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4 Methylthiazole Synthesis Essay


This review focuses on the syntheses of PI3K/Akt/mTOR inhibitors that have been reported outside of the patent literature in the last 5 years but is largely centered on synthetic work reported in 2011 and 2012. While focused on syntheses of inhibitors, some information on in vitro and in vivo testing of compounds is also included. Many of these reported compounds are reversible, competitive adenosine triphosphate (ATP) binding inhibitors, so given the structural similarities of many of these compounds to the adenine core, this review presents recent work on inhibitors based on where the synthetic chemistry was started, i.e. inhibitor syntheses which started with purines/pyrimidines are followed by inhibitor syntheses which began with pyridines, pyrazines, azoles, and triazines then moves to inhibitors which bear no structural resemblance to adenine: liphagal, wortmannin and quercetin analogs. The review then finishes with a short section on recent syntheses of phosphotidyl inositol (PI) analogs since competitive PI binding inhibitors represent an alternative to the competitive ATP binding inhibitors which have received the most attention.

Keywords: PI3K/Akt/mTor inhibitor, protein kinase inhibitor, anti-cancer drugs

1. Introduction

The PI3K/Akt/mTOR signaling pathways regulate cell proliferation, survival, and angiogenesis and hence are extremely important in cancer biology. A number of recent reviews of work 2 performed to understand and inhibit these signaling pathways have appeared and there is increasing evidence that inhibitors of at least 2 signals in the pathways may be most desirable.1 None of the recent reviews referenced above focus on how these inhibitors were prepared, therefore this review will concentrate on the organic chemistry of the syntheses of inhibitors and include some selected in vitro and in vivo screening results. The literature reviewed includes reports that have appeared outside of the patent literature in the last 5 years but will concentrate on studies reported in 2011 and 2012 since approximately 100 reports of PI3K inhibitor syntheses have appeared in the last two years alone. Many of these reported compounds are reversible competitive ATP binding inhibitors and their synthetic preparation relies on chemistry which is initiated from purine (diazolopyrimidine)/ pyrimidine, pyridine, pyrazine, triazine or azole core structures. The first sections of this review article were organized by looking at where the syntheses started. In many cases, this meant what heterocycle did the chemists prepare first or purchase and start with, and that was defined as the core structure under which to file that synthesis, ie pyrimidines, pyridines, triazines, etc. Many of these inhibitors contain multiple heterocyclic rings so they could conceivably be placed under several of these categories if one just asked does it contain one of the heterocycles under the category being discussed.

Synthetic work also continues on inhibitors based on the steroidal and terpenoidal cores found in wortmannin, quercetin, and liphagal. Therefore, this review will present recent work on inhibitors based on purines/pyrimidines, followed by pyridines, pyrazines, azoles, and triazines then move to liphagal, wortmannin and quercetin analogs. Some synthetic work also continues on phosphotidyl inositol binding inhibitors and that work is presented last.

2. Pyrimidines and Quinazolines

Synthesis of pyrimidine containing PI3K inhibitors continues to be an area of intense interest. Compounds in this class were some of the first that were found to be selective PI3K α inhibitors.2 These initial reports have been followed in the last few years with a number of additional reports of the synthesis and testing of pyrimidine derivatives and, in particular, morpholino pyrimidine derivatives.

In early 2010, a number of new 4-morpholinopyrrolopyrimidines were reported.3 This work reported routes to pyrrolo[3,2-d]pyrimidines and pyrrolo[2,3-d]pyrimidines. The pyrrolo[3,2-d] pyrimidine core syntheses were initiated using 2,4-dichloro-6-methyl-5-nitropyrimidine (1) as a starting material (Figure 1). The 4-chloro (ortho to the nitro) group was replaced first via a SNAr reaction and then aromatic substitutents were added to the pyrimidine core in the 2 position via Suzuki cross coupling reactions of aryl boronic acids via the 2nd chloride (2-chloro) to produce 2. The pyrrolo[3,2-d]pyrimidine core was then formed via treatment with dimethylformamide dimethylacetal. This reagent forms methoxide and the iminium salt when heated so would be expected add a formyl group to the 6 methyl position. Reduction of the nitro group to an aniline then provided a substrate which cyclized to the pyrrolo[3,2-d]pyrimidine core (3). The enamine functional group within that core structure was then used to condense with aldehydes and ketones to add substituents to the 7 position (4).

Figure 1

Pyrrolo[3,2-d]pyrimidine Syntheses.

The pyrrolo[2,3-d]pyrimidine core was synthesized via condensation of 6-amino uracil (5) with chloroacetaldehyde (6) (Figure 2). Conversion of the hydroxyl groups to chlorides with POCl3 was then followed by sequential addition of morpholine and aryl boronic acids as described above for the regioisomeric nucleus to produce 8. The pyyrole nitrogen was alkylated with alkyl halides and when 4-aminophenyl boronic acid was used for the Suzuki coupling then that 4-amino group was further converted into a variety of ureas (9) via treatment with triphosgene followed by amines.

Figure 2

Pyrrolo[2,3-d]pyrimidine Syntheses.

These urea derivatives were synthesized to improve water solubility. These compounds inhibited PI3Kα and mTOR at low nanomolar concentrations. In vivo testing of 9 (R’ = CF3, R1 = -Ph-4’-C(O)N(Me)CH2CH2NMe2) in MDA-MB-361 breast cancer xenografts showed substantial inhibition of both p70S6 and Akt phosphorylation – signaling pathways downstream of PI3K, 8h after iv injection of 25mg/kg. This dose produced tumorostatic effects on MDA-MB-361 xenografts, whereas 50 mg/kg decreased tumor size.

2010 also saw the report of the syntheses of a number of triazoles that were PI3K and Akt inhibitors (Figure 3).4 These syntheses started with 4-chloro-6-methylpyrrolo[2,3-d]pyrimidine (10). The most active new compounds were prepared by displacement of the chloride with 3-pyrrolidinol followed by Parikh-Doering oxidation of the alcohol with SO3-pyridine. The pyrrolidinone was then used in a modified Strecker reaction where both dimethoxybenzylamine and TMS-CN were added to the ketone. These additions were followed by nitrile reduction with lithium aluminum hydride (LAH) to produce 11. The primary amine was then converted to different amides using hydroxybenzotriazole (HOBt) and a carbodiimide and the dimethoxybenzyl protecting group was removed with trifluoroacetic acid (TFA) to produce the final products (12).

