Synthesis of bisindolylmaleimides related to GF109203x and their efficient conversion to the bioactive indolocarbazoles

Sudipta Roy,a Alan Eastmanb and Gordon W. Gribble*a

Received 26th May 2006, Accepted 30th June 2006
First published as an Advance Article on the web 21st July 2006 DOI: 10.1039/b607504e

From a structure–activity relationship perspective, the new indolocarbazoles 11 and 12 have been synthesized and evaluated biologically as novel Chk1 inhibitors. Compounds 11 and 12 were synthesized in high yield from indole via bisindolylmaleimides 18 and 24.

Introduction
Cell cycle checkpoints are activated in response to DNA damage thereby delaying cell cycle progression in order to provide more time for DNA repair. Cell cycle arrest in G1 or S phase prevents replication of damaged DNA, while arrest at G2 prevents damaged chromosomes from being segregated in mitosis; thus preventing the propagation of genetic abnormalities. Inhibition of the G2 checkpoint has attracted widespread interest because most cancer cells have an inoperative G1 checkpoint. The activity of the G1 checkpoint is dependent on the p53 tumor suppressor protein which is deleted or mutated in more than 50% of all cancers. Although cells with defective p53 are unable to activate the G1 checkpoint in response to DNA damage, they retain the ability to arrest in S and G2. This provides the cells with an opportunity to repair their DNA and thereby survive and grow. The S and G2 checkpoints are regulated by various kinases among which checkpoint kinase 1 (Chk1) plays a major role. Inhibitors of Chk1 preferentially abrogate cell cycle arrest in p53-defective cells and selectively sensitize cancer cells with mutated p53 to killing by DNA-damaging agents. Therefore, combining a Chk1 inhibitor with a DNA damaging agent should selectively drive p53-defective cells into a premature and lethal mitosis.1
UCN-01 (1), the synthetic 7-hydroxy derivative of the non- selective PKC inhibitor staurosporine (2),2 generated considerable interest in our laboratory when it was found to be a potent inhibitor of DNA damage-induced S and G2 cell cycle checkpoints, which led to increased killing of tumor cells (Fig. 1).3 Although UCN-01 is well recognized as a protein kinase C inhibitor,4 this checkpoint inhibition was attributed to its ability to inhibit Chk1.5 Unfortu- nately, UCN-01 binds avidly to human serum proteins thereby compromising its potential therapeutic activity.6 Accordingly, we screened other indolocarbazoles to identify analogues with improved therapeutic potential. Initially, a K252a (3) analogue, ICP-1 (4), was synthesized and tested, and was found to overcome the problem of protein binding but it had considerably reduced potency.7
More recently, we found that G¨o6976 (5) is a very potent check- point inhibitor even in the presence of human serum,8 and this has also been attributed to the inhibition of Chk1.9 Additionally,

aDepartment of Chemistry, Dartmouth College, Hanover, NH 03755, USA. E-mail: [email protected]; Fax: 603-646-3946; Tel: 603-646-3118 bDepartment of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755, USA
G¨o6976 (5) abrogated S and G2 arrest at a concentration sub- stantially lower than that required to inhibit PKC. Interestingly, UCN-01 (1) did not demonstrate this selectivity for checkpoint inhibition. Accordingly, we initiated a synthetic program to develop novel analogues rationally designed to overcome the obstacles observed with the other analogues. During our screening to identify novel inhibitors of Chk1, we found that ICP-103 (6) is also a potent checkpoint inhibitor.10 Therefore, we have focused our investigation on this class of molecules as potential inhibitors of Chk1. We synthesized two nitrile analogues of ICP-103, ICP- 106 (7) and ICP-109 (8), with variable lengths of the nitrile arm to investigate the effect of the nitrile chain-length on Chk1 activity (Fig. 2).11 We find that ICP-106 (7) and ICP-109 (8) are less potent than ICP-103 (6) at abrogating DNA damage-induced cell cycle arrest. From this activity data for the nitrile analogues 7 and 8, it was found that a three-carbon nitrile chain provided maximum activity. We then synthesized a novel amide analogue of ICP-103, ICP-112 (9), bearing the same number of carbons in the amide arm.11 However, in this latter study we found that a cyano group is the more desirable functionality than an amide for activity since ICP-112 (9) was found to be less active than ICP-103 (6).
In the course of our structure–activity relationship studies on ICP-103 analogues, we have synthesized and tested two new amine analogues related to the known PKC inhibitor GF109203x12 (G¨o6850, 10), ICP-121 (11) and ICP-125 (12) (Fig. 3). We wanted to further explore the SAR by replacing the nitrile with an amine. We decided to maintain the same three-carbon spacer between the indole nitrogen and the functional group nitrogen as in ICP-103 (6) due to its high activity compared to ICP-106 (7) and ICP-109 (8).
We herein describe the synthesis of compounds 11 and 12, and briefly report the biological activity of these two novel indolocarbazoles.

