*2.3. In Vitro and In Vivo Biological Validation of [18F]1*

It is well demonstrated that several members of the MCT family are highly expressed in mammalian kidney, where over 95% of the lactate reabsorption takes place [1,3,45,46]. MCT1 mRNA and protein have clearly been detected on both the human kidney derived cell line HK-2 and human kidney cortex. In HK-2 cells it was found exclusively on the basal membrane [45]. Therefore, the specific binding of [18F]**1** to MCTs was initially proven by in vitro autoradiography using cryosections of the mouse kidney. As reflected by the autoradiographic images presented in Figure 4, co-incubation of ~1 nM [ 18F]**1** with 10 μM α-CCA-Na resulted in significantly lower binding of the radiotracer. Therefore, the binding of [18F]**1** in mouse kidney in vitro is highly specific.

**Figure 4.** In vitro autoradiography of [18F]**1** in transversal cryosections of the mouse kidney. Total (**A**) and non-specific (**B**) binding of ~1 nM [18F]**1** obtained without and with co-incubation with 10 μM α-CCA-Na.

To investigate the stability of [18F]**1** in vivo, samples of plasma and brain homogenates obtained from CD-1 mice at 30 min after intravenous injection of the radiotracer were analyzed for radiometabolites by using reversed-phase and micellar (MLC) radio-HPLC. MLC allows a direct injection of the samples into the HPLC system without the elimination of the tissue matrix as already described [47,48]. In general, the results obtained with both methods are comparable and the analyses revealed solely intact radiotracer and no detectable radiometabolites in plasma (Figure 5A–B) and brain (Supplementary Data, Figure S2) samples. Notably, in both samples two peaks a/b were detected by analytical HPLC (Figure 5/Figure S2) which are supposed to represent the neutral and deprotonated form of the radiotracer ([18F]**1a**/**b**). This finding suggests that the analytical HPLC conditions do not reflect the physiological milieu at which the neutral compound fraction would be negligible according to the Henderson–Hasselbalch equation. Research into the speciation of **1** under analytical-chemical conditions is subject to a future investigation.

**Figure 5.** Analytical UV- and radio-HPLC chromatograms representing two peaks a/b which are supposed to reflect the neutral and deprotonated form of the radiotracer ([18F]**1a**/**b**) in mouse plasma at 30 min p.i. measured under: (**A**) reversed phase (Reprosil-Pur C18-AQ, 250 × 4.6 mm, gradient with an eluent mixture of ACN/20 mM NH4OAc (aq.), 370 nm, 1.0 mL/min), and (**B**) micellar conditions (Reprosil-Pur C18-AQ, 250 × 4.6 mm, isocratic mode with water containing 50 mM sodium dodecyl sulfate/10 mM NaHPO4, 1.0 mL/min).

Pharmacokinetic studies of [18F]**1** were performed by dynamic PET imaging in mouse using a dedicated small animal PET/MR camera. The target-specificity of [18F]**1** was investigated by pre-administration of the blocking compound α-CCA-Na. Maximum intensity projections of PET studies from a representative control and α-CCA-Na treated animals and time-activity curves (TACs) from tissues of interest are presented in Figure 6. [18F]**1** cleared rapidly from the blood with an initial TAC peak standardized uptake value (SUV) of 7.3 and a SUV of 1.5 after 10 min followed by a slow blood clearance to a SUV of 0.9 after 60 min in the control group (Figure 6B). Pre-administration of the MCT inhibitor α-CCA-Na resulted in an initial TAC peak SUV of 6.9 which was comparable to the control group, whereas a higher SUV of 3.5 after 10 min and a SUV of 1.5 was reached after 60 min p.i., reflecting higher availability of the radiotracer in the blood (Figure 6B). This is expected to be caused by blocking the uptake of [18F]**1** in peripheral organs in vivo. In comparison to the control conditions, the pre-administration of α-CCA-Na significantly reduced the activity accumulation in the MCT1-expressing renal cortex [46] throughout the whole imaging period, which is shown by the SUV ratio (SUVR) of kidney cortex-to-blood (Figure 6D). Furthermore, the displacement study revealed 39.2% drop of the SUV, 20 min after i.v. injection of α-CCA-Na (Figure 6E), which implicates a reversible tissue uptake of [18F]**1** in the kidney cortex. Nevertheless, further studies are needed to clarify the exact mechanism of the radiotracer uptake. Regarding liver, where the highly expressed MCT1 transports L-lactate into the parenchymal cells for gluconeogenesis [1], a constantly increasing accumulation of activity can be observed under both control and blocking conditions, although at lower values under pre-administration of α-CCA-Na (Figure 6C).

