Synthesis of biotin and fluorescein labeled (–)-lentiginosine

The important proapoptotic activity of (–)-lentiginosine, the enantiomer of a natural glycosidase inhibitor, associated with its low cytotoxicity, suggests the study of the unknown receptor responsible for the triggering of the proapoptotic cascade. To this purpose derivatives of (–)- lentiginosine 7 and 8 , which contain the biotin moiety as an affinity label and fluorescein as fluorophore, have been synthesized. Significantly, the compounds maintain a good activity as the hydroxylentiginosine precursor.

Among the different iminosugars (+)-lentiginosine (1) (Figure 1), a natural alkaloid, 6 has attracted our interest for the simplified structure, with respect to other iminosugars, compared to the important glycosidase inhibitory activity, 7,8 and the more recently discovered activity as a good inhibitor of Hsp90 (Heat Shock Protein 90). 9 This interest was reinforced by the discovery that the non-natural enantiomer (-)-lentiginosine (2, Figure 1) is a potent proapoptotic agent against tumor cells with low toxicity towards normal cells. 10This activity is also shared by the corresponding synthetic 7S-OH derivative 3 (Figure 1). 11poptosis, a form of programmed cell death that proceeds by a highly regulated mechanism, plays an important role in numerous diseases including cancer.Accordingly, the development of new pro-apoptotic molecules and the knowledge of their mechanism of action are major challenges in organic chemistry and biomedical sciences.Initial studies have disclosed that the activity of 2 is caspase dependent and involves activation of the intrinsic pathway of apoptosis, 12 but still many aspects of the mechanism of action of 2 need to be investigated.In particular, the specific receptor of the molecule that is able to start the cascade of events leading to apoptosis has yet to be identified.
4][15] We therefore decided to synthesize labeled lentiginosines and verify the effect of the structural modification on bioactivity.

Figure 2. Lentiginosine derivatives
Since the 7-hydroxy substituted lentiginosine 3 retains the pro-apoptotic activity of the parent compound, we envisioned using the hydroxyl group as a handle to introduce biotin and fluorescein tags.The choice of the connecting moiety at indolizidine C-7 required a preliminary study.Our earlier effort to develop a biotin-labeled lentiginosine resulted in a triazole derivative 4 (Figure 2) that was inactive. 16The loss of activity in 4 could be ascribed to various factors such as the inverted configuration at C-7, the large biotin tag linked to the indolizidine via a simple triazole unit, and the triazole ring itself that is considered an isostere of the amide bond. 17The synthesis and biological evaluation of the model 1,2-dihydroxy-3-[(octylcarbamoyl)oxy]indolizidine 5 (Figure 2) revealed that an alkyl carbamate could be a suitable spacer between the indolizidine ring system and the chosen tags.Accordingly, a flexible C-10 carbon chain ,functionalized with an isocyanate and an amino group was chosen as linker.
Herein, we report on the synthesis of the common intermediate 6 and its coupling with biotin and fluorescein.The proapoptotic activity of deprotected indolizidines 7 and 8 was also evaluated.
The introduction of a carbamate group in the 7-position was performed by nucleophilic addition of the free hydroxy group of 9 to a suitable isocyanate.Intermediate 9b was reacted with the commercially available octyl isocyanate in the presence of TEA in anhydrous dichloromethane for 7 hours at room temperature.Simple removal of the solvent and treatment with the strongly basic resin Ambersep 900-OH in methanol to hydrolyze the benzoyl ester groups gave the deprotected carbamate 5 in 64% yield over two steps (Scheme 2).