Results and discussion
Towards the synthesis of 11, we alkylated the indole nitrogen with 1,3-dibromopropane13 in the presence of KOH to furnish 13 (Scheme 1). A small amount (10%) of 1-allylindole was also recovered from the reaction mixture. Compound 13 was then treated with di-tert-butyl-iminodicarboxylate and caesium carbonate to produce fully protected amine 14 in 99% yield. The key starting material 14 can be prepared alternatively using 15 with sodium hydride in DMF–THF. Compound 15 was synthesized

Fig. 1 Indolocarbazoles 1–4.

Fig. 2 G¨o6976 analogues.

Fig. 3 GF109203x analogues.

from 1,3-dibromopropane and di-tert-butyl-iminodicarboxylate in the presence of sodium hydride.14 N-Alkylation of indole-3- acetic acid using methyl iodide in the presence of excess sodium hydride gave 1-methylindole-3-acetic acid (16) in 94% yield.10
Amine 14 was treated with oxalyl chloride in dichloromethane to furnish the glyoxylyl chloride which was immediately treated with 1-methylindole-3-acetic acid (16) in the presence of triethylamine to produce anhydride 17 in 30% yield in two steps from 14 (Scheme 2).15 Loss of one Boc group was observed during this
reaction sequence. The anhydride 17 was subsequently converted to the imide 18 by exposure to HMDS and MeOH in DMF in 99% yield.16 During our synthesis of ICP-106 (7), we found that the final oxidative cyclization was quite challenging for the bisindolylmaleimide with substituents present on both N-12 and N-13.17 Low yields are registered in most cases and the isolation of the final product is difficult from the complex reaction mixture. However, in some cases, palladium(II) trifluoroacetate was found to be superior for this cyclization.11 To our delight, heating

Scheme 1

Scheme 2

bisindolylmaleimide 18 in DMF in the presence of palladium(II) trifluoroacetate gave 19 in 80% yield. Finally, deprotection of the Boc-group using an ethereal solution of 1M HCl in methanol gave the target compound 11 in essentially quantitative yield.
Our target compound 12 was synthesized in a similar fashion. Boc-Protection of 3-bromopropylamine hydrobromide furnished 20 in 92% yield (Scheme 3).18 Compound 20 was then used to alkylate indole to give compound 21. Compound 21 was methylated with iodomethane in the presence of sodium hydride to produce 22. Fully protected amine 22 was subjected to the coupling reaction with 1-methylindole-3-acetic acid (16) which produced the desired anhydride 23 in 32% yield. Conversion of anhydride 23 to imide 24 was achieved using HMDS and MeOH in 99% yield. Bisindolylmaleimide 24 was then subjected to the challenging oxidative cyclization reaction using palladium(II) trifluoroacetate. To our extreme satisfaction, we obtained the desired product 25 in excellent yield. This is the highest yield we have obtained so far for this otherwise capricious cyclization step. Finally, deprotection of the Boc group furnished the target compound 12.
In work to be reported separately, we find that ICP-125 (12) exhibits high potency in an assay using flow cytometry analysis. Thus, ICP-125 (12) abrogates S phase arrest at 100 nM indicating the compound is inhibiting Chk1. However, ICP-121 (11) was tested up to 10 lM and found to be inactive in the same assay. These values can be compared to the efficacy of G¨o6976 (5) of 30 nM and efficacy of ICP-103 (6) of 100 nM in the same assay.
In summary, we have synthesized ICP-125 (12) and find it to be a potent Chk1 inhibitor. We have found that Pd(II) catalyzed ox- idative cyclization is much more effective for bisindolylmaleimides bearing an amine group. Also, we find that a secondary amine or a nitrile are more desirable than a primary amine or amide on the chain. Work is in progress in our laboratory with other nitrogen- bearing functionalities and these will be reported in due course.
Experimental
Melting points were determined with a Mel-Temp Laboratory Device apparatus and are uncorrected. IR spectra were recorded
1 H-and 13 C-NMR spectra were recorded on either a Varian XL-300 or 500 Fourier transform NMR spectrometer. Both low- and high resolution mass spectra were carried out at the Mass Spectrometry Laboratory, School of Chemical Sciences, University of Illinois at Urbana Champaign. Anhydrous THF and CH2 Cl2 were prepared by a solvent purification system. All other solvents (analytical grade) including anhydrous solvents and reagents were used as received. All experiments were performed under a nitrogen atmosphere unless otherwise stated.

