Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline
derivatives, synthesis and biological activity
Ravi Chaniyara
a,d, Naval Kapuriya
a, Huajin Dong
b, Pei-Chih Lee
a, Sharda Suman
a, Bhavin Marvania
a,d,
Ting-Chao Chou
b, Te-Chang Lee
a, Rajesh Kakadiya
a, Anamik Shah
d, Tsann-Long Su
a,c,⇑a
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan
b
Preclinical Pharmacology Core Laboratory, Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021, USA
c
Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, Taiwan
d
Department of Chemistry, Saurashtra University, Rajkot, Gujarat, India
a r t i c l e
i n f o
Article history:
Received 14 September 2010 Revised 9 November 2010 Accepted 9 November 2010 Available online 6 December 2010 Keywords:
Pyrrolo[1,2-b]isoquinoline Structure–activity relationships DNA interstrand cross-linking Cell cycle
Antitumor agents
a b s t r a c t
A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitu-mor studies in animal models. The results show that this agent can induce complete tuantitu-mor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Among DNA alkylating agents, the naturally occurring antitu-mor alkylating agent, mitomycin C (MMC, 1,Fig. 1) is a clinically useful chemotherapeutic agent for treating various cancer patients.1,2MMC and its synthetic analogue indoloquinone EO9 (2),3which bears two reactive nucleophilic centers in the molecule, were reported to be capable of cross-linking with DNA. The qui-none moiety of these agents plays an important role in their anti-tumor activity. It requires bioreductive activation to switch on the nucleophilic centers on the pyrrole ring to allow interaction with DNA, which forms biadducts.4
Another class of bifunctional DNA alkylators, such as thioimidaz-oles (i.e., carmethizole, 3, Fig. 1),5 bis(hydroxymethyl)pyrrole derivatives (i.e., 46, 57and 68), 2,3-dihydroxy-6,7-bis(alkylcarba-mates)pyrrolizines [e.g., 7 (IPP) and 8]9were developed originally from the pyrrolizine alkaloids. Of the bis(alkylcarbamates)pyrroli-zine analogues, compound 7 was found to have significant antitu-mor activity against a broad range of experimental human tuantitu-mor xenografts.10It was demonstrated that the electronic properties of the substituent(s) on the phenyl ring as well as the lipophilicity and planarity of the molecule may affect the antitumor activity and toxicity of compounds belonging to this class.10Later, Anderson
et al. synthesized bis(carbamoylmethyl) derivatives of pyrrolo [2,1-a]isoquinolines (9) and pyrrolo[1,2-a]quinolines (10), which bear angular tricyclic structures to limit the deviation from co-pla-narity of the phenyl and pyrrolo rings.11The results showed that these agents exhibited a broad spectrum of antitumor activity against a wide range of tumors.
Recently, we have synthesized a series of bis(hydroxy-methyl)azacyclopenta[a]indenes and their bis(methylcarbamate) derivatives.12These agents can be considered as ‘benzologues’ of bis(hydroxymethyl)pyrrolizines and were able to cross-link to DNA double strands. These analogues exhibited potent cytotoxicity and antitumor activity against human lymphoblastic leukemia and various solid tumors.12 Remarkably, complete tumor remission (CR) in nude mice bearing human breast carcinoma MX-1 xenograft by bis(hydroxymethyl) derivatives (11 and 12,Fig. 1) and bis(meth-ylcarbamate) derivatives (13 and 14) and significant suppression against prostate adenocarcinoma PC3 xenograft by 12 were achieved.
One of the drawbacks of using DNA alkylating agents is that these drugs may lose antitumor activity because tumor cells possess DNA repair mechanisms to fix DNA damage. More recently, we found that the combination treatment of 13 and arsenic trioxide (ATO, DNA repair inhibitor) significantly suppressed human large cell lung carcinoma H460 xenograft (>82%) and cisplatin-resistant NTUB1/P human bladder carcinoma xenografts (>92%) in nude mice.13 These exciting results prompted us to continue designing and
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2010.11.030
⇑ Corresponding author. Tel.: +886 2 2789 9045; fax: +886 2 2782 5573. E-mail address:[email protected](T.-L. Su).
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Bioorganic & Medicinal Chemistry
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b m csynthesizing new bis(hydroxymethyl)pyrrolizine analogues for antitumor studies.
As mentioned previously, pyrrolo[2,1-a]isoquinolines (9) and pyrrolo[1,2-a]quinolines (10) bear an angular tricyclic ring system. To investigate whether derivatives with a linear tricyclic ring sys-tem also possess potent antitumor activity and/or have superior potency than the corresponding bis(hydroxymethyl)azacyclopen-ta[a]indenes, we have synthesized a series of linear 5,10-dihydro-pyrrolo[1,2-b]isoquinolines and their bis(alkylcarbamates) (15), all which were subjected to antitumor evaluation. The results show that the newly synthesized analogues exhibit significant antitumor activity and are able to induce DNA interstrand cross-linking. Here-in, we report the antitumor activity of these agents against various human tumor cell growths both in vitro and in vivo and mecha-nism of action studies.
2. Results and discussion 2.1. Chemistry
The synthetic route for bis(hydroxymethyl)-5,10-dihydropyr-rolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcar-bamate) derivatives (21–23) is shown in Scheme 1. The known 3-carboxy-1,2,3,4-tetrahydroisoquinoline (17) was synthesized by treating the commercially available D,L-phenylalanine (16) with
formaldehyde and concd HCl by following the procedure devel-oped by Dean.14 Compound 17 was N-acylated by treating with
various acid chlorides (R1COCl) in presence of 2 N NaOH to give N-acyl-3-carboxy-1,2,3,4-tetrahydroisoquinolines (18b–j) by fol-lowing the literature procedure.15,16Compounds 18b–j were then reacted with dimethyl acetylenedicarboxylate (DMAD) in acetic anhydride at 60–70 °C yielded 5,10-dihydropyrrolo[1,2-b]isoquin-oline-1,2-dicarboxylic acid dimethyl esters (19b–j). The methyl ester derivative 19a (R1= Me) was prepared in good yields directly from 17 by reacting with acetic anhydride and DMAD.17The ester function of 19a–j was reduced to bis(hydroxymethyl) derivatives 20a–j by treating with LiAlH4 in a mixture of ether/CH2Cl2 at 0–5 °C. Compounds 20a–j were then further treated with methyl-, ethyl- or iso-propylisocyanate in presence of triethyl-amine (TEA) to furnish the desired bis(alkylcarbamate)-5,10-dihy-dropyrrolo[1,2-b]isoquinoline-1-yl derivatives (21–23) in good to high yields.
2.2. Biological results 2.2.1. In vitro cytotoxicity
Table 1shows the antiproliferative activities of the newly syn-thesized bis(hydroxylmethyl)-5,10-dihydropyrrolo[1,2-b]isoquin-oline derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against human lymphoblastic leukemia (CCRF-CEM) and its drug-resistant sublines resistant to Taxol (CCRF-CEM/Taxol) and Vinblastine (CCRF-CEM/VBL) cell growth in vitro. It demon-strated that the newly synthesized conjugates possess significant cytotoxicity with IC50 in submicro molar range. In the series of
bis(hydroxymethyl) derivatives, it showed that 3-alkyl (Me or Et) substituted derivatives are more cytotoxic than the 3-phenyl substituted compounds. The order of their potency, for example, is 20a (3-Me) > 20b (3-Et) > 20c (3-Ph), indicating that the com-pounds having a smaller size of substituent at C3 have greater cytotoxicity. A similar observation is found in 3-phenyl deriva-tives: the cytotoxicity decreases when the number of the methoxy functions increases (20h vs 20i vs 20j). Furthermore, the cytotox-icity decreases when the number of the halo function increases in the halo substituted 3-phenyl derivatives (20d > 20f and 20e > 20g). In this study, one can observe that the C3-methoxy-phenyl derivatives are somewhat more cytotoxic than the corre-sponding halophenyl compounds. This suggests that the electron properties of the substituent(s) on the phenyl ring have very little influence over their potency.
In the series of bis(alkylcarbamates) derivatives, the alkylcarba-mate moiety may serve as a better leaving group than that of the OH group. Thus, the bis(alkylcarbamates) derivatives may easily generate nucleophilic cations on both methylene functions that al-low the cations to become more favorable targets of DNA. In the series of 3-phenyl substituted derivatives, the bis(alkylcarbamates) are generally more potent than their corresponding bis(hydroxy-methyl) derivatives, except for compound bis(ethylcarbamate) 22i, which is as potent as the corresponding bis(hydroxymethyl) 20i and more cytotoxic than bis(iso-propylcarbamates) 23i. How-ever, in the series of C3-alkyl derivatives, the bis(hydroxymethyl) derivatives (20a and 20b) are more potent than their correspond-ing bis(alkylcarbamates) (21a, 22a, and 23a; 22b and 23b, respectively).
Our previous research on the SAR studies of 1,2-bis(hydroxylm-ethyl)cyclopenta[a]indenes and their counterparts 1,2-bis(meth-ylcarbamate) derivatives (11–14, Fig. 1) demonstrated that the size and the electron property of the substituents at the C3 position affected the cytotoxicity of these agents.12However, we found that the cytotoxicity of pyrrolo[1,2-b]isoquinolines is mainly affected by the size of the substituents at the C3 position rather than the electron property in the current studies. In comparison with the potency of both these series, the bis(hydroxymethyl) derivatives of pyrrolo[1,2-b]isoquinoline are more cytotoxic than the corre-sponding cyclopenta[a]indenes. In contrast, the bis(alkylcarba-mate) derivatives of cyclopenta[a]indenes are more potent than the corresponding pyrrolo[1,2-b]isoquinolines.
Our previous report demonstrated that cyclopenta[a]indenes have no multi-drug resistance toward antitumor agents such as Taxol and Vinblastine. To realize whether the newly synthesized pyrrolo[1,2-b]isoquinoline derivatives also have no cross-resis-tance to these two agents, we evaluated their cytotoxicity against CCRF-CEM/Taxol and CCRF-CEM/VBL, which are subcell lines of CCRF-CEM cells that are 330-fold resistant to Taxol, and 680-fold resistant to Vinblastine, respectively. As shown in Table 1, the newly synthesized pyrrolo[1,2-b]isoquinolines have no cross-resis-tance to either Taxol or Vinblastine. This suggests that all deriva-tives are neither a good substrate of membrane multi-drug resistance transporters (i.e., p-glycoprotein) nor mutated tubulin.
