Nobilamide B, a TRPV1 antagonist, and a series of Ala-substituted analogues were synthesized and their neuroactivity was assessed in a primary culture of dorsal root ganglion (DRG) neurons. Analogues 4, 6, and 7 showed comparable activity, affecting capsaicin response in neurons, in contrast to 2, 3, and 5 which showed minimal blocking. Compounds 2, 3, and 5 correspond to analogues in which D-Phe, D-Leu and D-allo-Thr have been substituted with Ala, respectively. The observed loss of bioactivity in these three analogues highlights the importance of D amino acids in the primary structure of nobilamide B.