Synthesis and Spectroscopic Characterization of Novel Thiourea-Bearing Photoactivatable NADPH Mimics Targeting NO Synthases

Abstract

A new set of photoactivatable NADPH mimics bearing a thiourea linkage between a diarylbutadiene and an adenosine moiety functionalized by O-carboxymethyl groups has been designed and synthesized in a convergent strategy. These compounds display absorption and fluorescence emission maxima in DMSO (λ_max,abs = 390 nm and λ_max,em = 460 nm, respectively) consistent with the previously described analogues, with good fluorescence quantum yields (Φ_F = 0.35–0.36), as well as two-photon absorption (σ² = 10.1 GM at λ_max,exc = 780 nm). These molecules could be useful photosensitive tools for biological studies, especially for cellular studies of nitric oxide synthases.

Nitric oxide (NO) is known as a major signalling molecule in various biological processes, starting from Ignarro’s demonstration of its undistinguishable properties with respect to endothelium-derived relaxing factor (EDRF).[1] NO is involved in smooth muscle relaxation induced by nitrovasodilators, some of which have been clinically used for more than 150 years in the treatment of cardiac pathologies.[2] NO was shown to play a key role in a complex signal transduction pathway,[3] related to p53 upregulation and able to trigger apoptosis.[4] Numerous physiopathologies have been correlated with endogenous or exogenous release of NO, such as cardiovascular diseases, renal dysfunction, obesity, erectile dysfunction or cancer.[5] [6] In order to further elucidate the bioprocesses regulated by NO and their underlying mechanism, light-triggered approaches are the most promising, as light could achieve the most precise space–time control. Several strategies towards light-induced NO release have been developed,[7–9] but one of particular interest relies on photoactivatable mimics of NADPH, called nanotriggers (NT).[10] These compounds are able to competitively bind to the reductase domain of nitric oxide synthases (NOSs)[11] and trigger its catalytic transformation upon photoinduced electron transfer.[12] [13] Our group has recently developed a new generation of these analogues bearing a functionalized adenosine moiety for the recognition of the enzyme reductase domain alongside a diarylbutadiene conjugated structure as an electron-donating component, both linked by a rigid triazole moiety.[14] A carboxymethyl moiety at position 3′ was found to be a good bioisostere for the natural 2′-O-phosphate in NADPH.[14] [15] To increase the chemical diversity of photoactive NADPH mimics, we decided to synthesize new nanotriggers bearing a more flexible thiourea function between the electron-donating diarylbutadiene and the adenosine moiety (Figure [1]). Our new design based on a thiourea linker is motivated by its well-established common framework in a variety of drugs and bioactive compounds, its easy synthesis through an isothiocyanate intermediate compared to the urea analogue, and formation of stable hydrogen bonding with the biological partner.[16]

The target nanotriggers were obtained via convergent synthesis by formation of a thiourea linkage between O-alkylated adenosines and a diarylbutadiene moiety, bearing respectively an amine and an isothiocyanate functional group. In order to investigate the influence of the carboxymethyl group at positions 2′ and 3′, we prepared four derivatives with or without the carboxymethyl group at those positions.

The desired recognition moieties were synthesized as shown in Scheme [1]. Introducing an azido group at position 5′ of adenosine was achieved in 93% yield by an S_N2 reaction of the known chloride 1 [17] with NaN₃ in the presence of NaI in DMF at 110 °C. This method has two advantages compared to the method reported by Comstock and Rajski,[18] by using the Mitsunobu reaction to introduce the azido function on 2′,3′-O-isopropylideneadenosine 3, followed by the deprotection of 4: easy purification compared to tedious consecutive column chromatography steps to remove the triphenylphosphine oxide side product, and no risk of de-N-glycosylation during the acidic deprotection step. We have observed that the deprotection of the isopropylidene moiety in a mixture of water and TFA (1:10) gave a quantitative yield if the mixture was carefully evaporated at a temperature never rising above 20 °C. Under a few higher degrees, the yield would drop drastically (to around 50% for a temperature of 27 °C). Subsequent O-alkylation of 5′-azido-5′-deoxyadenosine 2 with tert-butyl bromoacetate led to a mixture of O-alkylated products 5a, 5b and 5c which can be easily separated by column chromatography over silica gel.[14] The four prepared 5′-azido-5′-deoxyadenosines were then reduced to their respective primary amines 6a, 6b, 6c and 6d by hydrogenation using palladium on carbon (for 6d) or by Staudinger reduction with supported triphenylphosphine in the presence of 11 equivalents of water in anhydrous THF (for 6a–c) in high yield.[19]

The diarylbutadiene moiety was prepared through the Wittig reaction of aldehyde 7 [20] with tributyl(1,3-dioxolan-2-ylmethyl)phosphonium bromide (8) under basic conditions, followed by selective cleavage of the isopropylideneacetal over silica gel in the presence of a catalytic amount of AcOH, leading to compound 9 [20] in 88% yield (Scheme [2]). Subsequent Horner–Wadsworth–Emmons olefination with phosphonate 10 [21] furnished diarylbutadiene 11 (93% yield) which was then converted into azide 12 via the mesylate intermediate. To facilitate the final one-step total deprotection, the benzoyl protecting group on the amine was transformed into the acid-labile Boc group through Boc protection of 12 with Boc₂O/DMAP followed by hydrazinolysis, affording Boc derivative 13 in 77% yield over two steps. For the formation of isocyanate, we chose a one-pot procedure employing supported PPh₃ in the presence of an excess of CS₂ (Staudinger’s reduction of the azide to iminophosphorane, followed by reaction with CS₂), affording isothiocyanate 14 in 92% yield. Then, the protected nanotriggers could be readily obtained through a simple coupling of the amines 6a, 6b, 6c and 6d with isothiocyanate 14 in THF. Compounds 15a, 15b, 15c and 15d could not be easily purified and required an optimized elution (a mixture of MeCN and H₂O) to be efficiently purified over C18 silica gel. Nonetheless, the cleavage of the protecting groups under acidic conditions could be performed all at once. Compounds 15a, 15b and 15c could be deprotected using TFA/CH₂Cl₂ (1:9) in 2 hours, while 15d required harsher conditions such as TFA/EtOH (9:1) for 2 hours, leading to the desired nanotriggers 16a, 16b, 16c and 16d, respectively.

The final objective would be to use these molecules in cellular experiments towards enzymatic studies, and as such the difference in their photophysical properties when enzyme-bound or free is crucial knowledge. Thus, the synthesized molecules were spectroscopically characterized in DMSO and Tris buffer pH 7.4, mimicking solvation of median and high polarity, respectively.[22] The four compounds displayed very similar properties in DMSO, as presented in Figure [2], with respect to their maxima of absorption (λ_max,abs = 390 nm) and fluorescence emission (λ_max,em = 462–463 nm), molar absorption coefficient ε (26000–29000 L·mol^–1·cm^–1) and fluorescence quantum yield Φ_F (0.35–0.36). The two-photon absorption property of 16c, bearing the carboxymethyl moiety at position 3′ position, was also measured in DMSO, with σ² = 10.1 GM at λ_max,exc = 780 nm (Figure [3]).

