Lamellarin D Bioconjugates II: Synthesis and Cellul ar Internalization of Dendrimer- and Nuclear Location Signal-Derivatives

The design and synthesis of Lamellarin D conjugates with a nuclear localization signal peptide and a poly(ethylene glycol)-based de ndrimer are described. Conjugates 1-4 were obtained in 8-84% overall yields from the correspon ding protected Lamellarin D. Conjugates 1 and 4 are 1.4 to 3.3-fold more cytotoxic than the parent compound against three human tumor cell lines (MDA-MB-231 breast, A-549 lung, and HT-29 colon). B esides, conjugates 3, 4 showed a decrease in activity potency in BJ skin fibroblasts, a norma l cell culture. Cellular internalization was analyzed and nuclear distribution pattern was obser ved for 4, which contains a nuclear localization signalling sequence.


INTRODUCTION
Actively mediated cellular delivery of biomolecules (1) has garnered great interest as a strategy for delivering cancer chemotherapeutics (2)(3)(4)(5). Conjugates of a drug and a macromolecular vehicle such as NLS 1 peptidic sequences (5)(6)(7)(8), PEG carriers (9) and dendrimers (10) may have better cellular internalization than the drug alone, and in some cases, may produce passive accumulation of the drug in tumors by the EPR effect (11). In addition, the therapeutic activity of these conjugates is associated to their capacity to release the drug at a specific subcellular target. Thus, the suitability of macromolecules as vehicles also extends to their propensity to deliver the drug to a predetermined intracellular location.
The marine alkaloid Lam-D (12-15) is a promising drug candidate due to its Topo I inhibition activity. Topoisomerases are nuclear enzymes crucial in cellular replication. They change the topology of DNA before and after the replication and transcription processes. Therefore, they are especially attractive targets for cancer therapy (16)(17)(18)(19). Lam-D is limited by its insolubility in common solvent media, especially in water. Therefore, it has been used to investigate its conjugation to macromolecules. In the previous paper we have described the first generation of Lam D-bioconjugates based on PEG esters such as 1 (9). In this paper we describe a second generation of Lam-D conjugates ( Figure 1) based on esterification with either a poly(ethylene glycol)-based dendrimer (in 2) or NLS oligopeptide sequences (in 3 and 4). The peptide NLS H-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-OH, which has been demonstrated (20) to shuttle compounds to the nucleus, was used in the present work.
The introduction of such oligomeric systems to Lam D demanded an integrated and robust synthetic scheme with a collection of suitable orthogonal protecting groups, in terms of selective removal and compatibility with the presence of other functional groups (21). HF (5 mL) at -196 ºC was poured over solid 11, 12 or 14 (0.05-0.08 mmol). The solution was stirred for 1 min and the solvent was immediately removed under vacuum at low temperature.
MeCN was added to the crude, and the deprotected compound was precipitated by addition of MTBE, cooling to 0 ºC and centrifugation (10 min, 2000 r.p.m.). The residue was dried in vacuo to give the final Lam-D conjugates 1-3.

B) General Method for MOMO deprotection
Me 3 SiI (142 µL, 3.00 mmol) was added at r.t. to a solution of 10a, or 10b (1.00 mmol) in CH 2 Cl 2 (225 mL), and the resulting orange solution was stirred at r.t. for 20 min. The solvent was removed in vacuo, and the residue was dissolved with EtOAc and then washed three times with sat. NH 4 Cl and brine. The organic phase was dried over anhydrous MgSO 4 and the solvent was removed in vacuo. Purification by column chromatography on silica gel by elution with hexane/EtOAc (80:20 to 60:40) gave the title compounds (84%-quant. yield). C) General Method for Esterification. Synthesis of Conjugates. DMAP (0.6 mmol) and 5 (1 mmol) in dry CH 2 Cl 2 (45 mL) were added to a solution of NHBocPEG 6 -OH, or Boc-NLS-Gly-OH (4 mmol), and EDC·HCl (4 mmol) in dry CH 2 Cl 2 (5 mL). The resulting solution was stirred at r.t. for 2 h. The reaction mixture was diluted with CH 2 Cl 2 and washed with sat. NaHCO 3 solution and brine. The organic phase was dried over anhydrous MgSO 4 and the solvent removed under vacuum, to provide the title compounds 11 and 13 (89%-quant. yield).

