**3. Targeting COX-2 enzyme**

There is considerable evidence to suggest that prostaglandins play an important role in the development and growth of cancer. The enzyme cyclooxygenase (COX) catalyses the

Remarks in Successful Cellular Investigations for

and will be discussed later on in this chapter.

et al., 2001; Takemura et al., 1997).

different substituent.

**4. Targeting dihydrofolate reductase (DHFR) enzyme** 

MCF-7 cell line.

Fighting Breast Cancer Using Novel Synthetic Compounds 93

As is shown in figure 6, both compounds have acceptable cytotoxicity effects with C1 being stronger. However, the same experiment has been conducted using the same cell line and the same concentrations of C1 and C2 compounds but using MTT assay. MTT failed to present any cytotoxicity for these compounds on MCF-7 cell line as is shown in figure 7.

 Fig. 7. MTT based cytotoxicity measurement of two novel COX-2 inhibitors of C1 and C2 on

Failure of one experiment using a technique in spite of success for the other technique in acquiring result is a considerable phenomena in cellular investigation on cytotoxic agents

Inhibitors of DHFR are classified as either 'classical' or 'non-classical' antifolates. The 'classical' antifolates are characterized by a *p*-aminobenzoylglutamic acid side-chain in the molecule and thus closely resemble folic acid itself. Methotraxate (MTX) is the most well known drug among the 'classical' antifolates. Compounds classified as 'non-classical' inhibitors of DHFR do not possess the *p*-aminobenzoylglutamic acid side-chain but rather have a lipophilic side-chain. MTX serves as an antimetabolite, which means that it has a similar structure to that of a cell metabolite, resulting in a compound with a biological activity that is antagonistic to that of the metabolite, which in this case is folic acid (Barnhart

New, more lipophilic antifolates have been developed in an attempt to circumvent the mechanisms of resistance, such as decreased active transport, decreased polyglutamation, DHFR mutations and so on (Assaraf, 2007; Gangjee et al., 2006; Takemura et al., 1997). In a series of synthesized compounds for this purpose in our center the pyrimidine ring remained (figure 8) and the side-chain attachment at the position 2 was replaced with

**NH2**

**N**

**H2N S**

Fig. 8. The central structure of novel DHFR inhibitors.

**N**

**R <sup>2</sup>**

conversion of arachidonic acid to prostaglandins (Abou-Issa et al., 2001). There has been a considerable amount of interest in recent years to take advantage of COX inhibitors specifically COX-2 inhibitors in prevention and treatment of malignancies (Talley et al., 2000; Zarghi et al., 2006). Majority of COX-2 inhibitors belong to a class of diaryl heterocycles that possess vicinal diaryl substitution attached to mono, bicyclic or tricyclic central rings (Penning et al., 1997; Prasit et al., 1999; Riendeau et al., 2001).

As a part of ongoing program to design new types of selective COX-2 inhibitors, our center has synthesized novel COX-2 inhibitor derivatives having a new tricyclic central ring scaffold and different substituents at the N-3 as is shown in figure 5.

Fig. 5. Central structure of novel COX-2 inhibitors.

The nature and size of substituent attached to N-3 influenced both selectivity and potency for COX-2 inhibitory activity. Two different compounds of C1 and C2 with different N-3 substituent have been applied to MCF-7 cell line for the evaluation of anticancer effects, using clonogenic assay. MCF-7 cells were seeded for the clonogenic assay in 12-well plates at 150 cells per well for 24 hours. These cells were then exposed to C1 and C2 derivatives for 24 hours. Media was then changed to fresh media without these compounds and plates remained in incubator for couple of days until most of colonies in the control wells contained more than 50 cells. Media was then excluded and cells were fixed with 96% ethanol and stained using trypan blue. Plates were washed and percentages of colonies in different wells were compared to controls (Shirazi et al., 2005).

Fig. 6. Cytotoxicity of two novel COX-2 inhibitors of C1 and C2 on MCF-7 cell line using clonogenic assay.

conversion of arachidonic acid to prostaglandins (Abou-Issa et al., 2001). There has been a considerable amount of interest in recent years to take advantage of COX inhibitors specifically COX-2 inhibitors in prevention and treatment of malignancies (Talley et al., 2000; Zarghi et al., 2006). Majority of COX-2 inhibitors belong to a class of diaryl heterocycles that possess vicinal diaryl substitution attached to mono, bicyclic or tricyclic

As a part of ongoing program to design new types of selective COX-2 inhibitors, our center has synthesized novel COX-2 inhibitor derivatives having a new tricyclic central ring

The nature and size of substituent attached to N-3 influenced both selectivity and potency for COX-2 inhibitory activity. Two different compounds of C1 and C2 with different N-3 substituent have been applied to MCF-7 cell line for the evaluation of anticancer effects, using clonogenic assay. MCF-7 cells were seeded for the clonogenic assay in 12-well plates at 150 cells per well for 24 hours. These cells were then exposed to C1 and C2 derivatives for 24 hours. Media was then changed to fresh media without these compounds and plates remained in incubator for couple of days until most of colonies in the control wells contained more than 50 cells. Media was then excluded and cells were fixed with 96% ethanol and stained using trypan blue. Plates were washed and percentages of colonies in

 Fig. 6. Cytotoxicity of two novel COX-2 inhibitors of C1 and C2 on MCF-7 cell line using

central rings (Penning et al., 1997; Prasit et al., 1999; Riendeau et al., 2001).

scaffold and different substituents at the N-3 as is shown in figure 5.

Fig. 5. Central structure of novel COX-2 inhibitors.

different wells were compared to controls (Shirazi et al., 2005).

clonogenic assay.

As is shown in figure 6, both compounds have acceptable cytotoxicity effects with C1 being stronger. However, the same experiment has been conducted using the same cell line and the same concentrations of C1 and C2 compounds but using MTT assay. MTT failed to present any cytotoxicity for these compounds on MCF-7 cell line as is shown in figure 7.

Fig. 7. MTT based cytotoxicity measurement of two novel COX-2 inhibitors of C1 and C2 on MCF-7 cell line.

Failure of one experiment using a technique in spite of success for the other technique in acquiring result is a considerable phenomena in cellular investigation on cytotoxic agents and will be discussed later on in this chapter.