The antiproliferative activity of the selected pyrrolo[1,2-b]iso-quinoline derivatives in inhibiting human solid tumors such as breast carcinoma MX-1, colon carcinoma HCT-116, non-small cell lung carcinoma H1299, prostate PC3, oral carcinoma OECM1 and glioma U87 cell growth in vitro (Table 2) were also investigated. Of these compounds, C3-Me derivative (20a) was found to have potent cytotoxicity in inhibiting MX-1 cell growth in vitro with IC50values of 0.66
l
M. Compounds 20a, 21e, and 21f exhibited po-tent inhibitory activity against human colon carcinoma HCT-116 with IC50values of 0.29, 0.04 and 0.36l
M, respectively. The tested compounds have good to moderate effects against H1299, PC3, OECM1 and U87 cell growth in vitro.2.2.2. In vivo antitumor activity
To investigate the antitumor activity of pyrrolo[1,2-b]isoquino-line derivatives in animal models, we selected compound 20a for evaluating its therapeutic efficacy in animal models since this agent has the most potent cytotoxicity among all of the com-pounds tested and exhibits a broad spectrum of antitumor activity in inhibiting both CCRF/CEM and other solid tumors in vitro. Nude mice implanted with human breast carcinoma MX-1 xenograft were given 30 mg/kg, every two days for two times (Q2D 2), intravenous injection (iv injection) on day 8 and 10 (Fig. 2). Remarkably, it shows that complete tumor remission against human breast carcinoma MX-1 in nude mice was achieved (Fig. 2A). This concentration was established from tolerability studies. Under this dosage, one can see that nude mice’s body weights recovered after cessation of the treatment, indicating the low toxicity of the compound to the host (Fig. 2B). In another experiment, we found that 20a was able to effectively suppress
Scheme 1. Reagents and conditions: (i) 37% formalin/concd HCl, reflux; (ii) acid chloride, 2 N NaOH/acetone, room temperature; (iii) DMAD/Ac2O, 60–70 °C; (iv) LiAlH4,
human ovarian tumor SK-OV-3 implanted in nude mice on day 22 at the dose of 20 mg/kg, every day for four times (QD 4), iv injec-tion (Fig. 3A and B). The results of MX-1 and SK-OV-3 xenografts studies show the potential utility of compound 20a in inhibiting the growth of both tumors.
2.2.3. DNA cross-linking study
To realize whether the newly synthesized compounds are capa-ble of cross-linking with DNA doucapa-ble strands, pEGFP-N1 plasmid DNA was treated with bis(hydroxymethyl) derivatives (20a and 20h) and their corresponding bis(alkylcarbamate) derivatives (22a and 23h, respectively) at various concentrations as indicated (1, 10, and 20
l
M) using alkaline agarose gel shifting assay (Fig. 4).18 Melphalan (1, 5, and 10l
M) was used as the positive control. As revealed inFigure 4, one can see that all of the tested compounds were able to induce DNA interstrand cross-linking,suggesting that DNA cross-linking may be the main mechanism of action for these agents.
2.2.4. Cell cycle inhibition
It is well known that DNA interacting agents can alter the cell cy-cle progression by arresting the cell cycy-cle at the G2/M phase.19 Pre-viously, we have demonstrated that 3a-aza-cyclopenta[a]indene derivatives were able to induce G2/M arrest.12We therefore studied the inhibitory effect of 20a on cell cycle distribution (Table 3). The human non-small lung carcinoma H1299 cells were treated with 20a at the concentrations of 1.25, 2.5, and 5
l
M for 24 h. The cells were harvested, stained with propidium iodide (PI) and analyzed with a flow cytometer. It clearly shows that 20a remarkably accu-mulated the cells at G2/M phase. Furthermore, increased sub-G1 populations were noticed in cells treated with 20a at each concentration.Table 1
The cytotoxicity of newly synthesized bis(hydroxymethyl)-5,10-dihydropyrrolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against human lymphoblastic leukemia (CCRF-CEM) and its drug-resistant sublines (CCRF-CEM/Taxol and CCRF-CEM/VBL)a
Compd R1
R2
Cell growth inhibition (IC50lM)
CCRF-C EM CCRF-CEM/Taxolb CCRF-CEM/VBLb 20a Me H 0.08 ± 0.02 0.10 ± 0.001 [1.22]c 0.09 ± 0.01 [1.1] 20b Et H 0.18 ± 0.01 0.21 ± 0.003 [1.15] 0.19 ± 0.02 [1.03] 20c C6H5 H 0.51 ± 0.01 0.50 ± 0.04 [0.99] 0.29 ± 0.02 [0.56] 20d 40-F-C 6H4 H 0.51 ± 0.01 0.88 ± 0.03 [1.72] 0.49 ± 0.002 [0.95] 20e 40-Cl-C 6H4 H 0.60 ± 0.002 0.77 ± 0.17 [1.28] 0.72 ± 0.18 [1.2] 20f 30,40-Di-F–C 6H3 H 1.14 ± 0.22 1.26 ± 0.26 [1.11] 0.95 ± 0.21 [0.84] 20g 30,40-Di-Cl–C 6H3 H 8.44 ± 0.06 4.82 ± 0.01 [0.57] 6.42 ± 0.05 [0.76] 20h 40-MeO–C 6H4 H 0.23 ± 0.03 0.25 ± 0.001 [1.02] 0.11 ± 0.001 [0.48] 20i 30,40-Di-MeO–C 6H3 H 1.10 ± 0.04 0.76 ± 0.02 [0.69] 0.62 ± 0.01 [0.56] 20j 30,40,50-Tri-MeO–C 6H2 H 1.97 ± 0.46 2.88 ± 0.05 [1.45] 2.78 ± 0.09 [1.40] 21a Me CONHMe 0.13 ± 0.01 0.11 ± 0.002 [0.86] 0.08 ± 0.001 [0.59] 21e 40-Cl–C 6H4 CONHMe 0.19 ± 0.01 0.22 ± 0.004 [1.18] 0.16 ± 0.05 [0.87] 21f 30,40-Di-F–C 6H3 CONHMe 0.49 ± 0.09 0.45 ± 0.20 [0.91] 0.47 ± 0.05 [0.97] 21h 40-MeO–C 6H4 CONHMe 0.18 ± 0.03 0.13 ± 0.03 [0.72] 0.09 ± 0.02 [0.49] 22a Me CONHEt 0.24 ± 0.002 0.11 ± 0.001 [0.48] 0.12 ± 0.003 [0.51] 22b Et CONHEt 0.44 ± 0.01 0.21 ± 0.01 [0.48] 0.24 ± 0.01 [0.53] 22c C6H5 CONHEt 0.26 ± 0.01 0.26 ± 0.03 [1.01] 0.23 ± 0.001 [0.90] 22d 40-F–C 6H4 CONHEt 0.31 ± 0.01 0.43 ± 0.05 [1.39] 0.57 ± 0.01 [1.85] 22e 40-Cl–C 6H4 CONHEt 0.59 ± 0.01 0.35 ± 0.01 [0.59] 0.57 ± 0.06 [0.97] 22f 30,40-Di-F–C 6H3 CONHEt 2.08 ± 0.02 1.25 ± 0.02 [0.60] 0.67 ± 0.003 [0.32] 22g 30,40-Di-Cl–C 6H3 CONHEt 2.37 ± 0.02 0.90 ± 0.002 [0.38] 1.19 ± 0.04 [0.50] 22h 40-MeO–C 6H4 CONHEt 0.37 ± 0.02 0.22 ± 0.003 [0.58] 0.35 ± 0.01 [0.93] 22i 30,40-Di-MeO–C 6H3 CONHEt 1.07 ± 0.001 0.81 ± 0.01 [0.75] 0.65 ± 0.02 [0.60] 22j 30,40,50-Tri-MeO–C 6H2 CONHEt 1.41 ± 0.02 2.14 ± 0.08 [1.52] 3.13 ± 0.03 [2.23] 23a Me CONH-i-Pr 0.27 ± 0.004 0.23 ± 0.002 [0.84] 0.14 ± 0.003 [0.50] 23b Et CONH-i-Pr 0.57 ± 0.01 0.32 ± 0.01 [0.56] 0.18 ± 0.0002 [1.42] 23c C6H5 CONH-i-Pr 0.45 ± 0.01 0.23 ± 0.02 [0.50] 0.25 ± 0.01 [0.55] 23d 40-F–C 6H4 CONH-i-Pr 0.76 ± 0.03 0.23 ± 0.01 [0.30] 0.23 ± 0.001 [0.31] 23e 40-Cl–C 6H4 CONH-i-Pr 0.54 ± 0.003 0.63 ± 0.01 [1.16] 0.57 ± 0.04 [1.06] 23f 30,40-Di-F–C 6H3 CONH-i-Pr 1.33 ± 0.01 0.78 ± 0.003 [0.58] 0.61 ± 0.05 [0.46] 23g 30,40-Di-Cl–C 6H3 CONH-i-Pr 3.55 ± 0.02 2.47 ± 0.07 [0.69] 1.35 ± 0.04 [0.38] 23h 40-MeO–C 6H4 CONH-i-Pr 0.34 ± 0.002 0.19 ± 0.002 [0.56] 0.16 ± 0.001 [0.47] 23i 30,40-Di-MeO–C 6H3 CONH-i-Pr 1.29 ± 0.10 0.60 ± 0.02 [0.46] 0.77 ± 0.05 [0.59] 23j 30,40,50-Tri-MeO–C 6H2 CONH-i-Pr 0.89 ± 0.01 1.20 ± 0.01 [1.35] 1.42 ± 0.05 [1.59] Taxol 0.003 ± 0.0003 0.43 ± 0.05 [330] 1.27 ± 0.05 [980] Vinblastine 0.0007 ± 0.001 0.08 ± 0.01 [106.2] 0.50 ± 0.12 [679.5]
aCell growth inhibition was measured by the XTT assay20for leukemic cells after 72-h incubation using a microplate spectrophotometer as described previously.22Similar
in vitro results were obtained by using the Cell Counting Kit-8 for the CCK-8 assays as described by technical manual of Dojindo Molecular Technologies, Inc. (Gaithersburg, MD; Website:www.dojindo.com). IC50values were determined from dose-effect relationship at six or seven concentrations of each drug by using the CompuSyn software by
Chou and Martin24
based on the median-effect principle and plot using the serial deletion analysis.25,26
Ranges given for Taxol and vinblastine were mean ± SE (n = 4).
b
CCRF-CEM/Taxol and CCRF-CEM/VBL are subcell lines of CCRF-CEM cells that are 330-fold resistant to Taxol, and 680-fold resistant to vinblastine, respectively, when comparing with the IC50of the parent cell line.
