Effect of substitution at N 00 -position of N 0 -hydroxy-N-amino guanidines on tumor cell growth Arijit Basu a,⇑ , Barij Nayan Sinha a , Philipp Saiko b , Thomas Szekeres b a Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835215 , India b Department of Medical and Chemical Laboratory Diagnostics, Vienna General Hospital, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna, Austria article info Article history: Received 19 January 2012 Revised 9 June 2012 Accepted 15 June 2012 Available online 21 June 2012 Keywords: Hydroxyguanidine Ribonucleotide reductase Lead modification abstract Structural modification of one of our earlier reported lead molecule (ABNM13) has been carried out to study the effect of different substituents at the N 00 -position of N-hydroxy-N 0 -amino guanidines (HAGs) on their anticancer activity. Compounds with electron donating substituents were found to be less active. In contrast, those with electron withdrawing groups were found favorable for anticancer activity. The obtained results provide significant SAR information that may be useful for further drug designing with HAGs. Ó 2012 Elsevier Ltd. All rights reserved. Hydroxyaminoguanidines (HAGs), 1–5 have been reported as anticancer agents, primarily against L1210 murine, and HL60 hu- man leukemia cell lines. They were found to inhibit DNA synthesis by inhibiting the R2 subunit of the enzyme Ribonucleotide reduc- tase (RR). 6,7 Earlier, we have identified 5,8 an anticancer lead mole- cule (ABNM13) with IC 50 (HL60 cell line) of 11 lM, CC 50 >100 lM that is a selectivity index of more than ten. It is a potent inhibitor of RR, and an arabinofuranosylcytosine (Ara-C) synergist. In our earlier studies we have also emphasized the need for its further structural modifications. ABNM13 has been identified through a virtual screening (VS) experiment, by screening an in-house ligand library, on a developed pharmacophore based QSAR model. To fur- ther expand these studies, we have explored different structural modifications of ABNM13, on anticancer activity. Lead modification process often involves the application of structure or/and ligand based drug designing. We could not apply either, due to the following reasons. (a) Reasons for precluding the already developed QSAR model for further lead design: ABNM13 has been identified by pharma- cophore based virtual screening of an in-house library. The training set for developing such a model consisted of differ- ent HAGs with aromatic substitutions at the N 0 -position. Therefore, the developed model revealed designing informa- tion pertaining only to this position, and nowhere else. Therefore, our earlier developed model will be out of scope for predicting newer compounds with substitution at differ- ent positions (Fig. 1). (b) Reasons for precluding structure based designing: HAGs inhibit the M2 subunit of the enzyme RR. Mode of action of most of these analogs have been deduced by cell line assays, dif- ferent spectroscopic studies, and enzyme assays. No crystallo- graphic information of the ligand bound M2 subunit has been reported till date. In absence of these information, structure based drug designing on this target has been a serious concern, and therefore cannot be undertaken for the current work. We have resorted to a classical drug designing approach. A few analogs with both electron donating and withdrawing substituents were synthesized, and tested for their anticancer activity. For the current work, we have used a synthetic modification strategy on our earlier developed lead molecule (Fig. 1). The synthesis of the compounds are presented in Schemes 1 and 2. We report a method for synthesizing N 00 substituted HAGs. The first step involved the synthesis of methyl dithiocarbazinate (1), which was prepared as per our earlier reported methodology. 9–11 Synthesis of compound 2 is straight forward nucleophilic addition between the amine and the aldehyde, with good yield of around 85%. The third step in the synthesis involved nucleophilic attack by different amines on –C@S, carbon of compound 2. We used ex- cess amine to drive the reaction in forward direction. Excess amine, and the use of high temperature (>120 °C) favors the hydrolysis of compound 2 to corresponding aldehyde, which was monitored by TLC co-spotting with anthraldehyde. This unavoidable side reac- tion makes the purification difficult for compounds 3–10, and leads to poor yield. We managed to purify the crude product by using automated flash chromatography (BUCHI Sepacore, Switzerland), and employing a gradient elution technique. 0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2012.06.048 ⇑ Corresponding author. E-mail address: arijit4uin@gmail.com (A. Basu). Bioorganic & Medicinal Chemistry Letters 22 (2012) 4934–4938 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl