**Structure-activity Studies of Biological Effectiveness in Drug Design and Therapeutic Use**

**Chapter 4**

**Provisional chapter**

**Indomethacin from Anti-Inflammatory to Anticancer**

**Indomethacin from Anti-Inflammatory to Anticancer** 

The chapter "Indomethacin from Anti-inflammatory to Anticancer Agent" covers the recent reports regarding the implication of COX-2/PGE2 in multiple cancer cell proliferation to emphasize the anticancer potential of COX-inhibitors including indomethacin and to reveal that the reduction of PGE2 production interferes with the cancer cell proliferation belongs to multiple cancer cell types. Impressively, indomethacin is involved in antiproliferative and apoptotic actions against cancer cell types via COX-2-independent mechanisms to highlight indomethacin as promising anticancer agent with dual actions to control the cancer cell proliferation. The cardiovascular complications result from diaryl heterocycle sulfonamide/methylsulfone selective COX-2 inhibitors upon reduction in PGE2 and PGI2 production that affects the vascular tone limits the use of Celecoxib as chemopreventive agent against recurrence of colorectal carcinoma cells. Kinetic profile of indomethacin against COX-2 showed obvious difference from that of selective COX-2 inhibitors in which it recovered completely from the enzyme after long time of incubation while COX-2 inhibitors did not recover to impress that this might be implicated in the cardiovascular toxicity of the selective inhibitors. This raised the concern to develop the indomethacin from nonselective COX- to selective COX-2-inhibitors and to assert whether the cardiac complications are from pharmacological class effect or chemical class effect. **Keywords:** indomethacin, COX-2-independent mechanism, apoptosis, antiproliferative,

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Indomethacin is indole-3-acetic acid derivative, classified as nonsteriodal anti-inflammatory drug (NSAID). The drug is primarily used for the treatment of painful inflammatory conditions

DOI: 10.5772/intechopen.79677

**Agent**

**Agent**

Shaymaa Emam Kassab

Shaymaa Emam Kassab

**Abstract**

kinetic profile

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79677

#### **Indomethacin from Anti-Inflammatory to Anticancer Agent Indomethacin from Anti-Inflammatory to Anticancer Agent**

DOI: 10.5772/intechopen.79677

#### Shaymaa Emam Kassab Shaymaa Emam Kassab

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79677

#### **Abstract**

The chapter "Indomethacin from Anti-inflammatory to Anticancer Agent" covers the recent reports regarding the implication of COX-2/PGE2 in multiple cancer cell proliferation to emphasize the anticancer potential of COX-inhibitors including indomethacin and to reveal that the reduction of PGE2 production interferes with the cancer cell proliferation belongs to multiple cancer cell types. Impressively, indomethacin is involved in antiproliferative and apoptotic actions against cancer cell types via COX-2-independent mechanisms to highlight indomethacin as promising anticancer agent with dual actions to control the cancer cell proliferation. The cardiovascular complications result from diaryl heterocycle sulfonamide/methylsulfone selective COX-2 inhibitors upon reduction in PGE2 and PGI2 production that affects the vascular tone limits the use of Celecoxib as chemopreventive agent against recurrence of colorectal carcinoma cells. Kinetic profile of indomethacin against COX-2 showed obvious difference from that of selective COX-2 inhibitors in which it recovered completely from the enzyme after long time of incubation while COX-2 inhibitors did not recover to impress that this might be implicated in the cardiovascular toxicity of the selective inhibitors. This raised the concern to develop the indomethacin from nonselective COX- to selective COX-2-inhibitors and to assert whether the cardiac complications are from pharmacological class effect or chemical class effect.

**Keywords:** indomethacin, COX-2-independent mechanism, apoptosis, antiproliferative, kinetic profile

#### **1. Introduction**

Indomethacin is indole-3-acetic acid derivative, classified as nonsteriodal anti-inflammatory drug (NSAID). The drug is primarily used for the treatment of painful inflammatory conditions

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

that involves gout and osteoarthritis [1]. The mechanistic role of indomethacin in inhibition of pain has been verified by being nonselective inhibitor to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes [2]. The enzymatic activity of COX involves bis-oxygenation of arachidonic acid to (prostaglandin G2) PGG2, which then reduced to PGH2 in a peroxidase reaction by the same protein [3]. COX-1 is constitutively expressed in most tissues, to which the production of prostaglandins is attributed to; and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, is implicated to the elevated levels of prostaglandins during the inflammation. Prostaglandins are hormone-like mediators involved in the induction of pain, fever and inflammation [2]. The inhibition of indomethacin to the two COX isozymes with minimal selectivity to COX-2 made the drug have serious complications such as gastric ulcers and renal toxicity upon long-term oral administration [4, 5].

disorders except for rofecoxib and lumiracoxib (the only carboxylic coxib) that were withdrawn due to observation of cardiovascular complications from the recommended daily dose with rofecoxib [27] and observation of liver failure with lumiracoxib [28]. Selective COX-2 inhibitors were launched to treat the individuals who cannot tolerate severe gastrointestinal responses of NSAIDs. A few years later, extensive preclinical and clinical data generated to report the role of COX-2 in tumor growth and/or metastasis [29]. Studies on experimental animals showed that selective COX-2 inhibitors including celecoxib block the formation, growth and metastases of multiple tumor types [30]. Consistently, celecoxib demonstrated dramatic chemopreventive efficacy against colon polyps and reduced the incidence of recurrent adenomas of any type by 45% and of high risk lesions by 66% over a 400 mg dose twice daily for 3 years [31, 32]. One of the complications that should be tackled in the near future for selective COX-2 inhibitors celecoxib in specific is the cardiovascular complications that comes after administration of 400 mg twice daily to be the same as the dose recommended for chemopreventive effect to control the recurrence of CRC [32, 33]. The magnitude of cardiovascular complications of celecoxib limits its use for colon cancer prevention since the development of colon cancer is a slow process, so, the patients with polyps would need to take celecoxib for a long period of time to achieve the target protective effect. Accordingly, a question should be admitted, and should have an evidenced answer: Does the cardiovascular problems of selective COX-2 inhibitors class of anti-inflammatory agent come out of pharmacological class effect or chemical class effect? To my knowledge, we cannot confirm that it is pharmacological class effect and not chemical class effect because the chemical structure of COX-2 inhibitors that share the CVS side effects are Y-shaped diaryl-heterocycle sulfonamide/methylsulfonyl. Thus, it is required to develop new chemical class of selective COX-2 inhibitors help us be provided with verified answer to such important question. The answer of the question would raise the concern to the main reason(s) of CVS complications to tackle and eventually modify the strategy toward generation of selective COX-2 inhibitors with chemopreventive benefits against CRC and other cancer cell types. Based on the above findings indomethacin, as nonselective COX-inhibitor could be considered strategic lead compound that worth it studying and developing to line it among the chemotherapeutic agents used against cancer to be either prophylactic or therapeutic treatment and/or even adjuvant therapy upon combination with other anticancer agents to

Indomethacin from Anti-Inflammatory to Anticancer Agent

http://dx.doi.org/10.5772/intechopen.79677

47

**Figure 1.** Diarylsulfonamide/methylsulfone selective COX-2 inhibitors.

Indomethacin and the other NSAIDs were found to have significant anticancer activity against wide variety of cancer cell types, *in vitro* and *in vivo* [6–10]. Moreover, epidemiological studies reported that the use of such type of drugs is linked to the reduction of cancer risk [11–13]. Indomethacin performs its anticancer activity in different fashions, inhibits proliferation via induction of apoptic death of tumor cells [6, 9, 10], reduces tumorigenesis by enhancing the immune response [14, 15] and inhibiting the angiogenesis [16, 17] as well.

Interestingly, to mention that the mechanism to which the anticancer activity of NSAIDs including indomethacin attributed is the reduction of PGE2; a type of prostaglandins generated from the bis-oxygenation of arachidonic acid by COX-2. PGE2 contributes to the cell proliferation, cell cycle proliferation and cell cycle progression through various cell signaling mechanisms leads to induction of oncogenic genes and eventually overexpression of proliferative proteins [18–22]. Recently, extensive studies on various cancer cell types including colorectal carcinoma (CRC) justified the efficacy of indomethacin to reduce the levels of antiapoptotic proteins and progressive cell proliferation represented by tumor size by COXindependent mechanisms [23–26].

After emerge and marketing of celecoxib; selective COX-2 inhibitor in December 1998, rofocoxib was released in 1999 worldwide then lumiracoxib and etoricoxib (**Figure 1**) that are marketed in Europe. Those inhibitors are still marketed for the treatment of inflammatory disorders except for rofecoxib and lumiracoxib (the only carboxylic coxib) that were withdrawn due to observation of cardiovascular complications from the recommended daily dose with rofecoxib [27] and observation of liver failure with lumiracoxib [28]. Selective COX-2 inhibitors were launched to treat the individuals who cannot tolerate severe gastrointestinal responses of NSAIDs. A few years later, extensive preclinical and clinical data generated to report the role of COX-2 in tumor growth and/or metastasis [29]. Studies on experimental animals showed that selective COX-2 inhibitors including celecoxib block the formation, growth and metastases of multiple tumor types [30]. Consistently, celecoxib demonstrated dramatic chemopreventive efficacy against colon polyps and reduced the incidence of recurrent adenomas of any type by 45% and of high risk lesions by 66% over a 400 mg dose twice daily for 3 years [31, 32]. One of the complications that should be tackled in the near future for selective COX-2 inhibitors celecoxib in specific is the cardiovascular complications that comes after administration of 400 mg twice daily to be the same as the dose recommended for chemopreventive effect to control the recurrence of CRC [32, 33]. The magnitude of cardiovascular complications of celecoxib limits its use for colon cancer prevention since the development of colon cancer is a slow process, so, the patients with polyps would need to take celecoxib for a long period of time to achieve the target protective effect. Accordingly, a question should be admitted, and should have an evidenced answer: Does the cardiovascular problems of selective COX-2 inhibitors class of anti-inflammatory agent come out of pharmacological class effect or chemical class effect? To my knowledge, we cannot confirm that it is pharmacological class effect and not chemical class effect because the chemical structure of COX-2 inhibitors that share the CVS side effects are Y-shaped diaryl-heterocycle sulfonamide/methylsulfonyl. Thus, it is required to develop new chemical class of selective COX-2 inhibitors help us be provided with verified answer to such important question. The answer of the question would raise the concern to the main reason(s) of CVS complications to tackle and eventually modify the strategy toward generation of selective COX-2 inhibitors with chemopreventive benefits against CRC and other cancer cell types.

Based on the above findings indomethacin, as nonselective COX-inhibitor could be considered strategic lead compound that worth it studying and developing to line it among the chemotherapeutic agents used against cancer to be either prophylactic or therapeutic treatment and/or even adjuvant therapy upon combination with other anticancer agents to

**Figure 1.** Diarylsulfonamide/methylsulfone selective COX-2 inhibitors.

that involves gout and osteoarthritis [1]. The mechanistic role of indomethacin in inhibition of pain has been verified by being nonselective inhibitor to cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) isozymes [2]. The enzymatic activity of COX involves bis-oxygenation of arachidonic acid to (prostaglandin G2) PGG2, which then reduced to PGH2 in a peroxidase reaction by the same protein [3]. COX-1 is constitutively expressed in most tissues, to which the production of prostaglandins is attributed to; and COX-2, which is induced by cytokines, mitogens and endotoxins in inflammatory cells, is implicated to the elevated levels of prostaglandins during the inflammation. Prostaglandins are hormone-like mediators involved in the induction of pain, fever and inflammation [2]. The inhibition of indomethacin to the two COX isozymes with minimal selectivity to COX-2 made the drug have serious complications

such as gastric ulcers and renal toxicity upon long-term oral administration [4, 5].

Indomethacin and the other NSAIDs were found to have significant anticancer activity against wide variety of cancer cell types, *in vitro* and *in vivo* [6–10]. Moreover, epidemiological studies reported that the use of such type of drugs is linked to the reduction of cancer risk [11–13]. Indomethacin performs its anticancer activity in different fashions, inhibits proliferation via induction of apoptic death of tumor cells [6, 9, 10], reduces tumorigenesis by enhancing the

Interestingly, to mention that the mechanism to which the anticancer activity of NSAIDs including indomethacin attributed is the reduction of PGE2; a type of prostaglandins generated from the bis-oxygenation of arachidonic acid by COX-2. PGE2 contributes to the cell proliferation, cell cycle proliferation and cell cycle progression through various cell signaling mechanisms leads to induction of oncogenic genes and eventually overexpression of proliferative proteins [18–22]. Recently, extensive studies on various cancer cell types including colorectal carcinoma (CRC) justified the efficacy of indomethacin to reduce the levels of antiapoptotic proteins and progressive cell proliferation represented by tumor size by COX-

After emerge and marketing of celecoxib; selective COX-2 inhibitor in December 1998, rofocoxib was released in 1999 worldwide then lumiracoxib and etoricoxib (**Figure 1**) that are marketed in Europe. Those inhibitors are still marketed for the treatment of inflammatory

immune response [14, 15] and inhibiting the angiogenesis [16, 17] as well.

independent mechanisms [23–26].

46 Medicinal Chemistry

synergize the chemotherapeutic effect [25]. The subjected insight in this book chapter regarding indomethacin could be easily justified on scientific bases: (1) indomethacin is the most NSAID that is intensively studied as chemopreventive and chemotherapeutic agent against multiple cancer cell types among the other drugs of the same class to show observable results. (2) Indomethacin as a different chemical class when compared to selective COX-2 inhibitors, developing indomethacin-based selective COX-2 inhibitor to excel celecoxib would benefit in asserting whether the cardiovascular system (CVS) problems are originated from chemical class effect. In case, the reason of the cardiovascular system (CVS) complications is attributed to the chemical class effect and indomethacin-based developed structures are devoid of the complications, the patients would be largely benefited from such class of compounds, and it would be chemopreventive agents used for long time without developing cardiovascular system (CVS) complications. (3) Kinetic profile of indomethacin inhibition to COX-2 shows recovery after long time of tight binding to the enzyme [34], and on the other hand, selective COX-2 inhibitors' kinetic profile shows no recovery even after long time of tight binding [35]. This obvious difference between indomethacin and selective COX-2 inhibitors in performing the functionally irreversible inhibition effect to COX-2 has to lead us highlighting indomethacin as promising base to build upon it the developed structures in a way to generate selective COX-2 inhibitors with minimized serious side effects observed with the diaryl heterocycle sulfonamide/methylsulfone class of compounds.

It was reported for celecoxib to be effective after Helicobacter Pylori eradication therapy in improving gastric precancerous lesions and stops progression into cancer. The therapeutic effect of celecoxib is explained in the study by measuring the expression and activity of COX-2 for patients with gastric precancerous lesions received celecoxib up to 3 months to be compared with those received placebo for the same period of time. The measurements were determined by immunostaining and PGE2 assay, cell proliferation by Ki-67 immunostaining, apoptosis by TUNEL staining and angiogenesis by microvascular disease (MVD) assay using CD31 staining. The results showed that there was a significant elevation in COX-2 protein expression in gastric precancerous lesions when compared with that resulted from chronic gastritis with consequent increase in cell proliferation and angiogenesis. Patients who were treated with celecoxib showed significant improvement in gastric precancerous lesions (sites of dysplasia) with 84.6% regressed dysplasia, while those treated with placebo showed 60% suggesting that celecoxib was effective on the regression of dysplasia. On the other hand, celecoxib effectively suppressed cell proliferation, induced cell apoptosis and inhibited angiogenesis exhibited by decreased MVD. Interestingly, COX-2 inhibition was accompanied by up-regulation of PPARγ

Indomethacin from Anti-Inflammatory to Anticancer Agent

http://dx.doi.org/10.5772/intechopen.79677

49

expression that is protective protein with reported antineoplastic effects [36].

of PGE2 should be regarded for future strategies targeting HNSCC prevention [19].

carcinogenesis [20].

beside COX-2 and PGE2 [21].

Another study defined a critical mechanism to justify the role of PGE2 in promoting CRC cell division in which prometastatic adaptor protein human enhancer filamentation 1 (HEF1) links between PGE2 and cell cycle machinery in CRC cells. PGE2 induces expression of HEF1 mRNA and protein in CRC. Knockdown of HEF1 suppresses PGE2-induced cell proliferation and cell cycle progression. CRC cells were examined and found that there is 50% elevated levels of HEF1 when compared to normal tissues. Further, HEF1 promotes cell cycle progression of colorectal carcinogenesis via interaction with and activation of cell cycle kinase Aurora A to report that PGE2 is inducer to crucial downstream mediator, HEF1 in colorectal

Small noncoding RNA, microRNAs (miRNAs) have a key role in stopping the translation and accelerate the degradation of mRNA that regulates the cellular growth and survival through gene suppression. miRNA has a significant contribution in controlling disease progression in pancreatic cancer cells (PaCa). Elevated levels of COX-2 were observed with PGE2 and decrease in miRNA increased the cancer growth and metastases of PaCa. Restoration of miRNA-143 (miR-143) in human PaCa cells reduced COX-2 and inhibited cell proliferation. Mitogen activated kinase (MAPC) was correlated to not detecting miR-143 in some pancreatic cancer cell subtypes to justify the implication of MAPC activation in regulating miR-143

Overexpression of COX-2 frequently occurred in head and neck squamous cell carcinoma (HNSCC). COX-2 promotes the release of pro-inflammatory mediator PGE2 which binds to cell surface G-protein coupled receptors EP1–4 to exert its pharmacological effects. Upon studying the biochemical functions of PGE2 and its cell receptors in HNSCC cellular proliferation, it was found that COX-2 and cell receptors EP1, EP2 and EP3 were constitutively expressed in tumoral lesions of HNSCC. An important finding was declared in the study states that small concentration of selective COX-2 inhibitors succeed to suppress PGE2 without inhibition of cell proliferation. However, exogenous addition of EP3-specific agonists with PGE2 induces DNA synthesis in all HNSCC cell lines. Thus, it could be suggested that EP3 receptor subtype

The book chapter covers progressively and in detail some critical topics served in concluding future trends in regard to developing indomethacin to be effective chemopreventive and treatment of various cancer cell types without induction of severe cardiovascular system (CVS) complications: *the implication of COX-2/PGE2 in the anticancer activity of COX-inhibitors*, *COX-2-independent mechanisms of anticancer activity of indomethacin*, and *significance of indomethacin's anticancer activity over the other NSAIDs and selective COX-2 inhibitors.*

## **2. Implication of COX-2/PGE2 in anticancer activity of COX-inhibitors**

PGE2 implicated in promoting cell proliferation of human esophageal squamous cell carcinoma. The study started with observation of expression and upregulation of c-Myc, an oncogenic transcription factor, and then a link was expected to exist between PGE2 and c-Myc but requires a reliable elucidation. Deeper studies revealed that PGE2 substantially increased the proliferation of cultured esophageal squamous cell carcinoma cells and increased mRNA and protein expression of c-Myc. Moreover, knockdown of c-Myc by RNA interference significantly attenuated PGE2-induced cell proliferation. Furthermore, a mechanistic study described that stability and nuclear accumulation of c-Myc oncogenic protein is attributed PGE2 via phosphorylation on serine 62 that induced by extracellular signal regulated kinase (ERK)-dependent manner and this was confirmed when PGE2 activation of ERK was fully abolished by protein kinase C (PKC) inhibitors. Consistently, PGE2 receptor (EP2) agonist resulted in the same effect on expression of c-Myc as PGE2 and knockdown of EP2 receptor by EP2 small interfering RNA (siRNA) delayed PGE2-induced c-Myc expression to verify the association of PGE2 to c-Myc protein expression in esophageal squamous cancer cell proliferation [18].

