Identification and Preclinical Evaluation of SC144, a Novel Pyrroloquinoxaline Derivative with Broad-Spectrum Anticancer Activity
Fedora Grande1*, Francesca Aiello1, Antonio Garofalo1 and Nouri Neamati2
1Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, 87036 Arcavacata di Rende (Cs), Italy; 2Department of Medicinal Chemistry, Translational Oncology Program, University of Michigan, Ann Arbor, MI 48109, USA
Abstract: Design and discovery of new classes of anticancer agents with unique mechanisms of action is an urgent medical need. During the past several years, a series of salicylhydrazide class of compounds were reported to possess remarkable potency in a large panel of cancer cell lines from different tumor origins. In particular, the optimized lead compound, SC144, was further investigated and selected as a valuable drug candidate endowed with favorable pharmacokinetic and anti- proliferative properties in various in vitro and in vivo xenograft models. This lead compound is active
in cells resistant to conventional chemotherapies, synergistic with several standard-of-care drugs, and possesses an unique mechanism acting through the inhibition of the gp130-STAT3-survivin axis. Because of this novel mechanism, clinical development of SC144 will provide new therapeutic options for diverse cancers.
Keywords: Anticancer agents, cytotoxicity, gp130, hydrazides, IL-24, pyrroloquinoxaline, survivin.
Herein, we provide a brief summary of previous studies focusing on the identification, synthesis, and pharmacological evaluation of a series of salicylhydrazide class of compounds leading to the discovery and selection of a pyrroloquinoxaline hydrazide, SC144, for further development as a novel anticancer agent.
Previously, it was showed that certain HIV-1 integrase inhibitors exhibit significant cytotoxicity due to lack of selectivity for integrase [1-5]. In fact, the similarities between retroviral integrases and topoisomerases prompted the evaluation of topoisomerase I and II poisons as potential integrase inhibitors. Topoisomerases were afterwards adopted as a potential counter screen for integrase inhibitors identification [6-9]. It was also shown that select integrase inhibitors were able to significantly inhibit RAG1/2 enzymes that are essential for VDJ recombination . All these enzymes share a similar chemistry of DNA binding, cleavage, and recombination that require a divalent metal (Mn2+ or Mg2+) as a cofactor . Because integrase belongs to the polynucleotidyl transferases , and more in general to the polymerases superfamily, it is plausible that certain HIV inhibitors could target other DNA-processing enzymes. Subsequently, a database of 10,000 compounds consisting of reported and patented integrase inhibitors was
*Address correspondence to this author at the Università della Calabria, Dipartimento di Farmacia e Scienze della Salute e della Nutrizione, 87036 Arcavacata di Rende (Cs), Italy; Tel: +39-0984-493019; Fax: +39-0984- 493118; E-mail: [email protected]
constructed and interrogated to select compounds that were nonspecific for integrase to be repurposed as anticancer agents.
Using such a database, various pharmacophore models were developed, followed by ADMET prediction and cluster analysis, to separate cytotoxic agents from a majority of antiviral compounds .
On the basis of these pharmacophores, some salicylhydrazide based compounds were identified as potential leads to be included in an anticancer drug discovery program. Pursuing development of this class of compounds, a multiconformational database of 4.5 million compounds was searched and >2200 compounds that possess common structural features and pharmacophore fragments were identified. About a thousand analogues satisfying ADMET properties calculations were then acquired and subjected to MTT cytotoxicity assay for an initial screen, which led to the identification of salicylhydrazides showing IC50 < 0.1 µM .
IDENTIFICATION OF PYRROLOQUINOXALINE BASED CYTOTOXIC AGENTS
The investigation of novel anticancer agents was pursued by the design of new derivatives, bearing a hydrazide moiety along with a pyrroloquinoxaline nucleus, preliminarily tested for their antiviral properties. Two of these compounds revealed only a weak antiviral activity, while possessing basic prerequisites for further evaluation of their anticancer potential. These derivatives retained the hydrazide moiety characterizing the salicylhydrazide reference compounds,
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while one out of the two cyclic portions was replaced by a pyrroloquinoxaline and the other one by a pyridine or a pyrazine nucleus. Both compounds showed a moderate activity in the MTT assay .
