Next Article in Journal
Seven Conformations of the Macrocycle Cyclododecanone Unveiled by Microwave Spectroscopy
Previous Article in Journal
Induction of G2/M Cell Cycle Arrest via p38/p21Waf1/Cip1-Dependent Signaling Pathway Activation by Bavachinin in Non-Small-Cell Lung Cancer Cells
Previous Article in Special Issue
Challenges and Progress in Nonsteroidal Anti-Inflammatory Drugs Co-Crystal Development
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 26
Issue 17
10.3390/molecules26175160
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs

by
Mateja Đud
1,†,
Markéta Tichotová
1,2,†,
Eliška Procházková
2,* and
Ondřej Baszczyňski
1,2,*
1
Faculty of Science, Charles University, 128 43 Prague, Czech Republic
2
Institute of Organic Chemistry and Biochemistry, The Czech Academy of Sciences, 166 10 Prague, Czech Republic
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Molecules 2021, 26(17), 5160; https://doi.org/10.3390/molecules26175160
Submission received: 23 July 2021 / Revised: 12 August 2021 / Accepted: 20 August 2021 / Published: 25 August 2021
(This article belongs to the Special Issue Drug Delivery Systems for Anti-infectious, Anti-inflammatory and Anti-cancer Agents)
Browse Figures
Versions Notes

Abstract

:
Amine-containing drugs often show poor pharmacological properties, but these disadvantages can be overcome by using a prodrug approach involving self-immolative linkers. Accordingly, we designed l-lactate linkers as ideal candidates for amine delivery. Furthermore, we designed linkers bearing two different cargos (aniline and phenol) for preferential amine cargo release within 15 min. Since the linkers carrying secondary amine cargo showed high stability at physiological pH, we used our strategy to prepare phosphate-based prodrugs of the antibiotic Ciprofloxacin. Therefore, our study will facilitate the rational design of new and more effective drug delivery systems for amine-containing drugs.

1. Introduction

Drugs containing an amino group are key pharmaceutical agents, covering a broad spectrum of biological actions and displaying anti-inflammatory [1], anticancer [2,3], antimicrobial [4,5], and pain-relieving properties [6]. Currently, 542 drugs containing an amino group have already been approved for the EU market, according to the drug bank online (https://go.drugbank.com, accessed on 15 July 2021). This number does not include many other biologically active compounds—potential leads—or compounds from natural resources, such as alkaloids. However, amine drugs often show poor pharmacological properties, such as low aqueous solubility and poor membrane permeability due to ionization of the amino group [7] under physiological conditions. Nevertheless, amines are generally susceptible to derivatization. Thus, a prodrug strategy [8,9,10] can be used to overcome these drawbacks [7,11].
Prodrug strategies rely on a structural modification (masking) of the active pharmaceutical agent—a drug—with a suitable protecting group (promoiety) to modulate its pharmacokinetic properties. Such a change helps to facilitate drug delivery to the target site (e.g., tissues, cells, cell compartments, or organs). One of the most rapidly developing prodrug strategies consists of using self-immolative (SI) linkers [12,13] to control drug release [14].
SI linkers are covalent assemblies that couple an active compound (drug) to a protecting group. After external stimuli, either chemical or enzymatic, a cascade of spontaneous reactions [15] leads to linker fragmentation and consequently to drug release. The two main classes of SI linkers are (1) carbamates and (2) phosphate-based systems. Phosphorus-based SI linkers stand above the “classical” carbamate linkers because they make it possible to attach an additional substituent, which can help fine-tune the SI rate or provide a double cargo option.
Phosphorus-based SI linkers have been introduced as suitable drug-delivery systems for several drugs. A paradigmatic example of a phosphorus-based SI linker application is the methoxymethylphosphonic acid (MMPA) drug delivery vehicle for the oral delivery of propofol [16]. Other examples include pro-nucleotide prodrugs (ProTides) [17], which have been used to treat various viral infections, such as HIV [18], hepatitis B [19], or SARS-CoV-2 (COVID-19) [20]. Considering their success as drug-delivery systems, phosphate-based SI linkers may find broader applications in drug discovery and materials science through systematic studies.
This study reports the development of new phosphate-based SI linkers designed to release amine-containing cargos (Figure 1). For this purpose, we searched for a suitable spacer responsible for SI. Although SI of ethylene glycol linkers 1–6 did not lead to amine release in a reasonable time, our lactate linkers 7–9 showed successful cargo release. After screening a wide range of linkers bearing various amines (10–16), representing model drugs, we prepared a prodrug of the FDA-approved drug Ciprofloxacin, which is a broad-spectrum fluoroquinolone antibiotic.

2. Results

2.1. Ethylene Glycol Phosphate-Based Linkers

Ethylene glycol-based linkers provide stable cyclic intermediates traceable by NMR spectroscopy, which indicates that the cargo is released via SI [22]. For proof of concept, we prepared glycol-based linkers 1–6 (Scheme 1) bearing phenethylamine, piperidine, and aniline (LG-1, LG-2 and LG-3, respectively) as representatives of primary, secondary, and aromatic amines, respectively.
Target linkers were synthesized in two consecutive phosphorylation steps, starting from commercially available phosphorodichloridates 20–21 (Scheme 1). Carbonate 19 was prepared in a reaction between ethylene glycol and dimethoxynitrobenzyl (DMNB) chloroformate in THF, using pyridine as the base [22]. The reaction of equimolar amounts of dichloridates 20–21 and DMNB carbonate 19 in the presence of triethylamine (TEA) in toluene gave intermediates 22 and 23. This reaction was monitored by 31P-NMR spectra (signal at δP 4.7 ppm for 22 and δP 0.08 ppm for 23, in CDCl3), and intermediates 22 and 23 were directly used for the second phosphorylation step. The reaction of 22 and 23 with one equivalent of the corresponding amine and TEA as a base afforded the final linkers 1–6. The isolated yields in the series bearing ethoxy substituent (1–3) were moderate (23–44%), whereas compounds 4–6 were isolated in low yields (4–12%) despite multiple flash chromatography (silica gel and C18).
The self-immolation of 1–6 was triggered photochemically (365 nm), and the reaction was monitored by 31P-NMR spectroscopy. Surprisingly, the successful photoactivation of 1–3 yielded intermediates (1–3)-I, with no further spectral change over several days, thus indicating that the cargo was not released (Figure S1 in Supplementary Materials).
In contrast to 1–3, linkers 4 and 6 afforded cyclic intermediates 4-cyc-I and 6-cyc-I within 5 min of irradiation (Figure 2). In compound 5, bearing a secondary amine, only a trace of 5-cyc-I was detected overnight. However, the downfield shifted 31P-NMR signals of cyclic intermediates 4-cyc-I, 5-cyc-I, and 6-cyc-I (δP 28.0, 26.5, 21.7 ppm, respectively) indicated that the amine cargo was still attached to the phosphorus and that phenol was released instead.
31P-NMR spectra recorded overnight suggested three different scenarios: (1) preferential release of phenol as 4-cyc-I and 4-P2 were detected; (2) formation of the stable intermediate 5-I, which released phenol in several days; (3) sequential release of phenol in minutes (6-cyc-I) and aniline overnight (6-cyc-P). The formation of phospholane intermediates (4-6)-cyc-I could be useful for other applications, such as preparing functional biopolymers for controlled drug delivery systems [23], but linkers 1–6 did not release amine successfully. Therefore, we altered the spacer structure to find an effective system for releasing amine-containing cargos.

2.2. Lactate Phosphate-Based Linkers

To stimulate amine cargo release, we altered the glycol spacer in 1–3 to an l-lactate spacer, thus preparing linkers 7–9 (Scheme 2). Despite promoting a slow release of phenolic compounds, [25] the lactate spacer could be suitable for amines. Compounds 7–9 were synthesized from DMNB ester 24 via acid-catalyzed esterification in refluxing toluene [25], and intermediate 25 was generated in a reaction of 24 with dichloridate 20 in toluene. The reaction was monitored by 31P-NMR, and a new pair of 31P-NMR signals at δP 4.6 ppm and δP 4.1 ppm (ca. 1:1 ratio), corresponding to two diastereoisomers of 25, was observed due to the new stereogenic center on phosphorus. Intermediate 25 was directly used for amine phosphorylation, and the final products 7–9 were isolated with good yields (42–52%) as 1:1 mixtures of diastereoisomers, as shown by the two sets of NMR signals.
The SI of 7–9, monitored by 31P-NMR, showed successful amine release, which provided the final product P in 15 min (Figure S2 in the Supplementary Materials). However, in 7 and 9, we also detected a new 31P signal (δP −1.6 ppm) belonging to the undesired product of alternative decomposition (7-X and 9-X). Interestingly, linker 8, bearing a secondary amine as a cargo, did not form the undesired product 8-X and followed the expected reaction course.
Given the unexpected reactivity of 7 and 9 in the CACO/DMSO mixture (1/1, v/v), we optimized the solvent system. For this purpose, we performed irradiation experiments on 7, in various solvent mixtures (Figure S3 in the Supplementary Materials), and we found that the formation of side product 7-X can be suppressed by either decreasing the pH of the cacodylate buffer (to pH = 5) or changing the buffer itself. HEPES buffer or an unbuffered system can suppress the formation of 7-X. Lastly, we selected the HEPES (pH 7.4)/DMSO system (1:1, v/v) for further SI investigation.
We monitored the SI of 7–9 in the HEPES/DMSO solvent mixture (Figure 3). In 5 min, we detected the final product P in all cases (δP −1.1 ppm). Linkers 7 and 8 did not provide any intermediate 7-I and 8-I, respectively, as they undergo fast cyclization, and photoactivation is a rate-limiting step. In turn, the SI of 9 was slow, and we did detect intermediate 9-I (Figure 3, right) or traces of the undesired product 9-X (δP −1.6 ppm). Considering the overall limited stability of 7 and 9, we investigated the formation of the unknown side product X and performed stability tests in 7–9. The results showed the limited stability of 7 and 9, which were significantly decomposed within 7 days (Figures S6 and S7 in the Supplementary Materials, details therein).

2.3. Characterization of the Undesired Product X

To identify the alternative decomposition pathway, we characterized the undesired product X. The significant upfield shift (δP < 0) suggested that the P-NH bond had been cleaved. In addition, one singlet 31P-NMR signal indicated the lack of a stereogenic center on the phosphorus atom. 31P-NMR chemical shifts of 7-X and 9-X differed slightly (δP −1.66 and −1.58 ppm, respectively), as found in the starting compounds 7 and 9 (differing in LGs). Combined, these findings demonstrate that 7-X and 9-X also differ in the amine moiety, which may be explained by the intramolecular rearrangement proposed in Figure 4. Additional 2D NMR experiments performed on linker 7 suggested the formation of carboxamide 7-X. We found the key interaction between the phenethylamine alkyl chain and lactate carbonyl in the HMBC spectrum (Figure 4). The proposed structure of 7-X was confirmed by HR-MS (Figure S64 in the Supplementary Materials).
Only the linkers containing the NH group in the phosphoramidate bond underwent the proposed intramolecular rearrangement (linker 8 without NH did not form 8-X). Indeed, N-methylation of 9 yielded linker 10, which did not form the undesired product 10-X (Figure S4 in the Supplementary Materials). This intramolecular rearrangement has already been reported by the Mulliez group in 1985 [26].

2.4. Amine Screening—Application Scope

Based on the successful SI observed in linkers 7–9, we examined the synthetic scope and application feasibility of lactate linkers by altering the structure of the cargo. Accordingly, we prepared linkers 10–16 (Scheme 3).
Linkers with aliphatic amines (morpholine and benzylamine) were synthesized as final products 11 and 12, respectively. Aromatic amines provided only two linkers bearing N-methylaniline and 2-aminopyrimidine (10 and 13, respectively). Other heterocyclic amines, such as imidazole, indoline, 2-aminobenzothiazole, 1- and 2-aminobenzoimidazole, and 2-aminobenzoxazole, yielded complex reaction mixtures, as shown in 31P-NMR spectra (Figure S9 in the Supplementary Materials), which we were unable to separate.
Since cargo release was faster in 4–6, phenyl-lactyl phosphate analogs 14–16 were also prepared with a phenyl instead of an ethyl group attached to the phosphorus.
Then, we subjected 10–16 to irradiation NMR experiments (Figure 5 and Figure 6). Compounds 10–12 afforded the final product P within 5 min, and product P was a major component in the reaction mixtures in 15 min. In contrast, 2-aminopyrimidine derivative 13 showed a slow formation of intermediate 13-I without any further spectral change in 15 min. Lastly, the pyrimidine cargo was fully released from 13-I within 19 days.
Linkers 14–16 released their amine cargos slightly faster than their ethyl counterparts 7–9. Linkers 14–16 afforded the final product P, which emerged as one singlet 31P signal at δP −6 ppm (Figure 6). Although 14 and 15 released the corresponding amines within 5 min, linker 16 did so overnight.

