Synthesis of Novel Drugs for Prostate Cancer
Purpose of the research
The goal of the research project was to develop a method to synthesize small
molecules that would lead to a potential drug which would treat prostate cancer. It will
take multiple steps to make the chemical. My project was to develop the initial steps in
the synthesis of the chemical.
Rationale for the research / Pertinent scientific literature
Prostate cancer is one of the most frequently diagnosed cancers among men and
the second most common cause of cancer-related deaths in most Western societies
(second to lung cancer)1. The highest risk groups are men with a family history of
prostate cancer and African American men1. 37,000 men per year die from prostate
cancer. Prostate tumor cells generally grow slowly, so traditional drugs don’t work well
since these drugs affect rapidly growing cancer cells. Prostate cancer is not curable thus
there is a need to develop new treatments for prostate cancer.
The prostate gland is located in the male genitourinary system (see Figure 1) 1.
Prostate cancer can either be benign or malignant1. If the cancer is benign, the cancer
cells will remain confined to a small area in the prostate, while in a malignant cancer cell,
the cancer cells spread throughout the body1.
1
Figure 1. A schematic diagram of the prostate gland.
A marker for prostate cancer2 is the Prostate Specific Membrane Antigen (PSMA),
which is a transmembrane protein in the cell membrane of prostate cancer cells2. PSMA
is an enzyme that catalyzes a reaction that generates glutamate3 (see Figure 2). The cell
membrane of normal prostate cells contains little PSMA2, so a PSMA inhibitor would
selectively affect the cancer cells. The biological function of PSMA is uncertain, but a
nonselective PSMA inhibitor has antitumor activity against prostate cancer4, thus PMSA
inhibitor is a new treatment for prostate cancer.
COOH
O
N
H
OH
N
N
H2N
N
N
H
COOH
O
PSMA
H
N
COOH
N
N
O
COOH
N
O
N
H
OH
H2N
N
N
H
H
N
COOH
+
N
O
N
H
H2N
COOH
O
OH
COOH
COOH
n-1
n
Folate Polyglutamate
Glutamate
Figure 2: Enzymatic Activity of PSMA.
2
The chemical structure of the phosphonamidate derivatives of glutamate (Figure
3a) is similar to the substrate of PSMA (See Figure 2) thus these derivatives may be a
competitive inhibitor of PSMA (part of both chemicals look like glutamate). The final
purpose of the project is the synthesis of these possible PSMA inhibitors. The synthesis
of these inhibitors will require many steps. In order to make the compound, the first step
is to make allylphosphonate (figure 3b). The remainder of a proposed synthetic pathway
to generate the PSMA inhibitors is shown in the conclusion section of this report (figure
6).
3
a.
X = 4-Cl; 4-OMe; 3,4-dichloro; 4-OPh; 3-Cl; 4-tBu; 4-CF3; 4-Br; 3-CF3-4-Cl; 3Me; 4-N(Me)2; 3-N(Me)2; 3-CF3; 2,4-dichloro; 4-NO2; 3-CF3-4-NO2; 4-NH2; 4OH; 3-OMe; 4-OPro; 2-Cl; 2-Me; 2-OMe; 4-F; 3,5-dichloro; 3-NO2; 4-OCH2Ph.
b.
Figure 3. Chemical Structures of Interest in this Project.
a. Phosphonamide derivatives of glutamate are possible PSMA inhibitors.
b. Allylphosphonate, an intermediate in the synthesis of the various PSMA
inhibitors.
Preliminary results showed that a compound without X bound to a phenyl group
(see figure 3A) inhibited PSMA (mentor; personal communication). This observation
suggests that there is a nearby hydrophobic binding site to the catalytic site of PSMA.
The basis of the functional group attached to this phenyl group will be based upon the
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Topliss approach5, an algorithm based on electronic and hydrophobic considerations to
optimize binding to the suspected hydrophobic binding site in PSMA, which could
increase the specificity of the PSMA inhibitor.
Methods.
There are two possible methods to make allylphosphonate, an intermediate in the
synthesis of the PSMA inhibitors, which are summarized in the figure 4.
Method 1. Method using potassium hexamethyl disilazane.
