Journal of Chemistry
Volume 2015 (2015), Article ID 263072, 6 pages
http://dx.doi.org/10.1155/2015/263072

Research Article

Synthesis of PAMAM Dendrimers Loaded with Mycophenolic Acid to Be Studied as New Potential Immunosuppressants

Lorenzo Guazzelli,1,2 Felicia D’Andrea,¹ Francesco Giorgelli,¹ Giorgio Catelani,¹ Alessandra Panattoni,³ and Andrea Luvisi³

¹Department of Pharmacy, University of Pisa, Via Bonanno 33, 56126 Pisa, Italy
²School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland
³Department of Agriculture, Food and Environment, University of Pisa, Via del Borghetto 80, 56124 Pisa, Italy

Received 17 February 2015; Accepted 19 April 2015

Academic Editor: Albert Demonceau

Copyright © 2015 Lorenzo Guazzelli et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The terminal N-Boc protected diamino PAMAM 7 was condensed (EDC-DMAP) with two units of mycophenolic acid (MPA) giving the N-Boc protected dendron 8 in a good yield (76%). The ammonium trifluoroacetate 9 was prepared from 8 by acid treatment (TFA-THF-H₂O) and was split into two equal parts. The first half was treated with di-2-pyridyl thionocarbonate (DPT) in the presence of Et₃N to give the corresponding isothiocyanate 10. This was reacted with the second half of 9 providing the symmetrical dendrimer 11 (68% yield), exposing four MPA units around the thioureido-PAMAM core.

1. Introduction

Mycophenolic acid (MPA, 1) is a potent inhibitor of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo synthesis of guanine nucleotide [1]. MPA exhibits antiproliferation activity and has been established as an anticancer agent, an immunosuppressant [1, 2] as well as an antiviral drug against some plant viruses [3–5]. Among these promising properties of MPA, the immunosuppressant property is by far the most important. MPA is a cornerstone immunosuppressant used to prevent rejection after organ transplantation. Currently, two MPA derivatives are used clinically for this purpose (Figure 1): mycophenolate sodium (MPS, Myfortic, 2) and mycophenolate mofetil (MMF, CellCept, 3). Several structural modification of MPA were carried out during the last twenty years to advance the elucidation of the structure-activity relationship of MPA in its interaction with IMPDH and contributed to the understanding of the relevant sites required for drug activity. Analogues which displayed similar or better immunosuppressive activity were reported. These derivatives present modifications only at the terminal carboxylic acid [6–11].

Figure 1: Mycophenolic acid (1) and its marketed derivatives (2) and (3); schematic representation of the structure of a generic dendrimer (4) and of a single PAMAM branch (4a).

In the search of new immunosuppressants, we planned the preparation of a new family of MPA derivatives linking more units of MPA to a scaffold by means of the carboxylic group. Among the possible scaffolds, we decided to use dendrimers as a drug delivery system (4, Figure 1) [12]. These are nanostructured macromolecules characterized by a tree-like architecture with exponential numbers of discrete dendritic branches radiating out from a common core. One of the most commonly known dendrimeric structure is the poly(amidoamine) (PAMAM) dendrimer which was introduced and developed by Tomalia and Fréchet [13]. PAMAM dendrimers are monodispersed, biocompatible, and synthetic macromolecules with well-defined structural architecture and composition. Here, we present the synthesis of PAMAM derivatives decorated with MPA units. The antiviral activity of these compounds has been recently tested in Nicotiana tabacum L. cv. Xanthi explants infected with a cucumber mosaic virus [14].

2. Experimental

Melting points were determined with a Kofler hot-stage apparatus and are uncorrected. Optical rotations were measured with a Perkin-Elmer 241 polarimeter at °C. ¹H NMR and ¹³C NMR spectra were recorded in appropriate solvents with a Bruker Avance II spectrometer operating at 250.12 MHz (¹H) and 62.9 MHz (¹³C). Spin resonances were reported as chemical shifts (δ) in part per million (ppm) and the references to the residual peak of the solvent employed as follows: CD₃CN 1.94 ppm (¹H NMR, central band) and 1.28 ppm (¹³C NMR, central band), CD₃OD 3.31 ppm (¹H NMR, central band) and 49.0 ppm (¹³C NMR, central band). Spin multiplicity was indicated by s = singlet, d = doublet, t = triplet, bt = broad triplet, q = quartet, m = multiplet. Coupling constants were reported in Hertz (Hz). The assignments were made, when possible, with the aid of DEPT, HETCOR, COSY, and HSQC experiments. Low resolution mass spectra were recorded on a LCQ Advantage ThermoFinnigan spectrometer equipped with an ion trap analyzer (Thermo Electron Company, San Jose, CA, USA). All reactions were followed by TLC on Kieselgel 60 F₂₅₄ with detection by UV light and/or with ethanolic 10% phosphomolybdic or sulfuric acid and heating. Kieselgel 60 (E. Merck, 70–230 and 230–400 mesh, resp.) was used for column and flash chromatography. Solvents were dried and purified by distillation according to standard procedure [15] and stored over 4 Å molecular sieves activated for at least 24 h at 200°C. MgSO₄ was used as the drying agent for solutions. Compounds 5 and 7 were prepared according to the reported procedures [16].