It was reported for celecoxib to be effective after Helicobacter Pylori eradication therapy in improving gastric precancerous lesions and stops progression into cancer. The therapeutic effect of celecoxib is explained in the study by measuring the expression and activity of COX-2 for patients with gastric precancerous lesions received celecoxib up to 3 months to be compared with those received placebo for the same period of time. The measurements were determined by immunostaining and PGE2 assay, cell proliferation by Ki-67 immunostaining, apoptosis by TUNEL staining and angiogenesis by microvascular disease (MVD) assay using CD31 staining. The results showed that there was a significant elevation in COX-2 protein expression in gastric precancerous lesions when compared with that resulted from chronic gastritis with consequent increase in cell proliferation and angiogenesis. Patients who were treated with celecoxib showed significant improvement in gastric precancerous lesions (sites of dysplasia) with 84.6% regressed dysplasia, while those treated with placebo showed 60% suggesting that celecoxib was effective on the regression of dysplasia. On the other hand, celecoxib effectively suppressed cell proliferation, induced cell apoptosis and inhibited angiogenesis exhibited by decreased MVD. Interestingly, COX-2 inhibition was accompanied by up-regulation of PPARγ expression that is protective protein with reported antineoplastic effects [36].

synergize the chemotherapeutic effect [25]. The subjected insight in this book chapter regarding indomethacin could be easily justified on scientific bases: (1) indomethacin is the most NSAID that is intensively studied as chemopreventive and chemotherapeutic agent against multiple cancer cell types among the other drugs of the same class to show observable results. (2) Indomethacin as a different chemical class when compared to selective COX-2 inhibitors, developing indomethacin-based selective COX-2 inhibitor to excel celecoxib would benefit in asserting whether the cardiovascular system (CVS) problems are originated from chemical class effect. In case, the reason of the cardiovascular system (CVS) complications is attributed to the chemical class effect and indomethacin-based developed structures are devoid of the complications, the patients would be largely benefited from such class of compounds, and it would be chemopreventive agents used for long time without developing cardiovascular system (CVS) complications. (3) Kinetic profile of indomethacin inhibition to COX-2 shows recovery after long time of tight binding to the enzyme [34], and on the other hand, selective COX-2 inhibitors' kinetic profile shows no recovery even after long time of tight binding [35]. This obvious difference between indomethacin and selective COX-2 inhibitors in performing the functionally irreversible inhibition effect to COX-2 has to lead us highlighting indomethacin as promising base to build upon it the developed structures in a way to generate selective COX-2 inhibitors with minimized serious side effects observed with the diaryl heterocycle

The book chapter covers progressively and in detail some critical topics served in concluding future trends in regard to developing indomethacin to be effective chemopreventive and treatment of various cancer cell types without induction of severe cardiovascular system (CVS) complications: *the implication of COX-2/PGE2 in the anticancer activity of COX-inhibitors*, *COX-2-independent mechanisms of anticancer activity of indomethacin*, and *significance of indomethacin's* 

PGE2 implicated in promoting cell proliferation of human esophageal squamous cell carcinoma. The study started with observation of expression and upregulation of c-Myc, an oncogenic transcription factor, and then a link was expected to exist between PGE2 and c-Myc but requires a reliable elucidation. Deeper studies revealed that PGE2 substantially increased the proliferation of cultured esophageal squamous cell carcinoma cells and increased mRNA and protein expression of c-Myc. Moreover, knockdown of c-Myc by RNA interference significantly attenuated PGE2-induced cell proliferation. Furthermore, a mechanistic study described that stability and nuclear accumulation of c-Myc oncogenic protein is attributed PGE2 via phosphorylation on serine 62 that induced by extracellular signal regulated kinase (ERK)-dependent manner and this was confirmed when PGE2 activation of ERK was fully abolished by protein kinase C (PKC) inhibitors. Consistently, PGE2 receptor (EP2) agonist resulted in the same effect on expression of c-Myc as PGE2 and knockdown of EP2 receptor by EP2 small interfering RNA (siRNA) delayed PGE2-induced c-Myc expression to verify the association of PGE2 to c-Myc

sulfonamide/methylsulfone class of compounds.

**COX-inhibitors**

48 Medicinal Chemistry

*anticancer activity over the other NSAIDs and selective COX-2 inhibitors.*

**2. Implication of COX-2/PGE2 in anticancer activity of** 

protein expression in esophageal squamous cancer cell proliferation [18].

Overexpression of COX-2 frequently occurred in head and neck squamous cell carcinoma (HNSCC). COX-2 promotes the release of pro-inflammatory mediator PGE2 which binds to cell surface G-protein coupled receptors EP1–4 to exert its pharmacological effects. Upon studying the biochemical functions of PGE2 and its cell receptors in HNSCC cellular proliferation, it was found that COX-2 and cell receptors EP1, EP2 and EP3 were constitutively expressed in tumoral lesions of HNSCC. An important finding was declared in the study states that small concentration of selective COX-2 inhibitors succeed to suppress PGE2 without inhibition of cell proliferation. However, exogenous addition of EP3-specific agonists with PGE2 induces DNA synthesis in all HNSCC cell lines. Thus, it could be suggested that EP3 receptor subtype of PGE2 should be regarded for future strategies targeting HNSCC prevention [19].

Another study defined a critical mechanism to justify the role of PGE2 in promoting CRC cell division in which prometastatic adaptor protein human enhancer filamentation 1 (HEF1) links between PGE2 and cell cycle machinery in CRC cells. PGE2 induces expression of HEF1 mRNA and protein in CRC. Knockdown of HEF1 suppresses PGE2-induced cell proliferation and cell cycle progression. CRC cells were examined and found that there is 50% elevated levels of HEF1 when compared to normal tissues. Further, HEF1 promotes cell cycle progression of colorectal carcinogenesis via interaction with and activation of cell cycle kinase Aurora A to report that PGE2 is inducer to crucial downstream mediator, HEF1 in colorectal carcinogenesis [20].

Small noncoding RNA, microRNAs (miRNAs) have a key role in stopping the translation and accelerate the degradation of mRNA that regulates the cellular growth and survival through gene suppression. miRNA has a significant contribution in controlling disease progression in pancreatic cancer cells (PaCa). Elevated levels of COX-2 were observed with PGE2 and decrease in miRNA increased the cancer growth and metastases of PaCa. Restoration of miRNA-143 (miR-143) in human PaCa cells reduced COX-2 and inhibited cell proliferation. Mitogen activated kinase (MAPC) was correlated to not detecting miR-143 in some pancreatic cancer cell subtypes to justify the implication of MAPC activation in regulating miR-143 beside COX-2 and PGE2 [21].

α7 Nicotinic acetylcholine receptor (nAChR) protein is significantly biosynthesized via cholinergic signaling in nonsmall cell lung cancer (NSCLC) beside COX-2-driven PGE2. The mechanism by which PGE2 promoted NSCLC cell proliferation over α-7 nAChR induction showed the positive effect of PGE2 on α7 nAChR expression, promotor activity and cell signaling pathways. The association of the two stimulatory factors to cell growth of NSCLC cells was confirmed upon attenuation of PGE2-induced cell proliferation via α7 nAChR siRNA or acetylcholine transferase. Moreover, PGE2 induced α7 nAChR production was blocked by EP4 receptor antagonist and EP4 siRNA. Furthermore, it was recorded that blocking c-Jun, critical transcription factor, activated by c-Jun N-terminal kinase (JNK), phosphoinositol 3-kinase (PI3K) and protein kinase A (PKA), led to abolishing the PGE2-induced α7 nAChR production and consequent cell growth. It is worthy to mention that activation of JNK, PI3K and PKA resulted from acting of PGE2 on EP4 receptor subtype [22].

inhibit the CRC growth. The total proteins from indomethacin-treated and untreated cancer cells were separated by immobilized pH gradient-based two-dimensional gel electrophoresis. The different proteins produced throughout the test were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry. The results revealed that indomethacin induced HCT116 apoptosis and inhibited cell growth by downregulation Wnt1-inducible signaling pathway protein 1, Bcl-2-related

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c-AMP activates PKA in and c-AMP-response element binding (CREB) protein in melanogenesis. CREB plays an important role in binding to the promotor of the microphthalmiaassociated transcription factor (Mitf) gene and consequently activates Mitf gene transcription [40, 41]. Thus, Mitf has a crucial role in transcription of melanogenic genes and activates melanogenic gene transcription of tyrosinase as well. Indomethacin was studied to investigate the effect on melanogenesis in B16F1 melanoma cells. The study resulted in indomethacin inhibited α-melanocyte stimulating hormone that enhances melanin synthesis in melanoma cells., suppressed tyrosinase and Mitf protein levels, reduced tyrosinase promoter activity,

AMP-protein kinase (AMPK) is a key factor of master regulation of cellular energy homeostasis [42]. When AMPK is activated, it induces block of cell cycle and apoptotic cell death in different types of cancer cells including gliomas, the primary tumors of central nervous system [43–47]. The apoptotic death and inhibition of growth of cancer cell actions of AMPK are mediated by one of the signaling pathway in which mammalian target of rapamycin (mTOR) is inhibited. It is worthy to note that mTOR is a catalytic core for formation of two definite complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2) and both are sensitive targets to rapamycin, allosteric inhibitor of the complexes [48]. mTORC1 has a supporting role in protein synthesis and cell proliferation. mTOR performs its biological functions by phosphorylating ribosomal p70S6 kinase and translational repressor 4E-BP1 [48]. AMPK phosphorylates raptor and/or tuberous sclerosis complex-mediated inhibition of mTOR stimulator Ras homolog enriched in brain (RHEB) [49]. Beside the role of mTORC1 in cell proliferation, it causes major and observable negative regulation to intracellular degradation of unnecessary and dysfunctional cellular components through lysosomal machinery which is a kind of cytotoxic mechanism [50]. Indomethacin was reported as growth inhibitor to CRC cells by mTOR inhibition [51]. For the glioma cells U251, indomethacin showed superior *in vitro* antiglioma action and restriction to cell proliferation to the other COX-inhibitors when tested against the same cancer cells. The antiproliferative and proapoptotic actions of indomethacin against U251 was evidenced by G2M cell cycle arrest P21 associated with caspase-3/9 activation, DNA fragmentation that were displayed by indomethacin-treated glioma cells [52]. Also, indomethacin stimulates AMPK phosphorylation in glioma cells and the implication of this pathway in antiglioma effect of indomethacin was confirmed by knockdown of AMPK via RNA interference to alter the AMPK activity and antiglioma actions of indomethacin. Generally, AMPK seemed not sufficient antiproliferative pathway to restrict glioma cell proliferation. Therefore, it is expected that treating glioma cells with indomethacin had a synergistic effect came from its AMPK-dependent and -independent

protein A1 and mitogen-activated protein kinase [24].

lowered mRNA of melanogenic genes, including Mitf gene [23].

pathways in inhibiting the cancer cell growth and proliferation of gliomas.

Chemopreventive effects of indomethacin was observed for 4-hydroxybutyl(butyl) nitrosamine(OH-BBN)-induced urinary bladder cancers in mice. The study came over conducting three experiments in which the indomethacin was continually administered prior to week 1 or following week 13 OH-BBN dosing for 32 weeks, 1 week after intake of OH-BBN at week 13 for 12 weeks and 30 weeks, and 1 week after intake of OH-BBN at week 13 for 61 weeks, respectively. The chemopreventive effect of indomethacin was observably impressive to show development of palpable bladder masses 3% of animals in case of experiment 1, 77% decrease in palpable masses and 82% decrease in palpable and microscopic masses in case of experiment 2, 26% developed palpable mass under treatment of indomethacin and 66% in control group in case of experiment 3 [26].

## **3. COX-2-independent mechanisms of anticancer activity of indomethacin**

Apoptosis is a programmed cell death induced intrinsically by mitochondrial-mediated or extrinsically by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated signals [37]. Caspase activation is generally accompanied by apoptosis that is dependent on mitochondrial mediated or classical extrinsic TRAIL- or death receptor (DR)-mediated signaling [25]. Tse et al. reported the capability of indomethacin to make tumor cells responsive to TRAIL-mediated apoptosis signals through upregulation of TRAIL receptor (DR 5) and down-modulation of survivin, antiapoptic protein [38]. The report provided convincing mechanism to the indomethacin-induced process to overcome TRAIL-resistant melanomas. It is well known that indomethacin enhances mitochondrial oxidative stress and the production of reactive oxygen species (ROS) that modulate mitochondrial-mediated signaling [39]. ROS induces the transcription factor, C/enhancer-binding homologous protein that leads to upregulation of DR 5 on tumor cells. Moreover, ROS has a role in down-modulation of surviving via inhibition of transcription of the known regulator, NF-kB [37]. The report suggests that indomethacin could successfully sensitize TRAIL-resistant melanoma cells.

The ability of indomethacin to work against HCT116 human CRC cells does not express COX was reported using proteomic approach to identify the mechanism by which indomethacin inhibit the CRC growth. The total proteins from indomethacin-treated and untreated cancer cells were separated by immobilized pH gradient-based two-dimensional gel electrophoresis. The different proteins produced throughout the test were identified by peptide mass fingerprint (PMF) based on matrix-assisted laser desorption/ionization time of flight mass spectrometry. The results revealed that indomethacin induced HCT116 apoptosis and inhibited cell growth by downregulation Wnt1-inducible signaling pathway protein 1, Bcl-2-related protein A1 and mitogen-activated protein kinase [24].

α7 Nicotinic acetylcholine receptor (nAChR) protein is significantly biosynthesized via cholinergic signaling in nonsmall cell lung cancer (NSCLC) beside COX-2-driven PGE2. The mechanism by which PGE2 promoted NSCLC cell proliferation over α-7 nAChR induction showed the positive effect of PGE2 on α7 nAChR expression, promotor activity and cell signaling pathways. The association of the two stimulatory factors to cell growth of NSCLC cells was confirmed upon attenuation of PGE2-induced cell proliferation via α7 nAChR siRNA or acetylcholine transferase. Moreover, PGE2 induced α7 nAChR production was blocked by EP4 receptor antagonist and EP4 siRNA. Furthermore, it was recorded that blocking c-Jun, critical transcription factor, activated by c-Jun N-terminal kinase (JNK), phosphoinositol 3-kinase (PI3K) and protein kinase A (PKA), led to abolishing the PGE2-induced α7 nAChR production and consequent cell growth. It is worthy to mention that activation of JNK, PI3K

Chemopreventive effects of indomethacin was observed for 4-hydroxybutyl(butyl) nitrosamine(OH-BBN)-induced urinary bladder cancers in mice. The study came over conducting three experiments in which the indomethacin was continually administered prior to week 1 or following week 13 OH-BBN dosing for 32 weeks, 1 week after intake of OH-BBN at week 13 for 12 weeks and 30 weeks, and 1 week after intake of OH-BBN at week 13 for 61 weeks, respectively. The chemopreventive effect of indomethacin was observably impressive to show development of palpable bladder masses 3% of animals in case of experiment 1, 77% decrease in palpable masses and 82% decrease in palpable and microscopic masses in case of experiment 2, 26% developed palpable mass under treatment of indomethacin and

Apoptosis is a programmed cell death induced intrinsically by mitochondrial-mediated or extrinsically by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated signals [37]. Caspase activation is generally accompanied by apoptosis that is dependent on mitochondrial mediated or classical extrinsic TRAIL- or death receptor (DR)-mediated signaling [25]. Tse et al. reported the capability of indomethacin to make tumor cells responsive to TRAIL-mediated apoptosis signals through upregulation of TRAIL receptor (DR 5) and down-modulation of survivin, antiapoptic protein [38]. The report provided convincing mechanism to the indomethacin-induced process to overcome TRAIL-resistant melanomas. It is well known that indomethacin enhances mitochondrial oxidative stress and the production of reactive oxygen species (ROS) that modulate mitochondrial-mediated signaling [39]. ROS induces the transcription factor, C/enhancer-binding homologous protein that leads to upregulation of DR 5 on tumor cells. Moreover, ROS has a role in down-modulation of surviving via inhibition of transcription of the known regulator, NF-kB [37]. The report suggests

and PKA resulted from acting of PGE2 on EP4 receptor subtype [22].

**3. COX-2-independent mechanisms of anticancer activity of** 

that indomethacin could successfully sensitize TRAIL-resistant melanoma cells.

The ability of indomethacin to work against HCT116 human CRC cells does not express COX was reported using proteomic approach to identify the mechanism by which indomethacin

66% in control group in case of experiment 3 [26].

**indomethacin**

50 Medicinal Chemistry

c-AMP activates PKA in and c-AMP-response element binding (CREB) protein in melanogenesis. CREB plays an important role in binding to the promotor of the microphthalmiaassociated transcription factor (Mitf) gene and consequently activates Mitf gene transcription [40, 41]. Thus, Mitf has a crucial role in transcription of melanogenic genes and activates melanogenic gene transcription of tyrosinase as well. Indomethacin was studied to investigate the effect on melanogenesis in B16F1 melanoma cells. The study resulted in indomethacin inhibited α-melanocyte stimulating hormone that enhances melanin synthesis in melanoma cells., suppressed tyrosinase and Mitf protein levels, reduced tyrosinase promoter activity, lowered mRNA of melanogenic genes, including Mitf gene [23].

AMP-protein kinase (AMPK) is a key factor of master regulation of cellular energy homeostasis [42]. When AMPK is activated, it induces block of cell cycle and apoptotic cell death in different types of cancer cells including gliomas, the primary tumors of central nervous system [43–47]. The apoptotic death and inhibition of growth of cancer cell actions of AMPK are mediated by one of the signaling pathway in which mammalian target of rapamycin (mTOR) is inhibited. It is worthy to note that mTOR is a catalytic core for formation of two definite complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2) and both are sensitive targets to rapamycin, allosteric inhibitor of the complexes [48]. mTORC1 has a supporting role in protein synthesis and cell proliferation. mTOR performs its biological functions by phosphorylating ribosomal p70S6 kinase and translational repressor 4E-BP1 [48]. AMPK phosphorylates raptor and/or tuberous sclerosis complex-mediated inhibition of mTOR stimulator Ras homolog enriched in brain (RHEB) [49]. Beside the role of mTORC1 in cell proliferation, it causes major and observable negative regulation to intracellular degradation of unnecessary and dysfunctional cellular components through lysosomal machinery which is a kind of cytotoxic mechanism [50]. Indomethacin was reported as growth inhibitor to CRC cells by mTOR inhibition [51]. For the glioma cells U251, indomethacin showed superior *in vitro* antiglioma action and restriction to cell proliferation to the other COX-inhibitors when tested against the same cancer cells. The antiproliferative and proapoptotic actions of indomethacin against U251 was evidenced by G2M cell cycle arrest P21 associated with caspase-3/9 activation, DNA fragmentation that were displayed by indomethacin-treated glioma cells [52]. Also, indomethacin stimulates AMPK phosphorylation in glioma cells and the implication of this pathway in antiglioma effect of indomethacin was confirmed by knockdown of AMPK via RNA interference to alter the AMPK activity and antiglioma actions of indomethacin. Generally, AMPK seemed not sufficient antiproliferative pathway to restrict glioma cell proliferation. Therefore, it is expected that treating glioma cells with indomethacin had a synergistic effect came from its AMPK-dependent and -independent pathways in inhibiting the cancer cell growth and proliferation of gliomas.