On the other hand, their absorption properties displayed a massive discrepancy in Tris buffer pH 7.4 at 20 mM, as shown in Figure [4]. The concentration of the compounds drastically modified the absorption for 16b, 16c and 16d. The notable isosbestic point at 335 nm would suggest the existence of an equilibrium of free and discrete molecular associates, whose relative amount would depend on charge properties of the compounds. Dynamic light scattering experiments at different concentrations (up to 80 μM) did not show the presence of any high molecular weight species or aggregates.[23]

The self-association of 16d, which does not bear any charged moiety at pH 7.4, is supported by the large dependency of its absorption spectrum on concentration. The presence of one carboxylate group in 16b and 16c significantly reduces the effect of concentration, which could be assigned to the electrostatic repulsion between the negatively charged moieties, thereby disfavoring the formation of molecular associates. This hypothesis is confirmed by the observation that the absorption spectrum of 16a, containing two carboxylate moieties, remains independent on concentration, suggesting its sole existence as a free molecular species. This latter feature would not be an issue towards cellular experiments as it is a reversible equilibrium, especially with the concentrations used (below 10 μM). The fluorescence quantum yield measured for 16a in Tris buffer is a tenth of the one measured in DMSO, enabling enzymatic imaging as described for previous nanotriggers.[15] Additionally, a 32 nm increase in Stokes shift supports a higher rate of vibrational relaxation and energy dissipation via solvent interaction, decreasing Φ_F compared to DMSO. The spectroscopic characterizations on 16a, 16b, 16c and 16d in DMSO and Tris buffer are summarized in Table [1].

In summary, four new photoactivatable NADPH mimics bearing a thiourea linkage were obtained through convergent synthesis, and their photophysical properties were investigated in DMSO and Tris buffer. Like the second generation of nanotriggers, these new compounds display good one-photon fluorescence properties in DMSO under excitation at 390 nm, and two-photon absorption properties compatible with studies in the cellular context. Their good spectroscopic properties combined with the presence of a more flexible thiourea linker suggest that these new compounds could constitute interesting photoactivatable NOS-dependent NO generators. Photooxidation properties of these new analogues upon NOS binding as well as their photoactivation effects in the cellular context will be further studied.

Commercial solvents and reagents were used without purification. Reactions conducted under anhydrous conditions were carried out under an argon or nitrogen atmosphere with glassware dried in a 110 °C oven or with a heat gun. Anhydrous solvents were obtained via an MBraun MB-SPS-800 purification system, giving access to anhydrous CH₂Cl₂, DMF, MeCN, THF and toluene. TLC analysis was conducted on SDS silica gel or C18 silica gel coated aluminum plates (Merck) with a fluorescent indicator absorbing at 254 nm. Visualization was performed by either irradiation with a UV lamp at 254 nm or 365 nm, or dipping in solutions of sulfuric acid, ninhydrin, vanillin or potassium permanganate, alongside the use of a heat gun to fully visualize the plates. R_f values were measured as a ratio of the length between the highest intensity within a spot over the total distance of eluent migration. Purifications by column chromatography were carried out on SDS silica gel 40–63 μm, or on a CombiFlash Rf apparatus (Teledyne ISCO) with the respective pre-packed silica gel columns. Pre-columns were packed with 70–200 μm silica gel (Macherey-Nagel). The O-alkylated compounds 5a, 5b, 5c and 5d were prepared as previously described.[14] NMR spectra were recorded on a JEOL ECS400 spectrometer (400 MHz for ¹H and 101 MHz for ¹³C). Chemical shifts, δ, are expressed in ppm using TMS as a reference if available or the deuterated solvent residual peak.[26] Deuterated solvents were obtained from Eurisotop. HRMS analysis was carried out externally by Cyril Colas, HRMS platform, ICOA, Orléans (FR2708 CBM-ICOA). Absorbance spectra were recorded on a Cary-100, Cary-4000 or Cary-5000 spectrophotometer (Agilent). Fluorescence spectra were recorded on a Fluoromax-3, Fluoromax-4 or Fluorolog-3 spectrofluorometer (Jobin Yvon). Solvents used were spectroscopic grade. The weights of samples were measured with a MYA 4Y.P Plus microbalance (Radwag). Fusion temperatures were measured with a Wagner & Munz Kofler WME system, calibrated with Reichert test substances. FTIR spectra were recorded on a Thermo Fisher FTIR Nexus 670 spectrometer equipped with an ATR module. Absorption bands were interpreted using published references;[27] the following abbreviations are used: str (stretching), asym str (asymmetric stretching), def (deformation). Samples 16a, 16b, 16c and 16d were also characterized by DLS (dynamic light scattering) on a Malvern Zetasizer Nano S analyzer at concentrations ranging from 60 μM to 80 μM. Measurements were done at two time points, right after the sample preparation and 5 hours later. The two-photon absorption cross-section (σ²) of 16c was measured according to Nguyen et al.[14] The two-photon excitation spectrum was monitored by measuring the fluorescence emission spectrum of 16c in the 400–700 nm range (λ_max,em = 463 nm) as a function of NIR wavelength (from 740 to 880 nm). The concentration of 16c was 20 μM in DMSO. The σ² value of 16c was calculated using coumarin-102 in MeOH as a reference (Φ_F = 0.88 and σ² _{780 nm} = 26).[28]

Amines 6 by Reduction of O-Alkylated 5′-Azido-5′-deoxyadenosines

To a solution of 4 in EtOH (0.1 M) was added Pd/C (10 wt %). Hydrogen (1 bar) was introduced in the flask via 3 cycles of vacuum/hydrogen, and the reaction mixture was stirred overnight.

To a solution of 5a, 5b or 5c in anhydrous THF (0.075 M) was added supported PPh₃ (1.2 equiv) and H₂O (11 equiv) under argon.

The reaction progress was monitored by TLC (CH₂Cl₂/EtOH, 95:5) and, upon completion, the reaction mixture was filtered and solvents were removed under reduced pressure to obtain 6a (100%), 6b (100%), 6c (85%) or 6d (99%), which were used without further purification.

5′-Amino-2′,3′-di-O-(tert-butoxycarbonylmethyl)-5′-deoxyadenosine (6a)

R_f = 0.09 (CH₂Cl₂/EtOH, 95:5), 0.32 (CH₂Cl₂/EtOH, 80:20).

T _f = 65 °C (dec).

IR (ATR): 3329 (N–H str), 3183 (N–H str), 1741 (C=O ester str), 1644 (C–N= str), 1598 cm^–1 (N–H ArNH₂ def).