These initial evaluations led to an in-depth lead optimization campaign resulting in several potent cytotoxic analogs. The common chemical features characterizing this series were therefore a hydrazine moiety between a tricyclic pyrroloquinoxaline system and a heteroacyl residue. After the initial cytotoxicity screens, some of the newly synthesized compounds showed remarkable activity in a panel of tumor cell lines .
SYNTHESIS OF PYRROLOQUINOXALINE HYDRAZIDES
Two synthetic approaches for the preparation of compounds SC144 and SC153-SC166 are outlined in Fig. 1A. The synthesis of compounds SC155-SC159 was
accomplished starting from lactams (1), in turn obtained from 1-(2-aminophenyl)pyrroles or 1-(2-fluorophenyl)pyrroles following known procedures . Lactams were subsequently transformed into 4-chloro-1H-pyrrolo[1,2- a]quinoxalines (2) by treatment with phosphoryl chloride. Reaction of compounds (2) with hydrazine monohydrate afforded 4-hydrazinopyrrolo[1,2-a]quinoxalines, which were in turn condensed with the appropriate carboxylic acid to give the desired compounds SC155-SC159.
Alternatively, the chloro-derivative intermediates (2) were directly reacted with preformed acylhydrazides . This latter sequence directly yielded final compounds SC144 and SC160-SC166 in the form of hydrochloride salts.
Subsequently, additional hydrazides (e.g. SC167-SC180) were designed and synthesized to better establish structure- activity relationships and identify key features important for cytotoxicity (Fig. 1B).
N N N Ar H
SC155 H 2-indolyl SC156 H 6-indolyl SC157 H 5-indolyl SC158 H 3-indolyl
SC159 H 2-methoxyphenyl
SC144 7-F pyrazin-2-yl SC160 7-F pyridin-2-yl
1 2 N
N N Ar H
SC161 9-F pyrazin-2-yl SC162 7-F pyridin-3-yl SC163 7-F 2-fluorophenyl SC164 7-F thiophen-2-yl SC165 7-F furan-2-yl SC166 7-F quinoxalin-2-yl
R H O
N N N Ar H
N Y .HCl
R X Y Ar
SC167 7-F CH CH 2-aminophenyl SC168 7-F CH CH 4-aminophenyl SC169 7-F CH CH 4-hydroxyphenyl SC170 7-F CH CH phenyl
SC171 H N CH phenyl
SC172 H N CH 4-hydroxyphenyl
SC173 H N CH pyrazin-2-yl
SC174 H N CH pyridin-3-yl
SC175 7-F CH N 4-hydroxyphenyl
SC176 7-F CH N pyrazin-2-yl
SC177 7-F CH N pyridin-3-yl
SC178 7-F CH CH pyrroloquinoxalin-4-yl SC179 H N CH pyrroloquinoxalin-4-yl SC180 7-benzyloxy CH CH pyrazin-2-yl
Ar SC181 pyrazin-2-yl SC182 pyridin-3-yl
SC184 thiophen-2-yl SC185 furan-2-yl
Fig. (1). A: Synthetic pathways to pyrroloquinoxalinhydrazides. B: Chemical structures of compounds SC167-SC185.
Several chemical modifications to the former compounds were made by varying the fused-ring system and/or the heteroacyl moiety. For example, the benzo-fused ring was replaced by pyridine, the pyrrole ring was replaced by imidazole, together with various alterations of the heteroacyl moiety. The introduction of an extra nitrogen atom of pyridine or imidazole offered the possibility of forming conjugate salts to improve water solubility. A second series of compounds based on a N′-heteroacyl-9H-pyrrolo[1,2- a]indol-9-hydrazone structure SC181-SC185, envisaged as 5-deaza analogues of the pyrroloquinoxaline based reference compounds, was also prepared. However, after initial cytotoxicity evaluation, compounds belonging to this last series did not show any significant improvement in terms of potency and activity spectrum, compared to pyrroloquinoxaline based parent compounds, and therefore were no longer pursued .
