Development of ¹⁸F-Labeled Picolinamide Probes for PET Imaging of Malignant Melanoma

Abstract

Melanoma is an aggressive skin cancer with worldwide increasing incidence. Development of positron emission tomography (PET) probes for early detection of melanoma is critical for improving the survival rate of melanoma patients. In this research, ¹⁸F-picolinamides based PET probes were prepared by direct radiofluorination of the bromopicolinamide precursors using no-carrier-added ¹⁸F-fluoride. The resulting probes, ¹⁸F-1, ¹⁸F-2 and ¹⁸F-3 were then evaluated in vivo by small animal PET imaging and biodistribution studies in C57BL/6 mice bearing B16F10 murine melanoma tumors. Noninvasive small animal PET studies demonstrated excellent tumor imaging contrasts for all probes, while ¹⁸F-2 showed higher tumor to muscle ratios than ¹⁸F-1 and ¹⁸F-3. Furthermore, ¹⁸F-2 demonstrated good in vivo stability as evidenced by the low bone uptake in biodistribution studies. Collectively, these findings suggest ¹⁸F-2 as a highly promising PET probe for translation into clinical detection of melanoma.

Introduction

Malignant melanoma is one of the most aggressive and lethal cancers that has increasing incidence world widely, especially in the Caucasian population.^1–2 Its strong tendency to metastasize and absence of effective treatment for advanced diseases result in a poor overall survival.^3–4 Therefore, development of novel and accurate molecular imaging techniques to detect melanoma at its earliest stages is critical for improving the survival of patients that have malignant melanoma.

Over the last a few decades, noninvasive molecular imaging of malignant melanoma with various modalities has been extensively studied; these modalities include radionuclide imaging with positron emission tomography (PET) and single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), computed tomography (CT), and ultrasound (US) imaging. The higher sensitivity of PET and SPECT than the traditional imaging methods such as MRI, CT, and US has attracted more and more research interests on developing nuclear imaging agents for melanoma detection.⁵ Especially, ¹⁸F-Fluoro-deoxyglucose (¹⁸F-FDG) has demonstrated much higher sensitivity and specificity than those obtained by CT, ultrasound, radiography, and liver function tests and histology or clinical follow-up.⁶ Furthermore, ¹⁸F-FDG PET was shown to detect malignant melanoma up to 6 months earlier than those conventional techniques.^7–8 However, because the uptake and cellular retention of ¹⁸F-FDG involve increased glucose metabolism, which also occurs in many other tumor types, or in surgical wounds, pneumonia, and infection/inflammation conditions, ¹⁸F-FDG thus lacks high specificity for melanoma imaging, and false-positive detections often happen.^{6–7, 9}

Nuclear imaging modality (SPECT and PET) heavily rely on probes for monitoring specific molecular targets or pathways in vivo. Nuclear probes with high specificity based on different targeting or molecular recognition mechanisms help to increase the effectiveness of PET or SPECT for melanoma detection. Therefore, many radiolabeled probes for imaging different molecular targets or processes associated with malignant melanoma have been designed and evaluated for melanoma imaging, such as monoclonal antibodies against melanoma-associated antigens,^10–11 iodoamphetamine,¹² α-melanocyte–stimulating hormone peptides,^13–17 benzamide (BZA)-based compounds.^18–20 So far, BZA analogs have been shown to be among the most promising melanoma targeting agents for both diagnosis and therapeutic applications. In vitro cell study demonstrates the high binding affinity of BZA analogs to melanin that presents in melanoma.²¹ Moreover, a clinical study using ¹²³I-labeled BZA for detecting malignant melanoma and its metastases revealed 81% diagnostic sensitivity, 87% accuracy, and 100% specificity.²² Consequently, the ¹⁸F-labeled BZA (¹⁸F-FBZA, Figure 1D) has been developed for PET imaging of melanoma and its metastasis using mice bearing B16F10 melanoma tumors.^23–24 It was reported that the B16F10 tumor uptake at 2 h post injection (p.i.) reached 5.94 ± 1.83 percentage injected dose per gram (%ID/g).²³ However, the multistep radiosynthesis of ¹⁸F-FBZA could be a bottleneck for its large production and potential clinical translation.

Figure 1.

The chemical structures of ¹⁸F-1, ¹⁸F-2, ¹⁸F-3 (¹⁸F-MEL050) and ¹⁸F-FBZA.