¹H NMR (400 MHz, DMSO-d₆): δ = 8.47 (s, 1 H, H₂), 8.13 (s, 1 H, H₈), 7.32 (bs, 4 H, 2 NH₂), 6.08 (d, J = 5.7 Hz, 1 H, H_1′), 4.87 (dd, J = 5.7, 4.5 Hz, 1 H, H_2′), 4.36–4.27 (m, 2 H, H_3′, H_4′), 4.25–4.21 (m, 2 H, CH₂), 4.21–4.17 (m, 2 H, CH₂), 3.49–3.42 (m, 2 H, 2 H_5′), 1.43 (s, 9 H, t-Bu), 1.33 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, DMSO-d ₆): δ = 169.26 (C_q), 168.80 (C_q), 156.20 (C_q), 152.60 (CH), 149.07 (C_q), 140.11 (CH), 119.46 (C_q), 86.20 (CH), 80.94 (C_q), 79.01 (CH), 77.71 (CH), 67.10 (CH₂), 60.73 (CH₂), 60.30 (CH₂), 27.77 (3 CH_{3 t-Bu}), 27.58 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₂H₃₅N₆O₇: 495.2562; found: 495.2564; m/z [M + Na]⁺ calcd for C₂₂H₃₄N₆NaO₇: 517.2381; found: 517.2380.

5′-Amino-2′-O-(tert-butoxycarbonylmethyl)-5′-deoxyadenosine (6b)

R_f = 0.06 (CH₂Cl₂/EtOH, 95:5), 0.16 (CH₂Cl₂/EtOH, 80:20).

T _f = 89 °C (dec).

IR (ATR): 3331 (O–H, N–H str), 3181 (N–H str), 1740 (C=O ester str), 1646 (C–N= str), 1599 cm^–1 (N–H ArNH₂ def).

¹H NMR (400 MHz, DMSO-d ₆): δ = 8.45 (s, 1 H, H₂), 8.13 (s, 1 H, H₈), 7.30 (bs, 4 H, 2 NH₂), 6.10–6.04 (m, 1 H, H_1′), 5.34–5.28 (m, 1 H, H_2′), 4.74–4.64 (m, 1 H, H_3′), 4.39–4.29 (m, 1 H, H_4′), 4.18–4.08 (m, 2 H, CH₂), 3.52–3.41 (m, 2 H, 2 H_5′), 1.31 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CD₃OD): δ = 171.89 (C_q), 157.55 (C_q), 153.94 (CH), 150.77 (C_q), 142.55 (CH), 121.12 (C_q), 89.23 (CH), 86.79 (CH), 83.42 (C_q), 83.41 (CH), 71.90 (CH), 69.68 (CH₂), 44.56 (CH₂), 28.31 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₆H₂₅N₆O₅: 381.1881; found: 381.1885; m/z [M + Na]⁺ calcd for C₁₆H₂₄N₆NaO₅: 403.1700; found: 403.1707.

5′-Amino-3′-O-(tert-butoxycarbonylmethyl)-5′-deoxyadenosine (6c)

R_f = 0.05 (CH₂Cl₂/EtOH, 95:5), 0.11 (CH₂Cl₂/EtOH, 80:20).

¹H NMR (400 MHz, CD₃OD): δ = 8.27 (s, 1 H, H₂), 8.17 (s, 1 H, H₈), 5.95 (d, J = 5.5 Hz, 1 H, H_1′), 4.88–4.84 (m, 1 H, H_2′), 4.30–4.18 (m, 4 H, CH₂, H_3′, H_4′), 3.04–2.97 (m, 2 H, 2 H_5′), 1.51 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, DMSO-d ₆): δ = 169.64 (C_q), 156.11 (C_q), 152.56 (CH), 149.44 (C_q), 140.09 (CH), 119.30 (C_q), 87.28 (CH), 84.08 (CH), 80.90 (C_q), 79.22 (CH), 72.37 (CH), 67.70 (CH₂), 43.61 (CH₂), 27.79 (3 CH_{3 t-Bu}).

5′-Amino-2′,3′-O-isopropylidene-5′-deoxyadenosine (6d)

R_f = 0.11 (EtOAc/EtOH, 70:30).

T _f = 160 °C (dec).

IR (ATR): 3299 (N–H str), 3147 (N–H str), 1646 (C–N= str), 1602 cm^–1 (N–H ArNH₂ def).

¹H NMR (400 MHz, CD₃OD): δ = 8.28 (s, 1 H, H₂), 8.21 (s, 1 H, H₈), 6.19–6.06 (m, 1 H, H_1′), 5.45–5.40 (m, 1 H, H_2′), 5.07–4.92 (m, 1 H, H_3′), 4.24–4.08 (m, 1 H, H_4′), 2.81–2.67 (m, 2 H, 2 H_5′), 1.60 (s, 3 H, CH₃), 1.38 (s, 3 H, CH₃).

¹³C NMR (101 MHz, DMSO-d ₆): δ = 156.15 (C_q), 152.73 (CH), 148.96 (C_q), 139.99 (CH), 119.17 (C_q), 113.17 (C_q), 89.12 (CH), 86.68 (CH), 82.71 (CH), 81.60 (CH), 43.50 (CH₂), 27.06 (CH₃), 25.24 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₁₃H₁₉N₆O₃: 307.1513; found: 307.1514; m/z [M + 2 H]²⁺ calcd for C₁₃H₂₀N₆O₃: 154.0793; found: 154.0798.

N-(4-((1E,3E)-4-(4-(Methyl(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)amino)phenyl)buta-1,3-dien-1-yl)phenyl)benzamide (11)

Aldehyde 9 (2.00 g, 6.91 mmol) and phosphonate 10 (2.64 g, 7.61 mmol) were added to a flask under an argon atmosphere, then anhydrous THF (70 mL) was added. NaH (60%; 1.10 g, 27.5 mmol) was then slowly added by portion, waiting for hydrogen release to slow down before each addition. The reaction mixture was stirred for 4 d until completion, and the reaction progress was monitored by TLC (petroleum ether/EtOAc, 70:30) after extracting a small amount of the mixture with CH₂Cl₂ and a saturated solution of NH₄Cl. Upon completion, a saturated solution of NH₄Cl (25 mL) was slowly added to the reaction mixture, which was then stirred until the hydrogen release stopped. Organic solvents were then removed under reduced pressure, and deionized H₂O (25 mL) was added to redissolve some precipitated NH₄Cl. CH₂Cl₂ was added to the mixture, the flask was sonicated, then phases were separated, and this operation was repeated until no solid residue was left. Organic layers were combined (750 mL), dried over MgSO₄, and filtered on cotton, and solvents were removed under reduced pressure, yielding 3.11 g (93%) of compound 11 as a brown solid.

R_f = 0.35 (petroleum ether/EtOAc, 70:30).

T _f = 177 °C.

IR (ATR): 3299 (N–H str), 3148 (N–H str), 1678, 1646 (C–N= str), 1603 cm^-1 (N–H Ar-NH₂ def).