CYTOTOXICITY EVALUATION AND EFFECTS ON CELL CYCLE
All compounds that satisfied the Lipinski’s rule-of-five as calculated using ADMET predictions were subjected to cytotoxicity evaluation in MTT assay . Two of the compounds synthesized in the first batch (SC144 and SC161) showed potent cytotoxicity with IC50 values ranging from 0.3 to 4 M against five human cancer cell lines: MDA-MB-435 (breast cancer), LNCaP (prostate cancer), HCT116 p53+/+, HCT116 p53-/- and HT29 (colon cancer). Colony formation assays were also performed to confirm activity and, at doses above 1 M, cell growth was completely abolished .
All compounds subsequently synthesized were tested in six human cancer cell lines from different origins: MDA- MB-435 and SKBR-3 (breast cancer), HCT116 p53+/+, HCT116 p53-/- and HT29, and HEY (cisplatin-resistant ovarian cancer) . Among the new analogs, compounds SC173 and SC176 also showed significant potency with IC50 values ranging from 0.3 to 0.5 μM. Their activity was further assessed by colony formation assay, in which both compounds demonstrated to be highly active at 1 μM.
Flow cytometry analysis demonstrated that both compounds arrested cells in G0/G1 phase in HCT116 and SKOV3 (ovarian cancer) cell lines. Their properties to induce cell
cycle arrest made them suitable agents for combination therapies with drugs acting at different stages of cell cycle, such as paclitaxel and camptothecin . Flow cytometry and fluorescence microscopy assays indicated that a possible mechanism of action for these compounds is the inhibition of superoxide dismutase (SOD) enzymes and, subsequently, accumulation of reactive oxygen species (ROS) with induction of superoxide-mediated apoptosis.
The in vivo efficacies of compounds SC173 and SC176 were evaluated in a nude mice xenograft model of human ovarian cancer by intraperitoneal injection. Compound SC176, in particular, significantly reduced tumor burden. Both compounds were less active than paclitaxel, which was included as a positive control. Treatments were well tolerated and did not result in any drug-related deaths or changes in body weight. Moreover, histopathological examination of the organs from the treated mice did not reveal obvious toxic effects.
Because of their noteworthy activity these two latter compounds, together with previously identified SC144 and SC161 were selected for further studies (cytotoxicity of the four selected compounds is reported in Table 1).
Structure-activity relationship for the whole class of compounds, showed the crucial role played by the pyrazinoylhydrazine fragment: any alteration to this moiety led to less active or completely inactive derivatives. A number of modifications of the tricyclic system was instead possible, provided that the core motif is retained. In fact, the activity was retained for compound SC173 and SC176 in which the benzo-fused ring and the pyrrole were bioisosterically replaced by a pyridine and an imidazole, respectively. A fluorine atom at 7-position (SC144 and SC176) or 9-position (SC161) was introduced for enhanced metabolic stability and favorable PK properties .
THE LEAD COMPOUND: SC144
After careful evaluation of potency, drug-like properties, broad effectiveness and synthetic feasibility, compound SC144 was chosen for a more in-depth anticancer evaluation. To confirm the potential of such compound as the lead, its cytotoxic profile was investigated in a broad panel of 16 cell lines from different human tumor origins,
Table 1. Cytotoxic activity of selected compounds in a panel of cancer cell lines.