Recently the pyridine-based precursor, ¹⁸F-6-fluoro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide (¹⁸F-MEL050, Figure 1C), was successfully synthesized in single step with high radiochemical yield (RCY). This novel PET probe displays excellent performance for imaging of primary and metastatic melanoma.^25–27 In pigmented melanoma B16-F0 xenografts, ¹⁸F-MEL050 exhibits high tumor uptake and tumor-to-background ratio of approximately 50:1 at 2 h p.i. of the probe. The excellent in vivo performance of ¹⁸F-MEL050, plus its easy radiosynthesis, encouraged us to design and biologically evaluate more ¹⁸F-MEL050 analogs for melanoma imaging. More specifically, N-(2-(diethylamino)ethyl)-¹⁸F-4-fluoropicolinamide (shortened as ¹⁸F-1, Figure 1A), and N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide (shortened as ¹⁸F-2, Figure 1B) were synthesized and side-by-side compared with ¹⁸F-MEL050 (shorted as ¹⁸F-3,, Figure 1C) by small animal PET imaging and biodistribution studies in B16F10-tumor bearing mice.

Results

Chemistry and Radiochemistry

The authentic ¹⁹F-fluorine compounds (¹⁹F-1, ¹⁹F-2, and ¹⁹F-3), and their precursors (4, 5, and 6, Scheme 1) for ¹⁸F-fluorination were prepared by condensation of a bromopicolinic acid or bromoniconitic acid with N,N-diethylethylenediamine (DEED) via O-(N-succinimidyl)-1,1,3,3-tetramethyl-uronium tetrafluoroborate (TSTU) activation in the presence of diisopropylethylamine (DIPEA) (Scheme 1 and Supplemental Data). The azeotropically dried ¹⁸F-fluoride can replace the bromo leaving group in each precursor to make the corresponding product in one step. Hence, all of the ¹⁸F-labeled products were prepared within 1 h, including reversed phase high performance liquid chromatography (RP-HPLC) purification and product formulation for further biological evaluation. The resultant products, ¹⁸F-1, ¹⁸F-2, and ¹⁸F-3, were prepared in radiochemical yields of 24.5 ± 6.7%, 9.5 ± 1.9%, and 21.5 ± 15.5% (n = 3, non-decay corrected), respectively. All the ¹⁸F probes were produced in more than 95% radiochemical purity and free of the corresponding bromo-precursors, as demonstrated by quality control analysis (RP-HPLC). The specific activity for these PET probes was in the range of 100–150 GBq/μmol.

Scheme 1.

Synthetic schemes for preparation of the precursors for ¹⁸F-fluorination and ¹⁹F authentic standards.

Small Animal PET Imaging Studies

The tumor-targeting efficacy and imaging property of ¹⁸F-1 and ¹⁸F-2 were evaluated in B16F10 tumor-bearing mice and the results were compared with ¹⁸F-3 which was examined in the same tumor model. In static small animal PET scans, representative coronal images of B16F10 tumor bearing mice (n = 4) at different times after intravenous injection of about 3.7 MBq (100 μCi) of ¹⁸F-1, ¹⁸F-2, or ¹⁸F-3 are shown in Figure 2. B16F10 tumors were clearly visualized with high tumor-to-background contrast at all time points from 0.5 to 2 h. The highest uptakes observed in the kidneys at early time points suggested that these PET probes were mainly excreted through the renal system. Quantification analysis of tumors and other major organ activity accumulation in PET images was done by analyzing the regions of interests (ROIs) that circle the entire organ on the coronal images. The tumor uptakes of ¹⁸F-1 were determined to be 9.52 ± 1.27, 8.97 ± 1.76, 9.73 ± 1.41 %ID/g at 0.5, 1, and 2 h. The tumor uptakes of ¹⁸F-3 were determined to be 7.54 ± 1.60, 7.94 ± 0.96, and 8.47 ± 1.35 %ID/g at 0.5, 1, and 2 h. ¹⁸F-1 and ¹⁸F-3 had much lower tumor uptake compared with ¹⁸F-2, which were 12.74 ± 1.70, 16.61 ± 2.60, and 16.87 ± 1.23 %ID/g at 0.5, 1, and 2 h, respectively (Figure 3 A, B, C).

Figure 2.

Decay-corrected whole-body coronal small animal PET images of C57BL/6 mice bearing B16F10 murine melanomas from a static scan at 0.5, 1, and 2 h after injection of ¹⁸F-1, ¹⁸F-2, and ¹⁸F-3. Tumors are indicated by arrows.

