One-step template-directed synthesis of acridine-based rigid cyclophanes

Tetrahedron 68 (2012) 8773e8782

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One-step template-directed synthesis of acridine-based rigid cyclophanes
Ferna
ndez, Lourdes Gude, Antonio Lorente * Sara Sierra, Katerina Duskova, María-Jose
nica, Universidad de Alcala
, 28871-Alcala
de Henares, Madrid, Spain Departamento de Química Orga

a r t i c l e i n f o

a b s t r a c t

Article history: Received 13 June 2012 Received in revised form 27 July 2012 Accepted 1 August 2012 Available online 9 August 2012

Cyclo-bisintercalands are macrocyclic systems containing aromatic subunits, which are commonly used as hosts for aromatic molecules and as DNA intercalators. In this article, a step-by-step synthesis of a series of cyclo-bisintercalands containing 3,6-diaminoacridines as aromatic units, and connected by rigid spacers of different lengths, is reported. In addition, we describe herein a more efficient synthetic alternative, involving single-step template-directed processes. The new routes allow the easy synthetic access to these macrocyclic systems with acceptable yields. The synthesized cyclo-bisintercalands and their precursors have been structurally characterized by UVevisible and NMR methods. Preliminary biological activity assays performed on the bisintercalands and cyclo bisintercalands revealed interesting cytotoxic properties against different tumor cell lines, especially in the case of the bisintercalands, highlighting their potential as cancer chemotherapeutic agents. Ó 2012 Elsevier Ltd. All rights reserved.


L. Soto on the Dedicated to Professor Dr. Jose occasion of his 80th anniversary

Keywords: Cyclophanes Template effect Acridine Cyclo-bisintercalands

1. Introduction Cyclo-bisintercalands are macrocyclic receptors of the cyclophane-type1,2 containing two planar polycyclic arenes (such as acridine,3 phenanthridine,4 quinacridine,5 flavin,6 phenazine,7 pyrene and diazapyrene,8 porphyrin,9 and anthracene or imidazolium,10 among others). Most of these systems incorporate in their structure aromatic subunits of adequate surface, separated at a suitable distance to facilitate the intercalation of flat aromatic guests. The template effect, either by ions or uncharged organic molecules, is a very useful tool for the synthesis of macrocyclic compounds.11e20 The macrocycle ring size strongly depends on the size of the template. Moreover, the conformational orientation fixed by the template significantly increases the yield of the cyclic product. € nig and co-workers have observed an organic guest template Hu effect in the synthesis of a cyclophane with diphenylmethane subunits in the presence of phenanthrene.21 Dispersive forces (donoreacceptor interactions) and ‘edge-to-face’ interactions (T-type) are responsible for the templated synthesis of the cyclophanes. In this article, we describe the synthesis of cyclo-bisintercalands containing 3,6-diaminoacridine subunits linked by rigid spacers, as potential receptors of flat aromatic molecules and as DNA cyclo-

bisintercalands. The polycyclic arenes employed in this work are derivatives of the 3,6-diaminoacridine ring, which extended aromatic surface may favor p-stacking interactions with aromatic substrates. Indeed, 3,6-diaminoacridines are well-known to interact very efficiently with nucleic acids, mainly through intercalation.22,23 Moreover, the designed cyclo-bisintercalands include rigid linking chains in their structure, based on the hypothesis that improved binding characteristics would be expected for systems that are highly preorganized. As a matter of fact, bisintercalands containing rigid linkers have shown enhanced DNA affinities compared to those containing flexible chains, since selfstacking between the aromatic units is effectively prevented.24e28 Although the cyclo-bisintercalands described in this work can be prepared following a step-by-step procedure,29 here we report a more efficient, one-step synthesis, in which the use of the template effect constitutes an alternative to the typical high-dilution conditions required for the preparation of these macrocyclic systems. This approach results in a significant simplification in the synthetic routes that give access to these cyclophanes, with acceptable overall yields compared to the step-by-step methodologies previously reported.29 2. Results and discussions 2.1. Stepwise syntheses of cyclo-bisintercalands

* Corresponding author. Tel.: þ34 91 885 4762; fax: þ34 91 885 4686; e-mail address: [email protected] (A. Lorente). 0040-4020/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2012.08.003

Firstly, we carried out the stepwise syntheses (procedure A) of cyclo-bisintercalands 3 (Scheme 1) with cavities of lateral

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S. Sierra et al. / Tetrahedron 68 (2012) 8773e8782

Scheme 1.

dimensions of ca. 7.5  A and vertical dimensions ranging from 3.4  A to 6  A. The rigid linker for these systems was either a benzene ring disubstituted at ortho, meta or para or a naphthalene ring connected through the 20 and 60 positions. In the first step of the synthetic procedure, reaction of (6-amino-3-acridinyl)carbamic acid methyl ester and the corresponding dibromide afforded bisintercalands 1aed, which by treatment with methyl chloroformate yielded the tetracarbamate derivatives 2. Cyclization of compounds 2 with 1 equiv of the corresponding dibromide in highdilution conditions gave rise to cyclophanes 3aec, with an overall yield around 34% (three steps). We also considered the synthesis of cyclo-bisintercaland 9 containing diacetylenic linkers (Scheme 2). The choice of this spacer was made based on the C1eC6 distance (6.77  A), which is suitable for complexation of aromatic substrates and for intercalation with DNA. Thus, cyclo-bisintercaland 9 resembles an open-ended box having a cavity with a lateral dimension of ca. 7.5  A and a vertical dimension of ca. 5.2  A (measured on a CPK model). Synthesis of 9 (Scheme 2) was accomplished through the use of a propargylic derivative of 3,6-acridinediylbiscarbamic acid dimethyl ester (5), which by a Glasser oxidative coupling, afforded the bisintercaland 6. Propargylation and subsequent cyclization in high-dilution conditions yielded cyclo-bisintercaland 9 and cyclotrisintercaland 8 as a minor product. These results are similar to those described by Whitlock co-workers30 for the synthesis of naphthalenophanes with diacetylenic bridges. 2.2. One-step syntheses of cyclo-bisintercalands The procedure described in the preceding section afforded cyclo-bisintercalands 3aec and 9 with low to moderate yields.

However, this route constitutes a tedious and time-expensive methodology for the preparation of these cyclo-bisintercalands. Therefore, as a first approach to circumvent this problem, we attempted the syntheses of cyclo-bisintercalands 3 from 3,6acridinediylbiscarbamic acid dimethyl ester and the corresponding dibromide in high-dilution conditions. The application of the dilution principle normally allows transforming the polyfunctionalized starting compounds into the cyclophane compounds with acceptable yields. However, in our case, a complex mixture of products was obtained from which the corresponding cyclo-bisintercalands 3 were isolated in low yield (3a: 26%; 3b: 8%; 3c: 15%). For this reason, we now describe the synthesis of this type of molecular receptors following one-step procedures based on templated macrocyclization reactions. As it is shown in Scheme 3, one-step synthesis of the cyclobisintercalands 3 from 3,6-acridinediylbiscarbamic acid dimethyl ester and the corresponding dibromide can be easily accomplished by using the commercially available p-rich naphthalene derivative 2,7-dimethoxynaphthalene as template. The reaction proceeded with a relatively good yield for the ortho-disubstituted benzene ring (42%), and lower yields (w22%) for the meta- and para-related systems. The template effect can be explained by the existence of stabilizing electron donoreacceptor interactions between the 2,7dimethoxynaphthalene and the acridine subunits in the bisintercaland intermediate of the macrocyclization process (Fig. 1). When the same procedure was applied to prepare cyclobisintercaland 3d, containing 2,6-naphthalenediyl linkers, only traces of the desired macrocyclic derivative were detected, probably due to the longer chain of the naphthalene linker in comparison to the chain containing the benzene ring. Therefore, it was

S. Sierra et al. / Tetrahedron 68 (2012) 8773e8782

MeO 2C

80% NaH/ DMF N H

MeO 2C

NH2 r.t./ 2 h (93%)

N

+

N

N

4

Br H2C C CH

MeO2C

N

N

N H

Cu(AcO)2 py-MeOH 45ºC/ 96 h (88%) MeO2C

NH2

N

H N

N

ClCO 2CH3 Na2CO3 acetone 5 h reflux (74%)

MeO2C N

N

NH CO2Me

5

CO 2Me

MeO 2C

N

N

N

CO2Me

BrH2C C CH NaOH/ DMF

Cu(AcO)2

RT/ 1.5 h (67%)

py-CH2Cl2 reflux / H-D 120 h

CO 2Me

MeO 2C

N

N

6

N

CO2Me

7

MeO 2C

CO 2Me

N

MeO2C

8775

N

MeO2C N

N

N

N

N

CO 2Me

N CO2Me

+ N

N

MeO 2C

N

N

N CO2Me

N

N

9 (22%)

CO 2Me

MeO2C

8 (5%)

Overall yield: 9% Scheme 2.

