**3. Coumarins**

BH3-only members function upstream of Bax and Bak. It is shown that members of the BH3 only subfamily are required for the activation of proapoptotic Bax/Bak function. But it should

In summary, as it is shown in **Figure 6**, in a viable cell antiapoptotic proteins like Bcl-2 antagonize Bax/Bak. In response to an apoptotic stimulus, BH3-only proteins are activated. Activated BH3-only proteins prevent antiapoptotic Bcl-2 members from inhibiting proapoptotic members. Therefore, Bax/Bak are activated and form pores in the mitochondrial membrane. In consequence, cytochrome C and other proapoptotic factors are released from the inner mitochondrial membrane into the cytosol. They cause the formation of the apoptosome and the

Mcl-1 is one of the Bcl-2-related survival proteins but is somewhat structurally distinct and probably lacks a "classical" BH4 domain. It was first discovered in differentiating myeloid cells where Mcl-1 is thought to play a transient role in promoting cell survival, but it has been expressed in various malignant cells, like CLL. Overexpression of Mcl-1 in CLL cells associ-

Mcl-1 protein has a rapid turnover, and it has a short half-life (a few hours). Mcl-1 has a critical role in regulating apoptosis in response to rapidly changing environmental cues. During apoptosis, Mcl-1 is a very efficient substrate for caspases [43–46]. While Mcl-1 is an antiapoptotic protein, its cleavage by caspases converts it into a cell-death-promoting molecule [43]. Therefore, Mcl-1

ated with a failure to achieve complete remission following cytotoxic therapy [42].

be noted that prosurvival members Bcl-2 and Bcl-XL have a role in this way [41].

subsequent activation of the caspase cascade [26].

**Figure 6.** Regulation of apoptosis by the Bcl-2 family [26].

**2.3. Mcl-1 and CLL**

96 Cytotoxicity

Coumarins (2H-1-benzopyran-2-one) consist of a large class of phenolic substances found in plants and all of which consist of a benzene ring joined to a pyrone ring. More than 1300 coumarins have been identified as secondary metabolites from plants, bacteria, and fungi. The prototypical compound is known as 1,2 benzopyrone or, less commonly, as -hydroxycinnamic acid and lactone. Coumarins were initially extracted in *tonka* bean (*Dipteryx odorata* Wild) and are reported in about 150 different species distributed over nearly 30 different families, of which a few important ones are *Rutaceae*, *Umbelliferae* (*Apiaceae*), *Clusiaceae*, *Guttiferae*, *Caprifoliaceae*, *Oleaceae*, and *Nyctaginaceae* [53]. They are found at high levels in some essential oils, particularly in *cinnamon* bark oil, *cassia* leaf oil, and *lavender* oil. Coumarin is also found in fruits (e.g., bilberry and cloudberry), green tea, and other foods such as chicory. The richest sources of most coumarins among the higher plants are *Rutaceae* and *Umbelliferone*. The coumarins occur at the highest levels in the fruits, followed by the roots, stems, and leaves although they are distributed throughout all parts of the plant. Environmental conditions and seasonal changes can influence the occurrence in diverse parts of the plant [54].

### **3.1. Classification**

Based on the chemical structure of their compounds, natural coumarins are classified into six groups (**Table 1**).

Coumarin and its derivatives are principal oral anticoagulants. Coumarin is water insoluble; however, 4-hydroxy substitution confers weakly acidic properties to the molecule that makes it water soluble under slightly alkaline conditions (**Figure 7**) [54].

