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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Bioorg Med Chem Lett. Author manuscript; available in PMC 2010 December 1.
Published in final edited form as:
PMCID: PMC2783978
NIHMSID: NIHMS156366

Isoquinoline-based analogs of the cancer drug clinical candidate tipifarnib as anti-Trypanosoma cruzi agents

Abstract

We developed a synthetic route to prepare isoquinoline analogs of the cancer drug clinical candidate tipifarnib. We show that these compounds kill Trypanosoma cruzi amastigotes grown in mammalian host cells at concentrations in the low nanomolar range. These isoquinolines represent new leads for the development of drugs to treat Chagas disease.

Keywords: enzyme inhibitors, anti-parasite therapeutics, Chagas disease, lanosterol 14α-demethylase, sterol biosynthesis

Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi). Approximately 8–11 million people in Latin America are infected with this parasite, and 30% of those can be expected to develop complications ranging from mild to terminal1. There is no effective treatment for Chagas disease at this time.

We have previously shown that the protein farnesyltransferase inhibitor tipifarnib (compound 1, Figure 1) kills T. cruzi parasites by binding to lanosterol 14α-demethylase2. This enzyme is required for production of ergosterol, a required component of the parasite’s membranes, which cannot be replaced by mammalian host cell derived cholesterol3. Using the x-ray structure of tipifarnib bound to mammalian protein farnesyltransferase and a homology model of T. cruzi lanosterol 14α-demethylase with tipifarnib docked into the active site2, we designed compound 2 (Figure 1) which no longer inhibits protein farnesyltransferase (IC50 > 5,000 nM versus ~1 nM for tipifarnib) and is about 10-fold more potent at killing T. cruzi amastigotes (clinically relevant parasite life cycle stage that grows in mammalian host cells) (EC50 = 0.6 nM) than is our lead compound tipifarnib (EC50 = 4 nM)4. The extra methyl group present in 2 clashes with the surface of protein farnesyltransferase. In addition, the amino group of tipifarnib is part of a hydrogen bond network in the active site of protein farnesyltransferase, which does not exist in the sterol demethylase4. Compound 2 contains a OMe group in place of this amino group. We are hopeful that these tipifarnib analogs can be taken into clinical development for Chagas disease because they are highly potent at killing the parasite, and they are expected to have the desireable oral bioavailability and pharmacokinetics that tipifarnib has.

Figure 1
Structure of tipifarnib 1 and analog 2.

Our modeling studies of tipifarnib docked into the active site of T. cruzi lanosterol 14α-demethylase shows that the carbonyl and N-Methyl portions of the amide of the quinoline ring are in contact with enzyme surface residues only (Figure 2). Since such interactions are usually not strong because of the inherent flexibility of protein surface residues, we considered making a new series of analogs in which the quinolone ring is replaced with a quinoline ring. However, quinolines are well known to be susceptible to oxidative metabolism in which carbon-2 is hydroxylated leading to the quinolone ring after tautormerization of the 2-hydroxyquinoline (Figure 3). Thus, we decided to make isoquinoline analogs of the general core structure 3 (Figure 3).

Figure 2
Stereo diagram of tipifarnib docked into the active site of the homology model of T. cruzi lanosterol 14α-demethylase. Tipifarnib is in red, the heme is in yellow, and the heme-iron is in cyan.
Figure 3
Metabolic oxidation of a quinoline to a quinolone. Compound 3 is the core structure of isoquinoline-based tipifarnib analogs prepared in this study.

