Romidepsin is a natural product produced by fermentation that inhibits histone deacetylase (HDAC) enzymes, altering gene expression in cancer cells. It is metabolized to its active form which interacts with zinc ions in HDAC enzymes. This prevents cancer cell proliferation and induces apoptosis. Its synthesis involves multiple steps, including Mukaiyama aldol addition, ester hydrolysis, nucleophilic substitution, and disulphide bond formation.

1. The Modes of Action and Synthesis of Romidepsin
Abstract
Romidepsin is a natural product produced by the fermentation of bacteria. Years after discovery, it
was found to have anticancer properties. It is a prodrug which is metabolised by glutathione in the
liver. It is an enzyme modulator which is used to stop tumour growth and induce apoptosis in cancer
cells. This is achieved via inhibition of histone deacetylase enzymes. Romidepsin alters the gene
expression of cancer cells to cause cell cycle arrest, halt cell proliferation, and induce apoptosis of
cancer cells. Synthesis includes multiple steps including Mukaiyama aldol addition, ester hydrolysis,
nucleophilic substitution, and oxidation to form disulphide bonds.
Introduction
Romidepsin, branded Istodax, is an anticancer prodrug discovered in 19941
which is used to treat
malignant tumours, peripheral T-cell lymphoma (PTCL) and cutaneous T-cell lymphoma (CTCL).1, 2
Romidepsin was discovered as a fermentation product of the bacterium Chromobacterium
violaceum (C. violaceum). It is a cyclic peptide, as shown below in figure 1, which was later
discovered to have anticancer and antitumour properties in 1998. It was found to be a very potent
inhibitor of histone deacetylase (HDAC) enzymes.1
Shown below, in figure 1, is the skeletal structure
of romidepsin.
Figure 1- The Skeletal Structure of Romidepsin
Discovery
Romidepsin, or FK228, is a natural product formed via fermentation of C. violaceum in 1994. In 1998,
it was discovered that romidepsin inhibited HDAC enzymes which prompts apoptosis of cancer cells.
During clinical trials, it was found to be effective for a variety of malignant cancers.Romidepsin
displays a much higher therapeutic effect than some more frequently used anticancer drugs.

2. Although it is found to be more effective than other drugs, the mechanism of which romidepsin
produces its cytotoxic effects is not fully understood.3
Modes of Action/ Metabolism
The ras oncogene aids the formation of a tumour. Romidepsin has been found to reverse the
development of Ha-ras oncogenes. It was also found to have cytotoxic properties.4
Romidepsin is a potent inhibitor of HDAC enzymes. As romidepsin is a prodrug it needs to be
metabolised into its active form after administration. This is achieved via cellular reduction of the
disulphide bond.1,5,6,7
It is presumed to be metabolised in the liver by cytochrome P450 (CYP450)
enzymes and glutathione to produce a monocyclic dithiol.1,4,6,7
The molecule below in figure 2,
displays sulfhydryl functional groups present on the active metabolite of romidepsin.4
Figure 2- Reduced romidepsin4
Histones can be acetylated or deacetylated. Whichever state the histone is currently in will
determine the alteration of chromatin during gene transcription. Increased levels of acetylated
histones H3 and H4 open chromatin for gene transcription. The mechanism of which romidepsin
inhibits HDAC enzymes is not fully understood but it is thought to target class I, II and IV HDAC
enzymes. This is due to the active sites of these HDAC classes having zinc active sites whilst class III
HDAC enzymes have NAD+
active sites.1,7
Once reduction of the disulphide bond occurs, the
sulfhydryl groups now present on the metabolised romidepsin molecules will interact with the zinc
active sites present on class I HDAC enzymes.1
It is thought to be inhibited via the sulphur atoms
interacting with the zinc ions through water molecules.4
Although romidepsin can inhibit class II and
IV HDAC enzymes, it would require a much higher dose to produce a therapeutic effect. This
prevents the deacetylation of histones, altering the structure of chromatin during gene
transcription.1
This will inhibit cell proliferation, preventing the tumour from growing. HDAC enzyme inhibition
increases the upregulation of cyclin dependant kinase (CDK) inhibitor p21. This is thought to be
caused by the acetylation of the transcription factor protein Sp1 as it is a class I HDAC. As it is
acetylated it cannot bind to p21 promoters. This allows Sp3 (another transcription factor protein) to
bind to p21 promoters which can edit gene transcription. This will increase expression of p21
proapoptotic proteins. CDK inhibitor p21 binds to cyclins involved in the G1 phase of the cell cycle
preventing them from binding to CDK halting the cell cycle.1,4
Apoptosis of cancer cells occur due to

3. the levels of acetylated histone levels increasing. This causes a downregulation of antiapoptotic
genes and an upregulation of proapoptotic genes. The cell cycle may also be inhibited at G2 phase.
Romidepsin can reduce the levels of Chk1 proteins and lowers inhibitory phosphorylation of cell
cycle regulatory proteins. Angiogenesis is also prevented stopping the tumour from receiving a
sufficient oxygen supply by decreasing the levels of VEGF expressed.1
Immune responses are modulated preventing an excessive immune response from damaging the
healthy tissues close to the tumour. Cellular differentiation will also increase.1
Romidepsin has also been found to have a greater therapeutic affect when used in combination with
proteasome inhibitors such as bortezomib. When used together, they have been found to increase
apoptosis of myeloma cells.1
Synthesis Steps
Scheme 1- Total synthesis of romidepsin8
Scheme 1 displays a synthetic route for synthesising romidepsin. There are multiple steps in this
synthesis.

4. Scheme 2- Mukaiyama aldol addition of molecules 4 (CAS#81171-44-0) and 5 (CAS#180973-22-2)
followed by a desilylation reaction
The Mukaiyama aldol reaction is a catalytic cycle which involves the addition of a trimethylsilyl group
to the ketone of molecule 5 using a Lewis acid. After Mukaiyama aldol addition is complete, a
desilylation reaction will occur with the use of a fluoride ion followed by a H+
ion to protonate the
oxygen atom. Hydrochloric acid was used in scheme 2 above. This produces molecule 6
(CAS#180973) of scheme 1.

5. Scheme 3- ester hydrolysis of CAS#180973-41-65
In this step of the synthesis, methanol (MeOH) is used for the ester hydrolysis of CAS#180973-41-65.
It is used to break the ester bond using the hydroxide ion of methanol.

6. Scheme 4- Nucleophilic substitution followed by the formation of a disulphide bond
Iodine ions are nucleophiles. Nucleophilic substitution must be carried out on molecule 9 to allow a
disulphide bond to form via oxidation. This produces romidepsin.
Conclusion
To conclude, romidepsin is a prodrug which is metabolised in the liver by CYP450 and glutathione.
The metabolite produced uses the newly formed sulfhydryl functional groups to inhibit the zinc
active sites of class I, II and IV HDAC enzyme groups. This produces the therapeutic affects of
romidepsin. Inhibition of HDAC enzymes prevent cancer cells from proliferating and causes cell
apoptosis.
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