Synthesis and cancer growth inhibitory activities of 2-fatty-alkylated pyrrolidine-3,4-diol derivatives

A series of new amphiphilic pyrrolidines containing dodecyl and oleyl apolar side chains were prepared and evaluated for their ability to inhibit the growth of pancreatic ductal adenocarcinoma cells in vitro . The new compounds are shown to exhibit anticancer activity at concentrations in the  M range.


Introduction
In recent years, several compounds with amphiphilic structure have been proposed as a new type of potential anticancer agents due to the lipophilic nature of their lipid chains.Series of such compounds isolated from natural sources have indeed shown promising anticancer activities.For instance, Ircinamine B, isolated from marine sponge Dactylia, 1 and Melophlins Q 2 isolated from Palauan marine sponge are active against mouse leukemia cell lines in the M range.Alkylglycerols 3 from shark liver also show anticancer activity in vivo in the M range with no toxicity on human umbilical vein endothelial cells, the best compounds bearing unsaturated 16 and 18 carbon alkyl chains.Motuporamine C from a marine Sponge, 4 interferes with the migration of human breast carcinoma and glioma cells in culture with low toxicity in vitro.Sphingosine 4-sulfate 5 from the sponge Spirastrella abata shows cytotoxic effects on several human tumor cell lines.The glycosyl ceramide, Turbostatin 1,2,3,4, 6 from Asian marine mollusk Turbo stenogyrus, is active against leukemia cells in the low M range.Notably, synthetic amphiphilic compounds have also shown interesting anticancer properties.As shown by Gajate and Mollinedo, [7][8][9] edelfosine (Figure 1), a lipid analogue of lysophosphatidyl-choline induces apoptosis in several tumor cell lines through its lipid ether moiety.Once edelfosine is inside the cell, it induces selective apoptosis in cancer cells through the activation of the Fas death receptor located on the surface of cells without the participation of the corresponding Fas ligand. 10

Figure 1
Other compounds such as Jaspines, having also amphiphilic character by combining a polar head (amino and hydroxy moieties) with aliphatic long chains, have shown good anticancer activities as well.Among the different diastereoisomeric Jaspines evaluated against human alveolar cell lines, Jaspine B (Figure 1) was the most active, showing cytotoxicity at nanomolar concentrations in in vitro assays. 11ue to their amphiphilic nature, glycolipids and their analogues also show therapeutic potential.Numerous glycolipid derivatives have been proposed as antibacterial/antiviral agents, 12,13 cell adhesion mediators, 14 and oral drug carriers. 15In the past few years, glycolipids have also been proposed as new potential anticancer agents due to the lipophilic nature of their aglycones.A variety of synthetic glycolipids have been prepared and their anticancer properties evaluated.Thus, oleyl N-acetyl-α-and β-D-glucosaminides and their thioglycosyl analogues 1a and 1b (Figure 2) exhibited antimitotic activity on rat glioma (C6) and human lung carcinoma (A549) cell cultures in the M range. 16,17Triazologlycolipids 2 showed cytotoxicity against a panel of cancer cell lines in the M range. 18