2.5. Application

Ultimately, we prepared two model prodrugs of Ciprofloxacin (Figure 7), which is a known fluoroquinolone antibiotic containing a secondary amino group, to demonstrate the applicability of lactate phosphate-based linkers. To avoid side reactions during the synthesis of 17 and 18, we protected the carboxylic group of Ciprofloxacin by methylation, which should not decrease the antibiotic activity as reported previously [27]. Then, Ciprofloxacin methyl ester 27 was phosphorylated, following the procedure that had been used for model linkers 7–9 (Scheme 2). Two-step phosphorylation starting from DMNB ester 24 and ethyl dichlorophosphate 20 afforded photoactivable compound 17. In addition to 17, we also prepared its enzymatically activable analog 18. Both compounds were obtained in moderate isolated yields (43–44%) as ca. 1:1 mixtures of diastereoisomers, as confirmed by the presence of two sets of NMR signals.
The photoactivation of linker 17 resulted in Ciprofloxacin release in 5 min, which was supported by the formation of product 17-P (Figure 8a). Compound 18 was activated by a lipase from Candida Antarctica. We detected 18-P after 3 h, with ca. 30% cargo release in 24 h. Most Ciprofloxacin (97%) was released in 6 days (Figure 8b). Enzymatic cargo release was relatively slow, which was presumably due to the substrate specificity of the lipase that was used in the experiment.
Furthermore, we performed a biological screening of 18 and 27, which showed that Ciprofloxacin methylation at the carboxylic moiety inhibits the antibiotic activity of this fluoroquinolone (Table S1 in the Supplementary Materials), despite previous reports stating otherwise [27]. Nevertheless, we believe that phosphate-based linkers may be used to design secondary amine drug delivery systems.

3. Discussion

Our study demonstrates that a universal spacer for delivering all types of amines will unlikely ever be designed given the sensitivity of the phosphorus atom to substitution. An ethylene glycol spacer, which was previously identified as the best linker for phenolic cargo delivery [22], proved inefficient in delivering amines, as shown in 1–6. An alteration in the electron density of phosphorus caused by oxygen substitution (from phenol—previous work [22]) for nitrogen (from amine) could explain the inefficiency of 1–6. Although installing phenol (4–6) as the second cargo slightly accelerated the SI, when compared to the ethyl analogs 1–3, phenol was preferentially released instead of the amine cargos. Surprisingly, the l-lactate spacer was the most suitable for aliphatic amines, releasing the cargo in 15 min (7, 8, 11, 12, 14, 15). In contrast, linkers with aromatic amine cargos (aniline (9 and 16), N-methylaniline (10), and 2-aminopyrimidine (13)) released their cargos more slowly than linkers bearing aliphatic amines, as indicated by the formation of higher amounts of intermediate I.
Slower SI, especially in 13 (13-I), could be partly explained by the low pKa of 2-aminopyrimidine (pKa 3.54 [28]). Although there is no clear correlation between the pKa of amine and the SI rate, pKa plays a key role in amine release [29]. N-protonation in phosphoramidates facilitates the nucleophilic attack of water (a carboxylate group in our case) to the phosphorus atom. Imbach [30] has shown that phosphoramidates consisting of low pKa amines are stable, whereas those containing amines with a higher pKa (more than 9) show the fastest hydrolysis, which has also been described by Wagner [31]. Nevertheless, differences in cargo release rate could not be easily explained by the various pKa of amines. Based on Mayr’s extensive studies of amine behavior in solution, the amine leaving group can be affected by attributes other than pKa, such as nucleophilicity [32], hydration energy (amine stabilization by solvation in water), polarity, and solvent pH, in addition to structural (cyclic vs. acyclic) or sterical effects. Therefore, predicting the optimal spacer for a specific cargo is a difficult task, and designing purposeful drug delivery systems requires studying structure-activity relationships in detail.