2.1. Synthesis of Compound 6

A solution of mycophenolic acid (1) (125 mg, 0.390 mmol, 1.2 eq) and amine 5 (57 mg, 0.354 mmol) in dry DMF (1.5 mL) containing Et₃N (148 μL, 1.06 mmol, 3.0 eq) was treated at room temperature with 1-ethyl-3-(3-(dimethylamino)-propyl)-carbodiimide hydrochloride (EDC, 90.4 mg, 0.472 mmol, 1.34 eq) and N-hydroxybenzotriazole (HOBt, 49.2 mg, 0.364 mmol, 1.03 eq). The solution was stirred at room temperature until TLC analysis (9 : 1 CH₂Cl₂-MeOH, 21 h) revealed the complete disappearance of amine 5 and the formation of a major product ( = 0.85). The mixture was concentrated under diminished pressure and the purification of the residue by flash chromatography over silica gel (2 : 8 hexane-EtOAc) gave pure 6 (120 mg, 73% calculated from 5) as a solid foam; = 0.22 (2 : 8 hexane-EtOAc); ¹H NMR (250.12 MHz, CD₃CN-CDCl₃): δ 1.38 [s, 9H, C(CH ₃)₃], 1.92 (s, 3H, =CCH ₃), 2.10 (s, 3H, Ar-CH ₃), 2.18–2.24 (m, 4H, =C(CH ₂) ₂CO), 2.98 (q, 2H, = 5.7 Hz, CH ₂NHCO), 3.10 (q, 2H, = 5.7 Hz, CH ₂NHCOO), 3.34 (d, 2H, = 7.0 Hz, ArCH ₂CH=), 3.73 (s, 3H, CH ₃), 5.20 (s, 2H, ArCH ₂OCO), 5.17 (t, 1H, = 7.0 Hz, CH=C), 5.42 (bt, 1H, NHCO), 6.53 (bt, 1H, NHCOO), 7.74 (s, 1H, OH); ¹³C NMR (62.9 MHz, CD₃CN-CDCl₃): 11.6 (Ar-CH ₃), 16.3 (=CCH ₃), 23.1 (ArCH ₂CH=), 28.5 [C(CH ₃) ₃], 35.4, 35.8 (=C(CH ₂) ₂CO), 40.0, 40.9 [HN(CH ₂) ₂NH], 61.5 (OCH ₃), 70.8 (ArCH ₂O), 79.3 [C(CH₃)₃], 107.2 (C=), 123.3 (CH=), 116.0, 122.5, 135.3, 145.8 (4 × Ar-C), 157.0 (O-C=O), 153.8, 164.2 (2 × Ar-C-O), 173.2, 173.6 (2 × C=O). ESIMS: calcd for C₂₄H₃₄N₂O₇Na [M + Na]⁺ 485.5, found 485.3. Anal. Calcd for C₂₄H₃₄N₂O₇: C, 62.32; H, 7.41; N, 6.06. Found: C, 62.34; H, 7.44; N, 6.08.

2.2. Synthesis of Compound 8

Method A (EDC, HOBT, Et ₃N, and DMF). A solution of mycophenolic acid (1) (369 mg, 1.151 mmol, 1.2 eq) and amine 7 (204 mg, 0.525 mmol) in dry DMF (6.0 mL) containing Et₃N (48 μL, 3.48 mmol, 3.0 eq) was treated at room temperature with EDC (296 mg, 0.514 mmol, 1.33 eq) and HOBt (161 mg, 1.19 mmol, 1.03 eq) and the solution was stirred at room temp. After 48 h, TLC analysis (8 : 2 CH₂Cl₂-MeOH) revealed the complete disappearance of amine 7 and the formation of a major product ( = 0.71). The mixture was concentrated under diminished pressure and the purification of the residue by flash chromatography over silica gel (88 : 12 CH₂Cl₂-MeOH) gave pure 8 (298 mg, 57% calculated from 7).