c Numbers in the brackets are fold of cross-resistant determined by comparison with the corresponding IC
Table 2
The cytotoxicity of newly synthesized bis(hydroxymethyl)-5,10-dihydropyrrolo[1,2-b]isoquinolin-1-yl derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against human solid tumors (breast carcinoma MX-1, colon carcinoma HCT-116, lung carcinoma H1299, prostate carcinoma PC3, oral carcinoma OECM1 and glioma U87) cell growth in vitro
Compd Cell growth inhibition (IC50lM)
MX-1a HCT-116a H1299b PC3b OECM1b U87b 20a 0.66 ± 0.03 0.29 ± 0.01 2.48 ± 1.22 6.07 ± 1.70 6.10 ± 1.11 27.91 ± 3.90 20d 4.01 ± 0.05 2.46 ± 0.08 8.76 ± 1.15 5.93 ± 1.52 6.99 ± 0.12 23.25 ± 0.26 20e 3.28 ± 0.01 3.06 ± 0.46 24.73 ± 8.16 9.85 ± 0.79 12.05 ± 0.67 26.68 ± 5.79 20f 6.99 ± 0.35 3.21 ± 0.21 NDc ND ND ND 20h 1.99 ± 0.07 1.13 ± 0.07 13.94 ± 0.83 10.93 ± 1.21 6.12 ± 0.21 10.57 ± 0.23 21a 1.68 ± 0.01 1.17 ± 0.02 ND ND ND ND 21e 1.18 ± 0.01 0.04 ± 0.002 ND ND ND ND 21f 2.73 ± 0.05 0.36 ± 0.001 ND ND ND ND 21h 1.33 ± 0.02 1.07 ± 0.05 ND ND ND ND 22a 2.27 ± 0.01 0.90 ± 0.03 19.42 ± 0.42 15.45 ± 1.84 11.34 ± 2.07 13.89 ± 4.90 22d 1.51 ± 0.03 0.79 ± 0.05 3.94 ± 1.13 9.59 ± 1.31 12.52 ± 1.70 28.36 ± 4.86 22e 1.69 ± 0.06 0.62 ± 0.10 37.25 ± 0.89 10.98 ± 1.14 25.32 ± 0.18 33.59 ± 3.91 22h 1.27 ± 0.05 0.75 ± 0.004 27.61 ± 1.65 18.54 ± 0.82 10.48 ± 0.13 17.06 ± 1.78 23a 1.28 ± 0.01 1.38 ± 0.02 9.77 ± 3.75 16.87 ± 7.42 11.80 ± 2.58 26.61 ± 4.21 23d 1.17 ± 0.19 0.63 ± 0.001 8.13 ± 1.34 12.29 ± 1.34 11.05 ± 2.61 36.87 ± 5.98 23e 1.70 ± 0.09 0.69 ± 0.01 25.15 ± 4.05 9.97 ± 1.32 26.10 ± 7.86 22.72 ± 2.03 23h 0.98 ± 0.03 0.97 ± 0.04 13.16 ± 1.94 6.79 ± 1.01 8.83 ± 0.74 21.04 ± 0.67 Taxol 0.035 ± 0.00514 0.0013 ± 0.0005 ND ND ND ND Vinblastine 0.0029 ± 0.0002 0.0018 ± 0.0004 ND ND ND ND Cisplatin ND ND 4.95 ± 0.60 26.65 ± 4.19 ND ND
a Cell growth inhibition was measured by the SRB assay21for solid tumor cells after 72-h incubation using a microplate spectrophotometer as described previously.22
b Cell growth inhibition was determined by the Alamar blue assay23in a 72 h incubation using a microplate spectrophotometer as described previously.
c
Not determined.
Figure 2. Therapeutic effects of 20a in nude mice bearing MX-1 human mammary xenograft (iv injection, n = 4). (A) Average tumor size changes. (B) Average body weight changes.
Figure 3. Therapeutic effects of 20a in nude mice bearing ovarian adenocarcinoma SK-OV-3 xenograft (iv injection, n = 4). (A) Average tumor size changes. (B) Average body weight changes.
3. Conclusion
In the present studies, we have synthesized a series of linear pyrrolo[1,2-b] isoquinoline derivatives for antitumor evaluation. Both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show potent antitumor activity in inhibiting various human tumor xenografts in vitro. Among these analogues, we discovered com-pound 20a, which was selected for antitumor studies in animal models, exhibits potent therapeutic efficacy against human breast MX-1 xenograft in nude mice, as complete tumor remission was observed. This agent is also able to significantly suppress human ovarian tumors implanted in nude mice. The results reported here-in warrant further here-investigation to optimize the schedule and dos-age to get greater suppression of other human tumor growth in animal models. Additional, the evaluation of the antitumor activity of 20a in combination with DNA repair inhibitor (e.g., ATO) is cur-rently undergoing in our laboratory.
4. Experimental section
4.1. General methods and materials
All commercial chemicals and solvents were reagent grade and were used without further purification unless otherwise specified. Melting points were determined on a Fargo melting point appara-tus and are uncorrected. Thin-layer chromatography was per-formed on Silica Gel G60 F254(Merck) with short-wavelength UV light for visualization. All reported yields are isolated yields after
chromatography or crystallization. Elemental analyses were done on a Heraeus CHN-O Rapid instrument. 1H NMR spectra were recorded on a 600 MHz, Brucker AVANCE 600 DRX and 400 MHz, Brucker Top-Spin spectrometers in the indicated solvent. The chemical shifts were reported in ppm (d) relative to TMS and cou-pling constants (J) in Hertz (Hz) and s, d, t, m, br s, refer to singlet, doublet, triplet, multiplet, broad, respectively. High performance liquid chromatography analysis for checking purity of synthesized compounds were recorded on a Hitachi D-2000 Elite instrument: column, Mightysil RP-18 GP 250–4.6 (5
l
m); mobile phase, MeCN/THF (50:50 v/v); flow rate, 1 mL/min; injected sample 10l
L, column temp, 27 °C; wavelength, 254 nm. The purity of all tested compounds was P95% based on analytical HPLC.4.2. 1,2,3,4-Tetrahydroisoquinoline-3-carboxylic acid hydrochloride (17)
A mixture ofD,L-phenylalanine (16, 50 g, 3.03 mol), concd HCl
(325 mL) and 37% formalin (110 mL) was heated to a gentle reflux with vigorous stirring. After 30 min, another portion of formalin (50 mL) and concd HCl (110 mL) was added. The reaction mixture was further stirred and heated for 4 h and cooled to room temper-ature. The white solid separated out was filtered and washed with methanol (30 mL) to give 17 64.1 g, yield 98%; mp >280 °C (lit.14 mp 286–290 °C). 1H NMR (DMSO-d
6) d 3.15 (1H, m, CH), 3.32 (1H, m, CH), 4.30 (2H, m, CH2), 4.43 (1H, m, CH), 7.20–7.35 (4H, m, 4 ArH), 9.89 (1H, br s, exchangeable, NH); 10.06 (1H, br s, exchangeable, COOH).
Figure 4. Representative DNA cross-linking gel shift assay for bis(hydroxymethyl) derivatives (20a and 20h) and their corresponding bis(alkylcarbamate) derivatives (22a and 23h, respectively) at various concentrations as indicated. Control lane shows single-stranded DNA (SS), while CL shown in all tested lanes is DNA double-stranded cross-linking. Melphalan (1, 5, and 10lM) was used as a positive control.
Table 3
Effects of compound 20a on cell cycle progress in human non-small cell lung adenocarcinoma H1299 Concentration (lM) 0 1.25 2.5 5 Sub G1 14.1 ± 0.7 21.6 ± 4.2 26.6 ± 0.7 26.5 ± 3.0 G1 42.9 ± 0.8 5.6 ± 0.6 7.7 ± 0.7 0.5 ± 0.3 S 22.5 ± 0.3 32.7 ± 5.9 18.3 ± 8.4 24.7 ± 4.6 G2/M 20.5 ± 0.9 40.1 ± 6.5 47.3 ± 2.1 48.4 ± 2.2
4.3. 2-Propionyl-1,2,3,4-tetrahydroisoquiniline-3-carboxylic acid (18b)
To a suspension of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic-acid hydrochloride (17, 10 g, 46.8 mmol) in acetone (60 mL) was added 2 N NaOH (40 mL) solution at room temperature. The clear solution obtained then added dropwise into a solution of propionyl chloride (5.2 g, 57 mmol) in acetone (20 mL) at room temperature, simultaneously 2 N NaOH was added dropwise and pH maintained above 10. The reaction mixture was stirred at room temperature for 2 h, the solvent was evaporated under reduce pressure. The solution was acidified to pH 5–6 with 3 N HCl. The white solid sep-arated, filtered it and dried to give 18b 9 g, yield 87%; mp 173– 174 °C.1H NMR (DMSO-d
6) d 1.03 (3H, m, Me), 2.45 (2H, m, CH2), 3.13 (2H, m, CH2), 4.52 (2H, m, CH2), 5.09 (1H, m, CH), 7.16–7.20 (4H, m, 4 ArH), 12.63 (1H, br s, exchangeable, COOH). Anal. Calcd for (C13H15NO3): C, 66.94; H, 6.48; N, 6.00. Found: C, 66.64; H, 6.48; N, 5.89.
By following the same synthetic procedure as that for 18b, the following compounds were synthesized.
4.3.1. 2-Benzoyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18c)
Compound 18c was prepared from 17 (8.0 g, 37.4 mmol) and benzoyl chloride (6.43 g, 45.6 mmol). Yield, 9.9 g (77%); mp 168– 169 °C (lit.15mp 168–169 °C).1H NMR (DMSO-d
6) d 3.18 (2H, m, CH2), 4.52 (2H, m, CH2), 5.08 (1H, m, CH), 7.14–7.22 (4H, m, 4 ArH), 7.41–7.49 (5H, m, 5 ArH), 12.85 (1H, br s, exchange-able, COOH).
4.3.2. 2-(4-Fluorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18d)
Compound 18d was prepared from 17 (10.0 g, 46.8 mmol) and 4-fluorobenzoyl chloride (9.91 g, 57.0 mmol). Yield, 13.5 g (97%); mp 179–180 °C; 1H NMR (DMSO-d
6) d 3.20 (2H, m, CH2), 4.52 (2H, m, CH2), 5.06 (1H, m, CH), 7.19–7.33 (6H, m, 6 ArH), 7.48– 7.52 (2H, m, 2 ArH), 12.76 (1H, br s, exchangeable, COOH). Anal. Calcd for (C17H14FNO3): C, 68.22; H, 4.71; N, 4.68. Found: C, 68.08; H, 4.57; N, 4.42.