## **4. Significance of indomethacin and its developed structures' anticancer activity over the other NSAIDs and selective COX-2 inhibitors**

According to the literature scan had been done on indomethacin as nonselective COX-inhibitor and antiproliferative agent, it could be observed that indomethacin is the most NSAID that attracted the interest of researchers to study, investigate, identify more about the definite mechanisms and cellular signaling pathways involved in the antiproliferative and apoptotic effects of indomethacin. This might be attributed to two apparent points: one is the observable inhibition of cell growth, reduction of tumor size and implication in the programmed cell death of multiple tumor cell types including glioma and glioblastomas that require lipophilicity for cell penetration. The second is that indomethacin exhibited its anticancer activity against wide variety of cancer cell types by COX-2/PGE2-dependent and -independent mechanisms. This adds great advantage to indomethacin over the other nonselective COX-inhibitors because in that way, indomethacin has dual actions by which it could exert its cytotoxic activity effectively.

drug's selective inhibition of human COX-2 but impressively the inhibitor did not show successful recovery even upon dialysis but the inhibitor is freed to inhibit another enzyme under the effect of denaturation to confirm that the tight binding of inhibitor to the enzyme was not

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The detrimental differences in regard to the kinetic profile of indomethacin, nonselective COX-inhibitor and DuP 697, selective COX-2 inhibitor for inhibition of COX-2 raised my concern with that emerge of developed selective COX-2 inhibitors based on indomethacin would

**i.** *Is the cardiovascular toxicity of selective COX-2 inhibitors pharmacological class effect of chemical class effect?* The change of chemical class of selective COX-2 inhibitors from the traditional diaryl heterocycle sulfonamide/methylsulfone to indomethacin-based structures and identifying the kinetic profile of the new class of selective inhibitors would provide a strong evidence on the real reasons of cardiovascular problems after administration of selective COX-2 inhibitors to discover whether it lies behind the kinetic mode of enzyme inhibition which depends on the chemical structure or it lies behind the selective action of the drug against COX-2. In case of similar kinetic profile for the generated new inhibitors to the traditional class and no recovery to the inhibitor is shown, so, it will imply that it is a general feature to the selective inhibitors whatever is the chemical structure. It could be drawn that the inability of selective inhibitors to get recovered from COX-2 enzyme might be a significant reason to the CVS complications of the inhibitors. In case, the new structures saved the kinetic profile of the original lead compound, indomethacin, so, it would be worth it monitoring the development of CVS problems after long-term administration of the newly developed indomethacin-based compounds. There are two reports based on epidemiological studies stated clearly that prolonged use of NSAIDs is associated by small increase in CVS risk [56, 57]. This attracted our attention to comment on that this happens though NSAIDs inhibit COX-2 with the same efficacy as selective

based on formation of covalent bond [35].

definitely help us answer two important questions:

Regarding the selective COX-2 inhibitors, the prophylactic actions of celecoxib against recurrence of colon polyps is defined by researchers as dramatic to indicate the capability of celecoxib and other COX-2 inhibitors to block the cancer cell growth and metastases as well. One serious complication that is developed upon long-term therapy of celecoxib that limits its use as chemopreventive therapy for CRC is the cardiovascular toxicity that results from critical reduction in PGE2 and prostacyclin (PGI2) production. Those types of prostaglandins are COX2-dependent product responsible for regulating vascular tone and atherosclerosis [53]. The problem is absolutely the same as all the selective COX-2 inhibitors since they share the same pharmacological action in which production of prostaglandins is significantly diminished.

Some differential points related to the kinetic profile of both indomethacin [34] and selective COX-2 inhibitors [35] were worth it stopping at to help us draw a future plan to develop indomethacin's chemical structure in a way to enhance COX-2 inhibition activity like selective COX-2 inhibitors and be devoid of cardiovascular complications as well. Nonselective inhibitors including indomethacin perform its inhibition action against the enzyme through 2-step inhibition mechanism involving slow and time-dependent step due to tight reversible binding to the enzyme to be considered as functionally irreversible. But selective COX-2 inhibitors inhibit the enzyme through 3-step inhibition mechanism involving time-dependent step that represents the tightly bound complex of inhibited enzyme. The observation that should be highlighted for both types of COX-inhibitors while monitoring the kinetic model of inhibition mechanism is that indomethacin carboxylic acid is not essential for the tight binding and time-dependent step of enzyme inhibition because the esterified counterpart did not abolish this step or even reduce the tightness of binding to human COX-2 and the formation of the complex maintained functionally irreversible [54, 55]. Further, indomethacin recovered intact after prolonged time of incubation with the enzyme, this suggests that enzyme inhibition came over conformational change not covalent bond formation [34]. On the other hand, DuP 697, selective COX-2 inhibitor showed the same time-dependent step that was responsible for drug's selective inhibition of human COX-2 but impressively the inhibitor did not show successful recovery even upon dialysis but the inhibitor is freed to inhibit another enzyme under the effect of denaturation to confirm that the tight binding of inhibitor to the enzyme was not based on formation of covalent bond [35].

**4. Significance of indomethacin and its developed structures' anticancer activity over the other NSAIDs and selective COX-2** 

According to the literature scan had been done on indomethacin as nonselective COX-inhibitor and antiproliferative agent, it could be observed that indomethacin is the most NSAID that attracted the interest of researchers to study, investigate, identify more about the definite mechanisms and cellular signaling pathways involved in the antiproliferative and apoptotic effects of indomethacin. This might be attributed to two apparent points: one is the observable inhibition of cell growth, reduction of tumor size and implication in the programmed cell death of multiple tumor cell types including glioma and glioblastomas that require lipophilicity for cell penetration. The second is that indomethacin exhibited its anticancer activity against wide variety of cancer cell types by COX-2/PGE2-dependent and -independent mechanisms. This adds great advantage to indomethacin over the other nonselective COX-inhibitors because in that way, indomethacin has dual actions by which it could exert its cytotoxic activity effectively. Regarding the selective COX-2 inhibitors, the prophylactic actions of celecoxib against recurrence of colon polyps is defined by researchers as dramatic to indicate the capability of celecoxib and other COX-2 inhibitors to block the cancer cell growth and metastases as well. One serious complication that is developed upon long-term therapy of celecoxib that limits its use as chemopreventive therapy for CRC is the cardiovascular toxicity that results from critical reduction in PGE2 and prostacyclin (PGI2) production. Those types of prostaglandins are COX2-dependent product responsible for regulating vascular tone and atherosclerosis [53]. The problem is absolutely the same as all the selective COX-2 inhibitors since they share the same pharmacological action in which production of prostaglandins is significantly

Some differential points related to the kinetic profile of both indomethacin [34] and selective COX-2 inhibitors [35] were worth it stopping at to help us draw a future plan to develop indomethacin's chemical structure in a way to enhance COX-2 inhibition activity like selective COX-2 inhibitors and be devoid of cardiovascular complications as well. Nonselective inhibitors including indomethacin perform its inhibition action against the enzyme through 2-step inhibition mechanism involving slow and time-dependent step due to tight reversible binding to the enzyme to be considered as functionally irreversible. But selective COX-2 inhibitors inhibit the enzyme through 3-step inhibition mechanism involving time-dependent step that represents the tightly bound complex of inhibited enzyme. The observation that should be highlighted for both types of COX-inhibitors while monitoring the kinetic model of inhibition mechanism is that indomethacin carboxylic acid is not essential for the tight binding and time-dependent step of enzyme inhibition because the esterified counterpart did not abolish this step or even reduce the tightness of binding to human COX-2 and the formation of the complex maintained functionally irreversible [54, 55]. Further, indomethacin recovered intact after prolonged time of incubation with the enzyme, this suggests that enzyme inhibition came over conformational change not covalent bond formation [34]. On the other hand, DuP 697, selective COX-2 inhibitor showed the same time-dependent step that was responsible for

**inhibitors**

52 Medicinal Chemistry

diminished.

The detrimental differences in regard to the kinetic profile of indomethacin, nonselective COX-inhibitor and DuP 697, selective COX-2 inhibitor for inhibition of COX-2 raised my concern with that emerge of developed selective COX-2 inhibitors based on indomethacin would definitely help us answer two important questions:

**i.** *Is the cardiovascular toxicity of selective COX-2 inhibitors pharmacological class effect of chemical class effect?* The change of chemical class of selective COX-2 inhibitors from the traditional diaryl heterocycle sulfonamide/methylsulfone to indomethacin-based structures and identifying the kinetic profile of the new class of selective inhibitors would provide a strong evidence on the real reasons of cardiovascular problems after administration of selective COX-2 inhibitors to discover whether it lies behind the kinetic mode of enzyme inhibition which depends on the chemical structure or it lies behind the selective action of the drug against COX-2. In case of similar kinetic profile for the generated new inhibitors to the traditional class and no recovery to the inhibitor is shown, so, it will imply that it is a general feature to the selective inhibitors whatever is the chemical structure. It could be drawn that the inability of selective inhibitors to get recovered from COX-2 enzyme might be a significant reason to the CVS complications of the inhibitors. In case, the new structures saved the kinetic profile of the original lead compound, indomethacin, so, it would be worth it monitoring the development of CVS problems after long-term administration of the newly developed indomethacin-based compounds. There are two reports based on epidemiological studies stated clearly that prolonged use of NSAIDs is associated by small increase in CVS risk [56, 57]. This attracted our attention to comment on that this happens though NSAIDs inhibit COX-2 with the same efficacy as selective COX-2 inhibitors. Thus, it is suggested that chemical structure and/or binding mode most likely play a significant role in determining the kinetic mode of enzyme inhibition. But, we are still in serious need to an evidence results from experimental studies to assertively answer the above question.

**ii.** *Would the anticancer activity of indomethacin be enhanced with the new indomethacin-based compounds?* Improvement of the selective inhibition of COX-2 in comparison to indomethacin is supposed to potentiate the antiproliferative and apoptotic activities upon enhanced diminishing to COX-2/PGE2, combining this important signaling pathway with the COX-2-independent mechanisms of anticancer activities that previously described in this book chapter for indomethacin in Section 2 "COX-2-independent mechanisms of anticancer activity of indomethacin".

Several attempts for generation of indomethacin-based analogs of selective COX-2 inhibition activity [58–60], but the publication that I had to put it in focus in this regard is that described the design and synthesis of indomethacin-based analogs of potentially selective COX-2 inhibition activity and observed diminishing to PGE2 [61]. The successful generation of developed indomethacin structures with selective COX-2 inhibition activity was iteratively reported in the literature but picking this publication to comment on among the others was based on the obvious selectivity index of the generated analogs that excelled celecoxib, dramatic lowering to plasma levels of PGE2 when compared to indomethacin, the innovative perspective upon

which the design and modification of the analogs are designed, and above all of this the biological profiling through multiple *in vitro* and *in vivo* tests that done for the analogs to impress the discovery of interesting tetrahydrocarbazole candidates. Accordingly, the newly selective COX-2 inhibitors worth it promoting to study how far it is implicated in CVS toxicity upon

**Figure 3.** Schematic representation of SC-558 (COX-2 selective inhibitor) binding to COX-2 (a) and indomethacin

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55

The new tetrahydrocarbazole selective COX-2 inhibitors **(1)** and **(2)** (**Figure 2**) generated were based on enlarging the size of indomethacin indole ring to occupy the wider catalytic pocket of COX-2 than COX-2 by 25% [62] and reducing the opportunity of the ringextended candidates to interact with COX-1 to raise the selectivity. Introduction of methyl sulfonyl group to replace methoxy group of indomethacin at position 6 to enhance the interaction of the designed inhibitor **(2)** (**Figure 3**) with the polar side pocket of COX-2 (selective pocket) that is critical for COX-2 inhibition activity [63]. Deletion of carboxylic acid from the new candidates **(1)** and **(2)** reduce the possibility of the inhibitor to interact with COX-1 via formation of salt bridge with Arg120 amino acid (**Figure 3**) [64] that is critical for conformational change and inhibition of the isoenzyme. Impressively, the biological results throughout *in vitro* testing represented by enzymatic assays against human COX-1 (hCOX-1) and hCOX-2 and *in vivo* testing represented by % inhibition of plasma PGE2 and others revealed the successful verification of the proposed hypothesis suggested to enhance the COX-2 inhibition selectivity. Methoxy derivative **(1)** (**Figure 2**) gave selectivity index against COX-2 (207.2165) to excel both that of indomethacin (0.98859) and celecoxib (333.3333) standard drugs. For the methylsulfone derivative **(2)** (**Figure 2**), it excelled the standard materials at much higher value (452.1739). Moreover, the diminishing of plasma levels of PGE2 was very observable in comparison to indomethacin (98.29%) and celecoxib (77.25%) (**Figure 2**). Thus, kinetic profile of the enzyme inhibition of the new candidate **(2)** in the near future would answer the questions described in this section on the book and eventually be able to judge on the chemopreventive of the new selective COX-2 inhibitor

long-term therapy against either cancer or inflammatory diseases.

and antiproliferative activity as well.

(nonselective inhibitor) (b).

**Figure 2.** Biological data of the new indomethacin-based selective COX-2 inhibitors 1 and 2 in comparison with indomethacin and celecoxib.

COX-2 inhibitors. Thus, it is suggested that chemical structure and/or binding mode most likely play a significant role in determining the kinetic mode of enzyme inhibition. But, we are still in serious need to an evidence results from experimental studies to assertively

**ii.** *Would the anticancer activity of indomethacin be enhanced with the new indomethacin-based compounds?* Improvement of the selective inhibition of COX-2 in comparison to indomethacin is supposed to potentiate the antiproliferative and apoptotic activities upon enhanced diminishing to COX-2/PGE2, combining this important signaling pathway with the COX-2-independent mechanisms of anticancer activities that previously described in this book chapter for indomethacin in Section 2 "COX-2-independent mechanisms of

Several attempts for generation of indomethacin-based analogs of selective COX-2 inhibition activity [58–60], but the publication that I had to put it in focus in this regard is that described the design and synthesis of indomethacin-based analogs of potentially selective COX-2 inhibition activity and observed diminishing to PGE2 [61]. The successful generation of developed indomethacin structures with selective COX-2 inhibition activity was iteratively reported in the literature but picking this publication to comment on among the others was based on the obvious selectivity index of the generated analogs that excelled celecoxib, dramatic lowering to plasma levels of PGE2 when compared to indomethacin, the innovative perspective upon

**Figure 2.** Biological data of the new indomethacin-based selective COX-2 inhibitors 1 and 2 in comparison with

answer the above question.

54 Medicinal Chemistry

indomethacin and celecoxib.

anticancer activity of indomethacin".

**Figure 3.** Schematic representation of SC-558 (COX-2 selective inhibitor) binding to COX-2 (a) and indomethacin (nonselective inhibitor) (b).

which the design and modification of the analogs are designed, and above all of this the biological profiling through multiple *in vitro* and *in vivo* tests that done for the analogs to impress the discovery of interesting tetrahydrocarbazole candidates. Accordingly, the newly selective COX-2 inhibitors worth it promoting to study how far it is implicated in CVS toxicity upon long-term therapy against either cancer or inflammatory diseases.

The new tetrahydrocarbazole selective COX-2 inhibitors **(1)** and **(2)** (**Figure 2**) generated were based on enlarging the size of indomethacin indole ring to occupy the wider catalytic pocket of COX-2 than COX-2 by 25% [62] and reducing the opportunity of the ringextended candidates to interact with COX-1 to raise the selectivity. Introduction of methyl sulfonyl group to replace methoxy group of indomethacin at position 6 to enhance the interaction of the designed inhibitor **(2)** (**Figure 3**) with the polar side pocket of COX-2 (selective pocket) that is critical for COX-2 inhibition activity [63]. Deletion of carboxylic acid from the new candidates **(1)** and **(2)** reduce the possibility of the inhibitor to interact with COX-1 via formation of salt bridge with Arg120 amino acid (**Figure 3**) [64] that is critical for conformational change and inhibition of the isoenzyme. Impressively, the biological results throughout *in vitro* testing represented by enzymatic assays against human COX-1 (hCOX-1) and hCOX-2 and *in vivo* testing represented by % inhibition of plasma PGE2 and others revealed the successful verification of the proposed hypothesis suggested to enhance the COX-2 inhibition selectivity. Methoxy derivative **(1)** (**Figure 2**) gave selectivity index against COX-2 (207.2165) to excel both that of indomethacin (0.98859) and celecoxib (333.3333) standard drugs. For the methylsulfone derivative **(2)** (**Figure 2**), it excelled the standard materials at much higher value (452.1739). Moreover, the diminishing of plasma levels of PGE2 was very observable in comparison to indomethacin (98.29%) and celecoxib (77.25%) (**Figure 2**). Thus, kinetic profile of the enzyme inhibition of the new candidate **(2)** in the near future would answer the questions described in this section on the book and eventually be able to judge on the chemopreventive of the new selective COX-2 inhibitor and antiproliferative activity as well.

## **5. Conclusions**

Generation of indomethacin-based analogs to indomethacin aiming at enhancing the selective COX-2 inhibition would definitely help us answer an important question concerning the real reason of cardiovascular toxicity of selective COX-2 inhibitors, whether it is pharmacological class effect or chemical class effect. Moreover, enhancing the selectivity of indomethacin against COX-2 among the other NSAIDs providing a candidate privileged with potential antiinflammatory activity devoid of gastrointestinal side effects and what is more important is obtaining newly developed structure carries effective antiproliferative and apoptotic activity standing for the dual actions reported for indomethacin as a lead compound based on that it performs its anticancer activity by both COX-2-dependent and COX-2-independent mechanisms. Further, the CVS toxicity is expected to be minimized upon enhancing the selective COX-2 inhibition of indomethacin due to observation that there might be a difference in the kinetic mode of enzyme inhibition between diaryl heterocycle sulfonamide/methylsulfone chemical class of selective COX-2 inhibitors and the new indomethacin-based chemical class of compounds that may permit successful recovery of the new inhibitors from the enzyme after long incubation period.

PI3K phosphoinositol 3-kinase

OH-BBN 4-hydroxybutyl(butyl)nitrosamine

CREB c-AMP-response element binding AMPK adenosine monophosphate kinase mTOR mammalian target of rapamycin RHEB Ras homolog enriched in brain

TRAIL tumor necrosis factor-related apoptosis-inducing ligand

Indomethacin from Anti-Inflammatory to Anticancer Agent

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57

Address all correspondence to: shaymaa.kassab@pharm.dmu.edu.eg

tanoid biosynthesis. Chemical Reviews. 2011;**111**:5821-5865

Faulty of Pharmacy, Pharmaceutical Chemistry Department, Damanhour University,

[1] Suleyman H, Albayrak A, Bilici M, Cadirci E, Halici Z. Different mechanisms in formation and prevention of indomethacin-induced gastric ulcers. Inflammation. 2010;**33**:224-234

[2] Guo YC, Chang CM, Hsu WL, Chiu SJ, Tsai YT, Chou YH, Hou MF, Wang JY, Lee MH, Tsai KL, Chang WC. Indomethacin inhibits cancer cell migration via attenuation of cel-

[3] Smith WL, Urade Y, Jakobsson PJ. Enzymes of the cyclooxygenase pathways of pros-

[4] Zarghi A, Arfaei S. Selective COX-2 inhibitors: A review of their structure-activity rela-

[5] Peesa JP, Yalavarthi PR, Rasheed A, Mandava VBR. A perspective review on role of novel NSAID prodrugs in the management of acute inflammation. Journal of Acute Disease.

lular calcium mobilization. Molecules (Basel, Switzerland). 2013;**18**:6584-6596

tionships. Iranian Journal of Pharmaceutical research: IJPR. 2011;**10**:655-683

PKA protein kinase A

DR death receptor

**Author details**

**References**

Shaymaa Emam Kassab

2016;**5**:364-381

Damanhour, El-Buhaira, Egypt

ROS reactive oxygen species PMF protein mass fingerprint

## **Nomenclature**



## **Author details**

**5. Conclusions**

56 Medicinal Chemistry

after long incubation period.

COX cyclooxygenase PG prostaglandin

CVS cardiovascular

PKC protein kinase C

MVD microvascular d

miRNA microRNA

siRNA small interfering RNA

CRC colorectal carcinoma

NSAID nonsteroidal anti-inflammatory drug

ERK extracellular signal regulated kinase

HNSCC head and neck small cell carcinoma

HEF1 human enhancer filamentation 1

MAPC mitogen activated kinase NSCLC nonsmall cell lung cancer JNK c-Jun N-terminal kinase

**Nomenclature**

Generation of indomethacin-based analogs to indomethacin aiming at enhancing the selective COX-2 inhibition would definitely help us answer an important question concerning the real reason of cardiovascular toxicity of selective COX-2 inhibitors, whether it is pharmacological class effect or chemical class effect. Moreover, enhancing the selectivity of indomethacin against COX-2 among the other NSAIDs providing a candidate privileged with potential antiinflammatory activity devoid of gastrointestinal side effects and what is more important is obtaining newly developed structure carries effective antiproliferative and apoptotic activity standing for the dual actions reported for indomethacin as a lead compound based on that it performs its anticancer activity by both COX-2-dependent and COX-2-independent mechanisms. Further, the CVS toxicity is expected to be minimized upon enhancing the selective COX-2 inhibition of indomethacin due to observation that there might be a difference in the kinetic mode of enzyme inhibition between diaryl heterocycle sulfonamide/methylsulfone chemical class of selective COX-2 inhibitors and the new indomethacin-based chemical class of compounds that may permit successful recovery of the new inhibitors from the enzyme

Shaymaa Emam Kassab

Address all correspondence to: shaymaa.kassab@pharm.dmu.edu.eg

Faulty of Pharmacy, Pharmaceutical Chemistry Department, Damanhour University, Damanhour, El-Buhaira, Egypt

## **References**


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**Chapter 5**

**Provisional chapter**

**1,4-Benzodiazepines and New Derivatives: Description,**

Benzodiazepines are widely used drugs for several indications. This study provides, on the other hand, a global vision of the family starting for their fortuitous discovery, the synthesis of their derivatives, their mechanism of action widely known nowadays, the actual classification according to the chemical structure and pharmacokinetic properties, and their uses and indications, the traditional and the new ones. On the other hand,the study is focused in the mainly problems of benzodiazepines, depedence, and tolerance, many times led by a misuse of the patient, wrong prescriptions, or extended treatments. A withdrawal program is proposed that includes the important factors or criteria to success, with a slow and gradual reduction of these drugs, avoiding relapse or severe adverse effects. New lines of research related to benzodiazepines are taken into account, which not only include the new therapeutic uses but also the adverse effects in short and long term. They are also analyzed the new discoveries concerning the nonbenzodiazepine

drugs due to the close relation they have with benzodiazepines.

