*2.2. Radiosynthesis, Stability, and Determination of log D7.4*

As shown in Scheme 3, [18F]**1** was synthesized on the basis of an SNAr reaction via substitution of the NO2 leaving group of **15** by [18F]fluoride in the presence of Kryptofix® (K2.2.2) and K2CO3. In dimethylsulfoxide (DMSO), the SNAr reaction proceeded smoothly and resulted in high radiochemical yields of 73 <sup>±</sup> 12% (n = 4, non-isolated, radio-HPLC) for [18F]**<sup>1</sup>** after 15 min conventional heating at 130 ◦C. Besides unreacted [18F]F<sup>−</sup>, radioactive by-products accounted for less than 5%. [18F]**1** was isolated by semi-preparative HPLC (Supplementary Data, Figure S1A), trapped on a pre-conditioned Sep-Pak C18 light cartridge, eluted with ethanol, and formulated in isotonic saline containing 10% of EtOH (*v*/*v*) for better solubility. Analytical radio- and UV-HPLC analyses of the final product co-eluted with the reference **1**, confirmed the identity of the radiotracer (Supplementary Data, Figure S1B). Finally, [18F]**<sup>1</sup>** was obtained with radiochemical yields of 51 <sup>±</sup> 11% (n <sup>=</sup> 3, decay-corrected to the end of the bombardment) in a total radiosynthesis time of about 90 min, at a radiochemical purity of ≥ 98% and with molar activities in the range of 180–200 GBq/μmol (n = 3, end of synthesis) using starting activities of 2–3 GBq.

**Scheme 3.** Radiosynthesis of [18F]**1**.

The stability of the radiotracer was investigated by incubation of [18F]**1** in *n*-octanol, saline, phosphate-buffered saline (PBS) and ethanol. Samples were analyzed by radio-thin-layer chromatography (TLC) and radio-HPLC and no degradation or defluorination was observed in any of the solvents after 60 min incubation at 40 ◦C.

A variety of physicochemical parameters affects the brain permeability of different brain-targeting radiotracers [41]. Lipophilicity, often, but not necessarily, correlates with the ability to cross the BBB, and is considered as an important physicochemical property. In Table 1, calculated bioavailability-related parameters are listed for α-CCA, FACH and selected structural analogs. Accordingly, the new derivatives **1** and **2** show the desired higher hydrophobicity (log *K*ow = logarithmic *n*-octanol/water partition coefficient [42], log D7.4 = log *K*ow corrected for ionization at pH 7.4 [43,44]) as compared to FACH. Nevertheless, Table 1 also shows that the predicted brain-blood partition coefficients (log *K*BB [43]) of **1** (–0.49) and **2** (–1.05) are below the one of FACH (–0.10).

**Table 1.** Calculated physicochemical parameters of our drug candidates and structurally related compounds.1


<sup>1</sup> The decadic logarithms of the *n*-octanol/water partition coefficient (log *K*ow) have been calculated with EPI Suite [42], and these values have been employed for predicting the p*K*a-pH-corrected *n*-octanol/water distribution coefficients, log D7.4. For the latter, the ACD approach log *K*ow (ionized) = log *K*ow (unionized) − 3.75 (applicable for the relevant range of log *K*ow data) [43,44] with D7.4 = *f* <sup>u</sup> × *K*ow + (1 − *f* u) × *K*ow (ionized) has been employed (see also [44]). Note that the ACD-calculated log *K*ow data are lower by 0.5–1.5 log units except for a slightly larger value for cinnamic acid, resulting in correspondingly lower calculated log D7.4 data. Moreover, *f* <sup>u</sup> denotes the compound fraction unionized at pH 7.4 according to the Henderson-Hasselbalch relationship, and p*K*<sup>a</sup> as well as the brain-blood partition coefficient (*K*BB) have been calculated with the ACD software.

Note further that FACH is both more lipophilic and more BBB-permeable than α-CCA. Regarding the Michael-acceptor unit mentioned above, comparison of cinnamic acid and its α-CN derivative shows that the α-CN substitution decreases the p*K*<sup>a</sup> value by 3.7 units, most likely because of its combined inductive and mesomeric electron-withdrawing effect. Accordingly, all α-CCA derivatives are significantly acidic with p*K*a values below 1, indicating for all of them that the dissociated carboxylate form is prevalent under physiological conditions. Experimental investigation of the lipophilicity of [ 18F]**1** through employing the shake-flask method using *n*-octanol and PBS (pH 7.4) resulted in a log D7.4 value of 0.820 ± 0.003 (n = 4). This value agrees pretty well with its calculated counterpart of 1.08 (Table 1), which holds correspondingly for the FACH log D7.4 value (0.42 experimental [28] vs. 0.69 calculated) as well as for the respective difference in log D7.4 values.
