Periodic Reporting for period 1 - SHAPECHANGE (Framework Editing of Macrocycles Through Catalytic Olefin Metathesis)
Reporting period: 2023-01-15 to 2025-01-14
Summary of the context and overall objectives of the project
Macrocyclic natural products are valuable to drug discovery and development. Through chemical synthesis, many of these often-complex molecules have been synthesized. The goal of our project was to devise a set of protocols that allow for generating, in a controllable and programmable fashion, a variety of precisely altered skeletal analogs. The latter compounds can then be used to screen for better efficacy, reduced toxicity, and enhanced physicochemical properties. Through the use of anti-cancer agent epothilone C, as the model macrocyclic natural product, we show that three different types of catalytic olefin metathesis, ring-opening/cross-metathesis, cross-metathesis and ring-closing metathesis, can be merged with olefin transposition, catalytic stereoselective boryl substitution and catalytic E-selective alkene isomerization to generate virtually any new expanded, contracted or distorted macrocyclic analog. Accordingly, we have been able to present a worksheet for how countless analogs of a large variety of macrocyclic natural products may be prepared in short order and exactly which catalysts and reagents would be needed for doing so.
A recent study by our research group illustrated that through ground-up programmable skeletal alteration, the three dimensional contours a small number of skeletal analogs of a naturally occurring bridged polycyclic indole alkaloid, originally found to be only mildly antimalarial, can result in identification of new leads (~3mM activity) for anticancer drug discovery.
The exciting outcomes of the above project led us to wonder whether we could design a set of practical and efficient strategies through which provide facile access to precisely altered analogs of other important classes of bioactive compounds. This led us to focus on macrocycles, a set of organic molecules that continue to emerge as compelling therapeutic candidates. Macrocycles are particularly relevant to difficult-to-drug or “non-druggable” targets. This is because they can offer the necessary blend of conformational rigidity and flexibility, which allows them to adapt to and interact effectively with a range of biological receptors.
At the heart of our approach, which we referred to as late-stage diverted total synthesis, is a class of compounds that are present towards the end of many total syntheses of macrocycles, namely, linear dienes that are subjected to macrocyclic ring-closing metathesis (MRCM). The macrocyclic alkene thus formed is either the final target and the olefin can be further functionalized (for example, by hydrogenation or epoxidation). Our approach is founded on the principle that such dienes are considerably more valuable realized, that is, merely as precursors to a natural product. Rather, they can also serve as hubs for generating analogs that are expanded or contracted at a specific location by no more than two or three additional steps. What is more, if the macrocyclic alkene can be accessed easily, the diene hub can be obtained simply by a single transformation: ring-opening/cross-metathesis (ROCM) involving ethylene (ethenolysis).
We show that ring-expanded analogs can be obtained by extending the length of a diene hub through catalytic cross-metathesis (CM) followed by a substitution reaction and then performing MRCM to generate either a one- or two-unit expanded macrocycle that contains either a Z- or an E-alkene. The corresponding ring-contracted analogs can be secured by iterative methylene deletion comprised of two catalytic transformations: alkene isomerization and ethenolysis. Again, ensuing MRCM leads to the desired smaller ring analogs in either stereoisomeric form.
As the model platform, we chose epothilone C, a naturally occurring macrocyclic alkene with considerable anti-tumor activity. There were several other reasons why we opted for epothilone C. (1) It is commercially available and can be generated in significant quantities through fermentation. Furthermore, it has been prepared by catalytic stereoselective MRCM of a diene hub that can be accessed by well-established total synthesis routes. (2) Previous investigations have led to the identification of more active epothilone C analogs derived from aziridination of the cyclic alkene (even more so than the epoxide, which is epothilone B). (3) It contains various polar functional units and a heterocyclic ring, thus allowing us to develop a set of protocols that tolerate these frequently occurring moieties.
We targeted six distinct types of remodeling operations. One of our goals was to probe the feasibility of converting the natural Z-disubstituted macrocyclic alkene to its E isomer (Twist). Another aim was to use our approach to expand the macrocycle through site-specific insertion of a methylene unit (Expand-I or Expand-II). Similarly, we targeted contracted analogs such as Contract-I and Contract-II. Finally, methylene insertion and deletion could be merged to generated “distorted” frameworks (Distort).
A recent study by our research group illustrated that through ground-up programmable skeletal alteration, the three dimensional contours a small number of skeletal analogs of a naturally occurring bridged polycyclic indole alkaloid, originally found to be only mildly antimalarial, can result in identification of new leads (~3mM activity) for anticancer drug discovery.
The exciting outcomes of the above project led us to wonder whether we could design a set of practical and efficient strategies through which provide facile access to precisely altered analogs of other important classes of bioactive compounds. This led us to focus on macrocycles, a set of organic molecules that continue to emerge as compelling therapeutic candidates. Macrocycles are particularly relevant to difficult-to-drug or “non-druggable” targets. This is because they can offer the necessary blend of conformational rigidity and flexibility, which allows them to adapt to and interact effectively with a range of biological receptors.