**Keywords:** benzodiazepines, withdrawal program, nonbenzodiazepine drugs,

Many of the drugs that had represented a great advance in many therapeutic approaches were not a result of a rational design but of a consequence of casual observations, fortuitous discoveries, or serendipity. Way back then, a rational design did not guarantee the exit because the knowledge of the biological systems was not clear or complete. That happened in the beginning of the past century, and many of the drugs used nowadays come from this type of

**1,4-Benzodiazepines and New Derivatives: Description,** 

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.79879

**Analysis, and Organic Synthesis**

**Analysis, and Organic Synthesis**

Elisabet Batlle, Enric Lizano, Miquel Viñas and

Elisabet Batlle, Enric Lizano, Miquel Viñas and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79879

biological activities, side effects

**1. Discovery and history**

Maria Dolors Pujol

Maria Dolors Pujol

**Abstract**


#### **1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis 1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis**

DOI: 10.5772/intechopen.79879

Elisabet Batlle, Enric Lizano, Miquel Viñas and Maria Dolors Pujol Elisabet Batlle, Enric Lizano, Miquel Viñas and Maria Dolors Pujol

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79879

#### **Abstract**

[55] Kalgutkar AS, Crews BC, Rowlinson SW, Marnett AB, Kozak KR, Remmel RP, Marnett LJ. Biochemically based design of cyclooxygenase-2 (COX-2) inhibitors: Facile conversion of nonsteroidal antiinflammatory drugs to potent and highly selective COX-2 inhibitors. Proceedings of the National Academy of Sciences of the United States of America.

[56] Hippisley-Cox J, Coupland C. Risk of myocardial infarction in patients taking cyclooxygenase-2 inhibitors or conventional non-steroidal anti-inflammatory drugs: Population based nested case-control analysis. BMJ (Clinical research ed.). 2005;**330**:1366 [57] Garcia Rodriguez LA, Gonzalez-Perez A. Long-term use of non-steroidal anti-inflammatory drugs and the risk of myocardial infarction in the general population. BMC

[58] Blobaum AL, Uddin MJ, Felts AS, Crews BC, Rouzer CA, Marnett LJ. The 2′-Trifluoromethyl analogue of indomethacin is a potent and selective COX-2 inhibitor. ACS Medicinal

[59] Lau CK, Black WC, Belley M, Chan C, Charleson S, Denis D, Gauthier JY, Gordon R, Guay D, Hamel P, Kargman S, Leblanc Y, Mancini J, Ouellet M, Percival D, Prasit P, Roy P, Skorey K, Tagari P, Vickers P, Wong E. From indomethacin to a selective COX-2 inhibitor. Development of indolalkanoic acids as potent and selective cyclooxygenase-2

[60] Estevao MS, Carvalho LC, Freitas M, Gomes A, Viegas A, Manso J, Erhardt S, Fernandes E, Cabrita EJ, Marques MM. Indole based cyclooxygenase inhibitors: Synthesis, biological evaluation, docking and NMR screening. European Journal of Medicinal Chemistry.

[61] Kassab SE, Khedr MA, Ali HI, Abdalla MM. Discovery of new indomethacin-based analogs with potentially selective cyclooxygenase-2 inhibition and observed diminishing to

[62] Luong C, Miller A, Barnett J, Chow J, Ramesha C, Browner MF. Flexibility of the NSAID binding site in the structure of human cyclooxygenase-2. Nature Structural Biology. 1996;

[63] Dwivedi AK, Gurjar V, Kumar S, Singh N. Molecular basis for nonspecificity of nonsteroidal anti-inflammatory drugs (NSAIDs). Drug Discovery Today. 2015;**20**:863-873 [64] Estevão MS, Carvalho LCR, Freitas M, Gomes A, Viegas A, Manso J, Erhardt S, Fernandes E, Cabrita EJ, Marques MMB. Indole based cyclooxygenase inhibitors: Synthesis, biological evaluation, docking and NMR screening. European Journal of Medicinal Chemistry.

PGE2 activities. European Journal of Medicinal Chemistry. 2017;**141**:306-321

inhibitors. Advances in Experimental Medicine and Biology. 1997;**407**:73-78

2000;**97**:925-930

62 Medicinal Chemistry

Medicine. 2005;**3**:17

2012;**54**:823-833

**3**:927-933

2012;**54**:823-833

Chemistry Letters. 2013;**4**:486-490

Benzodiazepines are widely used drugs for several indications. This study provides, on the other hand, a global vision of the family starting for their fortuitous discovery, the synthesis of their derivatives, their mechanism of action widely known nowadays, the actual classification according to the chemical structure and pharmacokinetic properties, and their uses and indications, the traditional and the new ones. On the other hand,the study is focused in the mainly problems of benzodiazepines, depedence, and tolerance, many times led by a misuse of the patient, wrong prescriptions, or extended treatments. A withdrawal program is proposed that includes the important factors or criteria to success, with a slow and gradual reduction of these drugs, avoiding relapse or severe adverse effects. New lines of research related to benzodiazepines are taken into account, which not only include the new therapeutic uses but also the adverse effects in short and long term. They are also analyzed the new discoveries concerning the nonbenzodiazepine drugs due to the close relation they have with benzodiazepines.

**Keywords:** benzodiazepines, withdrawal program, nonbenzodiazepine drugs, biological activities, side effects

#### **1. Discovery and history**

Many of the drugs that had represented a great advance in many therapeutic approaches were not a result of a rational design but of a consequence of casual observations, fortuitous discoveries, or serendipity. Way back then, a rational design did not guarantee the exit because the knowledge of the biological systems was not clear or complete. That happened in the beginning of the past century, and many of the drugs used nowadays come from this type of

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

discovery, from the curiosity of many investigators that decided to study the reason why they were not achieving their goals.

Discovery starts with chemist Leo Sternbach and his research group, working in the Hoffmann-La Roche laboratories in Nutley, New Jersey. They were trying to find new tranquilizers, but due to the limited knowledge of the processes occurring in the brain, they were taking an empirical approach: to search for a new class of drugs purely guided by modifications in the known chemical synthesis [1]. In 1957, they serendipitously identified the first benzodiazepine (BZD), *chlordiazepoxide*, while they were studying the activity of *quinazoline oxide*. They saw that the compound obtained was not a quinazoline-*N*<sup>3</sup> -oxide but a benzodiazepine-*N*<sup>4</sup> -oxide. With a posterior investigation, Sternbach himself managed to explain what happened [2].

By 1960, Hoffmann-La Roche introduced the *chlordiazepoxide* in clinical treatment under the brand name Librium®, and it pursued molecular modifications to improve its activity. By the time of its introduction, it was felt that an explanation of the BZDs mechanism of action might be really helpful to understand the basis of anxiety. *Diazepam* (Valium®) followed in 1963, which was considered for a long time as head of the family.

An important improvement was their lack of respiratory depression, a safety concern they had with barbiturates [3].

Medical professionals accepted benzodiazepines enthusiastically at first, increasing their popularity and patient demand. BZDs were prescribed frequently and often long term for various conditions. Soon they became the pharmacological family *par excellence* in the treatment of anxiety disorders and so initiating "the benzodiazepine saga" [4].

It took 15 years for the researchers to associate benzodiazepines and their effect with their high-affinity receptor complex as a mechanism of action. They did it in 1977, and it was the major turning point in the research [2].

> It is important to note that the variation of the dose changes the effects: a hypnotic BZD administered in low doses produces anxiety-relieving effects, whereas a BZD marketed as an

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

http://dx.doi.org/10.5772/intechopen.79879

65

To understand their mechanism of action, it is necessary to know the physiology and function of the *gamma*-aminobutyric acid (GABA) neurotransmitter. They are neurotransmitters in the central nervous system (CNS) that increment or decrease the excitability of neurons and so regulate the brain activity. GABA functions as the principal inhibitory neurotransmitter, and

**The GABAA receptor** is a protein complex located in the synapses of neurons. It belongs to a family of receptors associated to ionic channels, formed by combinations of protein subunits with high selectivity for chloride ion (Cl−). They conduct chloride ions across neuronal cell membranes. The receptor is formed by five subunits arranged around the central chloride: two alphas, two betas, and one gamma. There are also multiple isoforms of each subunit: six

antianxiety drug at higher doses induces sleep.

**Table 1.** Principal actions and uses of BZDs [5].

Anxiolytic Anxiety and panic/phobias, alcohol withdrawal

Amnesic Intraoperatively or pre-surgery medication

Muscle relaxant Muscle spasms, spasticity caused by CNS pathologies

Anticonvulsive Attacks caused by drug intoxications, some forms of epilepsy

**1.2. Mechanism of action**

**Figure 1.** BZD structure.

**Action Clinical uses**

Hypnotic Insomnia

BZDs potentiate that function.

#### **1.1. Benzodiazepines (BZDs)**

Benzodiazepines are a structural class of compounds that are used as hypnotics, anxiolytics, anticonvulsants, and muscle relaxants. Their core chemical structure is formed by the fusion of a benzene ring and a diazepine ring (**Figure 1**). Different compounds have different side groups attached to this central structure in position 1, 2, 5, or 7. The different side groups affect the binding of the molecule to the GABAA receptor and so can modulate the pharmacological properties, the potency of the effect, and the pharmacokinetic conditions (duration of the effect, distribution, etc.).

BZDs have proven to be excellent drugs for the known pharmacological properties they present, as shown in **Table 1**.

In humans, benzodiazepines are also recognized to have anterograde amnestic effects, providing amnesia for events that occur subsequent to the administration of the drug [6]. Another important use they have is in alcohol withdrawal syndrome (AWS). They are generally considered to provide no analgesia.

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis http://dx.doi.org/10.5772/intechopen.79879 65

**Figure 1.** BZD structure.

discovery, from the curiosity of many investigators that decided to study the reason why they

Discovery starts with chemist Leo Sternbach and his research group, working in the Hoffmann-La Roche laboratories in Nutley, New Jersey. They were trying to find new tranquilizers, but due to the limited knowledge of the processes occurring in the brain, they were taking an empirical approach: to search for a new class of drugs purely guided by modifications in the known chemical synthesis [1]. In 1957, they serendipitously identified the first benzodiazepine (BZD), *chlordiazepoxide*, while they were studying the activity of *quinazoline oxide*. They saw that

By 1960, Hoffmann-La Roche introduced the *chlordiazepoxide* in clinical treatment under the brand name Librium®, and it pursued molecular modifications to improve its activity. By the time of its introduction, it was felt that an explanation of the BZDs mechanism of action might be really helpful to understand the basis of anxiety. *Diazepam* (Valium®) followed in 1963,

An important improvement was their lack of respiratory depression, a safety concern they

Medical professionals accepted benzodiazepines enthusiastically at first, increasing their popularity and patient demand. BZDs were prescribed frequently and often long term for various conditions. Soon they became the pharmacological family *par excellence* in the treat-

It took 15 years for the researchers to associate benzodiazepines and their effect with their high-affinity receptor complex as a mechanism of action. They did it in 1977, and it was the

Benzodiazepines are a structural class of compounds that are used as hypnotics, anxiolytics, anticonvulsants, and muscle relaxants. Their core chemical structure is formed by the fusion of a benzene ring and a diazepine ring (**Figure 1**). Different compounds have different side groups attached to this central structure in position 1, 2, 5, or 7. The different side groups affect the binding of the molecule to the GABAA receptor and so can modulate the pharmacological properties, the potency of the effect, and the pharmacokinetic conditions (duration of

BZDs have proven to be excellent drugs for the known pharmacological properties they pres-

In humans, benzodiazepines are also recognized to have anterograde amnestic effects, providing amnesia for events that occur subsequent to the administration of the drug [6]. Another important use they have is in alcohol withdrawal syndrome (AWS). They are generally con-

posterior investigation, Sternbach himself managed to explain what happened [2].

ment of anxiety disorders and so initiating "the benzodiazepine saga" [4].



were not achieving their goals.

64 Medicinal Chemistry

had with barbiturates [3].

major turning point in the research [2].

**1.1. Benzodiazepines (BZDs)**

the effect, distribution, etc.).

sidered to provide no analgesia.

ent, as shown in **Table 1**.

the compound obtained was not a quinazoline-*N*<sup>3</sup>

which was considered for a long time as head of the family.


**Table 1.** Principal actions and uses of BZDs [5].

It is important to note that the variation of the dose changes the effects: a hypnotic BZD administered in low doses produces anxiety-relieving effects, whereas a BZD marketed as an antianxiety drug at higher doses induces sleep.

#### **1.2. Mechanism of action**

To understand their mechanism of action, it is necessary to know the physiology and function of the *gamma*-aminobutyric acid (GABA) neurotransmitter. They are neurotransmitters in the central nervous system (CNS) that increment or decrease the excitability of neurons and so regulate the brain activity. GABA functions as the principal inhibitory neurotransmitter, and BZDs potentiate that function.

**The GABAA receptor** is a protein complex located in the synapses of neurons. It belongs to a family of receptors associated to ionic channels, formed by combinations of protein subunits with high selectivity for chloride ion (Cl−). They conduct chloride ions across neuronal cell membranes. The receptor is formed by five subunits arranged around the central chloride: two alphas, two betas, and one gamma. There are also multiple isoforms of each subunit: six alpha subtypes (α1,2,3,4,5,6), four beta (β1,2,3,4), three gamma (γ1,2,3), and one delta (δ). These receptors are heterogeneous and can consist of different mixtures of different polypeptide classes (alpha, beta, gamma, etc.)

There are two GABA binding sites in the receptor and a single binding site for the BZDs which is located in the pairing (interphase) between an α subunit and a β subunit (**Figure 2**).

The binding of a BZD to its binding site cause an increment of the GABA affinity for its own binding site. They act as a **positive allosteric modulator**: the union of the BZD to the receptor does not alter the GABA union, but it increases the total conduction of chloride ions across the neuronal cell membrane. This increment of chloride ions leads to a hyperpolarization of the neuron and, as a result, a decrease of the neuronal activity [8].

The advantage of the BZDs comparing to other drugs that act in the same receptor and decrease the activity of neurons is that BZDs are the only drugs that give GABA more affinity for its receptor and act as an allosteric modulator. For the same reason, BZDs are not able to provide a higher activation than GABA itself, and this is what explains the elevated therapeutic index (toxic/therapeutic dose ratio), superior than barbiturates.

This last group, barbiturates, in low doses helps to maintain the chloride channel opened by acting in the GABA. However, in high doses they open directly the chloride cannel, which can lead to toxicity.

#### **1.3. Specific BZD receptors**

The BZD receptor has been classified into different types, based on α subunit isoforms and clinical effects related to each type [8, 9]. In addition, each BZD has different affinity to the GABAA receptor and its subunits:

can also be in 1,5 or 2,3. Normally the benzodiazepines used in clinical are 1,4-dinitrogenated

**Figure 2.** The GABAA receptor. On the left, a side complete view of the receptor: the subunits and the chloride ion channel, with the BZDs binding sites. On the right, a top view of the receptor, illustrating the most common combination

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

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67

By analyzing the structure, we can see the substitutions at the different positions of and the

(O or N) derived from carboxyl first generation of BZDs. Although it can also be non-

• Substitution at position 3: If it is not substituted or has an −OH: ↑ polarity glucuronida-

• Substituted in *ortho* by **Cl, F**: ↑ activity (electron-attracting group). Example: *flurazepam*

Favorable position to ↑ activity, specially by an electron-attracting group: CF<sup>3</sup> > NO<sup>2</sup> >

• Substitution at position 1: ↑ Activity by alkylation (prodrug). Example: *diazepam*

• Replaced by another cycle. Example: cyclohexenyl (tetrazepam).

systems.

of *α*, β, and *γ* subunits [7].

consequences that have on the activity:

substituted. Example: medazepam

(F) and *clonazepam* (Cl).

: Hypnotic action.

Br > Cl > OCH3 > R.

• NO<sup>2</sup>

• Substitution at position 2: Electronegative atom

tion faster elimination. Example: lorazepam

• Benzene ring at position 5: Optimal for activity.

• Substitution at position 7: Establish the potency.


The different effects of the BZDs are explained by their interaction and binding with the different receptors (the isoform, the affinity of the binding, and the location of the receptor in the CNS). According to this, all the effects should be expected for those BZDs that interact indiscriminately with all the receptors. Others, nonbenzodiazepines or Z-drugs, for example, only interact with one type of receptor (BZ1 in this case) so they are going to be used with more specificity.

#### **1.4. Chemical structure and structure-activity relationship (SAR)**

As introduced before, BZDs have a cyclic structure that includes one benzene cycle (*benzo*) plus a heterocycle where two atoms are nitrogen (−*diaza*-) normally in 1 and 4 positions but which 1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis http://dx.doi.org/10.5772/intechopen.79879 67

**Figure 2.** The GABAA receptor. On the left, a side complete view of the receptor: the subunits and the chloride ion channel, with the BZDs binding sites. On the right, a top view of the receptor, illustrating the most common combination of *α*, β, and *γ* subunits [7].

can also be in 1,5 or 2,3. Normally the benzodiazepines used in clinical are 1,4-dinitrogenated systems.

By analyzing the structure, we can see the substitutions at the different positions of and the consequences that have on the activity:


(O or N) derived from carboxyl first generation of BZDs. Although it can also be nonsubstituted. Example: medazepam

	- Substituted in *ortho* by **Cl, F**: ↑ activity (electron-attracting group). Example: *flurazepam* (F) and *clonazepam* (Cl).
	- Replaced by another cycle. Example: cyclohexenyl (tetrazepam).

Favorable position to ↑ activity, specially by an electron-attracting group: CF<sup>3</sup> > NO<sup>2</sup> > Br > Cl > OCH3 > R.

• NO<sup>2</sup> : Hypnotic action.

alpha subtypes (α1,2,3,4,5,6), four beta (β1,2,3,4), three gamma (γ1,2,3), and one delta (δ). These receptors are heterogeneous and can consist of different mixtures of different polypeptide classes

There are two GABA binding sites in the receptor and a single binding site for the BZDs which is located in the pairing (interphase) between an α subunit and a β subunit (**Figure 2**). The binding of a BZD to its binding site cause an increment of the GABA affinity for its own binding site. They act as a **positive allosteric modulator**: the union of the BZD to the receptor does not alter the GABA union, but it increases the total conduction of chloride ions across the neuronal cell membrane. This increment of chloride ions leads to a hyperpolarization of the

The advantage of the BZDs comparing to other drugs that act in the same receptor and decrease the activity of neurons is that BZDs are the only drugs that give GABA more affinity for its receptor and act as an allosteric modulator. For the same reason, BZDs are not able to provide a higher activation than GABA itself, and this is what explains the elevated therapeu-

This last group, barbiturates, in low doses helps to maintain the chloride channel opened by acting in the GABA. However, in high doses they open directly the chloride cannel, which

The BZD receptor has been classified into different types, based on α subunit isoforms and clinical effects related to each type [8, 9]. In addition, each BZD has different affinity to the

of the GABAA receptors. This receptor is highly concentrated in the cortex, thalamus, and cerebellum, and it is responsible for sedative effects and anterograde amnesia, explaining

is a widespread receptor, it is believed that those located in the spinal cord and motor

isoform, and the BZ3

The different effects of the BZDs are explained by their interaction and binding with the different receptors (the isoform, the affinity of the binding, and the location of the receptor in the CNS). According to this, all the effects should be expected for those BZDs that interact indiscriminately with all the receptors. Others, nonbenzodiazepines or Z-drugs, for example, only interact with

As introduced before, BZDs have a cyclic structure that includes one benzene cycle (*benzo*) plus a heterocycle where two atoms are nitrogen (−*diaza*-) normally in 1 and 4 positions but which

one type of receptor (BZ1 in this case) so they are going to be used with more specificity.

receptor contains the α1 subunit isoform, which represents approximately the 60%

contains the α<sup>3</sup>

isoform. Although the

receptor, and those located in the

neuron and, as a result, a decrease of the neuronal activity [8].

tic index (toxic/therapeutic dose ratio), superior than barbiturates.