Taking into consideration the high activity concentrations persistently accumulated in the kidney and liver, the blocking effect of α-CCA-Na in both tissues will result in a strong increase in the fraction of available tracer in blood as reflected by the higher SUV in blood observed in the blocking experiments (Area Under the Curve (AUC)0–60 min = 140 SUV × minutes) compared to the control experiments (AUC0–60 min = 75 SUV × minutes).

**Figure 6.** (**A**) Maximum intensity projections (MIPs) of small animal positron emission tomography (PET) images (45–60 min p.i., n = 1) of female CD-1 mice depicting the differential distribution of [18F]**1** in peripheral organs without and with pre-administration of α-CCA-Na; (**B**–**D**) Time-activity curves representing the SUV of blood, as well as the standardized uptake value (SUV) ratio (SUVR) from liver-to-blood and kidney cortex-to-blood; (**E**) displacement study: i.v. application of α-CCA-Na 20 min after tracer injection (n = 2), MIPs of the left kidney (left: 15–20 min p.i. and right: 45–60 min p.i.).

According to Figure 7A, [18F]**1** shows a similar brain uptake as [18F]FACH despite a somewhat higher lipophilicity. Note, however, that this is in accordance with our ACD predictions regarding both log D7.4 and log *K*BB values (Table 1). The slightly higher lipophilicity (log *K*ow and log D7.4) of **1** is accompanied by a slightly lower BBB penetration (log *K*BB), demonstrating further that passive brain uptake is not governed alone by log D7.4. Moreover, a selective uptake into a particular brain region could not be verified for both radiotracers (Figure 7, C and D). The SUVR (brain-to-blood) of [18F]**1** was reduced by α-CCA-Na by only 25% compared to the control animal (Figure 7B), which is much less than the reduction of kidney uptake and indicates that the uptake of [18F]**1** into the brain is partially mediated by MCT1 expressed at the endothelial cells of the BBB [49,50], and partly by non-specific diffusion mediated by lipophilicity.

**Figure 7.** (**A**) Time-activity curve of [18F]**1** (n = 1) and [18F]FACH (n = 3) representing the SUVs without and with pre-administration of α-CCA-Na; (**B**) The SUVR of [18F]**1** showing the brain to blood uptake ratio (n = 1); (**C**) horizontal section of representative PET image (averaged time frames from 0–60 min p.i.) of mouse head; (**D**) SUVs of different brain regions over time after injection of [18F]**1**.

**WP LQ**

Brain capillary endothelial cells are tightly bound to each other and constitute the permeability barrier of the BBB/Neurovascular Unit (NVU), which serves to restrict the transport of compounds into the brain. The permeation of compounds across the BBB/NVU is determined not only by lipophilicity, ionic feature and molecular size, but also by various transporters expressed on the endothelial cell membrane [51]. For example, the radiotracer [11C]choline has initially been demonstrated a comparably low brain accumulation with 0.08% of injected dose/g brain (%ID/g) 10 min after injection in mice [52] and 0.15% ID/g at 15 min after injection in rats [53]. This low uptake of [11C]choline from blood is mediated by a choline-specific transport system of brain endothelial cell membranes [54]. Despite the low BBB/NVU permeability of [11C]choline, it is a clinically relevant radiopharmaceutical for brain tumor imaging with high and specific accumulation in proliferating cancer cells [55,56]. Notably, in our control experiment using the MCT radioligand [18F]**1,** a moderate brain accumulation of 0.4% ID/g at 10 min after injection was observed. It has been shown that MCT1 is expressed along with MCT4 in the cerebral microvascular endothelium cells suggesting a key role in transporting endogenous monocarboxylates into and out of the brain [50,57,58]. Together with the important role of MCT1 and MCT4 for glioma metabolism [5,32,33,59], it provides evidence that [18F]FACH and/or [18F]**1** might be suitable for brain tumor imaging.