4.3.3. 2-(4-Chlorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18e)
Compound 18e was prepared from 17 (10.0 g, 46.8 mmol) and 4-chloroenzoyl chloride (10.1 g, 57.0 mmol). Yield, 13.8 g (93%), mp 77–79 °C;1H NMR (DMSO-d
6) d 3.29 (2H, m, CH2), 4.49 (2H, m, CH2), 5.02 (1H, m, CH), 7.01–7.17 (4H, m, 4 ArH), 7.40–7.58 (4H, m, 4 ArH), 12.81 (1H, br s, exchangeable, COOH). Anal. Calcd for (C17H14ClNO3): C, 64.67; H, 4.47; N, 4.44. Found: C, 64.52; H, 4.56; N, 4.33.
4.3.4. 2-(3,4-Difluorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18f)
Compound 18f was prepared from 17 (10.0 g, 46.8 mmol) and 3,4-difluorobenzoyl chloride (10.2 g, 57.0 mmol). Yield, 14.2 g (95%); mp 89–91 °C; 1H NMR (DMSO-d
6) d 3.18 (2H, m, CH2), 4.48 (2H, m, CH2), 5.02 (1H, m, CH), 7.09–7.15 (4H, m, 4 ArH), 7.33 (1H, s, ArH), 7.46–7.54 (2H, m, 2 ArH), 12.84 (1H, br s, exchangeable, COOH). Anal. Calcd for (C17H13F2NO3): C, 64.35; H, 4.13; N, 4.41. Found: C, 64.21; H, 4.26; N, 4.56.
4.3.5. 2-(3,4-Dichlorobenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18g)
Compound 18g was prepared from 17 (10.0 g, 46.8 mmol) and 3,4-dichlorobenzoyl chloride (12.1 g, 57.0 mmol). Yield, 14.5 g (89%); mp 101–102 °C;1H NMR (DMSO-d
6) d 3.27 (2H, m, CH2),
4.45 (2H, m, CH2), 4.99 (1H, m, CH), 7.08–7.13 (4H, m, 4 ArH), 7.44–7.46 (1H, m, ArH), 7.66–7.70 (2H, m, 2 ArH), 12.67 (1H, br s, exchangeable, COOH). Anal. Calcd for (C17H17Cl2NO3): C, 58.31; H, 3.74; N, 4.00. Found: C, 58.19; H, 4.04; N, 3.87.
4.3.6. 2-(4-Methoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18h)
Compound 18h was prepared from 17 (10.0 g, 46.8 mmol) and 4-methoxybenzoyl chloride (9.8 g, 57.0 mmol). Yield, 12.6 g (86%); mp 178– 180 °C;1H NMR (DMSO-d
6) d 3.28 (2H, m, CH2), 3.85 (3H, s, MeO), 4.60 (2H, m, CH2), 5.02 (1H, m, CH), 6.90–6.94 (3H, m, 3 ArH), 7.18–7.22 (3H, m, 3 ArH), 7.42–7.48 (2H, m, 2 ArH), 12.66 (1H, br s, exchangeable, COOH). Anal. Calcd for (C18H17NO4): C, 69.44; H, 5.50; N, 4.50. Found: C, 69.12; H, 5.62; N, 4.27.
4.3.7. 2-(3,4-Dimethoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18i)
Compound 18i was prepared from 17 (10.0 g, 46.8 mmol) and 3,4-dimethoxybenzoyl chloride (12.5 g, 57 mmol). Yield, 11.9 g (61%); mp 235–236 °C;1H NMR (DMSO-d
6) d 3.19 (2H, m, CH2), 3.77 (6H, s, 2 MeO), 4.56 (2H, m, CH2), 5.04 (1H, m, CH), 6.99– 7.04 (4H, m, 4 ArH), 7.17–7.23 (3H, m, 3 ArH), 12.88 (1H, br s, exchangeable, COOH). Anal. Calcd for (C19H19NO5): C, 66.85; H, 5.61; N, 4.10. Found: C, 66.65; H, 5.66; N, 3.86.
4.3.8. 2-(3,4,5-Trimethoxybenzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (18j)
Compound 18j was prepared from 17 (7.0 g, 32.7 mmol) and 3,4,5-trimethoxybenzoyl chloride (9.3 g, 39.9 mmol). Yield, 12.0 g (98%); mp 186–189 °C;1H NMR (DMSO-d
6) d 3.19 (2H, m, CH2), 3.70 (3H, s, MeO), 3.80 (6H, s, 2 MeO), 4.59 (2H, m, CH2), 5.02 (1H, m, CH), 6.70 (2H, s, 2 ArH), 7.13–7.28 (4H, m, 4 ArH), 12.72 (1H, br s, exchangeable, COOH). Anal. Calcd for (C20H21NO6): C, 64.68; H, 5.70; N, 3.77. Found: C, 64.44; H, 5.50; N, 3.44. 4.4. 3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19a)
Dimethyl acetylenedicarboxylate (6.39 g, 45.0 mmol) was added into a mixture of 1,2,3,4-tetrahydroisoquinoline-3-carbox-ylic acid (17, 10 g, 46.8 mmol) in acetic anhydride (70 mL) and the reaction mixture was heated at 70 °C with stirring for 1.5 h. The reaction mixture was evaporated to dryness in vacuo. The res-idue was recrystallized from MeOH to give 19a, 11.0 g (74%); mp 152–154 °C (lit.17 mp 140–142 °C). 1H NMR (DMSO-d
6) d 2.40 (3H, s, Me), 3.70 (3H, s, COOMe), 3.72 (3H, s, COOMe), 4.18 (2H, s, CH2), 5.06 (2H, s, CH2), 7.21–7.32 (2H, m, 2 ArH), 7.35–7.39 (2H, m, 2 ArH).
4.4.1. 3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19b)
A mixture of dimethyl acetylenedicarboxylate (DMAD) (6.39 g, 45.0 mmol) and 18b (7.0 g, 30.0 mmol) in acetic anhydride (50 mL) was heated at 65 °C with stirring for 1.5 h. The reaction mixture was evaporated to dryness in vacuo and the residue was recrystallized from MeOH to give 19b, 8.77 g (93%); mp 88– 89 °C.1H NMR (DMSO-d
6) d 1.15 (3H, t, J = 7.6 Hz, Me), 2.85 (2H, q, J = 7.6 Hz, CH2), 3.71 (6H, s, 2 COOMe), 4.19 (2H, s, CH2), 5.12 (2H, s, CH2), 7.29–7.33 (2H, m, 2 ArH), 7.38–7.43 (2H, m, 2 ArH). Anal. Calcd for (C18H19NO4): C, 68.99; H, 6.11; N, 4.47. Found: C, 68.96; H, 6.05; N, 4.38.
By following the same synthetic procedure as that for 19b, the following compounds were synthesized.
4.4.2. 3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19c)
Compound 19c was prepared from DMAD (6.0 g, 41.0 mmol) and 18c (8.0 g, 28.4 mmol). Yield, 7.0 g (68%); mp 137–138 °C.1H NMR (DMSO-d6) d 3.58 (3H, s, COOMe), 3.76 (3H, s, COOMe), 4.33 (2H, s, CH2), 4.98 (2H, s, CH2), 7.21–7.32 (3H, m, 3 ArH), 7.41–7.53 (6H, m, 6 ArH). Anal. Calcd for (C22H19NO4): C, 73.12; H, 5.30; N, 3.88. Found: C, 72.91; H, 5.30; N, 3.53.
4.4.3. 3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19d)
Compound 19d was prepared from DMAD (10.7 g, 65.0 mmol) and 18d (15.0 g, 50.1 mmol). Yield, 11.1 g (58%); mp 149–150 °C. 1H NMR (DMSO-d
6) d 3.58 (3H, s, COOMe), 3.76 (3H, s, COOMe), 4.32 (2H, s, CH2), 4.96 (2H, s, CH2), 7.23–7.30 (1H, m, ArH), 7.32– 7.36 (4H, m, 4 ArH), 7.40–7.42 (1H, m, ArH), 7.49–7.52 (2H, m, 2 ArH). Anal. Calcld for (C22H18FNO4): C, 69.65; H, 4.78; N, 3.69. Found: C, 69.54; H, 4.82; N, 3.78.
4.4.4. 3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19e)
Compound 19e was prepared from DMAD (6.8 g, 47.0 mmol) and 18e (10.0 g, 31.6 mmol). Yield, 8.0 g (64%); mp 164–166 °C. 1H NMR (DMSO-d
6) d 3.60 (3H, s, COOMe), 3.76 (3H, s, COOMe), 4.32 (2H, s, CH2), 4.98 (2H, s, CH2), 7.20–7.26 (1H, m, ArH), 7.27– 7.33 (2H, m, 2 ArH), 7.39–7.43 (1H, m, ArH), 7.48 (2H, d, J = 8.5 Hz, 2 ArH), 7.57 (2H, d, J = 8.5 Hz, 2 ArH). Anal. Calcd for (C22H18ClNO4): C, 66.75; H, 4.58; N, 3.54. Found: C, 66.52; H, 4.85; N, 3.66.
4.4.5. 3-(3, 4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19f)
Compound 19f was prepared from DMAD (6.71 g, 47.0 mmol) and 18f (10.0 g, 31.5 mmol). Yield, 8.4 g (67%); mp 142–144 °C. 1H NMR (DMSO-d
6) d 3.61 (3H, s, COOMe), 3.76 (3H, s, COOMe), 4.31 (2H, s, CH2), 5.00 (2H, s, CH2), 7.22–7.42 (5H, m, 5 ArH), 7.54–7.61 (2H, m, 2 ArH). Anal. Calcd for (C22H17F2NO4): C, 66.50; H, 4.31; N, 3.52. Found: C, 66.15; H, 4.44; N, 3.36.
4.4.6. 3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19g)
Compound 19e was prepared from DMAD (8.5 g, 60.0 mmol) and 18g (14.0 g, 40.0 mmol). Yield, 11.0 g (63%); mp 169–170 °C. 1H NMR (DMSO-d
6) d 3.61 (3H, s, COOMe), 3.77 (3H, s, COOMe), 4.31 (2H, s, CH2), 5.02 (2H, s, CH2), 7.22–7.33 (3H, m, 3 ArH), 7.40–7.47 (2H, m, 2 ArH), 7.76–7.78 (2H, m, 2 ArH). Anal. Calcd for (C22H17Cl2NO4): C, 61.41; H, 3.98; N, 3.26. Found: C, 61.33; H, 3.95; N, 2.89.