Figure 3.

A, B, C Small animal PET images quantification of tumors and major organs at 0.5 h, 1 h, and 2 h after injection of ¹⁸F-1, ¹⁸F-2, and ¹⁸F-3 respectively (~100 μCi/mouse, n = 4). D. Time–activity curves of tumor and major organs of C57BL/6 mice bearing B16F10 murine melanoma tumors from 35 min dynamic scans after intravenous injection of ¹⁸F-2 (~100 μCi/mouse, n = 4).

The higher tumor uptake of ¹⁸F-2 encouraged us to perform a 35-min dynamic small animal PET scans for this novel probe (n = 4). As shown in Figure 3 D, ¹⁸F-2 was rapidly cleared from renal system as determined by ROI analysis of the kidneys. At 5 min after tail vein injection of ¹⁸F-2, radioactivity was rapidly accumulated in kidneys (24.91 ± 5.29 %ID/g) and decreased to 9.44 ± 4.04 %ID/g at 35 min p.i. In contrast, tumor uptake reached 5.28 ± 1.54 %ID/g at 5 min p.i. and gradually increased to the highest 10.52 ± 1.29 %ID/g at the end of the 35 min dynamic scan. During the whole dynamic scan frames, low levels of liver and muscle uptakes were observed.

Biodistribution Studies

We also performed a biodistribution experiment by direct-sampling tumors and tissues of interest. The results are shown in Table 1. The B16F10 tumor uptakes for ¹⁸F-1 were 7.54 ± 1.64 and 8.66 ± 1.42 % ID/g at 1 and 2 h, respectively, while for ¹⁸F-3, they were 9.54 ± 3.44 and 9.29 ± 2.49 %ID/g at 1 and 2 h p.i.. Both ¹⁸F-1 and ¹⁸F-3 have lower uptake than ¹⁸F-2, which were 15.20 ± 3.37 %ID/g and 16.97 ± 3.28 %ID/g at 1 and 2 h p.i. respectively. Of note, because of the high melanin concentration in C57BL/6 mouse eyes, the uptake of eyes remained at high levels for all three probes. Interestingly, the bone uptakes of ¹⁸F-2 (2.40 ± 0.26 and 1.72 ± 0.45 % ID/g at 1 and 2 h, respectively, n = 4) and ¹⁸F-3 (3.48 ± 0.80 and 2.12 ± 0.38 % ID/g at 1 and 2 h, respectively, n = 4) were significantly lower than those of ¹⁸F-1 (11.29 ± 2.88 and 10.27 ± 3.59 % ID/g at 1 and 2 h, respectively, n = 4) (P < 0.05), which indicated that ¹⁸F-2 and ¹⁸F-3 have better in vivo stability against defluorination. In comparison, ¹⁸F-1 showed significant defluorination in vivo as demonstrated by its high bone uptakes. Moreover, because of the rapid clearance of these ¹⁸F labeled picolinamide probes from normal non-targeted organs, most of the tumor-to-normal tissue ratios increase with time. For examples, the tumor to muscle ratio of ¹⁸F-2 was 13.84 ± 2.38 and 36.79 ± 5.21 at 1 and 2 h, respectively; while for ¹⁸F-3, it was 11.24 ± 2.76 and 15.22 ± 3.91 at 1 and 2 h p.i., respectively (Table 1).

Table 1.

Biodistributions of ¹⁸F-1, ¹⁸F-2, and ¹⁸F-3 in C57BL/6 mice bearing B16F10 murine melanoma tumors at 1 and 2 h p.i. Data are expressed as normalized accumulation of activity in %ID/g ± SD (n = 4).