¹H NMR (400 MHz, CDCl₃): δ = 7.86 (s, 1 H, NH), 7.85–7.81 (m, 2 H, 2 H_Bz), 7.59 (d, J = 8.5 Hz, 2 H, 2 H_Ar), 7.56–7.50 (m, 1 H, H_Bz), 7.50–7.43 (m, 2 H, 2 H_Bz), 7.40 (d, J = 8.5 Hz, 2 H, 2 H_Ar), 7.30 (d, J = 8.9 Hz, 2 H, 2 H_Ar), 6.89 (dd, J = 15.3, 10.4 Hz, 1 H, Ar-CH=CH-), 6.75 (dd, J = 15.3, 10.4 Hz, 1 H, Ar-CH=CH-), 6.67 (d, J = 8.9 Hz, 2 H, 2 H_Ar), 6.58 (d, J = 15.4 Hz, 1 H, Ar-CH=CH-), 6.53 (d, J = 15.4 Hz, 1 H, Ar-CH=CH-), 4.59–4.54 (m, 1 H, O-CH_THP), 3.93–3.83 (m, 1 H, N-CH ₂-CH₂-O), 3.83–3.75 (m, 1 H, N-CH ₂-CH₂-O), 3.62–3.51 (m, 3 H, N-CH₂-CH ₂-O, O-CH_{2 THP}), 3.49–3.42 (m, 1 H, N-CH₂-CH ₂-O), 3.01 (s, 3 H, N-CH₃), 1.78–1.73 (m, 1 H, CH_{2 THP}), 1.72–1.63 (m, 1 H, CH_{2 THP}), 1.56–1.44 (m, 4 H, CH_{2 THP}).

¹³C NMR (101 MHz, CDCl₃): δ = 165.81 (C_q), 148.94 (C_q), 136.92 (C_q), 135.14 (C_q), 134.54 (C_q), 133.31 (CH), 132.01 (CH), 129.74 (CH), 129.63 (CH), 128.96 (CH), 127.78 (CH), 127.22 (CH), 126.89 (CH), 125.71 (C_q), 125.16 (CH), 120.47 (CH), 112.21 (CH), 99.19 (CH), 64.99 (CH₂), 62.35 (CH₂), 52.50 (CH₂), 39.18 (CH₃), 30.80 (CH₂), 25.57 (CH₂), 19.55 (CH₂).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₁H₃₅N₂O₃: 483.2642; found: 483.2642; m/z [M + Na]⁺ calcd for C₃₁H₃₄N₂NaO₃: 505.2462; found: 505.2464.

N-(4-((1E,3E)-4-(4-((2-Azidoethyl)(methyl)amino)phenyl)buta-1,3-dien-1-yl)phenyl)benzamide (12)

To a solution of 11 (2.01 g, 4.16 mmol) in i-PrOH (100 mL) under argon atmosphere was added PTSA monohydrate (1.55 g, 8.15 mmol). The reaction mixture changed color from yellow to red wine and was then heated at 55 °C and stirred overnight. Reaction progress was monitored by TLC (CH₂Cl₂/EtOH, 95:5), and upon completion solvents were removed under reduced pressure. A saturated solution of NaHCO­₃ (150 mL) was slowly added to the residue which changed color from purple to pine green as CO_2(g) was released. The suspension was sonicated and filtered on a sintered glass funnel, and the precipitate was washed with deionized H₂O (100 mL) and dried under vacuum to give 1.50 g (90%) of the corresponding deprotected alcohol as a pine green solid.

To a stirred solution of the latter (780 mg, 1.96 mmol) in CH₂Cl₂ (10 mL) under argon atmosphere was added NEt₃ (0.82 mL, 5.9 mmol), before cooling to 0 °C using an ice bath. MsCl (0.30 mL, 3.9 mmol) was added dropwise, and the ice bath was removed 10 min after completing the addition. The reaction progress was monitored by TLC (petroleum ether/EtOAc, 50:50; identical R_f but different fluorescence at 365 nm). MeOH (5 mL) was added upon completion and the reaction mixture was stirred 10 min before addition of H₂O (15 mL) and CH₂Cl₂ (20 mL), and phase separation. Further rinsing of the organic phase was done and organic phases were combined, dried over MgSO₄, and filtered, and solvents were thoroughly removed under reduced pressure. The dry residue and NaN₃ (637 mg, 9.8 mmol) were then dissolved in DMF (10 mL) under argon atmosphere, before heating the reaction mixture to 90 °C for 2.5 h. The reaction progress was monitored by TLC (petroleum ether/EtOAc, 50:50) after extracting a small amount of the mixture with EtOAc and distilled H₂O. Upon completion, solvents were removed under reduced pressure and the residue was thoroughly washed with deionized H₂O before filtration on a sintered glass funnel, giving 767 mg (92% over 2 steps or 83% over 3 steps) of compound 12 as a yellow solid.

R_f = 0.35 (petroleum ether/EtOAc, 70:30), 0.70 (CH₂Cl₂/EtOAc, 70:30).

T _f = 105 °C (dec).

IR (ATR): 2098 cm^–1 (N₃ asym str).

¹H NMR (400 MHz, CDCl₃): δ = 7.87 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 7.86 (bs, 1 H, NH), 7.61 (d, J = 8.5 Hz, 2 H, 2 H_Ar), 7.56 (t, J = 6.7 Hz, 1 H, H_Ar), 7.49 (dd, J = 8.4, 6.7 Hz, 2 H, 2 H_Ar), 7.42 (d, J = 8.4 Hz, 2 H, 2 H_Ar), 7.35 (d, J = 8.5 Hz, 2 H, 2 H_Ar), 6.91 (dd, J = 15.4, 10.5 Hz, 1 H, CH=C), 6.79 (dd, J = 15.2, 10.5 Hz, 1 H, CH=C), 6.69 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 6.60 (d, J = 15.2 Hz, 1 H, CH=C), 6.56 (d, J = 15.4 Hz, 1 H, CH=C), 3.57 (t, J = 6.0 Hz, 2 H, N-CH₂-CH₂-N), 3.47 (t, J = 6.0 Hz, 2 H, N-CH₂-CH₂-N), 3.04 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CDCl₃): δ = 165.71 (C_q), 148.15 (C_q), 136.98 (C_q), 135.16 (C_q), 134.48 (C_q), 133.00 (CH), 132.11 (CH), 130.02 (CH), 129.62 (CH), 129.05 (CH), 127.91 (CH), 127.19 (CH), 127.01 (CH), 126.56 (C_q), 125.74 (CH), 120.37 (CH), 112.28 (CH), 52.05 (CH₂), 49.04 (CH₂), 39.13 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₆H₂₆N₅O: 424.2132; found: 424.2138; m/z [M + Na]⁺ calcd for C₂₆H₂₅N₅NaO: 446.1951; found: 446.1957.

tert-Butyl (4-((1E,3E)-4-(4-((2-Azidoethyl)(methyl)amino)phenyl)buta-1,3-dien-1-yl)phenyl)carbamate (13)

To 12 (2.37 g, 5.60 mmol), di-tert-butyl dicarbonate (4.27 g, 19.6 mmol) and DMAP (137 mg, 1.12 mmol) was added THF (50 mL), and the mixture was stirred overnight. The reaction progress was monitored by TLC (petroleum ether/EtOAc, 70:30) and upon completion, MeOH (5 mL) was added to the reaction medium which was then stirred for 15 min. Hydrazine hydrate (1.10 mL, 22.4 mmol) was then added and a precipitate started forming. The reaction progress was monitored by TLC under the same conditions and showed completion after an hour. A saturated solution of NH₄Cl (10 mL) was added and organic solvents were removed under reduced pressure until a precipitate was left in a clear aqueous solution, which was then filtered and dried under reduced pressure. The crude residue was purified over silica gel (CH₂Cl₂) to give 1.81 g (77%) of compound 13 as a yellow solid.

