marine drugs Article

Design and Synthesis of Analogues of Marine Natural Product Galaxamide, an N-methylated Cyclic Pentapeptide, as Potential Anti-Tumor Agent in Vitro Jignesh Lunagariya 1,† , Shenghui Zhong 1,† , Jianwei Chen 1 , Defa Bai 2 , Poonam Bhadja 3 , Weili Long 1 , Xiaojian Liao 1, *, Xiaoli Tang 4, * and Shihai Xu 1, * 1

2 3 4

* †

Department of Chemistry, Life Science School, Jinan University, Guangzhou 510632, China; [email protected] (J.L.); [email protected] (S.Z.); [email protected] (J.C.); [email protected] (W.L.) College of Pharmacy, Jinan University, Guangzhou 510632, China; [email protected] Institute of Biomineralization and Lithiasis Research, Department of Chemistry, Life Science School, Jinan University, Guangzhou 510632, China; [email protected] Alpert Medical School, Brown University, 55 Claverick St. 4th Floor, Providence, RI 02903, USA Correspondence: [email protected] (X.L.); [email protected] (X.T.); [email protected] (S.X.); Tel.: +86-20-85-221-346 (S.X.) These authors contributed equally to this work.

Academic Editor: Paul Long Received: 2 August 2016; Accepted: 26 August 2016; Published: 3 September 2016

Abstract: Herein, we report design and synthesis of novel 26 galaxamide analogues with N-methylated cyclo-pentapeptide, and their in vitro anti-tumor activity towards the panel of human tumor cell line, such as, A549, A549/DPP, HepG2 and SMMC-7721 using MTT assay. We have also investigated the effect of galaxamide and its representative analogues on growth, cell-cycle phases, and induction of apoptosis in SMMC-7721 cells in vitro. Reckon with the significance of conformational space and N-Me aminoacid (aa) comprising this compound template, we designed the analogues with modification in N-Me-aa position, change in aa configuration from L to D aa and substitute one Leu-aa to D / L Phe-aa residue with respective to the parent structure. The efficient solid phase parallel synthesis approach is employed for the linear pentapeptide residue containing N-Me aa, followed by solution phase macrocyclisation to afford target cyclo pentapeptide compounds. In the present study, all galaxamide analogues exhibited growth inhibition in A549, A549/DPP, SMMC-7721 and HepG2 cell lines. Compounds 6, 18, and 22 exhibited interesting activities towards all cell line tested, while Compounds 1, 4, 15, and 22 showed strong activity towards SMMC-7221 cell line in the range of 1–2 µg/mL IC50 . Flow cytometry experiment revealed that galaxamide analogues namely Compounds 6, 18, and 22 induced concentration dependent SMMC-7721 cell apoptosis after 48 h. These compounds induced G0/G1 phase cell-cycle arrest and morphological changes indicating induction of apoptosis. Thus, findings of our study suggest that the galaxamide and its analogues 6, 18 and 22 exerted growth inhibitory effect on SMMC-7721 cells by arresting the cell cycle in the G0/G1 phase and inducing apoptosis. Compound 1 showed promising anti-tumor activity towards SMMC-7721 cancer cell line, which is 9 and 10 fold higher than galaxamide and reference DPP (cisplatin), respectively. Keywords: anti-tumor; apoptosis; cyclic pentapeptide; galaxamide analogues; macrocyclisation

1. Introduction The marine environment is the vital source of living organisms [1,2], which provides diversity of natural products that pave the way for medicinal scientists to discover well-suited bioactive compounds Mar. Drugs 2016, 14, 161; doi:10.3390/md14090161