The structure of coumarin nucleus (**Figure 8**) mimics A and B rings of the steroid hormone and binds to the aromatase-binding site with a superior affinity. Upon tactically extending the structure to the tricyclic system, it mimics the steroid hormones that act as SERM/SERD (selective estrogen receptor modulator/selective estrogen receptor downregulator) and thus enhancing the receptor interaction, leading to a development of a potent pharmacophore. 17b-HSD3 (17b-hydroxysteroid dehydrogenase type3), cell division cycle protein, and NF-kB inhibitory activity are potentiated by structural extension with sulfur linked at the C-4 position [55]. It has also been shown that the anticancer activity of coumarins is potentiated by the substitution of imidazole, 1,2,3-triazol, piperidine purine, benzothiazole, substituted phenyl

ring, aryl acrylic acid, and chalcone at the fourth position of coumarin nucleus by a linker

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**Figure 7.** 4-hydroxy substitution of coumarin makes it water soluble in alkaline conditions.

Induction of apoptosis in leukemic cell lines by coumarins and their derivatives is demonstrated in different in vitro studies. Coumarin compounds have antiproliferative and/or cytotoxic activity on cancer cells, depending on their substitution pattern [56–58]. It is shown that while long alkyl substitution at C7 position increased the cytotoxic activity against the leukemia cancer cell lines [59], the presence of two hydroxyl groups at C7 and C8 positions seems to improve the potency of methylcoumarins as cytotoxic agents. It is also shown that among 7,8- DHMC (dihydroxy-4-methylcoumarin) derivatives, the longer the C3 alkyl chain, the higher was the activity. This effect of the alkyl group on the cytotoxicity is presumably due to the enhanced lipophilicity of the longer alkyl chains that consequently enhances cell membrane penetration ability of the test compounds. Bromo groups substituted at C4 and C6 positions for DHMCs increased the cytotoxic activity in all the cell lines (**Figure 9A**) [60]. In another study, it was shown that 7-hydroxycoumarin analog containing carboxymethyl ester group on cinnamoyl moiety (**Figure 9B**) showed good antiproliferative activity against leukemic cell lines [61]. It is worth noting that the cinnamoyl moiety at C3 is more effective than alkyl chain moiety for increased cytotoxic effect against leukemic cell line K562 (IC50 = 4.4 μM vs. 40.8 μM). Moreover, studies showed that molecular hybridization of coumarins increased their cytotoxicity against leukemic cell lines. For example, the hybrids with ortho-dihydroxy groups or ortho-hydroxy-methoxy group on the aromatic A ring exhibit superior antiproliferative activity in comparison with those with such groups on the aromatic B ring. Specially, a new hybrid, 6-methoxy-7-hydroxy-3-(4`-hydroxyphenyl)coumarin, emerged as an important lead compound with excellent antiproliferative, apoptosis-inducing, and cell cycle arrest activities

such as methylene and oxygen [55].

against HL-60 cell line (IC50 = 5.2 ± 0.6 μM) (**Figure 10**) [62].

**3.2. Coumarins and leukemia**

**Figure 8.** Structure of simple coumarin.


**Table 1.** Classification of natural coumarins based on their chemical structure.

**Figure 7.** 4-hydroxy substitution of coumarin makes it water soluble in alkaline conditions.

**Figure 8.** Structure of simple coumarin.

**3.1. Classification**

98 Cytotoxicity

groups (**Table 1**).

Simple coumarins

Furano coumarins

Phenyl coumarins

Bicoumarins

Dihydrofurano coumarins

Pyrano coumarins (linear types)

Pyrano coumarins (angular types)

Based on the chemical structure of their compounds, natural coumarins are classified into six

Coumarin and its derivatives are principal oral anticoagulants. Coumarin is water insoluble; however, 4-hydroxy substitution confers weakly acidic properties to the molecule that makes

The structure of coumarin nucleus (**Figure 8**) mimics A and B rings of the steroid hormone and binds to the aromatase-binding site with a superior affinity. Upon tactically extending the structure to the tricyclic system, it mimics the steroid hormones that act as SERM/SERD (selective estrogen receptor modulator/selective estrogen receptor downregulator) and thus enhancing the receptor interaction, leading to a development of a potent pharmacophore. 17b-HSD3 (17b-hydroxysteroid dehydrogenase type3), cell division cycle protein, and NF-kB inhibitory activity are potentiated by structural extension with sulfur linked at the C-4 position [55]. It has also been shown that the anticancer activity of coumarins is potentiated by the substitution of imidazole, 1,2,3-triazol, piperidine purine, benzothiazole, substituted phenyl

it water soluble under slightly alkaline conditions (**Figure 7**) [54].