1-(3-Bromopropyl)-1H -indole (13)

To a stirred solution of indole (1.17 g, 10 mmol) and freshly powdered KOH (88%, 0.64 g, 10 mmol) in DMF (25 mL) was added 1,3-dibromopropane (6.06 g, 30 mmol) in DMF (25 mL) in one portion. The mixture was stirred at rt for 24 h. Water (100 mL) was added and extracted with ether (3 × 75 mL). The organic phase was washed with water (100 mL), dried (MgSO4 and concentrated in vacuo. The residue was purified by column chromatography on silica gel (initially pet. ether; then 15 : 1 pet. ether : ether) to furnish the desired product (1.21 g, 51%) as a colorless oil: IR (thin film): 3051, 2939, 1611, 1511, 1461, 1314,
1 ; 1 H-NMR (CDCl3 ): d 7.71–7.73 (m, 1H), 7.45 (d, 1H, J = 8.3 Hz), 7.29–7.32 (m, 1H), 7.19–7.22 (m, 2H),
6.58(d, 1H, J = 2.9 Hz), 4.38 (t, 2H, J = 6.3 Hz), 3.35 (t, 2H,
13 C-NMR (CDCl3 ): d 135.9, 128.8, 128.1, 121.8, 121.2, 119.6, 109.4, 101.6, 44.0, 32.8, 30.7.

Scheme 3

A small amount of 1-allylindole was recovered as a colorless oil19 (0.16 g, 10%) which came in earlier fractions during column
1 H-NMR (CDCl3 ): d 7.78–7.82 (m, 1H), 7.44– 7.47 (m, 1H), 7.33–7.38 (m, 1H), 7.24–7.30 (m, 1H), 7.20 (d, 1H, J = 3.2 Hz), 6.04–6.16 (m, 1H), 5.29–5.34 (m, 1H), 5.16–
13 C-NMR (CDCl3 ): d 136.2, 133.6, 128.8, 128.0, 121.7, 121.1, 119.6, 117.3, 109.7, 101.5, 48.9.

Bis(1,1-dimethylethyl)(3-bromopropyl)imidodicarbonate (15)

To a stirred solution of di-tert-butyl-iminodicarboxylate (1.09 g,
5mmol) in THF : DMF (40 mL, 3 : 1) was added sodium hydride (60% dispersion in mineral oil, 0.21 g, 5.25 mmol). The mixture was heated at 65 ◦ C for 2.5 h, 1,3-dibromopropane (2.3 mL, 22.5 mmol) was added, and the mixture was stirred for 3 h. It was cooled to 0 ◦ C and ether (50 mL) was added. Excess hydride was destroyed by the dropwise addition of water. The organic phase was washed with water (2 × 50 mL) and dried (MgSO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (initially 98 : 2 hexanes : ether; then 1 : 1 hexanes : ether) to yield the desired product (1.06 g, 63%) as a colorless oil: IR (thin
1 ; 1 H-NMR
(CDCl3 ): d 3.68–3.73 (m, 2H), 3.39 (t, 2H, J = 6.7 Hz), 2.08–2.18
13 C-NMR (CDCl3 ): d 152.6, 82.7, 45.4, 32.3, 30.6, 28.2.

Bis(1,1-dimethylethyl)[3-(1H -indolyl)propyl]imidodicarbonate (14) From 13. Compound 13 (0.26 g, 1.1 mmol), di-tert-butyl-
iminodicarboxylate (0.22 g, 1 mmol) and caesium carbonate (0.33 g, 1 mmol) in DMF (10 mL) were stirred for 6 h at 70 ◦ C. Water (40 mL) was added and extracted with ethyl acetate (3 × 25 mL). The organic phase was washed with water (2 × 25 mL) and dried (Na2 SO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (4 : 1 hexanes : ethyl acetate) to furnish the desired product (0.37 g, 99%) as a colorless oil.
From 15. To a stirred suspension of NaH (60% dispersion in mineral oil, 0.22 g, 5.5 mmol) in DMF (15 mL) at 0 ◦ C was added dropwise a solution of indole (0.41 g, 3.5 mmol) in DMF (10 mL). After stirring the mixture at 0 ◦ C for 30 min, a solution of 15 (1.59 g, 4.7 mmol) in DMF : THF (20 mL, 1 : 1) was added. The mixture was allowed to reach rt and stirring was continued for 36 h. It was then cooled to 0 ◦ C and water (50 mL) was added very slowly. The aqueous layer was extracted with ethyl acetate (2 × 50 mL). The organic phase was washed with water (3 × 50 mL) and brine (50 mL), and dried (Na2 SO4 ). The crude product was

purified by column chromatography on silica gel (4 : 1 hexanes : ethyl acetate) to yield the desired product (0.65 g, 50%) as a colorless oil.
IR (thin film): 2977, 2933, 1787, 1745, 1696, 1366, 1146, 1126,
1 ; 1 H-NMR (CDCl3 ): d 7.62–7.64 (m, 1H), 7.33 (d, 1H, J = 8.3 Hz), 7.19–7.22 (m, 1H), 7.13 (d, 1H, J = 2.9 Hz), 7.08–7.12 (m, 1H), 4.16 (t, 2H, J = 7.1 Hz), 3.65 (t, 2H, J = 7.1 Hz), 2.14
13 C-NMR (CDCl3 ): d 152.6, 136.0, 128.8, 127.8, 121.6, 121.2, 119.4, 109.3, 101.4, 82.7, 44.3,
+ ), 218, 201, 173, 144,