4. Materials and Methods

Unless otherwise indicated, all chemicals were purchased from commercial suppliers (Sigma Aldrich, Merck, EU; Fluorochem, UK; Acros Organics, Thermo Fisher Scientific, EU) and used without further purification. All reactions sensitive to air or moisture were performed under an inert atmosphere of argon in dry solvents. Thin layer chromatography (TLC) was performed on TLC aluminium sheets (silica-gel 60 F254; Merck, EU) and visualized by UV fluorescence. The reaction was monitored by TLC and/or 31P-NMR spectroscopy in CDCl3. Flash-column chromatography was performed on a Compact (ECOM s.r.o., EU) chromatography system using silica-gel or C18 silica-gel 230–400 mesh, 60 Å (Merck KGaA, EU).
NMR spectroscopy. NMR spectra were recorded on a Bruker Avance III spectrometer operating at 400 MHz for 1H and 101 MHz for 13C equipped with a probe with an ATM module (5 mm BBFO BB-19F/1H/D Z-GRD). For NMR signal assignment, standard Bruker pulse sequences were used for 1D (1H, 13C-APT, 31P, 19F) and 2D (COSY, ROESY, HSQC, HMBC) NMR experiments at a corrected temperature of 25 °C. NMR spectra coupled with UV irradiation were recorded on a Bruker Avance III spectrometer with a broad-band cryo probe with an ATM module (5 mm CPBBO BB-1H/19F/15N/D Z-GRD) operating at 500 MHz for 1H and 125.7 MHz for 13C. All NMR data were interpreted using Topspin 3.5. For reference, the following solvent signals were used: DMSO-d6: 2.50 (1H) and 39.7 (13C) ppm or CDCl3: 7.28 (1H) and 77.0 (13C) ppm. The 31P-NMR spectra were referenced to H3PO4 with 0 ppm.
For NMR experiments with in situ irradiation, a light emitting diode (LED; Thorlabs, EU) was used at 365 nm. The light was guided into the spectrometer, directly into the NMR tube via a multimode silica optical fiber with 1 mm diameter, 0.39 NA, and a high amount of OH (Thorlabs, EU).
Mass spectrometry. Mass spectra were measured on a LTQ. Orbitrap XL (Thermo Fisher Scientific, EU) using electrospray ionization (ESI).
All products were viscous oils, semi-solids or non-crystalline solids. The reaction conditions were not optimized for the highest possible yields.
4,5-dimethoxy-2-nitrobenzyl (2-hydroxyethyl) carbonate (19) was prepared according to a literature procedure [22], using 1.0 g of 4,5-dimethoxy-2-nitrobenzyl chloroformate and obtained (0.98 g) as a yellow solid (90%).
l-4,5-dimethoxy-2-nitrobenzyl 2-hydroxypropanoate (24) was prepared according to a literature procedure [25], using 2.13 g of 4,5-dimethoxy-2-nitrobenzyl alcohol and obtained (0.94 g) as a light pink solid (33%).
General Procedure for the One-Pot Synthesis of Phosphate-Based Linkers. For each experiment, a DMNB-containing photoarm 19 or 24 (0.5 mmol, 1.0 eq.) was dissolved in 2.5 mL of dry toluene under argon at 25 °C, adding dry TEA (90.6 μL, 0.65 mmol, 1.3 eq.) followed by the corresponding dichlorophosphate (0.5 mmol, 1.0 eq.). The reaction mixture was stirred at 25 °C for 16 h, and the formation of the intermediates was confirmed by 31P-NMR (22: δP 4.7 ppm; 23: δP 0.08 ppm; 25: δP 4.6 and 4.1 ppm; 26: δP −0.08 and −0.17 ppm) before phosphorylating the amines. The corresponding amine (0.5 mmol, 1.0 eq.) was added, followed by dry TEA (69.7 μL, 0.5 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature until completing the reaction (monitored by 31P-NMR). After evaporating the solvent, pure products were isolated by Flash silica gel chromatography, which was followed by reverse-phase chromatography, as described for each compound.
4,5-dimethoxy-2-nitrobenzyl (2-((ethoxy(phenethylamino)phosphoryl)oxy)ethyl) carbonate (1) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→90:10, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→50:50, v/v), which afforded 1 as a dense yellow oil (58.6 mg, 23%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.74 (s, 1H, 3″), 7.30 (m, 2H, 3′), 7.25–7.20 (m, 1H, 4′), 7.20 (m, 2H, 2′), 7.07 (s, 1H, 6″), 5.60 (s, 1H, 4a), 5.60 (s, 1H, 4b), 4.40 (m, 2H, 2), 4.25–4.10 (m, 2H, 1), 4.10–3.99 (m, 2H, OCH2CH3), 3.98 and 3.97 (-s, 6H, 4″-OCH3, 5″-OCH3), 3.25–3.16 (m, 2H, NHCH2), 2.80 (m, 1H, CH2Ph), 1.32 (td, 3H, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.59 (3), 153.52 and 148.38 (4″, 5″), 139.68 (2″), 138.53 (1′), 128.88 (2′), 128.64 (3′), 126.59 (4′), 126.52 (1″), 109.96 (6″), 108.20 (3″), 67.08 (d, J2-P = 7.0 Hz, 2), 66.55 (4), 63.57 (d, J1-P = 5.2 Hz, 1), 62.66 (d, JCH2-P = 5.7 Hz, OCH2CH3), 56.55, and 56.44 (4″-OCH3, 5″-OCH3), 42.65 (NHCH2), 37.85 (d, JCH2-P = 6.2 Hz, CH2Ph), 16.19 (d, JCH3-P = 6.9 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 9.1 ppm. HR-MS (ESI) calculated for C22H30O10N2P 513.16326, found [M + H]+ 513.16284.
4,5-dimethoxy-2-nitrobenzyl (2-((ethoxy(piperidin-1-yl)phosphoryl)oxy)ethyl) carbonate (2) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→90:10, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→50:50, v/v), which afforded 2 as a dense yellow oil (103.7 mg, 0.22 mmol, 44%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.74 (s, 1H, 3″), 7.09 (s, 1H, 6″), 5.62–5.60 (m, 2H, 4), 4.46–4.35 (m, 2H, 2), 4.28–4.12 (m, 2H, 1), 4.09–3.98 (m, 2H, OCH2CH3), 4.00 (s, 3H, 5″-OCH3), 3.97 (s, 3H, 4″-OCH3), 3.14–3.05 (m, 4H, 1′), 1.62–1.47 (m, 6H, 2′, 3′), 1.31 (td, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.58 (3), 153.73 (5″), 148.35 (4″), 139.65 (2″), 126.62 (1″), 109.89 (6″), 108.18 (3″), 67.14 (d, J2-P = 7.4 Hz, 2), 66.47 (4), 63.38 (d, J1-P = 5.2 Hz, 1), 62.39 (d, JCH2-P = 5.9 Hz, OCH2CH3), 56.55 (5″-OCH3), 56.43 (4″-OCH3), 45.36 (d, J1′-P = 2.3 Hz, 1′), 26.02 (d, J2′-P = 5.1 Hz, 2′), 24.39 (d, J3′-P = 1.5 Hz, 3′), 16.17 (d, JCH3-CH2 = 7.0 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 9.10 ppm. HR-MS (ESI) calculated for C19H30O10N2P 477.16326, found [M + H]+ 477.16340.
4,5-dimethoxy-2-nitrobenzyl (2-((ethoxy(phenylamino)phosphoryl)oxy)ethyl) carbonate (3) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→90:10, v/v), followed by two reverse-phase chromatographies using a gradient (H2O/MeCN 100:0→50:50, v/v), which afforded 3 as a dense yellow oil (82.8 mg, 34%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.75 (s, 1H, 3″), 7.24 (m, 2H, 3′), 7.06 (s, 1H, 6″), 7.00 (m, 2H, 2′), 6.95 (m, 1H, 4′), 5.75 (d, JNH-P = 9.0 Hz, NH), 5.56 (m, 2H, 4), 4.42 (m, 2H, 2), 4.41–4.25 (m, 2H, 1), 4.25–4.08 (m, 2H, OCH2CH3), 3.98–3.97 (m, 6H, 4″-OCH3, 5″-OCH3), 1.33 (td, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.49 (3), 153.76 and 148.34 (4″, 5″), 139.61 (2″), 139.16 (1′), 129.35 (3′), 126.58 (1″), 121.97 (4′), 117.45 (d, J2′-P = 7.2 Hz, 2′), 109.82 (6″), 108.18 (3″), 66.78 (d, J2-P = 7.3 Hz, 2), 66.55 (4), 64.00 (d, J1-P = 4.6 Hz, 1), 63.31 (d, JCH2-P = 5.0 Hz, OCH2CH3), 56.57 and 56.43 (4″-OCH3, 5″-OCH3), 16.07 (d, JCH3-P = 7.0 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 2.31 ppm. HR-MS (ESI) calculated for C20H26O10N2P 485.13196, found [M + H]+ 485.13189.
4,5-dimethoxy-2-nitrobenzyl (2-(((phenethylamino)(phenoxy)phosphoryl)oxy)ethyl) carbonate(4) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v) and preparative HPLC chromatography using a gradient (H2O/MeCN 95:5 -> 20:80, v/v), which afforded 4 as a dense yellow oil (10.2 mg, 4%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.74 (s, 1H, 3″), 7.35–7.12 (m, 10H, 2′, 3′, 4′, 2‴, 3‴, 4‴), 7.06 (s, 1H, 6″), 5.60 (s, 1H, 4a), 5.60 (s, 1H, 4b), 4.47–4.37 (m, 2H, 2), 4.35–4.21 (m, 2H, 1), 3.97 (s, 3H, 4″-OCH3), 3.94 (s, 3H, 5″-OCH3), 3.33–3.24 (m, 2H, NHCH2), 2.93–2.82 (m, 1H, NH), 2.77 (t, 2H, JCH2Ph-CH2NH = 6.8 Hz, CH2Ph). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.55 (3), 153.73 (5″), 150.78 (d, J1‴-P = 6.8 Hz, 1‴), 148.37 (4″), 139.65 (2″), 138.27 (1′), 129.69 (3′ or 3‴), 128.87 (2′), 128.67 (3′ or 3‴), 126.65 (4′), 126.50 (1″), 124.87 (4‴), 120.13 (d, J2‴-P = 4.8 Hz, 2‴), 109.94 (6″), 108.19 (3″), 66.87 (d, J2-P = 7.4 Hz, 2), 66.58 (4), 64.18 (d, J1-P = 5.2 Hz, 1), 56.54 (5″-OCH3), 56.43 (4″-OCH3), 42.78 (NHCH2), 37.69 (d, JCH2-P = 6.0 Hz, CH2Ph). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 4.42 ppm. HR-MS (ESI) calculated for C26H30O10N2P 561.16326, found [M + H]+ 561.16318.
4,5-dimethoxy-2-nitrobenzyl (2-(((piperidin-1-yl)(phenoxy)phosphoryl)oxy)ethyl) carbonate(5) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v) and preparative HPLC chromatography using a gradient (H2O/MeCN 95:5→0:100, v/v), which afforded 5 as a dense yellow oil (31.9 mg, 12%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.75 (s, 1H, 3″), 7.32 (m, 2H, 3‴), 7.22 (m, 2H, 2‴), 7.14 (m, 1H, 4‴), 7.09 (s, 1H, 6″), 5.62 (s, 1H, 4a), 5.62 (s, 1H, 4b), 4.47–4.43 (m, 2H, 2), 4.35–4.28 (m, 2H, 1), 3.99 (s, 3H, 5″-OCH3), 3.98 (s, 3H, 4″-OCH3), 3.20–3.12 (m, 4H, 1′), 1.60–1.51 (m, 2H, 3′), 1.51–1.44 (m, 4H, 2′). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.57 (3), 153.75 (5″), 150.98 (d, J1‴-P = 6.8 Hz, 1‴), 148.36 (4″), 139.64 (2″), 129.60 (3‴), 126.61 (1″), 124.62 (4″), 120.07 (d, J2‴-P = 5.2 Hz, 2‴), 109.90 (6″), 108.18 (3″), 66.96 (d, J2-P = 7.3 Hz, 2), 66.53 (4), 63.98 (d, J1-P = 5.3 Hz, 1), 56.55 and 56.44 (4″-OCH3, 5″-OCH3), 45.49(d, J1′-P = 2.3 Hz, 1′), 25.82 (d, J2′-P = 4.7 Hz, 2′), 25.26 (3′). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 4.27 ppm. HR-MS (ESI) calculated for C23H30O10N2P 525.16326, found [M + H]+ 525.16278.