Ar IC ( M)
a MDA- MB-435 b HCT116
p53+/+ b HCT116
p53-/- b HT29 c LNCaP d HEY a SkBr-3
SC144 7-F CH CH Pyrazyn-2-yl 4±0.1 0.6±0.07 0.9±0.04 0.9±0.06 0.4±0.06 1±0.1 3,6±0.5
SC161 5-F CH CH Pyrazyn-2-yl 3±2 0.4±0.01 0.3±0.1 0.3±0.1 0.4 - -
SC173 H N CH Pyrazyn-2-yl 0.3±0.03 0.4±0.03 0.4±0.15 0.4±0.1 - 0.3±0.1 0.5±0.1
SC176 7-F CH N Pyrazyn-2-yl 0.3±0.03 0.4±0.03 0.4±0.15 0.4±0.1 - 0.3±0.1 0.5±0.1
aBreast. bColon. cProstate. dOvarian.
wherein the compound showed excellent activity, with IC50 values ranging from 0.5 to 4.0 μM (Table 2). Cytotoxicity of SC144 was time- and dose-dependent, while it appeared to be independent of HR, p53, pRb, p21, HER-2 and p16 status. Considering its activity in drug-resistant ovarian cancer NCI/ADR-RES and HEY cells as well as oxaliplatin- resistant colorectal HTOXAR3 cells, SC144 demonstrated promising therapeutic potentials for treatment of human cancers that are refractory to current treatment regimes. Cell cycle analysis by flow cytometry indicated that SC144 induced cell cycle arrest in G0/G1-phase in both breast (MDA-MB-435) and colorectal (HT29) cancer cell lines. Flow cytometry studies also confirmed that SC144 is able to induce apoptosis at its IC50 value concentration in MDA- MB-435 cells. The in vivo efficacy of SC144 was then evaluated in nude mice xenograft models of human breast and colon cancers. Compared to control, treated tumors were smaller in size and poorly vascularized. The treatment was also well tolerated and did not result in any body weight loss or drug-related deaths even at the highest dose of 150 mg/kg .
ACTIVITY OF SC144 IN COMBINATION WITH CONVENTIONAL ANTITUMOR AGENTS
Since combination therapy represents an effective approach to delay the emergence of resistance, the activity of SC144, in association with several conventional cytotoxic agents, using cytotoxicity assay and cell cycle analysis, was evaluated. The growth inhibitory effect of SC144 in combination with 5-fluorouracil and oxaliplatin in HT29 cells, the current standard chemotherapeutic agents for colon cancer, was examined. A synergistic effect was observed with these agents. The cell growth inhibition was evaluated with different combination treatments of SC144 and oxaliplatin in HT29 and HTOXAR3 (an oxaliplatin-resistant colorectal cancer cell line). While in HT29 cells the cytotoxicity elicited by the drug combination was noted to be independent of the treatment schedule, in HTOXAR3 cells the pre-exposure to SC144 seemed to sensitize resistant cells towards oxaliplatin effects (from 25% cytotoxicity for
oxaliplatin alone to 35% after oxaliplatin → SC144 treatment and 70% after SC144 → oxaliplatin treatment, respectively).
To further explore the therapeutic potential of the compound in different cancer models, its effect on the growth inhibition of MDA-MB-435 cells was investigated after various treatment schedules in combination with paclitaxel. Interestingly, the anticancer activity of SC144/ paclitaxel seemed to be sequence dependent. In fact, the administration after paclitaxel revealed synergism at all the fractions attempted (90% fractional inhibition), while the effects of the other treatment schedules was only additive (< 40% fractional inhibition after simultaneous administration,
< 50% after paclitaxel → SC144 sequential treatment). Furthermore, the in vivo efficacy of this combination treatment was ascertained in a mouse xenograft model. As a result, the treatment significantly reduced tumor burden, delayed tumor growth in a dose-dependent manner and was well tolerated [19, 21].
SC144 UPREGULATES IL-24 IN COLON CANCER
More detailed investigations were carried out to better understand SC144 mechanism of action in three different forms of cancer: colon, ovarian and prostate.