4.4.7. 3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19h)
Compound 19h was prepared from DMAD (7.5 g, 53.0 mmol) and 18h (10.0 g, 35.1 mmol). Yield, 8.4 g (66%); mp 155–158 °C. 1H NMR (DMSO-d
6) d 3.58 (3H, s, COOMe), 3.75 (3H, s, COOMe), 3.82 (3H, s, MeO), 4.31 (2H, s, CH2), 4.95 (2H, s, CH2), 7.05–7.07 (2H, m, 2 ArH), 7.21–7.24 (1H, m, ArH), 7.28–7.31 (2H, m, 2 ArH), 7.37–7.42 (3H, m, 3 ArH). Anal. Calcd for (C23H21NO5): C, 70.58; H, 5.41; N, 3.58. Found: C, 70.25; H, 5.44; N, 3.49. 4.4.8. 3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19i)
Compound 19i was prepared from DMAD (5.6 g, 39.0 mmol) and 18i (9.0 g, 26.4 mmol). Yield, 9.0 g (81%); mp 202–203 °C.1H NMR (DMSO-d6) d 3.60 (3H, s, COOMe), 3.66 (3H, s, MeO), 3.78 (3H, s, COOMe), 3.82 (3H, s, MeO), 4.32 (2H, s, CH2), 5.00 (2H, s, CH2), 6.99 (1H, dd, J = 2.0 and 8.0 Hz, ArH), 7.02 (1H, d, J = 2.0 Hz,
ArH), 7.07 (1H, d, J = 8.0 Hz, ArH), 7.22–7.25 (1H, m, ArH), 7.25– 7.32 (2H, m, 2 ArH), 7.40–7.42 (1H, m, ArH). Anal. Calcd for (C24H23NO6): C, 68.40; H, 5.50; N, 3.32. Found: C, 68.02; H, 5.53; N, 2.94.
4.4.9. 3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-dicarboxylic acid dimethyl ester (19j)
Compound 19i was prepared from DMAD (5.7 g, 40.0 mmol) and 18j (10.0 g, 26.9 mmol). Yield, 6.2 g (51%); mp 165–166 °C. 1H NMR (DMSO-d
6) d 3.63 (3H, s, COOMe), 3.74 (6H, s, COOMe and MeO), 3.80 (6H, s, 2 MeO), 4.32 (2H, s, CH2), 5.00 (2H, s, CH2), 6.75 (2H, s, 2 ArH), 7.22–7.26 (1H, m, ArH), 7.28–7.32 (1H, m, ArH), 7.35–7.37 (1H, m, ArH), 7.40–7.42 (1H, m, ArH). Anal. Calcd for (C25H25NO7): C, 66.51; H, 5.58; N, 3.10. Found: C, 66.25; H, 5.59; N, 3.01.
4.5. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20a)
A solution of 19a (8.0 g, 26.7 mmol) in anhydrous dichloro-methane (20 mL) was added dropwise in to a stirred suspension of LiAlH4 (2.5 g, 66.0 mmol) in anhydrous diethyl ether (50 mL) at 0 to 5 °C. The reaction mixture was further stirred for 15 min after the addition was completed. The excess hydride was de-stroyed by the sequential addition of water (2.5 mL), 15% aqueous NaOH (2.5 mL), and water (2.5 mL) at 0 °C. The mixture was fil-tered through a pad of Celite, the solid residue was washed with dichloromethane. The combined filtrate and washings were evap-orated to dryness in vacuo. The residue was recrystallized from ether to give 20a, 4.2 g (64.0%); mp 99–101 °C.1H NMR (DMSO-d6) d 2.22 (3H, s, Me), 3.96 (2H, s, CH2), 4.33 (2H, m, CH2), 4.38 (4H, m, CH2 and exchangeable, 2 OH), 4.93 (2H, s, CH2), 7.24– 7.28 (2H, m, 2 ArH), 7.34–7.36 (2H, m, 2 ArH). Anal. Calcd for (C15H17NO2): C, 74.05; H, 7.04; N, 5.76. Found: C, 74.24; H, 6.97; N, 5.71.
By following the same synthetic procedure as that for 20a, the following compounds were synthesized.
4.5.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20b)
Compound 20b was prepared from 19b (5.0 g, 15.9 mmol) and LiAlH4 (1.5 g, 39.0 mmol). Yield, 3.15 g (76%) as syrup; 1H NMR (DMSO-d6) d 1.10 (3H, t, J = 7.6 Hz, Me), 2.66 (2H, q, J = 7.6 Hz, CH2), 3.95 (2H, s, CH2), 4.33 (3H, m, CH2and exchangeable, OH), 4.39 (3H, m, CH2and exchangeable, OH), 4.96 (2H, s, CH2), 7.23– 7.28 (2H, m, 2 ArH), 7.33–7.39 (2H, m, 2 ArH).
4.5.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20c)
Compound 20c was prepared from 19c (7.0 g, 19.4 mmol) and LiAlH4 (1.8 g, 48.0 mmol). Yield, 4.7 g (80%); mp 150–152 °C. 1H NMR (DMSO-d6) d 4.06 (2H, s, CH2), 4.28 (2H, d, J = 4.8 Hz, CH2), 4.50 (2H, d, J = 4.8 Hz, CH2), 4.54 (2H, br s, exchangeable OH), 4.98 (2H, s, CH2), 7.18 (1H, t, J = 7.2 Hz, ArH), 7.26 (2H, t, J = 7.2 Hz, 2 ArH), 7.36–7.39 (2H, m, 2 ArH), 7.45–7.51 (4H, m, 4 ArH). Anal. Calcd for (C20H19NO2): C, 78.66; H, 6.27; N, 4.59. Found: C, 78.48; H, 6.42; N, 4.41.
4.5.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20d)
Compound 20d was prepared from 19d (10.0 g, 26.4 mmol) and LiAlH4(2.5 g, 65.0 mmol). Yield, 6.36 g (74%); mp 135–136 °C.1H NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.26 (2H, d, J = 4.8 Hz, CH2), 4.49 (2H, d, J = 4.8 Hz, CH2), 4.53 (2H, t, J = 4.8 Hz, exchangeable, OH), 4.96 (2H, s, CH2), 7.17–7.21 (1H, m, ArH), 7.26–7.34 (4H, m, 4 ArH), 7.38 (1H, m, ArH), 7.48–7.52 (2H, m, 2 ArH). Anal.
Calcd for (C20H18FNO2): C, 74.29; H, 5.61; N, 4.33. Found: C, 74.38; H, 5.68; N, 3.98.
4.5.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20e)
Compound 20e was prepared from 19e (7.0 g, 17.7 mmol) and LiAlH4 (1.63 g, 44 mmol). Yield, 3.1 g (52%); mp 170–172 °C.1H NMR (DMSO-d6) d 4.06 (2H, s, CH2), 4.28 (2H, d, J = 4.8 Hz, CH2), 4.50 (2H, d, J = 4.8 Hz, CH2), 4.55 (2H, m, exchangeable, OH), 4.99 (2H, s, CH2), 7.18–7.20 (1H, m, ArH), 7.21–7.27 (2H, m, 2 ArH), 7.29 (1H, d, J = 7.6 Hz, ArH), 7.38 (2H, d, J = 7.2 Hz, 2 ArH), 7.48 (2H, d, J = 7.2 Hz, 2 ArH). Anal. Calcd for (C20H18ClNO2): C, 70.69; H, 5.34; N, 4.12. Found: C, 70.42; H, 5.37; N, 4.05.
4.5.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20f)
Compound 20f was prepared from 19f (7.5 g, 18.0 mmol) and LiAlH4 (1.73 g, 47 mmol). Yield, 5.3 g (83%); mp 145–146 °C.1H NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.27 (2H, d, J = 5.2 Hz, CH2z), 4.48 (2H, d, J = 5.2 Hz, CH2), 4.53 (1H, t, J = 5.2 Hz, exchangeable, OH), 4.60 (1H, t, J = 5.2 Hz, exchangeable, OH), 5.01 (2H, s, CH2), 7.18–7.33 (4H, m, 4 ArH), 7.37–7.39 (1H, m, ArH), 7.52–7.54 (2H, m, 2 ArH). Anal. Calcd for (C20H17F2NO2): C, 70.37; H, 5.02; N, 4.10. Found: C, 70.08; H, 5.01; N, 3.76.
4.5.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20g)
Compound 20g was prepared from 19g (9.0 g, 20.9 mmol) and LiAlH4(1.9 g, 52.0 mmol). Yield, 5.7 g (72%); mp 150–151 °C. 1H NMR (DMSO-d6) d 4.05 (2H, s, CH2), 4.26 (2H, d, J = 4.8 Hz, CH2), 4.48 (2H, d, J = 4.8 Hz, CH2), 4.53 (1H, t, J = 4.8 Hz, exchangeable OH), 4.63 (1H, t, J = 4.8 Hz, exchangeable, OH), 5.03 (2H, s, CH2), 7.18–7.21 (1H, m, ArH), 7.23–7.27 (2H, m, 2 ArH), 7.28–7.30 (1H, m, ArH), 7.38–7.40 (1H, m, ArH), 7.46–7.48 (2H, m, 2 ArH). Anal. Calcd for (C20H17Cl2NO2): C, 64.18; H, 4.58; N, 3.74. Found: C, 63.89; H, 4.40; N, 3.87.
4.5.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20h)
Compound 20h was prepared from 19h (7.5 g, 19.0 mmol) and LiAlH4 (1.77 g, 47 mmol). Yield, 4.3 g (67%); mp 174–176 °C.1H NMR (DMSO-d6) d 3.82 (3H, s, MeO), 4.04 (2H, s, CH2), 4.26 (2H, s, CH2), 4.48 (4H, m, CH2 and exchangeable, OH), 4.93 (2H, s, CH2), 7.04–7.06 (2H, m, 2 ArH), 7.17–7.21 (1H, m, ArH), 7.24– 7.27 (2H, m, 2 ArH), 7.36–7.39 (3H, m, 3 ArH). Anal. Calcd for (C21H21NO3): C, 75.20; H, 6.31; N, 4.18. Found: C, 75.36; H, 6.30; N, 4.13.
4.5.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20i)
Compound 20i was prepared from 19i (8.0 g, 18.9 mmol) and LiAlH4(1.75 g, 47 mmol). Yield, 5.34 g (77%); mp 145–146 °C.1H NMR (DMSO-d6) d 3.79 (6H, s, 2 MeO), 4.04 (2H, s, CH2), 4.29 (2H, s, CH2), 4.52 (4H, br s, CH2and exchangeable, OH), 4.97 (2H, s, CH2), 6.96–6.98 (1H, m, ArH), 7.05–7.07 (2H, m, 2 ArH), 7.19–7.28 (3H, m, 3 ArH), 7.37–7.39 (1H, m, ArH). Anal. Calcd for (C22H23NO4): C, 72.31; H, 6.34; N, 3.83. Found: C, 72.25; H, 6.34; N, 3.95.