1,1-Dimethylethyl(3-{3-[2,5-dihydro-4-(1-methyl-1H -indol-3-yl)- 2,5-dioxo-1H -pyrrol-3-yl]-1H -indol-1-yl}propyl)carbamate (18)
To a stirred solution of 17 (0.25 g, 0.5 mmol) in DMF (2.5 mL) at rt was added HMDS (1.1 mL, 5 mmol) and methanol (0.1 mL, 2.5 mmol). The flask was tightly sealed and the mixture was stirred at rt for 24 h. The mixture was poured into water (25 mL) and extracted with ethyl acetate (2 × 25 mL) and dried (MgSO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (95 : 5 dichloromethane : methanol)

130 (100%); HRMS (EI): calcd for C 374. 2197.

21

30

2
4 : 374.2206, found:
to yield the desired product (246 mg, 99%) as a dark red solid: mp 112–114 ◦ C; IR (thin film): 2969, 1755, 1703, 1610, 1531, 1333,

1-Methyl-1H -indole-3-acetic acid (16)

To a stirred suspension of NaH (6.0 g, 150 mmol, 60% mineral oil dispersion) in THF (125 mL) at 0 ◦ C was added a solution
1 ; 1 H-NMR (DMSO-d6 ): d 10.93 (s, 1H), 7.86 (s, 1H), 7.76 (s, 1H), 7.45 (d, 1H, J = 8.2 Hz), 7.40 (d, 1H, J = 8.2 Hz), 6.97–7.04 (m, 3H), 6.89 (d, 1H, J = 7.9 Hz), 6.61–6.70 (m, 3H), 4.23 (t, 2H, J = 6.7 Hz), 3.86 (s, 3H), 2.90 (q, 2H, J = 6.1 Hz), 1.83 (qn, 2H, J = 6.6 Hz, 1.38 (s, 9H); LRMS (ESI+): m/z 521

of indole-3-acetic acid (5.25 g, 30 mmol) in THF (50 mL). After stirring the mixture for 30 min at 0 ◦ C, a solution of methyl iodide (14.2 g, 100 mmol) in THF (50 mL) was added dropwise. The mixture was allowed to slowly reach rt and stirring was continued
+ , 499 [M + H]+ ; HRMS (ESI+): calcd for C29
[M + H]: 499.2345, found: 499.2341.
31
4
4

for 16 h. The reaction mixture was then cooled to 0 ◦ C and excess hydride was carefully destroyed by slow addition of MeOH (5 mL) with vigorous stirring, followed by cold water until a clear yellow solution resulted. Ether (100 mL) was added. The aqueous phase was separated, acidified with 6 N HCl and extracted with dichloromethane (3 × 100 mL). The combined dichloromethane extracts were dried (Na2 SO4 and concentrated to about 40– 50 mL. Pet. ether was then added slowly until a brownish colored solid completely precipitated out. The crude solid was recrystallized from ethanol to give the desired product (5.33 g, 94%) as a pale brown solid: mp 127–128 ◦ C (lit20 127–129 ◦ C); IR
1 ; 1 H-NMR (CDCl3 ): d 7.64–7.62 (m, 1H), 7.34–7.26 (m, 2H), 7.19–7.15 (m, 1H), 7.07 (s,
13 C-NMR (CDCl3 ): d 178.8, 137.0, 128.1, 127.7, 122.0, 119.5, 119.1, 109.5, 106.2, 32.9, 31.2.