Method B (EDC, DMAP, and DMF). A solution of amine 7 (98.4 mg, 0.253 mmol) and mycophenolic acid (1) (179 mg, 0.558 mmol, 2.2 eq) in dry DMF (3 mL) was treated with EDC (130 mg, 0.676 mmol, 2.67 eq) and 4-dimethylaminopyridine (DMAP, 9.3 mg, 0.0759 mmol, 0.3 eq). The mixture was stirred at room temperature and after 48 h the TLC analysis (9 : 1 CH₂Cl₂-MeOH) revealed the complete disappearance of amine 7 and the formation of a major product ( = 0.40). The mixture was concentrated under diminished pressure and the purification of the residue by flash chromatography over silica gel (CH₂Cl₂-MeOH 92 : 8) gave pure 8 (191 mg, 76%).

Compound 8 was a solid foam; = 0.12 (92 : 8 CH₂Cl₂-MeOH); ¹H NMR (250.12 MHz, CD₃OD-CDCl₃): 1.40 [s, 9H, C(CH ₃)₃], 1.80 (s, 6H, 2 × =CCH ₃), 2.13 (s, 6H, 2 × Ar-CH ₃), 2.30 (bt, 4H, = 6.3 Hz, 2 × NCH₂CH ₂CONH), 2.32 (bs, 8H, 2 × =C(CH ₂) ₂CO), 2.50 (t, 2H, = 6.8 Hz, NCH ₂CH₂NHCOO), 2.75 (bt, 4H, = 6.3 Hz, 2 × NCH ₂CH₂CONH), 3.15 (t, 2H, = 6.8 Hz, NCH₂CH ₂NHCOO), 3.30 [m, 8H, 2 × CONH(CH ₂) ₂NHCO], 3.36 (d, 4H, = 6.6 Hz, 2 × ArCH ₂CH=), 3.75 (s, 6H, 2 × OCH ₃), 5.41 (m, 6H, 2 × CH=C, 2 × ArCH ₂OCO); ¹³C NMR (62.9 MHz, CD₃OD-CDCl₃): 11.6 (2 × ArCH ₃), 23.5 (2 × ArCH ₂CH=), 16.3 (2 × =CCH ₃), 33.4 (2 × NCH₂CH ₂CONH), 28.7 [C(CH ₃)₃], 35.6, 36.3 (2 × =C(CH ₂) ₂CO), 39.8, 39.7 [2 × HN(CH ₂) ₂NH], 38.4 (NCH₂CH ₂NHCOO), 50.8 (2 × NCH ₂CH₂CONH), 53.1 (NCH ₂CH₂NHCOO), 61.5 (2 × OCH ₃), 70.7 (2 × ArCH ₂O), 80.1 [C(CH₃)₃], 107.5 (2 × C=), 123.9 (2 × CH=), 117.7, 123.3, 134.8, 145.8 (8 × Ar-C), 158.1 (O-C=O), 154.5, 164.6 (4 × Ar-C-O), 173.6, 174.3, 175.6 (6 × C=O). ESIMS: calcd for C₅₁H₇₂N₂O₁₄Na [M + Na]⁺ 1015.5, found 1015.4. Anal. Calcd for C₅₁H₇₂N₆O₁₄: C, 61.68; H, 7.31; N, 8.46. Found: C, 61.70; H, 7.33; N, 8.47.