Figure 3

Syntheses of Pyrrolidine Substituted Pyrrolopyrimidines.

Twelve final compounds were evaluated for selectivity for Akt versus PKA kinases, and one compound was chosen for subsequent testing. Akt inhibitor (12) (R1 = Et, R = 2,4-difluorophenyl) reportedly inhibited Akt phosphorylation and decreased PC3 tumor growth by 25, 51 and 75% when administered orally bid for 10 days at 25, 75 or 100mg/kg, yet figures illustrating these effects were not presented.

Following these reports of the preparation and screening of pyrrolopyrimidines, a report appeared on preparation of dihydrothieno- and dihydrofuranopyrimidines which were more selective as Akt inhibitors but also functioned as PI3K inhibitors.5 The synthetic scheme used here started with the syntheses of the dihydrothiophene or dihydrofuran cores followed by construction of the pyrimidine (Figure 4). The five member rings were synthesized by Michael addition of 2-hydroxy or 2-mercapto methyl acetate (13) onto methyl crotonate (14) followed by Dieckmann condensation to yield the keto ester (16). Reaction of the keto ester with formamidine yielded the pyrimidinol which was converted to the chloride with POCl3. The chloride was replaced with a Boc protected piperazine. The NBoc was then removed and the free amine (18) was acylated with a variety of different amino acids to produce the targets (19).

Figure 4

Preparation of Dihydrothieno- and Dihydrofuranopyrimidines.

One compound (19) (X = S, R = (S)-CH2NHisopropyl, Ar = 4-ClPh) with favorable PK/PD parameters was selected for in vivo studies. In tissue culture of LNCaP cells 50% nhibition of Akt at 137nM of 19 was reported. In U87 xenografts at dose –dependent inhibition of phosphorylation of Akt substrate PRAS40 was reported that platoed at 75–200mg/kg oral dose for 4 hours based on. Inhibition of PC3 prostate cancer xenografts growth was reported at a daily dose of 200 mg/kg. These reports were not supported by original data showing PRAS40 phosphorylation. It is also somewhat surprising that effects on Akt activity and tumor growth were not reported in the same models.

Pecchi and co-workers reported both solid and solution phase syntheses of a number of 2-morpholino-6-arylpyrimidines in 2010 (Figure 5).6 The solution phase syntheses started with aryl malonate esters (20) which were cyclized with morpholino guanidine (21) to provide the pyrimidine core. The hydroxyl group on the pyrimidine was converted to a triflate and then subjected to Pd catalyzed C-N cross coupling to produce the final products (23).

Figure 5

Syntheses of Morpholino Aryl Pyrimidines.

The optimized compounds (23) were tested in the A2780 cell line (ovarian carcinoma, PTEN deleted), and inhibition of Akt at 7nM and proliferation at 0.37µM was reported (23, R = 3’-OH, R2 = H, R3 = 4’-OMe-3’-pyridyl).

In the summer of 2010, McDonald and co-workers reported the syntheses of a number of trisubstituted pyrimidines by a route very similar to that reported by Pecchi and co-workers (Figure 6).7 6-Aryl substituted pyrimidines were prepared via condensation of aryl β-ketoesters (24) with arylamidines (25) to generate pyrimidinones in which the hydroxyl was converted to a chloride then displaced with morpholine to produce the products (27). In this same report, alcohol and amine substituted pyrimidines were prepared starting with 2,4,6-trichloropyrimidine which was treated with the alcohol or amine of interest followed by morpholine. The monochloro pyrimidine products of these 2 steps were then cross coupled with aryl boronic acids using Pd(dppf)Cl2, Pd(PPh3)4, or in several cases a Pd(II) palladacyle catalyst. Tests in IGROV-1 ovarian cancer cells showed inhibition of S473 phosphorylation after 24 h of exposure.

Figure 6

Additional Syntheses of Morpholino Aryl Pyrimidines.

Additional 4,6-disubstituted-2-morpholinopyrimidines were reported by Burger, Pecchi, and co-workers at Novartis Institutes for Biomedical Research in early 2011 (Figure 7).8 One synthetic route started from tribromopyrimidine (28) and the amine was added first and the morpholine second. Suzuki cross coupling of the mono bromo pyrimidine (29) with heteroaromatic boronates then produced the final 4,6-disubstituted-2-morpholino pyrimidines (30). Alternatively, morpholine formamidine hydrobromide (32) was condensed with diethyl malonate (31) and the pyrimidinol thus produced was converted to the dichloropyrimidine (33) using POCl3. The chlorines were then sequentially replaced using Suzuki cross coupling and Buchwald-Hartwig C-N bond formation to produce products (35).

Figure 7

Syntheses of Amino Morpholino Aryl Pyrimidines.

Additional morpholino amino pyrimidinyl pyrimidines were also reported in early 2011 by Ohwada and co-workers (Figure 8).9 The morpholino pyrimidine core structures of these compounds were also prepared via a condensation of morpholino amidine (32) with a β-ketoester, in this case a β-ketolactone (36). Pyrimidinol chlorination was followed by a Pd catalyzed C-N bond formation to produce 38. In the cases where the syntheses originated with a morpholino dichloropyrimidine (39) rather than a morpholino monochloropyrimidine, the C-N bond formation could be performed first via SNAr chemistry and then the heteroaromatic could be added last via a Suzuki cross coupling to yield 40. Optimized compound CH5132799 (40, Ar = 2’-amino-5’-pyrimidinyl, R = SO2Me) showed inhibition of KPL-4 breast cancer xenografts at 12.6 mg/kg. In the follow-up experiments with KPL-4 cells in tissue culture Akt phosphorylation at S473 and T308 as well as of Akt substrate PRAS40 was inhibited at 10nM and of FOXO transcriptional factors at 100nM.10

Figure 8

Syntheses of Bicyclic Morpholino Pyrimidines.

Early 2011 also saw the report of syntheses of some benzothiazole substituted pyrimidines which are PI3K/mTOR dual inhibitors (Figure 9).11 Syntheses of all of these compounds started with the use of dihalopyrimidines or dihalopyridines (41) which were cross coupled to the pinacol boronate ester or tetrafluoroborate of the benzothiazole (42). The pyrimidine or pyridine cores were further modified by a base catalyzed SNAr reaction with sulfur or oxygen nucleophiles or a Pd catalyzed C-N bond formation in the case of nitrogen nucleophiles whereas the cross coupling yielded the final products for the pyridines (43).

