4-Amidinoquinolines (4-AMQ) and 10-Amidinobenzonaphthyridines (10-AMB) as new classes of antimalarials with high potency in vitro and in vivo

Vasiliy N. Korotchenko,† Ramadas Sathunuru,† Lucia Gerena, Diana Caridha, Qigui Li, Thu‑Lan Luong, Delaney Hettithantrige, Raul Olmeda, Liang Zhang, Sean Marcsisin, Victor Zottig, Mara Kreishman‑Deitrick, Philip L. Smith and Ai J. Lin

Experimental Therapeutics Branch, Military Malaria Research Program, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910

† equal contributions

Chloroquine (CQ) has been used as a first line malaria therapeutic drug for decades. Emergence of CQ drug-resistant Plasmodium falciparum (Pf) malaria throughout endemic areas of the world has limited its clinical value. Mefloquine (MQ) has been used as an effective malaria prophylactic drug, due to its long action and high potency against blood stage Pf. However, serious CNS toxicity of MQ has compromised its clinical value as a prophylactic drug. Therefore, new antimalarial drugs with no cross-resistance to CQ and no CNS toxicity are urgently needed to combat this deadly human disease. Azacrin (Benzo[b][1,5]naphthyridine analog of CQ) was extensively studied in the 1950’s as an active antimalarial compound. It showed no cross-resistance with CQ and was efficacious as a schizonticidal drug against Pf and P.  vivax. However, it was not superior to CQ and further studies were not pursued.

Material has been reviewed by the Walter Reed Army Institute of Research. There is no objection to its presentation and/or publication. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense. Research was conducted in compliance with the Animal Welfare Act and other federal statutes and regulations relating to animals and experiments involving animals and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, NRC Publication, 2011 edition

BACKGROUND

Figure 1. Structures of 4-AMQ, 10-AMB, chloroquine (CQ) and azacrin

DISCLAIMER

SYNTHESIS OF 4-AMQ AND 10-AMB ANALOGS

ACKNOWLEDGEMENTS

REFERENCES

RESULTS

We developed two synthetic approaches to the 4-AMQ and 10-AMB analogs. Originally, 4‑AMQ analogs were prepared by a 4-step Route A from 4,7-dichloroquinoline 1 via 4-amino-7-chloroquinoline 2 and amides 3. Amides 3 were converted to corresponding thioamides 4 using the Lawesson’s reagent. We found that this key step works fine for aliphatic amides (acetamide and isobutyramide), but the reaction failed for benzamide derivative (R1 = phenyl). Finally, thioamide intermediates were treated with corresponding diamines in the presence of either mercury oxide or DCC providing 4-AMQ analogs 5 in good yield.

Route A: Thioamide approach to 4-AMQ Route B is a new and facile 2-step procedure to synthesize the target compounds while

allowing for a wider choice of substituents at the R1 position than Route A. In addition, Route B avoids the use of hazardous HgO or tedious DCC as a coupling agents. The route B starts with arylation of substituted amidines R1-C(=NH)-NH2 with 4,7-dichloroquinoline 1, followed by alkylation of intermediates 6 with appropriate N,N-disubstituted 3-chloropropylamines. "

Route B can be applied to synthesis of 10-AMB analogs 9 as well, starting with 7,10-dichloro-2-methoxybenzo[b][1,5]naphthyridine 7, followed by alkylation of intermediates 8. N-Arylation of amidines with precursors 1 or 7 required K2CO3 as base for R1 = methyl or isopropyl, whereas R1 = aryl required stronger bases (KOt-Bu or NaH). "

As a side products in this reaction, minor amounts of bis-N-alkylated derivatives 10 and 11 were isolated when 1 : 1 ratio of intermediates 6 or 8 to 3-chloropropylamines was used; higher ratio of 3-chloropropylamines to 6 or 8 yielded mainly the bis-alkylation products 10, 11. "

[1] Korotchenko, V. N., Sathunuru, R., Gerena, L., Caridha, D., Li, Q., KreishmanDeitrick, M., Smith, P., Lin, A. J. Journal of Medicinal Chemistry, 2015, 58 (8), 3411-3431

Funding for this project was provided by the Military Infectious Diseases Research Program (MIDRP)

CONCLUSIONS

Route B: Synthesis of 4-AMQ and 10-AMB analogs

Synthesized 4-AMQ and 10-AMB analogs (approx. 50 compounds total) were tested for in vitro potency against three strains of Pf (D6, W2 and TM21C235), in vitro metabolic stability and in vitro cytotoxicity. Compounds with IC50 < 100 nM (D6) and t1/2 > 30 min were tested for in vivo antimalarial efficacy (Thompson Test, P. berghei infected mice model). Most of the new analogs have no cross-resistance with CQ (IC50 ratio for CQ sensitive strain D6 and CQ resistant strain W2 is less than four). Results for selected compounds presented in Table 1; full set of data collected for these molecules discussed in our recent publication [1].

Two new classes of potent antimalarials: 4‑amidinoquinoline (4-AMQ) and 10‑amidinobenzonaphthyridine (10‑AMB) derivatives were discovered in our laboratory. The new molecules displayed high activity in vitro and in vivo, have little or no cross‑resistance to CQ, and no toxicity in mice up to 80 mpk x 3. The best analog WR910397 show nanomolar in vitro potency against D6, W2 and C235 Pf clones, low inhibitory activity in the hERG potassium channel blockage test, no mutagenicity in the Ames test, and cure 5 out of 5 mice infected with P. berghei at 15 mpk x 3 (Thompson Test). These results suggest that 4-AMQ and 10-AMB derivatives are potentially promising classes to develop new antimalarial drugs. Further characterization, development and optimization of the most promising candidates from the 4-AMQ and 10-AMB classes are ongoing

We designed two new structural classes of potential antimalarials by introducing an additional nitrogen to the side chains in the chloroquine and azacrin molecule, providing 4-amidinoquinoline (4-AMQ) and 10‑amidinobenzonaphthyridine (10‑AMB) structures, respectively (Figure 1). Through conjugation with the amidine group, the basicity of the ring nitrogen and the 4(10)-amino group of the new molecules is expected to be significantly altered. Furthermore, the additional amino group could provide a potential additional binding site to the drug’s biological receptors, which could result in significant changes in pharmacological profiles compared to azacrin and chloroquine. In this study, a series of new and 4-AMQ and 10-AMB derivatives were designed, prepared and assessed in search for new antimalarial agents."

ABSTRACT

TABLE 1: BEST 4-AMQ and 10-AMB analogs Compounds

IC50 (Pf), nM

D6, W2, C235

Tx2 IC50 (uM)

Met Stab Human liver microsomes

(min)

TT (mg/kg x

3)

Plasma half-life

Mice Tox (mpk x 3)

hERG IC50 (uM)

Ames Test

WR826809 22, 42, 26 > 67 > 60 160, 4/5C 6.7 h TBD TBD TBD

WR910376 3, 10, 5 14 > 60 TBD TBD TBD 5.7 Negative

WR910415 3, 4, 1 4.8 48 TBD 5.6 h TBD TBD Negative

WR909831 2, 10, 5 5.8 > 60 120, 5/5C 60, 4/5C 47.5 h > 120 > 10

Negative

WR909890 2, 2, 2 12.9 18 60, 5/5C TBD > 120 TBD TBD

WR910397 3, 7, 4 5.2 32 15, 5/5C 69.7 h > 60 TBD Negative