Figure 2
Derivatization of iminosugars with aliphatic side chains has been extensively studied in order to improve their biological activity, lipophilicity and bioavailability. 19For instance N-nonyl-1-deoxynojirimycin inhibits hepatitis B virus in cell based assays. 20-1-C-Alkyl-1deoxynojirimycin derivatives have shown inhibitory activity toward intestinal isomaltase and their potency was highly dependent on the alkyl chain length. 21N-alkylated-D-fagomine derivatives 3 and N-alkylated hydroxylated pyrrolidine 4 (Figure 3) bearing a long alkyl chain, not only presented an improved inhibitory selectivity towards α-D-glucosidase and -Lfucosidase, respectively, but also exhibited enhanced cytotoxic activities on a panel of cancer cell lines compared to their non-alkylated progenitors. 22Other N-alkylated pyrrolidine derivatives such as 5 23 and 6 24,25 presented interesting inhibition against Nacetylglucosaminidases and -mannosidases, respectively.3) were evaluated as enzyme inhibitors and for their toxicity on solid and hematological malignancies showing activities in the M range. 26,27Even though these compounds are not inhibitors of α-mannosidases, they show promising cytotoxicity on solid and hematological malignancies, their activity depending on the length of the alkyl side chain.The oleyl derivative 9 and its C-18 analogues, linoleyl 10, linolenyl 11 and octadecyl 12, exhibited the most potent anti-cancer activity, with oleyl-conjugate 9 being the most efficient in killing tumor cells.These results indicated that the side chain and the number and position of the unsaturations play a role in determining the biological properties of this class of compounds.The anticancer activities increased with the length of the alkyl chain, in accordance with the reported data for other iminosugars 22 and with the hypothesis that a higher lipophilicity would facilitate compound transport through the cell membrane.
Compound 9 was chosen as representative of the new class of compounds because of its interesting anticancer activities against breast, lung, prostate cancer, and glioblastoma cells in the M range (IC50 between 2 and 21 M). 26,27he promising results shown by compound 9 and derivatives, prompted us to explore further structural variations in order to improve their anticancer properties, in particular in pancreatic cancer cell lines.Thus, different series of compounds bearing amphiphilic character were prepared for structure-activity relationship studies by combining polyhydroxylated pyrrolidines with 2-fatty-alkyl moieties.
We report herein the synthesis of a series of this type of compounds starting from different sugars as source of chirality and the results of their evaluation in terms of toxicity in pancreatic cancer cell lines.The structural variations of the new compounds are shown in Figure 4.The diversity at the new structures will allow us to examine the role of different configurations at the stereogenic centers, the type of linker (X), the distance between the side chain and the pyrrolidine skeleton and the role of different functionalities at the nitrogen atom on their anticancer activity.Thus, compound 19 was prepared from D-ribose as outlined in Scheme 1. Starting from the known D-ribose derivative 13, 28 reaction with ethyl triphenylphosphoranylidene acetate in dichloromethane at reflux afforded a mixture of alkenes 14 and 15 in 86% yield and a ratio E:Z 1:3.6.The stereoselectivity in the Wittig reaction was in accordance with that described in the literature for other ribo-lactols. 29Mesylation of the free OH group in both alkenes followed by treatment with ammonia in ethanol led to pyrrolidine 16 as unique product (94% yield), through tandem conjugate addition-internal SN2 displacement.Reductive amination with butanal, ester hydrolysis and amide coupling with oleyl amine in the presence of PyBOP and DIPEA afforded compound 18 in 30% yield (two last steps).Deprotection with TBAF led to pyrrolidine 19 in quantitative yield.Attempts to deprotect 19 with cold TFA aq. were unsuccessful.The preparation of other acetonide protected pyrrolidines with the same configuration have been reported. 30cheme 1. Synthesis of 19.
The effect of a triazole linker between the pyrrolidine skeleton and the side chain was also studied.These compounds were prepared by click chemistry upon reaction of the corresponding pyrrolidine azides with tetradec-1-yne.Differently configurated pyrrolidine azides were obtained from D-mannose, D-gulono-lactone and D-arabinose (Figure 5).
The preparation of the azido-pyrrolidines is outlined in Scheme 2. Starting from known aldehyde 20, 31 reduction and tosylation gave derivative 21, which after displacement with sodium azide in DMF furnished azido derivative 22.Similarly, derivative 25 was obtained by azido displacement of the corresponding tosyl derivative 24 32 , obtained through oxidation, reduction and tosylation of known diol 23. 33Derivative 28 was prepared from pyrrolidine ester 26, 34 by Cbz protection and reduction with LiAlH4 followed by mesylation and azido displacement.

Scheme 2. Synthesis of azido-pyrrolidines.
The preparation of these azido derivatives requires benzyloxycarbonyl as N protecting group.The use of other carbamates as protecting groups for the preparation of the azido pyrrolidines gave unexpected results.Thus, tosylation of Boc-protected alcohol 29 did not lead to the corresponding tosyl derivative as expected.Instead, we mostly obtained cyclic carbamate 30 (Scheme 3) as confirmed by its spectral data.The 1 H-and 13 C NMR spectra of 30 did not show the typical signals for the tosyl and tert-butyl groups.However, a signal at 152.9 ppm was observed in the 13 C NMR spectra, corresponding to the carbonyl group of the carbamate.

Scheme 3. Participation of the N-protecting group.
The formation of cyclic carbamate 30 depends on the protecting group used in the introduction of the azide group at C-1' of the pyrrolidine.Boc protecting group as well as Fmoc exhibit a hydrogen atom at the β position with respect to the oxygen atom, which is not the case in the Cbz group.Under mild basic conditions, the H at  position is released with the concomitant displacement of the leaving group, promoting the formation of the cyclic carbamate, as outlined in Scheme 4. Compounds bearing a triazole moiety were prepared by copper-catalyzed dipolar cycloaddition [35][36][37] of pyrrolidine azides (22, 25 and 28) and tetradec-1-yne in the presence of CuI and DIPEA.Subsequent deprotection reactions led to derivatives 31, 32 and 33 in good overall yields, and their structures were confirmed by NMR and HRMS spectra (Scheme 5).

Scheme 5. Preparation of triazole derivatives.
Biological evaluation.The cytotoxic activity of the new amphiphilic derivatives has been evaluated on the pancreatic cancer cell lines MiaPaCa2 and PK9.The results are summarized in Table 1.They all exhibited antiproliferative activity in the M range.
Although undoubtedly much research needs to be done in this area, the results shown here indicate that the amphiphilic compounds combining unsaturated alkyl chains and iminoalditols of defined configurations might be of interest for the definition of new anticancer agents to be used in the treatment of pancreatic cancer.

Scheme 4 .
Scheme 4. Participation of carbamates on leaving group departure.