At the heart of our approach, which we referred to as late-stage diverted total synthesis, is a class of compounds that are present towards the end of many total syntheses of macrocycles, namely, linear dienes that are subjected to macrocyclic ring-closing metathesis (MRCM). The macrocyclic alkene thus formed is either the final target and the olefin can be further functionalized (for example, by hydrogenation or epoxidation). Our approach is founded on the principle that such dienes are considerably more valuable realized, that is, merely as precursors to a natural product. Rather, they can also serve as hubs for generating analogs that are expanded or contracted at a specific location by no more than two or three additional steps. What is more, if the macrocyclic alkene can be accessed easily, the diene hub can be obtained simply by a single transformation: ring-opening/cross-metathesis (ROCM) involving ethylene (ethenolysis).
We show that ring-expanded analogs can be obtained by extending the length of a diene hub through catalytic cross-metathesis (CM) followed by a substitution reaction and then performing MRCM to generate either a one- or two-unit expanded macrocycle that contains either a Z- or an E-alkene. The corresponding ring-contracted analogs can be secured by iterative methylene deletion comprised of two catalytic transformations: alkene isomerization and ethenolysis. Again, ensuing MRCM leads to the desired smaller ring analogs in either stereoisomeric form.
As the model platform, we chose epothilone C, a naturally occurring macrocyclic alkene with considerable anti-tumor activity. There were several other reasons why we opted for epothilone C. (1) It is commercially available and can be generated in significant quantities through fermentation. Furthermore, it has been prepared by catalytic stereoselective MRCM of a diene hub that can be accessed by well-established total synthesis routes. (2) Previous investigations have led to the identification of more active epothilone C analogs derived from aziridination of the cyclic alkene (even more so than the epoxide, which is epothilone B). (3) It contains various polar functional units and a heterocyclic ring, thus allowing us to develop a set of protocols that tolerate these frequently occurring moieties.
We targeted six distinct types of remodeling operations. One of our goals was to probe the feasibility of converting the natural Z-disubstituted macrocyclic alkene to its E isomer (Twist). Another aim was to use our approach to expand the macrocycle through site-specific insertion of a methylene unit (Expand-I or Expand-II). Similarly, we targeted contracted analogs such as Contract-I and Contract-II. Finally, methylene insertion and deletion could be merged to generated “distorted” frameworks (Distort).
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
We have developed a three-phase editing platform for programmable and controllable alteration of macrocyclic alkene frameworks. The three phases of the approach consist of:
I. Accessing the diene hub. The hub can either be obtained by multistep synthesis, as has been countless times for various total syntheses, or by ROCM if the natural products is readily accessible.
IIa. Modifying the diene hub. Extending. An extended diene can be obtained through Z-selective CM, which affords a separable mixture of two single extended and one doubly extended chain. Stereocontrol limits the number of generated products, rendering purification and access to all three isomers more straightforward. This is followed by alkene transposition or catalytic allylic substitution involving an altered diene. These protocols can be performed iteratively for efficient and programmable generation of more extended dienes.
IIb. Modifying the diene hub. Clipping. A clipped diene is generated by a two-step protocols comprised of catalytic isomerization and catalytic ethenolysis. As with chain extension, this might occur site selectively or at both termini and a combination thereof; in the latter case the three isomers may be separated and further modified individually. These protocols can also be performed iteratively.
III. Reclosure to skeletally altered analogs. The foregoing extended and clipped dienes can be cyclized by Z- and/or E-selective MRCM to generate the desired macrocyclic alkene, which can be further modified if desired.
The approach relies on just a few types of transformations and require the availability of only a few metal complexes and reagents, many of which are purchasable and all can be prepared by established procedures. Different types of catalytic olefin metathesis are pivotal: (i) ROCM (ethenolysis) for cleaving an existing macrocyclic alkene. (ii) Stereoselective or stereoretentive CM for extending an acyclic diene hub. (iii) Ethenolysis (CM with ethylene) for clipping an isomerized olefin. (iv) MRCM to generate the altered macrocyclic alkene. Only three other types of transformation are needed: alkene transposition, catalytic stereoselective boryl substitution and alkene isomerization. Thus, through the use of four different Ru-based, one W-based, two Mo-based and a Cu-based catalyst and a reagent, assorted skeletally altered macrocyclic alkenes can be accessed through sequences that typically require two or three additional operations involving a diene hub. Preliminary in vitro activity screening indicates that several analogs possess comparable activity as the natural framework (AC50 as low as 0.04 μM vs. as low as 0.02 μM for epothilone C). Epothilone C is only a case in point. The approach may be used to remodel many other bioactive cyclic alkenes.
I. Accessing the diene hub. The hub can either be obtained by multistep synthesis, as has been countless times for various total syntheses, or by ROCM if the natural products is readily accessible.
IIa. Modifying the diene hub. Extending. An extended diene can be obtained through Z-selective CM, which affords a separable mixture of two single extended and one doubly extended chain. Stereocontrol limits the number of generated products, rendering purification and access to all three isomers more straightforward. This is followed by alkene transposition or catalytic allylic substitution involving an altered diene. These protocols can be performed iteratively for efficient and programmable generation of more extended dienes.
