Matthew C. Powell

Research Summary

Our laboratory has developed a strategy to selectively target cells overexpressing nuclear receptors and

to generate predominantly cytotoxic 3-MeA adducts in these targeted cells. The implementation of this strategy

led to the design and synthesis of compound 1 shown in

Figure 4. In this molecule, the estradiol unit can

selectively bind to the estrogen receptor and therefore

enable the targeting of cells overexpressing this receptor.

The bispyrrole triamide core of this molecule is a known

DNA-binding motif that selectively binds to the minor

groove of DNA at A/T-rich regions (1)

. This site- specific

binding of such compounds would then place the reactive

methyl sulfonate unit in the vicinity of the minor groove

of DNA at A/T-rich regions where the most

nucleophilic site is the N3 position of adenine, which

would then get methylated forming the desired 3-MeA adduct.

Compounds 1, 2, and 3 have been synthesized and tested. They all produce predominantly 3-MeA

adducts, show formidable binding to the estrogen receptor-alpha (ER-α) and initiate transcription of ER-α

response genes, and are selective cytotoxicity to MCF-7 (ER-α+) breast cancer cells. However, each of the

three compounds varied in toxicity with compound 2 showing substantially greater toxicity.

Objectives of the current research

I. Cause of variation in toxicity/DNA methylation rates

It is necessary to determine the reason for the variation in the methylation ability of compounds 1, 2 and

3 in order to identify design features that will lead to more potent molecules. From computational analysis, it

was hypothesized that the variation in 3-MeA formation by the compounds is due to differences in the DNA-

binding ability of the three compounds. To test this hypothesis, I have performed DNA-binding studies of these

compounds. Typically, DSC/ITC is used to determine binding constants, but due to the limited water solubility

of these compounds, fluorescent intercalator displacement assay (FID) was performed. Along, with having

higher DNA methylation levels and greater toxicity, compound 2 exhibits stronger DNA binding.

Compound DNA RBA (molar

eq.)

3MeAdenine Formation

(µmol/molDNABP)

1 4.72 794 +/-28

2 2.54 11448 +/- 127

3 4.43 1716 +/- 38

Figure 1. DNA binding strength and methylation capacity.

II. Adapting Strategy to Target other Nuclear Receptors

Given structural and functional similarities in the nuclear receptor family, it should be possible to target

other endocrinal cancer cells that overexpress their respective nuclear receptor. By adapting the strategy used

for producing the estradiol-conjugated compounds, new molecules functionalized with dihydrotestosterone,

testosterone, progesterone, and cortisol have been, or are currently being synthesized. Upon completion, these

linkable derivatives are coupled (EDCI or T3P) to a DNA bind moiety.

A. Generating Cell Targeting Units for Androgen Receptor (+) cells

The figure below shows the reaction scheme I followed to successfully synthesize a 7α-hexylamino-

testosterone derivative that is readily coupled to various functionalized molecules. Also the scheme depicts the

adapted method working toward the production of a DHT conjugate.(2)

Figure 2. Synthetic approach to DHT/Testosterone derivatives.

I have successfully adapted this synthesis to form a 7α-hexylamino-progesterone derivative and early

steps towards a cortisol derivative have been completed as well.

Figure 3. A. 7α-hexylamino-progesterone, B. Progress in cortisol derivitization.

For several reasons, supported by recent publications, I decided to design a dual targeted drug conjugate

inspired by the anti-cancer prodrug cyano-nilutamide. This idea offers a way of targeting the AR in which

subsequent gene transcription is impossible (unlike steroid tethered versions) while still making use of the

nuclear translocation ability of ligand bound AR complex which is necessary for DNA methylation.

Figure 4. Synthetic route used to the form the cyano-nilutamide derivatives.

B. Synthesis of DNA Binding Unit

Figure 4. Synthesis of the A/T rich minor groove DNA binding moiety with

various head groups.

