The molecular and solid-state structures of methyl [2-(acridin-9-ylimino)-3-( tert-butylamino)-4-oxothiazolidin-5-ylidene]acetate ( 4) were determined by X-ray diffraction, 1H NMR, 13C NMR, CI mass spectroscopy and B3LYP calculations using the basis sets 6-31G(d, p), 6-311+G(d, p) and 6-311++G(2d, 2p). In particular, calculations of long-range heteronuclear coupling constants were found to be extraordinarily indicative for the E/ Z configuration of a distal segment. Compound 4 (C 23H 22N 4O 3S, M r = 434.52) crystallized in the P 1 ¯ space group wherein: a = 11.973(4), b = 12.398(3), c = 15.289(4) Å and α = 85.80(2)°, β = 73.70(3)°, γ = 87.93(2)°, V = 2172.2(11) Å 3, Z = 4 and calculated density = 1.329 g cm −3. The formation of dimers, chains and the crystal packing between the molecules by means of intermolecular hydrogen bonds and π–π interactions between neighboring acridine moieties are discussed. NMR spectral analysis and quantum chemical calculations were found to be in good agreement with the results of the X-ray study. The observation of 4 indicates the dominate role that the N-2 nitrogen of the isothiosemicarbazide intermediate plays in its formation via the reaction of 4-(acridin-9-yl)-1-( tert-butyl)thiosemicarbazide with dimethyl acetylenedicarboxylate (DMAD). This dominance is a result of the steric influences of the substituents on the competing nitrogen atoms.