Figure 9

Preparation of Benzothiazole Substituted Pyrimidines and Pyridines.

A pyridine benzothiazole biaryl (43, R = 4-F) with optimized pharmacokinetics that inhibited S473Akt phosphorylation in U87-MG cells with IC50 6,3nM was selected for in vivo tests. Inhibition of the PI3K/Akt pathway induced in liver by iv injection of HGF was observed starting at 0.1 mg/kg with maximal inhibition at 1mg/kg. A time course study showed that at 3mg/kg maximal inhibition lasted for 6h and significant inhibition lasted for 24 hours. Experiments in U87-MG (PTEN null) glioblastoma, A549 (KRAS mutant) lung adenocarcinoma and HCT11618,57 (KRAS and PI3KR mutant) colon adenocarcinoma tumor xenografts showed dose-dependent tumorostatic effect with ED 0.26 mg/kg. However subsequent in vivo experiments demonstrated accumulation in liver of a deacylated metabolite that inhibits PI3Kα at nM concentrations, which precluded further development of this compound.

In early 2011, Hong, Hong, and co-workers reported the syntheses of some PI3K inhibitors based on a xanthine core (Figure 10).12 This core was picked to produce inhibitors in which the sub cellular localization in live cells could be monitored by fluorescence microscopy. The fluorescent inhibitors screened here were prepared by a Cu(II) catalyzed N-arylation yielding 46 using aryl boronic acids followed by a Pd and Cu catalyzed C8 arylation (47) of the xanthine core.

Figure 10

Preparation of Substituted Xanthines.

A 2011 follow up publication13 on furano and thienopyrimidines that showed good PI3Kα to β selectivity used the same synthetic scheme which had been reported in 20082b for the preparation of these new analogs. The amino furanyl or thioenyl ester was converted to the furano or thieno morpholino mono chloro pyrimidine then the chlorine was replaced via Suzuki coupling of a boronic acid or ester. In addition to these extensions of previous work, this paper outlined the syntheses of some thienobenzoxepin PI3K inhibitors (Figure 11). To prepare these compounds a keto benzoxepin core (48) was condensed with the β-chlorovinylaldehyde generated via treatment of DMF with POCl3 and that condensation product was treated with methyl thioglycolate to produce 49 then with the appropriate aniline to produce 50.

Following on earlier reports of the activity of morpholino pyridinyl or pyrimidinyl pyrimidines, the synthesis of a very active bismorpholino pyridinyl pyrimidine (BKM-120) (53) was reported in the late summer of 2011 (Figure 12).14 The synthesis of this compound involved treatment of 2,4,6-trichloropyrimidine (51) with excess morpholine to generate the chloro dimorpholino pyrimidine (52) which was separated from the isomeric 2-chloro-4,6-dimorpholino pyrimidine and then cross coupled with the pyridinyl pinacol boronic ester to generate the final product (53).

In vitro kinase assays, demonstrated inhibition of all class I PI3K (isoforms α β δ γ at 52, 166, 116 and 262 nM respectively) with over 50 fold specificity over mTOR and vps34 and high µM range against other tested kinases. In vitro tests were supported by tissue culture experiments in cells with ectopical expression of myristoylated (constitutively active) PI3K α β and δ that show inhibition of Akt with IC50 at 100, 200 and 500nM. Further tests in tissue culture showed convincing inhibition of constitutive Akt phosphorylation in PTEN-deficient U87 cells at 1 µM and PDGF-induced Akt phosphorylation at 250nM in SF268 cells. Experiments in Rat-1 xenografts that ectopically express myr-PI3K α showed Akt inhibition starting at 1h and lasted for 16h after oral administration of 60mg/kg of BKM120. Inhibition of Rat-1 xenografts that ectopically express myr-PI3K α was demonstrated with 40mg/kg. This dose also delayed growth of HCT116 or BT474 xenografts whereas combination with either MEK inhibitor AZD6244 or heceptin dramatically improved anti-tumor efficacy. Substantial inhibition of tumor neo vascularisation and reduction of tumor vacular permeability was also noted.

Favorable pharmacokinetics and pharmacodynamics profiles made BKM120 a component of choice for experiments that address the consequences of PI3K inhibition in mouse models of cancers. Phase I clinical trials showed that overall BKM120 was well tolerated (MTD-100mg half-life 40h with little interpatient variability). Dose-limiting toxicities were associated with hyperglycemia, mood alterations and skin rash.15 Phase II clinical trials are currently ongoing.

Several recent publications emphasized the enhanced antitumor effects in mouse models when BKM120 was combined with inhibitors of other signaling pathways, whereas when used alone or in combination with inhibitors of the PI3K/mTOR pathway it showed tumorostatic effects.16 Somewhat surprisingly, BKM120 showed better efficacy against metastases compared to primary xenograft tumors MDA-MB-453 and BT474.17

New analogs of the amino pyrimidinyl thieno pyrimidine (GDC 0941) were reported in late 2011 (Figure 13).18 These compounds were prepared using a method similar to this group’s earlier report.2b Boc protected piperazine was condensed with the aldehyde and the intermediate iminium ion was reduced to the amine using Na(OAc)3BH to produce 54. The aminopyrimidine was added via a Suzuki reaction, the Boc amino protecting group was removed and the terminal nitrogen of the piperazine was acylated to obtain 55.

Figure 13

Preparation of Amino Pyrimidinyl Thieno Pyrimidines.

The most active new compound GDC-0980 (55, R = (S)-2-hydroxypropanoyl) inhibits both class I PI3K isoforms with IC50s of 5, 27, 7, and 14 nM for PI3Kα, β, δ, and γ, respectively; and inhibits mTOR with a Ki of 17 nM. At 7.5mg/kg daily dose compound GDC-0980 showed tumorostatic effects in PC3 and MCF-7neo/HER2. Subsequent experiments in tissue culture models demonstrated inhibition of Akt and PRAS40 phosphorylation at 0.1–0.5 mcM.19 Experiments in xenograft models showed delay or inhibition of tumor growth at 5 mg/kg. Analysis of pharmacodynamics in PTEN deficient PC3 xenografts showed inhibition of Akt phosphorylation by 75% at 24h after administration of 10 mg/kg, which caused tumor regression. Tumor regression was also demonstrated in breast cancer models MX-1 and MCF7-neo/HER2 and in NSCLC model A549. Currently GDC-0980 is in phase II clinical trials.