1,1-Dimethylethyl(3-{3-[2,5-dihydro-4-(1-methyl-1H -indol-3-yl)- 2,5-dioxo-3-furanyl]-1H -indol-1-yl}propyl)carbamate (17)
To a stirred solution of 14 (0.37 g, 1.0 mmol) in dichloromethane (10 mL) at 0 ◦ C was added dropwise oxalyl chloride (0.09 mL, 1.05 mmol). After stirring for 45 min at 0 ◦ C, the solvent was evap- orated in vacuo. The residue was redissolved in dichloromethane (10 mL) and added dropwise to a stirred solution of 16 (0.19 g, 1.0 mmol) and triethylamine (0.28 mL, 2 mmol) in dichloromethane (5 mL) at rt. The mixture was stirred at rt for 10 h. The solvent was evaporated and the crude residue was purified by column chromatography on silica gel (98 : 2 dichloromethane : methanol) to furnish the desired product (149 mg, 30%) as a dark red solid: mp 99–101 ◦ C; IR (thin film): 2977, 1817, 1750, 1704,
1 ; 1 H-NMR (DMSO-d6 ): d 7.98 (s, 1H), 7.88 (s, 1H), 7.52 (d, 1H, J = 8.2 Hz), 7.47 (d, 1H, J = 8.2 Hz), 7.07–7.11 (m, 2H), 6.97–7.00 (m, 2H), 6.78 (t, 1H, J = 7.6 Hz), 6.69–6.73 (m, 2H), 4.25 (t, 2H, J = 6.7 Hz), 3.89 (s, 3H), 2.90 (q, 2H, J = 6.4 Hz), 1.83 (qn, 2H, J = 6.7 Hz), 1.38 (s, 9H); LRMS
+ ), 425, 399 (100%), 356, 312, 283, 269, 249, 191, 158, 107, 77; HRMS (EI): calcd for C29 29 3 5 : 499.2107, found: 499.2112.
1,1-Dimethylethyl[12-(3-aminopropyl)-12,13-dihydro-13-methyl- 5H -indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(6H )- dione]carbamate (19)
A mixture of 18 (25 mg, 0.05 mmol) and palladium(II) triflu- oroacetate (84 mg, 0.25 mmol) in DMF (3 mL) was heated at 90 ◦ C for 2.5 h. The mixture was cooled to rt and ethyl acetate (25 mL) was added. The organic phase was washed with 0.5 N HCl (50 mL), water (50 mL) and dried (Na2 SO4 ). The solution was filtered through Hyflo and purified by column chromatography on silica gel (95 : 5 dichloromethane : methanol) to yield the desired product (20 mg, 80%) as a yellow fluorescent solid: mp 213–215 ◦ C (dec); IR (thin film): 3203, 1755, 1698, 1575, 1450, 1317, 1163,
1 ; 1 H-NMR (DMSO-d6 ): d 11.12 (s, 1H), 9.10–9.14 (m, 2H), 7.87 (d, 1H, J = 8.2 Hz), 7.75 (d, 1H, J = 8.2 Hz), 7.60–7.67 (m, 2H), 7.38–7.43 (m, 2H), 6.76 (t, 1H, J = 5.3 Hz), 4.79 (t, 2H, J = 7.5 Hz), 4.21 (s, 3H), 2.65 (q, 2H, J = 6.1 Hz), 1.67 (qn,
+ ; HRMS (ESI+): calcd for C29 29 4 4 [M + H]: 497.2189, found: 497.2169.

12-(3-Aminopropyl)-12,13-dihydro-13-methyl-5H -indolo[2,3-a]- pyrrolo[3,4-c]carbazole-5,7-(6H )-dione hydrochloride (11)
To a solution of 19 (15 mg, 0.03 mmol) in methanol (2 mL) at rt was added dropwise 1 M HCl in ether (9 mL). The mixture was stirred at rt for 5 h. The solvent was evaporated and the residue was recrystallized from methanol to furnish the desired product
1H-NMR (DMSO-d6 ): d 11.18 (s, 1H), 9.16 (d, 1H, J = 7.6 Hz), 9.13 (d, 1H, J = 7.9 Hz), 7.97 (d, 1H, J = 8.5 Hz), 7.80 (d, 1H, J = 8.2 Hz), 7.65–7.70 (m, 5H), 7.44 (t, 2H, J = 7.6 Hz), 4.89 (t, 2H, J = 7.3 Hz), 4.25 (s, 3H), 2.46–2.49 (m, 2H), 1.78 (qn, 2H, J = 7.6 Hz); 13 C-NMR (DMSO-d6 ): d 170.8, 170.7, 144.9, 143.7, 133.3, 131.8, 127.7, 127.5, 124.9, 124.5, 122.9, 121.8, 121.5, 121.2, 121.1, 120.1, 119.6, 118.7, 112.4, 111.4, 45.6, 36.8; LRMS (ESI+): m/z 397
+ ; HRMS (ESI+): calcd for C24 21 4 2 [(M - HCl) + H]: 397.1665, found: 397.1662.

1,1-Dimethylethyl(3-bromopropyl)carbamate (20)

To a stirred solution of 3-bromopropylamine hydrobromide (2.20 g, 10 mmol) and Boc-anhydride (2.18 g, 10 mmol) in dichloromethane (50 mL) at 0 ◦ C was added dropwise triethy-

J = 8.3 Hz), 7.24–7.30 (m, 1H), 7.14–7.20 (m, 2H), 6.56 (d, 1H, J = 2.7 Hz), 4.18 (t, 2H, J = 7.2 Hz), 3.32 (t, 2H, J = 6.8 Hz), 2.88 (s,
13 C-NMR (CDCl3 ): d 156.1, 136.1, 129.0, 127.9, 121.8, 121.3, 119.6, 109.5, 101.5, 79.9, 46.7,
+ ), 232, 215, 156, 144, 131

lamine (2.89 mL, 20 mmol). The solution was stirred at the same temperature for 15 min. The mixture was allowed to reach rt and stirred for 7 h. The mixture was washed with water (2 × 50 mL)
(100%), 117; HRMS (EI): calcd for C17 288.1844.