The synthesis of the two fragments to make the isoquinoline tipifarnib analogs is shown in Figure 4. Reaction of substituted benzaldehyde 4 with trimethylsilyl cyanide followed by reduction with LiAlH4 gives hydroxyamine 5. Reductive amination with p-bromobenzaldehyde gives secondary amine 6. Friedel-Crafts alkylation leads to ring closure, and the compound is tritylated on the nitrogen to give 7 in preparation for the next step. The synthesis of the second fragment, methanone 11, is shown in Figure 4 (panel b). These are made by reacting the appropriate Weinreb amide 9 with a silylated and lithiated N-methylimidazole. Figure 5 shows the joining of the two fragments to give the target compounds. Compond 7 is converted to the aryl lithium by lithium-halide exchange, and treatment with methanone 11 gives tertiary alcohol 12. Alcohol 12 is detritylated and oxidized to isoquinoline 13 with MnO2. Conversion of the tertiary alcohol to the chloride and then to the target compound 14 is carried out with SOCl2 followed by treatment with methanol. It should also be possible to heat 13 in the presence of tosic acid and methanol to give 14 in 1 step5, but this was not attempted. Compound 15 was made by the same route. To make analog 16, which lacks the phenyl substitutent at the 4-position of the quinoline ring, we converted the commercially available 6-bromoisoquinoline to the aryl lithium and carried through the same sequence shown in Figure 5.

Figure 4
Synthesis of two fragments for isoquinoline tipifarnib analogs. (Panel a) a) TMSCN, ZnI2, CH2Cl2, 0°C-rt; b) LiAlH4, THF, 0°C-rt, 70% (2 steps); c) p-bromobenzaldehyde, Et3N, MeOH, then NaBH4, 75%; d) AlCl3, CH2Cl2, 70°C, 90%; ...
Figure 5
Synthesis of isoquinoline tipifarnib analogs. a) n-BuLi, THF, −78°C, 56%; b) TFA, CH2Cl2, 85%; c) MnO2, dioxane, 90°C, 64%; d) SOCl2, rt; e) MeOH, 90°C, 80% (2 steps).

We tested the isoquinolines for inhibition of growth of T. cruzi amastigotes (Tulahuen strain) inside of mammalian cells (3T3 cells). The parasites stably express the β-galactosidase from E. coli, and this enzyme converts yellow colored chlorophenol red β-D-galactopyranoside into a red colored product, which is readily measured by spectrophotometry5. This allows the number of parasites in the cutulre to be readily quantified without resorting to tedious parasite cell counting using microscopy. Data in Table 1 are provided as values of EC50, the concentration of compound that reduces parasite growth by 50%. It can be seen that compound 14 is as potent as the corresponding quinolone 2. Thus, the carbonyl and N-methyl groups of 2 are not required for efficacy in this parasite killing assay. Compound 15 with a 2,6-difluorophenyl replacing the 3-chlorophenyl substituent is also potent in this assay. Compound 16 lacks this aryl substituent and is not active against T. cruzi. This is consistent with our modeling showing that the phenyl substituent packs well into the active site of T. cruzi lanosterol 14α-demethylase. We also studied the lanosterol 14α-demethylase inhibitor posaconazole as a comparator compound. The data in Table 1 show that the new isoquinolines reported in this study are nearly as potent as posaconazole. The latter has been shown to cure mice suffereing from a chronic infection with T. cruzi3. Posaconaozle is an approved drug for treatment of fungal infections. There is now discussion of the use of this agent to treat Chagas disease. The new isoquinolines reported here offer the advantage that manufacturing cost is expected to be well below that of posaconazole. Given the outstanding potency of these compounds against T. cruzi growth, detailed pharmacokinetic and Chagas disease efficacy studies in rodents are warranted.

Table 1
Growth arrest of T. cruzi amastigotes by isoquinoline-based tipifarnib analogs.1

Supplementary Material

Acknowledgements

This work was supported by a grant from the National Institutes of Health (AI070218).

Footnotes

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Supplemental Material. Full details for the synthesis and characterization of the isoquinolines are provided as Supplemental Material.

References

1. Division of Parasitic Diseases. Chagas Disease. http://www.cdc.gov/chagas/
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3. Urbina JA, Payares G, Moling J, Sanoja C, Liendo A, Piras MM, Piras R, Perez N, Wincker P, Ryley JF. Science. 1996;273:969. [PubMed]
4. Kraus JM, Verlinde CLMJ, Karimi M, Lepesheva GI, Gelb MH, Buckner FS. J. Med. Chem. 2008;52:1639. [PMC free article] [PubMed]
5. Buckner FS, Verlinde CLMJ, La Flamme AC, Van Voorhis WC. Anti. Agents Chemother. 1996;40:2592. [PMC free article] [PubMed]