Styryl quinazolinones and its ethynyl derivatives induce myeloid differentiation

Abstract

The tumor suppressor transcription factor CCAAT enhancer-binding protein α (C/EBPα) expression is down-regulated in myeloid leukemias and enhancement of C/EBPα expression induces granulocytic differentiation in leukemic cells. Previously we reported that Styryl quinazolinones induce myeloid differentiation in HL-60 cells by upregulating C/EBPα expression. To identify more potent molecule that can induce leukemic cell differentiation we synthesized and evaluated new series of styryl quinazolinones, ethynyl styryl quinazolinones, styryl quinolinones and thienopyrimidinones. Thienopyrimidinones were found toxic and styryl quinolinones were found inactive. Ethynyl styryl quinazolinone 39 and styryl quinazolinone 5 were found active on par with the earlier reported analogues 1 and 2 suggesting that the 5-nitro furan-2-yl styryl quinazolinones find a real promise in leukemic cell differentiation. The improved potency of 5 suggested that further modifications in the 5-nitro furan-2-yl styryl quinazolinones can be at the phenyl substitution at the 3-position of the quinazolinone ring apart from the 5-position of the heteroaryl ring.

Introduction

Long-term survival of acute myeloid leukemia (AML) patients is low and drug makers are developing novel targeted therapeutics to treat AML.¹ Hindrance in differentiation is a common observation in all AML subtypes and promise lies in the induction of myeloid differentiation. At present, all trans retinoic acid (ATRA) is administered as the first line therapy to induce differentiation in patients with acute premyelocytic leukemia (APL).² Though ATRA treatment induces remission and constitutes a cure in nearly 70% of APL patients, it has no effect on other myeloid leukemias.³

Our group has studied the role of CCAAT enhancer-binding protein α (C/EBPα),⁴ transcription factor essential for differentiation of cells in liver, lung, adipose tissues, and bone marrow and is required for granulocytic or monocytic differentiation. We proposed that increased C/EBPα expression and/or activity in AML can lead to myeloid differentiation and demonstrated that styryl quinazolinone analogue (1), induces C/EBPα activity which in turn enhances differentiation and leads to growth arrest and apoptosis of leukemic cells.⁵ We further explored a series of styryl quinazolinones and identified 2 as potent C/EBPα inducer.⁶ Among various heteroaryls in development as drugs quinazolinone⁷ also finds significance.

Styryl quinazolinones are well studied for applications as anti-bacterials⁸ and as anticancer drugs.⁹ The interesting aspect of styryl quinazolinones is that they were explored as Heat shock protein 90 (HSP90) inhibitors,¹⁰ tubulin polymerization inhibitors,^11,12 RAD51 inhibitors,^13,14 and also cause shortening of telomeres.¹⁵ From our high throughput screen⁵ and subsequent development,⁶ we found that styryl quinazolinones induce C/EBPα expression in HL-60 cells and there by induce myeloid differentiation. We hypothesized that there may be connectivity between all these protein targets and styryl quinazolinones. We were driven to postulate the exact mechanism and pathway of the drug action and hence we screened a series of structurally variant styryl quinazolinones such as styryl quinazolinones, ethynyl styryl quinazolinones, thienopyrimidinones, styryl quinolinones to see same phenotypic myeloid differentiation. Herein we present our observation of the C/EBPα expression levels and subsequent myeloid differentiation capacity of various distinct styryl quinazolinones.

Styryl quinazolinones and thienopyrimidinones were synthesized¹⁶ according to the reported synthetic protocols.¹⁵ Briefly, 5-substituted benzoxazinone derivative (ii) was obtained from corresponding anthranilic acid (i) upon cyclisation using acetic anhydride. 5-Substituted benzoxazinone (ii) was treated with respective aniline under reflux to yield quinazolinone derivative (iii). Finally styryl derivatives 1 to 10 were obtained from the respective intermediate (iii) by heating with particular aldehydes in acetic acid (Scheme 1).

Scheme 1.

Representative synthetic route for styryl quinazolinones.

Thienopyrimidinones were synthesized directly from the corresponding derivative (v) upon heating with 5-nitro-furan-2-aldehyde in acetic acid solvent (Scheme 2). Ethynyl styryl quinazolinones and styryl quinolinones were procured from the earlier synthesis.¹⁵

Scheme 2.

Representative synthetic procedure for styryl thienopyrimidinones.

All the 49 styryl quinazolinone derivatives (10 styryl quinazolinones, 5 thienopyrimidinones (Table 1) 24 ethynyl phenyl substituted styryl quinazolinones (Table S1) and 10 Styryl Quinolinones (Table S2)) were screened using wright-giemsa staining and NBT reduction assay at 10 μM concentration similar to control.⁵ From the initial differentiation and apoptosis assay⁵ we found that among the styryl quinazolinones (1–10) screened derivatives 1,⁵ 2,⁶ 5, and 6 showed significant differentiation of HL-60 cells (Table 1). All thienopyrimidinones showed significant toxicity and minor differentiation was observed in the case of 11 (at 1 μM), 12 (at 3 μM), 13 (at 3 μM), and 14 (at 3 μM). At higher concentrations (more than 3 μM), all the thienopyrimidinones exhibited toxicity. Among the ethynyl styryl quinazolinones (Table S1), only 19 and 39 found to differentiate the HL-60 cells. Also all the quinolinones (Table S2) were found inactive towards differentiation or apoptosis. When we measured the increase in CD11b expression levels (Fig. 1) with these leads (5, 6 and 39) along with the controls (ATRA, 1, 2) we found that only compound 5 exhibited 89% increase of CD11b expression at 10 μM concentration compared to ATRA showing 96% increase at 1 μM concentration. Compound 6 showed 59% increase at 10 μM concentration.