24

2
2: 288.1838, found:

and dried (Na2 SO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (initially hexanes; then 1 : 1 hexanes : ethyl acetate) to furnish the desired product (2.19 g, 92%) as a colorless oil which slowly solidified at
-4 ◦ C: mp: 38–40 ◦ C (lit.21 33–34 ◦ C); IR (thin film): 3252, 2969,
1 ; 1 H-NMR (CDCl3 ): d
3.44 (t, 2H, J = 6.6 Hz), 3.27 (t, 2H, J = 6.6 Hz), 2.04 (qn, 2H,
13 C-NMR (CDCl3 ): d 156.1, 32.9, 31.0, 28.6, 27.6.

1,1-Dimethylethyl[3-(1H -indol-1-yl)propyl]carbamate (21)
To a stirred solution of NaH (60% dispersion in mineral oil, 0.24 g,
6mmol) in DMF (25 mL) at 0 ◦ C was added dropwise indole (1.19 g, 5 mmol) dissolved in DMF (15 mL). After the addition, the mixture was heated at 80 ◦ C for 1 h. It was then cooled to 0 ◦ C. Then, a solution of 20 (1.31 g, 5.5 mmol) in DMF (10 mL) was added dropwise and stirred at 0 ◦ C for 30 min. The mixture was then allowed to reach rt and stirring was continued for 18 h. The DMF was evaporated and the oily residue was dissolved
1,1-Dimethylethyl(3-{3-[2,5-dihydro-4-(1-methyl-1H -indol-3-yl)- 2,5-dioxo-3-furanyl]-1H -indol-1-yl}propyl)methylcarbamate (23)
To a stirred solution of 22 (1.44 g, 5 mmol) in dichloromethane (50 mL) at 0 ◦ C was added dropwise oxalyl chloride (0.45 mL, 5.1 mmol). The mixture was stirred at the same temperature for 20 min. Then the solvent was evaporated and the residue was redissolved in dichloromethane (50 mL) and added dropwise to a stirred solution of 16 (0.95 g, 5 mmol) and triethylamine (1.52 mL, 10 mmol) in dichloromethane (20 mL) at rt. The solution was stirred for 12 h. The solvent was evaporated and the residue was purified by column chromatography on silica gel (98 : 2 dichloromethane : methanol) to furnish the desired product (0.81 g, 32%) as a dark-red solid: mp 81–83 ◦ C; IR (thin film):
1 ; 1 H- NMR (DMSO-d6 ): d 7.99 (s, 1H), 7.85 (s, 1H), 7.50 (t, 2H, J = 9.3 Hz), 7.06–7.14 (m, 2H), 7.01 (d, 1H, J = 8.1 Hz), 6.79 (t, 1H, J = 7.5 Hz), 6.67–6.73 (m, 2H), 4.22 (t, 2H, J = 6.8 Hz), 3.89 (s, 3H), 3.14 (m, 2H), 2.74 (s, 3H), 1.93 (m, 2H), 1.28–1.39 (m, 9H);
+ , 514 [M + H]+ ; HRMS (ESI+):

in ethyl acetate (100 mL). The organic phase was washed with water (3 × 50 mL) and brine (50 mL), and dried (Na2 SO4 ). The
calcd for C30

32

3
5 [M + H]: 514.2342, found: 514.2336.

solvent was evaporated and the residue was purified by column chromatography on silica gel (2 : 1 hexanes : ethyl acetate) to furnish the desired product (1.03 g, 75%) as a colorless oil: IR (thin
1 ; 1 H-NMR (CDCl3 ): d 7.74 (d, 1H, J = 8.1 Hz), 7.38–7.41 (m, 1H), 7.28–7.33 (m, 1H), 7.19–7.24 (m, 1H), 7.16 (d, 1H, J = 3.3 Hz),
6.59(d, 1H, J = 2.9 Hz), 4.78 (brs, 1H), 4.16 (t, 2H, J = 6.9 Hz),
13 C-NMR (CDCl3 ): d 156.1, 135.9, 128.7, 127.9, 121.6, 121.1, 119.4, 109.3,
+ ), 218, 201, 156, 144, 130 (100%), 117; HRMS (EI): calcd for C16 22 2 2 274.1681, found: 274.1685.