The DNA binding unit portion of this molecule has been synthesized

according to the procedure shown above. I have synthesized three

variations (shown right Fig. 5) of this structure and maintain multi-

gram quantities of compounds A, which will serve as a non-toxic

control, and compound B, the active precursor, using known

procedures in our lab in and overall yield of 76% and 80%,

respectively.(2)

Conjugating these molecules (EDCI coupling) with the

appropriate cell targeting unit leads to reactive methylating compounds

(B.) and nonreactive control analogues (A.). Compound C has been

synthesized in fair yield and it is used as a fluorescent probe for cellular

translocation studies after EDCI coupling to its respective cell targeting

unit. The coupling reaction is shown below in Figure 6.

Figure 5. DNA binding moieties

Figure 6. Coupling of the DNA binding moiety to the cell targeting unit.

C. Final Steps in active Compound Synthesis

Figure 7. Final activation sequence for reactive methyl-sulfonate synthesis

Total synthesis of these molecules has been achieved for the testosterone as well as cyano-nilutamide classes

of molecules. This includes a reactive methyl-sulfonate, a non-reactive sulfone analogue, as well as a

fluorescent NBD probe for each targeting unit. Bioorganic studies are currently underway. These studies

include testing compounds ability to form 3-MeA on genomic DNA, receptor binding/DNA binding assays, and

cell toxicity studies. Selective toxicity was shown for the reactive compounds in LnCap vs HEK293 cells,

sufficient DNA binding with the non-reactive analogues have been shown via FID, and DNA methylation has

shown to be substantial for some of these reactive molecules by HPLC. Remaining studies include establishing

the binding affinity of these conjugates to the AR, various cell based assays, and mode of toxicity assays.

Figure 8. Examples of completed testosterone teathered AR-targeting molecules

III. Other Work in Our group

A. Generating a library of anti-neuro-inflammatory

Our group works in conjunction with a bio-pharmaceutical company developing many anti-neuro-

inflammatory derivatives of thalidomide in regards to Alzheimer’s disease. This work is patent protected, so I

am unfortunately unable to elaborate but it utilizes a wide variety of organic reactions and we have produced a

large library of compounds and metabolites.

B. Optimizing the methylating moiety

The methylsulfonate alkylating moiety of these compounds efficiently reacts with the N3-adenine sites to

produce the desired 3-MeA adducts. However, these compounds also undergo slow hydrolysis in aqueous

media, and therefore, a significant proportion of these compounds can

be rendered ineffective in biological systems. Here we aim to fine tune

the reactivity of the methylating moiety. In doing so, the hope is to

maintain reactivity to nitrogen nucleophiles while lowering reactivity

with oxygen nucleophiles. This area of research requires synthesis of

the “war heads” shown to the right. The relative reactivity of these

compounds will be determined via kinetic 1H-NMR. If any of them exhibit significant improvement to the

methylsulfonate, they will be incorporated into our compounds. Intermediates a. and the control molecule have

been synthesized and progress has been made on the other structures, primarily, structure C. Example synthetic

routes are shown below.

1.

2.

C. Other Synthesis

Auxiliary work in this lab includes intermediate synthesis for a host of side/colleague’s projects. Some

examples are shown below.

References

1. Franza, Gilberto, and Barry Gold. "The Biological Effects of N3-methyladenine." Journal of Cellular

Biochemistry 19.2 (2004): 250-257. Web. Aug. & Sept. 2013.

2. Luppa, Peter, Christine Bruchner, Ingrid Shwabb, Sabine Hauk, Stefan Schmidmayr, Christian

Birkmayer, Birgit Paulus, and Hagen Hauptmann. "7α-Biotinylated Testosterone Derivatives as Tracers

for a Competitive Chemiluminescence Immunoassay of Testosterone in Serum." Clinical

Chemistry 43.12 (1997): 2345-2352. Web.

3. Kishton, Rigel, Sean E. Miller, Heather Perry, Tera Lynch, Mayur Patel, Vinayak K. Gore, and Giridhar

R. Akkaraju. "DNA Site-specific N3-adenine Methylation Targeted to Estrogen Receptor-positive

Cells." Biorganic and Medicinal Chemistry 195093-5102. Web. Aug. & Sept. 2013.