Add dibenzylphosphite to a round bottom flask containing toluene with a
magnetic stir bar and place the flask in an ice bath. Potassium hexamethyl disilazane was
added drop-wise and the mixture was stirred for 1 hour. The mixture was removed from
the ice bath, then allyl bromide was added drop-wise and the mixture was stirred
overnight at various temperatures. The reason to try different temperatures was to shift
the equilibrium to the formation of the products. If the reaction is exothermic, it will need
to be cooled to increase the formation of the products. If the reaction is endothermic, it
will need to be heated to increase the formation of the products
Method 2. Method using potassium tert-butoxide. Add dibenzylphosphite to a
round bottom flask containing THF or DMSO with a magnetic stir bar. Potassium tertbutoxide (powder) was added and the mixture was stirred at room temperature for 1 hour.
Then allyl bromide was added drop-wise and stirred overnight at room temperature.
5
a.
Step 1
O
H
P
O
CH3
O
+HC
3
Si
(-)
N
H3C
CH3
Si
K+
O
Toluene
CH
CH3 3
:P
(-) K+
0oC
O
CH3
CH3
+
O
Si
Si
H3C
N
H3C H
Step 2
O
Br
O
P
Neat
O
Various Temperature
O
P
+
Unwanted Compound
Vinylphosphonate
O
O
Wanted
Compound
Allylphosphonate
b.
Step 1
O
H
P
O
O
O
O
+
:P
THF or DMSO
: (-) K+
(-) K+
O
O
Room Temperature
+
OH
Step 2
O
O
Br
Neat
Room Temperature
P
P
O
O
+
UnwantedVinylphosphonate
Compound
O
O
+ KBr
Wanted
Compound
Allylphosphonate
Figure 4. There are two possible synthetic pathways to produce allylphosphonate which
is an intermediate in the synthesis of the PSMA inhibitors. All reactions took place in an
argon atmosphere.
a. Method using potassium hexamethyldisilazane
b. Method using potassium tert-butoxide
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CH
CH3 3
After completing the reaction above, the reaction mixture was put into a
separatory funnel. Then ethyl acetate and water were added to the mixture.
The
separatory funnel was shaken and the bottom aqueous layer discarded. Then the organic
layer was sequentially washed with aqueous solutions containing 10% HCl, 10%
NaHCO3, and saturated NaCl, where the aqueous layer was discarded each time. Na2SO4
powder was added to the organic layer to absorb any water, then the suspension was
filtered to remove Na2SO4 and the solvent was evaporated. The content of the reaction
mixture was dissolved in CDCl3 to obtain a proton NMR spectrum which determined the
amount of reactants and products in the mixture (see figure 5).
Results:
The goal of my project was to determine the best method to make
Allylphosphonate, an intermediate in the synthesis of the various PSMA inhibitors. A
comparison of the % yields (table 1) using the two methods described above (see figure 4)
shows that while both methods produced the desired product (allylphosphonate), the
method using potassium tert-butoxide had a significant greater yield than the method
using potassium hexamethyldisilazane (75% versus 8 – 14%, respectively), while
producing none of the undesired product (vinylphosphonate). Based on these results, the
method of using potassium tert-butoxide is significant better to using potassium
hexamethyldisilazane to synthesize the desired product, allylphosphonate, which is an
intermediate in the synthesis of the PSMA inhibitors.
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Table 1. Comparison of synthetic pathways to produce allylphosphonate.
% yield
Method using potassium hexamethyldisilazanea
limiting
reactant*
AllylVinylphosphonate phosphonate
0
0
8
92
25
0
14
86
40
0
10
90
25
75
0
Temperature (ºC)
Method using potassium tert-butoxideb
25
* dibenzylphosphite
a
In this reaction, 2.25 mmol of dibenzylphosphite, 4.5 mmol potassium
bis(trimethylsilyl)-amide, and 4.5 mmol allyl bromide were used in the reaction. All of
the limiting reactant was converted to products.
b
In this reaction, 2.25 mmol of dibenzylphosphite, 6.75 mmol potassium tert-
butoxide, and 13.5 mmol allyl bromide were used in the reaction. Notice that
dibenzylphosphite is the limiting reactant in both synthesis methods.
