Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides as potential

5.1) Introduction

For years, chemically modified nucleoside analogues have been outstanding life‐saving medicines.13,20,37,41,190‐197

This pharmacologically manifold compound class, which contains structural features of the skeleton of natural nucleosides, is frequently used for cancer therapy and the treatment of viral infections.20,41,81,190‐196,198‐200

Along with human immunodeficiency virus (HIV) and hepatitis virus (HV); herpes simplex virus (HSV) and varicella‐zoster virus (VZV) are prominent pathogens. In addition to the approved drugs brivudine, acyclovir, and vidarabine, HSV and VZV are also medicated with the prototype of antiviral drugs 2’‐deoxy‐5‐iodouridine (also known as iodoxuridine) N1 since over 40 years (Figure 27).20,190,192,193,196,201

N1, and 2’‐deoxy‐5‐iodocytidine N2. Figure was adapted from literature ²⁰.

Drug N1, launched for example as Herples®, Stoxil®, Iodoxene®, and Virodox®, targets the DNA replication of the viruses.193,196,201

Thereby, N1 acts as an antagonist of thymidine, its

natural nucleoside counterpart, and targets the thymidylate phosphorylase and the viral DNA polymerase ‐ the workhorse of the DNA replication.41,193,196,201

In general, 5‐substituted‐

2´‐deoxycytidines are appreciably more selective, but equally or slightly less potent in their anti‐HSV activity than the corresponding 5‐substituted‐2´‐deoxyuridines.196,197,202,203

Thus, the antiviral spectrum of 2’‐deoxy‐5‐iodocytidine N2 (Figure 27), marketed as Cebeviran® or Cuterherpes®, is similar to N1 to which medicine N2 is turned over by enzymatic deamination.196,197,202,204

Besides 5‐halopyrimidine nucleosides, 4’‐C‐modified nucleosides aroused scientific interest, because a couple of derivatives of this interesting compound class showed antiviral activity.145,146,148‐153

In doing so, 4’‐C‐modified nucleosides functions as nucleoside reverse transcriptase inhibitors (NRTIs)145‐147,154,205

and showed even activity against multi‐drug resistant virus strains.^151,154 The evolution of viral resistance boosts the urgent need for new effective drugs and therapies against viral infections.^88,89 Because there is a great demand for the development of drugs2,12,13,88,90,91 and consequently also for novel nucleoside analogues, a synthetic route for 4´‐C‐alkylated‐5‐iodo‐2´‐deoxyuridines N3a‐c and 4´‐C‐alkylated‐5‐iodo‐2´‐deoxycytidines N4a‐c was designed and developed (Figure 28). The resulting novel entities N3a‐c and 4a‐c could be of great pharmaceutical interest, because they fuse the structural features of the marketed drug N1 or 2, and 4´‐C‐alkylated nucleosides in one small molecule. In addition, the intermediates on synthesis route to N3a‐c and 4a‐c, or the developed compounds itself could function as key building blocks in order to open up the way to several further 4´‐C‐modified‐5‐substituted nucleoside analogues of scientific and pharmaceutical interest.

Figure 28. Chemical structures of the designed 4’‐C‐alkylated‐5‐iodo‐2’‐deoxyuridines N3a‐c and 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c.

It is noteworthy to mention that 4´‐C‐modifications of nucleosides always comprise the generation of quaternary carbon centres including the obstacles associated with the

respective chemistry.²⁰⁶ Three main methodologies have been evolved for the synthesis of 4´‐C‐modified nucleosides (Figure 29). In the first methodology a 4´‐C‐branch is attached to 2´‐C‐deoxynucleosides;^206,207 in the second methodology the asymmetric SAMP/RAMP‐

hydrazone α‐alkylation and diastereoselective nucleophilic 1,2‐addition with Grignard and organocerium reagents is capitalized;^208,209 and in the third methodology suitable 4‐C‐ribose glycosyl donors¹⁶⁴ are synthesized for the nucleoside formation using Vorbrüggen`s method^210,211 (Figure 29, 30).²⁰⁶

