XU Xue-Mei LUO Zai-Gang HAN Xin-Xin LIU Qian-Nan LI Rui

(College of Chemical Engineering, Anhui University of Science and Technology, Huainan 232001, China)

ABSTRACT The target compounds 3 (C14H15NO4) and 4 (C20H17NO8) were synthesized and structurally determined by single-crystal X-ray diffraction. The crystal of 3 is in the monoclinic system, space group P21/c with a = 7.3017(3), b = 7.8737(5), c = 22.4227(11) ?, β = 94.837(5)°, C14H15NO4, Mr = 261.27, Dc = 1.351 g/cm3, V = 1284.51(11) ?3, Z = 4, F(000) = 552, μ(MoKa) = 0.828 mm–1, T = 289.12(10) K, 2236 independent reflections with 1827 observed ones (I > 2σ(I)), R = 0.0515 and wR = 0.1394 with GOF = 1.044 (R = 0.0605 and wR = 0.1548 for all data). The crystal of compound 4 is in the monoclinic system, space group P21/c with a = 7.1269(3), b = 12.6518(6), c = 20.7540(8) ?, β = 96.941(4)°, C20H17NO8, Mr = 399.35, Dc = 1.428 g/cm3, V = 1857.61(14) ?3, Z = 4, F(000) = 832.0, μ(MoKa) = 0.950 mm–1, T = 288.81(10) K, 3216 independent reflections with 2253 observed ones (I > 2σ(I)), R = 0.0612 and wR = 0.1548 with GOF = 1.055 (R = 0.0824 and wR = 0.1790 for all data). The skeleton of 1,4-dihydroquinoline 3 is noncoplanar, while pyrrolo[1,2-a]-quinoline 4 owns a coplanar frame structure. One-dimensional interaction model of compound 4 was formed by the one kind of π-π interaction between the two adjacent molecules at upper and lower levels. And the inhibition to the strand transfer process of HIV-1 integrase of the title compounds was also evaluated.

Keywords: 1,4-dihydroquinoline, pyrrolo[1,2-a]quinoline, synthesis, crystal structure, HIV-1 integrase;

1 INTRODUCTION

1,4-Dihydroquinoline derivatives, one kind of nitrogen- containing heterocycles, are an important class of biologically active compounds, which are widely used in organic synthesis and pharmaceutical chemistry[1-3]. In addition, among various N-heterocycles, pyrrolo[1,2-a]-quinoline derivatives also have received much attention for their unique properties in functional materials[4]and biological activities[5]as well as in natural products[6]. Therefore, many kinds of well-established methods for the synthesis of these nitrogen-containing heterocycles are available in the literature in the last several decades[7-12]. Although these reported methods have made a significant contribution to the preparation of these N- heterocycles, the development of a simple, efficient, and environmentally friendly protocol for the synthesis of 1,4-hydroquinolines and pyrrolo[1,2-a]quinolines is still needed.

Over the last three decades, numerous small-molecule HIV-1 integrase inhibitors have been described[13,14]. Recently, the formation of N-containing heterocycles has also been extensively investigated using N-methyl anilines as cheap, safe, and widely accessible starting materials[15-17]. In the present study, in order to gain new molecular entities with potential biological activities, we used 1,4-dihydroquinoline and pyrrolo[1,2-a]quinoline skeleton as the platforms to design two new class of integrase inhibitors. Herein, we report the synthesis of dimethyl 1-methyl-1,4-dihydroquinoline-3,4- dicarboxylate (3) and tetramethyl pyrrolo[1,2-a]quinoline- 2,3,4,5-tetracarboxylate (4) using N,N-dimethyl aniline and dimethyl but-2-ynedioate as the starting materials in the presence of dicumyl peroxide (DCP) as the oxidant in one-pot, as shown in Scheme 1. The structures of them were confirmed via1H NMR,13C NMR and HRMS. Meanwhile, the crystal structures of compounds (3) and (4) were determined by X-ray single-crystal diffraction analysis. And the inhibition to the strand transfer process of HIV-1 integrase of the title compounds was also evaluated.

Scheme 1. Syntheses of 1,4-dihydroquinoline (3) and pyrrolo[1,2-a]quinoline (4)

2 EXPERIMENTAL

2. 1 Instruments and reagents

The melting point was measured on a SGW X-4 mono- cular microscope melting point apparatus with thermometer unadjusted.1H NMR and13C NMR spectra were acquired on a 500 MHz Bruker Avance spectrometer with CDCl3as solvent. H RMS was obtained by ESI on a TOF mass analyzer. X-ray diffraction was performed using a Bruker Smart Apex CCD diffractometer.

Unless otherwise noted, all materials were obtained from commercial suppliers and purified by standard procedures. Column chromatography was performed with silica gel (200～300 mesh, Qingdao Haiyang Chemical Co., Ltd, China).