### **3. Materials and Methods**

#### *3.1. Organic Synthesis*

The syntheses of **1**, **2** and **15** (nitro precursor) were implemented by slight modifications of the previously reported procedures [26,36–39]. All final compounds described here meet the purity requirements determined by HPLC, NMR, and HR-MS analyses.

#### 3.1.1. General Information

Unless otherwise noted, moisture-sensitive reactions were conducted under dry nitrogen or argon. Pd(OAc)2, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethylxanthene), and Cs2CO3 were purchased from Sigma-Aldrich (SIGMA-ALDRICH Chemie GmbH, Schnelldorf, Germany). Other chemicals and reagents were purchased from commercial sources and were used without further purification. For thin-layer chromatography (TLC), Silica gel 60 F254 plates (Merck KGaA, Darmstadt, Germany) were used. Flash chromatography (fc) was performed using Silica gel 60, 40–64 μm (Merck). Room temperature was 21 ◦C. 1H, 13C, and 19F spectra were recorded on Varian "MERCURY plus 300/400" (Varian, Palo Alto, CA, USA) and Bruker Advance DRX-400 (Bruker, Billerica, MA, USA); δ in ppm related to tetramethylsilane; coupling constants (*J*) are given with 0.1 Hz resolution. Multiplicities of NMR signals are indicated as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), dd (doublet of doublets), ddd (doublet of doublet of doublets), td (triplet of doublets), dq (doublet of quartets) and h (hexet (sextet)). High-resolution mass spectra (HR-MS) were recorded on an FT-ICR APEX II spectrometer (Bruker Daltonics; Bruker Corporation, Billerica, MA, USA) using electrospray ionization (ESI) in positive ion mode.

#### 3.1.2. General Procedures

General Procedure A: The Buchwald-Hartwig Aryl Amination Reaction.

Substituted halide (2.5 mmol, 1.0 eq.) was dissolved in dry dioxane (10 mL) in a dry Schlenk tube, under an argon atmosphere. Pd(OAc)2 (28 mg, 0.125 mmol, 0.05 eq.), Xantphos (72 mg, 0.125 mmol, 0.05 eq.), and Cs2CO3 (2.04 g, 6.25 mmol, 2.5 eq.) were added afterwards and the mixture was stirred at 75 ◦C. After 20 min, amine (3.0 mmol, 1.2 eq.) was added, and the reaction mixture was conducted at 105 ◦C under an argon atmosphere. The reaction monitored by TLC and it was completed in 30 min. After cooling to room temperature, the reaction mixture was diluted with diethyl ether (Et2O), the solids were filtered and washed by Et2O. The solvents were evaporated under *vacuum* and the oily residue was then purified by column chromatography.

### General Procedure B

To a solution of substituted amine (2.0 mmol, 1.0 eq.) in *N,N*-dimethylformamide (DMF, 10 mL), NaH (26.0 mmol of a 60% dispersion in mineral oil, 13.0 eq.) was added in small portions under argon. 1-Iodopropane (0.488 mL, 5.0 mmol, 2.5 eq.) was thereafter added to the mixture. After stirring for 1 h at room temperature, the mixture was slowly poured on an ice-water mixture and stirred for 5 min. The mixture was extracted with ethyl acetate (EtOAc, 2 × 25 mL), the extracts were combined, dried with anhydrous MgSO4, and concentrated by evaporation of the solvents under vacuum. The residue was then purified by column chromatography.

#### General Procedure C

POCl3 (1.03 mL, 11.0 mmol, 1.1 eq.) was added dropwise to DMF (4.6 mL, 60.0 mmol, 6.0 eq.) at 0 ◦C and the mixture was stirred 30 min at room temperature. To this solution, *N,N*-disubstituted aniline (10.0 mmol, 1.0 eq.) was added and the reaction mixture was heated up to 80 ◦C. After 2–4 h, the reaction was quenched by addition of a mixture of ice water, stirred for additional 5 min. The pH was thereafter adjusted to 6–7 by using aqueous 1 M NaOH. The residue was extracted with chloroform

(CHCl3, 3 × 15 mL), and the organic phases were combined, washed with H2O and brine. The extracts were dried with anhydrous MgSO4 and the corresponding *N,N*-disubstituted benzaldehyde was purified by column chromatography after evaporation of the solvents.