4,5-dimethoxy-2-nitrobenzyl (2-(((phenylamino)(phenoxy)phosphoryl)oxy)ethyl) carbonate(6) was prepared from 19 (150.5 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v) and preparative HPLC chromatography using a gradient (H2O/MeCN 95:5→0:100, v/v), which afforded 6 as a dense yellow oil (18.1 mg, 7%). 1H-NMR (400 MHz, CDCl3, 25 °C) δ 7.74 (s, 1H, 3″), 7.28–7.22 (m, 4H, 3′, 3‴), 7.17–7.11 (m, 3H, 2‴, 4‴), 7.07 (m, 2H, 2′), 7.05 (s, 1H, 6″), 6.99 (m, 1H, 4′), 6.25 (d, JNH-P = 9.8 Hz, NH), 5.62–5.52 (m, 2H, 4), 4.49–4.33 (m, 4H, 1, 2), 3.97 (s, 3H, 4″-OCH3), 3.93 (s, 3H, 5″-OCH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 154.47 (3), 153.77 (5″), 150.15 (d, J1‴-P = 6.1 Hz, 1‴), 148.32 (4″), 139.56 (2″), 138.79 (1′), 129.70 (3‴), 129.39 (3′), 126.58 (1″), 125.30 (4‴), 122.33 (4′), 120.38 (d, J2‴-P = 4.8 Hz, 2‴), 117.87 (d, J2′-P = 7.4 Hz, 2′), 109.77 (6″), 108.16 (3″), 66.61 (d, J2-P = 7.0 Hz, 2), 66.59 (4), 64.53 (d, J1-P = 4.6 Hz, 1), 56.53 (5″-OCH3), 56.42 (4″-OCH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ −1.97 ppm. HR-MS (ESI) calculated for C24H26O10N2P 533.13196, found [M + H]+ 533.13188.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(phenethylamino)phosphoryl)oxy)propanoate (7) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 50:50→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeOH 100:0→0:100, v/v), which afforded 7 as a dense yellow oil (129.3 mg, 52%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.73 and 7.72 (s, 2H, 3″), 7.33–7.13 (m, 10H, 2′, 3′, 4′), 7.07 and 7.03 (s, 2H, 6″), 5.66–5.53 (m, 4H, 4), 5.02–4.88 (m, 2H, 1), 4.14–3.94 (m, 4H, OCH2CH3), 4.00 and 3.98 (s, 6H, 5″-OCH3), 3.96 and 3.96 (s, 6H, 4″-OCH3), 3.26–3.16 (m, 4H, NHCH2), 2.83–2.67 (m, 6H, CH2Ph, NH), 1.61 (d, 3H, J2-1 = 6.8 Hz), 1.56 (d, 3H, J2-1 = 6.9 Hz, 2), 1.32 (td, 3H, JCH3-CH2 = 7.0 Hz, JCH3-P = 0.8 Hz, OCH2CH3), 1.28 (td, 3H, JCH3-CH2 = 7.2 Hz, JCH3-P = 0.7 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.68–170.47 (m, 3), 153.65 and 153.63 (5″), 148.40 and 148.37 (4″), 139.84 (2″), 138.55 and 138.50 (1′), 128.86 and 128.83 (2′), 128.65 and 128.58 (3′), 126.53 and 126.43 (4′), 126.32 and 126.29 (1″), 110.47 and 110.35 (6″), 108.26 and 108.23 (3″), 70.57 (d, J1-P = 7.6 Hz, 1), 70.52 (d, J1-P = 7.6 Hz, 1), 64.09 and 64.06 (4), 62.93 (d, JCH2-P = 5.7 Hz, OCH2CH3), 62.79 (d, JCH2-P = 5.7 Hz, OCH2CH3), 56.68 and 56.62 (4″-OCH3), 56.42 (5″-OCH3), 42.65 and 42.54 (NHCH2), 37.88–37.70 (m, CH2Ph), 19.42 (d, J2-P = 5.5 Hz, 2), 19.41 (d, J2-P = 5.3 Hz, 2), 16.18 (d, JCH3-P = 7.0 Hz, OCH2CH3), 16.13 (d, JCH3-P = 7.0 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 9.07 and 8.39 ppm. HR-MS (ESI) calculated for C22H29O9N2NaP 519.15029, found [M + Na]+ 519.14986.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(piperidin-1-yl)phosphoryl)oxy)propanoate (8) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→80:20, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v), which afforded 8 as a dense yellow oil (97.0 mg, 42%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.74 and 7.73 (s, 2H, 3″), 7.10 and 7.09 (s, 2H, 6″), 5.64 (dd, 1H, JGem = 14.8 Hz, J4a-6″ = 0.6 Hz, 4a), 5.63 (dd, 1H, JGem = 14.8 Hz, J4a-6″ = 0.6 Hz, 4a), 5.59 (dd, 1H, JGem = 14.8 Hz, J4b-6″ = 0.6 Hz, 4b), 5.58 (dd, 1H, JGem = 14.8 Hz, J4b-6″ = 0.6 Hz, 4b), 5.00–4.88 (m, 1H, 1), 4.12–3.99 (m, 2H, OCH2CH3), 4.02 and 4.02 (s, 6H, 5″-OCH3), 3.97 and 3.97 (s, 6H, 4″-OCH3), 3.19–3.01 (m, 4H, 1′), 1.61 (d, J2-P = 6.9 Hz, 3H, 2), 1.59 (d, J2-P = 6.9 Hz, 3H, 2), 1.57–1.44 (m, 10H, 2′, 3′), 1.33 (td, 3H, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3), 1.28 (td, 3H, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.71 (d, J3-P = 4.4 Hz, 3), 170.51 (d, J3-P = 5.7 Hz, 3), 153.70 and 153.68 (5″), 148.28 (4″), 139.74 (2″), 126.66 and 126.60 (1″), 110.37 and 110.24 (6″), 108.17 (3″), 70.33 (d, J1-P = 5.1 Hz, 1), 70.18 (d, J1-P = 5.2 Hz, 1), 63.92 and 63.89 (4), 62.60 (d, JCH2-P = 5.9 Hz, OCH2CH3), 62.42 (d, JCH2-P = 5.8 Hz, OCH2CH3), 56.66 and 56.61 and 56.39–56.36 (m) (4″-OCH3, 5″-OCH3), 45.34 and 45.32 (1′), 25.96 (d, J2′-P = 4.7 Hz, 2′), 25.84 (d, J2′-P = 5.4 Hz, 2′), 24.33 (d, J3′-P = 1.5 Hz, 3′), 24.29 (d, J3′-P = 1.5 Hz, 3′), 19.45 (d, J2-P = 4.8 Hz, 2), 19.41 (d, J2-P = 5.2 Hz, 2), 16.17–16.01 (m, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 8.81 and 8.30 ppm. HR-MS (ESI) calculated for C19H29O9N2NaP 483.15029, found [M + Na]+ 483.14977.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(phenylamino)phosphoryl)oxy)propanoate (9) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v), which afforded 9 as a dense yellow oil (108.2 mg, 46%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.73 and 7.64 (s, H, 3″), 7.22 and 7.17 (m, 4H, 3′), 7.08 and 6.94 (s, 2H, 6″), 7.06–6.96 (m, 4H, 2′), 6.96–6.89 (m, 2H, 4′), 6.35 (d, 1H, JNH-P = 9.3 Hz, NH), 6.22 (d, 1H, JNH-P = 8.7 Hz, NH), 5.65 (dd, 1H, JGem = 14.9 Hz, J4a-6″ = 0.6 Hz, 4a), 5.57 (dd, 1H, JGem = 14.9 Hz, J4b-6″ = 0.5 Hz, 4b), 5.56–5.41 (m, 2H, 4a, 4b), 5.12 and 5.06 (m, 2H, 1), 4.29–4.07 (m, 4H, OCH2CH3), 3.97–3.95 (m, 6H, 4″-O-CH3), 3.95 and 3.90 (s, 6H, 5″-OCH3), 1.67 (d, 3H, J2-1 = 6.9 Hz, 2), 1.52 (d, 3H, J2-1 = 6.9 Hz, 2), 1.36–1.26 (m, 6H, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.13 (d, J3-P = 4.6 Hz, 3), 170.10 (d, J3-P = 5.4 Hz, 3), 153.64 and 153.55 (5″), 148.28 and 148.26 (4″), 139.69 and 139.67 (2″), 139.20 (d, J1′-P = 4.7 Hz, 1′), 129.19 and 129.10 (3′), 126.40 and 126.16 (1″), 121.88 and 121.84 (4′), 117.63 (d, J2′-P = 7.7 Hz, 2′), 117.55 (d, J2′-P = 7.3 Hz, 2′), 110.30 and 110.17 (6″), 108.15 and 108.11 (3″), 71.25 (d, J1-P = 4.6 Hz, 1), 70.92 (d, J1-P = 4.7 Hz, 1), 64.12 and 64.09 (4), 63.46 (d, JCH2-P = 5.4 Hz, OCH2CH3), 63.36 (d, JCH2-P = 5.6 Hz, OCH2CH3), 56.53, 56.40, 56.35, and 56.33 (4″-OCH3, 5″-OCH3), 19.30 (d, J2-P = 5.1 Hz, 2), 19.12 (d, J2-P = 6.0 Hz, 2),15.98 (d, JCH3-P = 7.3 Hz, OCH2CH3), 15.95 (d, JCH3-P = 7.3 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 2.14 and 1.86 ppm. HR-MS (ESI) calculated for C20H25O9N2NaP 491.11899, found [M + Na]+ 491.11862.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(N-methylphenyl)phosphoryl)oxy)propanoate (10) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by two reverse-phase chromatographies using a gradient (H2O/MeCN 100:0 -> 50:50, v/v), which afforded 10 as a dense yellow oil (44.5 mg, 18%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.74 and 7.73 (s, 2H, 3″), 7.33–7.30 (m, 2H, 2′), 7.30–7.22 (m, 6H, 2′, 3′), 7.14–7.04 (m, 2H, 4′), 7.10 and 7.00 (s, 2H, 6″), 5.65–5.62 and 5.56–5.54 (m, 4H, 4), 5.11–4.96 (m, 2H, 1), 4.23–4.00 (m, 4H, OCH2CH3), 3.99 and 3.98 and 3.98 (s, 9H, 4″-OCH3, 5″-OCH3), 3.91 (s, 3H, 5″-OCH3), 3.22 (d, 3H, JCH3-P = 9.0 Hz, NCH3), 3.17 (d, 3H, JCH3-P = 9.1 Hz, NCH3), 1.65 (d, 3H, J2-1 = 7.0 Hz, 2), 1.51 (d, 3H, J2-1 = 7.0 Hz, 2), 1.30 (td, 3H, JCH3-CH2 = 7.0 Hz, JCH3-P = 0.8 Hz, OCH2CH3), 1.28 (dt, 3H, JCH3-CH2 = 7.0 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.4* and 170.2* (3), 153.8* (5″), 148.3* (4″), 143.9* (1′), 139.8* and 139.7* (2″), 128.97 and 128.85 (3′), 126.67 and 126.65 (1″), 124.27 and 124.19 (4′), 123.13 (d, J2′-P = 4.6 Hz, 2′), 122.69 (d, J2′-P = 3.9 Hz, 2′), 110.33 and 110.11 (6″), 108.21 and 108.15 (3″), 71.15 (d, J1-P = 5.4 Hz, 1), 70.90 (d, J1-P = 4.9 Hz, 1), 64.08 and 64.04 (4), 63.34 (d, JCH2-P = 5.7 Hz, OCH2CH3), 62.96 (d, JCH2-P = 5.6 Hz, OCH2CH3), 56.69 and 56.58 and 56.49–56.33 (m, 4″-OCH3, 5″-OCH3), 37.35–36.95 (m, NCH3), 19.37 (d, J2-P = 4.6 Hz, 2), 19.16 (d, J2-P = 5.4 Hz, 2), 15.99 (d, JCH3-P = 6.7 Hz, OCH2CH3). * The 13C chemical shift was extracted from HMBC. 31P-NMR (162 MHz, CDCl3, 25 °C) δ 5.82 and 5.55 ppm. HR-MS (ESI) calculated for C21H27O9N2NaP 505.13464, found [M + Na]+ 505.13472.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(morpholin-1-yl)phosphoryl)oxy)propanoate (11) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by two reverse-phase chromatographies using a gradient (H2O/MeCN 100:0→60:40, v/v), which afforded 11 as a dense yellow oil (80.0 mg, 35%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.72–7.70 (m, 2H, 3″), 7.04 and 7.02 (s, 2H, 6″), 5.64–5.51 (m, 4H, 4), 5.00–4.87 (m, 2H, 1), 4.13–4.01 (m, 4H, OCH2CH3), 3.99 and 3.99 (s, 6H, 5″-OCH3), 3.96–3.94 (m, 6H, 4″-OCH3), 3.64 and 3.59 (m, 8H, 2′), 3.19–3.04 (m, 8H, 1′), 1.59 (dd, 6H, J2-1 = 6.9 Hz, J2-P = 1.0 Hz, 2), 1.32 (td, 3H, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.9 Hz, OCH2CH3), 1.28 (td, 3H, JCH3-CH2 = 7.1 Hz, JCH3-P = 0.8 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.41 (d, J3-P = 4.6 Hz, 3), 170.27 (d, J3-P = 4.9 Hz, 3), 153.55 and 153.52 (5″), 148.37 and 148.33 (4″), 139.85 and 139.82 (2″), 126.21 and 126.08 (1″), 110.53 and 110.49 (6″), 108.20 and 108.15 (3″), 70.49 (d, J1-P = 5.0 Hz, 1), 70.40 (d, J1-P = 5.3 Hz, 1), 66.83 (d, J2′-P = 5.8 Hz, 2′), 66.73 (d, J2′-P = 6.1 Hz, 2′), 64.02 and 64.00 (4), 62.95 (d, JCH2-P = 6.1 Hz, OCH2CH3), 62.74 (d, JCH2-P = 6.1 Hz, OCH2CH3), 56.56 and 56.52 (5″-OCH3), 56.35–56.30 (m, 4″-OCH3), 44.49–44.43 (m, 1′), 19.34 (d, J2-P = 4.7 Hz, 2), 19.33 (d, J2-P = 5.5 Hz, 2), 16.03 (d, JCH3-P = 7.0 Hz, OCH2CH3), 15.98 (d, JCH3-P = 6.7 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 7.68 and 7.13 ppm. HR-MS (ESI) calculated for C18H28O10N2P 463.14761, found [M + H]+ 463.14720.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(benzylamino)phosphoryl)oxy)propanoate (12) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 50:50→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeOH 100:0→0:100, v/v), which afforded 12 as a dense yellow oil (101 mg, 42%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 7.73 and 7.71 (s, 2H, 3″), 7.36–7.22 (m, 10H, 2′, 3′, 4′), 7.07 and 7.02 (s, 2H, 6″), 5.63 (d, 1H, JGem = 14.6 Hz, 4a), 5.58 (d, JGem = 14.6 Hz, 1H, 4b), 5.56 (d, 1H, JGem = 14.7 Hz, 4a), 5.51 (d, 1H, JGem = 14.7 Hz, 4b), 5.04 and 4.97 (m, 2H, 1), 4.17–4.05 (m, 8H, NHCH2, OCH2CH3), 4.00 and 3.98 (s, 6H, 5″-OCH3), 3.96 and 3.96 (s, 6H, 4″-OCH3), 3.12 (m, 2H, NH), 1.63 (d, 3H, J2-1 = 6.9 Hz, 2), 1.54 (d, 3H, J2-1 = 6.9 Hz, 2), 1.32 (tm, JCH3-CH2= 7.1 Hz, 3H, OCH2CH3), 1.29 (tm, JCH3-CH2= 7.1 Hz, 3H, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.69–170.57 (m, 3), 153.66 and 153.61 (5″), 148.35 and 148.32 (4″), 139.80 (2″), 13942–139.30 (m, 1′), 128.56 and 128.48 (3′), 127.41, 127.30, and 127.21 (2′, 4′), 126.43 and 126.23 (1″), 110.46 and 110.37 (6″), 108.19 (3″), 70.67 (d, J1-P = 4.7 Hz, 1), 70.60 (d, J1-P = 5.1 Hz, 1), 64.07 and 64.04 (4), 63.06 (d, JCH2-P = 5.4 Hz, OCH2CH3), 62.91 (d, JCH2-P = 5.4 Hz, OCH2CH3), 56.63 and 56.55 (5″-OCH3), 56.37 (4″-OCH3), 45.23 (d, JCH2-P = 6.2 Hz, NHCH2), 19.34 (d, J2-P = 5.4 Hz, 2), 19.28 (d, J2-P = 5.9 Hz, 2), 16.08 (d, JCH3-P = 7.4 Hz, OCH2CH3), 16.04 (d, JCH3-P = 6.9 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 8.61 and 7.87 ppm. HR-MS (ESI) calculated for C21H27O9N2NaP 505.13464, found [M + Na]+ 505.13456.