Synthesis and In vitro anticancer activity of some novel substituted carbazole carbamate derivatives against human glioma U87MG cell line

Nitin Kumar, Neetika Lal, Vishal Nemaysh, Pratibha Mehta Luthra

Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi 110007, India.

*Corresponding Author E-mail: nitinvishnoi@gmail.com

ABSTRACT:

Tricyclic carbazole derivatives displayed excellent anticancer activities in literature. In this research work, we synthesized a series of some novel substituted carbazole carbamate derivatives and also evaluated their anticancer activity (IC₅₀) against human glioma U87MG cell line. Both 6^th position and N-ethyl substituted carbazole carbamate derivatives synthesized and tested for anticancer potential on human glioma U87MG cell line based on colorimetric MTT assay. SAR (structure activity relationship) study of all substituted carbazole carbamate derivatives also elaborated on the basis of in vitro anticancer activity (IC₅₀) results. Four carbazole integrated carbamates (27, 28, 30, 31) possesses excellent anticancer profile with IC₅₀ values 17.97 µM, 15.25 µM, 29.58 µM, and 23.80 µM respectively on U87MG glioma cell line. Both 6-substituted and N-ethyl based carbazole carbamate compounds displayed excellent in vitro anticancer activity on tested glioma cell line. The study of novel substituted carbazole tethered carbamate derivatives found potential anticancer profile against U87MG glioma cell line and can be fruitful for further development against brain tumour.

KEYWORDS: Brain tumour, Carbazole, Carbamate, Anticancer, Glioma cell line.

1.0 INTRODUCTION:

Glioblastoma (GBM) is categorized aggressive malignant primary brain tumor, with short life span around 12-14 months^1-3. Brain tumour size, shape location and image in individual patients can be checked by Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) for better diagnosis and also further treatment ⁴. Current treatment modalities of brain cancer incudes chemotherapy, surgery (if needed) and exposure of radiation therapy mainly ^{3, 5}.

However, sad part of treatment modalities only improves life survival of patient’s not complete cure. Various pharmacological mechanism is imparting role in anticancer resistance and its leading failure of drugs in cancer treatment specially GBM⁶.

Organic carbamates are structural elements of many approved therapeutic agents and widely utilized as a peptide bond surrogate in medicinal chemistry. Furthermore, organic carbamates serve a very important role as optimum protecting groups for amines and amino acids in organic synthesis and peptide chemistry⁷. Over the years, a variety of methods available for synthesis of carbamates^7,8 (Scheme 1) (Fig.1).

Method 1: Carbamates prepared by utilizing the Hofmann rearrangement of amides

Method 2: Carbamates prepared by the Curtius rearrangement of acyl azides

Method 3: Carbamates prepared by the reaction of alcohols with isocyanates

Method 4: Carbamates prepared by the reductive carbonylation of nitroaromatics

Fig. 1 Traditional synthetic method for carbamates

The formation of carbamates from isocyanates (Method 3, Scheme 1) is fundamentally important to polyurethane industries. The readily available alkyl chloroformates are the most frequently used reagents for the preparation of carbamates (Method 4, Scheme 1).

2.0. RATIONAL APPROACH:

Various authors’ introduced carbamates moiety in the active pharmacophores of structurally diverse natural products and displayed potential activity (Fig.2). The natural antibiotic fumagillin 1, inhibits endothelial cell proliferation in-vitro and tumor-induced angiogenesis in-vivo. Replacement of unsaturated ester chain at C-6 by an O-(chloroacetyl) carbamoyl moiety resulted in the potent anticancer compound 2 (TNP-470, IC₅₀ = 0.43 nM), it shows 50 times more active than fumagillin (IC₅₀= 0.63 nM)⁹. Paclitaxel (Taxol), 3 a diterpene alkaloid exhibits potential anticancer against breast and ovarian cancers¹⁰. Docetaxel 4 shows a similar biological activity compared to Taxol 3 ^10-13. Staurosporine derivatives (5) based carbamate derivative tested their kinase inhibiting activity in various cancer cells such as PKC, Trka, VEGFR, CDK5. Staurosporine carbamate compound 6 (IC₅₀ = 8 nM) showed most potent in Trka kinase inhibition activity¹³. Retrolactone (7and 8) derivatives showed IC₅₀=0.088 μM potent cytotoxicity against the L1210 cell line ^10-13.