To a solution of the substituted benzaldehyde (5.0 mmol, 1.0 eq.) in 20 mL acetonitrile (ACN), cyanoacetic acid (0.991 mL, 15.0 mmol, 3.0 eq.) and piperidine (0.494 mL, 5.0 mmol, 1.0 eq.) were added and refluxed overnight at 85 ◦C. Upon the completion of the reaction, as judged by TLC, the above solution was poured into a mixture of 3M HCl (10 mL) on ice. The solution was stirred for 30 min and the solids were filtered using a Büchner funnel and washed with ice-cold ACN. The solid was afterwards poured into adequate amount of *n*-hexane and stirred for 15 min to remove the remaining aldehyde. The final compounds were obtained in pure form after filtration of the solids and consequent washing with *n*-hexane.

*6-Fluoro-N-(3-methoxyphenyl)pyridin-2-amine (5).* The reaction was carried out according to the general procedure A. Column chromatography: silica, EtOAc/*n*-hexane, 1:2; Yellow oil: 96 % yield; TLC: (silica gel, EtOAc/*n*-hexane, 2:1), Rf = 0.8. 1H NMR (300 MHz, CDCl3) δ 7.56 (q, *J* = 8.0 Hz, 1H), 7.25 (m, 1H), 6.97 (t, *J* = 2.2 Hz, 1H), 6.90 (ddd, *J* = 8.0, 2.1, 0.8 Hz, 1H), 6.73 (dd, *J* = 8.1, 2.3 Hz, 1H), 6.66 (ddd, *J* = 8.3, 2.4, 0.8 Hz, 1H), 6.33 (dd, *J* = 7.8, 2.2 Hz, 1H), 3.83 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 162.87 (d, *J* = 238.2 Hz), 160.49, 154.92 (d, *J* = 16.1 Hz), 142.12 (d, *J* = 8.4 Hz), 140.82, 130.03, 113.09, 108.87, 106.67, 104.30 (d, *<sup>J</sup>* <sup>=</sup> 4.2 Hz),98.29 (d, *<sup>J</sup>* <sup>=</sup> 36.1 Hz), 55.27; 19F NMR (282 MHz, CDCl3) <sup>δ</sup> <sup>−</sup>69.08 (d, *J* = 8.1 Hz).

*6-Fluoro-N-(3-methoxyphenyl)-N-propylpyridin-2-amine (6)*. The reaction was carried out according to the general procedure B. Column chromatography: silica, EtOAc/*n*-hexane, 1:3; Yellow oil: 95% yield; TLC: (silica gel, EtOAc/*n*-hexane, 1:3), Rf = 0.75. 1H NMR (400 MHz, CDCl3) δ 7.35–7.27 (m, 2H), 6.95–6.65 (m, 3H), 6.12 (td, *J* = 7.6, 2.5 Hz, 2H), 3.85 (d, *J* = 7.6 Hz, 2H), 3.81 (s, 3H), 1.66 (m, 2H), 0.92 (t, *J* = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 162.85 (d, *J* = 234.7 Hz), 160.87, 157.87 (d, *J* = 16.1 Hz), 145.82, 140.72 (d, *J* = 8.3 Hz), 130.50, 120.09, 113.64, 111.95, 104.78 (d, *J* = 4.1 Hz), 95.17 (d, *J* = 37.4 Hz), 55.30, 51.76, 21.05, 11.26; 19F NMR (377 MHz, CDCl3) <sup>δ</sup> <sup>−</sup>69.25 (d, *<sup>J</sup>* <sup>=</sup> 8.3 Hz).