2. 2 Synthesis of the title compounds 3 and 4

General procedure for the synthesis of 1,4-dihydroquino- line and pyrrolo[1,2-a]quinoline is described as follows: A Schlenk tube equipped with a magnetic stirring bar was charged with N,N-dimethyl aniline 1 (0.2 mmol), dimethyl but-2-ynedioate 2 (0.4 mmol), DCP (0.4 mmol) and PhCl (2 mL). Then the tube was sealed and the resulting mixture was heated to 120 °C for 24 h. After cooling, the solvent was diluted with water (5 mL) and extracted with dichlorome- thane (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4and concentrated by a rotary evaporator, and the residue was purified by column chroma- tography on silica gel (V(petroleumether)/V(ethylacetate)= 10:1～6:1, 0.6 > Rf > 0.3) to provide the desired products 3 and 4. The details of synthetic work were also reported by us, recently[18].

Compound 3: dimethyl 1-methyl-1,4-dihydroquinoline- 3,4-dicarboxylate, white solid, m.p.: 124～125 °C, 30 mg, 57% yield.1H NMR (500 MHz, CDCl3, ppm) δ: 7.48 (s, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.23 (d, J = 7.5 Hz, 1H), 7.03 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.0 Hz, 1H), 4.89 (s, 1H), 3.73 (s, 3H), 3.68 (s, 3H), 3.32 (s, 3H);13C NMR (125 MHz, CDCl3, ppm) δ: 173.56, 167.78, 143.58, 138.09, 130.06, 128.69, 123.74, 120.66, 113.46, 96.76, 52.62, 51.41, 43.43. 39.41. HRMS (ESI) m/z: calcd. for C14H16NO4[M+H]+: 262.1079, found 262.1076.

Compound 4: tetramethyl pyrrolo[1,2-a]quinoline-2,3,4,5- tetracarboxylate, yellow solid, m.p.: 179～181 °C, 18 mg, 23% yield.1H NMR (500MHz, CDCl3, ppm) δ: 8.43 (s, 1H), 7.97 (t, J = 7.5 Hz, 2H), 7.69～7.72 (m, 1H ), 7.50 (t, J = 7.5 Hz, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H);13C NMR (125 MHz, CDCl3, ppm) δ: 166.32, 165.21, 165.13, 163.43, 132.21, 131.02, 127.86, 126.55, 126.20, 124.95, 123.21, 119.63, 119.31, 117.38, 114.85, 113.35, 52.97, 52.84, 52.35, 52.02; HRMS (ESI) m/z: calcd. for C20H18NO8[M+H]+: 400.1032, found 400.1026.

2. 3 Structure determination

X-ray crystallographic data of the colorless block crystal 3 and light-yellow crystal of 4 were collected and mounted on a glass fiber for measurement at room temperature, respec- tively. X-ray crystallographic data were collected at 289.12(10) K for 3 and 288.81(10) K for 4, respectively. All measurements of the title compounds were made on a Bruker Smart Apex CCD diffractometer equipped with graphite-monochromated MoKα radiation (λ = 1.54184 ? for 3 and 4). The structures of compounds 3 and 4 were solved by direct methods, and then the non-hydrogen atoms were refined anisotropically with SHELXS-97 by applying a full-matrix least-squares procedure based on F2values after they were located from the trial structure[19]. Moreover, the hydrogen atom positions were fixed geometrically at calculated distances. At the same time, they were allowed to ride on the parent atoms. For compound 3: A total of 3939 reflections were selected in the range of 3.96≤θ≤65.99o (h: -8～8, k: -6～9, l: -26～20) by using a ψ-ω scan mode, of which 2236 were independent with R = 0.0515 and 1827 were observed with I > 2σ(I). The final refinement showed R = 0.0515, wR = 0.1394 with GOF = 1.044 (w = 1/[σ2(Fo2) + (0.0917P)2+ 0.1161P], where P = (Fo2+ 2Fc2)/3), (Δρ)max= 0.223, (Δρ)min= -0.281 e/?3and (?/σ)max= 0.001. For compound 4: Out of the 5936 reflections collected in the range of 4.10≤θ≤66.01o (h: -8～5, k: -14～14, l: -23～24) by using a ψ-ω scan mode, 3216 were independent with R = 0.0612 and 2253 were observed with I > 2σ(I). The final refinement showed R = 0.0612, wR = 0.1548 with GOF = 1.055 (w = 1/[σ2(Fo2) + (0.0930P)2], where P = (Fo2+ 2Fc2)/3), (Δρ)max= 0.271, (Δρ)min= -0.344 e/?3and (?/σ)max= 0.000. Data were collected by Rapid Auto program[20,21]. The hydrogen atoms bound to carbon atoms were deter- mined by accurate theoretical calculation. The representative bond lengths and bond angles for compounds 3 and 4 are illustrated in Tables 1 and 2, respectively.

Table 1. Selected Bond Lengths (?) and Bond Angles (°) of Compound 3

Table 2. Selected Bond Lengths (?) and Bond Angles (°) of Compound 4

2. 4 HIV-1 integrase inhibitory assay

The inhibition effects of compounds 3 and 4 were measured by HIV-1 integrase strand transfer activity assay, which was carried out as described previously with some minor modifications[22,23]. Compounds diluted in DMSO were pre-incubated with 800 ng of integrase at 37.8 °C in the reaction buffer in the absence of Mn2+for 10 min. Subsequently, 1.5 pmol of donor DNA and 9 pmol of target DNA were added and the reaction was initiated by the addition of 10 mmol/L Mn2+into the final reaction volume. The reactions were carried out at 37.8 °C for 1 h and subsequent detection procedure was applied to detect the assay signals. Integrase inhibitor, baicalein, was used as the control compound (positive control), whereas no compound but only DMSO in the reaction mixture was set as the drug-free control (negative control). The inhibition effects of compounds 3 and 4 were calculated based on the positive and negative controls.