www.mdpi.com/journal/marinedrugs

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2 of 21  forMar. Drugs 2016, 14, 161  the biological systems [3]. Among them, peptides are very interesting target following their compounds for the biological systems [3]. Among them, peptides are very interesting target following  important role as hormones [4], neurotransmitters [5], growth factors [6], ion channel ligands [7], or their important role as hormones [4], neurotransmitters [5], growth factors [6], ion channel ligands  anti-infectives [8]. Nearly, more than 7000 naturally occurring peptides have been identified [9],   [7], or anti‐infectives [8]. Nearly, more than 7000 naturally occurring peptides have been identified including several bioactive peptides. Peptides and their homologous compounds (proteins and [9], including several bioactive peptides. Peptides and their homologous compounds (proteins and  antibodies) can be used in multiple pathologies, including allergy and asthma [10], arthritis [11], antibodies)  can  be  used  in diseases multiple [13], pathologies,  allergy  and  asthma  [10],  arthritis  [11],  cancer [12], cardiovascular diabetesincluding  [14], gastrointestinal dysfunction [15], HIV [16], cancer  [12],  cardiovascular  diseases  [13],  diabetes  [14],  gastrointestinal  dysfunction  [15],  HIV  [16],  infective diseases [17], inflammation [18], pain [19], and so on. infective diseases [17], inflammation [18], pain [19], and so on.  At present, around 100 peptide medicines on the market, and which is expected to grow At  present,  around  100  peptide  medicines  on  the  market,  and  which  is  expected  to  grow  considerably [20], with about 140 currently in clinical trials and more than 500 therapeutic peptides in considerably [20], with about 140 currently in clinical trials and more than 500 therapeutic peptides  preclinical development [9]. in preclinical development [9].  In recent time, approve drug and candidates from small molecules in clinical trials are decreasing, In  recent  time,  approve  drug  and  candidates  from  small  molecules  in  clinical  trials  are  which led scientists to pay more attention towards peptides and other alternatives [21]. The probability decreasing, which led scientists to pay more attention towards peptides and other alternatives [21].  of The probability of regulatory approval is 20% for the peptide drugs with respect to 10% for the small  regulatory approval is 20% for the peptide drugs with respect to 10% for the small molecule [22,23], as molecule [22,23], as the small molecules are not selective and can accumulate in specific organs such  the small molecules are not selective and can accumulate in specific organs such as the kidney and liver, resulting in severe toxic side effects [24]. as the kidney and liver, resulting in severe toxic side effects [24].  In In peptide drug portfolio, around half of the peptides in clinical trial have target indication in  peptide drug portfolio, around half of the peptides in clinical trial have target indication in oncology, metabolic, cardiovascular and infectious diseasesdiseases  [24,25]. In[24,25].  particular, pentapeptides oncology,  metabolic,  cardiovascular  and  infectious  In  cyclic particular,  cyclic  have been demonstrated as anti-tumor agent, and shown cytotoxicity towards several types of tumors, pentapeptides have been demonstrated as anti‐tumor agent, and shown cytotoxicity towards several  including pancreatic [26–28], colon [29–32], breast, prostate, and melanoma cancers [26,33,34], which types of tumors, including pancreatic [26–28], colon [29–32], breast, prostate, and melanoma cancers  [26,33,34],  which  proved  high  potential  in  targeting  cancer.  Following  of  natural  proved its high potential in its  targeting the cancer. Followingthe  discovery of natural discovery  product sansalvamide, product  sansalvamide,  a  cyclic  depsipeptide,  several  reports  have  been  demonstrated  anti‐tumor  a cyclic depsipeptide, several reports have been demonstrated anti-tumor activity of Sansalvamide A activity of Sansalvamide A analogues, including N‐methylsanslavamide A analogues as a variety of  analogues, including N-methylsanslavamide A analogues as a variety of anti-tumor agent by Silverman anti‐tumor agent by Silverman group [26]. Accordingly, McAlpine group also published numerous  group [26]. Accordingly, McAlpine group also published numerous articles on San A, such as, articles on San A, such as, analogues as treatment of MSS colon cancer [30], synthesis of analogues as  analogues as treatment of MSS colon cancer [30], synthesis of analogues as potential anti-tumor [34], potential anti‐tumor [34], and potent derivative against pancreatic cancer cell lines [27]. Along the  and potent derivative against pancreatic cancer cell lines [27]. Along the same line, galaxamide 1, line,  galaxamide  1,  an  N‐methylated  cyclic  pentapeptide,  has  been  discovered  from  marine  an same  N-methylated cyclic pentapeptide, has been discovered from marine algae Galaxaura filamentosa with algae Galaxaura filamentosa with structural determination and first total synthesis by our group [35].  structural determination and first total synthesis by our group [35]. Galaxamide 1and general structure Galaxamide  1and  general  structure  of  its  analogues  have  shown  in  Figure  1.  Galaxamide  and  its  of its analogues have shown in Figure 1. Galaxamide and its analogues have also shown potential anti analogues  have  also  shown  potential  anti  tumor  activity  [36].  All  of  these  analogues,  including  tumor activity [36]. All of these analogues, including sansalvamide analogues, have clearly suggested sansalvamide analogues, have clearly suggested that the incorporation of N‐Me and  D aa alter the  that the incorporation of N-Me and D aa alter the conformational space and hydrogen bonding, conformational  space  and  hydrogen  bonding,  which  consequently  play  a  crucial  role  in  the  which consequently playpharmacokinetic  a crucial role in property  the cytotoxicity and other pharmacokinetic property of the cytotoxicity  and  other  of  the  compound.  Hence,  in  continuation  of  our  compound. Hence, in continuation of our interest to find more potent lead compound as anti-tumor interest to find more potent lead compound as anti‐tumor agent on the basis of conformational space  agent on the basisbonding,  of conformational space and hydrogen bonding,of we the solid phase synthesis and  hydrogen  we  report  the  solid  phase  synthesis  26 report galaxamide  analogues  with  of modification in N‐Me‐aa position, change in aa configuration from  26 galaxamide analogues with modification in N-Me-aa position, Lchange in aa configuration from L  to D aa and substitute one Leu‐ to aa to D aa  D and substitute one Leu-aa to D / L Phe-aa residue with respective to the parent structure, and /L Phe‐aa residue with respective to the parent structure, and their in vitro anti‐tumor activity  their in vitro anti-tumor activity against A549, A549/DPP, SMMC-7721 and HepG2 cell lines using against A549, A549/DPP, SMMC‐7721 and HepG2 cell lines using MTT assay. We further investigated  MTT assay. We further investigated its mechanism of action by detecting cell morphology, cell-cycle its mechanism of action by detecting cell morphology, cell‐cycle progression, and apoptosis using  SMMC‐7721 as representative cell line model.  progression, and apoptosis using SMMC-7721 as representative cell line model.