*6-Fluoro-N-(3-methoxyphenyl)-N-phenylpyridin-2-amine (7).* The reaction was carried out according to the general procedure A. For this compound, higher amounts of Pd(OAc)2 (84 mg, 0.375 mmol, 0.15 eq.) and Xantphos (217 mg, 0.375 mmol, 0.15 eq.) were added stepwise (3 × 0.125 mmol) to the reaction mixture over 24 h. Column chromatography: silica, EtOAc/n-hexane, 1:3; Milky oil: 46% yield; TLC: (silica gel, EtOAc/n-hexane, 1:3), Rf = 0.85. 1H NMR (400 MHz, CDCl3) δ 7.48 (dt, *J* = 8.4, 7.9 Hz, 1H), 7.38–7.29 (m, 2H), 7.28–7.12 (m, 4H), 6.79 (ddd, *J* = 7.9, 2.0, 0.9 Hz, 1H), 6.77–6.69 (m, 2H), 6.50 (ddd, *J* = 8.1, 2.2, 0.5 Hz, 1H), 6.34 (ddd, *J* = 7.8, 2.9, 0.5 Hz, 1H), 3.75 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 162.43 (d, *J* = 237.8 Hz), 160.49, 157.50 (d, *J* = 15.1 Hz), 146.32, 145.06, 141.55 (d, *J* = 8.1 Hz), 130.02, 129.39, 126.62, 125.23, 119.07, 112.63, 110.74, 109.10 (d, *J* = 4.5 Hz), 99.41 (d, *J* = 37.0 Hz), 55.29; 19F NMR (377 MHz, CDCl3) δ −67.46 (d, *J* = 7.3 Hz).

*4-((6-Fluoropyridin-2-yl)(propyl)amino)-2-methoxybenzaldehyde (8).* The reaction was carried out according to the general procedure C. Column chromatography: silica, EtOAc/petroleum ether (PE), 1:2; Yellow oil: 57% yield; TLC: (silica gel, EtOAc/PE, 1:2), Rf = 0.55. 1H NMR (300 MHz, CDCl3) δ 10.35 (s, 1H), 7.82 (d, *J* = 8.4 Hz, 1H), 7.46 (dt, *J* = 8.5, 7.9 Hz, 1H), 6.88 (ddd, *J* = 8.4, 2.0, 0.7 Hz, 1H), 6.82 (d, *J* = 1.9 Hz, 1H), 6.55 (ddd, *J* = 8.1, 2.4, 0.5 Hz, 1H), 6.30 (ddd, *J* = 7.8, 2.9, 0.5 Hz, 1H), 3.98–3.89 (m, 2H), 3.88 (s, 3H), 1.69 (dq, *J* = 14.8, 7.4 Hz, 2H), 0.94 (t, *J* = 7.4 Hz, 3H); 13C NMR (75 MHz, CDCl3) δ 189.19, 163.05 (d, *J* = 244.3 Hz), 161.72, 159.68 (d, *J* = 15.1 Hz), 149.95, 140.12 (d, *J* = 8.1 Hz), 130.33, 118.77, 111.37 (d, *J* = 4.5 Hz), 106.62, 100.47 (d, *J* = 37.0 Hz), 101.76, 55.99, 49.89, 20.95, 11.38; 19F NMR (282 MHz, CDCl3) δ −68.09 (d, *J* = 8.6 Hz).

*4-((6-Fluoropyridin-2-yl)(phenyl)amino)-2-methoxybenzaldehyde (9)*. The reaction was carried out according to the general procedure C. Column chromatography: silica, EtOAc/*n*-hexane, 1:3; Yellow oil: 68% yield; TLC: (silica gel, EtOAc/*n*-hexane, 1:3), Rf = 0.60. 1H NMR (300 MHz, CDCl3) δ 10.32 (s, 1H), 7.73

(d, *J* = 8.5 Hz, 1H), 7.59 (q, *J* = 8.0 Hz, 1H), 7.46–7.37 (m, 2H), 7.33–7.25 (m, 1H), 7.24–7.16 (m, 2H), 6.78 (d, *J* = 2.0 Hz, 1H), 6.69 (ddd, *J* = 8.5, 2.0, 0.8 Hz, 1H), 6.60 (dd, *J* = 8.0, 1.7 Hz, 1H), 6.54–6.46 (m, 1H), 3.77 (s, 3H); 13C NMR (75 MHz, CDCl3) δ 188.37, 162.91 (d, *J* = 235.1 Hz), 162.63,156.61 (d, *J* = 14.9 Hz), 152.14, 144.37, 142.13 (d, *J* = 8.1 Hz), 130.06, 129.50, 127.66, 126.70, 120.65, 116.19, 111.59 (d, *J* = 4.6 Hz), 106.62, 101.86 (d, *<sup>J</sup>* <sup>=</sup> 36.6 Hz), 55.60; 19F NMR (282 MHz, CDCl3) <sup>δ</sup> <sup>−</sup>66.92 (d, *<sup>J</sup>* <sup>=</sup> 8.5 Hz).