In the first study, SC144 increased IL-24 protein levels in colon cancer cells. MDA-7 protein, also known as IL-24 (a member of IL-10 family), is a cytokine that acts as a tumor suppressor [22, 23]. Loss of IL-24 expression has been associated with the progression of several types of cancer [24-26], i.e. colon cancer. Recombinant IL-24 induces apoptosis in a broad range of human cancers, with no significant toxicity to normal cells. In addition, IL-24 inhibits angiogenesis, stimulates antitumor immune response, sensitizes cancer cells to radiation and other modalities of conventional therapies, and induces multipronged ‘bystander’ activity eliminating both primary and distant tumors in animal models [27, 28]. Thus, a potential innovative approach to colon cancer therapy would be the development of small-molecule drugs that can increase IL-24 expression in tumor cells and tumor
Table 2. Cytotoxicity of SC144 in various cancer cell lines.
Cell line Origin IC50 (µM) Cell line Origin IC50 (µM)
DU-145 Prostate 3.0±0.3 MDA-MB-468 Breast 0.7±0.1
LNCaP Prostate 0.39± 0.06 SKBR-3 Breast 3.6±0.5
LCCaP/Her2 Prostate 0.4±0.06 MCF-7 Breast 1.7±0.3
HEY Ovarian 1.0±0.1 NCI/ADR-RES Ovarian 0.14
CRL5908 Lung 3.5±0.7 HCT116 p53+/+ Colorectal 0.58±0.07
H1299 Lung 1.67±0.05 HCT116 p53-/- Colorectal 0.94±0.04
CRL5908 Lung 3.5±0.7 HT29 Colorectal 0.9±0.06
MDA-MB-435 Breast 4.0±0.1 HTOXAR3 Colorectal 0.5±0.1
microenvironment. The effectiveness of SC144 in blocking proliferation in a panel of human colon cancer cells was shown to be due to its ability in up-regulating the cellular level of IL-24 by over 2.5-fold, in a dose-dependent and p53- independent manner. DARTS assay  demonstrated that SC144 directly binds and stabilizes IL-24. The binding of SC144 to IL-24 was confirmed by molecular docking experiments. In the predicted model, SC144 properly fitted into only one site of the protein binding pocket with a GOLD fitness score of 46.
The ability of SC144 to arrest cell cycle in the G0/G1 phase in colon cancer cells was consistent with the previous discovery that recombinant IL-24 caused cell cycle arrest in the G0/G1 phase , indicating that SC144-induced cell cycle perturbation resulted from, at least partially, IL-24 upregulation. Considering the high cost of recombinant IL-24 and Ad-mda7, an IL-24 cDNA adenoviral vector, as well as their poor oral bioavailability, this lead was recognized as an alternative to IL-24-based low-cost therapeutic option. The compound might also have other cellular protein targets, resulting in multiple potential molecular mechanisms, and thus the anticancer potency might be a sum of these effects .
INHIBITION OF GP130 ACTIVITY BY SC144 IN OVARIAN CANCER
A potential target for SC144 in inhibiting cancer cell proliferation is the gp130/Stat3 signaling axis. In fact, gp130 is positioned at the junction of this oncogenic signaling network and is an attractive therapeutic approach for gp130- dependent cancers .
SC144 inhibited cell growth and colony formation in a panel of human ovarian cancer cell lines, including paclitaxel, doxorubicin, and cisplatin-resistant cell lines, with IC50 values in a submicromolar range suggesting the ability of this compound to overcome drug resistance in select cancers.