IIb. Modifying the diene hub. Clipping. A clipped diene is generated by a two-step protocols comprised of catalytic isomerization and catalytic ethenolysis. As with chain extension, this might occur site selectively or at both termini and a combination thereof; in the latter case the three isomers may be separated and further modified individually. These protocols can also be performed iteratively.
III. Reclosure to skeletally altered analogs. The foregoing extended and clipped dienes can be cyclized by Z- and/or E-selective MRCM to generate the desired macrocyclic alkene, which can be further modified if desired.
The approach relies on just a few types of transformations and require the availability of only a few metal complexes and reagents, many of which are purchasable and all can be prepared by established procedures. Different types of catalytic olefin metathesis are pivotal: (i) ROCM (ethenolysis) for cleaving an existing macrocyclic alkene. (ii) Stereoselective or stereoretentive CM for extending an acyclic diene hub. (iii) Ethenolysis (CM with ethylene) for clipping an isomerized olefin. (iv) MRCM to generate the altered macrocyclic alkene. Only three other types of transformation are needed: alkene transposition, catalytic stereoselective boryl substitution and alkene isomerization. Thus, through the use of four different Ru-based, one W-based, two Mo-based and a Cu-based catalyst and a reagent, assorted skeletally altered macrocyclic alkenes can be accessed through sequences that typically require two or three additional operations involving a diene hub. Preliminary in vitro activity screening indicates that several analogs possess comparable activity as the natural framework (AC50 as low as 0.04 μM vs. as low as 0.02 μM for epothilone C). Epothilone C is only a case in point. The approach may be used to remodel many other bioactive cyclic alkenes.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
Innovative chemical synthesis methods allowing for precise framework remodeling of bioactive complex natural products are highly desirable:
In this project, we have developed a programmable strategy for precise skeletal remodeling of macrocyclic alkenes through tactical use of a select number catalytic methods with emphasis on olefin metathesis as a bond cleaving and bond forming process. The approach can be applied to generating analogs of macrocyclic natural products regardless of whether they contain an alkene or not. As such, we demonstrate that if catalytic reactions are considered as parts of a larger collection and their relationships to one another are better appreciated, they can be utilized in new and unprecedented ways. Our investigations make it possible to synthesize in an efficient and practical manner otherwise difficult-to-access macrocyclic leads for drug discovery. At the same time, there are a number of expanded and contracted isomers that cannot be generated by our new approach. Additionally, our investigations bring to light several key shortcomings in the state of the art, such as the absence of efficient, programmable and iterative protocols for insertion and deletion of heteroatoms in a ring.
Precisely altered non-natural scaffolds can offer new opportunities for lead identification and may provide additional insights regarding structure-activity relationship (SAR) and mechanism of action (MOA) studies:
Classic lead identification relies heavily on the natural product and its peripherally modified analogues. Skeletally altered analogs are important because they present distinct contours to a receptor site; however, strategy that can be used to access such compounds remain lacking. Precisely altered skeletal analogues are usually not taken into account due to synthetic difficulties. We have been able to take an important step towards addressing this problem The relatively high hit rate of in vitro testing data with our compound collections supports the notion that precisely remodeled frameworks are attractive leads for therapeutic discovery. Moreover, the difference in biological behavior of natural product and the remodeled non-natural frameworks can shed valuable light on subtle structural changes that impact influence its interaction with a biological macromolecule.
In this project, we have developed a programmable strategy for precise skeletal remodeling of macrocyclic alkenes through tactical use of a select number catalytic methods with emphasis on olefin metathesis as a bond cleaving and bond forming process. The approach can be applied to generating analogs of macrocyclic natural products regardless of whether they contain an alkene or not. As such, we demonstrate that if catalytic reactions are considered as parts of a larger collection and their relationships to one another are better appreciated, they can be utilized in new and unprecedented ways. Our investigations make it possible to synthesize in an efficient and practical manner otherwise difficult-to-access macrocyclic leads for drug discovery. At the same time, there are a number of expanded and contracted isomers that cannot be generated by our new approach. Additionally, our investigations bring to light several key shortcomings in the state of the art, such as the absence of efficient, programmable and iterative protocols for insertion and deletion of heteroatoms in a ring.
Precisely altered non-natural scaffolds can offer new opportunities for lead identification and may provide additional insights regarding structure-activity relationship (SAR) and mechanism of action (MOA) studies:
Classic lead identification relies heavily on the natural product and its peripherally modified analogues. Skeletally altered analogs are important because they present distinct contours to a receptor site; however, strategy that can be used to access such compounds remain lacking. Precisely altered skeletal analogues are usually not taken into account due to synthetic difficulties. We have been able to take an important step towards addressing this problem The relatively high hit rate of in vitro testing data with our compound collections supports the notion that precisely remodeled frameworks are attractive leads for therapeutic discovery. Moreover, the difference in biological behavior of natural product and the remodeled non-natural frameworks can shed valuable light on subtle structural changes that impact influence its interaction with a biological macromolecule.