*(E)-2-Cyano-3-(4-((6-fluoropyridin-2-yl)(propyl)amino)-2-methoxyphenyl)acrylic Acid (1).* The reaction was carried out according to the general procedure C. Yellow solid: 95% yield; TLC: (silica gel, CHCl3/MeOH/acetic acid (AcOH), 95:5:0.1), Rf = 0.60. 1H NMR (400 MHz, CDCl3/CD3OD) δ 8.63 (s, 1H), 8.30 (d, *J* = 8.7 Hz, 1H), 7.46 (q, *J* = 8.2 Hz, 1H), 6.86 (dd, *J* = 8.7, 2.1 Hz, 1H), 6.74 (d, *J* = 2.1 Hz, 1H), 6.59 (dd, *J* = 8.1, 2.2 Hz, 1H), 6.31 (dd, *J* = 7.8, 2.8 Hz, 1H), 3.93–3.86 (m, 2H), 3.80 (s, 3H), 1.65 (h, *J* = 7.4 Hz, 2H), 0.90 (t, *J* = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3/CD3OD) δ 164.86, 162.72 (d, *J* = 237.5 Hz), 160.52, 156.27 (d, *J* = 15.2 Hz), 151.55, 148.32, 141.46 (d, *J* = 8.3 Hz), 133.13, 130.43, 116.63, 116.39, 108.28 (d, *J* = 4.3 Hz), 106.26, 100.01, 99.02 (d, *J* = 36.9 Hz), 55.72, 51.83, 21.14, 11.14; 19F NMR (376 MHz, CDCl3/CD3OD) δ -68.23 (dt, *J* = 8.3, 2.2 Hz).HR-MS (ESI) *m*/*z*:calcd. for [C18H17FN3O]<sup>+</sup> = 310.1350; found = 310.1332 [M–CO2H]<sup>+</sup>.

*(E)-2-Cyano-3-(4-((6-fluoropyridin-2-yl)(phenyl)amino)-2-methoxyphenyl)acrylic Acid (2).* The reaction was carried out according to the general procedure C. Orange solid: 98% yield; TLC: (silica gel, CHCl3/MeOH/AcOH), 95:5:0.1), Rf = 0.75.1H NMR (400 MHz, CDCl3) δ 8.75 (s, 1H), 8.37 (d, *J* = 8.8 Hz, 1H), 7.65 (q, *J* = 8.0 Hz, 1H), 7.47 (t, *J* = 7.7 Hz, 2H), 7.41–7.17 (m, 3H), 6.80 (d, *J* = 1.9 Hz, 1H), 6.73 (dd, *J* = 8.8, 1.9 Hz, 1H), 6.69–6.61 (m, 1H), 6.58 (dd, *J* = 7.9, 2.7 Hz, 1H), 3.79 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 167.91, 162.31 (d, *J* = 240.5 Hz), 160.76, 156.25 (d, *J* = 14.6 Hz), 152.17, 149.60, 143.99, 142.34 (d, *J* = 8.2 Hz), 130.35, 130.24, 127.87, 127.16, 116.26, 115.94, 115.58, 112.29 (d, *J* = 4.5 Hz), 105.47, 102.54 (d, *J* = 36.5 Hz), 97.31, 55.82; 19F NMR (377 MHz, CDCl3) δ -66.88 (d, *J* = 8.2 Hz).HR-MS (ESI) m/z: calcd. for [C21H15FN3O]<sup>+</sup>. = 344.1194; found = 344.1180 [M–CO2H]<sup>+</sup>.