2.3. Synthesis of Dendrimer 11

A solution of 8 (186 mg, 0.186 mmol) in CH₂Cl₂-TFA-H₂O 7.4 : 2.4 : 0.2 (2.0 mL) was stirred at room temperature until the TLC analysis (8 : 2 CH₂Cl₂-MeOH, 1 h) revealed the complete disappearance of the starting material and the formation of a major product ( = 0.36). The reaction mixture was concentrated under diminished pressure and repeatedly coevaporated with toluene (4 × 30 mL). The crude residue (184 mg) analyzed by NMR proved to be constituted only by the ammonium trifluoroacetate 9. The salt 9 is a solid foam, = 0.36 (8 : 2 CH₂Cl₂-MeOH); ¹H NMR (250.12 MHz, CD₃CN): 1.77 (s, 6H, 2 × =CCH ₃), 2.10 (s, 6H, 2 × Ar-CH ₃), 2.70 (bs, 4H, 2 × NCH ₂CH₂CONH), 2.18–2.30 (m, 12H, 2 × =C(CH ₂) ₂CO, 2 × NCH₂CH ₂CONH), 3.05 (m, 2H, NCH ₂CH₂), 3.32 (d, 4H, = 6.8 Hz, 2 × ArCH ₂CH=), 3.20, 3.40 [2m, each 4H, 2 × CONH(CH ₂) ₂NHCO], 3.44 (m, 2H, CH ₂), 3.72 (s, 6H, 2 × OCH ₃), 5.16 (m, 6H, 2 × CH=C, 2 × ArCH ₂OCO), 10.1 (bs, 4H, 4 × NHCO), 7.80 (s, 2H, 2 × OH); ¹³C NMR (62.9 MHz, CD₃CN): 11.6 (2 × Ar-CH ₃), 16.2 (2 × =CCH ₃), 23.3 (2 × ArCH ₂CH=), 29.1 (2 × NCH₂CH ₂CONH), 35.9, 36.4 (2 × =C(CH ₂) ₂CO), 38.7, 39.9 [2 × HN(CH ₂) ₂NH], 51.9 (2 × NCH ₂CH₂CONH), 36.4 (CH ₂NHCO), 50.1 (CH ₂), 61.6 (2 × OCH ₃), 71.0 (2 × ArCH ₂O), 123.8 (2 × CH=), 107.3 (2 × C=), 119.0, 122.7, 135.2, 146.2 (8 × Ar-C), 153.9, 164.3 (4 × Ar-CO), 173.0, 176.0, 173.4 (6 × C=O).

To a solution of crude 9 (92 mg, 0.0912 mmol) in dry CH₂Cl₂ (1.4 mL), Et₃N (100 μL) and 2-pyridyl thiocarbonate (DPT, 22 mg, 0.0912 mmol) were added at room temperature. After 6 h the TLC analysis (7 : 3 ) revealed the complete disappearance of the starting material and the formation of a major product ( = 0.24). The solution was concentrated under diminished pressure and the crude isothiocyanate intermediate 10 (86 mg, 0.0912 mmol) was solubilized in dry 1 : 1 CH₂Cl₂-DMF (1.0 mL) and a solution of crude 9 (92 mg, 0.0912 mmol) in dry 1 : 1 CH₂Cl₂-DMF (2.0 mL) containing Et₃N (0.2 mL) was added dropwise. The reaction mixture was stirred at room temperature for 43 h, until TLC analysis (7 : 3 ) revealed the formation of a major product at = 0.22. The reaction was concentrated at reduced pressure and the crude was subjected to a flash chromatography (EtOAc) on silica gel to give dendrimer 11 (114 mg, 68% from 9) as a foam solid; = 0.22 (7 : 3 ); ¹H NMR (250.12 MHz, CD₃OD-CDCl₃): 1.81 (s, 12H, 4 × =CCH ₃), 2.14 (s, 12H, 4 × Ar-CH ₃), 2.26 [bs, 16H, 4 × =C(CH ₂) ₂CO], 2.34 (bt, 8H, = 6.4 Hz, 4 × NCH₂CH ₂CONH), 2.72–2.86 (m, 12H, 2 × NCH ₂CH₂NHCS, 4 × NCH ₂CH₂CONH), 3.22 [m, 16H, 4 × CONH(CH ₂) ₂NHCO], 3.37 (d, 8H, = 7.0 Hz, 4 × ArCH ₂CH=), 3.58 (t, 4H, = 5.6 Hz, NCH₂CH ₂NHCS), 3.76 (s, 12H, 4 × OCH ₃), 5.21 (s, 8H, 4 × ArCH ₂OCO), 5.24 (t, 4H, = 7.3 Hz, 4×CH=C); ¹³C NMR (62.9 MHz, CD₃CN-D₂O): 11.5 (4 × Ar-CH ₃). 16.3 (4 × =CCH ₃), 23.5 (4 × ArCH ₂CH=), 34.8 (4 × NCH₂CH ₂CONH), 35.8, 36.4 [4 × =C(CH ₂) ₂CO], 39.9 [4 × HN(CH ₂) ₂NH], 44.3 (NCH₂CH ₂NHCS), 50.8 (4 × NCH ₂CH₂CONH), 54.2 (2 × NCH ₂CH₂NHCS), 61.5 (4 × OCH ₃), 70.7 (4 × ArCH ₂O), 107.5 (4 × C=), 117.6, 123.4, 134.9, 146.2 (16 × Ar-C), 124.3 (4 × CH=), 154.5, 164.5 (8 × Ar-C-O), 173.6, 174.7, 175.7 (12 × C=O), 179.8 (C=S). ESIMS: calcd for C₉₃H₁₂₆N₁₂O₂₄SNa [M + Na]⁺ 1849.9, found 1849.7. Anal. Calcd for C₉₃H₁₂₆N₁₂O₂₄S: C, 61.10; H, 6.95; N, 9.19. Found: C, 61.13; H, 6.98; N, 9.21.