Organ	18F-1		18F-2		18F-3
	1 h	2 h	1 h	2 h	1 h	2 h
Tumor	7.54 ± 1.64*	8.66 ± 1.42*	15.20 ± 3.37*†	16.97 ± 3.28*†	9.54 ± 3.44†	9.29 ± 2.49†
Blood	0.33 ± 0.08	0.15 ± 0.01*	0.42 ± 0.13	0.19 ± 0.02*	0.46 ± 0.07	0.20 ± 0.01
Heart	0.60 ± 0.07*	0.37 ± 0.04*	1.17 ± 0.26*	0.59 ± 0.04*†	1.54 ± 0.53	0.40 ± 0.06†
Lungs	0.88 ± 0.13*	0.59 ± 0.08	1.58 ± 0.33*	0.69 ± 0.07†	1.52 ± 0.43	0.92 ± 0.13†
Liver	2.07 ± 0.47*	1.41 ± 0.12*	4.71 ± 1.47*	3.32 ± 0.39*†	4.47 ± 2.27	1.07 ± 0.25†
Spleen	1.09 ± 0.87	0.31 ± 0.02	1.78 ± 0.49	1.13 ± 0.87	2.88 ± 1.63	0.56 ± 0.06
Pancreas	1.06 ± 0.36	0.44 ± 0.02*	1.61 ± 0.48	0.61 ± 0.04*	3.58 ± 1.69	0.71 ± 0.07
Stomach	2.24 ± 0.60	1.64 ± 0.59	4.26 ± 2.62	1.47 ± 0.53	5.03 ± 1.78	2.27 ± 0.33
Brain	0.49 ± 0.14*	0.24 ± 0.03*	1.28 ± 0.33*	0.42 ± 0.06*	1.56 ± 0.41	0.41 ± 0.07
Intestine	2.28 ± 0.36	0.98 ± 0.04	2.01 ± 0.47	0.88 ± 0.21	3.46 ± 1.26	0.89 ± 0.53
Kidneys	1.34 ± 0.28	0.69 ± 0.06*	3.32 ± 1.23	1.03 ± 0.09*	5.63 ± 1.72	1.20 ± 0.29
Skin	0.73 ± 0.16*	0.63 ± 0.35	1.30 ± 0.48*	0.68 ± 0.41	2.37 ± 0.64	0.71 ± 0.39
Muscle	0.60 ± 0.14*	0.20 ± 0.03	1.16 ± 0.29*	0.41 ± 0.04*	1.11 ± 0.18	0.52 ± 0.08
Bone	11.29 ± 2.88*	10.27 ± 3.59*	2.40 ± 0.26*	1.72 ± 0.45*	3.48 ± 0.80	2.12 ± 0.38
Eyes	35.96 ± 12.17	32.45 ± 3.67	37.47 ± 2.55	32.83 ± 5.25	34.00 ± 9.19	32.23 ± 7.73
Uptake Ratio
Tumor to Blood	28.32 ± 3.32*	44.33 ± 7.35*	41.44 ± 8.09*†	88.25 ± 9.77*†	20.44 ± 4.34†	39.20 ± 4.66†
Tumor to Lung	8.81 ± 2.44	9.46 ± 1.76*	9.94 ± 1.29†	24.92 ± 6.56*†	6.23 ± 0.84†	10.49 ± 4.33†
Tumor to Liver	3.85 ± 1.32	4.16 ± 1.25	3.79 ± 1.41	5.14 ± 1.03	2.24 ± 0.32	7.26 ± 3.33
Tumor to Muscle	13.12 ± 4.15	27.75 ± 4.24*	13.84 ± 2.38	36.79 ± 5.21*†	11.24 ± 2.76	15.22 ± 3.91†

^*P < 0.05, comparison of biodistribution between ¹⁸F-1 and¹⁸F-2.

^†P <0.05, comparison of biodistribution between ¹⁸F-2 and ¹⁸F-3.

Discussion and Conclusions

¹⁸F (t_1/2 = 109.7 min; β⁺, 99%) is an ideal PET radionuclide for labeling biologically active ligands such as small molecules, peptides or small proteins for PET probe development. The success of ¹⁸F-FDG has made PET a powerful tool in cancer diagnosis, patient stratification, and monitoring the treatment monitoring of cancer patients.²⁸ In the development of melanoma specific-imaging probes other than the generic tumor imaging probes such as ¹⁸F-FDG, ¹²³I labeled BZA compounds have been evaluated in melanoma patients with 100% specificity.²² Because of favorable physical properties of ¹⁸F-fluoride, efforts have been made to develop ¹⁸F-labeled BZA (¹⁸F-FBZA)^23–24 and ¹⁸F-labeled BZA-like ¹⁸F-MEL050 (¹⁸F-3).²⁷ It has been found that ¹⁸F-3 offers high tumor uptakes, fast clearance, and low background.²⁷ The syntheses of ¹⁸F-3 analogs have been optimized recently, however, in vivo tumor targeting efficacy was not reported therein²⁹. To further develop ¹⁸F-3 analogs with better improved in vivo performance, we designed and synthesized ¹⁸F-1 and ¹⁸F-2 herein and compared their in vivo tumor imaging properties with ¹⁸F-3 in the same B16F10 melanoma model.