**Type of coumarin General chemical structure**

**Table 1.** Classification of natural coumarins based on their chemical structure.

ring, aryl acrylic acid, and chalcone at the fourth position of coumarin nucleus by a linker such as methylene and oxygen [55].

#### **3.2. Coumarins and leukemia**

Induction of apoptosis in leukemic cell lines by coumarins and their derivatives is demonstrated in different in vitro studies. Coumarin compounds have antiproliferative and/or cytotoxic activity on cancer cells, depending on their substitution pattern [56–58]. It is shown that while long alkyl substitution at C7 position increased the cytotoxic activity against the leukemia cancer cell lines [59], the presence of two hydroxyl groups at C7 and C8 positions seems to improve the potency of methylcoumarins as cytotoxic agents. It is also shown that among 7,8- DHMC (dihydroxy-4-methylcoumarin) derivatives, the longer the C3 alkyl chain, the higher was the activity. This effect of the alkyl group on the cytotoxicity is presumably due to the enhanced lipophilicity of the longer alkyl chains that consequently enhances cell membrane penetration ability of the test compounds. Bromo groups substituted at C4 and C6 positions for DHMCs increased the cytotoxic activity in all the cell lines (**Figure 9A**) [60]. In another study, it was shown that 7-hydroxycoumarin analog containing carboxymethyl ester group on cinnamoyl moiety (**Figure 9B**) showed good antiproliferative activity against leukemic cell lines [61]. It is worth noting that the cinnamoyl moiety at C3 is more effective than alkyl chain moiety for increased cytotoxic effect against leukemic cell line K562 (IC50 = 4.4 μM vs. 40.8 μM).

Moreover, studies showed that molecular hybridization of coumarins increased their cytotoxicity against leukemic cell lines. For example, the hybrids with ortho-dihydroxy groups or ortho-hydroxy-methoxy group on the aromatic A ring exhibit superior antiproliferative activity in comparison with those with such groups on the aromatic B ring. Specially, a new hybrid, 6-methoxy-7-hydroxy-3-(4`-hydroxyphenyl)coumarin, emerged as an important lead compound with excellent antiproliferative, apoptosis-inducing, and cell cycle arrest activities against HL-60 cell line (IC50 = 5.2 ± 0.6 μM) (**Figure 10**) [62].

**Figure 9.** R2 is 4-(COOMe).

**Figure 10.** 6-methoxy-7-hydroxy-3-(4` hydroxyphenyl)coumarin) as a new hybrid.

Paul et al. showed that the synthesis of new conjugated coumarin-benzimidazole hybrids displayed appreciable antileukemic activities in vitro. They showed that the introduction of ethanolamine at position 7 of coumarin-benzimidazole hybrid (**Figure 11**) shows higher selectivity against leukemia cancer cells (CCRF-CEM, HL-60(TB), K-562, and RPMI-8226) [63].

Kim and colleagues studied the antileukemic effects of decursin (a pyranocoumarin from *Angelica gigas*) and its derivatives (**Figure 14**) on K562 and U937 cell lines. They studied the ability of these compounds as a tumor-suppressing PKC activator and as an antagonist to phorbol 12-myristate 13-acetate (PMA), a tumor-promoting PKC activator. Based on their results, the structure-activity relationship of decursin and its derivatives is as follows: (i) the coumarin structure is required for antileukemic activity and (ii) the side chain is a determi-

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In another study, Ahn et al. showed the apoptosis induction by decursin in leukemic KBM-5 cells. They showed that decursin activates caspases 9 and 3 and PARP in KBM-5 cells. They also reported that decursin induced apoptosis via downregulation of COX-2-dependent survivin pathway in KBM-5 myeloid leukemia. In KBM-5 cells, it was reported that targeting

Esculetin (**Figure 15**) is a simple coumarin found in some traditional medicines. Induction of apoptosis in various leukemic cell lines was shown in different studies. Chu and their colleagues are one of the first teams that reported the antileukemic effects of esculetin. They showed that esculetin inhibits the survival of human promyelocytic leukemia HL-60 cells in a concentrationdependent and time-dependent manner. Esculetin induced the release of cytochrome C from mitochondria into cytosol, reduced Bcl-2 protein expression, and increased caspase activation [71].