Br 60% NaH/ DMF/ 50 ºC, 24 h MeO2 C N H

N

N H

CO 2Me

+

H 3CO

OCH3

3a-c a (42%) b (22%) c (21%)

Br Scheme 3.

Glasser oxidative coupling of 3,6-acridinediylbis(2propynylcarbamic acid dimethyl ester), 11. This time, as indicated in Scheme 5, besides compound 9, the cyclo-trisintercaland 8 was obtained as the major product. Notwithstanding this observation, the synthetic route length was reduced from five steps to two, the reaction time in the cyclization step considerably diminished (from 120 h to 48 h), and the overall yield of the desired cyclobisintercaland was increased (from 9% to 15%). 2.3. Spectroscopic and structural studies Fig. 1. Preorganization of the acridine subunits by the template effect.

necessary to synthesize the compound 1,4-bis(7-methoxy-2naphthylmethyl)benzene (10) (Scheme 4), as a new template in order to achieve the one-step synthesis of this cyclo-bisintercaland. By using 10 as template, the synthesis of 3d was performed in a single step in 19% yield. Finally, using 2,7-dimethoxynaphthalene as the template, cyclobisintercaland 9 with diacetylenic linkers was synthesized by

In the absence of X-ray crystallographic structures for the synthesized compounds, we relied upon spectroscopic and molecular mechanics studies29a to infer the salient structural features of the bis- and cyclo-bisintercalands. The proton NMR spectra of cyclobisintercalands were recorded at 300 MHz and unequivocal assignment of the signals was performed by two-dimensional (ROESY, DQF-COSY, and TOCSY) experiments. The NeCH2 protons are equivalent in the bisintercalands as well as in the cyclo-

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S. Sierra et al. / Tetrahedron 68 (2012) 8773e8782

MeO2C MeO2C

N H

N

N H

+

N

CO2Me

N

N

60% NaH / DMF 50 ºC / 48 h

Br

O 3

1

8

OCH3 6

4

N

5

N

MeO2C

Br

CO 2Me

O

N

OCH3

CO2Me

10

3d (19%)

Scheme 4.

MeO2C

N H

N

N H

CO2Me

MeO2C

BrH2C C CH

N

H3CO

OCH3

8 (24%)

N

CO2Me

80% NaH / DMF (93%)

Cu(AcO)2 py-MeOH-CH2Cl2

N

+

11

9 (16%)

22%

15%

overall yield

Scheme 5.

bisintercalands (sharp and broad singlets in CD2Cl2 and DMSO-d6, respectively). Variable temperature proton NMR spectra of the cyclo-bisintercalands in CD2Cl2 ranging from 25  C to 85  C, showed no splitting of the NeCH2 singlets into AB quartets even at 85  C. These results can be explained by a rapid interconversion between limiting syn and anti conformations by rotation of the acridine units through the cavity, in contrast to observations made in other more rigid macrobicyclic receptor molecules containing acridine subunits.3a,e In the cyclo-bisintercaland 3a, the hydrogens 2(7) and 9 are shifted downfield relative to the corresponding protons in the dicarbamate, while the hydrogens 4(5) are shifted upfield. These effects can be attributed to the mutual shielding of the acridine units, by assuming a non-parallel arrangement of the acridine rings, with the C4eN10eC5 axes in closer proximity. This arrangement would explain the greater deshielding of protons 2(7) by the benzene rings in the linkers. In the macrocycles 3bed, protons 4(5) are shielded with respect to the same protons of the dicarbamate, especially in the case of compound 3b. On the contrary, protons 2(7) experience no significant changes in their chemical shifts. This suggests a face-to-face arrangement of the acridine rings with an almost parallel disposition, which was also confirmed by previous molecular mechanic calculations,29a indicating that, for compound 3b, there is a slight nearing of both C1eC9eC8 axes. These facts are in good agreement with electronic absorption spectra. UV spectra of the series of compounds 3aec present a hypochromic effect with respect to the corresponding bisintercalands 1aec. This hypochromicity is ascribed to the stacking interaction between the acridine rings in the macrocyclic compounds, which is especially significant in compound 3a (62% hypochromicity) because of the closer proximity of the acridine units compared to compounds 3b and 3c (36% and 16% hypochromicity, respectively). Interestingly, the opposite effect was observed for compound 3d, where the cyclophane showed an

increased absorption relative to bisintercaland 1d, which may be explained by the fact that the naphthalene bridges are more effective in isolating the acridine rings, thus preventing their interaction. In the case of the bisintercalands containing the diacetylenic linker, no interaction between the acridine subunits was apparently observed, as the chemical shifts of the aromatic hydrogens in compound 6 were practically identical to the ones recorded for monomer 5. Therefore, the length of the diacetylenic linker seems to preclude the interaction between the two acridines. However, in the cyclo-bisintercaland 9 (Scheme 2), all the aromatic protons have negative values of Ddcyc[Ddcyc¼dcyclophane(9)dhalf molecule(11)] ranging from 0.06 ppm for H-2(7) to 0.12 ppm for H-9. This result clearly indicates the existence of a shielding interaction between the acridine rings in the macrocycle. Moreover, UV spectra support these findings, as 9 (lmax¼268 nm, 3 ¼35,000; MeOH) has a larger hypochromicity with respect to the bisintercaland 6 (lmax¼270 nm, 3 ¼130,000; MeOH), where the larger extinction coefficient found for the corresponding bisintercaland indicates that the two acridine units do not interact appreciably. On the other hand, the spectrum of cyclo-trisintercaland 8 (lmax¼268 nm, 3 ¼100,000; MeOH) indicates the existence of weaker stacking interactions relative to compound 9, in accordance with a nonparallel arrangement of the acridine subunits.

2.4. Biological studies: in vitro cytotoxicity evaluation The cytotoxicity of the synthesized compounds was evaluated by measuring the growth inhibition of P-388 (mouse lymphoid neoplasm), A-549 (human lung carcinoma), HT-29 (human colon adenocarcinoma), and MEL-28 (human melanoma) cells. Table 1 shows biological activity results as IC50 values, expressed in micromolar units. The biological activity of several hydrochloride salts obtained from the free-base compounds is also reported.

S. Sierra et al. / Tetrahedron 68 (2012) 8773e8782

for the development of novel macrocyclic systems with potential therapeutic applications.

Table 1 Cytostatic activity of bisintercalands and cyclo-bisintercalands Compound

1a 1a$2HCl 1b 1b$2HCl 1d 1d$2HCl 6 6$2HCl 3a 3a$2HCl 3b 3b$2HCl 3d 8 8$3HCl 9$2HCl

8777

IC50 (mM) P-388

A-549

HT-29

MEL-28

7.8 3.5 1.6 1.4 7.3 1.6 >27 3.1 >23 5.4 >11 5.4 >10 3.1 3.8 5.7

7.8 7 1.6 1.4 14 3.3 >27 12 >23 10 >11 5.4 >10 12 >7.6 11

3.9 7 1.6 1.4 >14 6.6 >27 12 >23 >10 >11 5.4 >10 >12 >7.6 >11

3.9 7 1.6 1.4 >14 13 >27 12 >23 >10 >11 10.8 >10 >12 >7.6 >11

It is apparent from Table 1 that most of the compounds assayed (with the exception of 1a), were more potent growth inhibitors (lower IC50 values) in the mouse lymphoid neoplasm (P-388) cell line than in the A-549, HT-29, and MEL-28 cell lines. Furthermore, it was found that the compounds that were tested as the hydrochloride salts normally presented higher cytotoxic activities than the free-base compounds. This clearly indicates that protonation of acridine nitrogen N10 provides more effective antitumor compounds. On the other hand, and as a general trend, bisintercalands were found to be more cytotoxic than the corresponding cyclobisintercalands. Although the reasons for these results remain unclear, we hypothesize that the observed effect could be explained taking into account the higher rigidity of the cyclic systems, and that the linker’s length may not be long enough to allow DNA bisintercalation. On the contrary, in the open-chain precursors, one acridine unit may interact by intercalating between the DNA base pairs, whereas the second one could interact with the DNA grooves, and this could increase the interaction between the bisintercalands and DNA. Of particular interest is the family of compounds b, containing the meta-substituted benzene ring as linker for the aminoacridine subunits, which exhibited the highest activities as antitumor compounds in the series. This is particularly relevant in the case of compound 1b, which suggests an optimal minimum length for the spacer that is not present in the ortho-substituted systems. Further studies to synthesize novel derivatives with improved pharmacological properties, using the meta-substituted benzene linkers, are currently underway. 3. Conclusions In summary, we report a one-step, template-directed synthesis of a series of rigid cyclophanes containing 3,6-diaminoacridines as aromatic units, as an improved synthetic approach for the preparation of these rigid macrocyclic systems, in comparison to the stepwise methodology previously reported. The new route constitutes a significant advance, allowing a rapid and easy synthetic access to the cyclo-bisintercalands with low, but acceptable yields, taking into account the complex nature of intermolecular macrocyclization reactions. Moreover, spectroscopic methods performed on these systems have revealed interesting structural features. The bisintercalands and cyclo-bisintercalands have also been tested for their in vitro biological activity against different tumor cell lines, and showed micromolar potency. Thus, the compounds described in this article may serve as useful probes for the study of ligandeDNA interactions, and they may constitute a starting point