The authentic standards (1–3) and non-radioactive precursors (4–6) can be readily prepared in high yields from their corresponding acids. The compounds 1–6 were fully characterized using nuclear magnetic resonance (NMR) and electrospray ionization mass spectrometry (ESI-MS). The direct and single step ¹⁸F-fluorination of the precursors can be easily accomplished to achieve the targeted PET probes in good radiochemical yields. Because of the low reactivity of the 3-position, the RCYs of ¹⁸F-2 are lower than the other two radioligands (P < 0.05). However, the radiosynthesis of ¹⁸F-2 can still be easily automated for large quantity synthesis (3.7–37GBq) for potential clinical translation, because of the short synthetic time and the straightforward radiochemistry.

As expected, ¹⁸F-1, ¹⁸F-2, and ¹⁸F-3 all exhibit high tumor targeting efficiency, excellent tumor imaging contrasts, and desirable biodistribution patterns (Figure 2, Table 1). Particularly, ¹⁸F-2 shows significantly lower bone uptakes than that of ¹⁸F-1 and ¹⁸F-3 (P < 0.05) (Table 1). Because of the high reactivity of 2- and 4-positions of pyridine ring towards the nucleophilic aromatic substitution³⁰, some weak nucleophiles, such as water, amino acid or proteins, can potentially replace the ¹⁸F in these two positions. On the contrary, the relative low reactivity of 3-position in the pyridine ring increases the relative in vivo stability of ¹⁸F-2. It is of note that the bone uptake of ¹⁸F-1 is about 10 times higher than those of ¹⁸F-2 or ¹⁸F-3, which suggests ¹⁸F in the 4-position is very unstable (Table 1).

In our previous study, ¹⁸F-FBZA was developed as a melanin-targeting PET probe with a chemical structure similar to that of ¹⁸F-2.²³ However, ¹⁸F-FBZA has a much lower tumor uptake in the B16F10 melanoma tumors than that of ¹⁸F-2 (5.94 ± 1.83 %ID/g for ¹⁸F-FBZA at 2 h p.i. vs. 16.97 ± 3.28 %ID/g for ¹⁸F-2 at 2 h p.i.). Furthermore, a 3-step radiosynthesis of N-succinimidyl 4-¹⁸F fluorobenzoate (¹⁸F-SFB) is needed before direct coupling of the ¹⁸F-SFB with the amine compound to prepare ¹⁸F-FBZA, thus the total radiosynthesis time is typically more than 3 h, compared with only 1 h for preparation ¹⁸F-1 and ¹⁸F-2. In this study, we also show that ¹⁸F-2 is superior to ¹⁸F-3 in terms of tumor uptake, tumor to normal organ ratios (Table 1), and tumor imaging contrast (Figure 2). Overall, ¹⁸F-2 is demonstrated to be an excellent candidate for translation as a clinical PET probe for melanoma diagnosis in term of radiosynthesis, tumor targeting efficiency, and in vivo stability.

In conclusion, we designed and synthesized two novel PET probes and a reported PET probe for melanoma diagnosis based on the picolinamide structure. The small animal PET and biodistribution studies in murine melanoma xenografts resulted in excellent tumor imaging contrast using all of these probes. Especially ¹⁸F-2 shows high in vivo stability and favorable pharmacokinetic properties such as fast clearance from urinal system and almost background level of uptakes for all of the major organs at 2 h. The high selectivity and specificity of ¹⁸F-2, as evidenced by the high tumor-to-non tumor ratios, highlight that ¹⁸F-2 PET has high potential to improve the melanoma detection. All the desirable properties of ¹⁸F-2 warrant large scale production and potential clinical applications of this novel PET probe.

