The spirocyclopropylcyclohexadienone family, including yatakemycin (YTM, 1), CC-1065 (2), and duocarmycin SA (3), all contain a highly active cyclopropane moiety and exhibit potent antitumor activities (Figure 1) [19,20,21,22]. Duocarmycin-based antibody-drug conjugates (ADC, SYD985 (4), and MDX-1203 (5)) have entered clinical trials for the treatment of specific cancers as prodrugs (Figure 1) [23,24]. They can selectively bind AT-rich regions in the DNA minor groove by non-covalent interaction, then form a covalent bond with DNA in which the cyclopropanol group is attacked by the N-3 of adenine (Figure 2A) [25]. The YTM-producer was first identified as protecting itself with DNA glycosylases YtkR2 through the base-excision repair mechanism (Figure 2A). The homologous enzyme C10R5 exhibited a similar function in the CC-1065-producing strain. [26] Because the cyclopropane warhead exhibits strong potency, additional self-protection of their hosts can also be achieved by the cleavage of this moiety. A GyrI-like protein was verified to hydrolyze the cyclopropane moiety in YTM and CC-1065 to facilitate detoxification (Figure 2B) [27,28,29].

The benzodipyrrole scaffold in CC-1065 was derived from serine, methionine, and tyrosine-derived DOPA, and was revealed by isotopic feeding experiments (Figure 3) [30,31]. Wu et al. proposed the possible biosynthetic pathway of CC-1065. Tyrosine was first oxidized to DOPA which underwent intramolecular cyclization to afford 10. Next, 11 produced by the combination of serine and 10 was decarboxylated and cyclized to yield 13 which was further modified to form three different types of building blocks (17, 18, and 19). The assembly of these building blocks generated the final core structure 21 (Figure 4).

Strategies for the incorporation of cyclopropane have long fascinated chemists, since it is an important synthetic building block and a common pharmacophoric group. The chemical synthesis of cyclopropane in this family of NPs was mainly achieved by nucleophilic cyclopropanation [32]. In the biosynthesis of CC-1065, Jin et al. reported that a two-component cyclopropanase system consisting of a HemN-like radical S-adenosylmethionine (SAM) enzyme C10P and a methyltransferase C10Q was responsible for generating the essential cyclopropane moiety involving a unique enzymatic mechanism (Figure 5) [33,34]. To explain in detail, the highly active SAM methylene radical attacks the C-11 position of 22 to generate the radical intermediate 23, which subsequently abstracts hydrogen to yield the SAM-substrate adduct 24. Following this, the deprotonation of the phenolic hydroxyl group in virtue of His-138 residue in C10Q induced S_N2 cyclopropanation to produce CC-1065 with S-adenosylhomocysteine (SAH) as the leaving group. Additionally, 24 could also be converted to 25 by non-enzymatic reaction with the release of SAH, and the following isomerization produces the methylated compound 26.

As an important family of NPs, type II polyketides display various structurally diverse biological activities [35,36]. Anthracycline compounds such as daunomycin and nogalamycin exhibit antitumor activities by intercalating into grooves of DNA, while most of these compounds are unable to form a covalent bond with duplex DNA [37,38]. Nevertheless, pluramycin antibiotics including hedamycin (27) and altromycin B (28) (Figure 6A), which usually contain an epoxide moiety, can intercalate and alkylate DNA simultaneously. Similar to daunomycin, their anthraquinone ring was characterized as intercalating into DNA and binding saccharides in the minor or major groove, thereby contributing to the stabilization of the drug–DNA complex [39,40,41]. Furthermore, their epoxides could be opened via nucleophilic attack of N-7 of guanine, resulting in the formation of an adduct by covalent bond (Figure 6B).

Biosynthetically, the epoxides in hedamycin were formed on its non-acetyl starter unit generated by two separate type I polyketide synthases (PKSs, HedT, and HedU). HedU was proposed to catalyze two rounds of chain elongation employing the acetyl starter unit provided by HedT (Figure 7) [42,43,44]. The obtained unsaturated 2,4-hexadienyl unit was then transferred to the downstream type II PKS to produce the aromatic precursor. The following oxidation of the C2-alkyl side in intermediate 30 afforded the epoxide intermediate 31 which was further modified by methyltransferase and two C-glycosyltransferases to yield hedamycin (Figure 7).

Trioxacarcins (TXNs), firstly isolated from Streptomyces bottropensis NRRL 12051 in 1981, exhibit extraordinary antibacterial, antimalarial, and antitumor activities [45]. Among the TXNs, TXN A (35) showed the most potent antitumor activities with sub-nanomolar IC₅₀ values against various cancer cell lines (Figure 8A). The unique fused spiro-epoxide of TXN A is essential for its bioactivities because the epoxide can react with N-7 of guanine to form a DNA–TXN A complex (Figure 8B). The crystal structure of this complex revealed that glycosyl groups at C-4 and C-13 were docked with the minor and major groove, respectively [46]. It also displayed an unexpected flipping out of the base at the intercalation site, which might be important for DNA–protein interaction. Recently, the study of the TXN analog LL-D49194α1 (36) showed that the deglycosylated compounds (37 and 38) exhibited more potent anticancer activities than 36, possibly suggesting a new mode of interaction with DNA (Figure 8A) [47]. Furthermore, the DNA–TXN complex (39) could be cleaved to yield gutingimycin (40) involving a self-resistance mechanism of an excising base (Figure 8B) [48,49]. Recently, four DNA glycosylases, TxnU2, TxnU4, LldU1, and LldU5, were reported to be responsible for excising the intercalated guanine adducts [50].