SC144 inhibited gp130 cytokine signaling, as well as the downstream gene expression, and showed in vitro and in vivo potency in ovarian cancer. Treatment with SC144 substantially increased the phosphorylation of gp130 (S782) and its deglycosylation in OVCAR-8 and Caov-3 cells in a time- and dose-dependent manner, followed by gp130 internalization. In addition, Western-blotting experiments showed that the compound inhibited Stat3 activation and the expression of downstream genes, preventing the activation of the downstream signaling pathways stimulated by gp130 ligands. The binding of SC144 to gp130 has been proven by DARTS assay, suggesting that the compound might directly induce conformational changes in gp130 and affect its activity . The suppression of gp130/Stat3-mediated gene expression by SC144 resulted in inhibition of cell-cycle progression and angiogenesis leading to apoptosis and cell death. Furthermore, SC144 delayed tumor growth after oral or intraperitoneal administration in mice xenograft models without detectable toxicity to normal tissues. On the basis of these results, this lead compound could be considered a first- in-class small-molecule gp130 inhibitor with oral activity in ovarian cancer [33, 34].
INHIBITION OF SURVIVIN IN PROSTATE CANCER
A plausible model for the anticancer mechanism of SC144 in prostate cancer (PCa) was investigated in a more recent study . Through the inhibition of the IL-6/ gp130/Stat3 signaling axis, SC144 is a potent suppressant of survivin, a cancer-specific marker and a promising drug target [34, 36]. The upregulation of survivin was confirmed in clinical studies, and was significantly associated with patients’ decreased overall survival.
Encoded by the BIRC5 gene, survivin is a member of the IAP (inhibitor of apoptosis protein) family and is highly expressed in most human tumors and fetal tissues, while it is absent in almost all terminally differentiated cells. Survivin is a nodal protein able to promote tumorigenesis by inhibiting caspase activity and apoptosis . In addition, expression of survivin in PCa contributes to chemoresistance and cancer progression . Only a limited number of selective survivin inhibitors are known, although the inhibition of such protein represents a promising strategy for cancer treatment.
SC144 suppressed cells growth and colony formation in a panel of PCa cells of mouse (CE1, CE2, E2, E4 and E8) and human (LNCaP, DU145, and PC3) origins with IC50 < 1 µM. These results were consistent with a survivin expression suppressant activity. As expected, the compound reduced survivin expression after 24 h treatment in the same panel of cells.
Furthermore, when combined with clorgyline, a selective and irreversible MAOA inhibitor, SC144 showed substantial synergistic effects on the inhibition of PCa cell growth and migration with concomitant decrease in survivin levels . It was then demonstrated that survivin depletion delayed MAOA increase during PCa progression, whereas MAOA inhibition, in turn, enhanced survivin suppression.
The rapid development of targeted therapeutic agents in oncology during the past decade has provided an unprecedented opportunity to study rational combination therapies. In addition, multi-targeted drugs offer a promising approach in the pharmacological management of cancer.
SC144 represents a prototype of a new family of compounds with novel mechanism of action inhibiting the gp130-STAT3-survivin axis that is proven to be essential for the survival of cancer cells. Because of its unique pharmaceutical, PK, and drug-like properties coupled with cytotoxicity towards cells resistant to standard-of-care chemotherapies, SC144 represents a new class of compounds warranting clinical investigation. Moreover, it shows synergy with various chemotherapies as well as a previously used nontoxic MAOA inhibitor, chlorgyline, suggesting a safe and efficacious combination therapy. This compound has proven to be effective in animal models of ovarian, breast, colon, and prostate cancers. Identification of key biomarkers for patient selection and treatment efficacy is under investigation to position this compound for Phase I clinical studies.
LIST OF ABBREVIATIONS
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BIRC5 = Baculoviral inhibitor of apoptosis repeat- containing 5
cDNA = Complementary deoxyribonucleic acid DARTS = Drug affinity responsive target stability DNA = Deoxyribonucleic acid
GOLD = Genetic optimization ligand docking
gp130 = Glycoprotein 130
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IC50 = Inhibitory concentration in 50% population IL24 = Interleukin 24
MAOA = Monoamine oxidase A
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Stat3 = Signal transducer and activator of
VDJ = Variable, diverse, and joining gene
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The authors confirm that this article content has no conflict of interest.
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Received: November 28, 2014 Revised: April 24, 2015 Accepted: June 20, 2015