Experimental sections

General

All chemicals obtained commercially were of analytic grade and used without further purification. No-carrier-added ¹⁸F-fluoride was obtained from an in-house PETtrace cyclotron (GE Healthcare). Reversed-phase extraction C18 Sep-Pak cartridges were obtained from Waters and were pretreated with ethanol and water before use. The syringe filter and polyethersulfone membranes (pore size, 0.22 μm; diameter, 13 mm) were obtained from Nalge Nunc International. The semipreparative RP-HPLC using a Vydac protein and peptide column (218TP510; 5μm, 250 × 10 mm) was performed on a Dionex 680 chromatography system with a UVD 170U absorbance detector and model 105S single-channel radiation detector (Carroll & Ramsey Associates). The recorded data were processed using Chromeleon version 6.50 software. With a flow rate of 5 mL/min, the mobile phase was changed from 95% solvent A [0.1% trifluoroacetic acid (TFA) in water] and 5% B [0.1% TFA in acetonitrile (MeCN)] (0–2 min) to 35% solvent A and 65% solvent B at 32 min. Analytical RP-HPLC has the same gradient system except that the flow rate was 1 mL/min with a Vydac protein and peptide column (218TP510; 5 μm, 250 × 4.6 mm). The UV absorbance was monitored at 218 nm and the identification of the small molecules was confirmed based on the UV spectrum acquired using a PDA detector. All synthesized compounds All synthesized compounds showed more than 95% purity (RP-HPLC). Small animal PET scans were performed on a microPET R4 rodent model scanner (Concorde Microsystems Inc.). The scanner has a computer-controlled bed and 10.8-cm transaxial and 8-cm axial fields of view (FOVs). It has no septa and operates exclusively in the 3-dimensional (3D) list mode. Animals were placed near the center of the FOV of the scanner, where the highest image resolution and sensitivity are available.

Chemistry and Radiochemistry

Preparation of ¹⁹F-1, ¹⁹F-2, ¹⁹F-3 and their bromo-precursor 4, 5, 6 (Scheme 1 and Supporting Information)

The same protocol was used for preparation of ¹⁹F-1, ¹⁹F-2, and ¹⁹F-3. As an example, ¹⁹F-1 was synthesized as following. To a solution of 4-fluoropicolinica acid (5.0 mg, 35.5 μmol) in 200 μL of N,N-dimethylformamide (DMF) was added TSTU (10.0 mg, 33.0 μmol) and 20 μL of DIPEA. After incubating at 60 °C for 3 h, the reaction mixture was cooled down to room temperature, followed by addition of N, N-diethylethylenediamine (7.0 mg, 60 μmol). After 2 h, the mixture was diluted with 1 mL 5% acetic acid solution. The product ¹⁹F-1 was isolated by semi-preparative RP-HPLC. The collected fractions were combined and acetonitrile was removed under reduced pressure. The final product was obtained by lyophilization.

N-(2-(diethylamino)ethyl)-4-fluoropicolinamide (¹⁹F-1)

The product was obtained as white powder in the yield of 56% and with 98% purity as determined by RP-HPLC. ESI-MS: m/z 240.3 [M+H]⁺ (C₁₂H₁₉FN₃O, calculated molecular weight: 240.2); ¹H NMR (CDCl₃, 300 MHz): δ = 8.94 (br, 1H), 8.57 (dd, J = 5.2 Hz, 9.0 Hz, 1H), 7.83 (dd, J = 2.4 Hz, 9.0 Hz, 1H), 7.15 (dd, J = 2.4 Hz, 5.2 Hz, 1H), 3.93 (t, J = 6.1 Hz, 2H), 3.29 (t, J = 5.9 Hz, 2H), 3.22 (q, J = 8.6 Hz, 4H), 1.37 (t, J = 7.1 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 167.9, 164.6 (d, J_C,F = 101.8 Hz), 152.5, 151.2 (d, J_C,F = 6.6 Hz), 114.2 (d, J_C,F = 16.5 Hz), 110.4 (d, J_C,F = 18.7 Hz), 51.2, 47.3, 35.0, 8.4.

N-(2-(diethylamino)ethyl)-5-fluoropicolinamide (¹⁹F-2)

The product was obtained as white powder in the yield of 75% and with 98% purity as determined by RP-HPLC. ESI-MS: m/z 240.5 [M+H]⁺ (C₁₂H₁₉FN₃O, calculated molecular weight: 240.2); ¹H NMR (CDCl₃, 300 MHz): δ = 11.35 (br, 1H), 8.41 (d, J = 2.7 Hz, 1H), 8.17 (dd, J = 4.4, 8.2 Hz, 1H), 7.45 (dd, J = 2.7, 8.2 Hz, 1H), 3.88 (t, J = 6.1 Hz, 2H), 3.38–3.08 (m, 6H), 1.36 (t, J = 7.3 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 164.9, 161.5 (d, J_C,F = 38.5 Hz), 161.4 (d, J_C,F = 261.5 Hz), 145.2 (d, J_C,F = 4.5 Hz), 137.3 (d, J_C,F = 25.4 Hz), 124.0 (d, J_C,F = 5.4 Hz), 51.5, 47.2, 34.8, 8.3.