Fig. 2 Some important structures of carbamates moiety-based drugs showed potential anticancer activity (1-8)

Carbazole scaffold based derivatives has been very popular among the researchers due to a wide variety of biological activities of interest ^14-18. Carbazole structure contains indole a potential pharmacophore which also displayed potential biological activities in literature^19-21. Ellipticine (9) also contains I,4-dimethyl carbazole scaffold which showed outstanding anticancer profile (IC₅₀) against various types of cancer lines (Fig.3) ²². One carbazole dithiocarbamate derivative linked substituted piperazine (10) displayed potential cytotoxic value (IC₅₀) 5.9 µg/mL on glioma cell line ²³. Our groups, reported a series of novel Bis carbazoles (11) showed excellent in vitro anticancer profile against human glioma U87 MG cell line and interacted with CT-DNA through groove binding mode (Fig.3) ^{2, 18}. Recently, we also published substituted carbazole thiosemicarbazide derivatives (12) also displayed significant Invitro anticancer activity against human glioma U87 MG cell line using MTT assay (Fig.3) ²⁴. Recently, we published novel 1,4-dimethyl-9-H-carbazole linked methenamine derivatives and in which one carbazole based methenamine derivative (13) found excellent in vitro anticancer activity against human glioma U87 MG cell line (Fig.3) ²⁵. Plant derived anticancer drugs like Vincristine contains both indole and carbazole nucleus which is significantly used in the treatment of brain tumor and also other cancers ^26-28. Considering the importance of carbamates as anticancer agents we synthesized carbazole tethered carbamate derivatives. We synthesized different substituent’s on the 6th position of the carbazole moiety (R=H, Br, OCH₃) tethered to 2-chloro-ethyl carbamate group linked to the this carbazole moiety. 2-chloro-ethyl carbamate group attached in carbazole molecule which present in carmustine (BCNU) and lomustine drugs ^{2, 26} respectively.

Fig. 3 Potential anticancer molecules based on carbazole scaffold (9-13) and proposed substituted 1,4-dimethyl-9-H-carbazole tethered carbamate derivatives (26-31)

Scheme-1 A) NaBH₄, methanol, 0⁰C to rt for 30-60 minutes B) 2-chloroethyl isocyanate, TEA, DCM , RT, 24 h.

3.0 MATERIAL AND METHODS:

3.1 Chemicals and instruments:

All chemical were purchased from Sigma Aldrich, Spectrochem Pvt. Ltd. Proton NMR was recorded in 400 MHZ Bruker instrument and 400 MHZ JEOL instrument. All final carbazole compounds purified by column chromatography using silica gel (100 mesh) with ethyl acetate/petroleum ether solvent system. Mass spectra also recorded of all compounds. In vitro anticancer activity of all compounds performed in triplicate cell plate. Treatment of cells (5×10³ U87MG) with different concentrations (1µM to 250 µM) of compound (26-31) for 24 h time period based on MTT assay^2,24.

3.2 Synthesis of substituted 1,4-dimethyl-9-H-carbazole tethered to carbamates derivatives:

In work, the synthesis of 1,4-dimetyl carbazole aldehyde scaffold (14-19) was carried accoding according to our and previous reported known Vilsmeier-Haack Formylation method[2, 24, 29-31]. Reduction of aldehyde group at 3^rd position in substituted 1,4-dimethyl-9-H-carbazole compounds (14-16) and 17-19 (preapred in earlier publihsed articels) [2,24] carried with 1.5 equivalent of sodium borohydride hydride (NaBH₄) in methanol solvent at 0⁰ C to rt for 30-60 minutes gave 6-substituted and N-ethyl based 1,4-dimethyl-9-H-carbazole alcohol derivatives (20-25) and used without further purification and confirmed by NMR ^32,33. Both 6-substituted and N-Ethyl carbazole carbamate compounds (26-31) were prepared from reaction of carbazole alcohol compound 20-25 (0.05 mol)] and triethylamine (0.06 mol) in dichloromethane solvent with a constant stirring at 0–5°C for 5-10 minutes, added 2-chloroethyl isocyanate (0.06 mol) was added drop wise gradually to this solution[7, 34]. The reaction mixture was stirred at rt for 24h. After the solvent was evaporated to dryness and pure target compounds (26-31) were obtained by 20% ethyl acetate/petroleum ether solvent using column chromatography in 70-80 % yield.

Proton NMR and Mass data of Compound-1,4-dimethyl-9-H-carbazole-3-yl)methyl(2-chloroethyl) carbamate(26): Yield: 86%. White solid; melting point 197ºC.¹H NMR (CDCl₃): δ 8.169(d,1H),7.97(s,1NH), 7.36(m,2H),7.17(m,2H),5.26(s,2H),3.58(t,3CH₂),3.54(t, CH₃), 2.79(s, CH₃),2.46(s, CH₃). (Fig. 4) Mass: calculated: 330.11 observed: 329.11(- mode)

Proton NMR and Mass data of Compound 26-1,4-dimethyl-9-H-carbazole-3-yl)methyl(2-chloroethyl) carbamate(29) Yield: 86%. White solid; melting point: 231ºC. ¹H NMR (CDCl₃): δ 8.16(d,1H),7.35(q,2H), 7.17(dd,1H), 7.13(s,1H), 5.22(s,2H), 4.48(q,2H), 3.51(t,2H), 3.44(t,2H), 2.77 (s,3CH3), 2.70(s, CH₃), 1.31(t,CH₃). (Fig. 5).  Mass calculated: 358.14, observed: 357.14(- mode)