In late 2011, Blanchard and co-workers reported a series of 2-anilino-4-aryl-purine derivatives as inhibitors of PDK1 which is downstream of PI3K in cell signaling pathways (Figure 14).20 The preparation of these compounds involved protection of the NH in 2,4-dichloropurine (56) followed by Suzuki coupling at the 4 position (58) and a Buchwald-Hartwig coupling at the 2 position. The benzylated nitrogen was then deprotected using trifluoroacetic acid (TFA) to produce 59.

Figure 14

Synthesis of 2-Anilino-4-Aryl Purines.

The last kinase of the PI3K/Akt/mTOR pathway is the ribosomal protein s6 kinase (p70S6K) and Bussenius and co-workers reported a series of pyrazolopyrimidines as inhibitors of this kinase in late 2011 (Figure 15).21 The target inhibitors were prepared via base catalyzed condensation of 3-substituted-5-chloro-2-methylphenylpiperazines (60) with 4-chloropyrazolopyrimidines (61). This same general synthetic sequence was also used to prepare an optimized dual Akt/p70S6K inhibitor reported in early 2012.22

Meng and Yang and co-workers reported some morpholino pyrido pyrrolo triazines in late 2011/early 201223 using Piramid Pharma’s PI-103 (67, X = O, Y = C, furano rather than pyrrolo) as a lead compound (Figure 16).11 The compounds were prepared via initial condensation of chloro nitro pyridine (63) with the sodium salt of diethyl malonate. That product was completely decarboxylated and then deprotonated and condensed with diethyl oxalate to produce 64. Reduction of the nitro group and intramolecular condensation yielded the azaindole (65). Reaction of the indole nitrogen with NH2Cl followed by condensation with a 3-methoxybenzimidate produced the base of the target (66). Chlorination of the pyrimidinol and replacement of the Cl with morpholine followed by methyl ether cleavage with BBr3 completed the core preparation (67). The phenol was then acylated to produce several new compounds. Analysis of phosphorylation of Akt and p70S6 kinase (downstream substrates of PI3K and mTOR) showed that new compounds inhibited mTOR with similar potency yet inhibition of Akt phosphorylation was less potent compared to PI-103.

Figure 16

Preparation of Morpholino Pyrido Pyrrolo Triazines.

As a follow up to earlier reports on the PI3K inhibiting ability of pyridopyrimidones, Lin and co-workers at GSK reported a series of imidazo pyrimidinones which were selective for PI3K β inhibition (Figure 17).24 These compounds were made from 2-aminoimidazoles (68) which were condensed with diethyl malonate to make imidazopyrimidinediones (69) which were then converted to the dichlorides (70) with POCl3. The dichlorides were hydrolyzed back to the mono chloro imidazopyrimidones (71) which were N-alkylated and then the remaining chloride was replaced with morpholine via simple microwave heating to yield 72. Similarly to TGX-221 these compounds preferentially targeted PI3K β and δ. Experiments in MDA-MB468 cells showed inhibition of Akt phosphorylation at 0.1µM.

Figure 17

Preparation of Imidazopyrimidinones.

Synthesis of a family of triazolopyrimidinones (73) was reported by another GSK group in early 2012 and the synthetic route used to prepare these compounds was identical to the one described above except it started with a triazole rather than an imidazole (Figure 18).25

These compounds demonstrated improved inhibition of PI3Kβ, thus, compound 73 (R = Me, R1 = 2-CH3-3-CF3benzyl, R2 = Me) inhibited PI3Kβ at 0.3 nM in vitro and inhibited Akt phosphorylation in MDA-MB-468 cells with IC50=5nM. However, high clearance rates precluded testing these new compounds in cancer models in mice.

In April 2012, sulfonyl-morpholino-pyrimidines (77) were reported as mTOR selective kinase inhibitors (Figure 19).26 The compounds were synthesized via an initial condensation of an amidine or urea with a chloro β-keto ester (74). The pyrimidinols thus produced were chlorinated with POCl3 and then the chloride was displaced with morpholine. The sulfonyl groups were added via sulfinic acid salt displacement of the primary chloride or thiolate displacement of the primary chloride followed by oxidation of the resulting sulfide with oxone to produce 77 and 79.

Figure 19

Preparaton of Sulfonyl Morpholino Pyrimidines.

Morpholino pyrimidine derivatives which were selective PI3K β inhibitors were also reported in April27 and October 2012.28 The derivatives were prepared via condensation of aromatic diamines or aminophenols with the morpholino pyrimidine carboxylate salt (81)(Figure 20). The morpholino pyrimidine (80) was prepared via reaction of excess ethyl 3-amino-3-ethoxyacrylate with morpholine.

Figure 20

Preparation of a Morpholino Pyrimidine Salt.

A condensation product of 80 (compound 82) selected for in vivo studies showed selective inhibition of PI3K β and inhibited Akt in cell based assays with IC50 76nM (8-fold less than TGX221, IC50 10nM). Oral administration of 300 mg/kg inhibited Akt in PC3 xenografts y over 50% for 6h. Effective intratumoral concentration was calculated to be 2.1µM. When administered twice per day this compound inhibited Akt for 24h and delayed PC3 xenograft growth without evident toxicity.

Thienopyrimidines which are selective for PI3K δ inhibition were reported in mid 2012 (Figure 21).29 Their syntheses started from a morpholine substituted thieno chloro pyrimidinyl aldehyde which had been reported earlier.18 Reductive amination of the aldehyde using Na(OAc)3BH produced amino morpholino thieno pyrimidinyl chlorides (83). Depending on the aryl substituent desired, three different coupling sequences were used on the chloride. In some cases, the thienopyrimidinyl chloride (83) was converted to a SnBu3 reagent and used in a Stille coupling with aryl bromides. In some cases the chloride (83) was used in Suzuki cross couplings with aryl boronic acids or esters. With indole nitrogen nucleophiles C-N bond formation could be accomplished under base catalyzed conditions whereas if displacement of the Cl by a benzimidazole was desired then Buchwald-Hartwig conditions were used. Lead compounds produced here demonstrated inhibition of Akt phosphorylation in cell-based assays with IC50 between 0.1 µM and 0.4 µM and at least 19 fold specificity over α and β PI3K isoforms.

Figure 21

Preparation of Aryl Thieno Pyrimidines from Chloro Thieno Pyrimidines.