N-(2-(diethylamino)ethyl)-6-fluoronicotinamide (¹⁹F-3)

The product was obtained as white powder in the yield of 62% and with 97% purity as determined by RP-HPLC. ESI-MS: m/z 240.3 [M+H]⁺ (C₁₂H₁₉FN₃O, calculated molecular weight: 240.2); ¹H NMR (CDCl₃, 300 MHz): δ = 11.00 (br, 1H), 8.80 (d, J = 1.7 Hz, 1H), 8.33 (dd, J = 1.7, 8.5 Hz, 1H), 6.99 (dd, J = 1.7, 8.5 Hz, 1H), 3.82 (t, J = 3.9 Hz, 2H), 3.37 (t, J = 3.9 Hz, 2H), 3.23 (q, J = 7.3 Hz, 2H), 1.35 (t, J = 7.3 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 167.0, 164.6 (d, J_C,F = 137.4 Hz), 148.7 (d, J_C,F = 15.9 Hz), 140.4 (d, J_C,F = 9.3 Hz), 127.1 (d, J_C,F = 4.4 Hz), 109.5 (d, J_C,F = 37.4 Hz), 52.4, 48.6, 35.8, 8.6.

4-bromo-N-(2-(diethylamino)ethyl)picolinamide (4)

The product was obtained as white powder in the yield of 82% and with 97% purity as determined by RP-HPLC. ESI-MS: m/z 300.4 [M+H]⁺ (C₁₂H₁₉BrN₃O, calculated molecular weight: 300.1). ¹H NMR (CDCl₃, 300 MHz): δ = 8.97 (br, 1H), 8.40 (d, J = 5.2 Hz, 1H), 8.28 (d, J = 1.7 Hz, 1H), 7.61 (dd, J = 1.7, 3.2 Hz, 1H), 3.91 (t, J = 3.7 Hz, 2H), 3.39 (t, J = 3.7 Hz, 2H), 3.23 (q, J = 7.1 Hz, 4H), 1.34 (t, J = 7.1 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 164.5, 150.2, 149.5, 134.3, 129.8, 125.8, 51.2, 47.1, 34.9, 8.3.

5-bromo-N-(2-(diethylamino)ethyl)picolinamide (5)

The product was obtained as white powder in the yield of 90% and with 98% purity as determined by RP-HPLC. ESI-MS: m/z 300.4 [M]⁺ (C₁₂H₁₉BrN₃O, calculated molecular weight: 300.1). ¹H NMR (CDCl₃, 300 MHz): δ = 8.90 (br, 1H), 8.62 (s, 1H), 8.01–7.95 (m, 1H), 7.61 (dd, J = 1.7, 3.2 Hz, 1H), 3.89 (t, J = 5.8 Hz, 2H), 3.26 (t, J = 5.8 Hz, 2H), 3.25 (q, J = 7.3 Hz, 4H), 1.34 (t, J = 7.3 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 165.1, 149.9, 147.4, 140.0, 124.6, 123.5, 51.4, 47.3, 34.8, 8.3.

6-bromo-N-(2-(diethylamino)ethyl)nicotinamide (6)

The product was obtained as white powder in the yield of 78% and with 97% purity as determined by RP-HPLC. ESI-MS: m/z 300.2 [M+H]⁺ (C₁₂H₁₉BrN₃O, calculated molecular weight: 300.1). ¹H NMR (CDCl₃, 300 MHz): δ = 9.38 (br, 1H), 8.92 (s, 1H), 8.08 (d, J = 8.3 Hz, 1H), 7.55 (d, J = 8.3 Hz, 1H), 3.84 (m, 2H), 3.34 (m, 2H), 3.21 (m, 4H), 1.35 (t, J = 7.1 Hz, 6H). ¹³C NMR (CDCl₃, 75 MHz): δ = 165.8, 150.1, 145.7, 137.2, 128.0, 123.8, 52.3, 48.6, 35.7, 8.