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((ethoxy(pyrimidin-2-ylamino)phosphoryl)oxy)propanoate (13) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 20 (63.2 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→90:10, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→0:100, v/v), which afforded 13 as a dense yellow oil (67.0 mg, 29%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 8.53 (d, 1H, J2′-P = 4.9 Hz, 2′), 8.47 (d, 1H, J2′-P = 4.9 Hz, 2′), 7.73 and 7.71 (s, 2H, 3″), 7.13 and 7.07 (s, 2H, 6″), 6.88 (t, 2H, J3′-P = 4.9 Hz, 3′), 6.85 (t, 2H, J3′-P = 4.9 Hz, 3′), 5.64 (dd, 1H, JGem = 15.0 Hz, J4a-6″ = 0.5 Hz, 4a), 5.59 (dd, 1H, JGem = 15.0 Hz, J4b-6″ = 0.5 Hz, 4b), 5.56 (dd, 1H, JGem = 14.9 Hz, J4a-6″ = 0.5 Hz, 4a), 5.51 (dd, 1H, JGem = 14.9 Hz, J4b-6″ = 0.5 Hz, 4b), 5.43–5.29 (m, 2H, 1), 4.39–4.18 (m, 4H, OCH2CH3), 4.02 and 3.98 (s, 6H, 5″-O-CH3), 3.96 and 3.96 (s, 6H, 4″-OCH3), 1.66 (dd, 3H, J2-1 = 6.9 Hz, J2-P = 0.4 Hz, 2), 1.59 (dd, 3H, J2-1 = 7.0 Hz, J2-P = 0.5 Hz, 2), 1.35 (td, 3H, JCH3-CH2= 7.1 Hz, J CH3-P = 0.9 Hz, OCH2CH3), 1.34 (td, 3H, JCH3-CH2= 7.1 Hz, J CH3-P = 0.9 Hz, OCH2CH3). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.73 (d, J3-P = 3.9 Hz, 3), 170.40 (d, J3-P = 4.6 Hz, 3), 159.22 (d, J1′-P = 6.2 Hz, 1′), 159.11 (d, J1′-P = 5.9 Hz, 1′), 158.38 (d, J2′-P = 1.5 Hz, 2′), 158.30 (d, J2′-P = 1.5 Hz, 2′), 153.81 and 153.65 (5″), 148.28 and 148.22 (4″), 139.67 and 139.56 (2″), 126.70 and 126.27 (1″), 114.56 and 114.51 (3′), 110.28 and 110.11 (6″), 108.12 and 108.11 (3″), 72.29 (d, J1-P = 5.4 Hz, 1), 72.19 (d, J1-P = 5.1 Hz, 1), 64.06 and 64.03 (4), 63.94 (d, JCH2-P = 5.5 Hz, OCH2CH3), 56.76 and 56.59 (5″-OCH3), 56.39–56.32 (m, 4″-OCH3), 19.37 (d, J2-P = 5.9 Hz, 2), 19.05 (d, J2-P = 6.7 Hz, 2), 16.07 (d, JCH3-P = 7.1 Hz, OCH2CH3), 16.03 (d, JCH3-P = 7.1 Hz, OCH2CH3). 31P-NMR (162 MHz, CDCl3, 25 °C) δ −0.87 ppm. HR-MS (ESI) calculated for C18H24O9N4P 471.12754, found [M + H]+ 471.12723.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((phenoxy(phenethylamino)phosphoryl)oxy)propanoate(14) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 100:0→50:50, v/v), which afforded 14 as a dense yellow oil (79.4 mg, 29%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:0.8 ratio) δ 7.74 and 7.71 (s, 2H, 3″), 7.38–7.11 (m, 20H, 2′, 3′, 4′, 2‴, 3‴, 4‴), 7.04 and 7.02 (s, 2H, 6″), 5.63 (dd, JGem = 14.8 Hz, J4a-6″ = 0.5 Hz, 1H, 4a), 5.63 (dd, 1H, JGem = 14.7 Hz, J4a-6″ = 0.5 Hz, 4a), 5.58 (dd, 1H, JGem = 14.7 Hz, J4b-6″ = 0.5 Hz, 4b), 5.55 (dd, 1H, JGem = 14.8 Hz, J4b-6″ = 0.5 Hz, 4b), 5.11–5.01 (m, 2H, 1), 3.98 (s, 3H, 4″-OCH3), 3.97 (s, 3H, 5″-OCH3), 3.96 (s, 3H, 4″-OCH3), 3.89 (s, 3H, 5″-OCH3), 3.39–3.23 (m, 4H, NHCH2), 2.96–2.84 (m, 2H, NH), 2.80–2.74 (m, 4H, CH2Ph), 1.64 (d, 3H, J2-1 = 6.9 Hz, 2), 1.57 (dd, 3H, J2-1 = 7.0 Hz, J2-P = 0.3 Hz, 2). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.37 (d, J3-P = 4.5 Hz, 3), 170.15 (d, J3-P = 4.3 Hz, 3), 153.72 and 153.71 (5″), 150.8* (1‴), 148.44 and 148.32 (4″), 139.87 and 139.77 (2″), 138.29 and 138.18 (1′), 129.69 and 129.62 (3′), 128.86 and 128.84 (2′), 128.71 and 128.63 (3‴), 126.71 and 126.60 (4‴), 126.45 and 126.30 (1″), 124.97 and 124.92 (4′), 124.97 and 124.92 (2′), 120.27 (d, J2‴-P = 4.9 Hz, 2‴), 120.11 (d, J2‴-P = 5.1 Hz, 2‴), 110.40 and 110.35 (6″), 108.28 and 108.16 (3″), 71.27 (d, J1-P = 4.9 Hz, 1), 71.25 (d, J1-P = 5.3 Hz, 1), 64.22 and 64.12 (4), 56.64, 56.56, 56.44, and 56.41 (4″-OCH3, 5″-OCH3), 42.77 and 42.66 (NHCH2), 37.64 (d, JCH2-P = 5.9 Hz, CH2Ph), 37.61 (d, JCH2-P = 6.1 Hz, CH2Ph), 19.44 (d, J2-P = 5.5 Hz, 2) 19.21 (d, J2-P = 5.8 Hz, 2). * The 13C chemical shift was extracted from HMBC. 31P-NMR (162 MHz, CDCl3, 25 °C) δ 4.40 and 3.55 ppm. HR-MS (ESI) calculated for C26H29O9N2NaP 567.15029, found [M + Na]+ 567.14944.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((phenoxy(piperidin-1-yl)phosphoryl)oxy)propanoate(15) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→90:10, v/v), followed by two reverse-phase chromatographies using a gradient (H2O/MeCN 100:0→60:40, v/v), which afforded 15 as a dense yellow oil (75.5 mg, 30%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:0.7 ratio) δ 7.75 and 7.72 (s, 2H, 3″), 7.34 and 7.27 (m, 4H, 3‴), 7.23 and 7.18 (m, 4H, 2‴), 7.17–7.10 (m, 2H, 4‴), 7.09 and 7.05 (s, 2H, 6″), 5.67–5.55 (m, 4H, 4), 5.12–5.01 (m, 2H, 1), 4.00 (s, 3H, 5″-OCH3), 3.98 (s, 3H, 4″-OCH3), 3.96 (s, 3H, 4″-OCH3), 3.88 (s, 3H, 5″-OCH3), 3.24–3.12 (m, 8H, 1′), 1.66 (d, 3H, J2-1 = 6.9 Hz, 2), 1.58 (d, 3H, J2-1 = 6.9 Hz, 2), 1.59–1.52 (m, 4H, 3′), 1.52–1.40 (m, 8H, 2′). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.32 (d, J3-P = 4.7 Hz, 3), 170.27 (d, J3-P = 4.6 Hz, 3), 153.77 and 153.75 (5″), 150.94 (d, J1‴-P = 6.9 Hz, 1‴), 150.86 (d, J1‴-P = 6.6 Hz, 1‴), 148.36 and 148.25 (4″), 139.78 and 139.68 (2″), 129.60 and 129.53 (3‴), 126.66 and 126.59 (1″), 124.71 (d, J4‴-P = 1.5 Hz, 4‴), 124.65 (d, J4‴-P = 1.5 Hz, 4‴), 120.17 (d, J2‴-P = 4.6 Hz, 2‴), 120.02 (d, J2‴-P = 5.4 Hz, 2‴), 110.28 and 110.26 (6″), 108.23 and 108.12 (3″), 71.06 (d, J1‴-P = 5.4 Hz, 1‴), 70.86 (d, J1‴-P = 5.3 Hz, 1‴), 64.07 and 64.01 (4), 56.68 and 56.58 (5″-OCH3), 56.44 and 56.40 (4″-OCH3), 45.51 (d, J1′-P = 2.3 Hz, 1′), 45.50 (d, J1′-P = 2.2 Hz, 1′), 25.79 (d, J2′-P = 4.6 Hz, 2′), 25.68 (d, J2′-P = 4.7 Hz, 2′), 24.24–24.20 (m, 3′), 19.48 (d, J2-P = 5.4 Hz, 2), 19.26 (d, J2-P = 5.4 Hz, 2). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 3.96 and 3.47 ppm. HR-MS (ESI) calculated for C23H30O9N2P 509.16834, found [M + H]+ 509.16852.
4,5-dimethoxy-2-nitrobenzyl (2S)-2-((phenoxy(phenylamino)phosphoryl)oxy)propanoate(16) was prepared from 24 (142.6 mg, 0.5 mmol, 1.0 eq.) and 21 (74.7 μL, 0.5 mmol, 1.0 eq.) according to the general procedure and purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 100:0→0:100, v/v), followed by two reverse-phase chromatographies using a gradient (H2O/MeCN 100:0→60:40, v/v), which afforded 16 as a dense yellow oil (53.5 mg, 21%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 2:1 ratio) δ 7.73 and 7.71 (s, 2H, 3″), 7.32–6.96 (m, 20H, 2′, 3′, 4′, 2‴, 3‴, 4‴), 5.91 (d, 1H, JNH-P = 9.8 Hz, NH), 5.86 (d, 1H, JNH-P = 9.1 Hz, NH), 5.67 (dd, 1H, JGem = 14.8 Hz, J4a-6″ = 0.6 Hz, 4a), 5.58 (dd, 1H, JGem = 14.8 Hz, J4b-6″ = 0.6 Hz, 4b), 5.55 (dd, JGem = 14.5 Hz, J4a-6″ = 0.6 Hz, 4a), 5.51 (dd, JGem = 14.5 Hz, J4b-6″ = 0.6 Hz, 4b), 5.26–5.14 (m, 2H, 1), 3.97–3.96 (m, 6H, 4″-OCH3), 3.90 and 3.87 (s, 6H, 5″-OCH3), 1.67 (dd, 3H, J2-1 = 6.9 Hz, J2-P = 0.5 Hz, 2), 1.60 (dd, 3H, J2-1 = 6.9 Hz, J2-P = 0.3 Hz, 2). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 170.03 (d, J3-P = 5.0 Hz, 3), 169.75 (d, J3-P = 4.7 Hz, 3), 153.73 and 153.65 (5″), 150.29 (d, J1‴-P = 6.8 Hz, 1‴), 150.12 (d, J1‴-P = 6.5 Hz, 1‴), 148.42 and 148.34 (4″), 139.84 and 139.75 (2″), 138.63 and 138.58 (1′), 129.72 and 129.66 (3‴), 129.40 and 129.29 (3′), 126.37 and 126.06 (1″), 125.40–125.31 (4‴), 122.57 and 122.52 (4′), 120.43 (d, J2‴-P = 4.6 Hz, 2‴), 120.28 (d, J2‴-P = 4.7 Hz, 2‴), 118.24 (d, J2′-P = 7.3 Hz, 2′), 118.07 (d, J2′-P = 7.5 Hz, 2′), 110.39 and 110.33 (6″), 108.23 and 108.18 (3″), 72.00 (d, J1-P = 4.9 Hz, 1), 71.75 (d, J1-P = 4.8 Hz, 1), 64.37 and 64.30 (4), 56.54 and 56.51 (5″-OCH3), 56.45–56.39 (4″-OCH3), 19.23 (d, J2-P = 5.8 Hz, 2). 31P-NMR (162 MHz, CDCl3, 25 °C) δ −2.50 and −2.99 ppm. HR-MS (ESI) calculated for C24H26O9N2P 517.13704, found [M + H]+ 517.13695.
Methyl-1-cyclopropyl-7-(4-(((1-((4,5-dimethoxy-2-nitrobenzyl)oxy)-1-oxopropan-2-yl)oxy)(ethoxy)phosphoryl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (17). To a solution of 24 (71.3 mg, 0.25 mmol, 1 eq.) in 1.5 mL of dry toluene, 20 (31.6 μL, 0.25 mmol, 1.0 eq.), and TEA (45.4 μL, 0.325 mmol, 1.3 eq.) were added, and the reaction mixture was stirred at 25 °C for 16 h. The formation of the intermediate 25 was confirmed by 31P NMR (δP 4.6 and 4.1 ppm) before adding amine 27 (86.3 mg, 0.25 mmol, 1.0 eq.) and TEA (34.9 μL, 0.25 mmol, 1.0 eq.). The reaction mixture was stirred at room temperature until completion (1 h). After solvent evaporation, the crude residue was purified by Flash silica gel chromatography using a gradient (hexane/EtOAc/MeOH 50:50:0→0:100:0→0:50:50 v/v/v), which afforded 17 as a white solid (78.1 mg, 43%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 8.53 and 8.53 (s, 2H, 9′), 8.00 (d, 1H, J7′-F = 13.2 Hz, 7′), 8.00 (d, 1H, J7′-F = 13.1 Hz, 7′), 7.72 and 7.67 (s, 2H, 3″), 7.27 (d, 1H, J4′-F = 7.2 Hz, 4′), 7.26 (d, 1H, J4′-F = 7.1 Hz, 4′), 7.05 and 7.02 (s, 2H, 6″), 5.66–5.53 (m, 4H, 4), 5.06–4.92 (m, 2H, 1), 4.19–4.06 (m, 4H, OCH2CH3), 4.01 and 4.00 (s, 6H, 5″-OCH3), 3.97 and 3.93 (s, 6H, 4″-OCH3), 3.91–3.90 (m, 6H, 16′), 3.50–3.30 (m, 10H, 1′, 12′), 3.26–3.08 (m, 8H, 2′), 1.65–1.60 (m, 6H, 2), 1.43–1.28 (m, 10H, OCH2CH3, 13′ or 14′), 1.18–1.11 (m, 4H, 13′ or 14′). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 173.04 (d, J11′-F = 2.1 Hz, 11′), 170.00 (d, J11′-F = 2.2 Hz, 11′), 170.50 (d, J3-P = 4.6 Hz, 3), 170.43 (d, J3-P = 5.4 Hz, 3), 166.40 and 166.35 (15′), 153.38 (d, J8′-F = 248.6 Hz, 8′), 153.63 (5″), 148.58 and 148.55 (4″), 148.42 (9′), 144.55 (d, J3′-F = 10.5 Hz, 3′), 144.49 (d, J3′-F = 10.6 Hz, 3′), 140.06 (2″), 138.01 (d, J5′-F = 1.5 Hz, 5′), 137.99 (d, J5′-F = 1.5 Hz, 5′), 126.09 and 125.97 (1″), 123.36 (d, J6′-F = 7.3 Hz, 6′), 123.28 (d, J6′-F = 6.9 Hz, 6′), 113.40 (d, J7′-F = 23.5 Hz, 7′), 113.30 (d, J7′-F = 22.9 Hz, 7′), 110.94 and 110.85 (6″), 110.12 and 110.07 (10′), 108.30 and 108.26 (3″), 105.13 (d, J4′-F = 9.5 Hz, 4′), 105.12 (d, J4′-F = 9.1 Hz, 4′), 70.69 (d, J1-F = 4.8 Hz, 1), 70.65 (d, J1-F = 5.6 Hz, 1), 64.21 and 64.18 (4), 63.21 (d, JCH2-P = 6.0 Hz, OCH2CH3), 63.00 (d, JCH2-P = 6.0 Hz, OCH2CH3), 56.68 and 56.63 (5″-OCH3), 56.45 and 56.44 (4″-OCH3), 52.07 and 52.06 (16′), 50.26 and 50.09 (m, 2′), 44.40 and 44.38 (1′), 34.56 and 34.55 (12′), 19.50 (d, J2-P = 5.2 Hz, 2), 16.18 (d, JCH3-P = 7.0 Hz, OCH2CH3), 16.14 (d, JCH3-P = 7.0 Hz, OCH2CH3), 8.15 (m, 13′, 14′). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 7.87 and 7.17 ppm. HR-MS (ESI) calculated for C32H39O12N4FP 721.22806, found [M + H]+ 721.22796.