1,1-Dimethylethyl[3-(1H -indol-1-yl)propyl]methylcarbamate (22)

To a stirred solution of NaH (60% dispersion in mineral oil, 0.26 g, 6.4 mmol) in THF (20 mL) at 0 ◦ C was added dropwise a solution of 21 (0.88 g, 3.2 mmol) in THF (10 mL). The mixture was stirred at the same temperature for 45 min. Then iodomethane
1,1-Dimethylethyl(3-{3-[2,5-dihydro-4-(1-methyl-1H -indol-3-yl)- 2,5-dioxo-1H -pyrrol-3-yl]-1H -indol-1-yl}propyl)methylcarbamate (24)
To a stirred solution of anhydride 23 (257 mg, 0.5 mmol) in DMF (2 mL) were added HMDS (1.1 mL, 5 mmol) and methanol (0.1 mL, 2.5 mL). The reaction flask was tightly closed and the mixture was stirred for 36 h. It was poured into cold water (50 mL) and extracted with ethyl acetate (3 × 50 mL). The organic phase was washed with water (2 × 50 mL) and dried (Na2 SO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (95 : 5 dichloromethane : methanol) to furnish the desired product (253 mg, 99%) as an orange-red solid: mp 215–217 ◦ C; IR (thin film): 3218, 2972, 1755, 1701, 1532,
1 ; 1 H-NMR (DMSO-d6 ): d 10.94 (s, 1H), 7.86 (s, 1H), 7.74 (s, 1H), 7.39–7.46 (m, 2H), 6.99–7.06 (m, 2H), 6.94 (d, 1H, J = 8.1 Hz), 6.68–6.72 (m, 2H), 6.58–6.63 (m, 1H), 4.19 (t, 2H, J = 6.8 Hz), 3.85 (s, 3H), 3.13 (m, 2H), 2.73 (s, 3H), 1.92
+ ;

(0.32 mL, 5.1 mmol) was added dropwise. After the addition, the solution was slowly allowed to warm to rt and stirred for 20 h. The solution was cooled to 0 ◦ C and the excess hydride was destroyed
HRMS (ESI+): calcd for C30 513.2501.

33

4
4 [M + H]: 513.2502, found:

by the dropwise addition of ice-cold water. Dichloromethane (100 mL) was added and the organic phase was washed with water (2 × 50 mL) and brine (50 mL), and dried (Na2 SO4 ). The solvent was evaporated and the residue was purified by column chromatography on silica gel (2 : 1 hexanes : ethyl acetate) to furnish the desired product (0.77 g, 84%) as a yellowish oil: IR (thin film): 2974, 2930, 1694, 1482, 1464, 1393, 1365, 1170, 1147,
1 ; 1 H-NMR (CDCl3 ): d 7.69–7.71 (m, 1H), 7.39 (d, 1H,
1,1-Dimethylethyl{12-(3-aminopropyl)-12,13-dihydro-13-methyl- 5H -indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(6H )- dione}methylcarbamate (25)
A mixture of imide 24 (26 mg, 0.05 mmol) and palladium(II) trifluoroacetate (84 mg, 0.25 mmol) in DMF (3 mL) was heated at 90 ◦ C for 2 h. The mixture was cooled and poured into ethyl acetate (25 mL). The organic phase was washed with 0.5 N HCl (50 mL),

water (2 × 50 mL), dried (Na2 SO4 ) and filtered through Hyflo. The filtrate was concentrated and purified by column chromatography on silica gel (95 : 5 dichloromethane : methanol) to furnish the desired product (24 mg, 94%) as a yellow solid: mp 208–210 ◦ C (dec.); IR (thin film): 3221, 2973, 1755, 1716, 1694, 1450, 1316,
1 ; 1 H-NMR (acetone-d6 ): d 9.84 (s, 1H), 9.18–9.22 (m, 2H), 7.79 (d, 1H, J = 8.2 Hz), 7.69 (d, 1H, J = 8.2 Hz), 7.58–7.64 (m, 2H), 7.34–7.39 (m, 2H), 4.84 (m, 2H), 4.27 (s, 3H), 2.87 (m, 2H), 2.64 (s, 3H), 1.83–1.89 (m, 2H), 1.26–1.41 (m,

2(a) G. W. Gribble and S. J. Berthel, in Studies in Natural Product Chemistry, ed. Atta-ur-Rahman, Elsevier, Amsterdam, 1993, vol. 12, p. 365; (b) M. Prudhomme, Recent Pat. Anti-Cancer Drug Discov., 2006, 1, 55; (c) M. Prudhomme, Curr. Pharm. Des., 1997, 3, 265; (d) U. Pindur and T. Lemster, Recent Res. Dev. Org. Bioorg. Chem., 1997, 33; (e) U. Pindur, Y.-S. Kim and F. Mehrabani, Curr. Med. Chem., 1999, 6, 29.
3(a) R. T. Bunch and A. Eastman, Clin. Cancer Res., 1996, 2, 791; (b) R. T. Bunch and A. Eastman, Cell Growth Differ., 1997, 8, 779; (c) E. A. Kohn, N. D. Ruth, M. K. Brown, M. Livingstone and A. Eastman, J. Biol. Chem., 2002, 277, 26553.
4(a) I. Takahashi, K. Asano, I. Kawamoto and H. Nakano, J. Antibiot.,

(ESI+): calcd for C30 533.2169.