4.5.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]dimethanol (20j)
Compound 20j was prepared from 19j (6.0 g, 13.3 mmol) and LiAlH4(1.2 g, 33.0 mmol). Yield, 3.1 g (58%); mp 146–147 °C. 1H NMR (DMSO-d6) d 3.74 (3H, s, MeO), 3.82 (6H, s, 2 MeO), 4.05 (2H, s, CH2), 4.31 (2H, s, CH2), 4.50 (2H, s, CH2), 4.53 (2H, s, exchangeable, OH), 5.04 (2H, s, CH2), 6.73 (2H, s, 2 ArH), 7.21–
7.27 (2H, m, 2 ArH), 7.33–7.38 (2H, m, 2 ArH). Anal. Calcd for (C23H25NO5): C, 69.86; H, 6.37; N, 3.54. Found: C, 69.71; H, 6.37; N, 3.45.
4.6. General procedure for preparing bis(alkylcarbamate) derivatives (21–23)
To a solution of bis(hydroxymethyl) derivatives (20a–20j, 1.0 equiv) and triethylamine (2–3 equiv) in anhydrous dichloro-methane was added alkylisocyanate (5 equiv). The reaction mix-ture was stirred at ambient temperamix-ture (for 3–20 h) under an argon atmosphere. After the completion of the reaction, the reac-tion mixture was evaporated to dryness in vacuo. The residue was triturated with ether, and solid separated was collected by fil-tration. The desired product was either obtained by recrystalliza-tion or liquid chromatography.
4.6.1. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate) (21a)
Compound 21a was synthesized from 20a (0.80 g, 3.2 mmol), Et3N (1 mL) and methylisocyanate (1.14 g, 20 mmol). Yield, 0.53 g (44%); mp 174–176 °C. 1H NMR (DMSO-d
6) d 2.23 (3H, s, Me), 2.53 (6H, m, 2 Me), 3.99 (2H, s, CH2), 4.88 (2H, s, CH2), 4.92 (2H, s, CH2), 4.95 (2H, s, CH2), 6.74 (2H, br s, exchangeable, NH), 7.21–7.32 (2H, m, 2 ArH), 7.34–7.38 (2H, m, 2 ArH). Anal. Calcd for (C19H23N3O4): C, 63.85; H, 6.49; N, 11.76. Found: C, 63.54; H, 6.57; N, 11.64.
4.6.2.
[3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate) (21e)
Compound 21e was synthesized from 20e (1.0 g, 3.0 mmol), Et3N (1 mL), and methylisocyanate (0.85 g, 15 mmol). Yield, 1.1 g (82%); mp 194–196 °C. 1H NMR (DMSO-d
6) d 2.54 (6H, m, 2 Me), 4.09 (2H, s, CH2), 4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.03 (2H, s, CH2), 6.81 (2H, br s, exchangeable, NH), 7.17–7.23 (1H, m, ArH), 7.24–7.30 (2H, m, 2 ArH), 7.36–7.40 (1H, m, ArH), 7.45–7.47 (2H, m, 2 ArH), 7.55–7.57 (2H, m, 2 ArH). Anal. Cacld for (C24H24ClN3O4): C, 63.50; H, 5.33; N, 9.26. Found: C, 63.33; H, 5.35; N, 9.20.
4.6.3. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate) (21f)
Compound 21f was synthesized from 20f (1.0 g, 2.9 mmol), Et3N (1 mL), and methylisocyanate (0.85 g, 15 mmol). Yield, 1.0 g (77%); mp 190–192 °C; 1H NMR (DMSO-d
6) d 2.54 (6H, m, 2 Me), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 4.99 (2H, s, CH2), 5.03 (2H, s, CH2), 6.83 (2H, br s, exchangeable, NH), 7.18–7.23 (1H, m, ArH), 7.25–7.31 (3H, m, 3 ArH), 7.36–7.40 (1H, m, ArH), 7.52–7.61 (2H, m, 2 ArH). Anal. Calcd for (C24H23F2N3O4): C, 63.29; H, 5.09; N, 9.23. Found: C, 63.00; H, 5.12; N, 9.16.
4.6.4. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(methylcarbamate) (21h)
Compound 21h was synthesized from 20h (1.0 g, 2.9 mmol), Et3N (1 mL), methylisocyanate (1.6 g, 29 mmol). Yield, 1.2 g (87%); mp 182–184 °C. 1H NMR (DMSO-d6) d 2.53 (6H, m, 2 Me), 3.85 (3H, s, MeO), 4.08 (2H, s, CH2), 4.78 (2H, s, CH2), 4.93 (2H, s, CH2), 5.01 (2H, s, CH2), 6.81 (2H, br s, exchangeable, NH), 7.06–7.08 (2H, m, 2 ArH), 7.17–7.23 (1H, m, ArH), 7.24– 7.30 (2H, m, 2 ArH), 7.32–7.40 (3H, m, 3 ArH). Anal. Calcd for (C25H27N3O5): C, 66.80; H, 6.05; N, 9.35. Found: C, 66.72; H, 6.15; N, 9.24.
4.7. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis (ethylcarbamate) (22a)
Compound 22a was synthesized from 20a (1.0 g, 4.1 mmol), Et3N (0.5 mL), and ethylisocyanate (1.1 g, 16 mmol). Yield, 0.83 g (53%); mp 208–209 °C;1H NMR (DMSO-d
6) d 0.97 (6H, t, J = 8 Hz, 2 Me), 2.25 (3H, s, Me), 2.95 (4H, q, J = 8 Hz, CH2), 4.00 (2H, s, CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.96 (2H, s, CH2), 6.86 (2H, br s, exchangeable, NH), 7.23–7.29 (2H, m, 2 ArH), 7.34– 7.35 (2H, m, 2 ArH). Anal. Calcd for (C21H27N3O4): C, 65.44; H, 7.06; N, 10.90. Found: C, 65.29; H, 7.14; N, 10.89.
4.7.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis (ethylcarbamate) (22b)
Compound 22b was synthesized from 20b (0.5 g, 1.9 mmol), Et3N (0.5 mL), ethylisocyanate (0.55 g, 7.7 mmol). Yield, 0.39 g (50%); mp 127–129 °C. 1H NMR (DMSO-d
6) d 0.95 (6H, t, J = 7.2 Hz, 2 Me), 1.10 (3H, t, J = 7.2 Hz, Me), 2.69 (2H, q, J = 7.2 Hz, CH2), 2.97 (4H, m, CH2), 3.99 (2H, s, CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.99 (2H, s, CH2), 6.84 (2H, br s, exchange-able, NH), 7.23–7.29 (2H, m, 2 ArH), 7.34–7.39 (2H, m, 2 ArH). Anal. Calcd for (C22H29N3O4): C, 66.14; H, 7.32; N, 10.52. Found: C, 66.35; H, 7.53; N, 10.74.
4.7.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis (ethylcarbamate) (22c)
Compound 22c was synthesized from 20c (1.0 g, 3.2 mmol), Et3N (1.0 mL), and ethylisocyanate (1.2 g, 16.0 mmol). Yield, 1.17 g (81%); mp 170–171 °C;1H NMR (DMSO-d6) d 0.97 (6H, t, J = 7.2 Hz, 2 Me), 2.98 (4H, q, J = 7.2 Hz, CH2), 4.10 (2H, s, CH2), 4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.03 (2H, s, CH2), 6.94 (2H, br s, exchangeable, NH), 7.18–7.20 (1H, m, ArH), 7.26–7.29 (2H, m, 2 ArH), 7.36–7.40 (4H, m, 4 ArH), 7.42–7.44 (2H, m, 2 ArH). Anal. Calcld for (C26H29N3O4): C, 69.78; H, 6.53; N, 9.39. Found: C, 69.64; H, 6.54; N, 9.37.
4.7.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate) (22d)
Compound 22d was synthesized from 20d (1.0 g, 3.0 mmol), Et3N (1.2 mL), and (1.1 g, 15.0 mmol). Yield, 1.08 g (76%); mp 183–184 °C.1H NMR (DMSO-d6) d 0.98 (6H, t, J = 6.8 Hz, 2 Me), 2.97 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2), 4.79 (2H, s, CH2), 4.95 (2H, s, CH2), 5.02 (2H, s, CH2), 6.93 (2H, br s, exchangeable, NH), 7.20–7.22 (1H, m, ArH), 7.27–7.41 (5H, m, 5 ArH), 7.46– 7.48 (2H, m, 2 ArH). Anal. Calcd for (C26H28FN3O4): C, 67.08; H, 6.06; N, 9.03. Found: C, 67.24; H, 6.02; N, 8.88.
4.7.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate) (22e)
Compound 22e was synthesized from 20e (1.0 g, 2.9 mmol), Et3N (1.0 mL), and ethylisocyanate (0.84 g, 18.0 mmol). Yield, 0.92 g (64%); mp 191–192 °C.1H NMR (DMSO-d
6) d 0.98 (6H, t, J = 7.2 Hz, 2 Me), 2.97 (4H, q, J = 7.2 Hz, CH2), 4.09 (2H, s, CH2), 4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.02 (2H, s, CH2), 6.92 (2H, br s, exchangeable, NH), 7.18–7.22 (1H, m, ArH), 7.25–7.27 (2H, m, 2 ArH), 7.28–7.30 (1H, m, ArH), 7.37–7.39 (2H, m, 2 ArH), 7.44–7.46 (2H, m, 2 ArH). Anal. Calcd for (C26H28ClN3O4): C, 64.79; H, 5.86; N, 8.72. Found: C, 64.54; H, 5.84; N, 8.82.
4.7.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis (ethyl carbamate) (22f)
Compound 22f was synthesized from 20f (1.0 g, 2.9 mmol), Et3N (1 mL), and ethylisocyanate (0.8 g, 11.7 mmol). Yield, 1.0 g
(70%); mp 189–190 °C. 1H NMR (DMSO-d
6) d 0.98 (6H, t, J = 6.8 Hz, 2 Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 5.00 (2H, s, CH2), 5.02 (2H, s, CH2), 6.95 (2H, br s, exchangeable, NH), 7.19–7.21 (1H, m, ArH), 7.25–7.39 (4H, m, 4 ArH), 7.53–7.60 (2H, m, 2 ArH). Anal. Calcd for (C26H27F2N3O4): C, 64.59; H, 5.63; N, 8.69. Found: C, 64.50; H, 5.60; N, 8.69.