3. Results and Discussion

Derivative 6 was first prepared, as a positive control for biological evaluations (Scheme 1). The monoprotected N-Boc-ethylenediamine 5 [16], which is also used as the central core in the construction of PAMAM dendrons, was used as capping reagent. The same kind of additional functional groups will be present in all synthetic structures, thus ensuring similar interactions during the evaluation of immunosuppressant properties.

Scheme 1: Synthesis of compound 6.

1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), which is nowadays preferred to N,N ′-dicyclohexylcarbodiimide (DCC) in the formation of amide bonds because it is not a skin sensitizer and its urea by-product can be easily removed, was chosen as a coupling agent and the reaction was carried out at room temperature in the presence of N-hydroxybenzotriazole (HOBt) and triethylamine (Et₃N). Compound 6 was isolated after 12 h in a 73% yield after flash chromatography on silica gel. The two-branched structure 7 [16] was easily obtained via double Michael addition of methyl acrylate to N-Boc-ethylenediamine followed by amidation of the intermediate with a large excess of ethylenediamine. This was then reacted with 1 using the same coupling conditions seen above (Scheme 2). The reaction required longer time (48 h) to go to completion and afforded 8 in a poor 57% yield. Better results were obtained when N,N-dimethylaminopyridine (DMAP) was used instead of the mixture Et₃N-HOBt. In this case, the desired compound was isolated in a 76% yield after chromatographic purification.

Scheme 2: Synthesis of compound 8. Reaction conditions: EDC, DMPA, DMF, room temperature, 48 h (76% yield) or EDC, HOBt, DMF, Et₃N, room temperature, 48 h (57% yield).

Having obtained this structure, the synthesis of the symmetrical dendrimer 11 was possible (Scheme 3). First, the acid labile N-Boc protecting group was removed (trifluoroacetic acid-water, in CH₂Cl₂) giving the ammonium trifluoroacetate salt 9 in a quantitative yield. The structure and purity of 9 were confirmed by NMR analysis, which verified the complete removal of the t-butoxy carbonyl group. The latter compound was then reacted, without purification, with di-2-pyridyl thionocarbonate (DPT) and triethylamine. DPT [17, 18] is a commercially available, solid, nontoxic reagent that can be used in the preparation of isothiocyanates as a safe alternative to thiophosgene. The reaction proceeded smoothly and gave the complete conversion after 5.5 h. The intermediate isothiocyanate obtained after removal of the solvents was directly reacted with crude 9 in the presence of Et₃N in a mixture of CH₂Cl₂ and DMF.

Scheme 3: Synthesis of dendrimer 11.

Dendrimer 11 was obtained after flash chromatographic purification in a satisfactory 68% overall yield. The symmetrical structure of compound 11 and the formation of the thioureido bridge (diagnostic C=S signal) were fully confirmed by ¹H, ¹³C and 2D NMR experiments.

4. Conclusions

In conclusion, we reported the synthesis of poly (amidoamine) structures decorated with mycophenolic acid. The target structures were prepared in good overall yields and with a limited number of purification steps. Furthermore, the very same strategy could be used to prepare dendrimers of higher generation. On the other hand, the presence of the orthogonally protected amino group on the inner core could allow for the preparation of a library of asymmetrical structures coupling the dendron with different compounds rather than its symmetrical counterpart. The immunosuppressant properties of these compounds are currently under investigation and the results will be published in due course.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgment

Thanks are due to Dr. Stefania Fuselli who performed part of the experimental work in the frame of her undergraduate thesis.

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