R_f = 0.74 (petroleum ether/EtOAc, 70:30).

T _f = 177 °C.

IR (ATR): 2111 cm^–1 (N₃ asym str).

¹H NMR (400 MHz, CDCl₃): δ = 7.37–7.29 (m, 6 H, 6 H_Ar), 6.86 (dd, J = 14.9, 10.5 Hz, 1 H, CH=C), 6.78 (dd, J = 14.8, 10.5 Hz, 1 H, CH=C), 6.71–6.65 (m, 2 H, 2 H_Ar), 6.57 (d, J = 14.9 Hz, 1 H, CH=C), 6.53 (d, J = 14.8 Hz, 1 H, CH=C), 6.46 (bs, 1 H, NH), 3.57 (t, J = 5.7 Hz, 2 H, N-CH₂-CH₂-N), 3.47 (t, J = 5.7 Hz, 2 H, N-CH₂-CH₂-N), 3.04 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CDCl₃): δ = 153.60 (C_q), 152.81 (C_q), 148.52 (C_q), 137.42 (C_q), 133.03 (C_q), 132.68 (CH), 130.06 (CH), 128.96 (CH), 127.79 (2 CH), 126.94 (2 CH), 126.35 (C_q), 125.63 (CH), 118.75 (2 CH), 112.29 (2 CH), 82.52 (C_q), 63.80 (CH₂), 51.19 (CH₂), 38.92 (CH₃), 28.54 (CH₃), 27.91 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₃₀N₅O₂: 420.2394; found: 420.2398; m/z [M + Na]⁺ calcd for C₂₄H₂₉N₅NaO₂: 442.2213; found: 442.2216.

tert-Butyl (4-((1E,3E)-4-(4-((2-Isothiocyanatoethyl)(methyl)amino)phenyl)buta-1,3-dien-1-yl)phenyl)carbamate (14)

To 13 (620 mg, 1.48 mmol) in anhydrous CH₂Cl₂ (15 mL) was added supported PPh₃ (0.74 g, 2.2 mmol) and CS₂ (3.6 mL, 60 mmol) before flushing with argon. The mixture was then stirred for 4 d, and was monitored by TLC (CH₂Cl₂/EtOH, 95:5, or petroleum ether/EtOAc, 70:30). Upon completion, the reaction mixture was filtered on a paper filter and solvents were removed under reduced pressure to obtain 592 mg (92%) of compound 14 as a yellow solid.

R_f = 0.65 (petroleum ether/EtOAc, 70:30).

T _f = 191 °C.

IR (ATR): 3353, 2188 (N=C=S asym str), 2109 cm^–1 (N=C=S asym str).

¹H NMR (400 MHz, CDCl₃): δ = 7.42–7.28 (m, 6 H, 6 H_Ar), 6.85 (dd, J = 14.9, 10.5 Hz, 1 H, CH=C), 6.77 (dd, J = 14.9, 10.5 Hz, 1 H, CH=C), 6.70–6.64 (m, 2 H, 2 H_Ar), 6.57 (d, J = 14.9 Hz, 1 H, CH=C), 6.54 (d, J = 14.9 Hz, 1 H, CH=C), 6.47 (bs, 1 H, NH), 3.73–3.69 (m, 4 H, N-CH₂-CH₂-N), 3.07 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CDCl₃): δ = 152.79 (C_q), 147.43 (C_q), 137.49 (C_q), 132.87 (C_q), 132.29 (CH), 130.46 (CH), 128.75 (CH), 127.89 (CH), 126.99 (CH), 126.16 (CH), 118.70 (CH), 112.44 (CH), 80.81 (C_q), 52.42 (CH₂), 42.72 (CH₂), 39.51 (CH₃), 28.54 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₂₅H₃₀N₃O₂S: 436.2053; found: 436.2057; m/z [M + Na]⁺ calcd for C₂₅H₂₉N₃NaO₂S: 458.1873; found: 458.1881.

Thiourea Linkage Formation

Compound 14 (1 equiv) and the functionalized adenosine 6a, 6b, 6c or 6d (1 equiv) were dissolved in THF (0.075 M) and the mixture was stirred under argon atmosphere for 3 d. The reaction progress was monitored by TLC (CH₂Cl₂/EtOH, 95:5) and upon completion, solvents were removed under reduced pressure. The crude residue was purified over silica gel (CH₂Cl₂/EtOH, 100:0 to 90:10) then C18 silica gel (MeCN/H₂O, 50:50 to 90:10) to give, respectively, 15a (35–94%*), 15b (16–70%*), 15c (10–71%*) or 15d (44–88%*). * Indicative yields given before final purification over C18 silica gel, obtained over 3, 4, 7 and 4 independent batches, respectively.

Compound 15a

R_f = 0.47 (CH₂Cl₂/EtOH, 95:5).

T _f = 125 °C (dec).

IR (ATR): 1727 (C=O ester str), 1635 (C–N= str), 1604 (N–H str), 1154 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CDCl₃): δ = 8.27 (bs, 1 H, NH), 8.08 (bs, 1 H, H₂), 7.85 (s, 1 H, H₈), 7.36–7.29 (m, 4 H, 4 H_Ar), 7.25 (d, J = 8.7 Hz, 2 H, 2 H_Ar), 6.84 (dd, J = 15.3, 10.5 Hz, 1 H, CH=C), 6.73 (dd, J = 15.3, 10.5 Hz, 1 H, CH=C), 6.69 (d, J = 8.7 Hz, 2 H, 2 H_Ar), 6.57 (bs, 1 H, NH), 6.52 (d, J = 15.3 Hz, 1 H, CH=C), 6.52 (d, J = 15.3 Hz, 1 H, CH=C), 6.35–6.28 (m, 1 H, NH), 5.98 (d, J = 5.7 Hz, 1 H, H_1′), 5.85 (bs, 2 H, NH₂), 4.68 (t, J = 5.7 Hz, 1 H, H_2′), 4.49–4.44 (m, 1 H, H_3′), 4.38–4.34 (m, 1 H, H_4′), 4.28 (d, J = 16.9 Hz, 1 H, CH), 4.18 (d, J = 16.9 Hz, 1 H, CH), 4.09 (d, J = 16.3 Hz, 1 H, CH), 3.99 (d, J = 16.3 Hz, 1 H, CH), 3.91 (bs, 1 H, CH), 3.75–3.42 (m, 6 H, N-CH₂-CH₂-N, 2 H_5′), 2.92 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu), 1.50 (s, 9 H, t-Bu), 1.34 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CDCl₃): δ = 184.11 (C_q), 169.85 (C_q), 168.95 (C_q), 155.96 (C_q), 152.84 (C_q), 152.53 (CH), 149.15 (C_q), 148.91 (C_q), 141.19 (CH), 137.47 (C_q), 132.96 (C_q), 132.53 (CH), 130.22 (CH), 128.88 (CH), 127.74 (CH), 126.96 (C_q), 126.76 (CH), 125.83 (CH), 121.08 (C_q), 118.77 (CH), 112.85 (CH), 89.21 (CH), 82.62 (CH), 82.28 (C_q), 80.81 (C_q), 80.46 (CH), 78.75 (CH), 77.44 (CH), 68.47 (CH₂), 68.32 (CH₂), 51.92 (CH₂), 42.28 (CH₂), 38.51 (CH₃), 28.54 (3 CH_{3 t-Bu}), 28.34 (3 CH_{3 t-Bu}), 28.17 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₄₇H₆₄N₉O₉S: 930.4542; found: 930.4528; m/z [M + 2 H]²⁺ calcd for C₄₇H₆₅N₉O₉S: 465.7307; found: 465.7304; m/z [M + Na]⁺ calcd for C₄₇H₆₃N₉NaO₉S: 952.4367; found: 952.4346.