(alpha, beta, gamma, etc.)

66 Medicinal Chemistry

can lead to toxicity.

• The BZ1

• The BZ2

BZ2

**1.3. Specific BZD receptors**

GABAA receptor and its subunits:

this frequent side effect in the most of the BZDs.

neurons largely mediate myorelaxant effect, such as BZ<sup>3</sup>

**1.4. Chemical structure and structure-activity relationship (SAR)**

limbic system are responsible for the anxiolytic effect.

receptor contains the α<sup>2</sup>

Example: clonazepam, nitrazepam, and lormetazepam.

• X: Anxiolytic action. Example: lorazepam and alprazolam.

Any substitution on the other positions (6, 8, and 9) may decrease the activity. There are others who are fused with triazole or imidazole ring and so producing *triazolobenzodiazepines* or *imidazolobenzodiazepine* (or diazolobenzodiazepines), respectively [10]. From a chemical structure point of view, BZDs can be divided in three groups (**Figures 3** and **4**).

such as the central nervous system and adipose tissue. It is important to mention that the major factor in predicting amnesia risk is lipid solubility: the greater the lipid solubility, the greater the risk of amnesia. BZDs with high lipid solubility have higher absorption rates and faster onset of clinical effects than BZDs with low lipid solubility [8]. Most BZDs are metabolized by the cytochrome P450 enzymes (phase I) by oxidation, hydroxylation, or dealkylation and after conjugated with glucuronide or sulfate (phase II). At the end, the urine excretes them almost entirely. Some BZDs produce active metabolites during the process, as they are administered in a prodrug form. This supposes an important consideration when prescribing these agents. For example, *diazepam*, a long-acting BZD, produces the active metabolites *oxazepam*, *desmethyldiazepam*, and *temazepam.* A classification of the BZDs exists in basis of their half-live time for elimination, an estimation of the time needed to reduce the drug concentration in the plasma

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69

by half. After 5–7 h post-administration, a drug is eliminated from the body [8].

drug action, which can also have variations in the elimination half-life.

when disruption, as well as amnesia and dependence problems.

**2.1. Abuse and dependence: problem presentation**

**2. Classification of BZDs**

or long-acting (**Figure 5**):

lormetazepam).

clorazepate).

These previous reasons should be considered when administering BZDs in the elderly and in the patients with preexisting hepatic diseases: the metabolites further increase the duration of

BZDs are classified in terms of their elimination half-life in short-acting, intermediate-acting,

• Short-acting. Elimination half-life <5 h (*midazolam* and *triazolam*). Mainly used as hypnotic for their quick sleep onset. They have few residual effects and can cause rebound insomnia

• Intermediate-acting. Elimination half-life 5–24 h, normally they are used for anxiety purposes. Might have next-day residual effects if used as hypnotic (alprazolam, lorazepam,

• Long-acting. Elimination half-life >24 h, arriving to 100 h in diazepam. They present risk of accumulation, especially in the elderly or patients with metabolism disease (diazepam,

A huge number of BZDs have been synthetized over the years, but only a few had shown improved efficacy and are actually used in clinical. Today, approximately 35 benzodiazepine

BZDs became one of the most frequently prescribed drugs in the world around the 1970s, even though the potential abuse and dependence was quickly detected. As a result of many concerns about misuse, BZDs were placed on the Food and Drug Administration (FDA) restricted drug list in 1975. It was not until the 1980s that the dependence occurring with these drugs was confirmed, after several clinical trials and after many declarations coming from not only patients but also from clinicians. Despite recommendations of a treatment no longer than 4 weeks, many of

derivatives exist, 21 of which have been approved internationally by clinical use [7].

#### **1.5. Pharmacokinetics and pharmacodynamics**

Some of the pharmacokinetics properties change in function of the side groups (R) of each BZD. That will be decisive when prescribing them. Normally this family of drugs is taken by oral administration due to its good absorption. The intravenous administration presents a quick distribution to the brain and central nervous system, but it is reserved for emergencies like acute seizures.

BZDs and their metabolites are highly protein bound (90% union with albumin). These compounds are widely distributed in the body and preferentially accumulated in lipid-rich areas

**Figure 4.** General structure of various BZDs: (A) 5-Aryl-1,4-benzodiazepine; (B) *midazolam*, a diazolobenzodiazepine; and (C) *triazolam*, a triazolobenzodiazepine [11].

such as the central nervous system and adipose tissue. It is important to mention that the major factor in predicting amnesia risk is lipid solubility: the greater the lipid solubility, the greater the risk of amnesia. BZDs with high lipid solubility have higher absorption rates and faster onset of clinical effects than BZDs with low lipid solubility [8]. Most BZDs are metabolized by the cytochrome P450 enzymes (phase I) by oxidation, hydroxylation, or dealkylation and after conjugated with glucuronide or sulfate (phase II). At the end, the urine excretes them almost entirely.

Some BZDs produce active metabolites during the process, as they are administered in a prodrug form. This supposes an important consideration when prescribing these agents. For example, *diazepam*, a long-acting BZD, produces the active metabolites *oxazepam*, *desmethyldiazepam*, and *temazepam.* A classification of the BZDs exists in basis of their half-live time for elimination, an estimation of the time needed to reduce the drug concentration in the plasma by half. After 5–7 h post-administration, a drug is eliminated from the body [8].

These previous reasons should be considered when administering BZDs in the elderly and in the patients with preexisting hepatic diseases: the metabolites further increase the duration of drug action, which can also have variations in the elimination half-life.

## **2. Classification of BZDs**

Example: clonazepam, nitrazepam, and lormetazepam.

**1.5. Pharmacokinetics and pharmacodynamics**

and (C) *triazolam*, a triazolobenzodiazepine [11].

**Figure 3.** Compound numbering.

like acute seizures.

68 Medicinal Chemistry

• X: Anxiolytic action. Example: lorazepam and alprazolam.

Any substitution on the other positions (6, 8, and 9) may decrease the activity. There are others who are fused with triazole or imidazole ring and so producing *triazolobenzodiazepines* or *imidazolobenzodiazepine* (or diazolobenzodiazepines), respectively [10]. From a chemical

Some of the pharmacokinetics properties change in function of the side groups (R) of each BZD. That will be decisive when prescribing them. Normally this family of drugs is taken by oral administration due to its good absorption. The intravenous administration presents a quick distribution to the brain and central nervous system, but it is reserved for emergencies

BZDs and their metabolites are highly protein bound (90% union with albumin). These compounds are widely distributed in the body and preferentially accumulated in lipid-rich areas

**Figure 4.** General structure of various BZDs: (A) 5-Aryl-1,4-benzodiazepine; (B) *midazolam*, a diazolobenzodiazepine;

structure point of view, BZDs can be divided in three groups (**Figures 3** and **4**).

BZDs are classified in terms of their elimination half-life in short-acting, intermediate-acting, or long-acting (**Figure 5**):


A huge number of BZDs have been synthetized over the years, but only a few had shown improved efficacy and are actually used in clinical. Today, approximately 35 benzodiazepine derivatives exist, 21 of which have been approved internationally by clinical use [7].

### **2.1. Abuse and dependence: problem presentation**

BZDs became one of the most frequently prescribed drugs in the world around the 1970s, even though the potential abuse and dependence was quickly detected. As a result of many concerns about misuse, BZDs were placed on the Food and Drug Administration (FDA) restricted drug list in 1975. It was not until the 1980s that the dependence occurring with these drugs was confirmed, after several clinical trials and after many declarations coming from not only patients but also from clinicians. Despite recommendations of a treatment no longer than 4 weeks, many of

They can also produce problems in psychomotor performances (driving, incoordination, sometimes causing falls). There is sufficient evidence from epidemiologic and experimental studies to establish a strong causal connection between benzodiazepine and also Z-drug use to motor vehicle accidents, falls, and fractures as a consequence of psychomotor impairment [15]. In addition, taking into account their pharmacological properties, benzodiazepines can cause muscular hypotonia and respiratory difficulties, especially in patients presenting a respiratory deficiency. The intensity of the effects depends on the doses and is worst in patients with hepatic alterations and in the elderly. The physiological changes of aging in the liver result in prolonged clearance of drugs: by decreasing the metabolism, the half-life elimination increases. BZDs are eliminated slowly from the body, so repeated doses over a prolonged period can result in significant accumulation in fatty tissues. Thus, some symptoms of overmedication (impaired

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The side effects of BZDs are increased when paired with other drugs such as barbiturates, alcohol, narcotics, or tranquilizers. BZDs potentiate the sedative effects of opioids and are the most common combination in polydrug users, along with alcohol [6]. The risk of fatality via respiratory or nervous system depression from BZD overdose is barely inexistent, but if they are involved with other agents known to cause CNS and respiratory depressions, especially

Over the past few years, biomedical literature has emerged raising a tentative link between benzodiazepine and/or Z-drug exposure with adverse outcomes such as respiratory disease exacerbation, infections, dementia, pancreatitis, and cancer. Doubt persists in the biomedical community regarding this relatively new safety accusation against these drugs by pharmaco-

Based on the Hill criteria for causation, a list of the possible adverse outcome associations is

+ + − ± − ± −

Consistency + + ± ± ± − ± Strength + + + ± + ± ± Temporality + + − + − − − Specificity − − − − − − − Dose–response + + ± − ± − ± Coherence + + ± ± − ± −

Analogy + + − − ± + −

+ criteria fulfilled, ± criteria partially fulfilled or arguable either way, − criteria not fulfilled.

**Dementia Infections Pancreatitis Respiratory** 

**worsening**

**Cancer**

thinking, disorientation, confusion, slurred speech) can appear over time [8].

alcohol or opioids, the risk of harm substantially increases.

**Falls leading to fractures**

epidemiologic researchers.

**Traffic accidents**

**Table 2.** Criteria for BZD/Z-drug adverse events [16].

indicated in **Table 2**.

Experimental evidence

**Figure 5.** Examples of 1,4-benzodiazepines.

them continued to prescribe them for months or even years. Their use gradually declined after the mid-1980s as a result of growing information and concerns, and also with the discovery of other antianxiety medications like the selective serotonin reuptake inhibitors (SSRIs), which proved to be safer and more effective than BZDs. In fact, the total BZD use increased from 1999 to 2014, mainly caused by the augmentation of the long-term inappropriate users [12].

Intentional abusers of BZD usually have other substance abuse problems. Benzodiazepines are usually a secondary drug of abuse, used mainly to augment the "high" received from another drug or to offset the adverse effects of other drugs. Few cases of addiction originated from legitimate use of benzodiazepines. On August 31, 2016, FDA issued a drug safety communication about serious risks, including death, when opioid pain or cough medicines are combined with benzodiazepines. The safety announcement warned that "health care professionals should limit prescribing opioid pain medicines with benzodiazepines... only to patients for whom alternative treatment options are inadequate" [13].

The pharmacological dependence derived from a BZD, which is normally manifested in withdrawal symptoms when the treatment is suddenly interrupted, can happen even from a legitimate use. This response, caused by the constant action of drug after a long time, can be avoided, for example, with dose tapering and/or medication switching [14].

#### **2.2. Adverse effects**

In general, BZD are well-tolerated drugs if the use and administration are correct. The toxicological profile of BZDs is similar between compounds, although the frequency and gravity of the reactions can be different. In most of the cases, adverse reactions are a prolongation of the pharmacological action that affects the CNS.


They can also produce problems in psychomotor performances (driving, incoordination, sometimes causing falls). There is sufficient evidence from epidemiologic and experimental studies to establish a strong causal connection between benzodiazepine and also Z-drug use to motor vehicle accidents, falls, and fractures as a consequence of psychomotor impairment [15]. In addition, taking into account their pharmacological properties, benzodiazepines can cause muscular hypotonia and respiratory difficulties, especially in patients presenting a respiratory deficiency.

The intensity of the effects depends on the doses and is worst in patients with hepatic alterations and in the elderly. The physiological changes of aging in the liver result in prolonged clearance of drugs: by decreasing the metabolism, the half-life elimination increases. BZDs are eliminated slowly from the body, so repeated doses over a prolonged period can result in significant accumulation in fatty tissues. Thus, some symptoms of overmedication (impaired thinking, disorientation, confusion, slurred speech) can appear over time [8].

The side effects of BZDs are increased when paired with other drugs such as barbiturates, alcohol, narcotics, or tranquilizers. BZDs potentiate the sedative effects of opioids and are the most common combination in polydrug users, along with alcohol [6]. The risk of fatality via respiratory or nervous system depression from BZD overdose is barely inexistent, but if they are involved with other agents known to cause CNS and respiratory depressions, especially alcohol or opioids, the risk of harm substantially increases.

Over the past few years, biomedical literature has emerged raising a tentative link between benzodiazepine and/or Z-drug exposure with adverse outcomes such as respiratory disease exacerbation, infections, dementia, pancreatitis, and cancer. Doubt persists in the biomedical community regarding this relatively new safety accusation against these drugs by pharmacoepidemiologic researchers.

Based on the Hill criteria for causation, a list of the possible adverse outcome associations is indicated in **Table 2**.


+ criteria fulfilled, ± criteria partially fulfilled or arguable either way, − criteria not fulfilled.

**Table 2.** Criteria for BZD/Z-drug adverse events [16].

them continued to prescribe them for months or even years. Their use gradually declined after the mid-1980s as a result of growing information and concerns, and also with the discovery of other antianxiety medications like the selective serotonin reuptake inhibitors (SSRIs), which proved to be safer and more effective than BZDs. In fact, the total BZD use increased from 1999

Intentional abusers of BZD usually have other substance abuse problems. Benzodiazepines are usually a secondary drug of abuse, used mainly to augment the "high" received from another drug or to offset the adverse effects of other drugs. Few cases of addiction originated from legitimate use of benzodiazepines. On August 31, 2016, FDA issued a drug safety communication about serious risks, including death, when opioid pain or cough medicines are combined with benzodiazepines. The safety announcement warned that "health care professionals should limit prescribing opioid pain medicines with benzodiazepines... only to

The pharmacological dependence derived from a BZD, which is normally manifested in withdrawal symptoms when the treatment is suddenly interrupted, can happen even from a legitimate use. This response, caused by the constant action of drug after a long time, can be

In general, BZD are well-tolerated drugs if the use and administration are correct. The toxicological profile of BZDs is similar between compounds, although the frequency and gravity of the reactions can be different. In most of the cases, adverse reactions are a prolongation of the

• Frequent: somnolence (half of the patients experiment it during the first days of treatment), sedation, ataxia (especially in the elderly), fatigue, and anterograde amnesia (difficulty to

to 2014, mainly caused by the augmentation of the long-term inappropriate users [12].

patients for whom alternative treatment options are inadequate" [13].

**2.2. Adverse effects**

**Figure 5.** Examples of 1,4-benzodiazepines.

70 Medicinal Chemistry

remember recent facts)

pharmacological action that affects the CNS.

avoided, for example, with dose tapering and/or medication switching [14].

• Occasionally: dizziness, headache, depression, confusion, and dysphasia

• Exceptionally: rush or urticaria, pruritus, and visual and/or audition alterations

There is a lack of evidence to prove causality between BZD and Z-drugs to any of these conditions due to insufficient and conflicting evidence from both epidemiologic and experimental studies, except for fall leading to fractures, which has already been proved [15]. Anyway, there are reasons to associate them: there are clinical studies that are in process to verify it or that are proposed for future research about the subject.

considered a derivative from *1,4-benzodiazepine-N4*

**Scheme 2.** Mechanism preparation of *chlordiazepoxide* [17].

**Scheme 3.** Metabolism and synthesis of *diazepam* [2].

essential for the biological action.

used nowadays.

to a 7-atom ring (benzodiazepine) as a consequence (**Scheme 2**).

C-2 of the quinazoline was produced, with a rearrangement of the 6-atom ring (quinazoline)

The reaction was generalized for other primary amines, but none of the new obtained products was better than *chlordiazepoxide* after all. Later they found that *N*-oxide group was not

Thus, new anxiolytic drugs such as *diazepam*, *bromazepam*, *or nitrazepam* were found, widely

*-oxide (9).* An addition reaction in the carbon

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1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

## **3. Synthesis of benzodiazepines**

The first BZD, serendipitously founded, was *chlordiazepoxide*, and its synthesis started after the synthesis of the *quinazoline-N-oxide*3 as indicated in **Scheme 1**. From the 2-aminobenzophenone, the synthesis of BZDs can be raised as indicated below:

The 2-aminobenzophenone is treated with hydroxylamine to obtain the oxime **1**. The oxime can exist in the form of two stereoisomers *Z* and *E*, the stereoisomer *E* being the most stable due to steric problems. The reaction of this compound with *chloroacetylchloride* gives the chloroacetamide, which by treatment with NaOH leads to the found benzodiazepine-*N*-oxide **5**. The intramolecular cyclization reaction proceeds through the nitrogen atom of the oxime. The resulting *N*-oxide function can be reduced by treatment with PCl3 .

By treating this *quinazoline-N-oxide* with secondary amines (HNRR), a tertiary amine was obtained as an expected compound for the nucleophilic substitution (**6**). However, by treating it with a primary amine: *methylamine* (CH3 NH<sup>2</sup> ), the result was an unexpected compound

**Scheme 1.** Synthesis of [1, 4]-benzodiazepines [2].

considered a derivative from *1,4-benzodiazepine-N4 -oxide (9).* An addition reaction in the carbon C-2 of the quinazoline was produced, with a rearrangement of the 6-atom ring (quinazoline) to a 7-atom ring (benzodiazepine) as a consequence (**Scheme 2**).

The reaction was generalized for other primary amines, but none of the new obtained products was better than *chlordiazepoxide* after all. Later they found that *N*-oxide group was not essential for the biological action.

Thus, new anxiolytic drugs such as *diazepam*, *bromazepam*, *or nitrazepam* were found, widely used nowadays.

**Scheme 2.** Mechanism preparation of *chlordiazepoxide* [17].

There is a lack of evidence to prove causality between BZD and Z-drugs to any of these conditions due to insufficient and conflicting evidence from both epidemiologic and experimental studies, except for fall leading to fractures, which has already been proved [15]. Anyway, there are reasons to associate them: there are clinical studies that are in process to verify it or

The first BZD, serendipitously founded, was *chlordiazepoxide*, and its synthesis started after the synthesis of the *quinazoline-N-oxide*3 as indicated in **Scheme 1**. From the 2-aminobenzo-

The 2-aminobenzophenone is treated with hydroxylamine to obtain the oxime **1**. The oxime can exist in the form of two stereoisomers *Z* and *E*, the stereoisomer *E* being the most stable due to steric problems. The reaction of this compound with *chloroacetylchloride* gives the chloroacetamide, which by treatment with NaOH leads to the found benzodiazepine-*N*-oxide **5**. The intramolecular cyclization reaction proceeds through the nitrogen atom of the oxime. The

By treating this *quinazoline-N-oxide* with secondary amines (HNRR), a tertiary amine was obtained as an expected compound for the nucleophilic substitution (**6**). However, by treating

NH<sup>2</sup>

.