Radiochemistry

An aqueous ¹⁸F-fluoride solution (15 – 30 mCi) was added to a 10 mL vial containing anhydrous acetonitrile (1 mL), K_2.2.2 (15 mg) and K₂CO₃ (3 mg). The solvent was evaporated under a stream of argon at 100 °C under vacuum to produce the K¹⁸F-K_2.2.2 complex. This azeotropic drying was repeated twice by anhydrous acetonitrile (2 × 1 mL). The bromo-precursor (3, 4, or 5) (5 mg) was dissolved in anhydrous DMSO (200 μL) and added to the dried K¹⁸F-K_2.2.2 complex. The reaction was stirred and heated at 110 °C for 10 min and cooled down to room temperature. The mixture was then diluted with 1 mL of 5% acetic acid solution for RP-HPLC purification. The collected radioactive peak was dried using a rotary evaporator and the radiolabeled products were reconstituted in phosphate buffered saline (PBS, 0.1 M, pH = 7.4) and passed through a 0.22 μm Millipore filter into a sterile vial for in vitro and in vivo experiments. The radiochemical yields were calculated based on the obtained radioactive product divided by the activity loaded into the reaction vessel.

Cell Culture

B16F10 cells were cultured in Dulbecco’s modified Eagle high-glucose medium (Gibco Life Sciences) supplemented with 10% fetal bovine serum with penicillin and streptomycin. The cells were regularly maintained in a 37 °C, 5% CO₂ humidified incubator.

Animal Biodistribution Studies

Animal procedures were performed according to a protocol approved by the Stanford University Institutional Animal Care and Use Committee. All of the animals were purchased from Charles River Laboratory. Approximately, cultured B16F10 cells (1.0 × 10⁶) were suspended in PBS and subcutaneously implanted in the right shoulders of C57BL/6 mice. Tumors were allowed to grown to a size of 0.5 cm (~ 10 day) before use. For biodistribution studies, the tumor bearing mice (n = 4 for each group) were injected with about 3.7 MBq (100 μCi) of ¹⁸F-1, ¹⁸F-2, or ¹⁸F-3, through the tail vein and sacrificed at 1.0, and 2.0 h p.i.. Tumor and normal tissues of interest were removed and weighed, and their radioactivity was measured in a gamma-counter. The radioactivity uptake in the tumor and normal tissues was calculated as % ID/g.

Small Animal PET Imaging

For dynamic scan, B16F10 tumor–bearing mice (n = 4) were injected via the tail vein with approximately 3.7 MBq (100 μCi) of ¹⁸F-2, and scans (6 × 20 sec, 8 × 60 sec, 10 × 150 sec, total of 24 frames) were started roughly 2.0 min after the injection of the probe and continued for 35 min. For static scans, the mice bearing B16F10 (n = 4 for each probe), tumor xenografts were injected with about 3.7 MBq (100 μCi) of ¹⁸F-1, ¹⁸F-2, or ¹⁸F-3, via the tail vein. At 0.5, 1, and 2 h p.i., the mice were anesthetized with isoflurane (5% for induction and 2% for maintenance in 100% O₂) using a knock-down box. With the help of a laser beam attached to the scanner, the mice were placed in the prone position and near the center of the field of view of the scanner. The 3-min static scans were then obtained. Images were reconstructed using a 2-dimensional ordered-subsets expectation maximization (OSEM) algorithm. No background correction was performed. ROIs (5 pixels for coronal and transaxial slices) were drawn over the tumor on decay-corrected whole-body coronal images. The maximum counts per pixel per minute were obtained from the ROI and converted to counts per milliliter per minute using a calibration constant. On the basis of the assumption of a tissue density of 1 g/ml, the ROIs were converted to counts per gram per min. Image ROI-derived %ID/g values were determined by dividing counts per gram per minute by injected dose. No attenuation correction was performed.

Statistical Analysis

Quantitative data are expressed as mean ± SD. Means were compared using the Student t test. P values of < 0.05 were considered statistically significant.

Abbreviations

PET

positron emission tomography

BZA

benzamide

%ID/g

percentage injected dose per gram

¹⁸F-1

N-(2-(diethylamino)ethyl)-¹⁸F-4-fluoropicolinamide

¹⁸F-2

N-(2-(diethylamino)ethyl)-¹⁸F-5-fluoropicolinamide

¹⁸F-3(¹⁸F-MEL050)

¹⁸F-6-fluoro-N-[2-(diethylamino)ethyl] pyridine-3-carboxamide

RCYs

radiochemical yields