Compound 15b

R_f = 0.38 (CH₂Cl₂/EtOH, 95:5).

T _f = 135 °C (dec).

IR (ATR): 1725 (C=O ester str), 1637 (C–N= str), 1604 (N–H str), 1156 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CDCl₃): δ = 8.52 (bs, 1 H, NH), 8.05 (bs, 1 H, H₂), 7.79 (s, 1 H, H₈), 7.34–7.29 (m, 4 H, 4 H_Ar), 7.23 (d, J = 8.7 Hz, 2 H, 2 H_Ar), 6.83 (dd, J = 15.2, 10.6 Hz, 1 H, CH=C), 6.72 (dd, J = 15.2, 10.6 Hz, 1 H, CH=C), 6.67 (d, J = 8.7 Hz, 2 H, 2 H_Ar), 6.58 (bs, 1 H, NH), 6.51 (d, J = 15.2 Hz, 1 H, CH=C), 6.49 (d, J = 15.2 Hz, 1 H, CH=C), 6.32 (bs, 1 H, OH), 5.90 (d, J = 7.0 Hz, 1 H, H_1′), 5.82 (bs, 2 H, NH₂), 4.60 (s, 1 H, H_2′), 4.47 (dd, J = 6.8, 4.6 Hz, 1 H, H_3′), 4.42 (bs, 1 H, NH), 4.39–4.34 (m, 1 H, CH₂-O), 4.10–4.09 (m, 1 H, CH₂-O), 3.97–3.87 (m, 1 H, H_4′), 3.82–3.55 (m, 5 H, N-CH₂-CH₂-N, H_5′a), 3.52–3.43 (m, 1 H, H_5′b), 2.92 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu), 1.45 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CDCl₃): δ = 171.10 (C_q), 156.01 (C_q), 152.85 (C_q), 152.51 (C_q), 149.17 (C_q), 148.83 (C_q), 141.71 (C_q), 137.50 (C_q), 132.92 (C_q), 132.41 (CH), 130.34 (CH), 128.82 (CH), 127.71 (CH), 126.97 (CH), 125.95 (CH), 121.28 (C_q), 118.80 (CH), 112.98 (CH), 88.52 (CH), 85.15 (CH), 84.08 (CH), 83.96 (C_q), 80.83 (C_q), 77.44 (CH), 70.56 (CH), 70.01 (CH₂), 51.97 (CH₂), 42.38 (CH₂), 38.63 (CH₃), 28.54 (3 CH_{3 t-Bu}), 28.20 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₄₁H₅₄N₉O₇S: 816.3861, found: 816.3864; m/z [M + 2 H]²⁺ calcd for C₄₁H₅₅N₉O₇S: 408.6967; found: 408.6971; m/z [M + Na]⁺ calcd for C₄₁H₅₃N₉NaO₇S: 838.3681; found: 838.3684.

Compound 15c

R_f = 0.31 (CH₂Cl₂/EtOH, 95:5).

T _f = 135 °C (dec).

IR (ATR): 1724 (C=O ester str), 1637 (C–N= str), 1604 (N–H str), 1158 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CDCl₃): δ = 8.50 (bs, 1 H, NH), 8.06 (bs, 1 H, H₂), 7.79 (s, 1 H, H₈), 7.36–7.28 (m, 4 H, 4 H_Ar), 7.23 (d, J = 8.8 Hz, 2 H, 2 H_Ar), 6.82 (dd, J = 15.3, 10.6 Hz, 1 H, CH=C), 6.72 (dd, J = 15.3, 10.6 Hz, 1 H, CH=C), 6.67 (d, J = 8.8 Hz, 2 H, 2 H_Ar), 6.58 (bs, 1 H, NH), 6.51 (d, J = 15.3 Hz, 1 H, CH=C), 6.49 (d, J = 15.3 Hz, 1 H, CH=C), 6.40 (bs, 1 H, OH), 5.90 (d, J = 7.1 Hz, 1 H, H_1′), 5.81 (bs, 2 H, NH₂), 4.58 (s, 1 H, H_2′), 4.48 (dd, J = 6.9, 4.7 Hz, 1 H, H_3′), 4.42 (bs, 1 H, NH), 4.39–4.34 (m, 1 H, CH₂-O), 4.19–4.11 (m, 1 H, CH₂-O), 3.97–3.87 (m, 1 H, H_4′), 3.79–3.69 (m, 4 H, N-CH₂-CH₂-N), 3.68–3.55 (m, 1 H, H_5′a), 3.51–3.42 (m, 1 H, H_5′b), 2.92 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu), 1.45 (s, 9 H, t-Bu).

¹³C NMR (101 MHz, CDCl₃): δ = 184.16 (C_q), 171.25 (C_q), 155.98 (C_q), 152.85 (C_q), 149.17 (C_q), 148.65 (C_q), 141.33 (C_q), 137.46 (C_q), 132.99 (C_q), 132.49 (CH), 130.21 (CH), 128.93 (CH), 127.65 (CH), 126.97 (CH), 125.90 (CH), 121.27 (C_q), 118.78 (CH), 112.91 (CH), 91.51 (CH), 83.68 (CH), 82.63 (CH), 80.81 (C_q), 77.44 (CH), 73.12 (CH), 69.93 (CH₂), 51.85 (CH₂), 46.06 (CH₂), 42.37 (CH₂), 38.26 (CH₃), 28.55 (3 CH_{3 t-Bu}), 28.28 (3 CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₄₁H₅₄N₉O₇S: 816.3861; found: 816.3859; m/z [M + 2 H]²⁺ calcd for C₄₁H₅₅N₉O₇S: 408.6967; found: 408.6970; m/z [M + Na]⁺ calcd for C₄₁H₅₃N₉NaO₇S: 838.3681; found: 838.3681.

Compound 15d

R_f = 0.40 (CH₂Cl₂/EtOH, 95:5).

T _f = 149 °C (dec).