30

4

4
+ , 511 [M + H]+ ; HRMS
Na [M + Na]: 533.2165, found:
1989, 42, 564; (b) I. Takahashi, Y. Saitoh, M. Yoshida, H. Sano, H. Nakano, M. Morimoto and T. Tamaoki, J. Antibiot., 1989, 42, 571.
5(a) P. R. Graves, L. Yu, J. K. Schwarz, J. Gales, E. A. Sausville, P. M. O’Connor and H. Piwinica-Worms, J. Biol. Chem., 2000, 275, 5600; (b) E. C. Busby, D. F. Leistritz, R. T. Abraham, L. M. Karnitz and J. N.

12,13-Dihydro-12-methyl-13-{3-[(1-methyl)amino]propyl}-5H – indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7-(6H )-dione hydrochloride (12)
To a stirred solution of 25 (15 mg, 0.03 mmol) in methanol (2 mL) was added 1 M HCl in ether (9 mL). The mixture was stirred at rt for 5 h. Then the solvent was evaporated and the residue was purified by recrystallization from methanol to yield the desired
1 H- NMR (DMSO-d6 ): d 11.17 (s, 1H), 9.16 (d, 1H, J = 7.9 Hz), 9.12 (d, 1H, J = 7.9 Hz), 8.61 (brs, 2H), 7.97 (d, 1H, J = 8.2 Hz), 7.79 (d, 1H, J = 8.2 Hz), 7.64–7.69 (m, 2H), 7.44 (t, 2H, J = 7.5 Hz), 4.88 (t, 2H, J = 7.5 Hz), 4.25 (s, 3H), 2.67 (m, 2H), 2.35 (s, 3H),
13 C-NMR (DMSO-d6 ): d 170.8, 170.7, 144.8, 143.6, 133.2, 131.7, 127.7, 127.5, 124.9, 124.5, 122.9, 121.8, 121.5, 121.2, 121.1, 120.2, 119.6, 118.7, 112.4, 111.4, 45.5, 45.4, 36.8,
Sarkaria, Cancer Res., 2000, 60, 2108.
6E. Fuse, H. Tanii, N. Kurata, H. Kobayashi, Y. Shimada, T. Tamura, Y. Sasaki, Y. Tanigawara, R. D. Lush, D. Headlee, W. D. Figg, S. G. Arbuck, A. M. Senderowicz, E. A. Sausville, S. Akinaga, T. Kuwabara and S. Kobayashi, Cancer Res., 1998, 58, 3248.
7A. Eastman, E. A. Kohn, M. K. Brown, J. Rathman, M. Livingstone, D. H. Blank and G. W. Gribble, Mol. Cancer Ther., 2002, 1, 1067.
8E. A. Kohn, C. J. Yoo and A. Eastman, Cancer Res., 2003, 63, 31.
9Y. Ishimi, Y. Komamura-Kohno, H.-J. Kwon, K. Yamada and M. Nakanishi, J. Biol. Chem., 2003, 278, 24644.
10S. Roy, A. Eastman and G. W. Gribble, Synth. Commun., 2005, 35, 595.
11S. Roy, A. Eastman and G. W. Gribble, Tetrahedron, 2006, 62, 7838.
12(a) D. Toullec, P. Pianetti, H. Coste, P. Bellevergue, T. Grand-Perret, M. Ajakane, V. Baudet, P. Boissin, E. Boursier, F. Lucette Loriolle, L. Duhamel, D. Charon and J. Kirilovsky, J. Biol. Chem., 1991, 266, 15771; (b) D. Thomas, B. C. Hammerling, A.-B. Wimmer, K. Wu, E. Ficker, Y. A. Kuryshev, D. Scherer, J. Kiehn, H. A. Katus, W. Schoels and C. A. Karle, Cardiovasc. Res., 2004, 64, 467; (c) D. R. Alessi, FEBS Lett., 1997, 402, 121; (d) H. Sipma, L. Vanderzee, J. Vandenakker, A. Denhertog and A. Nelemans, Br. J. Pharmacol., 1996, 119, 730; (e) P.

(ESI+): calcd for C25 411.1817.

23

4

2
+ ; HRMS
[(M - HCl) + H]: 411.1821, found:
Hauss, F. Mazerolles, C. Hivroz, O. Lecomte, C. Barbat and A. Fischer, Cell. Immunol., 1993, 150, 439; (f) S. K. Lee and P. H. Stern, Biochem. Pharmacol., 2000, 60, 923.
13W. Dehaen and A. Hassner, J. Org. Chem., 1991, 56, 896.
14J. K. Dutton, J. H. Knox, X. Radisson, H. J. Ritchie and R. Ramage, J. Chem. Soc., Perkin Trans. 1, 1995, 2581.

Acknowledgements
This investigation was supported in part by the donors of the Petroleum Research Fund (PRF), administered by the American Chemical Society, the National Institutes of Health (CA 82220), and Wyeth.
GF109203X
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