2`-deoxynucleoside PGO R

Figure 29. Main methodologies for the synthesis of 4’‐C‐modified nucleosides analogues. In the first methodology a 4´‐C‐branch is attached to 2´‐C‐deoxynucleosides;^206,207 the second methodology the asymmetric SAMP/RAMP‐hydrazone α‐alkylation and diastereoselective nucleophilic 1,2‐addition is capitalized;^208,209 and in the third methodology flexible 4‐C‐ribose glycosyl donors are synthesized for the nucleoside formation using Vorbrüggen`s method^208‐211. R = modification, PG = protection group. Figure was adapted from literature ^206,212.

Over the past years Marx et al. designed and synthesized a series of 4´‐C‐modified nucleosides and nucleotides.147,164,213‐221

In the present work this profound knowledge was combined with literature known synthesis strategies for N1²²² and 12a¹⁵³ in order to open up the way towards N3a‐c. On the basis of the transformation of uridines or thymidines into the respective cytidine analogues,216,223,224

N3a‐c were planed to be converted into the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c. To evaluate whether the novel 4’‐C‐alkylated‐

5‐iodo‐2’‐deoxypyrimidines are utilizable as building blocks to get access to further modified

nucleosides, compound N12c with the biggest hydrophobic 4’‐C‐modification was investigated in a Sonagashira test reaction.

5.2) Synthesis of 4‐C‐modified carbohydrate building blocks

Suitable 4‐C‐modified glycosyl donors ‐ methodology three ‐ have been widely used as precursors for the synthesis of 4´‐C‐modified nucleosides and nucleotides, because they are producible in a multigram‐scale starting from D‐glucose or D‐allofuranose (Figure 29, 30).^206,225 After benzylation and selective acid hydrolysis of the 5,6‐O‐isopropylidene group of D‐allofuranose, periodate cleavage yielded an aldehyde intermediate.^206,225 Afterwards, the 4‐C‐hydroxymethyl branch was installed by using formaldehyde in an aldol reaction that was followed by a Cannizzaro reduction in one pot.^225,226 The selective silylation with tert‐butyldiphenylsilyl chloride (TBDPSCl) leaded to the versatile key ribose building block N6 (Figure 30).²²⁷

Figure 30. Retrosynthetic scheme for key building block N6 starting from D‐allofuranose.

Recently, Rangam et al. reported a nine‐steps reaction sequences for 4`‐C‐methyl‐ and 4`‐C‐ethyl‐substituted deoxyuridines N5a‐b.¹⁶⁴ According to these synthetic routes, nucleosides N5a‐b and the respective glycosyl donors N8a‐b were obtained.¹⁶⁴ In addition, the synthesis of the novel 4`‐C‐propyl‐substituted deoxyuridines N5c and the corresponding novel glycosyl donor N8c were investigated (Figure 31).

Figure 31. Synthesis of glycosyl donors N8a‐c. Reagents and Conditions: i) Ph3P, imidazole, I2, toluene, reflux;¹⁶⁴ ii) n‐Bu₃SnH, AIBN, toluene, reflux, 96% over 2 steps;¹⁶⁴ iii) DMP, CH₂Cl₂, r.t., 91%;¹⁶⁴ iv) MePPh₃Br, n‐BuLi, THF, r.t., 97%;¹⁶⁴ iv) EtPPh3Br, t‐BuOK, THF, r.t., 84%; vi) AcOH, Ac2O, H2SO4, r.t, 79% (N8a)¹⁶⁴, 90% (N8b)¹⁶⁴, 64%

(N8c).