Also in mid 2012, Zhai, Gong, and co-workers reported a series of new 4-morpholinothienopyrimidines bearing arylmethylenehydrazones at the 2-position (Figure 22).30 These compounds were prepared from a 2,4-dichlorothienopyrimidine and the morpholine was added first at the 4 position followed by the addition of hydrazine to the 2 position. Condensation of the hydrazines with aldehydes then lead to the target hydrazones (85).

Figure 22

Preparation of Morpholino Thieno Pyrimidines Bearing Aryl Methylene Hydrazones.

The importance of the indole or benzimidazole substituent on the morpholinopyrimidine core for PI3K δ selectivity was first reported in July of 2012 (Figure 23).31 The core structure used here was a pyridopyrimidine (86) which was then substituted by an amine. The indole or benzimidazole (linked to the pyrimidine via carbon) was then added via its boronic acid or ester via a Suzuki coupling to produce 87. A compound (87, amine = α,α-dimethyl-4-piperidinemethanol, R1 = 5-F) that inhibited Akt phosphorylation at 40nM in cell based assays and demonstrated over 200 fold selectivity toward PI3Kδ compared to other PI3K isoforms was chosen for in vivo testing. Since PI3Kδ is necessary for antigen-receptor signaling in B cells, selective inhibitors could be therapeutically useful for treatments of autoimmune conditions. Tests in rodents showed over 80% oral bioavailability and plasma half-life over 4h, which justified further tests in animal models with the ultimate goal of developing new drugs for rheumatoid arthritis and other autoimmune diseases.

Figure 23

Preparation of Indole Substituted Pyrido Pyrimidines.

Murray, Sweeney and co-workers reported the preparation of 4-morpholinopurines bearing benzimidazoles at the 2 position in the summer of 2012 (Figure 24).32 Initially, they noted that pyrimidines fused to nitrogen heterocycles rather than sulfur heterocycles provided much better selectivity for PI3Kδ inhibition over PI3Kα inhibition. A variety of nitrogen heterocycles were added to a formyl group at the 8 position of the purine nucleus and a compound bearing a tetrahydropyranylazetidine was taken on into screening in rats and dogs.

Figure 24

Syntheses of 4-Morpholino Purines Bearing Benzimidazoles.

In the late summer of 2012, Blake and co-workers reported the preparation of a series of piperazine substituted cyclopentapyrimidines which were selective ATP competitive Akt inhibitors (Figure 25).33 The 6-chlorocyclopentapyrimidines were prepared first via condensation of semicarbazide with α-carboethoxycyclopentanones followed by conversion of the pyrimidinol to the pyrimidinyl chloride (93). The pyrimidinyl chloride was reacted with Boc protected piperazine followed by Boc deprotection and amine condensation with a variety of substituted phenylalanine amino acids to produce the targets (95).

Figure 25

Syntheses of Piperazine Substituted Cyclopentapyrimidines.

Pyridonyl pyrimidines which were PI3Kα and mTOR dual inhibitors were prepared and reported in August 2012 (Figure 26).34 Synthesis of the optimum new compound reported commenced with 1-amino-3-chloro-5-methyl pyrimidine (96). This compound was brominated and then the free amine was cyclized to a pyyrole (97) using 2,4 hexanedione. The chloride was displaced with hydrazine and the free NH2 was doubly alkylated with 1,4-dibromobutane to produce 98. The pyridine ring was installed using Heck chemistry and this first required N-acylation with acryloyl chloride. The intramolecular Heck reaction proceeded in a fairly low yield but it produced the desired pyridinone (99). The pyrrole was then deprotected, the pyridine brominated, and the N-Boc protected pyrazole was added via Suzuki cross coupling to produce 101. One wonders here if Buchwald conditions35 might not have worked better since those conditions are known to be successful with heterocyclic substrates which tend to deactivate palladium catalysts and these authors had to take a low yield at the end of this synthetic scheme with these conditions.

Thienopyrimidines were known pan PI3K inhibitors and pyrimidinones were known to be more selective for PI3K β so researchers from GSK combined these 2 observations and reported preparation of thiazolopyrimidinones which were selective PI3K β inhibitors (Figure 27).36 These thiazolopyrimidinones were made by one of two routes. One route used a 4-amino-2-(4-morpholinyl)-1,3-thiazole-5-carboxylate (102) and the other used the 5-carbonitrile (104). For the route which utilized the methyl ester (102), the amine and ester were cyclized by condensation with formamide. The N-H was then deprotonated and alkylated with a benzyl halide. The pyrimidinone was hydrolyzed to the amino amido thiazole which was acylated with propionyl chloride and then subjected to base catalyzed ring closure to provide the final substituted pyrimidinone (103). In the route which utilized the carbonitrile (104), the amine was first acetylated then alkylated with a benzyl halide. The resulting N-acetyl carbonitrile (105) was then cyclized to the pyrimidinone (106) using basic aqueous sodium perborate.

Figure 27

Preparation of Thiazolopyrimidinones.

Also in August 2012, Liu and co-workers at Pfizer reported the synthesis of cyclic sulfones fused to the pyrimidine nucleus and these compounds were highly selective for mTOR kinase over PI3K α (Figure 28).37 3, 3’-Thiobispropanoate (107) was used in a Dieckmann condensation to produce a thiopyranone (108) which was cyclized with urea to produce the bicyclic pyrimidine in low yield (One wonders here if this synthesis could have been accomplished more efficiently using a benzenecarboximidaamide). The pyrimidine diol was then chlorinated to produce 109 and the optically active 2-methyl morpholine was added. The sulfide was oxidized to the sulfone (110) with oxone and the phenyl urea was added via a Suzuki cross coupling of the pinacol boronate to yield 111. One also wonders here if the Buchwald conditions which work better for heterocyclic substrates (although admittedly usually used for C-N bond formation) might not have also significantly improved yield for this C-C bond formation with reactants containing a host of heteroatoms.35a,38

Figure 28

Syntheses of Bicyclic Sulfonyl Pyrimidines.