Table 1.

Synthesized and active styryl quinazolinones in HL-60 cell differentiation.

Structure	Concentration	Apoptosis	WG	NBT
DMSOa	0.1%	−	−	−
ATRAb	1 μM	+++	+++	+++
	10 μM	+	+	+
	3 μM	++	++	++
	10 μM	−	−	−c
	10 μM	−	−	−c
	10 μM 3 μM	+++ ++	++ +	++ +
	10 μM 3 μM	+++ −	++ −	++ −
	10 μM	−	+	+
	10 μM	−	−	−
	10 μM	+	−	−
	10 μM	−	−	−
	10 μM	+	−	+
	10 μM	+	−	+
	10 μM	+	−	±
	10 μM	+	−	±
	10 μM	+	−	−
	10 μM	+	+	+
	10 μM	+	+	+

^aDMSO used as control.

^bATRA used as positive control.

^cCompounds 3 and 4 are purchased from the vendor and not synthesized.

Fig. 1.

HL-60 treated with drugs for 7 days Anti human/mouse CD11b antibody – APC, Rat IgG2b.

Further we examined gene expression levels of C/EBPα and its downstream target C/EBPε, which have important role in terminal granulocyte differentiation and maturation. When HL-60 cells were treated with ATRA, 1, 2, 5, and 6 mRNA expression levels of CEBPA were increased in a time-dependent manner. Though compound 11 and 12 increased the CEBPA levels (Table 2) they did not increase CD11b levels which is measure of granulocytic differentiation of HL-60 cells. Increase in CEBPE levels was observed in the case of 1, 2, 5 and 6.

Table 2.

Synthesized active styryl quinazolinones in CD11b and ↑CEBPA gene expression levels.

Structure	↑ In CD11b in comparison with DMSO	↑ Gene expression of CEBPA (fold increase)	↑ Gene expression of CEBPE (fold increase)
DMSO	3.94	No change	No change
ATRA	96.4% at 1 μM	3 at 1 μM	ND
1	51.2% at 10 μM	1.5 at 10 μM	2.1 at 10 μM
2	42.2 at 3 μM	2.5 at 3 μM	10.3 at 3 μM
5	94.2%	1.6 at 7 μM	2.6 at 7 μM
	ND
6	59.0%	1.8 at 10 μM	6.0 at 10 μM
	ND

ND – denotes Not Determined.

Initially we observed a strong correlation between various oncological targets and small molecule styryl quinazolinone (Fig. 2). This inspired us to explore various distinct and analogous derivatives of styryl quinazolinones (1–49) to ascertain their activity towards inducing myeloid differentiation and C/EBPα expression. From the preliminary screen of all the compounds, we found that styryl quinolinones were inactive and completely lost activity due to the change in the heteroaryl ring by removal of one nitrogen. The toxicity of thienopyrimidinones 11 and 12 can be due to the replacement of the adjacent phenyl ring by the thiophene ring. Further this significant structural change might be the cause for the increase in CEBPA gene expression levels without significant increase in the CD11b expression levels. Among 24 ethynyl styryl quinazolinones only 19 and 39 showed differentiation of HL-60 cells. This implies that the ethynyl substitution had little or no effect on the differentiation of the leukemic HL-60 cells. Only compound 39 showed minimal increase in CD11b expression, CEBPA and CEBPE gene expression levels.

Fig. 2.

Interactions of Styryl quinazolinones with various cancer targets.

This may due to preservation of structure other than ethynyl phenyl part and also due to increase in hydrophobicity at quinazolinone 3-position. Among the styryl quinazolinones 1–10, all the derivatives with phenyl substitution at the styryl part were inactive except 1, which is with the dihydroxy phenyl group. Compound 10 might have lost activity due to change in the position of methoxy to the para position from the original ortho position. Among the derivatives 2, 5, 6, 7 all are with nitro furan substitution replacing phenyl ring of styryl substitution. All these derivatives contributed to differentiation of HL-60 cells except 7, which induced only a differentiation and no apoptosis. This activity might be due to the slightly acidic carboxyl group at the phenyl ring R2, meta position. Compounds 2, 5, 6 with minor changes at the phenyl ring R2 and at quinazolinone 5-position were found to enhance differentiation and subsequent apoptosis to a greater level compared to all other derivatives except ATRA. 2, 5, and 6 enhanced CEBPA and CEBPE gene expression levels significantly compared to other derivatives. The 89% increase in CD11b expression level due to 7 μM 5 treatment was nearly similar to ATRA treatment and marked highest level of differentiation from the current set of styryl quinazolinones screened.

In conclusion, we found that distinct variations in the structure of styryl quinazolinones made the derivatives completely inactive towards inducing myeloid differentiation. The thieno derivatives led to toxicity. Analogous derivatives were found to retain the differentiation potential and compound 5 was found to be the potent derivative for inducing myeloid differentiation in HL-60 cells. Exploring derivatives with further changes at the 5-position of the quinazolinone ring and elucidating the exact mechanism of action of these molecules will be our future interest.