After conversion of N6 with triphenylphosphine (Ph3P) and iodine, to the 4‐C‐iodomethyl derivative, the iodo intermediate was subsequently reduced with tributyltin hydride (n‐Bu3SnH) to the 4‐C‐methyl derivative N7a.¹⁶⁴ Acetolysis of N7a led to the 4‐C‐methyl‐ bis‐

acetate N8a (Figure 31).^164,206

In order to synthesize the 4‐C‐vinylated and 4‐C‐(Z)‐prop‐1‐enylated bis‐acetates N8b‐c, compound N6 was oxidised with Dess‐Martin periodinane (DMP)¹⁶⁵ to yield the corresponding aldehyde.¹⁶⁴ Afterwards, Wittig reaction was used for C‐C‐bond formation to yield the 4‐C‐vinylated N7b¹⁶⁴ and 4‐C‐(Z)‐prop‐1‐enyl ribose analogs N7c. Wittig reaction afforded for N7b the introduction of the C1‐unit with small steric restraints. The reaction could be carried out with methyltriphenylphosphonium bromide (MePPh3Br) and n‐butyllithium (n‐BuLi) as base.¹⁶⁴ In contrast, bulky alkoxides have previously been reported to be the bases of choice in Wittig reactions involving sterically encumbered substrates.^218,228 Thus, the reaction was performed with potassium tert‐butoxide (t‐BuOK) and ethyltriphenylphosphonium bromide (EtPPh3Br) as C2‐synthon. In addition, these cis‐

selective conditions (JHC=CH for N7c = 11.8 Hz) had the positive side effect that the possible products in the resulting diastereomeric mixture were brought to a minimum. By protection group manipulations N7b and 7c were converted to the substituted ribosyl acetates N8b and 8c in good yields (Figure 31).¹⁶⁴

5.3) Synthesis of 4’‐C‐alkylated‐pyrimidine nucleosides

Holding the substituted 4‐C‐modified glycosyl donors N8a‐c in hand, the nucleobase uracil was fused with N8a‐c according to the Vorbrüggen glycosylation^210,211 (Figure 32). The reaction with bis(trimethylsilyl)uracil, which is formed as an intermediate by silylation of uracil with bis(trimethylsilyl)acetamide (BSA), and trimethylsilyl triflate (TMSOTf) as catalyst gave stereoselectively the β‐configurated 4´‐C‐methyl¹⁶⁴, 4´‐C‐(Z)‐vinyl¹⁶⁴, and (Z)‐prop‐

1‐enyl substituted nucleoside N9a‐c.

Figure 32. Synthesis of 2´‐deoxy‐4´‐C‐methyl‐, 2´‐deoxy‐4´‐C‐ethyl‐, and the new 2´‐deoxy‐4´‐C‐propyluridine N5a‐c. Reagents and conditions: i) Uracil, BSA, TMSOTf, MeCN, reflux, 77% (N9a)¹⁶⁴, 74% (N9b)¹⁶⁴, 71% (N9c); ii) NaOMe, MeOH, r.t.; iii) PhOCSCl, DMAP, MeCN, r.t.; iv) n‐Bu₃SnH, AIBN, toluene, reflux; 70% (N10a)¹⁶⁴, 72%

(N10b)¹⁶⁴, 83% (10c) over 3 steps;v) 10% Pd/C, H2, EtOH, r.t.; vi) TBAF, THF, r.t, 79% (N5a)¹⁶⁴ 89% (N5b)¹⁶⁴, 65%

(N5c) over 2 steps.

After deacetylation with sodium methoxide (NaOMe) and reaction with phenyl chlorothionoformate (PhOCSCl) in the presence of 4‐dimethylaminopyridine (DMAP) the thiocarbonate esters were obtained, which were successive reduced with tributyltin hydride (n‐Bu3SnH) to the 2´‐deoxyuridine derivates N10a‐c. Catalytic hydrogenation with Pd/C followed by desilylation with tetrabutylammonium fluoride (TBAF) furnished the 2´‐deoxy‐

4´‐C‐methyl‐, 2´‐deoxy‐4´‐C‐ethyluridines N5a‐b¹⁶⁴ and the new 2´‐deoxy‐4´‐C‐propyluridine N5c (Figure 32).