IR (ATR): 1704, 1636 (C–N= str), 1604 (N–H str), 1157 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CDCl₃): δ = 8.35 (bs, 1 H, NH), 8.09 (bs, 1 H, H₂), 7.71 (s, 1 H, H₈), 7.36–7.28 (m, 4 H, 4 H_Ar), 7.19 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 6.81 (dd, J = 15.3, 10.5 Hz, 1 H, CH=C), 6.68 (dd, J = 15.3, 10.5 Hz, 1 H, CH=C), 6.65 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 6.59 (bs, 1 H, NH), 6.49 (d, J = 15.3 Hz, 1 H, CH=C), 6.44 (d, J = 15.3 Hz, 1 H, CH=C), 6.26–6.17 (m, 1 H, NH), 5.84 (bs, 2 H, NH₂), 5.77 (d, J = 4.6 Hz, 1 H, H_1′), 5.10 (dd, J = 6.0, 4.6 Hz, 1 H, H_2′), 4.92 (dd, J = 6.0, 2.1 Hz, 1 H, H_3′), 4.55–4.46 (m, 1 H, H_4′), 3.96–3.79 (m, 2 H, N-CH₂), 3.65–3.50 (m, 4 H, N-CH₂, 2 H_5′), 2.92 (s, 3 H, CH₃), 1.63 (s, 3 H, CH₃), 1.52 (s, 9 H, t-Bu), 1.37 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CDCl₃): δ = 184.27 (C_q), 155.97 (C_q), 152.86 (C_q), 152.59 (C_q), 149.19 (C_q), 148.62 (C_q), 140.78 (C_q), 137.50 (C_q), 132.91 (C_q), 132.37 (CH), 130.23 (CH), 128.86 (CH), 127.65 (CH), 126.94 (CH), 126.73 (CH), 125.88 (CH), 121.07 (C_q), 118.81 (CH), 114.86 (C_q), 112.86 (CH), 92.69 (CH), 83.95 (CH), 82.50 (CH), 81.65 (CH), 80.82 (C_q), 77.44 (CH), 51.71 (CH₂), 46.27 (CH₂), 42.52 (CH₂), 38.30 (CH₃), 28.54 (CH_{3 t-Bu}), 27.79 (CH_{3 t-Bu}), 25.56 (CH_{3 t-Bu}).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₈H₄₈N₉O₅S: 742.3494; found: 742.3491; m/z [M + Na]⁺ calcd for C₃₈H₄₇N₉NaO₅S: 764.3313; found: 764.3300.

Cleavage of the Protecting Groups

15a, 15b or 15c was dissolved in CH₂Cl₂/TFA (9:1) and the mixture was stirred at room temperature for 2 h. Compound 16a, 16b or 16c, respectively, was recovered after removal of the solvents under reduced pressure.

15d was dissolved in TFA/EtOH (9:1) and the mixture was stirred at room temperature for 2 h. Compound 16d was recovered after removal of the solvents under reduced pressure.

Compound 16a

R_f = 0.59 (MeCN/H₂O, 60:40; C18 silica gel).

T _f = 126 °C (dec).

IR (ATR): 1671 (C–N= str), 1141 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CD₃OD): δ = 8.45 (s, 1 H, H₂), 8.40 (bs, 1 H, H₈), 7.60 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 7.38 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 7.33 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 7.06 (dd, J = 15.4, 10.5 Hz, 1 H, CH=C), 6.97 (d, J = 8.6 Hz, 2 H, 2 H_Ar), 6.86 (dd, J = 15.4, 10.5 Hz, 1 H, CH=C), 6.66 (d, J = 15.4 Hz, 1 H, CH=C), 6.64 (d, J = 15.4 Hz, 1 H, CH=C), 6.23 (d, J = 4.9 Hz, 1 H, H_1′), 4.85–4.77 (m, 1 H, H_2′), 4.49–4.43 (m, 1 H, H_3′), 4.43–4.20 (m, 5 H, 2 CH₂-O, H_4′), 3.90 (bs, 2 H, NH₂), 3.75–3.65 (m, 2 H, N-CH₂), 3.64–3.57 (m, 2 H, N-CH₂), 3.29–3.22 (m, 2 H, 2 H_5′), 3.06 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CD₃OD): δ = 174.00 (C_q, COOH), 173.56 (C_q, COOH), 152.39 (C_q), 149.96 (CH), 149.00 (C_q), 146.03 (C_q), 144.58 (C_q), 140.43 (CH), 135.49 (CH), 133.16 (CH), 131.70 (C_q), 130.64 (C_q), 130.05 (CH), 129.08 (CH), 128.73 (CH), 126.99 (CH), 124.34 (CH), 121.08 (C_q), 115.15 (CH), 89.77 (CH), 83.40 (CH), 82.12 (CH), 79.26 (CH), 68.40 (CH₂), 53.65 (CH₂), 42.54 (CH₂), 40.17 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₄H₄₀N₉O₇S: 718.2766; found: 718.2757; m/z [M + 2 H]²⁺ calcd for C₃₄H₄₁N₉O₇S: 359.6419; found: 359.6421; m/z [M + 3 H]³⁺ calcd for C₃₄H₄₂N₉O₇S: 240.0970; found: 240.0980.

Compound 16b

R_f = 0.56 (MeCN/H₂O, 60:40; C18 silica gel).

T _f = 104 °C (dec).

IR (ATR): 1673 (C–N= str), 1136 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CD₃OD): δ = 8.46 (s, 1 H, H₂), 8.42 (bs, 1 H, H₈), 7.65–7.56 (m, 2 H, 2 H_Ar), 7.42–7.35 (m, 2 H, 2 H_Ar), 7.35–7.30 (m, 2 H, 2 H_Ar), 7.06 (dd, J = 15.4, 10.4 Hz, 1 H, CH=C), 7.01–6.94 (m, 2 H, 2 H_Ar), 6.86 (dd, J = 15.4, 10.4 Hz, 1 H, CH=C), 6.66 (d, J = 15.4 Hz, 1 H, CH=C), 6.64 (d, J = 15.4 Hz, 1 H, CH=C), 6.23–6.17 (m, 1 H, H_1′), 4.73–4.59 (m, 1 H, H_2′), 4.50–4.44 (m, 1 H, H_3′), 4.32–4.16 (m, 3 H, CH₂-O, H_4′), 3.90 (bs, 2 H, NH₂), 3.75–3.68 (m, 2 H, N-CH₂), 3.65–3.56 (m, 2 H, N-CH₂), 3.38–3.32 (m, 2 H, 2 H_5′), 3.06 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CD₃OD): δ = 174.14 (C_q, COOH), 152.31 (C_q), 150.00 (CH), 145.93 (CH), 144.78 (C_q), 144.70 (CH), 140.45 (C_q), 135.33 (CH), 133.12 (CH), 130.60 (C_q), 130.28 (CH), 129.12 (CH), 128.77 (CH), 127.42 (CH), 124.39 (2 CH), 121.09 (C_q), 115.68 (2 CH), 89.63 (CH), 85.11 (CH), 83.98 (CH), 83.87 (CH), 69.56 (CH₂), 68.94 (CH₂), 54.10 (CH₂), 42.43 (CH₂), 40.58 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₂H₃₈N₉O₅S: 660.2711; found: 660.2704; m/z [M + 2 H]²⁺ calcd for C₃₂H₃₉N₉O₅S: 330.6392; found: 330.6398.

Compound 16c

R_f = 0.57 (MeCN/H₂O, 60:40; C18 silica gel).

T _f = 117 °C (dec).