Esculetin is a cell cycle-specific antineoplastic agent. It can inhibit the growth of HL-60 and U937 leukemic cells by G1 cell cycle arrest [72, 73]. It also leads to the release of cytochrome C, activation of caspases 3, 8, and 9, downregulation of Bcl-2 protein, and increased the phos-

nant of PKC activation and the cytotoxic mechanism in leukemia cells [69].

**Figure 12.** Hydrazide-hydrazone moiety and acrylohydrazide hybrid of coumarin.

survivin could overcome the resistance against imatinib [70].

phorylation of MEK/ERK and JNK [74–77].

**Figure 13.** Copper complexes with coumarin.

Other studies showed that hydrazide-hydrazone (─CO─NH─N═CH─) moiety and acrylohydrazide hybrid at position 3 could increase the cytotoxicity against leukemic cell lines (**Figure 12**) [64, 65].

In other studies, it has been shown that the copper complexes with coumarin derivatives could increase the antileukemic effect of coumarin in vitro (**Figure 13**).

Specifically, in some studies, the significant inhibitory activity of certain coumarins on the proliferation of leukemic cell lines [58, 66–68] has been reported. In addition, it has been described that such inhibitory effects could be related to either differentiating [58, 66] or proapoptotic activities [67, 68] of the compounds, depending on the distribution of their substituents in the coumarin ring.

**Figure 11.** NR1 R2 is ethanolamine.

**Figure 12.** Hydrazide-hydrazone moiety and acrylohydrazide hybrid of coumarin.

**Figure 13.** Copper complexes with coumarin.

Paul et al. showed that the synthesis of new conjugated coumarin-benzimidazole hybrids displayed appreciable antileukemic activities in vitro. They showed that the introduction of ethanolamine at position 7 of coumarin-benzimidazole hybrid (**Figure 11**) shows higher selectivity against leukemia cancer cells (CCRF-CEM, HL-60(TB), K-562, and RPMI-8226) [63].

Other studies showed that hydrazide-hydrazone (─CO─NH─N═CH─) moiety and acrylohydrazide hybrid at position 3 could increase the cytotoxicity against leukemic cell lines

In other studies, it has been shown that the copper complexes with coumarin derivatives

Specifically, in some studies, the significant inhibitory activity of certain coumarins on the proliferation of leukemic cell lines [58, 66–68] has been reported. In addition, it has been described that such inhibitory effects could be related to either differentiating [58, 66] or proapoptotic activities [67, 68] of the compounds, depending on the distribution of their substitu-

could increase the antileukemic effect of coumarin in vitro (**Figure 13**).

**Figure 10.** 6-methoxy-7-hydroxy-3-(4` hydroxyphenyl)coumarin) as a new hybrid.

(**Figure 12**) [64, 65].

**Figure 9.** R2 is 4-(COOMe).

100 Cytotoxicity

ents in the coumarin ring.

**Figure 11.** NR1

R2

is ethanolamine.

Kim and colleagues studied the antileukemic effects of decursin (a pyranocoumarin from *Angelica gigas*) and its derivatives (**Figure 14**) on K562 and U937 cell lines. They studied the ability of these compounds as a tumor-suppressing PKC activator and as an antagonist to phorbol 12-myristate 13-acetate (PMA), a tumor-promoting PKC activator. Based on their results, the structure-activity relationship of decursin and its derivatives is as follows: (i) the coumarin structure is required for antileukemic activity and (ii) the side chain is a determinant of PKC activation and the cytotoxic mechanism in leukemia cells [69].