4. Experimental section 4.1. General information € chi Melting points are uncorrected and were determined in a Bu SMP-20 and in an Electrothermal IA9100 apparatus, for mps above 260  C. Infrared spectra were recorded on a FT-IR PerkineElmer 1725X spectrophotometer. The 1H and 13C NMR spectra were recorded at 300 and 75 MHz, respectively, on a Varian Mercury spectrometer. Mass spectra (EI) were recorded on a HewlettPackard HP-5988a spectrometer at 70 eV. Mass spectra (ESI and APCI) were recorded on an Automass Multi GC/API/MS Finnigan spectrometer. FAB(MS) were registered on a V.G. Autoexpec spectrometer using 3-nitrobenzyl alcohol as matrix. Elemental analyses were performed with a Heraeus CHN analyzer. Merck silica gel 60 (230e400 ASTM mesh) was employed for flash column chromatography. 4.2. (6-Amino-3-acridinyl)carbamic acid methyl ester To a suspension of 3,6-diaminoacridine hydrochloride (3 g, 12.21 mmol) in dry acetone (100 mL), freshly distilled methyl chloroformate (9.4 mL, 122.1 mmol) was added. The reaction mixture was refluxed for 72 h and then concentrated up to dryness. The residue thus obtained was dissolved in methanol and the solution was basified with sodium methoxide in methanol. The crude product obtained after concentrating the solution in vacuo was purified by flash column (ؼ6 cm) chromatography by using acetoneeethyl acetate-triethylamine (5/5/1, v/v/v) as eluent, affording 2.25 g (69%) of product; mp 163e165  C (hexaneedichloromethane). Found: C, 67.52; H, 4.85; N, 15.91. C15H13N3O2 requires C, 67.41; H, 4.90; N, 15.72; IR (KBr): n 3334, 3215, 1717, 1645, 1551, 1488, 1462, 1398, 1318 cm1; 1H NMR (DMSO-d6) d 10.00 (s, 1H, NH), 8.51 (s, 1H, H-9), 8.06 (d, J¼2.1 Hz, 1H, H-4), 7.83 (d, J¼9.1 Hz, 1H, H-8), 7.72 (d, J¼9.1 Hz, 1H, H-1), 7.41 (dd, J¼9.1, 2.1 Hz, 1H, H-7), 6.99 (dd, J¼9.1, 2.1 Hz, 1H, H-2), 6.84 (d, J¼2.1 Hz, 1H, H-5), 6.22 (br s, 2H, NH2), 3.72 (s, 3H, CO2CH3); 13C NMR (DMSO-d6): d 154.2, 151.6, 151.1, 149.8, 140.5, 135.0, 129.6, 129.3, 120.7, 120.2, 117.6, 112.4, 103.0, 52.0; MS (EI) (calcd for C15H13N3O2, 267.28) m/z 267 (Mþ, 82%), 235 (100), 208 (24), 181 (20), 153 (7). 4.3. 3,6-Acridinediylbiscarbamic acid dimethyl ester To a suspension of 3,6-diaminoacridine (5 g, 23.9 mmol) and potassium carbonate (79.3 g, 573 mmol) in dry acetone (550 mL) freshly distilled methyl chloroformate (22.1 mL, 268.8 mmol) was added. The reaction mixture was refluxed for 72 h and then concentrated up to dryness. The obtained residue was taken in dichloromethane (150 mL) and washed with water (350 mL). The organic phase was dried over magnesium sulfate and concentrated at reduced pressure. The crude product was purified by flash column (ؼ6 cm) chromatography by using ethyl acetate and then ethyl acetateeacetone (8/2, v/v) as eluent, affording 4.12 g (53%) of product, mp >290  C (hexaneedichloromethane). Found: C, 62.60; H, 4.52; N, 13.20. C17H15N3O4 requires C, 62.76; H, 4.65; N, 12.92; IR (KBr): n 3345, 1722, 1623, 1544, 1442, 1333 cm1; 1H NMR (DMSOd6) d 10.10 (s, 2H, NH), 8.79 (s, 1H, H-9), 8.21 (d, J¼2.1 Hz, 2H, H-4 and H-5), 7.99 (d, J¼9.1 Hz, 2H, H-1 and H-8), 7.56 (dd, J¼9.1, 2.1 Hz, 2H, H-2 and H-7), 3.74 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 153.9, 149.7, 140.8, 135.2, 129.2, 122.0, 119.4, 112.4, 52.1; MS (EI) (calcd for C17H15N3O4, 325.10) m/z 326 (Mþþ1, 9%), 325 (Mþ, 41), 294 (13), 293 (61), 261 (91), 234 (21), 207 (61), 208 (15), 179 (23).

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4.4. Syntheses of bisacridines 1. General procedure To a solution of (6-amino-3-acridinyl)carbamic acid methyl ester (2.87 g, 10.76 mmol) in dry DMF (100 mL) under argon, 80% sodium hydride (339 mg, 11.3 mmol) was added. Then, the corresponding dibromide (5.38 mmol) was added and the reaction mixture was stirred at room temperature for 3 h. The solvent was removed in vacuo and the oily residue was dissolved in dichloromethane and washed three times with water. The organic phase was dried with magnesium sulfate and concentrated up to dryness. The resulting solid was purified by flash column chromatography. 4.4.1. [1,2-Phenylenebis(methylene)]bis[(6-amino-3-acridinyl)carbamic acid dimethyl ester] (1a). According to the general procedure, the crude product was purified by flash column (ؼ3 cm) chromatography by using ethyl acetateeacetoneetriethylamine (10/4/ 2, v/v/v) as eluent, affording 2.02 g (59%) of 1a, mp 168e170  C (hexaneedichloromethane). Found: C, 69.21; H, 5.42; N, 11.87. C38H32N6O4$0.5CH2Cl2$0.5C6H14 requires C, 69.01; H, 5.58; N, 11.64; IR (KBr): n 3410; 1696; 1619 ; 1446; 1399; 1199 cm1; UV (methanol): lmax 274 nm (3 ¼98,000); 1H NMR (DMSO-d6) d 8.52 (s, 2H, H9), 7.77 (d, J¼9.0 Hz, 2H, H-1), 7.75 (d, J¼9.0 Hz, 2H, H-8), 7.64 (d, J¼1.7 Hz, 2H, H-4), 7.25e7.19 (m, 4H, H-2, H-40 and H-50 ), 7.17e7.12 (m, 2H, H-30 and H-60 ), 7.05 (dd, J¼9.0, 1.7 Hz, 2H, H-7), 6.84 (d, J¼1.7 Hz, 2H, H-5), 6.10 (br s, 4H, NH2), 5.10 (s, 4H, CH2), 3.66 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.3, 151.3, 151.0, 148.6, 142.5, 135.0, 134.7, 129.4, 128.6, 127.3, 127.2, 122.7, 122.6, 121.9, 120.9, 120.8, 102.6, 53.2, 50.3. MS (FAB): (calcd for C38H32N6O4, 636.25) m/ z 637 (MþþH, 100%). 4.4.2. [1,3-Phenylenebis(methylene)]bis[(6-amino-3-acridinyl)carbamic acid dimethyl ester] (1b). According to the general procedure, the crude product was purified by flash column (ؼ3 cm) chromatography with ethyl acetateeacetoneetriethylamine (5/5/1, v/v/ v) as eluent, yielding 2.05 g (60%) of 1b, mp 165e6  C (hexaneedichloromethane). Found: C, 69.03; H, 5.48; N, 11.94. C38H32N6O4$0.5CH2Cl2$0.5C6H14 requires C, 69.01; H, 5.58; N, 11.64; IR (KBr): n 3364; 2926; 1703; 1639; 1461; 1377; 1226 cm1; UV (dichloromethane) lmax 272 nm (3 ¼77,000); 1H NMR (DMSO-d6) d 8.57 (s, 2H, H-9), 7.79 (d, J¼9.1 Hz, 2H, H-8), 7.74 (d, J¼9.0 Hz, 2H, H-1), 7.61 (d, J¼1.6 Hz, 2H, H-4), 7.23e7.16 (m, 4H, H-20 , H-40 , H-50 and H-60 ), 7.12 (dd, J¼9.0, 1.6 Hz, 2H, H-2), 7.08 (dd, J¼9.1, 2.1 Hz, 2H, H-7), 6.80 (d, J¼2.1 Hz, 2H, H-5), 6.48 (br s, 4H, NH2), 5.00 (s, 4H, CH2), 3.64 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.2, 152.4, 149.7, 143.6, 137.9, 136.5, 130.0, 128.9, 128.7, 126.0, 125.4, 122.9, 121.4, 121.0, 120.9, 100.5, 53.2, 52.9; MS (FAB) (calcd for C38H32N6O4, 636.25) m/z 637 (MþþH, 6%). 4.4.3. [1,4-Phenylenebis(methylene)]bis[(6-amino-3-acridinyl)carbamic acid dimethyl ester] (1c). According to the general procedure, the crude product was purified by flash column (ؼ3 cm) chromatography by using ethyl acetateeacetoneetriethylamine (5/5/1, v/v/v) as eluent, affording 2.22 g (65%) of 1c, mp 165e7  C (hexaneedichloromethane). Found: C, 69.10; H, 5.75; N, 11.38. C38H32N6O4$0.5CH2Cl2$0.5C6H14 requires C, 69.01; H, 5.58; N, 11.64; IR (KBr): n 3419, 3349, 3215, 2954, 1693, 1642, 1610, 1464, 1376, 1291, 1227, 1215, 770 cm1; UV (methanol) lmax 269 nm (3 ¼67,000); 1H NMR (DMSO-d6) d 8.66 (s, 2H, H-9), 7.84 (d, J¼9.1 Hz, 2H, H-8), 7.82 (d, J¼9.1 Hz, 2H, H-1), 7.63 (d, J¼2.1 Hz, 2H, H-4), 7.27 (dd, 9.1, 2.1 Hz, 2H, H-2), 7.20 (s, 4H, H-20 , H-30 , H-50 and H-60 ), 7.09 (dd, J¼9.1, 2.1 Hz, 2H, H-7), 6.82 (d, J¼2.1 Hz, 2H, H-5), 6.52 (br s, 4H, NH2), 4.99 (s, 4H, CH2), 3.66 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.2, 152.6, 149.6, 143.7, 136.6, 130.1, 129.0, 127.3, 123.1, 121.4, 121.0, 120.9, 100.4, 53.2, 52.8; MS (APCI) (calcd for C38H32N6O4, 636.25) m/z 638 (Mþþ2H, 77%), 637 (MþþH, 100%).