Boc-Pro-Pro-Lys(Boc)-Lys(Boc)-Lys(Boc)-Arg(Boc 2 )-Lys(Boc)-Val-Gly-OH (8):
The Boc protected peptide was synthesized manually on solid-phase in a polypropylene syringe fitted with a porous polyethylene disc. Solvents and soluble reagents were removed by suction. Washings between deprotection, coupling and subsequent deprotection steps were carried out with DMF (5 × 1 min) and CH 2 Cl 2 (5 × 1 min) using 10 mL solvent/g resin each time. Standard Fmoc/tBu chemistry and chlorotrityl resin (0.5 g, 1.5 mmol/g) were used. The resin was pre-swollen in anhydrous CH 2 Cl 2 and then in DMF. The first Fmoc-protected amino acid (Fmoc-L-Gly-OH) (155 mg, 0.7 equiv) was introduced in the presence of DIEA (635 µL, 5 equiv) in DMF. After one hour, MeOH (0.5 mL) was added and the mixture was stirred for 30 min. The resin was then washed with DMF and CH 2 Cl 2 , and the synthesis continued as described below. The peptide was elongated through successive iterations of Fmoc removal and amino acid coupling. The Fmoc protecting group was removed with several treatments of 20% piperidine in DMF (1× 1 min + 2 × 15 min).
The resin was then washed with DMF and CH 2 Cl 2 . The corresponding Fmoc-protected amino acid (5 equiv) was introduced using DIPCDI (310 µL, 5 equiv) and HOBt (305 mg, 5 equiv) as coupling agents. After 2h, the resin was washed with DMF and CH 2 Cl 2 , and the coupling was monitored using the Kaiser test. Re-couplings were done when needed. Boc-L-Pro-OH (430 mg, 5 equiv) was used as a last amino acid. The peptide was finally cleaved from the resin using 3% TFA in CH 2 Cl 2 , (5 x 1 min). Washes were collected in a flask containing 50 mL of water. The CH 2 Cl 2 was then evaporated under reduced pressure, MeCN (30 mL) was added to the aqueous solution, and the resulting mixture was then lyophilized. Peptide 8 (590 mg, 94%) was obtained as a white solid.

Synthesis of Protected Lamellarins 5 and 6:
The protecting groups OiPr/OBn used in earlier strategies (9) required harsh deprotection conditions incompatible with the synthesis of more complex Lam-D conjugates such as 4. 2 The previously described lamellarin 9 (26,27), for which three different and orthogonal protecting groups were employed (MOM, Bn and TBDPS), was used as the precursor to Lam derivatives 5 and 6 3 (Scheme 1). Lam 9 was prepared following Banwell's strategy for the total synthesis of Lam-K (28). As a side note, compounds 10a and 10c are privileged synthetic intermediates for the construction of additional mono-, and di-conjugates at positions 11-OH, and 3,11-OHs of Lam-D, respectively.

Synthesis of the peptide NLS 8
The protected peptide 8 was synthesized on chlorotrityl resin following standard Fmoc/tBu solidphase chemistry, with 20% piperidine-DMF for the deprotection steps, and DIPCDI and HOBt as coupling reagents.
N-α-Fmoc-N-ω,N-ω'-bis-tert-butoxycarbonyl-L-arginine was used for the synthesis of the NLS peptide sequence. Attempts to use Fmoc-N-ω-Pbf-L-arginine, resulted in harsher deprotection conditions and complex reaction crudes. The last amino acid used was Boc-L-Pro-OH (as the desired building block had to be completely protected). The peptide was cleaved from the resin using 3% TFA in CH 2 Cl 2 , and after solvent evaporation, it was lyophilized.