5.4) Synthesis of 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides

For the halogenation in 5‐position of the uridines, a literature known synthesis strategy for compound N1²²² and 12a¹⁵³ was followed. Therefore nucleosides N5a‐c were acetylated to yield compounds N11a‐c. Next, the diammonium cerium (IV) nitrate (CAN) mediated

iodination (N12a‐c), followed by deprotection furnished in good to excellent yields the desired 4´‐C‐methyl‐, 4´‐C‐ethyl‐ and 4´‐C‐propyl‐5‐iodo‐2´‐deoxyuridine analogues N3a‐c (Figure 33).

Figure 33. Synthesis of 4´‐C‐alkylated‐2´‐deoxy‐5‐iodouridine derivates N3a‐c. Reagents and conditions: i) Et3N, Ac2O, DMAP, MeCN, r.t., 63% (N11a), 74% (N11b), 93% (N11c); ii) I2, CAN, MeCN, reflux, 89% (N12a), 98%

(N12b), 89% (N12c); iii) NaOMe, MeOH, r.t., 91% (N3a), 97% (N3b), 97% (N3c).

The strategy to synthesize the 4´‐C‐alkylated‐5‐iodo‐2´‐deoxycytidines N4a‐c was based on the tranformation of uridines or thymidines into the corresponding cytidine derivates (Figure 34).216,223,224 In the first trail to make N4a‐c accessible, the acetylated 5‐iodo‐2´‐

deoxyuridines N12a‐c were treated unsuccessfully with the 2,4,6‐

triisopropylbenzenesulfonyl chloride (TPSCl)‐Et3N‐DMAP system followed by aminolysis with ammonium hydroxide (NH4OH) in order to generate the exocyclic amino functions and to deacetylate the sugar moieties.

Figure 34. Synthesis of 4´‐C‐alkylated‐2´‐deoxy‐5‐iodocytidine analogues N4a‐c. Reagents and conditions: i) TBDMSCl, imidazole, DMF, r.t., 81% (N13a), 90% (N13b), 91% (N13c); ii) 1.) TPSCl, DMAP, Et₃N, MeCN, r.t.;

2.) 28% NH4OH, 78% (N14a), 77% (N14b), 80% (N14c); iii) TBAF, THF, r.t., 92% (N4a), 89% (N4b), 81% (N4c).

Because no product could be isolated in the first trail, the protecting group strategy had to be changed and N3a‐c were silylated with TBDMSCl in the presence of imidazole to yield

compounds N13a‐c. After silylation, the conversion of N13a‐c into N14a‐c with the (TPSCl)‐

Et3N‐DMAP system followed by aminolysis with ammonium hydroxide (NH4OH) to install the exocyclic amino functions was successful. Finally, desilylation with TBAF yielded the 4´‐C‐

methyl‐, 4´‐C‐ethyl‐ and 4´‐C‐propyl‐ 5‐iodo‐2´‐deoxycytidine analogues N4a‐c (Figure 34).

5.5) Evaluation of the synthons ‐ exemplified by 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐

iodo‐4’‐C‐propyluridine N12c in a Sonagashira test‐reaction

Beside the usage of 5‐halopyrimidine nucleosides as antiviral drugs, they have been applied extensively as key building blocks for the synthesis of a wide range of biological probes and other modified nucleosides of great pharmaceutical interest.117,153,190,198,229‐236

To get access to further 5‐modified pyrimidines starting from 5‐halopyrimidines nucleosides and nucleotides, palladium catalysed cross‐coupling reactions emerged to popular reaction types.153,198,230‐233,235‐241

Figure 35. Evaluation of 3’,5’‐di‐O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N12c in a Sonagashira test‐

reaction. Reagents and conditions: i) 0.1 eq Pd(PPh₃)₄, 0.2 eq CuI, NEt₃, DMF, r.t., 57%; ii) NaOMe, MeOH, r.t., 97%.