Methyl-1-cyclopropyl-7-(4-(ethoxy((1-ethoxy-1-oxopropan-2-yl)oxy)phosphoryl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate (18). To a solution of (-)-Ethyl L-Lactate (28.7 μL, 0.25 mmol, 1 eq.) in 1.5 mL of dry toluene, 20 (31.6 μL, 0.25 mmol, 1.0 eq.) and TEA (45.4 μL, 0.325 mmol, 1.3 eq.) were added, and the reaction mixture was stirred at 25 °C for 16 h. The formation of the intermediate was confirmed by 31P-NMR (δP 4.7 and 3.7 ppm) before adding amine 27 (86.3 mg, 0.25 mmol, 1.0 eq.) and TEA (34.9 μL, 0.25 mmol, 1.0 eq.). The reaction mixture was stirred at 25 °C until completion (1 h). After evaporating the solvent, the crude residue was purified by Flash silica gel chromatography using a gradient (hexane/EtOAc 50:50→0:100, v/v), followed by reverse-phase chromatography using a gradient (H2O/MeCN 95:5→50:50, v/v), which afforded 18 as white solid (61.1 mg, 44%). 1H-NMR (400 MHz, CDCl3, 25 °C, 2 diastereoisomers in ca. 1:1 ratio) δ 8.60–8.57 (m, 2H, 9′), 8.08 (d, 1H, J7′-F = 13.2 Hz, 7′), 8.07 (d, 1H, J7′-F = 13.2 Hz, 7′), 7.31–7.27 (m, 2H, 4′), 4.95–4.83 (m, 2H, 1), 4.30–4.08 (m, 8H, 4, OCH2CH3), 3.95–3.93 (m, 6H, 16′), 3.53–3.36 (m, 10H, 1′, 12′), 3.28–3.20 (m, 8H, 2′), 1.60 (d, 3H, J2-P = 7.0 Hz, 2), 1.57 (d, 3H, J2-P = 6.9 Hz, 2), 1.40–1.29 (m, 16H, 13′ or 14′, 5, OCH2CH3), 1.19–1.13 (m, 4H, 13′ or 14′). 13C-NMR (101 MHz, CDCl3, 25 °C) δ 173.17–173.02 (m, 11′), 171.00 (d, J3-P = 4.5 Hz, 3), 170.91 (d, J3-P = 5.4 Hz, 3), 166.57–166.49 (m, 15′), 153.44 (d, J8′-F = 249.1 Hz, 8′), 148.45 (9′), 144.76–144.55 (m, 3′), 138.02 (5′), 123.63–123.24 (m, 6′), 113.50 (d, J7′-F = 23.1 Hz, 7′), 113.47 (d, J7′-F = 22.8 Hz, 7′), 110.32–110.05 (m, 10′), 105.05–104.96 (m, 4′), 70.78 (d, J1-P = 5.4 Hz, 1), 63.06 (d, JCH2-P = 6.1 Hz, OCH2CH3), 62.94 (d, JCH2-P = 6.2 Hz, OCH2CH3), 61.46 and 61.41 (4), 52.14 (16′), 50.41–50.14 (m, 2′), 44.43–44.34 (m, 1′), 34.54 (12′), 19.54–19.36 (2), 16.20 (d, JCH3-P = 6.9 Hz, OCH2CH3), 16.17 (d, JCH3-P = 6.6 Hz, OCH2CH3), 14.21 and 14.15 (5), 8.18 (m, 13′, 14′). 31P-NMR (162 MHz, CDCl3, 25 °C) δ 7.78 and 6.98 ppm. HR-MS (ESI) calculated for C25H34O8N3FP 554.20621, found [M + H]+ 554.20614.
Methyl-1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylate (27). To a suspension of Ciprofloxacin (1.0 g, 3.0 mmol, 1 eq.) in 30 mL of dry methanol, cooled to 0 °C, SOCl2 was added dropwise, and the resulting solution was refluxed for 16 h [33]. After evaporating the solvent, the crude residue was dissolved in CH2Cl2 and washed with a saturated solution of K2CO3, and the organic layer was dried over Na2SO4. After filtration and solvent removal, the crude residue was purified by Flash silica gel chromatography using a gradient (CH2Cl2/MeOH 100:0→70:30, v/v), to obtain methyl ester 27 as a white solid (0.78 g, 76%).
Characterization of intermediates (I), products (P) obtained after irradiation with UV light, and undesired products (X)
1-I. 13C-NMR (126 MHz, 50% CACO/DMSO, 25 °C) δ 140.73 (1′), 130.43 (2′ or 3′), 130.07 (3′ or 2′), 127.93 (4′), 68.73 (d, J2-P = 5.8 Hz, 2), 64.25 (d, J1-P = 5.4 Hz, 1), 61.94 (d, JCH2-P = 7.9 Hz, OCH2CH3), 43.81 (NH-CH2), 38.71 (d, JCH2-P = 6.1 Hz, CH2Ph), 17.25 (d, JCH3-P = 6.5 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 10.83 ppm. HR-MS (ESI-) calculated for C12H19O4NP 272.10572, found [M−H]− 272.10551.
2-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 68.79 (d, J2-P = 5.5 Hz, 2), 64.34 (d, J1-P = 5.6 Hz, 1), 61.92 (d, JCH2-P = 7.6 Hz, OCH2CH3), 46.33 (d, J1′-P = 2.1 Hz, 1′), 26.97 (d, J2′-P = 4.7 Hz, 2′), 25.15 (3′), 17.25 (d, JCH3-P = 6.6 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 9.81 ppm. HR-MS (ESI−) calculated for C9H19O4NP 236.10572, found [M−H]− 236.10585.
3-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 141.11 (1′), 131.01 (3′), 123.48 (4′), 119.19 (d, J2′-P = 7.6 Hz, 2′), 69.46 (d, J2-P = 5.9 Hz, 2), 65.11 (d, J1-P = 5.6 Hz, 1), 61.82 (d, JCH2-P = 7.9 Hz, OCH2CH3), 17.19 (d, JCH3-P = 6.9 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 3.62 ppm. HR-MS (ESI−) calculated for C10H15O4NP 244.07442, found [M−H]− 244.07419.
4-I. 13C-NMR (126 MHz, 50% HEPES/DMSO-d6, 25 °C) δ 151.73 (d, J1‴-P = 6.8 Hz, 1‴), 140.51 (1′), 131.54 (3‴), 130.40 (2′), 130.08 (3′), 127.98 (4′), 126.77 (4‴), 121.67 (d, J2‴-P = 4.6 Hz, 2‴), 69.41 (d, J2-P = 5.9 Hz, 2), 61.85 (d, J1-P = 7.8 Hz, 1), 44.04 (NHCH2), 38.58 (d, JCH2-P = 6.1 Hz, CH2Ph). 31P-NMR (202 MHz, 50% HEPES/DMSO-d6, 25 °C) δ 6.32 ppm. HR-MS (ESI) calculated for C16H20O4NNaP 344.10222, found [M + Na]+ 344.10212.
4-cyc-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 140.45 (1′), 130.41 (2′), 130.08 (3′), 127.98 (4′), 67.74−67.69 (m, 2, 1), 43.71 (NHCH2), 38.67 (d, JCH2-P = 5.2 Hz, CH2Ph). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 28.06 ppm. HR-MS (ESI−) calculated for C10H13O3NP 226.06385, found [M−H]− 226.06358.
4-P2. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 141.56 (1′), 130.38 (2′), 130.03 (3′), 127.69 (4′), 66.76 (d, J2-P = 5.3 Hz, 2), 62.90 (d, J1-P = 7.3 Hz, 1), 44.60 (NHCH2), 39.2* (CH2Ph). * The 13C chemical shift was extracted from HMBC. 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 8.05 ppm. HR-MS (ESI−) calculated for C10H15O4NP 244.07442, found [M−H]− 244.07457.
5-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 151.72 (d, J1‴-P = 6.8 Hz, 1‴), 131.58 (3‴), 126.83 (4‴), 121.64 (d, J2‴-P = 5.1 Hz, 2‴), 64.48 (d, J2-P = 6.0 Hz, 2), 61.84 (d, J1-P = 7.9 Hz, 1), 46.53 (d, J1′-P = 1.9 Hz, 1′), 26.77 (d, J2′-P = 4.5 Hz, 2′), 25.01 (3′). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 5.10 ppm. HR-MS (ESI) calculated for C13H21O4NP 286.12027, found [M + H]+ 286.12039.
5-cyc-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 67.87−67.83 (m, 2, 1), 46.54 (d, J1′-P = 3.1 Hz, 1′), 27.03 (d, J2′-P = 3.6 Hz, 2′), 25.07 (3′). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 26.48 ppm. HR-MS (ESI) calculated for C7H15O3NP 192.07841, found [M + H]+ 192.07829.
6-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 151.39 (d, J1‴-P = 6.7 Hz, 1‴), 140.63 (1′), 131.66 (3‴), 131.10 (3′), 127.18 (4‴), 123.92 (4′), 121.55 (d, J2‴-P = 4.7 Hz, 2‴), 119.50 (d, J2′-P = 7.7 Hz, 2′), 70.19 (d, J2-P = 5.8 Hz, 2), 61.76 (d, J1-P = 7.6 Hz, 1). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −0.94 ppm.
6-cyc-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 140.23 (1′), 131.15 (3′), 124.73 (4′), 120.98 (d, J2′-P = 7.3 Hz, 2′), 68.02−67.95 (m, 2, 1). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ 21.66 ppm.
9-I.HR-MS (ESI−) calculated for C11H15O5NP 272.06933, found [M−H]− 272.06926.
13-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 177.54 (d, J3-P = 4.5 Hz, 3), 160.21–160.10 (m, 2′), 160.05–159.93 (m, 1′), 116.69 and 116.67 (3′), 76.31 (d, J1-P = 6.3 Hz, 1), 76.20 (d, J1-P = 6.1 Hz, 1), 65.89 (d, JCH2-P = 6.1 Hz, OCH2CH3), 65.87 (d, JCH2-P = 6.2 Hz, OCH2CH3), 21.29 (d, J2-P = 5.7 Hz, 2), 21.08 (d, J2-P = 5.5 Hz, 2), 17.14 (d, JCH3-P = 7.3 Hz, OCH2CH3), 17.10 (d, JCH3-P = 6.7 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −0.13 and −0.75 ppm. HR-MS (ESI−) calculated for C9H13O5N3P 274.05983, found [M − H]− 274.05995.
16-I. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 153.93 (d, J3-P = 6.8 Hz, 3), 151.68–151.46 (m, 1‴), 140.89 (1′). 140.79 (1′), 131.55 (3‴ or 3′), 131.53 (3‴ or 3′), 131.03 (3′ or 3‴), 130.98 (3′ or 3‴), 127.03 (4‴), 123.66 (4′), 123.63 (4′), 121.69–121.60 (m 2‴), 119.36 (d, J2′-P = 8.0 Hz, 2′), 76.35 (d, J1-P = 5.9 Hz, 1), 21.31 (d, J2-P = 4.8 Hz, 2). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −2.21 and −2.63 ppm. HR-MS (ESI−) calculated for C15H15O5NP 320.06933, found [M − H]− 320.06956.
7-P (= 8-P, 9-P, 10-P, 11-P, 12-P, 13-P, 17-P, 10-P, 11-P, 12-P, 13-P, 17-P, 18-P). 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 179.92 (d, J3-P = 6.5 Hz, 3), 73.60 (d, J1-P = 5.6 Hz, 1), 62.56 (d, JCH2-P = 5.5 Hz, OCH2CH3), 21.60 (d, J2-P = 3.1 Hz, 2), 17.52 (d, JCH3-P = 7.4 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −1.09 ppm. HR-MS (ESI−) calculated for C5H10O6P 197.02205, found [M − H]− 197.02187.
14-P1. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 179.52 (d, J3-P = 7.1 Hz, 3), 153.92 (d, J1‴-P = 6.9 Hz, 1‴), 130.95 (3‴), 124.87 (4‴), 121.76 (d, J2‴-P = 4.6 Hz, 2‴), 74.33 (d, J1-P = 6.1 Hz, 1), 21.51 (d, J2-P = 2.9 Hz, 2). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −6.24 ppm. HR-MS (ESI−) calculated for C9H10O6NP 245.02205, found [M−H]− 245.02184.
7-X. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 175.44 (d, J3-P = 6.5 Hz, 3), 140.41 (1′), 130.32 (2′), 130.07 (3′), 127.96 (4′), 72.75 (d, J1-P = 5.5 Hz, 1), 62.90–62.78 (m, OCH2CH3), 41.61 (NH-CH2), 36.02 (CH2Ph), 21.10–21.01 (m, 2), 17.61–17.45 (m, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −1.76 ppm. HR-MS (ESI−) calculated for C13H19O5NP 300.10063, found [M − H]− 300.10069.
9-X. 13C-NMR (126 MHz, 50% CACO/DMSO-d6, 25 °C) δ 173.93 (d, J3-P = 6.2 Hz, 3), 138.54 (1′), 130.60 (3′), 126.60 (4′), 122.38 (2′), 73.05 (d, J1-P = 5.5 Hz, 1), 62.94 (d, JCH2-P = 5.8 Hz, OCH2CH3), 20.93 (d, J2-P = 3.3 Hz, 2), 17.52 (d, JCH3-P = 7.2 Hz, OCH2CH3). 31P-NMR (202 MHz, 50% CACO/DMSO-d6, 25 °C) δ −1.57 ppm. HR-MS (ESI−) calculated for C11H15O5NP 272.06933, found [M−H]− 272.06943.