), the result was an unexpected compound

that are proposed for future research about the subject.

phenone, the synthesis of BZDs can be raised as indicated below:

resulting *N*-oxide function can be reduced by treatment with PCl3

**3. Synthesis of benzodiazepines**

72 Medicinal Chemistry

it with a primary amine: *methylamine* (CH3

**Scheme 1.** Synthesis of [1, 4]-benzodiazepines [2].

**Scheme 3.** Metabolism and synthesis of *diazepam* [2].

**Scheme 3** shows also an alternative synthesis for *diazepam* from ketone **12**, starting with a cycliza-

However, d*iazepam* has other alternative synthesis (**Scheme 4**). Starting with the 2-amino-

*Midazolam* can be prepared from 4-chloroacetanilide (**15**) by treatment with 2-fluorobenzoyl chloride. The obtained ketone **16** is treated with 3-nitro-2-propanamine to obtain the intermediate benzodiazepine **17**. Next, the nitro derivative **17** is reduced, and ethyl orthoformate is added to obtain the tricyclic system **18**. Finally, the oxidation of **18** with DDQ (2,3-dichloro-

When research scientists could finally give an explanation for the mechanism of action to understand the results they were obtaining with BZDs, a breakthrough happened, not only in the knowledge of anxiety but also in other central phenomena such as sleep problems or seizures. An important advance was concerning the barbiturates. Barbiturate abuse—both prescription and illicit—peaked in the 1970s, but by the late 1980s, barbiturates had been largely replaced by benzodiazepines for treatment of anxiety and insomnia due to safety issues [19]. BZDs proved to be effective for the same purposes but with a superior therapeutic index and lower risk to cause respiration depression, the principal serious adverse effect that made barbitu-

Generally, it can be considered that all BZDs that are actually used in clinical are anxiolytics in low doses and hypnotic in high doses. Pharmacokinetic properties are what differentiate each compound and what define the use. Furthermore, all the treatments with BZDs should

Back then, the explanation of BZDs' mechanism of action supposed an important discovery in

BZDs should be seen as a symptom treatment for this condition, to facilitate the patients' adaptation or reaction to a difficult situation in their everyday life but not as a first-choice anxiety treatment. Treating anxiety should be a personalized combination of drugs and psychotherapy during the period of time the patient need, and BZDs should be only used for sporadic moments. Nowadays there are other drugs as a first choice for anxiety treatment that does not present any long-term use problem and that show good results (SSRIs or SNRIs). Anyway, BZDs are

be short term due to their probability to cause tolerance and dependence problems.

the knowledge of anxiety, which the biological basis was not completely clear.

, this group is introduced by addition of the amino group to the carbonyl,

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis


75

and the introduction of a

OH, we obtain the oxime **7**. Then by reacting

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tion of the corresponding keto-aniline with methyl 2-aminoacetate. Then with CH3

The last scheme is midazolam's synthesis, as an example of a *diazolobenzodiazepine*.

the introduction of a methyl group in the nitrogen of the amide leads to *diazepam*.

ready for the next steps: a cyclization by dehydration with NaHSO<sup>3</sup>

5,6-dicyanobenzoquinone) leads to *midazolam* (**Scheme 5**).

**4. Traditional uses and new discoveries**

rates a dangerous drug with restricted uses.

5-chlorobenzophenone **12** and reacting with NH<sup>2</sup>

NH<sup>2</sup>

methyl group to obtain the *diazepam* (**11**).

with ClCOCH2

**4.1. Anxiolitics**

**Scheme 4.** Alternative synthesis of *diazepam* [17].

**Scheme 5.** Synthesis of *midazolam* [18].

In the first line of the previous scheme, we can see how *diazepam* is formed by metabolism of *chlordiazepoxide* (**9**) (**Scheme 3**). The first step is an oxidative deamination then the reduction of *N*-oxide **10** with PCl3 following with *N-*alkylation using CH3 I/base, which introduces a methyl group by nucleophilic substitution, obtaining the metabolite *diazepam*.

**Scheme 3** shows also an alternative synthesis for *diazepam* from ketone **12**, starting with a cyclization of the corresponding keto-aniline with methyl 2-aminoacetate. Then with CH3 -I/base again the introduction of a methyl group in the nitrogen of the amide leads to *diazepam*.

However, d*iazepam* has other alternative synthesis (**Scheme 4**). Starting with the 2-amino-5-chlorobenzophenone **12** and reacting with NH<sup>2</sup> OH, we obtain the oxime **7**. Then by reacting with ClCOCH2 NH<sup>2</sup> , this group is introduced by addition of the amino group to the carbonyl, ready for the next steps: a cyclization by dehydration with NaHSO<sup>3</sup> and the introduction of a methyl group to obtain the *diazepam* (**11**).

The last scheme is midazolam's synthesis, as an example of a *diazolobenzodiazepine*.

*Midazolam* can be prepared from 4-chloroacetanilide (**15**) by treatment with 2-fluorobenzoyl chloride. The obtained ketone **16** is treated with 3-nitro-2-propanamine to obtain the intermediate benzodiazepine **17**. Next, the nitro derivative **17** is reduced, and ethyl orthoformate is added to obtain the tricyclic system **18**. Finally, the oxidation of **18** with DDQ (2,3-dichloro-5,6-dicyanobenzoquinone) leads to *midazolam* (**Scheme 5**).

## **4. Traditional uses and new discoveries**

When research scientists could finally give an explanation for the mechanism of action to understand the results they were obtaining with BZDs, a breakthrough happened, not only in the knowledge of anxiety but also in other central phenomena such as sleep problems or seizures.

An important advance was concerning the barbiturates. Barbiturate abuse—both prescription and illicit—peaked in the 1970s, but by the late 1980s, barbiturates had been largely replaced by benzodiazepines for treatment of anxiety and insomnia due to safety issues [19]. BZDs proved to be effective for the same purposes but with a superior therapeutic index and lower risk to cause respiration depression, the principal serious adverse effect that made barbiturates a dangerous drug with restricted uses.

Generally, it can be considered that all BZDs that are actually used in clinical are anxiolytics in low doses and hypnotic in high doses. Pharmacokinetic properties are what differentiate each compound and what define the use. Furthermore, all the treatments with BZDs should be short term due to their probability to cause tolerance and dependence problems.

#### **4.1. Anxiolitics**

In the first line of the previous scheme, we can see how *diazepam* is formed by metabolism of *chlordiazepoxide* (**9**) (**Scheme 3**). The first step is an oxidative deamination then the reduction of

I/base, which introduces a methyl

following with *N-*alkylation using CH3

group by nucleophilic substitution, obtaining the metabolite *diazepam*.

*N*-oxide **10** with PCl3

**Scheme 5.** Synthesis of *midazolam* [18].

**Scheme 4.** Alternative synthesis of *diazepam* [17].

74 Medicinal Chemistry

Back then, the explanation of BZDs' mechanism of action supposed an important discovery in the knowledge of anxiety, which the biological basis was not completely clear.

BZDs should be seen as a symptom treatment for this condition, to facilitate the patients' adaptation or reaction to a difficult situation in their everyday life but not as a first-choice anxiety treatment. Treating anxiety should be a personalized combination of drugs and psychotherapy during the period of time the patient need, and BZDs should be only used for sporadic moments.

Nowadays there are other drugs as a first choice for anxiety treatment that does not present any long-term use problem and that show good results (SSRIs or SNRIs). Anyway, BZDs are indicated in several anxieties for short-term management of anxiety. They can be also used as an adjunct in treatment for panic disorders (PD), generalized anxiety disorders (GAD), and social anxiety disorders (SAD) as adjuncts to SSRIs for treatment of obsessive–compulsive disorder or as adjuncts to antipsychotics for treatment of acute mania or agitation [8, 20].

**4.3. Muscle relaxant**

**4.4. Anticonvulsive**

**4.5. Amnesics**

comfort to the patients.

*discomfort during EGD* [24]*.*

respiratory depression in some cases.

**4.6. Other uses**

Benzodiazepines such as *diazepam* may be used short term as muscle relaxants reducing the tone

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

in a less extent) in the spinal cord and motor neurons [8]. They can also help relieve the pain of the spasticity caused by other CNS pathologies. High doses are used: 2–10 mg even 4 times a day, depending on the severity and the patient's age, so adverse effects must be considered.

*Clonazepam* is the benzodiazepine most frequently used for long-term control and prevention of chronic seizure disorders. For this purpose, it is used at high doses to achieve high brain concentrations. However, in general BZDs are not the first choice for long-term treatment for epilepsy due to the tolerance and dependence problems that they present. Traditional types

Despite that, all BZDs have anticonvulsant properties especially for seizures caused by toxic agents or due to alcohol withdrawal syndrome. For most types of acute or prolonged seizures or *status epilepticus*, an intravenous or rectal benzodiazepine would be the treatment of first choice.

It is important to note that in the perioperative setting, BZDs are used specifically for their amnesic properties, but in nearly all other instances, amnesia is an undesired side effect.

Their use can be advantageous as an adjunct to anesthesia to induce relaxation and amnesia (procedural memory loss) in cases of outpatient surgery or procedure that allows the patient to return home the same day, for example, endoscopy or colonoscopy, which can cause dis-

Intravenous *midazolam* is normally the preference in these cases due to its rapid onset and short duration of action. However, recent researchers have found that sublingual *alprazolam* is as effective and safe as oral midazolam for sedation during esophagogastroduodenoscopy (EGD): *they were similar in reducing procedural anxiety, and patients had similar tolerance and satisfaction with both treatments; however, sublingual alprazolam was accompanied with less pain/*

BZDs can be used in patients in the intensive care unit (ICU) in those with mechanical ventilation or those with acute pain, although they should be used carefully because of the possible

• They are proved to be first-line choice in AWS treatment. AWS results in people who are dependent on alcohol and either stopped drinking or reduced their alcohol consumption. Severe forms of AWS may be associated with generalized seizures, hallucinations, and delirium tremens, which can be fatal [25]. BZDs have proved to be the best studied and


77

http://dx.doi.org/10.5772/intechopen.79879

of skeletal muscle. The myorelaxant effect is mediated through α<sup>2</sup>

of seizure treatments should be used in first line for epilepsy.

The BZDs used for relieving anxiety are the ones with long half-lives, which are converted in other active metabolites that also have long half-lives. According to this, we achieve continuous drug concentrations and therefore a long duration of action and effects. Some of these drugs are *alprazolam*, *bromazepam*, *oxazepam*, *clorazepate*, *diazepam*, and *lorazepam*.

It is important to note that even if the different compounds are in the same family and are used for the same objectives, they have different potencies, and the doses can notably range between compounds. For example, alprazolam is presented in 0.25, 0.5, 1, and 2 mg doses; a dose of 0.5 mg of alprazolam is equivalent to 10 mg of diazepam. That can lead to administration mistakes if there is a change between these two BZDs, for example.

#### **4.2. Hypnotics**

The quality of a hypnotic drug is not judged only on sleep but also on the state of the subject on awakening and during day, somnolence or not, on the possibility of adverse effects, etc. BZDs are used for hypnotic purposes because they increase the total sleep time by decreasing the time to fall asleep and the number of awakenings. However, the architecture of sleep is significantly altered [21]: it is composed by four non-REM stages (of which the 1 and 2 are considered light-sleep phases, while 3 and 4 phases are associated with deep sleep) and a REM stage. BZDs reduce the 3 and 4 stages and decrease the REM sleep stage, known as "the most restful phase of sleep" [22]. That could be translated, in a long-term, as a worsening of sleep quality [23].

They are useful for treating occasional insomnia, in short treatments (they must be used only for 2–4 weeks) or with an intermittent use. The most used for this objective are *lormetazepam*, *triazolam*, *nitrazepam*, *loprazolam*, *flunitrazepam*, and *estazolam*.

Either short-acting or long-acting, BZDs can be used:


The duration of the action must be adapted to the sleep period: if it is too short, it might be insufficient, and if it is too long, the patient can have residual insomnia on the next day.

In many cases, there is no need of pharmacological treatment for insomnia. The following recommendations are proposed: to change the sleep habits, to avoid caffeine late in the day, or to limit the electronics devices (mobile phone, TV) in the bedroom. Exercise can often help to promote a more restful sleep as well. All these options must be tried before starting a BZD treatment.

#### **4.3. Muscle relaxant**

indicated in several anxieties for short-term management of anxiety. They can be also used as an adjunct in treatment for panic disorders (PD), generalized anxiety disorders (GAD), and social anxiety disorders (SAD) as adjuncts to SSRIs for treatment of obsessive–compulsive disorder or as adjuncts to antipsychotics for treatment of acute mania or agitation [8, 20].

The BZDs used for relieving anxiety are the ones with long half-lives, which are converted in other active metabolites that also have long half-lives. According to this, we achieve continuous drug concentrations and therefore a long duration of action and effects. Some of these

It is important to note that even if the different compounds are in the same family and are used for the same objectives, they have different potencies, and the doses can notably range between compounds. For example, alprazolam is presented in 0.25, 0.5, 1, and 2 mg doses; a dose of 0.5 mg of alprazolam is equivalent to 10 mg of diazepam. That can lead to administra-

The quality of a hypnotic drug is not judged only on sleep but also on the state of the subject on awakening and during day, somnolence or not, on the possibility of adverse effects, etc. BZDs are used for hypnotic purposes because they increase the total sleep time by decreasing the time to fall asleep and the number of awakenings. However, the architecture of sleep is significantly altered [21]: it is composed by four non-REM stages (of which the 1 and 2 are considered light-sleep phases, while 3 and 4 phases are associated with deep sleep) and a REM stage. BZDs reduce the 3 and 4 stages and decrease the REM sleep stage, known as "the most restful phase of sleep" [22]. That could be translated, in a long-term, as a worsening of sleep quality [23].

They are useful for treating occasional insomnia, in short treatments (they must be used only for 2–4 weeks) or with an intermittent use. The most used for this objective are *lormetazepam*,

• To treat insomnia characterized by a difficulty of falling sleep, this BZD will have a rapid onset and a short duration of action, with the objective to quickly achieve higher concentrations. Among hypnotic benzodiazepines, triazolam is one, which has the fastest effect, but

• In other cases, when the patient tends to awake in the middle of the night and is not able to

The duration of the action must be adapted to the sleep period: if it is too short, it might be insufficient, and if it is too long, the patient can have residual insomnia on the next day.

In many cases, there is no need of pharmacological treatment for insomnia. The following recommendations are proposed: to change the sleep habits, to avoid caffeine late in the day, or to limit the electronics devices (mobile phone, TV) in the bedroom. Exercise can often help to promote a more restful sleep as well. All these options must be tried before starting a BZD treatment.

it also causes adverse effects such as amnesia and dependence problems.

continue sleeping, intermediate or long action BZD is more useful.

drugs are *alprazolam*, *bromazepam*, *oxazepam*, *clorazepate*, *diazepam*, and *lorazepam*.

tion mistakes if there is a change between these two BZDs, for example.

*triazolam*, *nitrazepam*, *loprazolam*, *flunitrazepam*, and *estazolam*.

Either short-acting or long-acting, BZDs can be used:

**4.2. Hypnotics**

76 Medicinal Chemistry

Benzodiazepines such as *diazepam* may be used short term as muscle relaxants reducing the tone of skeletal muscle. The myorelaxant effect is mediated through α<sup>2</sup> -containing receptors (and α<sup>3</sup> in a less extent) in the spinal cord and motor neurons [8]. They can also help relieve the pain of the spasticity caused by other CNS pathologies. High doses are used: 2–10 mg even 4 times a day, depending on the severity and the patient's age, so adverse effects must be considered.

### **4.4. Anticonvulsive**

*Clonazepam* is the benzodiazepine most frequently used for long-term control and prevention of chronic seizure disorders. For this purpose, it is used at high doses to achieve high brain concentrations. However, in general BZDs are not the first choice for long-term treatment for epilepsy due to the tolerance and dependence problems that they present. Traditional types of seizure treatments should be used in first line for epilepsy.

Despite that, all BZDs have anticonvulsant properties especially for seizures caused by toxic agents or due to alcohol withdrawal syndrome. For most types of acute or prolonged seizures or *status epilepticus*, an intravenous or rectal benzodiazepine would be the treatment of first choice.

#### **4.5. Amnesics**

It is important to note that in the perioperative setting, BZDs are used specifically for their amnesic properties, but in nearly all other instances, amnesia is an undesired side effect.

Their use can be advantageous as an adjunct to anesthesia to induce relaxation and amnesia (procedural memory loss) in cases of outpatient surgery or procedure that allows the patient to return home the same day, for example, endoscopy or colonoscopy, which can cause discomfort to the patients.

Intravenous *midazolam* is normally the preference in these cases due to its rapid onset and short duration of action. However, recent researchers have found that sublingual *alprazolam* is as effective and safe as oral midazolam for sedation during esophagogastroduodenoscopy (EGD): *they were similar in reducing procedural anxiety, and patients had similar tolerance and satisfaction with both treatments; however, sublingual alprazolam was accompanied with less pain/ discomfort during EGD* [24]*.*

#### **4.6. Other uses**

BZDs can be used in patients in the intensive care unit (ICU) in those with mechanical ventilation or those with acute pain, although they should be used carefully because of the possible respiratory depression in some cases.

• They are proved to be first-line choice in AWS treatment. AWS results in people who are dependent on alcohol and either stopped drinking or reduced their alcohol consumption. Severe forms of AWS may be associated with generalized seizures, hallucinations, and delirium tremens, which can be fatal [25]. BZDs have proved to be the best studied and most effective drugs, especially to prevent severe symptoms and particularly the risk of seizures and delirium tremens. The most used oral BZDs for this pathology are diazepam, chlordiazepoxide, and lorazepam.

• BZDs can be used for abreaction, a technique applied to recover memories.

## **5. A new discovery: BET inhibitors**

A few years ago, BZDs started to be investigated by their possible action as BET protein inhibitors. These families of proteins (bromo- and extra-terminal domain, BET) are epigenetic reader proteins, involved in transcription regulation and chromatin remodeling. Each protein contains two domains (D1 and D2) that bind acetylated lysine on histones H3 and H4. This bind is produced in the hydrophobic pocket of BET by hydrogen bonding, where researchers found high-affinity small molecule ligands that block the binding with the histones. These BET protein inhibitors are the first successful example of inhibition of epigenetic readers, and they offer the opportunity to target cancer drivers, for example, the family of proto-oncogenes *MYC*. Thus, BET inhibitor treatment of cancer cells dependent on the oncogene c*-MYC* can result in significant antiproliferative and cytotoxic effects [25].

In view of the results, at least 10 BET inhibitors are in clinical trials today for the treatment of a range of hematological cancers (including leukemia, lymphoma, and myeloma), certain solid tumors, and atherosclerosis [26]. Most of these molecules are structurally based on the BZD family and have their pharmacological properties.

They showed potent antiproliferative activity in some specific leukemia and downregulation of oncogene *MYC*. They also tested them on primary mouse osteosarcoma (OS) cells: both compounds inhibited proliferation of primary OS cell types, showing the utility of

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

http://dx.doi.org/10.5772/intechopen.79879

79

This new line of study shows a different and interesting use for BZDs that needs to continue to be developed according to the actual interest in the different lines of cancer treatment research. It is an example of how drugs that already exists for a determinate purpose can

Nowadays, BZDs are mostly used for symptomatic treatment of anxiety and/or insomnia, anesthesia, and AWS. Many BZDs received FDA approval for the treatment of "anxiety states" or "anxiety disorders." Therefore, BZD treatment represents an off-label use (without

Serotonergic agents (SSRIs or serotonin and norepinephrine reuptake inhibitors [SNRIs]) are the first-line pharmacologic treatments for anxiety disorders. These antidepressants typically take 4–6 weeks before they exert clinical effect, even more in the treatment of anxiety symp-

Moreover, antidepressants are not necessarily effective at starting doses. During titration to an effective dose (by increasing it in a gradual way), a patient can remain symptomatic. Consequently, it can be months before anxiety relieves because of the antidepressant treatment. Theoretically, BZDs are commonly used as adjuncts during the first few weeks of starting a serotonergic agent

Unfortunately, there is no evidence to support this practice. This was verified in a cohort's study performed between 2001 and 2004 to patients with recent depression diagnosis and with no

toms. When this treatment is initiated, it is typical to co-administer BZDs [28].

with the hopes that once a therapeutic dose is achieved, the BZD can be discontinued.

1,2,3-*triazolobenzodiazepine* derivatives in cancer studies.

**Scheme 6.** Synthesis of 7 (1,2,3-*triazolobenzodiazepine*) [27].

become the main source for a study with very different new indications.

**5.1. Analysis of the reasons that lead to abuse and addiction**

FDA disease-specific approval) for most mental disorders.

On November 2017, a new study was published concerning the *design*, *synthesis*, *and biological activity of 1,2,3-triazolobenzodiazepines BET inhibitors* [27]. Starting from the previous recent discoveries, they focused in testing if the different molecules had acetyl lysine mimicking activity. Based on the bromodomain-binding framework, they developed a 1,2,3-*triazolobenzodiazepine* with the optimal conditions for hydrogen bounding: a diazepine ring for protection and high affinity with asparagine.