4.4.4. [2,6-Napthalenediylbis(methylene)]bis[(6-amino-3-acridinyl) carbamic acid dimethyl ester] (1d). The crude product was purified by flash column (ؼ2 cm) chromatography on silica gel using ethyl acetateetriethylamineemethanol (20/3/2, v/v/v) affording pure product in 77% yield, mp 210  C (decomp.) (hexaneedichloromethane). Found: C, 70.41; H, 5.60; N, 11.02. C42H34N6O4$0.5CH2Cl2$0.5C6H14 requires C, 70.76; H, 5.48; N, 10.88; IR (KBr): n 3424, 3352, 3238, 1694, 1648, 1610, 1498, 1456, 1412, 1378, 1300 cm1; UV (methanol) lmax 268 nm (3 ¼14,000); 1H NMR (DMSO-d6) d 8.56 (s, 2H, H-9), 7.82 (d, 2H, J¼9.0 Hz, H-1), 7.79 (d, 2H, J¼8.5 Hz, H-40 and H-80 ), 7.76 (d, 2H, J¼9.0 Hz, H-8), 7.68e7.70 (m, 4H, H-10, H-50 and H-4), 7.40 (dd, 2H, J¼8.5, 1.6 Hz, H30 and H-70 ), 7.28 (dd, 2H, J¼9.0, 2.1 Hz, H-2), 7.03 (dd, 2H, J¼9.0, 2.1 Hz, H-7), 6.81 (d, 2H, J¼2.1 Hz, H-5), 6.13 (s, 4H, NH2), 5.16 (s, 4H, CH2), 3.68 (s, 6H, CO2CH3); 13C NMR (DMSO-d6) d 155.3, 151.0, 149.1, 144.6, 135.3, 132.0, 130.1, 129.1, 128.1, 125.5, 125.3, 123.1, 121.0, 120.9, 102.6, 53.3, 53.2; MS (FAB) (calcd for C42H34N6O4, 686.26) m/z 687 (MþþH, 100%), 686 (Mþ, 18).

4.5. Syntheses of compounds 2: General procedure To a solution of the corresponding compound 1 (0.9 mmol) in dry acetone (200 mL), anhydrous potassium carbonate (3 g, 21.6 mmol) and freshly distilled methyl chloroformate (0.8 mL, 10.78 mmol) were added. The reaction mixture was heated at reflux for 24 h and then filtered. The filtrate was concentrated up to dryness at reduced pressure and dissolved in dichloromethane. The solution obtained was washed with a 10% solution of sodium hydrogencarbonate and then three times with water. The organic phase was dried over anhydrous magnesium sulfate and the crude product purified as indicated in each case. 4.5.1. [1,2-Phenylenebis(methylene)]bis[[6-[(methoxycarbonyl) amino]-3-acridinyl]carbamic acid dimethyl ester] (2a). The crude product obtained was purified by flash column (ؼ2 cm) chromatography on silica gel using ethyl acetateetriethylamine (20/1, v/ v) affording pure product in 80% yield, mp 202e204  C (decomp.) (hexaneedichloromethane). Found: C, 61.71; H, 4.40; N, 9.83. C42H36N6O8$CH2Cl2 requires C, 61.65; H, 4.57; N, 10.03; IR (KBr): n 3309, 2954, 1709, 1617, 1546, 1493, 1456, 1380, 1235, 1064, 768 cm1; 1H NMR (DMSO-d6) d 10.14 (s, 2H, NHCO2), 8.76 (s, 2H, H9), 8.23 (d, J¼2.1 Hz, 2H, H-5), 7.99 (d, J¼9.1 Hz, 2H, H-8), 7.89 (d, J¼9.1 Hz, 2H, H-1), 7.82 (d, J¼2.1 Hz, 2H, H-4), 7.61 (dd, J¼9.1, 2.1 Hz, 1H, H-7), 7.42 (dd, J¼9.1, 2.1 Hz, H-2), 7.26e7.14 (m, 4H, H-30 and H60 ), 5.16 (s, 4H, CH2), 3.75 (s, 6H, CO2CH3), 3.68 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.4, 154.1, 149.7, 148.9, 143.2, 141.1, 135.3, 135.0, 129.2, 128.7, 127.5, 127.4, 124.8, 123.4, 123.0, 122.8, 120.4, 112.5, 53.2, 52.0, 50.1; MS (FAB) (calcd for C42H36N6O8, 752.26) m/z 753 (MþþH, 96%). 4.5.2. [1,3-Phenylenebis(methylene)]bis[[6-[(methoxycarbonyl) amino]-3-acridinyl]carbamic acid dimethyl ester] (2b). Purification of crude product was performed by flash column (ؼ2 cm) chromatography with ethyl acetateetriethylamine (20/1, v/v) as eluent, and silica gel as adsorbent, affording 2b in 80% yield, mp 169e171  C (decomp.) (hexaneedichloromethane). Found: C, 61.89; H, 4.81; N, 10.21. C42H36N6O8$CH2Cl2 requires C, 61.65; H, 4.57; N, 10.03; IR (KBr): n 3418, 2919, 1710, 1616, 1455, 1377, 1232, 1063, 769 cm1; 1H NMR (DMSO-d6) d 10.17 (s, 2H, NHCO2), 8.73 (s, 2H, H-9), 8.24 (d, J¼2.1 Hz, 2H, H-5), 7.99 (d, J¼9.1 Hz, 2H, H-8), 7.81 (d, J¼9.1 Hz, 2H, H-1), 7.79 (d, J¼2.1 Hz, 2H, H-4), 7.60 (dd, J¼9.1, 2.1 Hz, 2H, H-7), 7.31 (dd, J¼9.1, 2.1 Hz, 2H, H-2), 7.23e7.10 (m, 4H, H-20 , H-40 , H-50 and H-60 ), 5.04 (s, 4H, CH2), 3.74 (s, 6H, CO2CH3), 3.65 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.5, 154.2, 149.8, 149.0, 143.4, 141.2, 138.2, 135.4, 129.4, 128.8, 128.7, 126.3, 125.6,

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125.2, 123.5, 123.1, 122.9, 120.5, 112.5, 53.3, 53.0, 52.2; MS (FAB) (calcd for C42H36N6O8 752.26) m/z 753 (MþþH, 92%). 4.5.3. [1,4-Phenylenebis(methylene)]bis[[6-[(methoxycarbonyl) amino]-3-acridinyl]carbamic acid dimethyl ester] (2c). The filtrate was concentrated up to dryness at reduced pressure and treated with dichloromethane affording a yellow precipitate that was purified by flash column (ؼ2 cm) chromatography with ethyl acetateetriethylamine (5/1, v/v) as eluent, and silica gel as adsorbent, affording 2c in 65% yield, mp 213e214  C (decomp.) (hexaneedichloromethane). Found: C, 61.75; H, 4.33; N, 10.31. C42H36N6O8$CH2Cl2 requires C, 61.65; H, 4.57; N, 10.03; IR (KBr): n 3319, 2953, 1965, 1617, 1456, 1378, 1233, 1063, 771 cm1; 1H NMR (DMSO-d6) d 10.18 (s, 2H, NHCO2), 8.81 (s, 2H, H-9), 8.26 (d, J¼2.2 Hz, 2H, H-5), 8.03 (d, J¼9.2 Hz, 2H, H-8), 7.91 (d, J¼9.2 Hz, 2H, H-1), 7.82 (d, J¼2.2 Hz, 2H, H-4), 7.61 (dd, J¼9.2, 2.2 Hz, 1H, H-7), 7.37 (dd, J¼9.2, 2.2 Hz, 2H, H-2), 7.20 (s, 4H, H-20 , H-30 , H-50 and H60 ), 5.01 (s, 4H, CH2), 3.74 (s, 6H, CO2CH3), 3.66 (s, 6H, CO2CH3); 13C NMR (DMSO-d6): d 155.3, 154.0, 149.6, 148.8, 143.3, 141.1, 136.6, 135.4, 129.3, 128.7, 127.4, 125.2, 123.4, 123.3, 122.8, 120.4, 112.3, 53.3, 52.8, 52.2; MS (APCI) (calcd for C42H36N6O8 752.26) m/z 753 (MþþH). 4.6. Syntheses of cyclo-bisintercaland 3a 4.6.1. Procedure A (stepwise synthesis). To a stirred solution of 2a (300 mg, 0.4 mmol) in dry DMF (55 mL) at 40  C under argon, 14 mg (0.47 mmol) of 80% sodium hydride was added. Once the salt of 2a was formed, a solution of 1,2-bis(bromomethyl)benzene (111 mg, 0.42 mmol) in dry DMF (60 mL) was added dropwise. The mixture was then allowed to stand at room temperature and then a new portion (14 mg, 0.47 mmol) of 80% sodium hydride was added. The mixture was stirred at room temperature for 48 h and the solid, which separated out was filtered off, washed with water and then three times with ether, affording pure 3a in 73% yield, mp 149e151  C (hexaneedichloromethane). Found: C, 62.61; H, 4.93; N, 7.81. C50H42N6O8$2CH2Cl2$0.5C6H14 requires C, 61.86; H, 5.0; N, 7.87; IR (KBr): n 2955, 1707, 1618, 1456, 1383, 1259, 1227, 1047, 970 cm1; UV (methanol) lmax 274 nm (3 ¼37,000); 1H NMR (DMSO-d6) d 8.96 (s, 2H, H-9), 8.03 (d, J¼9.2 Hz, 4H, H-1 and H-8), 7.81 (dd, J¼9.2, 1.9 Hz, 4H, H-2 and H-7), 7.43 (br s, 4H, H-4 and H5), 7.12e7.21 (m, 8H, H-30 -H-60 ), 5.02 (br s, 8H, CH2), 3.73 (s, 12H, CO2CH3); 1H NMR (CD2Cl2): d 8.68 (s, 2H, H-9), 7.93 (d, J¼9.1 Hz, 4H, H-1 and H-8), 7.77 (dd, J¼9.1, 1.4 Hz, 4H, H-2 and H-7), 7.57 (br s, 4H, H-4 and H-5), 7.25 (s, 8H, H30 -H-60 ), 5.12 (s, 8H, CH2), 3.76 (s, 12H, CO2CH3); 13C NMR (CDCl3) d: 155.4, 149.2, 143.6, 135.1, 134.2, 128.1, 127.8, 127.1, 124.7, 122.0, 53.4, 51.6; MS (FAB) (calcd for C50H42N6O8, 854.31) m/z 855 (MþþH, 100%). 4.6.2. Procedure B (templated synthesis). To a solution of 3,6acridinediylbiscarbamic acid dimethyl ester (318 mg, 0.99 mmol) in dry DMF (30 mL) under argon, 60% sodium hydride (108 mg, 2.73 mmol) was added. The mixture was stirred at room temperature for 30 min and then 2,7-dimethoxynaphthalene (132 mg, 0.7 mmol) and 1,2-bis(bromomethyl)benzene (267 mg, 0.99 mmol) were added. The reaction mixture was stirred at 50  C for 24 h and then the solvent was removed in vacuo. The crude product was purified by flash column (ؼ2 cm) chromatography with hexaneeethyl acetate (1/3, v/v) as eluent, and silica gel as adsorbent, affording pure 3a in 42% yield. 4.7. Syntheses of cyclo-bisintercaland 3b 4.7.1. Procedure A. Following the procedure described in the previous section, 3b was obtained in 71% yield, mp 273e274  C (hexaneedichloromethane). Found: C, 61.71; H, 5.11; N, 8.02.

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C50H42N6O8$2CH2Cl2$0.5C6H14 requires C, 61.86; H, 5.0; N, 7.87; IR (KBr): n 2952, 2923, 1713, 1617, 1455, 1379, 1229, 1049, 978, 771 cm1; UV (dichloromethane) lmax 268 nm (3 ¼65,000). 1H NMR (DMSO-d6eCD2Cl2, 1/1) d 8.63 (s, 2H, H-9), 7.84 (d, J¼9.0 Hz, 4H, H-1 and H-8), 7.56 (d, J¼9.4 Hz, 4H, H-2 and H-7), 7.38 (br s, 4H, H-4 and H-5), 7.21 (t, J¼7.4 Hz, 2H, H-50 ), 7.10e7.08 (m, 6H, H20 , H-40 and H-60 ), 5.03 (br s, 8H, CH2), 3.65 (s, 12H, CO2CH3); 1H NMR (CD2Cl2) d 8.49 (s, 2H, H-9), 7.81 (d, J¼9.1 Hz, 4H, H-1 and H8), 7.61 (d, J¼9.1 Hz, 4H, H-2 and H-7), 7.55 (s, 4H, H-4 and H-5), 7.26 (t, J¼7.6 Hz, 2H, H-50 ), 7.16e7.14 (m, 6H, H-20 , H-40 and H-60 ), 5.05 (s, 8H, CH2), 3.70 (s, 12H, CO2CH3); 13C NMR (CDCl3) d 155.7, 149.0, 144.0, 137.6, 134.9, 129.1, 128.0, 125.6, 125.4, 124.6, 124.2, 121.8, 53.8, 53.3; MS (FAB) (calcd for C50H42N6O8, 854.31) m/z 855 (MþþH, 6%). 4.7.2. Procedure B. Following the procedure described in the previous section, 3b was obtained after purification by flash column (ؼ2 cm) chromatography using hexaneeethyl acetate (1/3, v/v) as eluent, and silica gel as adsorbent, affording pure product in 22% yield. 4.8. Syntheses of cyclo-bisintercaland 3c 4.8.1. Procedure A. Following the procedure described above, 3c was obtained in 79% yield, mp >290  C (hexaneedichloromethane). Found: C, 62.04; H, 4.86; N, 7.47. C50H42N6O8$2CH2Cl2$0.5C6H14 requires C, 61.86; H, 5.0; N, 7.87; IR (KBr) n 2953, 1709, 1617, 1452, 1378, 1259, 1204, 1050, 970, 776, 752 cm1; UV (methanol) lmax 268 nm (3 ¼43,000); 1H NMR (CDCl3) d 8.64 (s, 2H, H-9), 7.91 (d, 4H, J¼9.1 Hz, H-1 and H-8), 7.78 (d, 4H, J¼2.0 Hz, H-4 and H-5), 7.65 (br d, 4H, J¼9.1 Hz, H-2 and H-7), 7.21 (s, 8H, H-20 , H-30 , H-50 , and H-60 ), 5.06 (s, 8H, CH2), 3.77 (s, 12H, CO2CH3); 13C NMR (CDCl3) d 155.7, 149.3, 144.4, 136.2, 135.1, 128.2, 126.7, 125.5, 124.4, 121.8, 53.9, 51.6; MS (APCI) (calcd for C50H42N6O8 854.31) m/z 856 (Mþþ2H, 55%), 855 (MþþH, 91), 391 (100). 4.8.2. Procedure B. To a solution of 3,6-acridinediylbiscarbamic acid dimethyl ester (318 mg, 0.99 mmol) in dry DMF (30 mL) under argon, 60% sodium hydride (108 mg, 2.73 mmol) was added. The mixture was stirred at room temperature for 30 min and then 2,7-dimethoxynaphthalene (132 mg, 0.7 mmol) and 1,4bis(bromomethyl)benzene (267 mg, 0.99 mmol) were added. The reaction mixture was stirred at 50  C for 24 h and then the solvent was removed in vacuo. The crude product was purified by flash column (ؼ2 cm) chromatography with hexaneeethyl acetate (1/3, v/v) as eluent, and silica gel as adsorbent, affording pure product in 21% yield. 4.9. Synthesis of template 1,4-bis(7-methoxy-2naphthyloxymethyl)benzene (10) To a solution of 7-methoxy-2-naphtol (252 mg, 1.45 mmol) in dry DMF (10 mL) under argon, powdered potassium hydroxide (329 mg, 5.87 mmol) was added. The reaction mixture was stirred at room temperature for 15 min and then 1,4-bis(bromomethyl) benzene (194 mg, 0.74 mmol) was added. After stirring for 24 h, water (20 mL) and dichloromethane (60 mL) were added. The organic phase was decanted and washed three times with water and dried over anhydrous magnesium sulfate. The crude product obtained after evaporating the solvent was purified by flash column (ؼ2 cm) chromatography with hexaneeethyl acetate (1/1, v/v) as eluent, and silica gel as adsorbent, affording pure product in 59% yield, mp 201e202  C (ethyl acetate). Found: C, 80.02; H, 5.70. C30H26O4 requires C, 79.97; H, 5.82; IR (KBr) n 1628, 1609, 1378, 1181, 1028, 1003, 836, 800 cm1; 1H NMR (CDCl3) d 7.68 (d, 2H, J¼8.8 Hz, H-4), 7.66 (d, 2H, J¼8.8 Hz, H-5), 7.52 (s, 4H, H-20 , H-30 , H-