3 RESULTS AND DISCUSSION

3. 1 Spectroscopy analysis

The structures for the target compounds 3 and 4 were confirmed by1H NMR,13C NMR and HRMS. For example, the1H NMR spectrum of 1,4-dihydroquinoline 3 shows the signals of 7.48 and 4.89 ppm as singlet due to the =CH and CH in the N-containing six-membered ring, and the signals of CH3at 3.73, 3.68 and 3.32 as singlet. The phenyl signals appeared at 7.39～6.86 ppm as three doublets and one triplet. The1H NMR spectrum of pyrrolo[1,2-a]quinoline 4 also shows the signals of 8.43 ppm as singlet due to the =CH in the N-containing five-membered ring, and the signals of CH3at 3.98, 3.93, 3.92 and 3.91 as singlet. The phenyl signals appeared at 7.97～7.50 ppm as two triplets and one multiplet. The13C NMR signals of 1,4-dihydroquinoline 3 and pyrrolo[1,2-a]quinoline 4 possess the normal value. And the HRMS spectra of 3 and 4 show molecular ion peaks and [M+H]+at m/z 262.1076 and 400.1026, respectively. Furthermore, the solid of compounds 3 and 4 was recrystalli- zed from dichloromethane/ethanol to give colorless single crystal of 3 and light-yellow crystal of 4 suitable for single-crystal X-ray diffraction. The crystals of the title compounds are stable in air at room temperature.

3. 2 X-ray crystallographic analysis

X-ray diffraction of the molecular structure of the bicyclic 1,4-dihydroquinoline 3 and polycyclic pyrrolo[1,2-a]quino- line 4 is demonstrated in Fig. 1. The bicyclic skeleton of 1,4-dihydroquinoline 3 is not coplanar (Fig. 1a). The torsion angles of C1–C6–C7–C8and C9–N1–C1–C6are 25.7(2)° and –12.4(2)°, respectively. The two ester groups connected with C7and C8are approximately perpendicular to each other, for the torsion angle of N1–C9–C8–C13is –179.28(16)°, while the torsion angle of C9–C8–C7–C11is 96.1(2)°. Obviously, the polycyclic skeleton of pyrrolo[1,2-a]quinoline 4 is coplanar (Fig. 1b), and the bond lengths and bond angles are within normal ranges. And the four bulky substituents, methoxy carbonyl groups connected with C7, C9, C12and C13of the molecular frame structure, are in a staggered reverse alignment manner. It means that the molecule has thermodynamically stable conformation. However, the intermolecular and intramolecular hydrogen bonds are not found in the two molecules.

Fig. 1. (a) Coordination environment of compound 3, (b) Coordination environment of compound 4. The selected atoms are omitted for clarity

Moreover, the crystal packing of 4 illustrated in Fig. 2 reveals that the overall packing has reverse staggered parallel arrangement. Besides, one kind of π-π interaction between the two adjacent molecules at upper and lower levels can be observed, which formed a one-dimensional interaction model. As can be detected by the packing diagram with stratified arrangement, the distance between the upper and lower levels is 3.5177 ?.

Fig. 2. Packing diagram of compound 4

3. 3 Inhibition of HIV integrase activity

Compounds 3, 4 and positive control compound baicalein were tested against purified integrase and the data are summarized in Table 3.

As shown in Table 3, the value of inhibition ratio of compound 3 is 48.52% at the concentration of 50 μM, which means it has a moderate inhibitory activity towards HIV integrase; while compound 4 owns a lower inhibitory activity than compound 3 with the inhibition ratio of 38.98%, which maybe correlates with bulky steric hindrance of the four adjacent ester groups blocking the interaction of the molecule and HIV integrase. Further work based on these structures is in progress.

Table 3. Inhibition of HIV-1 Integrase Strand Transfer Catalytic Activitiesa

4 CONCLUSION

In summary, a facile method for the synthesis of 1,4-dihydroquinoline 3 and pyrrolo[1,2-a]quinoline 4 using N,N-dimethyl aniline and dimethyl but-2-ynedioate as the starting materials in one-pot process was reported, and their structures were confirmed by1H NMR,13C NMR and HRMS. X-ray diffraction of the molecular structure disclosed that the skeleton of 1,4-dihydroquinoline 3 is noncoplanar, while pyrrolo[1,2-a]-quinoline 4 owns a coplanar frame structure. One-dimensional interaction model of compound 4 was formed by one kind of π-π interaction between the two adjacent molecules at upper and lower levels. The HIV-1 integrase strand transfer activity assay results showed that compound 3 has more potent inhibitory activity than 4. The mechanism study and applications of these nitrogen-containing heterocycles are underway in our lab.