4.7.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl)bis(methylene]bis (ethylcarbamate) (22g)
Compound 22g was synthesized from 20g (1.0 g, 2.6 mmol), Et3N (1.0 mL), and ethylisocyanate (0.85 g, 10.7 mmol). Yield, 1.15 g (83%); mp 208–209 °C. 1H NMR (DMSO-d
6) d 0.98 (6H, t, J = 6.8 Hz, 2 Me), 2.97 (4H, q, J = 6.8 Hz, CH2), 4.09 (2H, s, CH2), 4.82 (2H, s, CH2), 5.02 (4H, s, 2 CH2), 6.95 (2H, br s, exchangeable, NH), 7.19–7.22 (1H, m, ArH), 7.26–7.31 (2H, m, 2 ArH), 7.39–7.45 (2H, m, 2 ArH), 7.70–7.72 (2H, m, 2 ArH). Anal. Calcd for (C26H27Cl2N3O4): C, 60.47; H, 5.27; N, 8.14. Found: C, 60.34; H, 5.13; N, 8.10.
4.7.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate) (22h)
Compound 22h was synthesized from 20h (1.0 g, 2.9 mmol), Et3N (1.0 mL), and ethylisocyanate (0.84 g, 11.8 mmol). Yield, 0.92 g (65%); mp 165–166 °C.1H NMR (DMSO-d 6) d 0.99 (6H, t, J = 6.8 Hz, 2 Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.83 (3H, s, MeO), 4.09 (2H, s, CH2), 4.78 (2H, s, CH2), 4.94 (2H, s, CH2), 5.02 (2H, s, CH2), 6.92 (2H, br s, exchangeable, NH), 7.05–7.08 (2H, m, 2 ArH), 7.19–7.22 (1H, m, ArH), 7.27–7.29 (2H, m, 2 ArH), 7.35–7.39 (3H, m, 3 ArH). Anal. Calcd for (C27H31N3O5): C, 67.91; H, 6.54; N, 8.80. Found: C, 67.73; H, 6.86; N, 8.57.
4.7.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene) bis(ethylcarbamate) (22i)
Compound 22i was synthesized from 20i (1.0 g, 2.7 mmol), Et3N (1 mL), and ethylisocyanate (0.77 g, 10.9 mmol). Yield, 0.96 g (69%); mp 124–125 °C. 1H NMR (DMSO-d 6) d 0.98 (6H, t, J = 6.8 Hz, 2 Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.77 (3H, s, MeO), 3.82 (3H, s, MeO), 4.08 (2H, s, CH2), 4.79 (2H, s, CH2), 4.98 (2H, s, CH2), 5.02 (2H, s, CH2), 6.93 (2H, br s, exchangeable NH), 6.96– 6.98 (2H, m, 2 ArH), 7.07–7.09 (1H, m, ArH), 7.18–7.22 (1H, m, ArH), 7.25–7.28 (2H, m, 2 ArH), 7.30–7.37 (1H, m, ArH). Anal. Calcd for (C28H33N3O6): C, 66.26; H, 6.55; N, 8.28. Found: C, 66.34; H, 6.45; N, 8.11.
4.7.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b] isoquinoline-1,2-diyl]bis (methylene)bis(ethylcarbamate) (22j)
Compound 22j was synthesized from 20j (0.79 g, 2.0 mmol), Et3N (0.8 mL), and ethylisocyanate (0.57 g, 8.0 mmol). Yield, 0.94 g (99%); mp 170–171 °C. 1H NMR (DMSO-d 6) d 0.97 (6H, t, J = 6.8 Hz, 2 Me), 2.98 (4H, q, J = 6.8 Hz, CH2), 3.73 (3H, s, MeO), 3.80 (6H, s, 2 MeO), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 5.02 (2H, s, CH2), 5.05 (2H, s, CH2), 6.70 (2H, s, 2 ArH), 6.95 (2H, br s, exchangeable, NH), 7.19–7.21 (1H, m, ArH), 7.23–7.26 (1H, m, ArH), 7.27–7.29 (1H, m, ArH), 7.33–7.35 (1H, m, ArH). Anal. Calcd for (C29H35N3O7): C, 64.79; H, 6.56; N, 7.82. Found: C, 64.68; H, 6.42; N, 7.97.
4.8. [3-Methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23a)
Compound 23a was synthesized from 20a (0.5 g, 2.0 mmol), Et3N (0.4 mL), and isopropylisocyanate (0.69 g, 8.0 mmol). Yield,
0.55 g (65%); mp 215–218 °C.1H NMR (DMSO-d
6) d 0.99 (12H, d, J = 6.4 Hz, 4 Me), 2.25 (3H, s, Me), 3.54 (2H, m, CH), 3.99 (2H, s, CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.96 (2H, s, CH2), 6.78 (2H, br s, exchangeable, NH), 7.25–7.29 (2H, m, 2 ArH), 7.34– 7.35 (2H, m, 2 ArH). Anal. Calcd for (C23H31N3O4): C, 66.81; H, 7.56; N, 10.16. Found: C, 66.69; H, 7.66; N, 10.10.
4.8.1. [3-Ethyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23b)
Compound 23b was synthesized from 20b (0.26 g, 1.0 mmol), Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4.0 mmol). Yield, 0.25 g (58%); mp 155–158 °C.1H NMR (DMSO-d
6) d 1.00 (12H, d, J = 6.4 Hz, 4 Me), 1.10 (3H, t, J = 7.6 Hz, Me), 2.69 (2H, q, J = 7.6 Hz, CH2), 3.55 (2H, m, CH), 3.99 (2H, s, CH2), 4.89 (2H, s, CH2), 4.93 (2H, s, CH2), 4.99 (2H, s, CH2), 6.79 (2H, br s, exchange-able, NH), 7.25–7.29 (2H, m, 2 ArH), 7.33–7.39 (2H, m, 2 ArH). Anal. Calcd for (C24H33N3O4): C, 67.42; H, 7.78; N, 9.83. Found: C, 67.11; H, 7.55; N, 9.71.
4.8.2. [3-Phenyl-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23c)
Compound 23c was synthesized from 20c (0.5 g, 1.6 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.54 g, 6.4 mmol). Yield, 0.52 g (74%); mp 175–176 °C.1H NMR (DMSO-d
6) d 0.99 (12H, d, J = 6.8 Hz, 4 Me), 3.62 (2H, m, CH), 4.10 (2H, s, CH2), 4.80 (2H, s, CH2), 4.98 (2H, s, CH2), 5.03 (2H, s, CH2), 6.86 (2H, br s, exchange-able, NH), 7.18–7.22 (1H, m, ArH), 7.26–7.28 (2H, m, 2 ArH), 7.38–7.42 (4H, m, 4 ArH), 7.49–7.51 (2H, m, 2 ArH). Anal. Calcd for (C28H33N3O4): C, 70.71; H, 6.99; N, 8.84. Found: C, 70.59; H, 6.92; N, 8.67.
4.8.3. [3-(4-Fluorophenyl)-5,10-dihydropyrrolo[1,2- b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23d)
Compound 23d was synthesized from 20d (0.5 g, 1.5 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.51 g, 6.0 mmol). Yield, 0.62 g (81%); mp 190–191 °C.1H NMR (DMSO-d
6) d 0.99 (12H, d, J = 6.4 Hz, 4 Me), 3.60 (2H, m, CH), 4.09 (2H, s, CH2), 4.79 (2H, s, CH2), 4.95 (2H, s, CH2), 5.02 (2H, s, CH2), 6.86 (2H, br s, exchange-able, NH), 7.18–7.22 (1H, m, ArH), 7.26–7.29 (5H, m, 5 ArH), 7.31–7.33 (2H, m, 2 ArH). Anal. Calcd for (C28H32FN3O4): C, 68.14; H, 6.53; N, 8.51. Found: C, 68.31; H, 6.40; N, 8.60.
4.8.4. [3-(4-Chlorophenyl)-5,10-dihydropyrrolo[1,2- b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23e)
Compound 23e was synthesized from 20e (0.45 g, 1.3 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.42 g, 5.0 mmol). Yield, 0.43 g (64%); mp 191–192 °C.1H NMR (DMSO-d
6) d 1.01 (12H, d, J = 6.4 Hz, 4 Me), 3.57 (2H, m, CH), 4.09 (2H, s, CH2), 4.80 (2H, s, CH2), 4.97 (2H, s, CH2), 5.02 (2H, s, CH2), 6.86 (2H, br s, exchange-able, NH), 7.18–7.22 (1H, m, ArH), 7.25–7.30 (2H, m, 2 ArH), 7.37–7.39 (1H, m, ArH), 7.46 (2H, d, J = 8.0 Hz, 2 ArH), 7.55 (2H, d, J = 8.0 Hz, 2 ArH). Anal. Calcd for (C28H32ClN3O4): C, 65.94; H, 6.32; N, 8.24. Found: C, 65.76; H, 6.17; N, 8.35.
4.8.5. [3-(3,4-Difluorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23f)
Compound 23f was synthesized from 20f (0.5 g, 1.4 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.49 g, 5.8 mmol). Yield, 0.62 g (83%); mp 176–178 °C.1H NMR (DMSO-d
6) d 1.02 (12H, d, J = 6.4 Hz, 4 Me), 3.58 (2H, m, CH), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 5.00 (2H, s, CH2), 5.02 (2H, s, CH2), 6.87 (2H, br s, exchange-able, NH), 7.19–7.21 (1H, m, ArH), 7.26–7.30 (3H, m, 3 ArH), 7.37–7.39 (1H, m, ArH), 7.51–7.59 (2H, m, 2 ArH). Anal. Calcld
for (C28H31F2N3O4): C, 65.74; H, 6.11; N, 8.21. Found: C, 65.51; H, 6.05; N, 8.38.
4.8.6. [3-(3,4-Dichlorophenyl)-5,10-dihydropyrrolo[1,2-
b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23g)
Compound 23g was synthesized from 20g (0.5 g, 1.3 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.45 g, 5.3 mmol). Yield, 0.6 g (82%); mp 192–193 °C.1H NMR (DMSO-d
6) d 1.01 (12H, d, J = 6.4 Hz, 4 Me), 3.57 (2H, m, CH), 4.09 (2H, s, CH2), 4.81 (2H, s, CH2), 5.02 (4H, s, 2 CH2), 6.87 (2H, br s, exchangeable, NH), 7.19–7.21 (1H, m, ArH), 7.25–7.27 (2H, m, 2 ArH), 7.30–7.37 (1H, m, ArH), 7.43–7.45 (1H, m, ArH), 7.69 (1H, s, ArH) 7.73–7.75 (1H, m, ArH). Anal. Calcd for (C28H31N3O4Cl2): C, 61.77; H, 5.74; N, 7.72. Found: C, 61.84; H, 5.67; N, 7.56.