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50 and H-60 ), 7.14 (d, 2H, J¼2.5 Hz, H-1), 7.08 (dd, 2H, J¼8.8, 2.5 Hz, H-3), 7.04 (d, 2H, J¼2.5 Hz, H-8), 7.0 (dd, 2H, J¼8.8, 2.5 Hz, H-6), 5.20 (s, 4H, CH2), 3.91 (s, 6H, OCH3); 13C NMR (CDCl3) d 158.0, 157.1, 136.6, 135.6, 129.1, 129.0, 127.6, 124.3, 116.2, 116.1, 106.5, 105.2, 69.7, 55.3; MS (EI) (calcd for C30H26O4 450.18) m/z 450 (Mþ, 11%), 277 (35), 145 (100), 104 (63). 4.10. Synthesis of cyclo-bisintercaland 3d 4.10.1. Procedure B. To a solution of 3,6-acridinediylbiscarbamic acid dimethyl ester (204 mg, 0.64 mmol) in dry DMF (24 mL) under argon, 60% sodium hydride (104 mg, 2.58 mmol) was added. The mixture was stirred at room temperature for 30 min and then 1,4-bis(7methoxy-2-naphthyloxymethyl)benzene (198 mg, 0.44 mmol) and 2,6-bis(bromomethyl)naphthalene (202 mg, 0.64 mmol) were added. The reaction mixture was stirred at 40  C for 48 h and then the solvent was evaporated at reduced pressure. The solid thus obtained was purified by flash column (ؼ3 cm) chromatography with hexaneeethyl acetate (4/1, v/v) as eluent, and silica gel as adsorbent, affording pure product in 19% yield, mp 288e290  C (hexaneedichloromethane). Found: C, 68.41; H, 4.72; N, 7.83. C58H46N6O8$CH2Cl2 requires C, 68.14; H, 4.65; N, 8.08; IR (KBr) n 1711, 1615,1453,1374,1229 cm1; UV (methanol) lmax 268 nm (3 ¼25,000); 1 H NMR (CDCl3) d 8.62 (s, 2H, H-9), 7.88 (d, 4H, J¼9.2 Hz, H-1 and H-8), 7.82 (s, 4H, H-4 and H-5), 7.67e7.63 (m, 12H, naphthalene), 7.32 (d, 4H, J¼8.4 Hz, H-7 and H-2), 5.2 (s, 8H, CH2), 3.78 (s, 12H, CO2CH3); 13C NMR (CDCl3) d 155.9, 149.3, 144.6, 135.2, 134.8, 132.6, 128.3, 128.2, 125.7, 125.3, 124.9, 124.5, 121.9, 54.2, 53.3; MS (APCI) (calcd for C58H46N6O8 954.34) m/z 956 (Mþþ2H). 4.11. (6-Amino-3-acridinyl)-2-propynylcarbamic acid methyl ester (4) To a stirred solution of (6-amino-3-acridinyl)carbamic acid methyl ester (1 g, 3.74 mmol) in dry DMF (60 mL), 80% sodium hydride (138 mg, 4.6 mmol) was added. After 30 min with stirring at room temperature propargyl bromide (80% w/w in toluene) (668 mg, 4.48 mmol) was added. The mixture was stirred for additional 2 h and then the solvent was evaporated in vacuo. The resulting solid was dissolved in dichloromethane and washed three times with water. The organic phase was dried over anhydrous magnesium sulfate and the crude product obtained after evaporating the solvent was purified by column (ؼ3 cm) chromatography using neutral aluminum oxide as adsorbent, and acetone as eluent, affording 4 in 93% yield, mp 193e195  C (decomp.) (hexaneedichloromethane). Found: C, 71.22; H, 4.82; N, 13.44. C18H15N3O2 requires C, 70.81; H, 4.95; N, 13.76; IR (KBr): n 3474, 3370, 2956, 1690, 1611, 1460, 1377, 1234, 1139 cm1. 1H NMR (DMSO-d6) d 8.66 (s, 1H, H-9), 7.93 (d, J¼9.1 Hz, 1H, H-1), 7.81 (d, J¼9.1 Hz, 1H, H-8), 7.79 (br s, 1H, H-4), 7.33 (dd, J¼9.1, 2.2 Hz, 1H, H2), 7.07 (dd, J¼9.1, 2.2 Hz, 1H, H-7), 6.88 (d, J¼2.2 Hz, 1H, H-5), 6.15 (br s, 2H, NH2), 4.57 (d, J¼2.5 Hz, 2H, CH2), 3.68 (s, 3H, CO2CH3), 2.07 (t, J¼2.5 Hz, 1H, C^CH); 13C NMR (DMSO-d6): d 154.6, 151.4, 151.1, 148.7, 142.3, 134.9, 129.5, 128.8, 122.7, 122.2, 121.0, 120.9, 102.6, 80.1, 75.1, 53.3, 39.7; MS (EI) (calcd for C18H15N3O2, 305.12) m/z 305 (Mþ, 32%), 274 (100),246 (81), 229 (15), 219 (26), 208 (20), 191 (24), 181 (24). 4.12. [6-[(Methoxycarbonyl)amino]-3-acridinyl]-2propynylcarbamic acid methyl ester (5) To a solution of 4 (348 mg, 1.14 mmol) in dry acetone (20 mL) under argon, anhydrous potassium carbonate (1.89 g, 13.7 mmol) and freshly distilled methyl chloroformate (0.5 mL, 6.84 mmol) were added. The mixture was heated at reflux for 5 h and then the solid, which separated out was filtered off. The filtrate was