The synthesis of **25** (**Scheme 6**) was carried out in the following way: first formation of the diarylamine **21** from the 1,2-diiodobenzene **20** and the aniline by means of a Buchwald crosscoupling reaction. Then, an introduction of an alkyne under Sonogashira coupling reaction conditions. The acylation of **22** with the 2-chloroacetyl chloride leads to **23**. Subsequently the addition of sodium azide to **23** allows a 1,3-dipolar cycloaddition cascade that leads directly to the *triazolobenzodiazepine* **24** by heating at 150°C. The yield of this step was low (13%). Finally, the reduction of carbonyl group with BH3 provided **25**.

They assessed this compound by a binding assay (*AlphaScreen*), and it showed good activity against all bromodomains. After that, they optimize it and expand the series, obtaining a range of analogs.

BET inhibitors have been shown to have a remarkable effect on certain primary cells and cell lines, consequently of downregulation of oncogenes like c-*MYC*. From all of the analogs, and after the tests were done, they selected two of these compounds, both with excellent selectivity in BET domains, and tested them against a cancer cell panel to study their antileukemic effects.

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis http://dx.doi.org/10.5772/intechopen.79879 79

**Scheme 6.** Synthesis of 7 (1,2,3-*triazolobenzodiazepine*) [27].

most effective drugs, especially to prevent severe symptoms and particularly the risk of seizures and delirium tremens. The most used oral BZDs for this pathology are diazepam,

A few years ago, BZDs started to be investigated by their possible action as BET protein inhibitors. These families of proteins (bromo- and extra-terminal domain, BET) are epigenetic reader proteins, involved in transcription regulation and chromatin remodeling. Each protein contains two domains (D1 and D2) that bind acetylated lysine on histones H3 and H4. This bind is produced in the hydrophobic pocket of BET by hydrogen bonding, where researchers found high-affinity small molecule ligands that block the binding with the histones. These BET protein inhibitors are the first successful example of inhibition of epigenetic readers, and they offer the opportunity to target cancer drivers, for example, the family of proto-oncogenes *MYC*. Thus, BET inhibitor treatment of cancer cells dependent on the oncogene c*-MYC* can

In view of the results, at least 10 BET inhibitors are in clinical trials today for the treatment of a range of hematological cancers (including leukemia, lymphoma, and myeloma), certain solid tumors, and atherosclerosis [26]. Most of these molecules are structurally based on the BZD

On November 2017, a new study was published concerning the *design*, *synthesis*, *and biological activity of 1,2,3-triazolobenzodiazepines BET inhibitors* [27]. Starting from the previous recent discoveries, they focused in testing if the different molecules had acetyl lysine mimicking activity. Based on the bromodomain-binding framework, they developed a 1,2,3-*triazolobenzodiazepine* with the optimal conditions for hydrogen bounding: a diazepine ring for protection

The synthesis of **25** (**Scheme 6**) was carried out in the following way: first formation of the diarylamine **21** from the 1,2-diiodobenzene **20** and the aniline by means of a Buchwald crosscoupling reaction. Then, an introduction of an alkyne under Sonogashira coupling reaction conditions. The acylation of **22** with the 2-chloroacetyl chloride leads to **23**. Subsequently the addition of sodium azide to **23** allows a 1,3-dipolar cycloaddition cascade that leads directly to the *triazolobenzodiazepine* **24** by heating at 150°C. The yield of this step was low (13%). Finally,

 provided **25**. They assessed this compound by a binding assay (*AlphaScreen*), and it showed good activity against all bromodomains. After that, they optimize it and expand the series, obtaining a

BET inhibitors have been shown to have a remarkable effect on certain primary cells and cell lines, consequently of downregulation of oncogenes like c-*MYC*. From all of the analogs, and after the tests were done, they selected two of these compounds, both with excellent selectivity in BET domains, and tested them against a cancer cell panel to study their antileukemic effects.

• BZDs can be used for abreaction, a technique applied to recover memories.

chlordiazepoxide, and lorazepam.

78 Medicinal Chemistry

**5. A new discovery: BET inhibitors**

result in significant antiproliferative and cytotoxic effects [25].

family and have their pharmacological properties.

and high affinity with asparagine.

the reduction of carbonyl group with BH3

range of analogs.

They showed potent antiproliferative activity in some specific leukemia and downregulation of oncogene *MYC*. They also tested them on primary mouse osteosarcoma (OS) cells: both compounds inhibited proliferation of primary OS cell types, showing the utility of 1,2,3-*triazolobenzodiazepine* derivatives in cancer studies.

This new line of study shows a different and interesting use for BZDs that needs to continue to be developed according to the actual interest in the different lines of cancer treatment research. It is an example of how drugs that already exists for a determinate purpose can become the main source for a study with very different new indications.

#### **5.1. Analysis of the reasons that lead to abuse and addiction**

Nowadays, BZDs are mostly used for symptomatic treatment of anxiety and/or insomnia, anesthesia, and AWS. Many BZDs received FDA approval for the treatment of "anxiety states" or "anxiety disorders." Therefore, BZD treatment represents an off-label use (without FDA disease-specific approval) for most mental disorders.

Serotonergic agents (SSRIs or serotonin and norepinephrine reuptake inhibitors [SNRIs]) are the first-line pharmacologic treatments for anxiety disorders. These antidepressants typically take 4–6 weeks before they exert clinical effect, even more in the treatment of anxiety symptoms. When this treatment is initiated, it is typical to co-administer BZDs [28].

Moreover, antidepressants are not necessarily effective at starting doses. During titration to an effective dose (by increasing it in a gradual way), a patient can remain symptomatic. Consequently, it can be months before anxiety relieves because of the antidepressant treatment. Theoretically, BZDs are commonly used as adjuncts during the first few weeks of starting a serotonergic agent with the hopes that once a therapeutic dose is achieved, the BZD can be discontinued.

Unfortunately, there is no evidence to support this practice. This was verified in a cohort's study performed between 2001 and 2004 to patients with recent depression diagnosis and with no previous treatment. No significant differences were found between the group that only was taking antidepressant and the group that simultaneously started both antidepressant and BZD [29].

treatments might be considered previous to initiating BZD treatment if there is not a strong evidence of efficacy. In many cases of anxiety, psychotherapy or support would be advised,

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

http://dx.doi.org/10.5772/intechopen.79879

81

An other reason that should be considered when talking about possible addiction is, as previously mentioned, the elevated percentage of patients who continue to use BZDs for long term or self-medication. Even when the prescription instructions are followed, these drugs normally present difficulties when discontinued, mostly due to their properties such as the

To help prevent abuse and diversion of BZDs, prescribers should use appropriate precautions, similar to those used when prescribing other controlled substances such as opioids.

As introduced before, a new type of related drugs appeared in the 1990s specifically for

There are three approved: *zolpidem*, *zopiclone*, (*eszopiclone* as the active enantiomer), and *zaleplon*. They present the same mechanism of action than BZDs (facilitating the inhibitory effect of GABA) but showing more selectivity for BZ1, which affects specifically to sedation and also cause fewer adverse effects [19]. However, they do not have BZD chemical structure,

The synthesis of zolpidem is proposed in **Scheme 7**. The aminomethylation of the imidaz-

the quaternary ammonium salt **28**, which is then reacted with sodium cyanide to give the corresponding nitrile **29.** The acid hydrolysis of the nitrile yields the carboxylic acid **30**, which is activated with carbonyldiimidazole (CDI) and then treated with dimethylamine excess to

The adverse effects of traditional BZDs (like alteration of the sleep architecture, reduction of deep sleep (REM), and residual effects on daytime lead to dependence, tolerance, and with-

I to obtain

insomnia treatment: nonbenzodiazepines receptor agonists (NBRAs) or Z-drugs.

opyridine yields the 3-dimethylamino derivative **27**, which is alkylated with CH3

drawal) have driven the development of these alternative sedative-hypnotic drugs.

obtain the corresponding dimethylamide **31** (*zolpidem*) (**Figures 6** and **7**).

**Scheme 7.** Chemical structure of the three commercialized Z-drugs.

instead of starting a treatment with a high potential of risk.

**5.2. "Z-drugs" or nonbenzodiazepines**

not even the same between them.

quick onset and relief of the symptoms that are likely to cause addiction.

Despite conventional knowledge, BZDs do not make SSRIs more effective when prescribed simultaneously. There are no long-term benefits, but there is a long-term risk of physical dependence (tolerance and/or withdrawal) when these drugs are associated at the beginning of the treatment. Moreover, it is frequently for patients to continue BZDs long term in the presence or absence of the antidepressant. Despite many clinicians intending to interrupt them after the 4–6 weeks (when SSRIs begin to have their therapeutic effect), 12% of patients receiving this treatment and trialed at the study previously mentioned to continue BZDs for over 6 months—sometimes in the absence of SSRIs—likely indicating the difficulty of discontinuing BZDs once started [29, 20].

Despite these mentioned factors, the rate of physicians prescribing this way has not stopped growing in the last 25 years [28]. Because of the risks associated with BZD, this practice (simultaneous new use at antidepressant initiation) requires careful consideration.

The only mental disorders—not including alcohol/sedative-hypnotic withdrawal—for which there is an evidence basis for BZD treatment are PD, GAD, social anxiety disorder (SAD), and insomnia. For these four conditions, BZDs have only demonstrated efficacy for short-term durations (less than 2–4 weeks) and for treatment-resistant cases. Even for those conditions, which there are proofs of efficacy, there is no evidence for benefit in long-term treatment [20].

Nevertheless, BZDs are frequently overprescribed for other indications for which there is no evidence of efficacy, to individuals who have contraindicated comorbid conditions, for longer periods than are recommended, and before other first- and second-line treatments are tried or offered.

Apart from these four previously mentioned, there are no other mental disorders with an evidence basis for BZD treatment. To the contrary, this treatment in post-traumatic stress disorders (PTSD) is particularly concerning because BZDs have not proved to possess preventative value and may actually increase the risk in 2–5 times of developing PTSD among the patients with trauma. Moreover, PTSD is commonly comorbid with conditions that are contraindicated for BZDs (substance use disorders, traumatic brain injury, depression, etc.), and BZDs can inhibit trauma-focused psychotherapy by inhibiting the cognitive processing, which is extremely necessary for a good recovery [30].

It is common in many disorders to find patients receiving treatment not supported by evidence-based clinical practice guidelines (CPGs). Though the only FDA-approved medications for PTSD are *sertraline* and *paroxetine* (both antidepressants), of PTSD patients receiving pharmacotherapy: 65–90% receive antidepressants, 37–74% receive sedative-hypnotics (including BZDs), and 21–34% receive antipsychotics [20]. In fact, most of CPGs strongly recommend against the use of BZDs for PTSD, such as the guideline done by the Department of Veterans Affairs/Department of Defense (VA/DOD) [31].

Psychotherapy is the gold standard treatment for anxiety, while medications are generally considered adjunctive: only serotonergic agents (SSRI and SNRI) are considered first-line pharmacologic monotherapies [32]. The evaluation of the recovery should be based on the improvement of the normal functioning and not only based on the results of the sedation, which often does not relate with the patients' improvement. A variety of evidence-based treatments might be considered previous to initiating BZD treatment if there is not a strong evidence of efficacy. In many cases of anxiety, psychotherapy or support would be advised, instead of starting a treatment with a high potential of risk.

An other reason that should be considered when talking about possible addiction is, as previously mentioned, the elevated percentage of patients who continue to use BZDs for long term or self-medication. Even when the prescription instructions are followed, these drugs normally present difficulties when discontinued, mostly due to their properties such as the quick onset and relief of the symptoms that are likely to cause addiction.

To help prevent abuse and diversion of BZDs, prescribers should use appropriate precautions, similar to those used when prescribing other controlled substances such as opioids.

#### **5.2. "Z-drugs" or nonbenzodiazepines**

previous treatment. No significant differences were found between the group that only was taking antidepressant and the group that simultaneously started both antidepressant and BZD [29]. Despite conventional knowledge, BZDs do not make SSRIs more effective when prescribed simultaneously. There are no long-term benefits, but there is a long-term risk of physical dependence (tolerance and/or withdrawal) when these drugs are associated at the beginning of the treatment. Moreover, it is frequently for patients to continue BZDs long term in the presence or absence of the antidepressant. Despite many clinicians intending to interrupt them after the 4–6 weeks (when SSRIs begin to have their therapeutic effect), 12% of patients receiving this treatment and trialed at the study previously mentioned to continue BZDs for over 6 months—sometimes in the absence of SSRIs—likely indicating the difficulty of discon-

Despite these mentioned factors, the rate of physicians prescribing this way has not stopped growing in the last 25 years [28]. Because of the risks associated with BZD, this practice

The only mental disorders—not including alcohol/sedative-hypnotic withdrawal—for which there is an evidence basis for BZD treatment are PD, GAD, social anxiety disorder (SAD), and insomnia. For these four conditions, BZDs have only demonstrated efficacy for short-term durations (less than 2–4 weeks) and for treatment-resistant cases. Even for those conditions, which there are proofs of efficacy, there is no evidence for benefit in long-term treatment [20]. Nevertheless, BZDs are frequently overprescribed for other indications for which there is no evidence of efficacy, to individuals who have contraindicated comorbid conditions, for longer periods than are recommended, and before other first- and second-line treatments are tried or offered. Apart from these four previously mentioned, there are no other mental disorders with an evidence basis for BZD treatment. To the contrary, this treatment in post-traumatic stress disorders (PTSD) is particularly concerning because BZDs have not proved to possess preventative value and may actually increase the risk in 2–5 times of developing PTSD among the patients with trauma. Moreover, PTSD is commonly comorbid with conditions that are contraindicated for BZDs (substance use disorders, traumatic brain injury, depression, etc.), and BZDs can inhibit trauma-focused psychotherapy by inhibiting the cognitive processing,

It is common in many disorders to find patients receiving treatment not supported by evidence-based clinical practice guidelines (CPGs). Though the only FDA-approved medications for PTSD are *sertraline* and *paroxetine* (both antidepressants), of PTSD patients receiving pharmacotherapy: 65–90% receive antidepressants, 37–74% receive sedative-hypnotics (including BZDs), and 21–34% receive antipsychotics [20]. In fact, most of CPGs strongly recommend against the use of BZDs for PTSD, such as the guideline done by the Department

Psychotherapy is the gold standard treatment for anxiety, while medications are generally considered adjunctive: only serotonergic agents (SSRI and SNRI) are considered first-line pharmacologic monotherapies [32]. The evaluation of the recovery should be based on the improvement of the normal functioning and not only based on the results of the sedation, which often does not relate with the patients' improvement. A variety of evidence-based

(simultaneous new use at antidepressant initiation) requires careful consideration.

tinuing BZDs once started [29, 20].

80 Medicinal Chemistry

which is extremely necessary for a good recovery [30].

of Veterans Affairs/Department of Defense (VA/DOD) [31].

As introduced before, a new type of related drugs appeared in the 1990s specifically for insomnia treatment: nonbenzodiazepines receptor agonists (NBRAs) or Z-drugs.

There are three approved: *zolpidem*, *zopiclone*, (*eszopiclone* as the active enantiomer), and *zaleplon*. They present the same mechanism of action than BZDs (facilitating the inhibitory effect of GABA) but showing more selectivity for BZ1, which affects specifically to sedation and also cause fewer adverse effects [19]. However, they do not have BZD chemical structure, not even the same between them.

The synthesis of zolpidem is proposed in **Scheme 7**. The aminomethylation of the imidazopyridine yields the 3-dimethylamino derivative **27**, which is alkylated with CH3 I to obtain the quaternary ammonium salt **28**, which is then reacted with sodium cyanide to give the corresponding nitrile **29.** The acid hydrolysis of the nitrile yields the carboxylic acid **30**, which is activated with carbonyldiimidazole (CDI) and then treated with dimethylamine excess to obtain the corresponding dimethylamide **31** (*zolpidem*) (**Figures 6** and **7**).

The adverse effects of traditional BZDs (like alteration of the sleep architecture, reduction of deep sleep (REM), and residual effects on daytime lead to dependence, tolerance, and withdrawal) have driven the development of these alternative sedative-hypnotic drugs.

**Scheme 7.** Chemical structure of the three commercialized Z-drugs.

**Figure 6.** Synthesis of zolpidem via Mannich aminomethylation [33].

personality disorders, or drug abuse potential, because the sensitization of GABA receptors in

ER = extended release. Due to its duration of action, *zaleplon* does not present next-day drowsiness; it can be taken within

**Duration of action Insomnia indication**

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83

Other symptoms seen in the reports are bizarre and complex behavioral effects like sleeprelated complex behaviors [38], proved to be related with Z-drugs, particularly *zolpidem* [39]. There have also been some reports and posterior studies of suicidal attempts by zolpidem. In 2016 a study demonstrated a significant association between using *zolpidem* and suicide or

Reports of incidents related with these drugs had increased over the years, indicating that *zolpidem* and others may not be considered as risk-free and should be carefully prescribed,

Studies have seen that Z-drugs usually present the same problems that of BZD: they are prescribed for longer use with excessive doses, particularly in the elderly. This fact shows a relation with the high incidence of falls and risk of hip fracture among these patients [15]. There are also studies that support the lack of demonstrable improved efficacy of Z-drugs, which

The last aspect to consider these drugs is their potential recreational use. As what happens with BZD, by mixing high doses of drug with opioids or alcohol, a major CNS depression is obtained, producing euphoric "high" symptoms with anterograde amnesia on the next day. A study carried out in 2011 showed that when *zolpidem* was ingested with other medications or ethanol, admissions to the ICU were highly common. Despite its reported safety, these overdoses often required ICU admissions, which were results of the association with other drugs and/or alcohol [42].

Despite BZDs' successful use, tolerance was rapidly discovered and studied. A clinical trial in 1985 performed by the Medical College of Ohio showed the regional differences in downregulation of brain BZD receptors using a quantitative autoradiographic method because of

Clinical experience showed that benzodiazepines are frequently used for long-term treatment, and there are many reasons for this: prescribing tradition, patient preference, difficulties

some of these patients may predispose to the development of hallucinations [37].

Zolpidem ER 30 1.6–5.5 Intermediate Sleep onset and sleep maintenance

suicide attempt in people with or without comorbid psychiatric illnesses [40].

causes similar rates of adverse events compared to benzodiazepines [41].

**5.3. Tolerance, dependence, and withdrawal syndrome**

the chronic presence of this drug to its receptor locus [43].

dispensed, and used [19].

**Drug Onset (min) Half-life** 

**Table 3.** Principal Z-drugs and properties.

**(h)**

4–5 hours of wake time without the risk of hangover effect [34].

Zolpidem 30 1.4–4.5 Short Sleep onset

Zaleplon 20 0.5–1 Ultrashort Sleep onset

Eszopiclone 30 6–7 Intermediate Sleep maintenance

**Figure 7.** Flumazenil.

Z-drugs have significant hypnotic effects by reducing sleep latency and improving sleep quality, though duration of sleep may not be significantly increased. Their pharmacokinetics properties approach those of the "ideal hypnotic" with rapid onset within 30 min and short half-life (**Table 3**).

Initially clinical trials were promising due to their low adverse effects and improvements, reducing the potential of abuse. They possess short duration of action and half-life, do not disturb sleep architecture, and cause less residual effects during daytime hours, making them more clinically attractive than BZDs [22, 35].

Despite that, there are other kinds of side effects that are common among these drugs. During the first trails, the most reported side effects were nausea, dizziness, malaise, hallucination, nightmares, or agitation. Although *zolpidem* appeared to be well tolerated, there were cases of abuse, withdrawal, or tolerance in cases where the recommended dose of *zolpidem* was exceeded or with patients who had a history of substance abuse and/or a psychiatric disorder.

Later, cases of Z-drug reports causing visual hallucinations and amnesia in people with no history of mental disease appeared. Although the mechanism of action to describe these phenomena is not clear, it is speculated that *GABA receptor (α1 subunit) may be overexpressed, or they may be rapid activation after quick absorption in sensitive individuals* [36]. As seen in the reports, this is especially true for those patients with mental disease such bipolar disorder, borderline


ER = extended release. Due to its duration of action, *zaleplon* does not present next-day drowsiness; it can be taken within 4–5 hours of wake time without the risk of hangover effect [34].