Scheme 2. Synthesis of the model compound 5.
Having established the possibility of the reaction of 9 with a long chain isocyanate, and encouraged by the conservation of the biological activity in the model compound 5 (see below), the next step was to prepare a long chain carbon isocyanate with a terminal amino group to link the molecular probe on lentiginosine.To this end, the inexpensive 11-aminoundecanoic acid (12)  was protected with the Cbz group, 19 and then the free carboxylic group was converted into isocyanate through Curtius rearrangement of the corresponding acyl azide.
Following analogous approaches reported in the literature, [20][21][22] the formation of a carbamate in a one-pot reaction was attempted.Heating a solution of 9a and 13 in the presence of diphenylphosphoryl azide (DPPA) and a base afforded only traces of the desired product along with 7-(diphenoxyphosphoryloxy)-lentiginosine.][25] Fortunately, the stepwise approach was successful.Activation of 13 with ethyl chloroformate followed by treatment with sodium azide gave acyl azide 14 in good yield (Scheme 3).Scheme 3. Synthesis of the key intermediate 6.
Compound 14 was heated in toluene to generate the corresponding isocyanate by Curtius rearrangement and then alcohol 9a was added.Pure carbamate 15 was obtained in 95% yield after chromatography.Deprotection of 15 by hydrogenation (Pd/C 10%, rt) afforded amine 6, a suitable intermediate for coupling with different probes, in excellent yield (Scheme 3).
The reaction of amine 6 with fluorescein 5-isothiocyanate (FITC) occurred at room temperature in DMF/ Et3N to give the addition product that could not be purified and was in sequence treated with TFA to remove tert-butoxy protection quantitatively (Scheme 4).This product, that was submitted to tests as such, gave a correct ESI-MS (see Experimental Section), but an unresolved NMR spectrum, because of the broadening caused by the acid form.Removal of acid by basic treatment gave the free indolizidine base with correct NMR data.
The reaction of amine 6 with biotin under standard coupling reaction conditions afforded amide 16 in 79% yield (Scheme 4).Deprotection of biotinylated compound 16 by TFA, followed by basic treatment, gave the deprotected labelled (-)-lentiginosine 8, which was submitted as such for biological tests.

Biological testing
Selected data on the effects of 4, 5, and 8 on both cell viability and apoptosis of MOLT-3 tumor cells are reported in Table 1 (for dose-effect data see Supporting Information).The three lentiginosine derivatives 4, 5, and 8 were not cytotoxic toward MOLT-3, exhibiting CC50 (cytotoxic inhibitory concentration 50%) values >1000 μM.Conversely, the well-known cytotoxic chemotherapeutic agent 7-ethyl-10-hydroxycamptothecin (SN38), used as a positive control, was highly toxic.Apoptosis was assayed by flow cytometric quantification of hypodiploid nuclei. 26Derivative 4 was unable to induce apoptosis, whereas 5 and 8 induced, respectively, 23% and 71% hypodiploid nuclei at a concentration of 500 µM.The colored fluorescein derivative 7•TFA could be tested only at low concentration and different assays will be necessary to get accurate results, but preliminary tests indicated a good pro-apoptotic activity as well.

Conclusions
Lentiginosine derivative 6 decorated with a C-10 chain functionalized at the terminal position with an amino group was synthesised in good yield from 7-hydroxylentiginosine 9a and the amino acid 12. Intermediate 6 was coupled with FITC and biotin to get labeled (-)-lentiginosines 7 and 8 that showed an adequate pro-apoptotic activity on MOLT-3 tumor cell to be used as fluorescent and affinity probes in the identification of the molecular target of (-)-lentiginosine.The present study also validated the applicability of the carbamate group as a suitable tether at C-7 of lentiginosine with preservation of the pro-apoptotic activity and low cytotoxicity.

Table 1 .
Effects of lentiginosine derivatives and SN38 on viability and apoptosis in MOLT-3 CC50 ±SD (µM) a % hypodiploid nuclei (µM conc.)b Apoptosis was evaluated as a percentage hypodiploid nuclei by flow cytometry analysis after 18 h of incubation with lentiginosine derivatives and SN38 at the µM concentration reported in brackets.
25mm silica gel plates.Melting points (m.p.) were determined on a Thiele Electrothermal apparatus.Polarimetric measurements were performed on a JASCO DIP-370.NMR spectra were measured on Varian Gemini ( 1 H, 200 MHz,13C, 50 MHz), Varian Mercury ( 1 H, 400 MHz,13C, 100 MHz) and Varian INOVA ( 1 H, 400 MHz,13C, 100 MHz), nuclear magnetic resonance spectrometers; CDCl3 was used as solvent in NMR analyses.NMR data are reported in δ (ppm) from TMS at 25 °C and peak assignments were made on the basis of 1 H-1 H COSY, HMQC and HMBC experiments.IR spectra were recorded with a Perkin-Elmer Spectrum BX FT-IR System spectrophotometer on CDCl3 solutions.Elemental analyses were performed with a Perkin-Elmer 2400 analyzer.Accurate mass spectra were recorded on a LTQ-Orbitrap high-resolution mass spectrometer (Thermo, San Jose, CA, USA), equipped with a conventional ESI source.MS (ESI) were recorded on a LCQ Fleet Ion Trap Mass Spectrometer with Surveyor Plus LC System (Thermo Scientific) operating in positive ( + ESI) and negative ( -ESI) ion mode by direct infusion of a methanolic solution of the sample.