To evaluate whether the novel 5‐iodo‐2’‐deoxypyrimidine nucleosides with hydrophobic 4’‐

C‐modifications are utilizable as reactants in palladium catalysed coupling reactions, 3’,5’‐di‐

O‐acetyl‐2’‐deoxy‐5‐iodo‐4’‐C‐propyluridine N12c, was subjected to a Sonagashira test reaction (Figure 35). The coupling reaction between N12c and a representative terminal alkyne‐biotin conjuate N15²⁴² was carried out in the presence of the catalyst tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), cuprous iodide (CuI) and dry NEt3 to yield N16c in 57%. In an additional step the ribose moiety was deacetylated to furnish N17c in 97% yield. It turned out that the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxypyrimidine nucleosides are also valuable intermediates to open up the synthetic way to further 4’‐C‐modified nucleosides.

5.6) Discussion and Conclusion

In order to extend the chemical diversity of 4’‐C‐modified uridine and cytosine analogues with potential interesting pharmacological features, the well established methodology three (Figure 29) was applied to build up of several known and novel 4’‐C‐alkylated uridines and cytosines. By using methodology three, 4‐C‐alkylated glycosyl donors N8a‐c or rather the 4’‐

C‐alkylated uridines N9a‐c were synthesized in multistep reaction sequences, starting from relative cheap and commercially available D‐allofuranose. Afterwards, analogues N9a‐c were successfully transformed according to standard procedures into the 2’‐deoxygenated analogues N5a‐c and those in turn into the corresponding 4’‐C‐alkylated‐5‐iodo‐2’‐

deoxyuridines 3a‐c. In the end an exocyclic amino group could be installed effectually to yield the 4’‐C‐alkylated‐5‐iodo‐2’‐deoxycytidines N4a‐c.

The novel nucleoside analogues N3a‐c and 4a‐c are 4’‐C‐alkylated derivatives of the approved antiviral drugs 2’‐deoxy‐ 5‐iodouridine N1 and 2’‐deoxy‐5‐iodocytidine N2. Due to the fact, that several derivatives of 4’‐C‐modified nucleosides also showed antiviral activity, the here reported molecules are generally of great interest because they fuse the structural features of the marketed drug N1 or 2, and 4´‐C‐alkylated nucleosides in one small molecule.

Additionally, the reported molecules could serve as useful chemical building blocks for the development of further compounds of scientific and pharmaceutical interest. This fact could be verified by a Sonogashira test‐reaction. In doing so N12c was reacted with a

representative alkyne‐biotin conjuate N15 to yield the 4´‐C‐alkylated‐5‐alkynylated nucleoside N16c and so the herein reported molecules can also act as versatile synthetic tools to open up the synthetic way for further 4’‐C‐modified nucleosides.

Conclusion

The survival and development of each organism relies on the equal distribution of its genome during cell division. Errors in this process can lead to severe developmental defects, cancer, or even death.^15‐17 DNA polymerases are key enzymes to pass the exact genomic information down generations (see also chapter 2). Over 50 years ago, Kornberg et al.

discovered the first enzyme (DNA polymerase I), that catalyses the accurate replication of DNA.^18,19 Since this and other pioneering discoveries, it was assumed for a long period of time, that only six “classical” DNA polymerases (pol α, β, γ, δ, ε, and TdT) are responsible for DNA replication and repair in mammalian cells.15,17,20,21

For that reasons, the discovery of several “novel” specialized DNA polymerases (pol η, θ, κ, λ, μ, ν, ι, ζ, and REV1) was a real sensation in the last decades (Table 1).15,17,20,22

So far, at least 15 different human DNA polymerases are known.15,17,20,22

In the course of errors in replication or by environmental conditions, DNA mutations and damages occur.¹⁶ To maintain the genetic integrity of the genome, an elaborate set of sophisticated repair mechanisms have evolved. The set includes, amongst others, the “novel” specialized DNA polymerases.15‐17,20,37