5. Conclusions

In summary, we designed and synthesized phosphate-based SI linkers for amine- containing drug delivery. We found that the lactate spacer can release amines effectively within 15 min; moreover, it can release two cargos sequentially—the first amine cargo within minutes and the second phenolic cargo overnight. Surprisingly, this is exactly the opposite release order that we found when using an ethylene glycol SI spacer, whereby phenol is released preferentially [22]. Interestingly, the linkers bearing primary amines lack stability at physiological pH (pH = 7.4) due to an intramolecular rearrangement caused by the nucleophilic attack of NH nitrogen from LG on the carbonyl group of lactate. This alternative decomposition, which yields the undesired product X, can be suppressed by changing the buffer (e.g., HEPES instead of Cacodylate buffer, pH = 7.4), by decreasing the buffer pH to mildly acidic (pH = 5), or by N-methylation of phosphoramidate nitrogen. In turn, derivatives bearing secondary amines are stable in a range of pH 5–7.4. As such, our prodrug approach is the most suitable for the delivery of secondary amines. Further applicability was demonstrated by phosphorylation of the antibiotic Ciprofloxacin, whose phototriggerable and enzyme-triggerable prodrugs released Ciprofloxacin successfully. Overall, our results establish an experimental paradigm for the smart design of new self-immolative systems for the targeted delivery of various amine-containing drugs and their enhanced cellular uptake and activity, thus broadening the applications of prodrug technology. Moreover, phospholane amidates could lead to the design of new synthetic approaches in phosphorus chemistry.

Supplementary Materials

The Supplementary Materials are available online.

Author Contributions

Conceptualization, O.B.; Funding acquisition, O.B. and E.P.; Investigation, M.Đ. and M.T.; Methodology, E.P.; Supervision, E.P. and O.B.; Writing—original draft, E.P. and O.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Czech Science Foundation (O.B. 20-25137Y and E.P. 21-23014S) and Experientia Foundation (SG-2018-1).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to acknowledge Dr. Zdeněk Tošner (https://is.cuni.cz/webapps/whois2/osoba/1073964350590543/?lang=cs) from Charles University for setting the NMR parameters for self-service measurements of NMR stability tests. We thank Kvetoslava Kertisová from Mass Spectrometry department at IOCB for HR-MS analysis. We would like to acknowledge Prof. Milan Kolář (https://www.lf.upol.cz/o-fakulte/organizacni-struktura/dekanat/prof-mudr-milan-kolar-phd/), Dr. Renata Večeřová (https://www.lf.upol.cz/ustavy-a-kliniky/ustavy/ustav-mikrobiologie/) and Dr. Kateřina Bogdanová (https://www.inis.upol.cz/fcgi/verso.fpl?fname=upol_tel_sez&_pracoviste=1500304) from the Palacký University Olomouc for the biological screening of antibiotic activity. We also thank Dr. Carlos V. Melo (https://is.cuni.cz/webapps/whois2/osoba/1715745504135343/?lang=en) for editing the manuscript.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the study design, in the data collection, analysis and interpretation, in the manuscript writing, or in the decision to publish the results.

Sample Availability

Samples of the compounds are not available from the authors.