September 2012 saw the publication of more morpholino thieno pyrimidines by Heffron and co-workers from Genentech and these thienopyrimidines were designed to be selective for PI3Kα and capable of penetrating the blood brain barrier (Figure 29).39 Almost all of the compounds in this study were prepared from the previously reported morpholino thieno pyrimidine monochloride (112).18 To prepare the most active compounds that contained azetidine or ether substituents off the thiophene ring, the thiophene was first metalated with BuLi. Quenching the Li salt with iodine and cross coupling with an iodo azetidine yielded a pyrimidinyl chloride (113) which was subsequently cross coupled with a pinacol borate of the appropriate amino pyrimidine to produce 114. Quenching the Li salt with ketones yielded alcohols containing chloropyrimidines that were directly cross coupled to pinacol borates of amino pyridines or the alcohols were converted to ethers then cross coupled to yield 115.

Figure 29

Syntheses of Morpholino Thienopyrimidines Designed to Cross the Blood Brain Barrier.

A lead compound (115, R1 = R3 = Me, R2 = -CH2OCH2-) demonstrated 62% inhibition of pAkt in brain 1h afer 50mg/kg dose that achieved 3.6µM in mouse brain. Although the compound was not tested on intracranially implanted xenografts, the authors extrapolated that it will inhibit tumor growth based on tumorostatic effects in subcutaneously implanted U87 xenografts in which concentrations were at 2µM level.

In September 2012, Giordanetto and co-workers at AstraZeneca reported a series of morpholino-pyrimidinones which were selective PI3K β inhibitors (Figure 30).40 They started this synthetic work by treating 2,4,6-trichloropyrimidine (116) with p-methoxy benzylalcohol (117) to generate 2 PMBO isomers (118–119). They then treated that mixture of isomers with morpholine to replace a 2nd chloride and separated the three monochloro isomers (120–122) that were produced by silica chromatography. Buchwald-Hartwig Pd catalyzed C-N bond formation was then performed on each of the monochloro isomers followed by TFA catalyzed PMB ether cleavage to liberate the pyrimidinones (123–125).

Figure 30

Syntheses of Morpholino Pyrimidinones.

Following this work a little later in 2012, Giordanetto and co-workers published a follow up report of synthetic routes to two specific classes of pyrimidinones that are selective PI3K β inhibitors (Figure 31).41 One family of compounds was prepared by starting from 4,6-dichloropyrimidin-2-amine (126). The amine was first alkylated with 1-(bromomethyl)naphthalene and then the dichloride was hydrolyzed to the monochloropyrimidinone (128) using aqueous base. The chloride was then replaced with amines in SNAr reactions or in some cases cross coupled with aryl boronic acids to yield targets (129). In either case yields for this last step were generally poor making one wonder if the order of these last two steps couldn’t have been reversed to synthetic advantage.

Figure 31

Syntheses of Diamino Substituted Pyrimidinones.

The second family of compounds reported here were prepared in a one pot reaction from 4,6-dichloropyrimidin-2-ol (130) (Figure 32). One chlorine was replaced by addition of a napthyl amine and then the second chlorine was replaced by an addition of excess morpholine to produce 131. In vivo testing in dogs and rats did not show increased bleeding or inhibition of glucose uptake typical for PI3Kα inhibitors. However inhibition of PI3Kβ activity was not directly tested in vivo.

Figure 32

Syntheses of Amino Morpholino Pyrimidinones.

A one pot multicomponent synthesis of quinazolinones which were designed to be selective PI3K-δ inhibitors was also reported in Fall 2012 (Figure 33).42 To prepare these compounds a substituted 2-aminobenzoic acid (132) was first condensed with 2-chloroacetylchloride and then the quinazolinone core (134) was formed via reaction with an aniline. Adenine was then used to displace the primary chloride that remained thus producing a family of purine quinazolinone derivatives (135).

In 2010, Lin and co-workers at Pfizer reported a series of quinazolines which we include here rather than with the pyrazines even though they were based on a pteridinone core (which contains a pyrazine) and use an intramolecular hydrogen bonding scaffold to mimic the pteridinones (Figure 34).43 2-Bromoaniline (136) was condensed with trichloroacetaldehyde and hydroxylamine to give isonitrosoacetanilide which was subjected to intramolecular Friedel-Crafts acylation. Oxidative decarboxylation of the ketoamide (137) yielded the amino bromo methyl benzoate. The ester was hydrolyzed to 138 and treated with acetic anhydride and ammonium acetate to prepare the quinazoline core (139). One wonders here if the reaction of acetamidine with 2,3-dibromobenzoic acid could also be used to prepare the desired core. The hydroxyl group was then converted to the chloride with POCl3 and then displaced with ammonia. Carbonylation of the bromide lead to an ester (140) which was hydrolyzed and condensed with amines to generate the final target amides (141).

3. Pyridines, Quinolines and Indoles

Most compounds reported since 2010 with the pyridine/quinoline core structure have been designed to be selective for some target other than PI3Kα in the PI3K/mTOR kinase family. A compound containing a bis quinoline core (Torin 1) was reported in 2010 as a selective mTOR inhibitor.44 The Torin 1 core structure was further refined and Torin 2 which was based on a pyridinyl quinoline core and was about 1000 fold more selective for mTOR over PI3Kα was reported in early 2011 (Figure 35).45 The syntheses of all the compounds reported in this work commenced with 6-bromo-3-carboethoxy-4-chloro-quinoline (142). The chloride was replaced via a SNAr reaction with anilines to produce 143 and then the ethyl ester was reduced to the alcohol via a nitrogen assisted/directed NaBH4 reduction. The alcohol was oxidized to the aldehyde (144) and the carbon chain extended via Horner-Wadsworth-Emmons olefination. The ester generated by that reaction underwent intramolecular cyclization to produce the pyridinone (145). Suzuki cross coupling of the remaining aromatic bromide provided the final products (146). No yields were reported for these transformations in the text or the supplementary material though.

This same group of investigators also reported additional compounds in early 2011 based on the original (Torin 1) bis quinoline core but also containing a biaryl off the pyridinone.46 The target compounds were prepared via sequential Suzuki cross couplings off the bromo chloro compound shown below (147, R1 = Br, R2 = Cl) (Figure 36). The bromide on the CF3 substituted aromatic ring being most reactive and cross coupled first followed by the chloride. A lead compound prepared from 147 demonstrated improved in vivo half-life and bioavailability compared to Torin1 and preserved high specificity toward mTor. Pharmacodynamic studies showed that this compound blocked 80–90% phosphorylation of S6K (T389) and pAkt (S473) in liver and lung tissues even after 6 h at a dosage of 20 mg/kg.