Features of some of these specialized enzymes are known, but to understand in depth the task of the majority the exact roles still await clarification. For that reason, there is a great demand for appropriate methods and reagents (e.g. the chemical genetics approach with itssmall‐molecule probes, see also chapter 1)^4,5,7‐11 to dissect the cellular functions of DNA polymerases. The entire process of DNA metabolism takes minutes, and individual steps take place in seconds. Given their fast mode of action, cell‐permeable small‐molecules are ideally suited to interfere with the highly dynamic replication process.^4,5,7‐11

In addition, DNA polymerases serve not only as a target for molecular probes to dissect their cellular functions, but have been tried and tested for decades as prominent target for life‐

saving medicines.15,17,20,22,37,41 Numerous pathological states, like cancer, autoimmune disease, and many bacterial and viral infections can be traced back to uncontrolled DNA metabolism.15,17,20,37,41,51,79 Today, it is one of modern medicine's top priorities to combat those diseases with novel and innovative drugs. For that reasons, these novel small‐molecule probes not only might be of great value for basic science but also may open up novel avenues for the treatment of diseases related to genome integrity.

The main part of this work deals with a reverse chemical genetics approach, in order to discover and develop molecular tools to dissect the functions of the homologous BER enzymes pol λ and β (chapter 4, see also chapter 2.3). In previous surveys several small‐

molecule inhibitors of human pol λ were identified and evaluated by applying SYBR® Green I based screening and radioactive primer extension (PEX) techniques (see also chapter 3.2).^75,76 Some rhodanine‐based compounds, classified as a privileged drug scaffold,^156‐161 were found to be the most active inhibitor class. The most potent inhibitor obtained was compound SM1, with an IC50 value of 5.9 μ^M. Interestingly, SM1 is up to ten times less active against the highly homologous pol β (IC50 = 64.4 μM).^75,76 Additionally, SM1 did not inhibit the DNA polymerases 9°N, Therminator, Pfu, and Dpo4 (IC50 > 100 μM).⁷⁵ During the course of evaluating an initial compound library (1^st compound generation) further rhodanine‐based inhibitors (SM10, 16, 21, 23) were found whose properties are comparable with SM1 and there were early indications that some compounds (e.g. SM11, 12, 20) also showed activity against pol β.⁷⁶ Finally, preliminary basic structure‐activity relationships (SAR) could be established out of the in vitro data⁷⁶, and thus, the rhodanine‐

based small‐molecules could be seen as an appropriate starting point for the development of molecular probes to specifically investigate the biological functions of pol λ and β.

On purpose to design a molecular scaffold for the creation of the 2^nd small‐molecule generation, first, the initial screening and IC50 data of the 1^st compound generation were confirmed. Based on the reproduced in vitro data, a suitable molecular scaffold could be introduced for the design of the 2^nd small‐molecule generation. Additionally, it was ascertained that SM1 acts also dose‐dependently on the TdT function of pol λ with an IC50 value of 4.5 μM. Continuing experiments with constant SM1 and increasing dNTP concentrations suggested that SM1 inhibits pol λ without directly competing for the substrate binding site.

In the next steps, the chemical diversity of rhodanine‐based small‐molecules with potential interesting pharmacological features could be further expanded by systematic synthetic optimization starting from cheap and commercially available building blocks. The scaffold oriented synthesis of the drug‐like molecules SM12, 29‐58 was subdivided into two parts. In the first part, the precursor aldehydes SM59‐61, 63‐83, and for compound SM33 a precursor

acetophenone SM62 were built up using high‐yielding SNAr reactions under basic conditions.

Thereby activated halides were displaced at the aromatic core of the scaffold by the corresponding thiols. For the synthesis of precursor SM84, an elegant literature‐known three step synthesis sequence¹⁶³ was assigned starting from SM59. Therefore, SM59 was reduced with NaBH4 to yield the methanol intermediate in 98%. Afterwards the intermediate was refluxed together with H2O2 to give SM85. In the last step, SM85 was oxidized with DMP to furnish precursor SM84. By performing the Knoevenagel condensation in the second part, several precursor molecules were fused together with varying heterocycles. To obtain the Z‐

isomeres exclusively, SM12, 29‐33, and 35‐58 were built up under thermodynamic reaction control in good yields. For the synthesis of racemic SM34 the exocyclic double bond of SM1 was reduced by the Hantzsch ester on silica gel method¹⁷⁶.