4.0 ANTICANCER ACTIVITY RESULTS OF CARBAZOLE CARBAMATE DERIVATIVES AGAINST U87 MG GLIOMA CELL LINE:

In vitro cytotoxicity IC₅₀ values of these compounds calculated and its compared with drugs used in brain tumors like carmustine (BCNU) and temozolamide (TMZ) shown in Table 1[2, 18, 24]. Two series (A and B) of compounds (26-28) and (29-31) were prepared based on substitution at 6^th (26-28), 9^th(N-ethyl),(29-31) in Scheme 1. In series A Carbazole carbamates was substituted at 6th position,where R=H,Br,OCH₃(26-28) respectively. In series B carbazole carbamates was substituted at N-H position with ethyl,where R=H,Br,OCH₃,R₂=CH₂CH₃ (29-31) respectively. All six compounds showed IC₅₀ value in range 15.25 µM-141.8 µM. In series A IC₅₀ value of 6th substituted on carbazole tethered carbamates derivatives(R= H, Br,OCH₃) 26-28 were found 141.8 μM, 15.25 µM, 17.25 μM respectively compare to unsubstituted compound 26(141.8 µM). The IC₅₀ of 6-substituted carbazole carbamates compounds with electron withdrawing or electron donating group (27-28) found better anticancer activity approximately 8 to 9 fold times compared to unsubstituted compound 26(141.8 µM). All three compounds (26-28) showed better in vitro cytotoxicity (IC₅₀) value than standard drug Temozolomide (IC₅₀= 100 µM) and standard drug carmustine (IC ₅₀ =18.24µM) [2, 18, 24]. (Table 1.) In series B IC₅₀ value of three N-ethyl substituted carbazole tethered carbamates derivatives (R₁=H, Br, OCH₃, R₁=CH₂CH₃) 29-31 were found, 66.70 µM, 29.58 µM, 23.80 μM respectively. In series B compounds 29-31 displayed loss of in vitro cytotoxicity (IC₅₀) value than series A 6-substituted carbazole carbamate derivatives (27-28) on glioma cell line (Table 1). SAR study of all final carbazole carbamate derivatives suggested that varying the 6^th position substituent either bromo or methoxy and no alkylation (ethyl group) was found potent anticancer activity.

Table 1

S. No.	R	R1	IC50(µM)
26	H	----------	141.8 µM
27	Br	---------	17.97 µM
28	OCH3	----------	15.25 µM
29	H	CH2CH3	60.5 µM
30	Br	CH2CH3	29.58 µM
31	OCH3	CH2CH3	23.80 µM
Temozolomide	----------------	----------	100 µM
Carmustine (BCNU)	-----------------	----------	18.24 µM

Fig. 4 NMR spectra of compound 26

Fig. 5 Mass spectra of compound 26 and NMR spectra of compound 29

5.0 CONCLUSION:

In summary we synthesized substituted carbazole carbamate derivative (26-31). All carbazole integrated carbamate derivatives were well characterized by NMR and Mass spectroscopy techniques. All six target compounds (26-31) were screened for in vitro anticancer activity (IC₅₀) values on U87 MG glioma cell line based on colorimetric MTT assay for 24-hour time period. Two compound 27 and 28 showed best vitro cytotoxicity (IC₅₀ = 17.97 µM and 15.25 µM) compare to standard drug carmustine (IC₅₀= 18.00µM) and temozolomide (IC₅₀ = 100 µM) respectively against U87 MG cell line. Two N-Ethyl substituted carbazole tethered carbamates (30 and 31) also showed potent vitro cytotoxicity (IC₅₀ = 29.58 µM and 23.80 µM) on glioma cell line. The study supports that substituted carbazole carbamate displayed significant anticancer potential on tested glioma cell line and further need of investigation required against brain tumor disease.

6.0 CONFLICT OF INTEREST:

All authors declared not have any conflict of interest.

7.0 ACKNOWLEDGMENT:

Pratibha Mehta Luthra thankful to DST India for project grant (SR/SO/HS-40/2004) and Nitin Kumar to CSIR for fellowship support for the work. We also thankful to USIC instrument facility for carrying NMR and MASS data.

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Received on 06.06.2024      Revised on 11.07.2024 Accepted on 09.08.2024   Published on 17.12.2024 Available online on December 23, 2024 Asian Journal of Pharmaceutical Research. 2024; 14(4):363-368. DOI: 10.52711/2231-5691.2024.00057 ©Asian Pharma Press All Right Reserved
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