panels C and F), apoptosis by Annexin V staining and flow cytometry. Results are mean ± SEM for 3 different experiments performed in duplicate, *P < 0.05, compared to control.

Figure 3. Anti-inflammatory properties of NOSH-NAP and NOSH-SUL

Rat paw edema was induced by carrageenan injection. Panel A, all agents caused a significant reduction in paw volume at 4hr. Results are mean ± S.E.M. of three rats in each group, *P < 0.05 versus vehicle treated rats. Panel B, all agents caused a significant reduction in PGE ₂ levels in the paw exudate. Results are mean ± S.E.M. for three rats in each group, *P < 0.01versus vehicle

Figure 4. NO and H₂S levels in vivo after NOSH-NAP and NOSH-SUL administration

Plasma concentration of NO _x and H ₂S was quantified as detailed in Section 4.2.6. Results are mean ± S.E.M. of three rats in each group. ^†P < 0.001 versus vehicle and parent NSAID-treated animals.

Scheme-1. Synthetic scheme for the synthesis of NOSH-Naproxen (AVT-219)

a. HBr/AcOH, reflux, 4h; b. Tf₂O, t-BuOH, NH₄OH; c. 4-bromobutyric acid, DCC/DMAP, DCM, rt, 6h; d. AgNO₃/acetonitrile, 70°C, 6h; e. TFA/DCM, 30 min; f. ADT-OH, DCC/DMAP, DCM, rt, 6h.

Scheme-2. Synthetic scheme for preparation of NOSH-Sulindac (AVT-18A)

a. TBDMSCl/Imidazole, DCM, 18h; b. 4-bromobutyric acid,DCC/DMAP, DCM, rt, 6h; c. AgNO₃/acetonitrile, 70°C, 6h; d. NaH₂PO₄, H₂O₂/NaClO₂, 0°C, 2h; e. ADT-OH, DCC/DMAP, DCM, rt, 6h; f. TBAF/AcOH, THF, 30 min; g. Sulindac, DCC/DMAP, DCM, rt, 6h.