Synthesis of Mitomycin C and decarbamoylmitomycin C N6 deoxyadenosine-adducts.

Mitomycin C (MC), an anti-cancer drug, and its analog, decarbamoylmitomycin C (DMC), are DNA-alkylating agents. MC is currently used in the clinics and its cytotoxicity is mainly due to its ability to form Interstrand Crosslinks (ICLs) which impede DNA replication and, thereby, block cancer cells proliferation. However, both MC and DMC are also able to generate monoadducts with DNA. In particular, we recently discovered that DMC, like MC, can form deoxyadenosine (dA) monoadducts with DNA. The biological role played by these monoadducts is worthy of investigation. To probe the role of these adducts and to detect them in enzymatic digests of DNA extracted from culture cells treated by both drugs, we need access to reference compounds i.e. MC and DMC dA-mononucleoside adducts. Previous biomimetic methods used to generate MC and DMC mononucleoside adducts are cumbersome and very low yielding. Here, we describe the diastereospecific chemical synthesis of both C-1 epimers of MC and DMC deoxyadenosine adducts. The key step of the synthesis involves an aromatic substitution reaction between a 6-fluoropurine 2'-deoxyribonucleoside and appropriately protected stereoisomeric triaminomitosenes to form protected-MC-dA adducts with either an S or R stereochemical configuration at the adenine-mitosene linkage. Fluoride-based deprotection methods generated the final four reference compounds: the two stereoisomeric MC-dA adducts and the two stereoisomeric DMC-dA adducts. The MC and DMC-dA adducts synthesized here will serve as standards for the detection and identification of such adducts formed in the DNA of culture cells treated with both drugs.

Design, synthesis, and mode of action studies of a mitomycin tetramer inducing double activations with a single probe.

We report design, synthesis, and mechanistic studies of a new mitomycin tetramer 9 along with a new mitomycin dimer 10. Mitomycin 9 is a tetramer connected by the disulfide linker 11, and easily undergoes disulfide cleavage to provide two dimeric structures 9r that each contains a single thiol probe for activations. So, tetramer 9 as a precursor of 9r was specifically targeted to undergo double activations with a single probe. A tetramer 9 was synthesized using 1 and key intermediate 11, and a dimer 10 was synthesized from 1 and diamine 12. Activation studies revealed that 9 underwent effective double activations with a single probe by nucleophiles while the reference 10 did not. Evaluations of DNA ISC formations showed that 9 generated substantial levels of DNA ISC by nucleophilic activation while the references 10 and 2 did not. The effectiveness of 9 in activation and formation of DNA ISC per probe was verified by comparing with dimers 5-8 of double activations with two probes. These findings highlighted the role of a single thiol in 9r and demonstrated the intended double activations with a single probe, which marks the first case in mitomycin studies.

Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment.

Synthesis of a major mitomycin C DNA adduct via a triaminomitosene.

We report here the synthesis of two amino precursors for the production of mitomycin C and 10-decarbamoylmitomycin C DNA adducts with opposite stereochemistry at C-1. The triamino mitosene precursors were synthesized in 5 steps from mitomycin C. In addition synthesis of the major mitomycin C-DNA adduct has been accomplished via coupling of a triaminomitosene with 2-fluoro-O(6)-(2-p-nitrophenylethyl)deoxyinosine followed by deprotection at the N(2) and O(6) positions.

Convergent synthesis of a steroidal antiestrogen-mitomycin C hybrid using "click" chemistry.

A convergent synthesis of a novel estrogen receptor-targeted drug hybrid was developed based on structures of the potent anti-proliferative mitomycin C and the steroidal anti-estrogen RU 39411. The steroidal antiestrogen was prepared with an azido-triethylene glycoloxy linker while the mitomycin C derivative (porfirimycin) incorporated a complementary 7-N-terminal alkyne. The two components were ligated using the Huisgen [3 + 2] cycloaddition ("click") reaction. Preliminary biological assays demonstrated that the final hybrid compound retained both potent anti-estrogenic and anti-proliferative activities.