Due to the biochemical evaluation of the novel entities, 23 highly active compounds against pol λ (SM12, 29, 33, 37‐44, 46‐50, 53‐58) with IC50 values less than 10 μM were discovered.

Interestingly, ten of these small‐molecules (SM29, 39, 40, 42, 44, 46, 47, 51‐53) selectively inhibited pol λ in a low micromolar range but not pol β. The exact IC50 values were determined for the five most active compounds. Compounds SM39, 48, 49, 53, and 57 dose‐

dependently inhibited the polymerization function of pol λ with IC50 values of 5.7, 6.0, 3.9, 4.0, and 4.0 μM and are thus equally or even more active than lead compound SM1 (IC50 = 5.9 μM)^75,76. Moreover, SM39, 48, 49, 53, and 57 act also as inhibitor of the TdT function of pol λ. In addition, 14 novel small‐molecules (SM12, 33, 37, 38, 41, 43, 48‐50, 54‐

58) that target pol β were identified. The exact IC50 values were determined in turn for the four most active compounds. SM38, 41, 48, and 49 inhibited dose‐dependently the polymerization function of pol β with IC50 values of 38.7, 28.1, 29.8, and 18.2 μM. With the aid of the molecular scaffold and the novel PEX data, the initial reported initial SAR⁷⁶ could be extended and discussed in depth (chapter 4.4.5).

In order to draw comparisons between the discovered small‐molecule pol λ and β inhibitors (SM1, 49) and literature known inhibitors, a variety of reported inhibitors were explored in side‐by‐side comparisons using the same PEX conditions. SM1, 49 were much more active than the on natural products based inhibitors. In consequence, the rhodanines are currently the strongest inhibitors for pol λ and comparable to the best of previously reported pol β inhibitors.37,51,75,76,78,177,181

With the aim to further develop the rodanine based probes in a cellular environment, first the effect of the small‐molecules itself on human cell lines was investigated. Cell viability of cervix carcinoma (HeLa‐S3) and liver carcinoma (Hep‐G2) cancer cell lines was suppressed dose‐dependently by selected small‐molecules of the first generation (SM1, 10, 16, 21, and 32). Except SM10, the rhodanines showed moderate toxicities on both human cell lines. In addition, the results suggested that SM1, 16, 21, and 32 are suited small‐molecule probes for more extensive studies in a cellular context.

Next, the two representatives of the discovered compounds (SM1, SM49) were explored in co‐treatment experiments on colorectal cancer cells (Caco‐2). In these proof‐of‐concept studies, it was assayed, whether the discovered compounds sensitize cancer cells towards the artificial induction of DNA‐damage. Therefore, human Caco‐2 cells were co‐treated with the discovered probes (SM1, SM49) and the approved monoalkylating drug TMZ or the model ROS inducer H2O2. Importantly, SM1 and SM49 were pharmacologically active and sensitized Caco‐2 cells towards both genotoxic agents. Because chemotherapies depend frequently in part on the artificial induction of DNA damage, consideration of targeting DNA repair capacity is an ongoing concern in improving responses to treatments.15,17,20,37,51,74,78,79,100

Like other gene products involved in DNA damage repair, the

regulation of pol β and λ, which are overexpressed in cancer tissue,⁷² could be fundamental in cancer treatment.15,17,37,51,53,78,100

The herein reported cellular studies support this notion, as SM1 and SM49 enhanced the sensitivity of human colorectal cancer cells towards the genotoxic TMZ and H2O2 considerably. The additive or even synergistic effects indicate that

Im Dokument Small‐Molecule Inhibitors of DNA Polymerase Function (Seite 57-80)