Based on the work of Zhang et al., TXNs were also biosynthesized from anthraquinone-intermediate parimycin (47), similarly to the precursor of hedamycin, as revealed by gene-deletion experiments [45]. According to isotope-labelled precursor feeding experiments, the unusual starter unit 2-methylbutyryl of TXNs was derived from L-isoleucine through transamination. After a series of modifications, including condensation with acetyl-CoA and decarboxylation, this starter unit was incorporated into the polyketide chain in virtue of KSIII (Figure 9). The formation and subsequent cyclization of the polyketide chain provided intermediate 46, whose pyrone ring was formed by a CalC-like protein, TxnO9 [51]. The decarboxylated intermediate 47 underwent complex tailoring steps to afford intermediate 51 with a unique spiro-epoxide structure, but the specific enzymatic process and mechanism remained uncharacterized. Following that, the methylation of 51 at C-4 and C-13 yielded 52, whose C4-sugar was finally acetylated by the membrane-bound O-acetyltransferase TxnB11 to form 35 (Figure 9) [52]. Unlike TXNs, the C-16 and C-4 of 45 were glycosylated and methylated to produce LL-D49194α1.

Mitomycins (MMs, such as MMA, B, and C) are antitumor NPs discovered in Streptomyces. They all contain the quinone backbone and a unique azabicycle moiety (Figure 10A) [18,53]. Among these compounds, MMC has been used as a chemotherapeutic agent in the clinic for more than five decades. MMC can form inter-strand and intra-strand cross-linking with DNA at the selective sequence (5’-CG-3’) and resides in the minor groove [54]. Other compounds of the mitomycin family, such as FR900482 and FR66979, also showed potent DNA cross-linking activity as well as bioactivities against cancer cell lines (Figure 10A). FR900482 was superior to MMC in both efficacy and safety [55].

The mode of action of MMs is well studied. Firstly, a reductive pathway is required to activate the quinone moiety of MMC by either enzymatic or chemical means to form the hydroquinone intermediate 60 [18,53]. Subsequent elimination of methanol in 60 affords 61 which undergoes tautomerization and the ring-open reaction of aziridine ring to yield 65 (Figure 10B). The N-2 of guanine attacks the C-1 position to generate the DNA–compound complex, then the departure of carbamate produces the iminium intermediate 69, which is attached by the second guanine of DNA in the same way to form 71. Furthermore, the first reductive activation could be inhibited by a FAD-dependent oxidoreductase MCRA (encoded by mcrA) which enables the oxidization of the hydroquinone form to the quinone form to confer self-resistance [56,57,58,59]. Although the alkylating mechanism of FR900482 is similar to that of MMC, it is activated by cleaving the N-O bond to form 59 (Figure 10B).

Since these compounds possess excellent bioactivities and the common pharmacophoric group azabicycle, their synthesis has attracted extensive attention. In chemical synthesis, the azabicycle moiety of MMs is installed from benzazocane intermediates via intramolecular substitution [60], but their biosynthetic pathways are still not well elucidated. According to isotopic precursors feeding experiments conducted by Hornemann et al., the origins of the O-methyl group and the carbamate were methionine and L-citrulline, respectively, while the mitosane core was derived from 3-amino-5-hydroxybenzoic acid (AHBA, 81) and glucosamine [61,62]. The precursor AHBA was formed via the amino-shikimate pathway related to rifamycin and kanosamine biosynthesis [63,64]. After the formation of AHBA, it was firstly activated by acyl AMP-ligase MitE and was then loaded onto acyl carrier protein (ACP) MmcB (Figure 11). The glycosyltransferase MitB was verified to catalyze the glycosylation of AHBA-MmcB with UDP-GlcNAc [65,66,67]. Recently, Wang et al. traced all the ACP-channelled MM intermediates indicating that AHBA-MmcB-GlcNAc intermediate 85 should undergo the deacetylation by MitC to form 86 which was further transformed to 88 by MitF and MitD [68]. The epoxide intermediate might be cyclized to provide benzazocine 92. 92 then underwent oxidation and several uncovered modifications to generate hydroxyquinone intermediate 95 which was methylated to afford MMA, the direct precursor of MMC [69]. Sherman and co-workers also identified a methyltransferase MitM which methylated the nitrogen of aziridine in MMA rather than MMC to yield MMF (96) [70]. In addition, the epoxide of 88 could be opened by the nucleophilic attack to afford 89 which was the precursor of the MMs with α-C9. Moreover, the oxidation of the aniline amine in 89 facilitated forming the core structure of FR900482.

The antitumor antibiotics azinomycin A (98) and B (99) contain naphthoic acid (NPA) moiety, epoxide, and azabicyclohexane ring which all contribute to alkylating DNA (Figure 12) [71]. The electrophilic epoxide and aziridine can both be attacked by N-7 of guanine and the latter can even be opened by N-7 of adenine, leading to the formation of interstrand DNA cross-links (Figure 12) [72,73]. NPA moiety also plays an important role in the DNA alkylating activity by virtue of non-covalent interactions [74]. In 2011, the aminoglycoside transferase AziR was identified to mediate the self-resistance of azinomycin and reduce the DNA damage via binding azinomycin. Recently, a novel DNA glycosylase Orf1 and an endonuclease AziN were reported to repair the DNA damage to achieve self-protection [75,76,77,78].