*N-(3-Methoxyphenyl)-6-nitropyridin-2-amine (12).* The reaction was carried out according to the general procedure A. Column chromatography: silica, EtOAc/*n*-hexane, 1:1; Yellow solid: 80 % yield; TLC: (silica gel, EtOAc/*n*-hexane, 1:1), Rf = 0.65. 1H NMR (400 MHz, CDCl3) δ 7.78–7.70 (m, 1H), 7.63 (dd, *J* = 7.6, 0.6 Hz, 1H), 7.31–7.24 (m, 1H), 7.23–7.19 (m, 1H), 7.14 (dd, *J* = 8.2, 0.6 Hz, 1H), 6.88 (ddd, *J* = 7.9, 2.0, 0.8 Hz, 1H), 6.71 (ddd, *J* = 8.3, 2.5, 0.8 Hz, 1H), 3.86 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 160.29, 155.31, 155.06, 140.46, 139.94, 129.79, 114.77, 112.21, 109.24, 107.35, 105.65, 55.16.

*N-(3-Methoxyphenyl)-6-nitro-N-propylpyridin-2-amine (13).* The reaction was carried out according to the general procedure B. Column chromatography: silica, EtOAc/*n*-hexane, 1:3; Yellow oil: 93% yield; TLC: (silica gel, EtOAc/*n*-hexane, 1:3), Rf = 0.70. 1H NMR (400 MHz, CDCl3) δ 7.53–7.29 (m, 3H), 6.88 (dd, *J* = 8.3, 2.3 Hz, 1H), 6.82 (d, *J* = 7.9 Hz, 1H), 6.79–6.68 (m, 1H), 6.59 (d, *J* = 8.2 Hz, 1H), 3.95 (t, *J* = 7.4 Hz, 2H), 3.82 (s, 3H), 1.69 (h, *J* = 7.4 Hz, 2H), 0.95 (t, *J* = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 161.44, 157.92, 156.25, 145.36, 139.22, 131.23, 120.24, 114.28, 113.93, 112.86, 105.78, 55.72, 52.38, 21.21, 11.68.

*2-Methoxy-4-((6-nitropyridin-2-yl)(propyl)amino)benzaldehyde (14)*. The reaction was carried out according to the general procedure C. Column chromatography: silica, EtOAc/PE, 1:2; Yellow oil: 92% yield; TLC: (silica gel, EtOAc/PE, 1:2), Rf = 0.60. 1H NMR (400 MHz, CDCl3) δ 10.40 (s, 1H), 7.89 (d, *J* = 8.3 Hz, 1H), 7.63–7.53 (m, 2H), 6.94–6.89 (m, 2H), 6.88 (d, *J* = 1.9 Hz, 1H), 4.03 (t, *J* = 7.4 Hz, 2H), 3.91 (s, 3H), 1.74 (h, *J* = 7.4 Hz, 2H), 0.97 (t, *J* = 7.4 Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 188.37, 163.12, 156.66, 155.98, 151.33, 139.56, 130.32, 122.69, 118.05, 115.38, 108.86, 107.32, 55.84, 52.30, 21.08, 11.38.

*(E)-2-Cyano-3-(2-methoxy-4-((6-nitropyridin-2-yl)(propyl)amino)phenyl)acrylic Acid (15).* The reaction was carried out according to the general procedure C. Yellow solid: >98% yield; TLC: (silica gel, CHCl3/MeOH/AcOH), 95:5:0.1), Rf = 0.70. 1H NMR (400 MHz, CDCl3/CD3OD) δ 8.62 (s, 1H), 8.30 (d, *J* = 8.5 Hz, 1H),7.58 (t, *J* = 7.9 Hz, 1H), 7.51 (d, *J* = 7.6 Hz, 1H), 6.92 (d, *J* = 8.3 Hz, 1H), 6.89 (dd, *J* = 8.6, 1.7 Hz, 1H), 6.80 (d, *J* = 1.5 Hz, 1H), 3.98 (t, *J* = 7.4 Hz, 2H), 3.82 (s, 3H), 1.68 (h, *J* = 7.4 Hz, 2H), 0.91 (t, *J* = 7.3 Hz, 3H); 13C NMR (101 MHz, CDCl3/CD3OD) δ 164.39, 160.51, 156.43, 155.70, 150.30, 148.24, 139.62, 130.62, 118.14, 117.66, 116.35, 115.78, 107.90, 107.54, 101.63, 55.82, 52.13, 20.95, 11.16. HR-MS (ESI) *m*/*z* calcd. for [C18H17N4O3]<sup>+</sup>**.** = 337.1295; found = 337.1283 [M–CO2H]<sup>+</sup>**.** .