concentrated up to dryness and the residue dissolved in dichloromethane. The organic phase was washed with 5% sodium hydrogencarbonate and then three times with water. The organic extracts were dried over anhydrous magnesium sulfate and the crude product obtained after evaporation of the solvent was purified by flash column (ؼ2 cm) chromatography using silica gel as adsorbent and hexaneeethyl acetate (1/2, v/v) as eluent, affording 5 in 74% yield, mp 183e185  C (hexaneeethyl acetate). Found: C, 65.91; H, 4.76; N, 11.81. C20H17N3O4 requires C, 66.11; H, 4.72; N, 11.56; IR (KBr): n 3413, 3251, 2953, 2362, 1717, 1616, 1455, 1376, 1230, 1052 cm1; 1H NMR (DMSO-d6) d 10.19 (s, 1H, NH), 8.95 (s, 1H, H-9), 8.30 (d, J¼2.1 Hz, 1H, H-5), 8.09 (d, J¼9.1 Hz, 1H, H-8), 8.07 (d, J¼9.1 Hz, 1H, H-1), 8.0 (d, J¼2.1 Hz, 1H, H-4), 7.63 (dd, J¼9.1, 2.1 Hz, 1H, H-7), 7.53 (dd, J¼9.1, 2.1 Hz, 1H, H-2), 4.62 (d, J¼2.5 Hz, 2H, CH2), 3.75 (s, 3H, CO2CH3), 3.70 (s, 3H, CO2CH3), 3.29 (t, J¼2.5 Hz, 1H, C^CH); 13C NMR (DMSO-d6): d 154.5, 153.9, 149.7, 148.8, 142.9, 141.1, 135.5, 129.3, 128.8, 124.8, 123.6, 122.8, 120.5, 112.4, 80.0, 75.3, 53.4, 52.2, 39.8, MS (EI) (calcd for C20H17N3O4, 363.12) m/z 363 (Mþ, 9%), 300 (28), 273 (21), 272 (31). 4.13. 2,4-Hexadiyne-1,6-diylbis[[6-[(methoxycarbonyl) amino]-3-acridinyl]-2-propynylcarbamic acid methyl ester] (6) To a solution of 5 (1.05 g, 2.88 mmol) in a mixture of dry methanol (100 mL) and dry pyridine (9 mL), 3.45 g (17.3 mmol) of copper(II) acetate monohydrate was added. The reaction mixture was stirred at 45  C for 96 h and then the yellow precipitate was filtered and washed with methanol yielding 780 mg of 6. The mother liquors were concentrated at reduced pressure and the resulting residue was purified by flash column (ؼ3 cm) chromatography over silica gel as adsorbent and ethyl acetateetriethylamine (20/1, v/v) as eluent, affording an additional amount of product, yield: 88%, mp 165e168  C (hexaneedichloromethane). Found: C, 61.02; H, 4.12; N, 10.15. C40H32N6O8$CH2Cl2 requires C, 60.82; H, 4.23; N, 10.38; IR (KBr) n 3322, 2955, 1696, 1618, 1574, 1550, 1456, 1383, 1298, 1234, 1051, 769 cm1; UV (methanol) lmax 270 nm (3 ¼130, 000); 1H NMR (DMSO-d6) d 10.18 (s, 2H, NH), 8.92 (s, 2H, H-9), 8.30 (d, J¼2.1 Hz, 2H, H-5), 8.07 (d, J¼9.1 Hz, 2H, H-8), 8.06 (d, J¼9.1 Hz, 2H, H-1), 7.95 (d, J¼2.1 Hz, 2H, H-4), 7.64 (dd, J¼9.1, 2.1 Hz, 2H, H-7), 7.49 (dd, J¼9.1, 2.1 Hz, 2H, H-2), 4.76 (s, 4H, CH2), 3.75 (s, 6H, CO2CH3), 3.68 (s, 6H, CO2CH3), 13C NMR (DMSOd6): d 154.4, 153.9, 149.7, 148.8, 142.6, 141.1, 135.5, 129.3, 129.0, 124.6, 123.1, 122.9, 120.5, 112.4, 75.8, 67.6, 53.5, 52.2, 40.3; MS (FAB) (calcd for C40H32N6O8, 724.23) m/z 725 (MþþH, 4%). 4.14. 2,4-Hexadiyne-1,6-diylbis[[6-[(methoxycarbonyl)-2propynylamino]-3-acridinyl]carbamic acid methyl ester] (7) To a solution of 6 (300 mg, 0.42 mmol) in dry DMF (90 mL), 35 mg (0.84 mmol) of powdered sodium hydroxide was added producing a change of color from yellow to red. Then, propargyl bromide (80% w/w in toluene) (153 mg, 0.84 mmol) was added. The reaction mixture was stirred at room temperature for 90 min and then the solvent was evaporated at reduced pressure. The resulting residue was dissolved in dichloromethane, washed three times with water, and dried over anhydrous magnesium sulfate. The residue obtained after solvent evaporation was purified by flash column (ؼ2 cm) chromatography on silica gel and hexaneeethyl acetate (1/2, v/v) as eluent, affording 225 mg (67%) of 7, mp 125  C (decomp.) (hexaneedichloromethane). Found: C, 68.32; H, 5.05; N, 9.54. C46H36N6O8$0.5 CH2Cl2$C6H14 requires C, 67.63; H, 5.57; N, 9.10; IR (KBr) n 2955, 2924, 2853, 1717, 1639, 1615, 1452, 1376, 1234, 1051, 769 cm1; 1H NMR (DMSO-d6): d 9.07 (s, 2H, H-9), 8.15 (d, J¼9.1 Hz, 2H, H-8), 8.14 (d, J¼9.1 Hz, 2H, H-1), 8.08 (d, J¼2.1 Hz, 2H, H-5), 8.03 (d, J¼2.1 Hz, 2H, H-4), 7.63 (dd, J¼9.1, 2.1 Hz, 2H, H-7),

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7.59 (dd, J¼9.1, 2.1 Hz, 2H, H-2), 4.79 (s, 4H, CH2eC^Ce), 4.63 (d, J¼2.3 Hz, 4H, CH2eC^CH), 3.70 (s, 6H, CO2CH3), 3.69 (s, 6H, CO2CH3), 3.33 (t, J¼2.3 Hz, 2H, C^CH); 13C NMR (DMSO-d6): d 154.7, 154.6, 149.0, 148.9, 143.5, 143.2, 136.0, 129.2, 129.0, 125.9, 125.7, 124.5, 123.0, 122.8, 80.1, 75.9, 75.4, 67.6, 53.6, 53.5; MS (ESI) (calcd for C46H36N6O8 800.26) m/z 801.25 (MþþH). 4.15. Cyclization of 7: synthesis of cyclo-trisintercaland 8 and cyclo-bisintercaland 9 A solution of 7 (747 mg, 0.93 mmol) in dichloromethane (165 mL) was added simultaneously with a solution of cupric acetate monohydrate (1.02 g, 5.1 mmol) in a mixture of dichloromethaneepyridine (102 mL, 33/1, v/v) over a period of 4 h, to a boiling solution of vigorously stirred cupric acetate monohydrate (102 mg, 0.51 mmol) in 10 mL of dichloromethaneepyridine (37/1, v/v). The mixture was heated at reflux for 120 h with one further portion of cupric acetate monohydrate (499 mg, 2.5 mmol) being added during this period. Then concentrated aqueous ammonia (50 mL) was added to the reaction mixture and the organic phase was decanted and washed four times with water. The organic extracts were dried over anhydrous magnesium sulfate, and the residue obtained after solvent evaporation was purified by flash column (ؼ3 cm) chromatography on silica gel with ethyl acetateetriethylamine (20/1, v/v) as eluent, affording 8 in 5% yield, mp >290  C (hexaneedichloromethane). Found: C, 65.10; H, 5.37; N, 8.16. C69H51N9O12$2CH2Cl2$2C6H14 requires C, 64.72; H, 5.43; N, 8.18; IR (KBr) n 2955, 2923, 2852, 1716, 1617, 1448, 1372, 1223, 1047, 771, 687 cm1; UV (methanol) lmax 268 nm (3 ¼100,000); 1H NMR (CDCl3) d 8.65 (s, 3H, H-9), 8.07 (d, J¼1.5 Hz, 6H, H-4 and H-5), 7.94 (d, J¼9.1 Hz, 6H, H-1 and H-8), 7.57 (dd, J¼9.1, 2.1 Hz, 6H, H-2 and H-7), 4.64 (s, 12H, CH2), 3.79 (s, 18H, CO2CH3); 13C NMR (CDCl3) d 154.7, 148.9, 143.3, 135.5, 128.6, 125.5, 124.7, 123.0, 74.2, 68.9, 53.7, 41.0; MS (FAB) (calcd for C69H51N9O12, 1197.36) m/z 1198 (MþþH, 41%). Further elution with the same eluent afforded 9 in 22% yield, mp >290  C (hexaneedichloromethane). Found: C, 53.32; H, 4.14; N, 7.12. C46H34N6O8$4CH2Cl2$0.5C6H14 requires C, 53.87; H, 4.18; N, 7.11; IR (KBr) n 2954, 2852, 1714, 1616, 1448, 1373, 1226, 1047, 769, 689 cm1; UV (methanol) lmax 268 nm (3 ¼35,000); 1H NMR (CDCl3) d 8.62 (s, 2H, H-9), 8.05 (d, J¼2.1 Hz, 4H, H-4 and H-5), 7.89 (d, J¼9.2 Hz, 4H, H-1 and H-8), 7.53 (dd, J¼9.2, 2.1 Hz, 4H, H-2 and H-7), 4.61 (s, 8H, CH2), 3.78 (s, 12H, CO2CH3); 13C NMR (CDCl3) d 154.6, 148.8, 143.2, 135.5, 128.5, 125.4, 124.7, 122.9, 74.1, 68.8, 53.7, 40.9; MS (FAB) (calcd for C46H34N6O8, 798.24) m/z 799 (MþþH, 2%). 4.16. 3,6-Acridinediylbis(2-propynylcarbamic acid dimethyl ester) (11) To a solution of 3,6-acridinediylbiscarbamic acid dimethyl ester in dry DMF (25 mL), 80% sodium hydride (69 mg, 2,3 mmol) was added. Once the salt of dicarbamate was formed, 343 mg (2.3 mmol) of propargyl bromide (80% w/w in toluene) was added. The reaction mixture was stirred at room temperature for 5 h and then the solvent was evaporated until dryness. The resulting residue was dissolved in dichloromethane, washed with water, and dried over anhydrous magnesium sulfate. The crude product obtained after evaporating the solvent was purified by flash column (ؼ3 cm) chromatography on silica gel using hexaneeethyl acetate (1/1, v/v) as eluent, affording 11 in 93% yield, mp 159e160  C (ethanol). Found: C, 66.94; H, 4.96; N, 9.76. C23H19N3O4$CH3CH2OH requires C, 67.10; H, 5.63; N, 9.39; IR (KBr) n 3253, 2953, 1718, 1616, 1457, 1345, 1231 cm1; 1H NMR (CDCl3) d 8.74 (s, 1H, H-9), 8.17 (d, J¼2.1 Hz, 2H, H-4 and H-5), 7.97 (d, J¼9.1 Hz, 2H, H-1 and H-8), 7.59 (dd, J¼9.1, 2.1 Hz, 2H, H-2 and H-7), 4.61 (d, J¼2.5 Hz, 4H, CH2), 3.82 (s, 6H, CO2CH3), 2.33 (t, J¼2.5 Hz, 2H, C^CH); 13C NMR (CDCl3)