References

  1. Barbosa-Filho, J.M.; Piuvezam, M.R.; Moura, M.D.; Silva, M.S.; Lima, K.V.B.; da-Cunha, E.V.L.; Fechine, I.M.; Takemura, O.S. Anti-inflammatory activity of alkaloids: A twenty-century review. Rev. Bras. Farmacogn. 2006, 16, 109–139. [Google Scholar] [CrossRef] [Green Version]
  2. Ballout, F.; Habli, Z.; Monzer, A.; Rahal, O.N.; Fatfat, M.; Gali-Muhtasib, H. Anticancer Alkaloids: Molecular Mechanisms and Clinical Manifestations. In Bioactive Natural Products for the Management of Cancer: From Bench to Bedside; Springer: Singapore, 2019; pp. 1–35. [Google Scholar]
  3. Mohan, K.; Jeyachandran, R. Alkaloids as anticancer agents. Ann. Phytomed. 2012, 1, 46–53. [Google Scholar]
  4. Krishnan, N.; Devadasan, V.; Raman, P. Plant-derived alkaloids as anti-viral agents. Int. J. Res. Pharm. Sci. 2020, 11, 6174–6182. [Google Scholar] [CrossRef]
  5. Cushnie, T.P.T.; Cushnie, B.; Lamb, A.J. Alkaloids: An overview of their antibacterial, antibiotic-enhancing and antivirulence activities. Int. J. Antimicrob. Agents 2014, 44, 377–386. [Google Scholar] [CrossRef]
  6. Sayhan, H.; Beyaz, S.G.; Çeliktaş, A. The Local Anesthetic and Pain Relief Activity of Alkaloids. In Alkaloids—Alternatives in Synthesis, Modification and Application; InTechOpen: London, UK, 2017. [Google Scholar] [CrossRef] [Green Version]
  7. Krise, J.P.; Oliyai, R. Prodrugs of Amines. In Prodrugs; Stella, V., Borchardt, R., Hageman, M., Oliyai, R., Maag, H., Tilley, J.E., Eds.; Springer: New York, NY, USA, 2008; pp. 801–831. [Google Scholar]
  8. Albert, A. Chemical aspects of selective toxicity. Nature 1958, 182, 421–423. [Google Scholar] [CrossRef] [PubMed]
  9. Abet, V.; Filace, F.; Recio, J.; Alvarez-Builla, J.; Burgos, C. Prodrug approach: An overview of recent cases. Eur. J. Med. Chem. 2017, 127, 810–827. [Google Scholar] [CrossRef] [PubMed]
  10. Rautio, J.; Kumpulainen, H.; Heimbach, T.; Oliyai, R.; Oh, D.; Järvinen, T.; Savolainen, J. Prodrugs: Design and clinical applications. Nat. Rev. Drug Discov. 2008, 7, 255–270. [Google Scholar] [CrossRef] [PubMed]
  11. Simplício, A.L.; Clancy, J.M.; Gilmer, J.F. Prodrugs for amines. Molecules 2008, 13, 519–547. [Google Scholar] [CrossRef] [Green Version]
  12. Alouane, A.; Labruère, R.; Le Saux, T.; Schmidt, F.; Jullien, L. Self-immolative spacers: Kinetic aspects, structure-property relationships, and applications. Angew. Chem.-Int. Ed. 2015, 54, 7492–7509. [Google Scholar] [CrossRef]
  13. Gonzaga, R.V.; do Nascimento, L.A.; Santos, S.S.; Machado Sanches, B.A.; Giarolla, J.; Ferreira, E.I. Perspectives About Self-Immolative Drug Delivery Systems. J. Pharm. Sci. 2020, 109, 3262–3281. [Google Scholar] [CrossRef] [PubMed]
  14. Teicher, B.A.; Chari, R.V.J. Antibody conjugate therapeutics: Challenges and potential. Clin. Cancer Res. 2011, 17, 6389–6397. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  15. Egron, D.; Lefebvre, I.; Périgaud, C.; Beltran, T.; Pompon, A.; Gosselin, G.; Aubertin, A.M.; Imbach, J.L. Anti-HIV pronucleotides: Decomposition pathways and correlation with biological activities. Bioorg. Med. Chem. Lett. 1998, 8, 1045–1050. [Google Scholar] [CrossRef]
  16. Wei, Y.; Qiu, G.; Lei, B.; Qin, L.; Chu, H.; Lu, Y.; Zhu, G.; Gao, Q.; Huang, Q.; Qian, G.; et al. Oral Delivery of Propofol with Methoxymethylphosphonic Acid as the Delivery Vehicle. J. Med. Chem. 2017, 60, 8580–8590. [Google Scholar] [CrossRef] [PubMed]
  17. Mehellou, Y.; Rattan, H.S.; Balzarini, J. The ProTide Prodrug Technology: From the Concept to the Clinic. J. Med. Chem. 2018, 61, 2211–2226. [Google Scholar] [CrossRef]
  18. Ray, A.S.; Fordyce, M.W.; Hitchcock, M.J.M. Tenofovir alafenamide: A novel prodrug of tenofovir for the treatment of Human Immunodeficiency Virus. Antivir. Res. 2016, 125, 63–70. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  19. McQuaid, T.; Savini, C.; Seyedkazemi, S. Sofosbuvir, a significant paradigm change in hcv treatment. J. Clin. Transl. Hepatol. 2015, 3, 27–35. [Google Scholar]
  20. Siegel, D.; Hui, H.C.; Doerffler, E.; Clarke, M.O.; Chun, K.; Zhang, L.; Neville, S.; Carra, E.; Lew, W.; Ross, B.; et al. Discovery and Synthesis of a Phosphoramidate Prodrug of a Pyrrolo[2,1-f][triazin-4-amino] Adenine C-Nucleoside (GS-5734) for the Treatment of Ebola and Emerging Viruses. J. Med. Chem. 2017, 60, 1648–1661. [Google Scholar] [CrossRef] [Green Version]
  21. Klán, P.; Šolomek, T.; Bochet, C.G.; Blanc, A.; Givens, R.; Rubina, M.; Popik, V.; Kostikov, A.; Wirz, J. Photoremovable protecting groups in chemistry and biology: Reaction mechanisms and efficacy. Chem. Rev. 2013, 113, 119–191. [Google Scholar] [CrossRef]
  22. Procházková, E.; Šimon, P.; Straka, M.; Filo, J.; Majek, M.; Cigáň, M.; Baszczyňski, O. Phosphate Linkers with Traceable Cyclic Intermediates for Self-Immolation Detection and Monitoring. Chem. Commun. 2020, 57, 211–214. [Google Scholar] [CrossRef]
  23. Zhang, S.; Wang, H.; Shen, Y.; Zhang, F.; Seetho, K.; Zou, J.; Taylor, J.S.A.; Dove, A.P.; Wooley, K.L. A simple and efficient synthesis of an acid-labile polyphosphoramidate by organobase-catalyzed ring-opening polymerization and transformation to polyphosphoester ionomers by acid treatment. Macromolecules 2013, 46, 5141–5149. [Google Scholar] [CrossRef] [Green Version]
  24. Procházková, E.; Filo, J.; Cigáň, M.; Baszczyňski, O. Sterically-Controlled Self-Immolation in Phosphoramidate Linkers Triggered by Light. Eur. J. Org. Chem. 2020, 2020, 897–906. [Google Scholar] [CrossRef]
  25. Šimon, P.; Tichotová, M.; Gallardo, M.G.; Procházková, E.; Baszczynski, O. Phosphate-Based Self-Immolative Linkers for Tunable Double Cargo Release. Chem. A Eur. J. in press. [CrossRef]
  26. Mulliez, M.; Wolf, R. Contraste entre la phosphorylation des alcools et des amines par les 2-4 dioxo, oxa-1, aza-3 phospholanes-2. Bull. Soc. Chim. Fr. 1986, 101–108. [Google Scholar]
  27. Jafar, N.N.A.; Majeed, N.S. Microwave-assisted synthesis and biological activity of ester, carbothioate and carbohydrazide derivative compounds of the drug Ciprofloxacin. J. Chem. Pharm. Sci. 2017, 10, 515–521. [Google Scholar]
  28. Albert, A.; Goldacre, R.; Phillips, J. 455. The strength of heterocyclic bases. J. Chem. Soc. 1948, 2240–2249. [Google Scholar] [CrossRef]
  29. Dudkin, S.M.; Ledneva, R.K.; Shabarova, Z.A.; Prokofiev, M.A. Hydrolysis of uridine-5′ N-aryl and N-alkyl phosphoramidates by ribonucleoside-5′ phosphoramidase. FEBS Lett. 1971, 16, 48–50. [Google Scholar] [CrossRef] [Green Version]
  30. Beltran, T.; Egron, D.; Pompon, A.; Lefebvre, I.; Périgaud, C.; Gosselin, G.; Aubertin, A.M.; Imbach, J.L. Rational design of a new series of pronucleotide. Bioorg. Med. Chem. Lett. 2001, 11, 1775–1777. [Google Scholar] [CrossRef]
  31. Drontle, D. Designing a Pronucleotide Stratagem: Lessons from Amino Acid Phosphoramidates of Anticancer and Antiviral Pyrimidines. Mini Rev. Med. Chem. 2004, 4, 409–419. [Google Scholar] [CrossRef]
  32. Kanzian, T.; Nigst, T.A.; Maier, A.; Pichl, S.; Mayr, H. Nucleophilic Reactivities of Primary and Secondary Amines in Acetonitrile. Eur. J. Org. Chem. 2009, 6379–6638. [Google Scholar] [CrossRef]
  33. Sachin, K.; Kim, E.M.; Cheong, S.J.; Jeong, H.J.; Lim, S.T.; Sohn, M.H.; Kim, D.W. Synthesis of N 4′-[18F]fluoroalkylated ciprofloxacin as a potential bacterial infection imaging agent for PET study. Bioconjug. Chem. 2010, 21, 2282–2288. [Google Scholar] [CrossRef]
Molecules 26 05160 g001 550
Figure 1. Self-immolation (SI) of 1–16; (a) SI is initiated by UV light (365 nm), which cleaves a photosensitive DMNB group [21]; (b) the intermediate I then spontaneously cyclizes, releasing R1 (4–6) or LG (7–16); (c) hydrolysis of the lactate-based cyclic intermediate leads to the final product P, whereas glycol-based cyc-I hydrolyzes to the final product P2 or the cyclic product cyc-P.
Figure 1. Self-immolation (SI) of 1–16; (a) SI is initiated by UV light (365 nm), which cleaves a photosensitive DMNB group [21]; (b) the intermediate I then spontaneously cyclizes, releasing R1 (4–6) or LG (7–16); (c) hydrolysis of the lactate-based cyclic intermediate leads to the final product P, whereas glycol-based cyc-I hydrolyzes to the final product P2 or the cyclic product cyc-P.
Molecules 26 05160 g001
Molecules 26 05160 sch001 550
Scheme 1. Linkers 1–6 bearing ethylene glycol spacer were synthesized under the following reaction conditions: (a) ethyl (20) or phenyl (21) dichlorophosphate (0.5 mmol), TEA (0.65 mmol), toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), toluene, 25 °C, 1–6 h.
Scheme 1. Linkers 1–6 bearing ethylene glycol spacer were synthesized under the following reaction conditions: (a) ethyl (20) or phenyl (21) dichlorophosphate (0.5 mmol), TEA (0.65 mmol), toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), toluene, 25 °C, 1–6 h.
Molecules 26 05160 sch001
Molecules 26 05160 g002 550
Figure 2. 31P-NMR spectra of linkers 4–6 (5 mM in 50% CACO/DMSO) measured before and after irradiation with UV light (365 nm) at room temperature (25 °C), previously optimizing the reaction conditions (DMSO/cacodylate buffer (CACO), 0.1 M, pH = 7.4; 1/1, v/v) [24].
Figure 2. 31P-NMR spectra of linkers 4–6 (5 mM in 50% CACO/DMSO) measured before and after irradiation with UV light (365 nm) at room temperature (25 °C), previously optimizing the reaction conditions (DMSO/cacodylate buffer (CACO), 0.1 M, pH = 7.4; 1/1, v/v) [24].
Molecules 26 05160 g002
Molecules 26 05160 sch002 550
Scheme 2. Lactate-based linkers 7–9 were synthesized under the following reaction conditions: (a) ethyl dichlorophosphate 20 (0.5 mmol), TEA (0.65 mmol), dry toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), dry toluene, 25 °C, 1 h.
Scheme 2. Lactate-based linkers 7–9 were synthesized under the following reaction conditions: (a) ethyl dichlorophosphate 20 (0.5 mmol), TEA (0.65 mmol), dry toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), dry toluene, 25 °C, 1 h.
Molecules 26 05160 sch002
Molecules 26 05160 g003 550
Figure 3. 31P NMR spectra of linkers 7–9 (5 mM), measured before and after irradiation by UV light (365 nm) in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Figure 3. 31P NMR spectra of linkers 7–9 (5 mM), measured before and after irradiation by UV light (365 nm) in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Molecules 26 05160 g003
Molecules 26 05160 g004 550
Figure 4. The proposed mechanism of intramolecular rearrangement: part of the COSY and HMBC spectra recorded after UV irradiation of 7 (5 mM solution in CACO (pH 7.4)/DMSO) show cross-peaks between lactate C=O and phenethylamine NH-CH2 groups, confirming the structure of 7-X.
Figure 4. The proposed mechanism of intramolecular rearrangement: part of the COSY and HMBC spectra recorded after UV irradiation of 7 (5 mM solution in CACO (pH 7.4)/DMSO) show cross-peaks between lactate C=O and phenethylamine NH-CH2 groups, confirming the structure of 7-X.
Molecules 26 05160 g004
Molecules 26 05160 sch003 550
Scheme 3. Lactate-based linkers 10–16 were synthesized under the following reaction conditions: (a) ethyl or phenyl dichlorophosphate 20–21 (0.5 mmol), TEA (0.65 mmol), dry toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), dry toluene, 25 °C, 1–24 h.
Scheme 3. Lactate-based linkers 10–16 were synthesized under the following reaction conditions: (a) ethyl or phenyl dichlorophosphate 20–21 (0.5 mmol), TEA (0.65 mmol), dry toluene, 25 °C, 16 h; (b) the corresponding amine (0.5 mmol), TEA (0.5 mmol), dry toluene, 25 °C, 1–24 h.
Molecules 26 05160 sch003
Molecules 26 05160 g005 550
Figure 5. 31P-NMR spectra of linkers 10–13, measured before and after irradiation with UV light (365 nm) of 5 mM solutions in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Figure 5. 31P-NMR spectra of linkers 10–13, measured before and after irradiation with UV light (365 nm) of 5 mM solutions in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Molecules 26 05160 g005
Molecules 26 05160 g006 550
Figure 6. 31P-NMR spectra of linkers 14–16, measured before and after irradiation with UV light (365 nm) using 5 mM solutions in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Figure 6. 31P-NMR spectra of linkers 14–16, measured before and after irradiation with UV light (365 nm) using 5 mM solutions in a solvent mixture of 50% HEPES (pH = 7.4)/DMSO at room temperature (25 °C).
Molecules 26 05160 g006
Molecules 26 05160 g007 550
Figure 7. Chemical structures of the FDA-approved fluoroquinolone antibiotic Ciprofloxacin, its methyl ester 27, and the prodrugs activated by light (17) or by an enzyme (18) prepared in this work.
Figure 7. Chemical structures of the FDA-approved fluoroquinolone antibiotic Ciprofloxacin, its methyl ester 27, and the prodrugs activated by light (17) or by an enzyme (18) prepared in this work.
Molecules 26 05160 g007
Molecules 26 05160 g008 550
Figure 8. 31P-NMR spectra of Ciprofloxacin prodrugs 17 (a) and 18 (b) recorded in 50% HEPES (pH = 7.4)/DMSO, 25 °C, with linker 17 bearing a photoactivable DMNB group, before and after irradiation by UV light (365 nm), and linker 18, where SI is activated enzymatically by lipase, before and after lipase addition.
Figure 8. 31P-NMR spectra of Ciprofloxacin prodrugs 17 (a) and 18 (b) recorded in 50% HEPES (pH = 7.4)/DMSO, 25 °C, with linker 17 bearing a photoactivable DMNB group, before and after irradiation by UV light (365 nm), and linker 18, where SI is activated enzymatically by lipase, before and after lipase addition.
Molecules 26 05160 g008
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Đud, M.; Tichotová, M.; Procházková, E.; Baszczyňski, O. Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs. Molecules 2021, 26, 5160. https://doi.org/10.3390/molecules26175160

AMA Style

Đud M, Tichotová M, Procházková E, Baszczyňski O. Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs. Molecules. 2021; 26(17):5160. https://doi.org/10.3390/molecules26175160

Chicago/Turabian Style

Đud, Mateja, Markéta Tichotová, Eliška Procházková, and Ondřej Baszczyňski. 2021. "Phosphate-Based Self-Immolative Linkers for the Delivery of Amine-Containing Drugs" Molecules 26, no. 17: 5160. https://doi.org/10.3390/molecules26175160

Article Metrics

Back to TopTop