In another study, Ahn et al. showed the apoptosis induction by decursin in leukemic KBM-5 cells. They showed that decursin activates caspases 9 and 3 and PARP in KBM-5 cells. They also reported that decursin induced apoptosis via downregulation of COX-2-dependent survivin pathway in KBM-5 myeloid leukemia. In KBM-5 cells, it was reported that targeting survivin could overcome the resistance against imatinib [70].

Esculetin (**Figure 15**) is a simple coumarin found in some traditional medicines. Induction of apoptosis in various leukemic cell lines was shown in different studies. Chu and their colleagues are one of the first teams that reported the antileukemic effects of esculetin. They showed that esculetin inhibits the survival of human promyelocytic leukemia HL-60 cells in a concentrationdependent and time-dependent manner. Esculetin induced the release of cytochrome C from mitochondria into cytosol, reduced Bcl-2 protein expression, and increased caspase activation [71].

Esculetin is a cell cycle-specific antineoplastic agent. It can inhibit the growth of HL-60 and U937 leukemic cells by G1 cell cycle arrest [72, 73]. It also leads to the release of cytochrome C, activation of caspases 3, 8, and 9, downregulation of Bcl-2 protein, and increased the phosphorylation of MEK/ERK and JNK [74–77].

in U-937 cells, involving decreased phosphorylation levels of ERK and Akt, 50 μM toddaculin

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Umbelliprenin (**Figure 19**) is a prenylated coumarin found in *Ferula* species. Its antileukemic effect was first reported by Gholami and his colleagues. They found that umbelliprenin has cytotoxic and proapoptotic effects on Jurkat and Raji cell lines. They showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and

Auraptene (**Figure 20**) is another coumarin that has a structure close to that of umbelliprenin. The difference between the chemical structures of these compounds is that the length of the 7-prenyloxy chain of umbelliprenin is longer and contains 15 instead of 10 carbons. Apoptogenic activity of auraptene on jurkat cells was shown in detail. Apoptotic effect of auraptene on Jurkat T-cells was exerted by the ER stress-mediated activation of caspase-8 and the subsequent induction of mitochondria-dependent or -independent activation of caspase

Coumarins can regulate the expression of Mcl-1. Their regulation is time- and dose-dependent. The regulation of Mcl-1 expression by auraptene, umbelliprenin, imperatorin, galbanic acid,

exerted differentiating effects [86].

**3.3. Mcl-1 and coumarins**

and gut-70 was studied.

**Figure 16.** Structure of kayeassamin A.

**Figure 17.** Structure of osthole (A) and imperatorin (B).


cascade, which could be suppressed by Bcl-xL [89].

**Figure 14.** Structures of decursin (1) and its derivatives 2–12.

**Figure 15.** Structure of esculetin.

Tung et al. fractionated and chemically investigated the methanol extract of *Mammea siamensis* flower, an evergreen tree belonging to the family of Calophyllaceae, and distributed throughout Thailand, Myanmar, Laos, Cambodia, and Vietnam. They isolated and identified eight compounds. Among the isolated compounds, three structurally related coumarins kayeassamin A (**Figure 16**), surangin C, and theraphin B showed significant antiproliferative activity against human leukemia HL-60 cells. Activation of caspases 3 and 8 and sub-G1 arrest by kayeassamin A have been shown in this study and another one [78, 79].

Osthole (**Figure 17**) is another coumarin where its antileukemic effect has been investigated. It has been shown that osthole has the strongest cytotoxic activity among the coumarins extracted from *Cnidii monnieri Fructus* on HL-60 cell line. The structure-activity relationship established from the results indicated that the prenyl group has an important role in the cytotoxic effects and apoptosis induction [80].

In another study, osthole could increase intracellular drug accumulation, decreased the expression of multidrug resistance gene 1 (MDR1), and could suppress P-gp expression by inhibiting the PI3K/Akt-signaling pathway in myelogenous leukemia K562/ADM cells [81].