Studies on synthesis and activation mechanism of mitomycin dimers connected by 1,2-dithiolane and diol linkers.

We report the synthetic and mechanistic studies on a new cyclic disulfide mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiolanyl-3″,5″-dimethylenyl)bismitomycin C (8), and a diol mitomycin dimer, 7-N,7'-N'-(2″,4″-dihydroxy-1″,5″-pentanediyl)bismitomycin C (9). Mitomycin 8 is a dimer connected by a 1,2-dithiolane (a five-membered cyclic disulfide) linker, and was specifically designed to undergo nucleophilic activation and double DNA alkylations leading to efficient production of DNA interstrand cross-link (DNA ISC) adducts. Disulfide cleavage in 8 would generate two thiol groups that could serve as probes to activate two mitomycin rings. At first, the target mitomycin 8 was synthesized using mitomycin A (1) and the key intermediate, cyclic disulfide (10), which was prepared through a seven-step synthetic sequence. Diol mitomycin 9 was also synthesized from 1 and diamine salt 13. Next, kinetic studies using solvolysis reaction revealed that the activation rates of 8 were much higher than those of 9 and mitomycin C (2) under nucleophilic conditions provided by Et(3)P presumably due to the presence of a cyclic disulfide unit in 8. These findings led us to propose a nucleophilic activation pathway for 8. Then, DNA ISC experiments further revealed that the levels of DNA ISC caused by 8 in the presence of Et(3)P were much higher (97%) than those by 9 (5%) and 2 (4%). More importantly, mitomycin 8 underwent much faster activation and produced slightly higher levels of DNA ISC than the previously reported mitomycins 5-7. Overall, we concluded that 8 was highly efficient for both nucleophilic activation and corresponding DNA ISC formation, and that this differentiation came from the crucial function of the cyclic disulfide unit in 8.

Synthesis and mechanistic studies of a mitomycin dimer containing an eight-membered cyclic disulfide.

Dimeric DNA alkylating agents have drawn significant interest because these compounds are expected to provide at least two reactive sites and as a result, generate enhanced levels of DNA interstrand cross-link (DNA ISC) adducts compared to their monomeric agents. We report the synthesis and mechanistic studies of a novel mitomycin dimer, 7-N,7'-N'-(1″,2″-dithiocanyl-3″,8″-dimethylenyl)bismitomycin C (8) connected by an eight-membered cyclic disulfide. Mitomycins require prior activation (i.e., transformation to a good electrophile) for DNA adduction and therefore, 8 was aimed to undergo facile nucleophilic activation and produce enhanced levels of DNA ISC. At the core of this function lies a cyclic disulfide in 8. It was expected that disulfide cleavage by an appropriate nucleophile would successively produce two thiols that may trigger activation of two mitomycin rings in a dimer through intramolecular cyclization to quinine rings. Compound 8 was synthesized from mitomycin A (1) and the key intermediate, cyclic disulfide (11), along with the reference diol mitomycin 7-N,7'-N'-(2″,7″-dihydroxy-1″,8″-octanediyl)bismitomycin C (23) which does not contain the disulfide unit. We found that 8 underwent significantly enhanced nucleophilic activation in the presence of Et(3)P compared with 23, and that the disulfide unit in 8 played a key role for the nucleophilic activation. Based on these findings, we proposed a mechanism for nucleophilic activation of 8. We further demonstrated that 8 generated much higher levels of DNA ISC (94%) compared with 23 (4%) and 2 (3%) in the presence of Et(3)P (and L-DTT) leading to the conclusion that 8 is more efficient for DNA ISC processes than 23 and 2 due to the role of disulfide unit.

Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis.

Synthetic and biosynthetic studies on FR900482 and mitomycin C: an efficient and stereoselective hydroxymethylation of an advanced benzazocane intermediate.

