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  • Classical DHFR inhibitors such as methotrexate MTX


    Classical DHFR inhibitors such as methotrexate (MTX) bind tightly to the enzyme and possess adequate clinical pharmacokinetics. MTX bind to the DHFR binding site through the formation of hydrogen bonds with Asn64, Lys68, Arg28, Arg70, Val115 and Ile7 amino PD 151746 receptor residues as well as hydrophobic interactions [4]. Several problems were associated with the clinical use of MTX such as: the high levels of toxicity and, resistance [4]. This justified the need for nonclassical inhibitors, such as trimethoprim (TMP,1) and trimetrexate (TMQ, 2), which bind to the same receptor through Arg22, Phe34 and Lys68 (Fig. 1). Several compounds bearing thiazole heterocycle were reported as nonclassical DHFR inhibitors such as tiazofurin (3), 4-phenyl-thiazole-1,3,5-triazines (4), netropsin (5) and thia-netropsin (6) [5], [6]. Molecular modeling studies concluded that recognition with key amino acid Leu4, Glu30, Arg22 and Val115 are essential for DHFR binding [7]. The core structure of several nonclassical DHFR inhibitors consists of a flat aromatic or heteroaromatic ring systems e.g. thiazole moiety bearing different aliphatic or aromatic substituents. Thiourea moiety is known to act as an anchoring group to help the main thiazole structure to perform its activity as shown in 7; causing remarkable increase in the DHFR inhibition [6]. Attaching a phenoxy substituted triazole residue to the 2-amino-thiazoles enhanced also the DHFR inhibition as in 8–10 [8]. Recently, a new series of 2,4-substituted-1,3-thiazoles and thiazolo[4,5-d]pyridazine both bearing the 2-thioureido function were designed, synthesized and evaluated for their DHFR inhibition. The type of substituent at the thioureido- function and at positions 7- of the thiazolo[4,5-d]pyridazine affected the DHFR inhibition potency [9]. In view of these facts, and in continuation to our previous efforts [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], the present study targeted the design, synthesis and biological evaluation of novel series of 1,3-thiazole containing heterocycles acting at the DHFR enzyme active site. Several bioisosteric replacements of these heterocyclic scaffolds have been performed, maintaining the ability of the compounds to bind to the receptor site in the same manner as the lead compounds 1–10 (Fig. 1). According to the previously mentioned SAR studies of the lead compounds 1–10, thioureido-1,3-thiazole analogue fitted with phenoxy function (A) was designed; the aryl thiazole moiety was replaced with the cyclized carbohydrazide analogue thiazolo[4,5-d]pyridazin-4(5H)-one (B). Further modification included replacement of thiazole scaffold with imidazo[2,1-b]thiazoles (C) and their cyclized carbohydrazide analogue imidazo[2′,1′:2,3]thiazolo[4,5-d]pyridazine (D), Fig. 2. This investigation aimed to identify novel synthetic lead compound(s) and to define structure features that enhance selectivity for binding to DHFR receptor sites through ring annexation of 1,3-thiazole to probe the active site for binding into the bicyclic thiazolo[4,5-d]pyridazine, imidazo[2,1-b]thiazoles and the tricyclic imidazo[2′,1′:2,3]-thiazolo[4,5-d]pyridazin fused rings. The synthesized derivatives were tested for their in vitro DHFR inhibition. As an application and to realize the use of the new DHFR inhibitors, the compounds were further tested for their in-vitro antitumor activity.
    Results and discussion
    Structure activity correlation In regard to DHFR inhibition activity, the electronegativity of the substituent on the 3-[4-substituted-phenyl)-thioureido]- function affected the DHFR inhibitory activity, which is in consistency with the previous obtained results [9]. The new 4-phenoxy-phenyl-thioureido- derivative 13 (IC50 0.05 ± 0.001 μM) proved to be almost 1000 fold more active than the 4-methoxy-phenyl-thioureido- derivative (IC50 79.0 ± 3.98 μM); and 200 fold more active than the 4-chloro-phenyl- counterpart (IC50 9.5 ± 0.76 μM), previously mentioned in [9]. This evidence confirmed that electron withdrawing functions like phenoxy or chloro favor the DHFR inhibition potency rather than the electron donating methoxy group. The thioureido- analogue 13 was used to prepare the corresponding thiazolo[4,5-d]pyridazin-4(5H)-one derivatives 18 and 19. The type of substituent on thiazolo[4,5-d]pyridazine nucleus also manipulated the DHFR inhibition activity. Furthermore, the ethyl 5-(4-substituted-phenyl)-imidazo[2,1-b]thiazole-3-carboxylates (23–25) did not show any significant DHFR inhibition activity. Cyclization of 23–25 produced 3-(4-substituted-phenyl)-8-substituted-phenyl-imidazo[2′,1′:2,3]thiazolo[4,5-d]pyridazin-5(6H)-ones (43–45). Compound 43 with IC50 0.06 ± 0.002 μM is one of the most active members of this study. Again, the electronegativity of the substituents on the phenyl rings at positions 3- and 8- plays a crucial role controlling the activity. Generally, in case of the thiazolo[4,5-d]pyridazine analogues; 3-[(4-phenoxy-phenyl) thioureido]- series is more active than the 3-[(4-chloro-phenyl)-thioureido]- and the 3-[4-methoxy-phenyl)-thioureido]- counterparts. The type of substituent at positions 7- of the thiazolo[4,5-d]-pyridazine affected the potency. In the 3-[(4-phenoxy-phenyl) series, the order of activity was 4-BrPh > 4-CH3Ph. In case of imidazo[2′,1′:2,3]thiazolo[4,5-d]-pyridazin-5(6H)-one analogues; at position 3- the order of activity was 4-BrPh > 4-CH3Ph > 4-CH3OPh; while at position 8- the order of activity was Ph > 4-CH3Ph > 4-BrPh > 4-CH3OPh in most cases. Ring expansion of the active 1,3-thiazole ring analogue 13 (IC50 0.05 ± 0.001 μM) into the bicyclic thiazolo[4,5-d]pyridazine (18,19) or imidazo[2,1-b]thiazoles (23–25) decreased the activity; while the formation of the tricyclic imidazo[2′,1′:2,3]-thiazolo[4,5-d]pyridazine (43–54) increased the inhibition potency. Regarding the antitumor activity, the obtained results added another evidence which emphasize the order of activity which was concluded for the DHFR inhibition. Comparing the potency of the active antitumor compounds and their DHFR inhibition revealed that compounds 13, 18, 43 and 49 might exert their antitumor activity through DHFR inhibition.