Also in 2011, Nishimura and co-workers at Amgen published the preparation of some biaryls containing pyridine and benzothiazoles which were pan PI3K/mTor inhibitors (Figure 37).11 They refined those compounds and later in 2011 reported a series of pyridyl quinolones and pyridyl quinoxalines which are selective PI3K δ inhibitors.47 All of the compounds reported here (150) were prepared via Suzuki cross coupling reactions of pyridinyl halides or boronates (148) with quinolinyl or quinoxalinyl halides or boronates (149) as appropriate.

Figure 37

Preparation of Pyridinyl Quinolones and Quinoxalines.

In mid April 2011, a second Amgen group reported a different series of pyridinyl biaryls with the coupling partners to the pyridines in these cases all being 6,5-heterocyclic ring systems such as benzimidazoles, benzoxazoles, benzoisothiazoles, and triazolopyridines.48 Again all these compounds were prepared by coupling pyridinyl halides or boronates to the appropriate 6,5-heterocyclic halide or boronate similar to the 148 + 149 route to 150 shown above.

In the fall of 2011, Kendall and co-workers at the University of Auckland reported a series of pyrazolopyridines which were selective PI3Kα inhibitors (Figure 38).49 The compounds screened here were prepared via an initial N-amination of the pyridine ring using hydroxylamines with O substituents which make them good NH2 transfer reagents. These N-aminated pyridines were then used in 3 + 2 cycloaddition reactions with ethyl propiolate to produce 151. The ester was decarboxylated and then replaced with an aldehyde using a Vilsmeier reaction. Reaction of the aldehyde (152) with a hydrazine produced a hydrazone which was N-sulfonylated with an aromatic sulfonyl chloride to yield 153.

In late 2011, Hong and co-workers reported pyrimidinyl substituted pyridines which were selective PI3K β inhibitors (Figure 39).50 All the compounds in this study were prepared from 3-amino-5-bromopyridine (154) which was N-sulfonylated and then converted to the pinacol boronic ester (155). The pinacol boronate was then cross coupled to a variety of aromatic bromides, most of which were pyrimidinyl bromides to produce 156. Tissue culture assays showed inhibition of cell growth, but effects on the PI3K/Akt pathway were not presented.

Figure 39

Preparation of Pyrimidinyl Pyridines.

Likewise in late 2011, 11C and 18F labeled versions of GSK2126458 (R1 = R2 = F) were synthesized and used for imaging PI3K and mTOR in cancers (Figure 40).51 The 2-18F and 4-18F compounds were prepared as well as the OMe compound substituted with a 11C labeled CH3. The synthesis involved sulfonylation of the amine in the amino pyridine (157) followed by Suzuki coupling of 159 with the quinolinyl boronate to produce 160.

Figure 40

Synthesis of Labelled Pyridinyl Quinolines.

In work published in April52 and May of 201253, Ellard and co-workers at Cellzome disclosed a series of triazolopyridines (165) which were dual PI3K γ/δ inhibitors (Figure 41). To prepare these compounds, 5-bromopyridine-3-sulfonamides (161) were first cross coupled with amino pyridine boronic acid pinacol esters. The resulting amino pyridines (162) were then treated with ethoxycarbonyl isothiocyanate to produce a thiourea (163). Reaction of the thiourea with hydroxylamine produces an aminotriazole (164) and the free amine was converted into a number of different ureas (165).

These efforts resulted in identification of CZC24832 (164, R = t-Bu, R1 = F), a first selective inhibitor of PI3Kγ that showed significant dose-dependent effects in the mouse model of arthritis upon oral administration at 3 and 10 mg/kg for two weeks. Further elaborations on these structures produced lead compounds that showed rapid in vivo clearance, and were evaluated in a mouse model of LPS-induced pulmonary neutrophilia, as preliminary surrogate for asthma. At 0.5 mg dose as intratracheal inhalant compounds, they showed significant inhibition (37% and 34%) of neutrophil accumulation. Compound CZC24758 (164, R = t-Bu, R1 = H) showed significant dose-dependent effects in a clinical arthritis mouse model (30% and 42% improvement at oral doses of 3 and 10 mg/kg).

In the summer of 2012, a number of triazolopyridines which are selective PI3K γ inhibitors were reported (Figure 42).54 The starting material used here was 2-amino-5-bromopyridine (166). This compound was treated with ethoxy carbonyl isothiocyanate to produce an intermediate thiourea (167) which was cyclized to the triazolopyridine (168) using hydroxylamine. The free NH2 was then doubly acetylated and then hydrolyzed back to the monoacetate (169). The bromide was then cross coupled with aryl boronic acids or converted to the pinacol boronic ester and cross coupled with aryl bromides to yield the desired products (170). Two compounds with core structure (170) were given orally at 10mg/kg and showed 53% and 38% reduction of clinical symptoms in the mouse model of collagen-induced arthritis, however degree of inhibition and PI3K isoform selectivity was not assessed in vivo.

In work that was initiated with a starting material similar to 166, Choi, Hong and co-workers reported the preparation of imidazopyridine derivatives with enhanced selectivity for PI3Kα over Akt1 (Figure 43).55 To prepare these compounds the 5-bromopyridine-2-amine starting materials (171) (X = F in the most selective inhibitor) were converted to the amidine (172) which was alkylated and cyclized with bromoacetonitrile. The nitrile (173) was then converted to the oxadiazole (174

1. Introduction

Recent studies have shown a constant interest in thiazole compounds due to a wide spectra of biologic activities, such as the antimalarial activity of hydrazinyl-thiazoles [1], antiproliferative activity of steroidal[17,16-d]thiazole against gastric carcinoma cells [2], antitumor activity of thiazol-1H-pyrrolo-[2,3-b]pyridine in peritoneal mesothelioma experimental models [3], antiproliferative activity of thiazol-1H-indoles and thiazol-1H-7-azaindoles in MiaPaCa-2 cell line [4], CDK-1 inhibitory activity of thiazol-1H-pyrrolo[3,2-b]pyridine [5], antimicrobial activity of thiazole-oxadiazole derivatives [6], or anti-inflammatory properties of hydrazono-thiazole derivatives [7]. The thiazole rings can be found in a variety of pharmaceutical drugs, such as Ritonavir (anti-HIV) [8], Bleomycin [9] and Tiazofurin (antineoplastics) [10], Fanetizole and Meloxicam (anti-inflammatories) [11], which explains the interest in the development of new compounds containing this heterocyclic unit.