Imperatorin, a biologically active furanocoumarin, is another coumarin that is extracted from *Cnidii monnieri Fructus* also showed cytotoxic effect against leukemic cell lines [82–85].

Toddaculin (**Figure 18**) is another important coumarin where its antileukemic effect is revealed. Vazquez et al. found that toddaculin was the most potent cytotoxic agent among the series of six prenylated coumarins isolated from the stem bark of *Toddalia asiatica* (*Rutaceae*). They found that while toddaculin at 250 μM (IC50 = 51.38 ± 4.39) was able to induce apoptosis in U-937 cells, involving decreased phosphorylation levels of ERK and Akt, 50 μM toddaculin exerted differentiating effects [86].

Umbelliprenin (**Figure 19**) is a prenylated coumarin found in *Ferula* species. Its antileukemic effect was first reported by Gholami and his colleagues. They found that umbelliprenin has cytotoxic and proapoptotic effects on Jurkat and Raji cell lines. They showed that umbelliprenin activates intrinsic and extrinsic pathways of apoptosis by the activation of caspase-8 and -9, respectively. Inhibition of Bcl-2 was also shown [87, 88].

Auraptene (**Figure 20**) is another coumarin that has a structure close to that of umbelliprenin. The difference between the chemical structures of these compounds is that the length of the 7-prenyloxy chain of umbelliprenin is longer and contains 15 instead of 10 carbons. Apoptogenic activity of auraptene on jurkat cells was shown in detail. Apoptotic effect of auraptene on Jurkat T-cells was exerted by the ER stress-mediated activation of caspase-8 and the subsequent induction of mitochondria-dependent or -independent activation of caspase cascade, which could be suppressed by Bcl-xL [89].

#### **3.3. Mcl-1 and coumarins**

Tung et al. fractionated and chemically investigated the methanol extract of *Mammea siamensis* flower, an evergreen tree belonging to the family of Calophyllaceae, and distributed throughout Thailand, Myanmar, Laos, Cambodia, and Vietnam. They isolated and identified eight compounds. Among the isolated compounds, three structurally related coumarins kayeassamin A (**Figure 16**), surangin C, and theraphin B showed significant antiproliferative activity against human leukemia HL-60 cells. Activation of caspases 3 and 8 and sub-G1 arrest by

Osthole (**Figure 17**) is another coumarin where its antileukemic effect has been investigated. It has been shown that osthole has the strongest cytotoxic activity among the coumarins extracted from *Cnidii monnieri Fructus* on HL-60 cell line. The structure-activity relationship established from the results indicated that the prenyl group has an important role in the cyto-

In another study, osthole could increase intracellular drug accumulation, decreased the expression of multidrug resistance gene 1 (MDR1), and could suppress P-gp expression by inhibiting the PI3K/Akt-signaling pathway in myelogenous leukemia K562/ADM cells [81].

Imperatorin, a biologically active furanocoumarin, is another coumarin that is extracted from

Toddaculin (**Figure 18**) is another important coumarin where its antileukemic effect is revealed. Vazquez et al. found that toddaculin was the most potent cytotoxic agent among the series of six prenylated coumarins isolated from the stem bark of *Toddalia asiatica* (*Rutaceae*). They found that while toddaculin at 250 μM (IC50 = 51.38 ± 4.39) was able to induce apoptosis

*Cnidii monnieri Fructus* also showed cytotoxic effect against leukemic cell lines [82–85].

kayeassamin A have been shown in this study and another one [78, 79].

toxic effects and apoptosis induction [80].

**Figure 14.** Structures of decursin (1) and its derivatives 2–12.

**Figure 15.** Structure of esculetin.

102 Cytotoxicity

Coumarins can regulate the expression of Mcl-1. Their regulation is time- and dose-dependent. The regulation of Mcl-1 expression by auraptene, umbelliprenin, imperatorin, galbanic acid, and gut-70 was studied.