Esterification and Synthesis of Conjugates 1-4
To test the efficacy of the protecting scheme, the synthesis of bioconjugate 1 was repeated using the following strategy (21). An ester bond between 5 and BocNH-CH 2 (CH 2 OCH 2 ) 6 CH 2 COOH was formed using EDC·HCl with a catalytic amount of DMAP to afford compound 11 (Figure 1) in quantitative yield. Compound 11 was considered a good model for the deprotection assays, because it contains the critical protecting groups and the ester functional group. Hence, it was used for the optimization of procedures and to test the success of the deprotection steps. Initial assays of sequential TBAF-TFA, two-step deprotection led to complex crude products. Furthermore, purification on SiO 2 gave low yields of 1. The best results were obtained via simultaneous deprotection of both groups, using liquid HF at low temperature. Highly pure final product was obtained from the reaction crude by precipitation with MTBE. Notwithstanding, the scope of the reaction was limited to small amounts of starting material. 4 Compound 1 was synthesized in 84% overall yield from its precursor 5. This is a major improvement over previous OiPr/OBn strategies (45% yield, from 4',11-diisopropyl-Lam-D) (9).
Formation of an ester bond between 5 and 7 (29) afforded compound 12 ( Fig. 1) in 26% yield, using EDC·HCl with a catalytic amount of PS solid supported DMAP in CH 2 Cl 2 . Deprotection of compound 12 using HF provided 2 in 36% yield.
Ester bond formation between 8 and protected lamellarin 5 or 6 using EDC·HCl was unsuccessful.
The inaccessibility of the carboxylic acid in the N-Boc protected oligopeptide sequence 8, or steric hindrance of the free phenolic group in Lam-D building blocks 5 and 6, may have been decisive to the lack of reaction. Various attempts at ester bond formation between 8 and the scaffold 5 were also unsuccessful. 5 Therefore, taking advantage of the relatively easy amide bond formation (i.e. compared to ester bonds), an N-Boc-Gly-OH spacer was introduced at position 3 of 5 (affording 13 in 89% yield, Fig. 2) for subsequent amide bond formation with 8. N-Boc deprotection of 13 with 40% TFA followed by reaction with 8 in EDC·HCl, HOBt, and DIPEA as base, gave 14 (Fig. 1, 58% yield). Deprotection of 14 with HF under standard conditions afforded the NLS peptidic conjugate 3 in 38% yield.
The NLS conjugate at position 4' of Lam-D could not be formed using the same conditions employed for ester bond formation between 6 and 8. 6,7 Instead, pre-activation of 8 with TCFH (30) and NEt 3 , followed by the addition of a solution of 6 and DMAP were required, affording 15 ( Fig.  1) in 45% yield (relative to 40% transformation of 6). Elimination of the nine Boc protecting groups with 40% TFA in CH 2 Cl 2 gave compound 4 in 17% yield.
The ester bond of compound 3, which has a double Gly spacer, is more susceptible to nucleophilic attack by nucleophiles from the medium than that of compound 4, which has a single Gly spacer.
Thus, the final conjugate 3 (derived from 14) was water labile. The rapid degradation of 3 made biological tests with this compound impossible. 8 Cytotoxicity and Cellular Uptake.
The cytotoxicity of Lam-D and its analogs (1, 2, 4 and 15 FACS flow cytometry was used to measure cellular uptake quantification (9). The results are shown in Table 2. Interestingly, the cellular internalization quotient for the PEGylated derivatives 1 and 2 were higher than that of Lam-D in all cancer cell lines. Indeed, compound 4, with an NLS sequence, was 10 times more active than Lam-D in A-549 and MDA-MB-231 cell lines and retained CIQ, despite having the highly charged NLS peptide. CIQ of conjugates in BJ cellular culture were from 76.8 to 128.6%.

Cellular Distribution of Lam-D, 1 and 4. Tracking in GFP-Topo I Transfected Cell
Cultures.
Lam-D is a Topo I inhibitor. To determine whether Lam-D, 1 and 4 localize to the same subcellular compartment as Topo I, a cellular localization assay was performed. A functional chimera of the green fluorescent protein EGFP with full length Topo I (GFP-Topo I) was expressed in HT-29, A-549 and MDA-MD-231 cells, which were then treated with either Lam-D, 1 or 4. As described, GFP-Topo I was located in the nucleus (25) in all cell types (Fig.3, positive control).
Conjugate 4, carrying the NLS signal, was localized to the nucleus in HT-29 and A-549 cells (Fig.   3, j1, j2, k1 and k2), suggesting that its higher activity could correlate to subcellular co-localization with its target, Topo I.

DISCUSSION
The new Lam-D conjugates reported here are excellent candidates for further biological evaluation. The evaluation of conjugates 3, 4 in BJ skin fibroblasts as normal cells was used in the present study. In this cellular culture, no significative variation, or even less citotoxicity was Our results (9) 3 The ester bonds of the protected Lam-D conjugates were labile; hence, to avoid problems with hydrolysis, we minimized the deprotection steps after condensation. 4 The yield for the deprotection was 84% working on a 20-30 mg scale. However, the procedure could not be scaled up. The stability 1 was studied in DMEM supplemented with 10% FBS and 100 units/mL penicillin and streptomycin at 37 °C. HPLC analysis indicates 97% of Lam-D liberation after 360 min of incubation. 5 Esterification of 5 was tested with EDC·HCl, TCFH or N,N,N',N'-tetramethylchloroformamidyl chloride as activating agents. 6 N-Cbz-Gly-OH was anchored to 6 in quantitative yield. However, further Cbz deprotection could not be performed without concomitant hydrolysis of the conjugate ester bond. 7 Other coupling reagents as EDC·HCl, DIPCDI, TFFH, PyBOP with HOAt, and MSNT with NMI failed in ester bond formation. 8 Compound 3 quickly hydrolyzes and liberates Lam-D into the medium, even on the time scale of an HPLC analysis.         The UV emissions corresponding to Lam-D, 1 and 4 are arbitrarily represented in red tones. The test compounds were seeded at a concentration of 1 µM and then incubated for 12 h.