We report a simple, efficient, and stereoselective Mukaiyama aldol approach to install the key hydroxymethyl moiety into the benzazocane framework of FR900482. Synthetic investigations revealed that the reaction is highly dependent upon the electronics of the aromatic ring. This approach enabled the economical introduction of a [13C] label to study the biosynthesis of these structurally and biogenetically related natural products. Epimerization of the initially formed beta-hydroxy ketone may enable access to mitomycin C or FR900482 biosynthetic congeners.

Synthesis of an advanced intermediate en route to the mitomycin natural products.

[Structure: see text] An advanced intermediate in our planned synthesis of mitomycin C has been acquired in nine steps from tert-butyl glyoxylate. The aziridinyl pyrrolidine and quinone subunits are coupled regioselectively to arrive at an enamine that is prepared for C10 homologation.

Synthesis, DNA cross-linking activity, and cytotoxicity of dimeric mitomycins.

Dimeric DNA cross-linking compounds have emerged as important new antitumor agents. We report the synthesis and biochemical evaluation of a select set of dimeric mitomycins in which the two mitomycin units are tethered at either the mitomycin C(7) amino or the aziridine N(1a) positions. Significantly, mitomycin C (1) itself is the prototypical bioreductive DNA cross-linking agent. DNA cross-linking experiments using a denaturing-gel-electrophoresis-based assay showed that the extent of DNA cross-linking for select dimeric mitomycins can exceed that of the parent compound, mitomycin C, and that the reaction proceeds, in part, at the two distal C(1) sites in the mitomycins. The efficiency of DNA cross-linking depended on the nature of the linker and the position of linker unit's attachment. When we compared the efficiency of DNA cross-linking for the dimeric mitomycins with their in vitro cytotoxicities in cultured human tumor cells, we observed a poor correlation. The mitomycins that gave the highest levels of DNA cross-linked adducts displayed the weakest cytotoxicities. These findings determined that the denaturing-gel-electrophoresis-based assay was a poor predictor of cytotoxic activity.

Mitomycin dimers: polyfunctional cross-linkers of DNA.

The three dimers 3, 4, and 5 of mitomycin C (MC), a natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with -(CH(2))(4)-, -(CH(2))(12)-, and -(CH(2))(3)N(CH(3))(CH(2))(3)- tethers, respectively. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied in direct comparison with MC, which is itself an ICL agent. Evidence is presented that 3-5 multifunctionally alkylate and cross-link extracellular DNA and form DNA ICLs more efficiently than MC. Reductive activation, required for these activities, is catalyzed by the same reductases and chemical reductants that activate MC. Dimer 5, but not MC, cross-linked DNA under activation by low pH also. Sequence specificities of cross-linking of a 162-bp DNA fragment (tyrT DNA) by MC, 3, and 5 were determined using DPAGE. The dimers and MC cross-linked DNA with the same apparent CpG sequence specificity, but 5 exhibited much greater cross-linking efficacy than MC. Greatly enhanced regioselectivity of cross-linking to G.C rich regions by 5 relative to MC was observed, for which a mechanism unique to dimeric MCs is proposed. Covalent dG adducts of 5 with DNA were isolated and characterized by their UV and mass spectra. Tri- and tetrafunctional DNA adducts of 5 were detected. Although the dimers were generally less cytotoxic than MC, dimer 5 was highly and uniformly cytotoxic to all 60 human tumor cell cultures of the NCI screen. Its cytotoxicity to EMT6 tumor cells was enhanced under hypoxic conditions. These findings together verify the expected features of the MC dimers and warrant further study of the biological effects of dimer 5.

Enantioselective synthesis of a mitosane core assisted by diversity-based catalyst discovery.