**Figure 16.** Structure of kayeassamin A.

**Figure 17.** Structure of osthole (A) and imperatorin (B).

**Figure 18.** Structure of toddaculin.

**Figure 19.** Structure of umbelliprenin.

**Figure 20.** Structure of auraptene.

GUT-70 (**Figure 22**), a tricyclic coumarin derived from *Calophyllum brasiliense,* causes Mcl-1

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The effect of synthetic coumarins (RKS262, 5,7-dihydroxy-4-methyl-6-(3-methylbutanoyl) coumarin (DMAC), and 4-arylcoumarin analogs of combretastatin (**Figure 23**)) on Mcl-1 protein expression was studied. All of these compounds downregulate Mcl-1 protein dose- and

In conclusion, coumarins are one of the important cytotoxic agents. They could induce apoptosis and regulate Mcl-1 expression in CLL cell lines. We hope that they be one of the candi-

protein upregulation in mantle cell lymphoma (MCL) cell lines [95].

**Figure 23.** (A) RKS262, (B) DMAC, and (C) 4-arylcoumarin analogs of combretastatin.

time- dependently [96–98].

dates for chemotherapy of CLL in the future.

**4. Conclusion**

**Figure 22.** GUT-70.

**Figure 21.** Galbanic acid.

Gholami et al. studied and compared the expression of Mcl-1 gene after the Jurkat cells were incubated by umbelliprenin and auraptene. They showed that umbelliprenin increased the expression of Mcl-1 mRNA from 1 to 3 h of incubation, but this increase has a scale-down pattern. Auraptene decreased the expression of Mcl-1 mRNA for the same incubation times [90, 91]. This pattern is similar for Mcl-1 protein expression [91, 92].

Another natural coumarin where its effect on Mcl-1 expression was studied is galbanic acid (**Figure 21**). Galbanic acid downregulates the Mcl-1 protein expression dose dependently [93]. Imperatorin (**Figure 17B**), another natural coumarin like galbanic acid, decreased Mcl-1 protein level in a dose-dependent manner [94].

**Figure 22.** GUT-70.

**Figure 23.** (A) RKS262, (B) DMAC, and (C) 4-arylcoumarin analogs of combretastatin.

GUT-70 (**Figure 22**), a tricyclic coumarin derived from *Calophyllum brasiliense,* causes Mcl-1 protein upregulation in mantle cell lymphoma (MCL) cell lines [95].

The effect of synthetic coumarins (RKS262, 5,7-dihydroxy-4-methyl-6-(3-methylbutanoyl) coumarin (DMAC), and 4-arylcoumarin analogs of combretastatin (**Figure 23**)) on Mcl-1 protein expression was studied. All of these compounds downregulate Mcl-1 protein dose- and time- dependently [96–98].

#### **4. Conclusion**

Gholami et al. studied and compared the expression of Mcl-1 gene after the Jurkat cells were incubated by umbelliprenin and auraptene. They showed that umbelliprenin increased the expression of Mcl-1 mRNA from 1 to 3 h of incubation, but this increase has a scale-down pattern. Auraptene decreased the expression of Mcl-1 mRNA for the same incubation times [90,

Another natural coumarin where its effect on Mcl-1 expression was studied is galbanic acid (**Figure 21**). Galbanic acid downregulates the Mcl-1 protein expression dose dependently [93]. Imperatorin (**Figure 17B**), another natural coumarin like galbanic acid, decreased Mcl-1 pro-

91]. This pattern is similar for Mcl-1 protein expression [91, 92].

tein level in a dose-dependent manner [94].

**Figure 18.** Structure of toddaculin.

104 Cytotoxicity

**Figure 19.** Structure of umbelliprenin.

**Figure 20.** Structure of auraptene.

**Figure 21.** Galbanic acid.

In conclusion, coumarins are one of the important cytotoxic agents. They could induce apoptosis and regulate Mcl-1 expression in CLL cell lines. We hope that they be one of the candidates for chemotherapy of CLL in the future.