Regarding the synthesis of hydrazinyl-thiazoles, two procedures have been highlighted in the literature: the classical condensation of a carbonyl group with thiosemicarbazide followed by the cyclization of thiosemicarbazones with α-halocarbonyl derivatives [12,13,14,15], and a more recently reported one-step multi-component synthetic protocol [16,17].

The main goal of this work was to identify new possible chemotherapeutic agents based on organic heterocyclic derivatives, which are less harmful for the human body than the well-known platinum derivatives. In this paper, we present a two-step protocol for the synthesis of seven new arylidene-hydrazinyl-thiazoles 2c, 2f, 2h, 2j, 2l, 2m, 2p and nine previously reported thiazoles 2a, 2b, 2d, 2e, 2g, 2i, 2k, 2n, 2o, followed by the in vitro evaluation of the antiproliferative activity on two carcinoma cell lines, MDA-MB231 and HeLa. To identify a possible correlation between DNA damage and cytotoxicity, the interaction of the thiazole derivatives 2a, 2e, 2h, 2i with DNA was evaluated by electrophoresis.

2. Results and Discussion

2.1. Synthesis of Arylidene-Hydrazinyl-Thiazole Derivatives 2a–p

A series of arylidene-hydrazinyl-thiazole derivatives 2ap were synthesized in two steps: the condensation of aromatic aldehydes with hydrazinecarbothioamide, followed by the cyclization of aryliden-thiosemicarbazones 1ae with α-halocarbonyl derivatives (Scheme 1, Table 1). Both the condensation and cyclization reactions were performed in good yield by the Hantzsch protocol. Derivatives 2a, 2b, 2d, 2e, 2g, 2i, 2k, 2n and 2o have been previously prepared by other groups [17,18,19,20].

Scheme 1. Synthesis of arylidene-hydrazinyl-thiazoles 2ap.

Scheme 1. Synthesis of arylidene-hydrazinyl-thiazoles 2ap.

Table 1. Functional groups of the hydrazinyl-thiazole derivatives 2ap.

Compound 1abcde
Compodund 2abcdefgh
Compound 2ijklmnop

NMR and MS spectra were recorded for all the arylidene-hydrazinyl-thiazoles 2ap. The 1H-NMR spectra of arylidene-hydrazinyl-thiazoles 2ap present a similar pattern for the hydrazone unit. The most downfield singlet, around 12 ppm, corresponds to the hydrazinyl moiety (N-NH), which is only present in DMSO-d6 solutions, and otherwise missing due to the deuterium exchange. The singlet around 8.4~7.8 ppm is assigned to the azomethine proton (CH=N). The expected molecular ion (M+) is found in the mass spectra of all arylidene-hydrazinyl-thiazoles 2ap. Moreover, the fragmentation pathway involved the cleavage of the nitrogen-nitrogen bond from the hydrazinyl unit. For example, in the MS spectra of thiazole 2a, this fragmentation generates a peak at m/z 113 for the aza-thiazole ion, while for the thiazole 2b the corresponding aza-thiazole ion peak is observed at m/z 175, in accordance with the substitution of the thiazole heterocycle.

2.2. In Vitro Cytotoxicity Assay

The anti-proliferative activity of the sixteen arylidene-hydrazinyl-thiazole derivatives against two human carcinoma MDA-MB231 and HeLa cell lines was evaluated using MTT assays [14,21] after 24 h of treatment. According to the IC50 data (Table 2), five thiazole derivatives, 2a, 2e, 2f, 2h and 2i, have shown significant inhibition on both MDA-MB231 and HeLa cancer cell lines. Their activity is comparable or even better than that of the platinum drugs cisplatin and oxaliplatin, which were used as controls.

Having the IC50 values for thiazoles 2ap, we tried to establish a correlation between the cytotoxic activity and the molecular structure, by looking at the nature of the functional groups and their position on the arylidene-hydrazinyl-thiazole backbone. The presence of a methyl or phenyl group in position 4 (see Scheme 1) and a hydrogen or acetyl in position 5 on the thiazole ring, combined with phenyl, p-OH-phenyl or p-MeO-phenyl as the aromatic group attached to the hydrazinyl unit, led to compounds 2a, 2e, 2f, 2h and 2i, which exhibited the highest antiproliferative activity. On the other hand, the presence of chlorine atoms at the phenyl hydrazinyl unit and ethyl carboxylate group in position 5 on the thiazole ring 2mo decreased the antiproliferative efficiency (Table 2).

Table 2. IC50 values for thiazoles 2ap on the MDA-MB231 and HeLa cell lines.

CompoundIC50 (µg/mL)
2a3.92 ± 0.01511.4 ± 0.005
2b35.5 ± 0.003>100
2c>10064.87 ± 0.005
2e46.11 ± 0.00911.1 ± 0.009
2f16.25 ± 0.008>100
2h48.44 ± 0.01725.59 ± 0.010
2i18.54 ± 0.00820.04 ± 0.019
2j>10057.53 ± 0.011
2k81.02 ± 0.001>100
2l75.50 ± 0.009>100
2p64.95 ± 0.009>100
Cisplatin17.28 ± 0.00226.12 ± 0.010
Oxaliplatin14.09 ± 0.00123.17± 0.011

The viability of the breast cancer MDA-MB-231 cells and cervical cancer HeLa cells decreased with an increase in the concentration of the thiazole derivatives 2ap (Figure 1 and Figure 2). The profiles of the MDA-MB-231 cells survival viability, correlated to the thiazole doses, revealed a common trend for thiazoles 2a, 2f, 2i, as well as cisplatin and oxaliplatin (Figure 1). For the HeLa cell line, the same correlation is observed between compounds 2a, 2e, 2h, 2i and the chemotherapeutic drugs cisplatin and oxaliplatin (Figure 2).

Due to its significant antiproliferative activity on both MDA-MB-231 (IC50: 3.92 µg/mL) and HeLa (IC50: 11.4 µg/mL) cell lines, the 2-(2-benzyliden-hydrazinyl)-4-methylthiazole derivative 2a was studied further and used as a starting point for the development of new arylidene-hydrazinyl-thiazole compounds for the treatment of cancer. Additionally, 2-[2-(4-methoxybenzylidene) hydrazinyl]-4-phenylthiazole (2e), with an IC50 value of 11.1 µg/mL, was also considered as a potentially useful cytotoxic compound against the HeLa cell line.

2.3. DNA Intercalation Study

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