**Table 3.** Principal Z-drugs and properties.

Z-drugs have significant hypnotic effects by reducing sleep latency and improving sleep quality, though duration of sleep may not be significantly increased. Their pharmacokinetics properties approach those of the "ideal hypnotic" with rapid onset within 30 min and short

Initially clinical trials were promising due to their low adverse effects and improvements, reducing the potential of abuse. They possess short duration of action and half-life, do not disturb sleep architecture, and cause less residual effects during daytime hours, making them

Despite that, there are other kinds of side effects that are common among these drugs. During the first trails, the most reported side effects were nausea, dizziness, malaise, hallucination, nightmares, or agitation. Although *zolpidem* appeared to be well tolerated, there were cases of abuse, withdrawal, or tolerance in cases where the recommended dose of *zolpidem* was exceeded or with patients who had a history of substance abuse and/or a psychiatric disorder. Later, cases of Z-drug reports causing visual hallucinations and amnesia in people with no history of mental disease appeared. Although the mechanism of action to describe these phenomena is not clear, it is speculated that *GABA receptor (α1 subunit) may be overexpressed, or they may be rapid activation after quick absorption in sensitive individuals* [36]. As seen in the reports, this is especially true for those patients with mental disease such bipolar disorder, borderline

half-life (**Table 3**).

**Figure 7.** Flumazenil.

82 Medicinal Chemistry

more clinically attractive than BZDs [22, 35].

**Figure 6.** Synthesis of zolpidem via Mannich aminomethylation [33].

personality disorders, or drug abuse potential, because the sensitization of GABA receptors in some of these patients may predispose to the development of hallucinations [37].

Other symptoms seen in the reports are bizarre and complex behavioral effects like sleeprelated complex behaviors [38], proved to be related with Z-drugs, particularly *zolpidem* [39]. There have also been some reports and posterior studies of suicidal attempts by zolpidem. In 2016 a study demonstrated a significant association between using *zolpidem* and suicide or suicide attempt in people with or without comorbid psychiatric illnesses [40].

Reports of incidents related with these drugs had increased over the years, indicating that *zolpidem* and others may not be considered as risk-free and should be carefully prescribed, dispensed, and used [19].

Studies have seen that Z-drugs usually present the same problems that of BZD: they are prescribed for longer use with excessive doses, particularly in the elderly. This fact shows a relation with the high incidence of falls and risk of hip fracture among these patients [15]. There are also studies that support the lack of demonstrable improved efficacy of Z-drugs, which causes similar rates of adverse events compared to benzodiazepines [41].

The last aspect to consider these drugs is their potential recreational use. As what happens with BZD, by mixing high doses of drug with opioids or alcohol, a major CNS depression is obtained, producing euphoric "high" symptoms with anterograde amnesia on the next day. A study carried out in 2011 showed that when *zolpidem* was ingested with other medications or ethanol, admissions to the ICU were highly common. Despite its reported safety, these overdoses often required ICU admissions, which were results of the association with other drugs and/or alcohol [42].

#### **5.3. Tolerance, dependence, and withdrawal syndrome**

Despite BZDs' successful use, tolerance was rapidly discovered and studied. A clinical trial in 1985 performed by the Medical College of Ohio showed the regional differences in downregulation of brain BZD receptors using a quantitative autoradiographic method because of the chronic presence of this drug to its receptor locus [43].

Clinical experience showed that benzodiazepines are frequently used for long-term treatment, and there are many reasons for this: prescribing tradition, patient preference, difficulties associated with benzodiazepine withdrawal (even in patients taking low doses) because they have a rapid clinical onset of action, and good efficacy with few initial adverse effects. Long-term intake of a drug can induce tolerance of the secondary effects (because increased amounts are needed to achieve intoxication, or the effects are minimized with continued use) and physical dependence, a risk associated even at therapeutic doses [44]. There is no standard definition of long-term use, but the most common is 6–12 months. Tolerance to the sedating effects of benzodiazepines is rapid, but tolerance to the anxiolytic effects develops slowly and to a limited extent.

*5.3.2. Possible treatment of dependence to avoid withdrawal symptoms*

and proceeds at whatever rate he finds tolerable.

and safer the withdrawal would be [48].

*5.3.2.2. Switching to a long-acting BZD*

especially to BZDs.

*5.3.2.1. Dose tapering*

Based on several guidelines to avoid withdrawal symptoms, different steps are recommended when patients want to quit a BZD treatment. For the following recommendations, a specific guideline is consulted: *Benzodiazepines: how they work and how to withdraw* or commonly known as *The Ashton Manual* [46]. It is written by Professor C Heather Ashton, a psychopharmacologist from Newcastle, who has dedicated the majority of her career to psychotropic drugs, and

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Successful withdrawal strategies should combine gradual dosage reduction and sufficient psychological support. The precise rate of withdrawal is an individual matter and should be personalized, depending on many factors including the dose and type of BZD used, the duration of use, and the personality and the will of the patient. For patients without any motivation for withdrawal and those with a severe depressive episode or other major mental

Various authors suggest optimal times from 6 to 8 weeks to several months for the duration of withdrawal, but some patients may take a year or more if they have taken BZDs in prolonged use. The best results are achieved if the patient himself is in control of the rate of withdrawal

Sedative withdrawal symptoms can be avoided by slowly tapering down the dose of the BZD over several weeks and by managing the anxiety if needed. Under any circumstances it is recommended to suddenly stop the treatment. Abrupt withdrawal, especially from high doses, can precipitate convulsions, acute psychotic or confusional states, and panic reactions [47]. The ideal situation is one where the patient, with the help of the doctor, decides together the schedule, accepting that there will be readjustments to the time according to his progress. The length of time between each dose reduction should be based on the presence and severity of withdrawal symptoms. The longer the interval between reductions, the more comfortable

With short-acting BZDs, it is impossible to achieve a smooth decline in blood and tissue concentrations because of the way they are eliminated quickly from the body. In these cases, it is preferred to switch to a long-acting and slowly metabolized BZD such as *diazepam*. Due to its metabolites

The dose has a very important role: not only it has to be changed by the equivalent in *diazepam* but it also has to contemplate the properties of each BZD (if changed to an anxiolytic for a hypnotic, different symptoms can be expected). *Diazepam* is also good to switch to, because its

As indicated before, there is an equivalence of doses between different compounds depend-

and long half-life, it is easy to decrease the concentrations in a smooth and gradual way.

presentation (2 or 10 mg) makes the dose adaptation easier for every patient.

ing on the active metabolites and the potency (**Table 4**).

disorders, stabilization might be preferable before initiating withdrawal treatment [7].

Symptoms of withdrawal after long-term benzodiazepine use usually develop faster with shorter-acting drugs (within 2–3 days) than with longer-acting drugs (within 5–10 days). This is presented by physical symptoms (spasms, weakness, muscle tension, etc.) and psychological symptoms (anxiety and panic disorders, agitation, mood changes). Seizures are also quite common, especially if the agent is discontinued abruptly. Severe withdrawal symptoms include paranoid thoughts, hallucinations, and delirium [7].

#### *5.3.1. Intoxication and antidote*

Generally, BZDs are a safe family of drugs because they present a large therapeutic index. Patients may misuse them by self-medication or by increasing the therapeutic dose for recreational purposes [45]. Real risk comes when patients combine these drugs with other substances: the combined use of alcohol and benzodiazepines increases the risk of a fatal overdose. A similar fatal interaction can occur with opioids: BZDs are often misused by highrisk opioid users and are associated with morbidity and mortality among this group.

Misuse or abuse may lead to intoxication or a withdrawal syndrome, which may be fatal. Differential diagnosis of intoxication by these drugs could be polydrug use (toxicity is highly augmented by combination with other drugs), epilepsy, agitation, alcohol withdrawal delirium or respiratory depression, among others [7].

Fortunately, overdose with benzodiazepines and Z-drugs responds to an antagonist, *flumazenil*, although it has its limitations and potential adverse effects.

This benzodiazepine antagonist, *flumazenil*, is available for the treatment of acute benzodiazepine intoxication and has been shown to reverse also the sedative effects of all three Z-drugs [35]. Actually, it is a BZD with high affinity, which is able to displace other BZDs and has very short half-life, of approximately 1 hour.

It is used for:


However, it may not completely reverse respiratory depression, and it can provoke withdrawal seizures in patients with benzodiazepine dependence [9].

#### *5.3.2. Possible treatment of dependence to avoid withdrawal symptoms*

Based on several guidelines to avoid withdrawal symptoms, different steps are recommended when patients want to quit a BZD treatment. For the following recommendations, a specific guideline is consulted: *Benzodiazepines: how they work and how to withdraw* or commonly known as *The Ashton Manual* [46]. It is written by Professor C Heather Ashton, a psychopharmacologist from Newcastle, who has dedicated the majority of her career to psychotropic drugs, and especially to BZDs.

Successful withdrawal strategies should combine gradual dosage reduction and sufficient psychological support. The precise rate of withdrawal is an individual matter and should be personalized, depending on many factors including the dose and type of BZD used, the duration of use, and the personality and the will of the patient. For patients without any motivation for withdrawal and those with a severe depressive episode or other major mental disorders, stabilization might be preferable before initiating withdrawal treatment [7].

Various authors suggest optimal times from 6 to 8 weeks to several months for the duration of withdrawal, but some patients may take a year or more if they have taken BZDs in prolonged use. The best results are achieved if the patient himself is in control of the rate of withdrawal and proceeds at whatever rate he finds tolerable.

#### *5.3.2.1. Dose tapering*

associated with benzodiazepine withdrawal (even in patients taking low doses) because they have a rapid clinical onset of action, and good efficacy with few initial adverse effects. Long-term intake of a drug can induce tolerance of the secondary effects (because increased amounts are needed to achieve intoxication, or the effects are minimized with continued use) and physical dependence, a risk associated even at therapeutic doses [44]. There is no standard definition of long-term use, but the most common is 6–12 months. Tolerance to the sedating effects of benzodiazepines is rapid, but tolerance to the anxiolytic effects develops

Symptoms of withdrawal after long-term benzodiazepine use usually develop faster with shorter-acting drugs (within 2–3 days) than with longer-acting drugs (within 5–10 days). This is presented by physical symptoms (spasms, weakness, muscle tension, etc.) and psychological symptoms (anxiety and panic disorders, agitation, mood changes). Seizures are also quite common, especially if the agent is discontinued abruptly. Severe withdrawal symptoms

Generally, BZDs are a safe family of drugs because they present a large therapeutic index. Patients may misuse them by self-medication or by increasing the therapeutic dose for recreational purposes [45]. Real risk comes when patients combine these drugs with other substances: the combined use of alcohol and benzodiazepines increases the risk of a fatal overdose. A similar fatal interaction can occur with opioids: BZDs are often misused by high-

Misuse or abuse may lead to intoxication or a withdrawal syndrome, which may be fatal. Differential diagnosis of intoxication by these drugs could be polydrug use (toxicity is highly augmented by combination with other drugs), epilepsy, agitation, alcohol withdrawal delir-

Fortunately, overdose with benzodiazepines and Z-drugs responds to an antagonist, *flumaze-*

This benzodiazepine antagonist, *flumazenil*, is available for the treatment of acute benzodiazepine intoxication and has been shown to reverse also the sedative effects of all three Z-drugs [35]. Actually, it is a BZD with high affinity, which is able to displace other BZDs and has very

However, it may not completely reverse respiratory depression, and it can provoke with-

risk opioid users and are associated with morbidity and mortality among this group.

slowly and to a limited extent.

84 Medicinal Chemistry

*5.3.1. Intoxication and antidote*

include paranoid thoughts, hallucinations, and delirium [7].

ium or respiratory depression, among others [7].

short half-life, of approximately 1 hour.

• BZDs or Z-drugs intoxications

It is used for:

*nil*, although it has its limitations and potential adverse effects.

• To reverse the effects of anesthesia caused by a BZD

• Diagnosis of states of coma, which have an unknown origin

drawal seizures in patients with benzodiazepine dependence [9].

Sedative withdrawal symptoms can be avoided by slowly tapering down the dose of the BZD over several weeks and by managing the anxiety if needed. Under any circumstances it is recommended to suddenly stop the treatment. Abrupt withdrawal, especially from high doses, can precipitate convulsions, acute psychotic or confusional states, and panic reactions [47]. The ideal situation is one where the patient, with the help of the doctor, decides together the schedule, accepting that there will be readjustments to the time according to his progress. The length of time between each dose reduction should be based on the presence and severity of withdrawal symptoms. The longer the interval between reductions, the more comfortable and safer the withdrawal would be [48].

#### *5.3.2.2. Switching to a long-acting BZD*

With short-acting BZDs, it is impossible to achieve a smooth decline in blood and tissue concentrations because of the way they are eliminated quickly from the body. In these cases, it is preferred to switch to a long-acting and slowly metabolized BZD such as *diazepam*. Due to its metabolites and long half-life, it is easy to decrease the concentrations in a smooth and gradual way.

The dose has a very important role: not only it has to be changed by the equivalent in *diazepam* but it also has to contemplate the properties of each BZD (if changed to an anxiolytic for a hypnotic, different symptoms can be expected). *Diazepam* is also good to switch to, because its presentation (2 or 10 mg) makes the dose adaptation easier for every patient.

As indicated before, there is an equivalence of doses between different compounds depending on the active metabolites and the potency (**Table 4**).


The constant investigation concerning BZDs is an indication that the problems related with these drugs are an actual concern, not only as a medical issue but also as a social concern. On July 11, there is a **"**World Benzodiazepine Awareness day (W-BAD)," with the objective to educate the population, to offer support to the patients suffering from dependence, and to try to gain global awareness about the dependency this kind of drugs cause if they are not

1,4-Benzodiazepines and New Derivatives: Description, Analysis, and Organic Synthesis

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87

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Barcelona,

[1] Strenbachh L. The benzodiazepine story. Journal of Medicinal Chemistry. 1979;**22**:1-7

[3] Wick J. The history of benzodiazepines. The Consultant Pharmacist. 2013;**28**:538-548

Farmacos. Univ Santiago de Compostela; 2008. ISBN: mkt0003358332

[2] Rubira E, Medicamentos R. Un Viaje a Lo Largo de la Evolución Del Descubrimiento de

[4] López-Muñoz F, Álamo C, García P. The discovery of chlordiazepoxide and the clinical introduction of benzodiazepines: Half a century of anxiolytic drugs. Journal of Anxiety

[5] Ashton H. History of Benzodiazepines, Psychiatric Medication Awareness Group. Available from: https://www.psychmedaware.org/HistoryBenzodiazepines.html [Accessed:

[6] Harvey R, Silverstein D, Hopper K. Small Animal Critical Care Medicine. Saint Louis:

[7] Soyka M. Treatment of benzodiazepine dependence. The New England Journal of

[8] Griffin CE, Kaye AM, Bueno FR, Kaye AD. Benzodiazepine pharmacology and central

[9] Nutt DJ, Stahl SM. Searching for perfect sleep: The continuing evolution of GABAA receptor modulators as hypnotics. Journal of Psychopharmacology. 2010;**24**:1601-1612

[10] Structural activity relationships of benzodiazepines. Available from: https://egpat.com/ blog/structural-activity-relationships-of-benzodiazepines [Accessed: February 23, 2018]

nervous system–mediated effects. The Ochsner Journal. 2013;**13**:214-223

prescribed correctly, among others [49].

Disorders. 2011;**25**:554-562

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Medicine. 2017;**376**:1147-1157

February 20, 2018]

\*Address all correspondence to: mdpujol@ub.edu

Elisabet Batlle, Enric Lizano, Miquel Viñas and Maria Dolors Pujol\*

**Author details**

Barcelona, Spain

**References**

**Table 4.** Half-life and equivalent potencies of BZD anxiolytics [5].

Most potent drugs like *alprazolam*, *clonazepam*, or *lorazepam*, which has 10–20 times more potency that *diazepam*, are highly addictive; dependence develops rapidly, and they are particularly hard to leave. In addition, their dose presentations do not allow a gradual dosage reduction when withdrawal.

## **6. Conclusions**

Concerning the prescriptions, **guidelines have failed** to reduce the prescriptions: clinicians do not always adhere to recommendations to use BZDs as hypnotics and anxiolytics only for short term and only after trying psychological therapies. It has been difficult to accept the high risk and low benefits of the long term in most of the cases.

The **equivalence of doses** between different compounds had presented difficulties, leading to incorrect and excessive dose prescriptions in many situations. Prescriptions of most potent BZDs (as *alprazolam*, *clonazepam*, *or lorazepam*) with excessive dosage are the more problematic, partly of their addictive potential and partly of their dose presentation, that does not allow a gradual dosage reduction when withdrawal.

New lines of study related with BZDs as **BET inhibitor** compounds are an interesting way to change the direction of the therapeutic uses, especially long term. Other new uses, as perioperative, are a valuable way to use an adverse effect derived from the biological activity and apply it with a clinical purpose.

After analyzing the advantages and disadvantages of the **Z-drugs**, it can be concluded that even if they are not exactly as BZD, they must be treated with the same precaution due to the amount of adverse effect reports that had appeared over the recent years.

Despite the amount of biomedical literature on BZDs and Z-drugs, there is still a need to answer vital questions relevant to their effectiveness and safety in society, for example, the possibility of irreversible effects due to extended treatment, especially those associated to new safety accusations [16].

The constant investigation concerning BZDs is an indication that the problems related with these drugs are an actual concern, not only as a medical issue but also as a social concern. On July 11, there is a **"**World Benzodiazepine Awareness day (W-BAD)," with the objective to educate the population, to offer support to the patients suffering from dependence, and to try to gain global awareness about the dependency this kind of drugs cause if they are not prescribed correctly, among others [49].

## **Author details**

Elisabet Batlle, Enric Lizano, Miquel Viñas and Maria Dolors Pujol\*

\*Address all correspondence to: mdpujol@ub.edu

Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

## **References**

Most potent drugs like *alprazolam*, *clonazepam*, or *lorazepam*, which has 10–20 times more potency that *diazepam*, are highly addictive; dependence develops rapidly, and they are particularly hard to leave. In addition, their dose presentations do not allow a gradual dosage

**Benzodiazepine Half-life (h) (active metabolite) Oral dosages (mg)**

*Alprazolam* (Xanax) 6–12 0.5 *Clonazepam* (Klonopin) 18–50 0.5 *Lorazepam* (Ativan) 10–20 1 *Diazepam* (Valium) 20–100 10 *Chlordiazepoxide* (Librium) 5–30 25 *Clorazepate* (Tranxene) 36–200 15 *Oxazepam* (Serax) 4–15 20

Concerning the prescriptions, **guidelines have failed** to reduce the prescriptions: clinicians do not always adhere to recommendations to use BZDs as hypnotics and anxiolytics only for short term and only after trying psychological therapies. It has been difficult to accept the

The **equivalence of doses** between different compounds had presented difficulties, leading to incorrect and excessive dose prescriptions in many situations. Prescriptions of most potent BZDs (as *alprazolam*, *clonazepam*, *or lorazepam*) with excessive dosage are the more problematic, partly of their addictive potential and partly of their dose presentation, that does not allow a

New lines of study related with BZDs as **BET inhibitor** compounds are an interesting way to change the direction of the therapeutic uses, especially long term. Other new uses, as perioperative, are a valuable way to use an adverse effect derived from the biological activity and

After analyzing the advantages and disadvantages of the **Z-drugs**, it can be concluded that even if they are not exactly as BZD, they must be treated with the same precaution due to the

Despite the amount of biomedical literature on BZDs and Z-drugs, there is still a need to answer vital questions relevant to their effectiveness and safety in society, for example, the possibility of irreversible effects due to extended treatment, especially those associated to new

amount of adverse effect reports that had appeared over the recent years.

high risk and low benefits of the long term in most of the cases.

gradual dosage reduction when withdrawal.

apply it with a clinical purpose.

safety accusations [16].

reduction when withdrawal.

**Table 4.** Half-life and equivalent potencies of BZD anxiolytics [5].

**6. Conclusions**

86 Medicinal Chemistry


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**Section 3**

**Pharmacokinetic of Drugs, Effect of Compound**

**Interactions on Cytochrome P450 Activity**