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40.2; MS (EI) (calcd for C23H19N3O4, 401.14) m/z 401 (Mþ, 100%); 370 (75); 343 (62); 299 (24); 272 (64); 245 (25); 229 (14); 218 (14). 4.17. Cyclization of 11: synthesis of cyclo-trisintercaland 8 and cyclo-bisintercaland 9 4.17.1. Procedure A. A solution of 11 (1.01 g, 2.52 mmol) in a mixture of dichloromethaneemethanol (444 mL, 1/1, v/v) was added simultaneously with a solution of cupric acetate monohydrate (5.79 g, 29 mmol) in a mixture of dichloromethaneepyridine (305 mL, 21/1, v/v) over a period of 4 h, to a boiling solution of copper(II) acetate monohydrate (200 mg, 1 mmol) in dichloromethaneepyridine (10 mL, 21/1, v/v). The reaction mixture was heated at reflux for 120 h with one further portion of cupric acetate monohydrate (998 mg, 5 mmol) being added during this period. Then, concentrated aqueous ammonia (100 mL) was added to the reaction mixture. The organic phase was decanted and washed four times with water. The organic extract was dried over anhydrous magnesium sulfate and the residue obtained after evaporating the solvent purified by flash column (ؼ3 cm) chromatography on silica gel with ethyl acetateetriethylamine (20/1, v/v) as eluent, affording 8 in 17% yield. With the same eluent 9 was obtained in 5% yield. 4.17.2. Procedure B. To a mixture of cupric acetate monohydrate (638 mg, 3.2 mmol) and 2,7-dimethoxynaphthalene (42 mg, 0.22 mmol) in 10 mL of dichloromethaneemethanol (1/1, v/v), 1.6 mL of dry pyridine was added. The reaction mixture was stirred under argon and then a solution of 11 (106 mg, 0.26 mmol) in 10 mL of dichloromethaneemethanol (1/1, v/v) was added. The mixture was heated at reflux for 46 h and then concentrated aqueous ammonia (4 mL) was added to the reaction mixture and the organic phase separated off. The organic phase was washed four times with water and dried over anhydrous magnesium sulfate. The residue obtained after evaporating the solvent was purified by flash column (ؼ2 cm) chromatography on silica gel with ethyl acetateetriethylamine (20/1, v/v) as eluent, affording 8 (24% yield). With the same eluent 9 was obtained in 16% yield. 4.18. In vitro cytotoxicity evaluation Assays on cultured cells P-388, HT-29, A-549, and MEL-28 were carried out to study the antitumor activity of the synthesized compounds. All assays were performed in the PharmaMar Cambridge Laboratory (USA). 4.18.1. Cells that grow in suspension (P-388). Cells were harvested in 24-well cell culture plates (diameter 16 mm) at a concentration of 1104 cells per well, in 1 mL MEM 5FCS media aliquots. Tested compounds were added at different concentrations. Several wells contained harvested cells in the absence of compound, as controls for cellular growth. After 3 or 4 days of incubation at 37  C in a 10% CO2 atmosphere, plates were observed under the microscope and checked for compound activity by comparison of cellular growth in both sample containing wells and in control wells. To quantitate activity, cells from each well were counted using an electronic cell counter. Cell inhibition growth percentage was calculated, determining IC50 values (sample concentration needed to inhibit cell growth 50%) for each compound. 4.18.2. Cells that grow as monolayers (A-549, HT-29, and MEL28). Cells were harvested in 24-well cell culture plates (diameter 16 mm) at a concentration of 1104 cells per well, in 1 mL MEM 5FCS media aliquots. Tested compounds were added at different concentrations, and several wells contained harvested cells in the

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absence of compound, as control for cellular growth. After 3 or 4 days of incubation at 37  C in a 10% CO2 atmosphere, cells were stained with 0.1% crystal violet, and cellular growth was then compared with that observed in control wells, to determine the compound inhibitory activity by interference with cellular growth. To quantitate activity, after 3 or 4 days of incubation at 37  C and 10% CO2, cells were trypsinized, resuspended in media, and counted with an electronic cell counter. As with P-388 cells, cell inhibition growth percentage was calculated, determining IC50 values for each tested compound.

6. 7. 8.

9. 10.

Acknowledgements Financial support from Comunidad de Madrid-Universidad de
through project CAM-UAH CCG10-UAH/PPQ-5961 (M.-J. F.) Alcala and from MICINN through project CTQ2011-26822 (A.L.) is gratefully acknowledged. We also thank the company PharmaMar for performing the biological activity assays.

11.

12. 13. 14. 15.

References and notes
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din, A.; Mergny, J.-L.; Teulade-Fi2. Granzhan, A.; Monchaud, D.; Saettel, N.; Gue chou, M.-P. J. Nucleic Acids 2010, ID 460561. 3. (a) Claude, S.; Lehn, J.-M.; Vigneron, J.-P. Tetrahedron Lett. 1989, 30, 941e944; (b) Zimmerman, S. C.; Lamberson, C. R.; Cory, M.; Fairley, T. A. J. Am. Chem. Soc.
rez de Vega, M.-J.; Vigne1989, 111, 6805e6809; (d) Claude, S.; Lehn, J.-M.; Pe ron, J.-P. New J. Chem. 1992, 16, 21e28; (e) Teulade-Fichou, M.-P.; Vigneron, J.-P.; Lehn, J.-M. Supramol. Chem. 1995, 5, 139e147; (f) Slama-Schwok, A.; TeuladeFichou, M.-P.; Vigneron, J.-P.; Taillandier, E.; Lehn, J.-M. J. Am. Chem. Soc. 1995, 117, 6822e6830; (g) Teulade-Fichou, M.-P.; Vigneron, J.-P.; Lehn, J.-M. J. Chem. Soc., Perkin Trans. 2 1996, 2169e2175; (h) Cudic, P.; Vigneron, J.-P.; Lehn, J.-M.;
, T. Eur. J. Org. Chem. 1999, 2479e2484; (i) Teulade-Fichou, Cesario, M.; Prange M.-P.; Vigneron, J.-P.; Lehn, J.-M.; Berthet, N.; Michon, J.; Garcia, J.; Jourdan, M.; Lhomme, J. Nucleosides Nucleotides 1999, 18, 1351e1353; (j) Berthet, N.; Michon, J.; Lhomme, J.; Teulade-Fichou, M.-P.; Vigneron, J.-P.; Lehn, J.-M. Chem.dEur. J. 1999, 5, 3625e3630; (k) Issmaili, S.; Boyer, G.; Galy, J.-P. Synlett 1999, 641e643. 4. (a) Zinic, M.; Cudic, P.; Skaric, V.; Vigneron, J.-P.; Lehn, J.-M. Tetrahedron Lett. 1992, 33, 7417e7420; (b) Cudic, P.; Zinic, M.; Tomisic, V.; Simeon, V.; Vigneron, J.-P.; Lehn, J.-M. J. Chem. Soc., Chem. Commun. 1995, 1073e1075; (c) Piantanida, I.; Palm, B. S.; Cudic, P.; Zinic, M.; Schneider, H.-J. Tetrahedron Lett. 2001, 42, 6779e6783. 5. (a) Baudoin, O.; Teulade-Fichou, M.-P.; Vigneron, J.-P.; Lehn, J.-M. J. Org. Chem. 1997, 62, 5458e5470; (b) Baudoin, O.; Gonnet, F.; Teulade-Fichou, M.-

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