[reaction: see text]. Synthesis of mitosane 1 in optically pure form is reported. A retrosynthetic plan that proceeds through racemic allylic alcohol 3 was carried out. This intermediate served as a test substrate for a rapid screen of a small library (152 members) of peptide-based kinetic resolution catalysts. Peptide 9 was found to effect kinetic resolution with k(rel) = 27. Alcohol (-)-3 was then converted to optically pure (-)-1 in eight steps.

Use of the vicinal element effect for regiochemical control of quinone substitutions and its implication for convergent mitomycin construction.

[reaction--see text] Nucleophilic substitution reactions of 2-methoxy-3-alkyl-p-benzoquinones are described as they relate to the construction of the mitomycin backbone. Normally controlled by activating groups attached to the olefin, the observed regioselection in these cases is determined by the deactivating substituent. Approximation of carbonyl activating ability would not have predicted the behavior of two systems investigated in which the poorer of two leaving groups is substituted in each case.

A convergent approach to the mitomycin ring system.

[structure: see text]. A novel stereoselective approach to the ring system of the mitomycins is described. The synthesis was based on a convergent strategy involving a stereocontrolled addition of a beta-phenyl silyl enol ether to a pyrroline N-acyliminium ion followed by an intramolecular palladium-catalyzed aryl triflate amination to afford the (9R*,9aR*)-tetrahydropyrrolo[1,2-a]indole ring system.

Synthesis of the mitomycin and FR900482 ring systems via dimethyldioxirane oxidation.

[reaction: see text] Dimethyldioxirane oxidizes a 2,3-dihydo-1H-pyrrolo[1,2-a]indole unsubstituted at the C-9 position stereoselectively to form a hydroxy ketone with all the basic elements of the mitomycin ring system. On the other hand, a 2,3-dihydo-1H-pyrrolo[1,2-a]indole derivative substituted with an alkyl group at C-9 undergoes an oxidative ring expansion in the presence of dimethyldioxirane to give an FR900482 analogue.

Synthesis of a novel CS-g-MMCs conjugate and the inhabitation on the proliferation of Tenon's capsule fibroblasts in vitro.

A novel anti-proliferative macromolecular conjugate, CS-g-MMCs, was synthesized in order to decrease the cytotoxicity of Mitomycin C (MMC) which was a traditional anti-proliferative agent of fibroblast in trabeculectomy. The structure of CS-g-MMCs was characterized by (1)H NMR, FT-IR spectroscopy and GPC analysis. The grafting degree (dg) of MMC onto chitosan (CS) was determined to be in the range of 2.8-11.3%, which could be controlled by variation of the molar ratios of MMC to oxidized chitosan (CS-CHO). In the drug release profiles of CS-g-MMCs in vitro, an initial burst followed by slow leakage was observed, and addition of acid or lysozyme obviously accelerated the MMC release. The MTS assay indicated that CS-CHO of 8 mg/ml has no cytotoxicity against human Tenon's capsule fibroblasts (HTCFs). The inhibition of HTCFs proliferation by CS-g-MMCs increased along with increasing the dg of conjugate. The CS-g-MMCs also caused the apoptosis of HTCFs and interfered in the active DNA synthesis in HTCFs. Furthermore, the expression of a-SMA at gene and protein levels were obviously lower when HTCFs were treated with CS-g-MMCs, as compared to MMC or blend of MMC/CS-CHO (p<0.05). Our results primarily demonstrated that the CS-g-MMCs conjugates have low cytotoxicity and have the effect to inhibit fibroblast proliferation.

Stereoselective synthesis of complex polycyclic aziridines: use of the Brønsted acid-catalyzed aza-Darzens reaction to prepare an orthogonally protected mitomycin C intermediate with maximal convergency.

A concise synthesis of a highly functionalized intermediate lacking only C10 of the mitomycin backbone is described. The key to this development is the Brønsted acid-catalyzed aza-Darzens reaction used to forge the cis-aziridine. Additionally an oxidative ketalization fortuitously occurs during the quinone-enamine coupling step, leading to an orthogonally protected hydroquinone.