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Figure 5. Proton NMR spectrum of the crude sample in the method using potassium tertbutoxide, which was used to determine the amount of reactants and products. The proton
NMR spectrum of the purified sample was the same as the crude sample without the
signal due to allyl bromide and ethyl acetate.
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Discussion:
The allylphosphonate in Figure 3b may not be stable when using
hexamethyldisilazane as a reactant, because the allylphosphonate may be isomerized to
the vinylphosphonate due to the basic side product. Because the side product is not as
basic when using potassium tert-butoxide, the allylphosphonate was not contaminated by
its conversion to vinylphosphonate.
Conclusions:
I have established the initial steps in the synthesis of allyphosphate, an
intermediate in the synthesis of a family of new PSMA inhibitors. Subsequent work
would be to continue the development of a synthetic scheme to produce a family of
phosphonate derivatives of glutamate, potential PSMA inhibitors (figure 6 provides a
possible synthetic pathway).
After the successful synthesis of these PSMA inhibitors, they would be screened
for their ability to inhibit PSMA using a newly developed HPLC method (mentor;
personal communication). After this screening test, promising candidate drugs would be
tested in an in-vitro, followed by an in-vivo prostate cancer model system. Drugs that
pass these trails would be candidates for human clinical trails for the treatment of prostate
cancer. Based upon hypothetical arguments, the following two potential concerns should
be examined during the clinical trails.
First, the action involving the digestive system. The digestive system digests
proteins using various proteases, which could be inhibited by a PSMA inhibitor, thus it
may interfere with the absorption of proteins.
In addition, an inhibition of protein
digestion may produce diarrhea. Another concern regarding the digestive system is to
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examine if the digestive system degrades the PSMA inhibitor, i.e. is it an effective drug
when given orally?
For these reasons, the effectiveness of oral administration and
potential side-effects involving the digestive system should be monitored during the
clinical trails.
Second, an action involving the nervous system. PSMA is similar to another
membrane bound enzyme found in the rat brain, NAALADase (N-acetylated alpha-linked
L-amino dipeptidase)
3
that generates glutamate.
NAALADase is associated with
disorders that involve improper glutamatergic neurotransmission such as in schizophrenia,
seizures and other neuro-degenerative conditions such as Alzheimer’s and Huntington’s
disease.6 As PSMA and NAALADase have a very strong sequence homology, a PSMA
inhibitor may inhibit NAALADase, which could produce symptoms like schizophrenia,
seizures, Alzheimer’s and Huntington’s disease. For these reasons, such potential sideeffects on the nervous system should be monitored during the clinical trail of this novel
class of inhibitors. In the event of an adverse neural side-effect, the PSMA inhibitor may
be modified to retain its PSMA inhibitory action, while reducing its ability to cross the
blood-brain barrier.
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General Scheme
O
H
O
- +
O K
P
P
O
O
+
O
Pinacolborane
O
O
Br
O
O
B
P
O
1)Na
I2)Oxallyl
Cl
O
O
P
O
Cl
COObn
H2N
COObn
O
O
O
O
B
P
N
O H
PdCl2(dppf)
Base
O
I
O
X
X
O
O
O
P
N
O H
O
O
ACN
O
Pd/C/H2
X
O
O
OH
P
N
OHH
O
OH
Figure 6. Complete Synthetic Method of making PSMA inhibitor.
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References:
1. www.prostate.com (Accessed in July, 2004)
2. Jackson, P. F.; Cole, D. C.; Slusher, B. S.; Stetz, S. L.; Ross, L. E.; Donzanti, B.
A.; Trainor, D. A. J. Med. Chem. 39, 619-622, 1996.
3. Carter, R. E.; Feldman, A. R.; Coyle, J.T. Proc. Natl. Acad. Sci. USA. 93(2), 749753,1996.
4. Slusher, B.S.; Tiffany, C.W.; Merion, A.; Lapidus, R.G.; Jackson, P.F. Prostate.
44(1), 55-60, 2000.
5. Topliss, J.G.
J Med. Chem. 20(4),463-469, 1977.
6. Passani, L.A.; Vonsattel, J.P.; Carter, R.E.; Mol. Chem. Neuropathol. 31(2), 97118, 1997.
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