IR (ATR): 1673 (C–N= str), 1138 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CD₃OD): δ = 8.43 (s, 1 H, H₂), 8.39 (bs, 1 H, H₈), 7.64–7.56 (m, 2 H, 2 H_Ar), 7.44–7.36 (m, 2 H, 2 H_Ar), 7.35–7.30 (m, 2 H, 2 H_Ar), 7.14–6.95 (m, 3 H, 2 H_Ar, CH=C), 6.92–6.79 (m, 1 H, CH=C), 6.71–6.57 (m, 2 H, 2 CH=C), 6.05–5.99 (m, 1 H, H_1′), 4.82–4.71 (m, 1 H, H_2′), 4.40–4.24 (m, 4 H, CH₂-O, H_3′, H_4′), 3.90 (bs, 2 H, NH₂), 3.77–3.68 (m, 2 H, N-CH₂), 3.67–3.57 (m, 2 H, N-CH₂), 3.37–3.32 (m, 2 H, 2 H_5′), 3.08 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CD₃OD): δ = 174.78 (C_q, COOH), 152.28 (C_q), 149.94 (CH), 145.91 (C_q), 144.43 (C_q), 144.35 (C_q), 140.40 (CH), 136.90 (C_q), 135.11 (CH), 133.03 (CH), 130.62 (C_q), 130.50 (CH), 129.13 (CH), 128.79 (CH), 127.77 (CH), 124.42 (CH), 121.02 (C_q), 116.09 (CH), 91.03 (CH), 86.79 (CH), 83.40 (CH), 81.56 (CH), 74.94 (CH₂), 69.13 (CH₂), 52.75 (CH₂), 42.42 (CH₂), 40.92 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₂H₃₈N₉O₅S: 660.2711; found: 660.2707; m/z [M + 2 H]²⁺ calcd for C₃₂H₃₉N₉O₅S: 330.6392; found: 330.6394.

Compound 16d

R_f = 0.52 (MeCN/H₂O, 60:40; C18 silica gel).

T _f = 94 °C (dec).

IR (ATR): 1672 (C–N= str), 1135 cm^–1 (C=S thiourea str).

¹H NMR (400 MHz, CD₃OD): δ = 8.45 (s, 1 H, H₂), 8.40 (bs, 1 H, H₈), 7.64–7.58 (m, 2 H, 2 H_Ar), 7.47–7.38 (m, 2 H, 2 H_Ar), 7.38–7.29 (m, 2 H, 2 H_Ar), 7.15–7.01 (m, 3 H, 2 H_Ar, CH=C), 6.89 (dd, J = 15.4, 10.6 Hz, 1 H, CH=C), 6.66 (d, J = 15.4 Hz, 1 H, CH=C), 6.66 (d, J = 15.4 Hz, 1 H, CH=C), 6.02 (d, J = 5.2 Hz, 1 H, H_1′), 4.68 (t, J = 5.2 Hz, 1 H, H_2′), 4.35 (dd, J = 5.2, 4.0 Hz, 1 H, H_3′), 4.26–4.19 (m, 1 H, H_4′), 3.90 (bs, 2 H, NH₂), 3.76–3.70 (m, 2 H, N-CH₂), 3.69–3.61 (m, 2 H, N-CH₂), 3.38–3.31 (m, 2 H, 2 H_5′), 3.10 (s, 3 H, CH₃).

¹³C NMR (101 MHz, CD₃OD): δ = 152.39 (C_q), 149.94 (C_q), 148.75 (C_q), 146.04 (CH), 144.41 (CH), 140.35 (C_q), 135.33 (CH), 133.09 (CH), 130.67 (C_q), 130.18 (CH), 129.07 (CH), 128.73 (CH), 127.20 (CH), 124.37 (CH), 121.03 (C_q), 115.41 (CH), 91.19 (CH), 85.33 (CH), 75.58 (CH), 72.61 (CH), 70.70 (CH₂), 53.92 (CH₂), 42.51 (CH₂), 40.33 (CH₃).

HRMS (ESI): m/z [M + H]⁺ calcd for C₃₀H₃₆N₉O₃S: 602.2656; found: 602.2647; m/z [M + 2 H]²⁺ calcd for C₃₀H₃₇N₉O₃S: 301.6365; found: 301.6372; m/z [M + Na]⁺ calcd for C₃₀H₃₅N₉NaO₃S: 624.2476; found: 624.2469.

Conflict of Interest

The authors declare no conflict of interest.

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Corresponding Author

Publication History

Received: 26 December 2021

Accepted after revision: 28 January 2022

Article published online:
08 March 2022

© 2022. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

• References

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  CrossrefPubMedSearch in Google Scholar
• 2 Miller MR, Megson IL. Br. J. Pharmacol. 2007; 151: 305
  CrossrefPubMedSearch in Google Scholar
• 3 Murad F. Adv. Pharmacol. 1994; 26: 19
  CrossrefPubMedSearch in Google Scholar
• 4 Brüne B, von Knethen A, Sandau KB. Eur. J. Pharmacol. 1998; 351: 261
  CrossrefPubMedSearch in Google Scholar
• 5 Pacher P, Beckman JS, Liaudet L. Physiol. Rev. 2007; 87: 315
  CrossrefPubMedSearch in Google Scholar
• 6 Oliveira-Paula GH, Lacchini R, Tanus-Santos JE. Gene 2016; 575: 584
  CrossrefPubMedSearch in Google Scholar
• 7 Stoddard BL, Cohen BE, Brubaker M, Mesecar AD, Koshland DE. Nat. Struct. Biol. 1998; 5: 891
  CrossrefPubMedSearch in Google Scholar
• 8 Parisi C, Seggio M, Fraix A, Sortino S. ChemPhotoChem 2020; 4: 742
  CrossrefSearch in Google Scholar
• 9 Ieda N, Hotta Y, Yamauchi A, Nishikawa A, Sasamori T, Saitoh D, Kawaguchi M, Kimura K, Nakagawa H. ACS Chem. Biol. 2020; 15: 2958
  CrossrefPubMedSearch in Google Scholar
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  CrossrefSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
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  CrossrefSearch in Google Scholar
• 19 Xie J. Eur. J. Org. Chem. 2002; 3411
  Crossref
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  CrossrefSearch in Google Scholar
• 21 Bellucci C, Gualtieri F, Chiarini A. Eur. J. Med. Chem. 1987; 22: 473
  CrossrefSearch in Google Scholar
• 22 Reichardt C. Chem. Rev. 1994; 94: 2319
  CrossrefSearch in Google Scholar
• 23 Data not shown.
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  CrossrefSearch in Google Scholar
• 25 Rurack K, Spieles M. Anal. Chem. 2011; 83: 1232
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 27 Socrates G. Infrared and Raman Characteristic Group Frequencies: Tables and Charts, 3rd ed. Wiley; Chichester/New York: 2001
  Search in Google Scholar
• 28 Melnikov AS, Serdobintsev PY, Vedyaykin AD, Khodorkovskii MA. J. Phys.: Conf. Ser. 2017; 917: 062029
  CrossrefSearch in Google Scholar

• Supplementary Material