4.8.7. [3-(4-Methoxyphenyl)-5,10-dihydropyrrolo[1,2- b]isoquinoline-1,2-diyl]bis(methylene)bis(iso-propylcarbamate) (23h)
Compound 23h was synthesized from 20h (0.5 g, 1.4 mmol), Et3N (0.5 mL), and isopropylisocyanate (0.5 g, 5.9 mmol). Yield, 0.49 g (65%); mp 199–200 °C.1H NMR (DMSO-d 6) d 1.00 (12H, d, J = 6.0 Hz, 4 Me), 3.57 (2H, m, CH), 3.82 (3H, s, MeO), 4.07 (2H, s, CH2), 4.77 (2H, s, CH2), 4.92 (2H, s, CH2), 5.01 (2H, s, CH2), 6.85 (2H, br s, exchangeable, NH), 7.04–7.06 (2H, m, 2 ArH), 7.17– 7.21 (1H, m, ArH), 7.25–7.28 (2H, m, 2 ArH), 7.34–7.36 (3H, m, 3 ArH). Anal. Calcd for (C29H35N3O5): C, 68.89; H, 6.98; N, 8.31. Found: C, 68.93; H, 6.96; N, 8.14.
4.8.8. [3-(3,4-Dimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis(methylene)
bis(iso-propylcarbamate) (23i)
Compound 23i was synthesized from 20i (0.37 g, 1.0 mmol), Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4 mmol). Yield, 0.30 g (56%); mp 188–189 °C.1H NMR (DMSO-d
6) d 1.01 (12H, d, J = 6.0 Hz, 4 Me), 3.57 (2H, m, CH), 3.77 (3H, s, MeO), 3.82 (3H, s, MeO), 4.08 (2H, s, CH2), 4.78 (2H, s, CH2), 4.98 (2H, s, CH2), 5.01 (2H, s, CH2), 6.84 (2H, br s, exchangeable, NH), 6.95–6.97 (2H, m, 2 ArH), 7.06–7.08 (1H, m, ArH), 7.18–7.21 (1H, m, ArH), 7.25–7.29 (2H, m, 2 ArH), 7.30–7.37 (1H, m, ArH). Anal. Calcd for (C30H37N3O6): C, 67.27; H, 6.96; N, 7.84. Found: C, 67.27; H, 7.06; N, 7.62.
4.8.9. [3-(3,4,5-Trimethoxyphenyl)-5,10-dihydropyrrolo[1,2-b]isoquinoline-1,2-diyl]bis
(methylene)bis(iso-propylcarbamate) (23j)
Compound 23j was synthesized from 20j (0.39 g, 1.0 mmol), Et3N (0.4 mL), and isopropylisocyanate (0.34 g, 4 mmol). Yield, 0.32 g (57%); mp 189–190 °C.1H NMR (DMSO-d
6) d 1.01 (12H, d, J = 6.4 Hz, 4 Me), 3.57 (2H, m, CH), 3.73 (3H, s, MeO), 3.80 (6H, s, 2 MeO), 4.08 (2H, s, CH2), 4.79 (2H, s, CH2), 5.01 (2H, s, CH2), 5.05 (2H, s, CH2), 6.69 (2H, s, 2 ArH), 6.87 (2H, br s, exchangeable, NH), 7.19–7.29 (2H, m, 2 ArH), 7.33–7.39 (2H, m, 2 ArH). Anal. Calcld for (C31H39N3O7): C, 65.82; H, 6.95; N, 7.43. Found: C, 65.68; H, 6.74; N, 7.11.
4.9. Biological experiments 4.9.1. Cytotoxicity assays
The effects of the newly synthesized compounds on cell growth were determined in T-cell acute lymphocytic leukemia CCRF-CEM) and their resistant subcell lines (CCRF-CEM/Taxol and CCRF-CEM/ VBL) by the XTT assay20and human solid tumor cells (i.e., breast car-cinoma MX-1 and colon carcar-cinoma HCT-116) the SRB assay21in a 72 h incubation using a microplate spectrophotometer as described previously.22 After the addition of phenazine methosulfate-XTT
solution at 37 °C for 6 h, absorbance at 450 and 630 nm was detected on a microplate reader (EL 340; Bio-Tek Instruments Inc., Winooski, VT). The cytotoxicity of the newly synthesized compounds against non-small cell lung cancer H1299, human prostate cancer PC3, oral carcinoma OECM1 and human glioma U87 were determined by the Alamar blue assay23in a 72 h incubation using a microplate spectro-photometer as described previously. After the addition of Alamar blue solution, it was incubated at 37 °C for 6 h. Absorbance at 570 and 600 nm was detected on a microplate reader. IC50values were determined from dose-effect relationship at six or seven concentra-tions of each drug by using the CompuSyn software by Chou and Martin24based on the median-effect principle and plot.25,26Ranges given for Taxol and vinblastine were mean ± SE (n = 4).
4.9.2. In vivo studies
Athymic nude mice bearing the nu/nu gene were used for human breast tumor MX-1 and human ovarian adenocarcinoma SK-OV-3 xenograft. Outbred Swiss-background mice were obtained from the National Cancer Institute (Frederick, MD). Male mice 8 weeks old or older weighing about 22 g were used for the experiments. Drug was administrated via the tail vein by iv injection.22Tumor vol-umes were assessed by measuring length width height (or width) by using caliper. Vehicle used was DMSO (50
l
L) and Tween 80 (40l
L) in saline (160l
L). The maximal tolerable dose of the tested compound was determined and applied for the in vivo antitu-mor activity assay. All animal studies were conducted in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Animals and the protocol approved by the Memorial Sloan-Kettering Cancer Center’s Institutional Animal Care and Use Committee.4.9.3. Alkaline agarose gel shift assay
Formation of DNA cross-linking was analyzed by alkaline aga-rose gel electrophoresis. In brief, purified pEGFP-N1 plasmid DNA (1.5
l
g) was mixed with various concentrations (1–20l
M) of 20a, 22a, 20h and 23h in 40l
L binding buffer (3 mM sodium chlo-ride/1 mM sodium phosphate, pH 7.4, and 1 mM EDTA). The reac-tion mixture was incubated at 37 °C for 2 h. At the end of reacreac-tion, the plasmid DNA was linearized by digestion with BamHI and fol-lowed by precipitation with ethanol. The DNA pellets were dis-solved and denatured in alkaline buffer (0.5 N NaOH–10 mM EDTA). An aliquot of 20l
L of DNA solution (1l
g) was mixed with a 4l
L of 6 X alkaline loading dye and then electrophoretically resolved on a 0.8% alkaline agarose gel with NaOH–EDTA buffer at 4 °C. The electrophoresis was carried out at 18 V for 22 h. After staining the gels with an ethidium bromide solution, and the DNA was then visualized under UV light.4.9.4. Flow cytometric analysis
The effects of 20a on cell cycle distribution were analyzed with a flow cytometer as previously described.12 Briefly, human non-small cell lung carcinoma H1299 cells were treated with 20a at 1.25, 2.5, and 5
l
M for 24 h. The attached cells were thentrypsinized, washed with phosphate buffer saline (PBS), and fixed with ice-cold 70% ethanol for 30 min. The cells were stained with 4
l
g/ml propidium iodide (PI) in PBS containing 1% Triton X-100 and 0.1 mg/ml RNase A. The stained cells were then analyzed using the FACS SCAN flow cytometer (Becton Dickinson, San Joes, CA, USA). The percentage of the cells in each cell cycle phase was determined using the ModFit LT 2.0 software based on the DNA histograms.Acknowledgments
We are grateful to the National Science council (Grant No. NSC 99-2320-B-001-014) and Academia Sinica (Grant No. AS-96-TP-B06) for financial support. T.-C. Chou was supported by the Sloan-Kettering Institute General Fund. The NMR spectra of synthe-sized compounds were obtained at High-Field Biomolecular NMR Core Facility supported by the National Research Program for Genomic Medicine (Taiwan). We would like to thank Dr. Shu-Chuan Jao in the Institute of Biological Chemistry (Academia Sinica) for providing the NMR service and the National Center for High-performance computing for computer time and facilities. References and notes
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3. Oostveen, E. A.; Speckmap, W. N. Tetrahedron 1987, 43, 255. 4. Tomasz, M.; Palom, Y. Pharmacol. Ther. 1997, 76, 73.
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12. Kakadiya, R.; Dong, H.; Lee, P.-C.; Kapuriya, N.; Zhang, X.; Chou, T.-C.; Lee, T.-C.; Kapuriya, K.; Shah, A.; Su, T.-L. Bioorg. Med. Chem. 2009, 17, 5614.
13. Lee, P.-C.; Kakadiya, R.; Su, T.-L.; Lee, T.-C. Neoplasia 2010, 12, 376. 14. Dean, R. T.; Rapoport, H. J. Org. Chem. 1978, 43, 2115.
15. Shinkai, H.; Toi, K.; Kumashiro, I.; Seto, Y.; Fukuma, M.; Dan, K.; Toyoshima, S. J. Med. Chem. 1988, 31, 2092.
16. Grunewald, G. L.; Romero, F. A.; Criscione, K. R. J. Med. Chem. 2005, 48, 134. 17. Anderson, W. K.; McPherson, H. L.; New, J. S. J. Heterocycl. Chem. 1980, 17, 513. 18. Hartley, J. A.; Berardini, M. D.; Souhami, R. L. Anal. Biochem. 1991, 193, 131. 19. O’Connell, M. J.; Walworth, N. C.; Carr, A. M. Trends Cell Biol. 2000, 10, 296. 20. Scudiero, D. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Tierney, S.; Nofziger,
T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R. Cancer Res. 1988, 48, 4827. 21. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
Warren, J. T.; Bokesch, H.; Kenny, S.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1107.
22. Chou, T.-C.; O’Connor, O. A.; Tong, W. P.; Guan, Y.; Zhang, Z.-G.; Stachel, S. J.; Lee, C.; Danishefsky, S. J. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8113.
23. Al-Nasiry, S.; Hanssens, M.; Luyten, C.; Pijnenborn, R. Hum. Reprod. 2007, 22, 1304.
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![Table 1 shows the antiproliferative activities of the newly syn- syn-thesized bis(hydroxylmethyl)-5,10-dihydropyrrolo[1,2-b]isoquin-oline derivatives (20) and their bis(alkylcarbamate) derivatives (21–23) against human lymphoblastic leukemia (CCRF-CEM) an](https://thumb-ap.123doks.com/thumbv2/9libinfo/8978250.281051/2.892.118.754.97.645/antiproliferative-activities-hydroxylmethyl-dihydropyrrolo-derivatives-alkylcarbamate-derivatives-lymphoblastic.webp)