  Figure 1. Structure of galaxamide and general structure of its derivative.  Figure 1. Structure of galaxamide and general structure of its derivative.

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Mar. Drugs 2016, 14, 161  2. Results

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2. Results  2.1.2. Results  Chemistry

2.1. Chemistry  Recently, in most cases, synthesis of cyclo pentapeptide was employed on segment based solution 2.1. Chemistry  Recently,  in  most  cases,  synthesis  of  cyclo  was itsemployed  on have segment  phase synthetic strategy [27,30,34–36]. Synthesis of pentapeptide  galaxamide and analogues been based  reported Recently,  in  most  cases,  synthesis  of  cyclo  pentapeptide  was  employed  on  segment  based   [27,30,34–36]. Synthesis of galaxamide and its analogues have been  solution phase synthetic strategy by our group with a similar strategy [35,36].  [27,30,34–36]. Synthesis of galaxamide and its analogues have been  solution phase synthetic strategy reported by our group with a similar strategy [35,36].  In present study, we synthesized linear pentapeptide using Fmoc peptide synthetic strategy with reported by our group with a similar strategy [35,36].  2-chloro In present study, we synthesized linear pentapeptide using Fmoc peptide synthetic strategy with  trityl resin solid phase (Scheme 1), which is very proficient in a way to efficient, speed, easy In present study, we synthesized linear pentapeptide using Fmoc peptide synthetic strategy with  2‐chloro trityl resin solid phase (Scheme 1), which is very proficient in a way to efficient, speed, easy  to handle and high purity of crude product. Synthesis of D/L N-Me Leu aa residue were performed 2‐chloro trityl resin solid phase (Scheme 1), which is very proficient in a way to efficient, speed, easy  to handle and high purity of crude product. Synthesis of  D/L N‐Me Leu aa residue were performed  as to handle and high purity of crude product. Synthesis of  per reported method by Zhang et al. [37] (Scheme 2). Briefly, N-Fmoc protected aa was converted D/L N‐Me Leu aa residue were performed  as per reported method by Zhang et al. [37] (Scheme 2). Briefly, N‐Fmoc protected aa was converted  into 5-oxazolidinones derivative using para formaldehyde in the presence of catalytic amount of as per reported method by Zhang et al. [37] (Scheme 2). Briefly, N‐Fmoc protected aa was converted  into 5‐oxazolidinones derivative using para formaldehyde in the presence of catalytic amount of p‐ p-toluenesulfonic under azeotropic  azeotropicwater  waterremoval  removal condition, followed into 5‐oxazolidinones derivative using para formaldehyde in the presence of catalytic amount of p‐ toluenesulfonic acid acid (PTSA) (PTSA) in in toluene toluene  under  condition,  followed  by  by reduction with triethylsilane in the presence of lewis acid to provide N-Me aa. This method is toluenesulfonic  acid  (PTSA)  in  toluene  under  azeotropic  water  removal  condition,  followed more by  reduction with triethylsilane in the presence of lewis acid to provide N‐Me aa. This method is more  convenient over N-Me with sodium hydride/methyl iodide according to Benoiton’s protocol [38]. reduction with triethylsilane in the presence of lewis acid to provide N‐Me aa. This method is more  convenient over N‐Me with sodium hydride/methyl iodide according to Benoiton’s protocol [38]. 

convenient over N‐Me with sodium hydride/methyl iodide according to Benoiton’s protocol [38]. 

  Scheme 1. Reaction scheme for linear peptide on solid phase. Reagents and conditions: a, (i) dichloro  Scheme 1. Reaction scheme for linear peptide on solid phase. Reagents and conditions:   a, methane  (DCM),  N,N‐Diisopropylethylamine  (DIPEA)  4  eq.,  room  temperature  (rt);  (ii)  (i) Scheme 1. Reaction scheme for linear peptide on solid phase. Reagents and conditions: a, (i) dichloro  dichloro methane (DCM), N,N-Diisopropylethylamine (DIPEA) 4 eq., room temperature (rt); DCM/MeOH/DIPEA (4/5/1), 30 min, rt; b, (i) 20% piperidine in N,N‐Dimethylformamide (DMF) (2 ×  (ii)methane  DCM/MeOH/DIPEA (4/5/1), 30 min, rt; b, (i) 20% piperidine in N,N-Dimethylformamide (DCM),  N,N‐Diisopropylethylamine  (DIPEA)  4  eq.,  room  temperature  (rt);  (DMF) (ii)  10 min); (ii) DIPEA 2 eq., DEPBT 2 eq., DCM/DMF (1/1), 3 h, rt; c, 1% TFA in DCM for (2 × 30 min), rt.  (2 DCM/MeOH/DIPEA (4/5/1), 30 min, rt; b, (i) 20% piperidine in N,N‐Dimethylformamide (DMF) (2 ×  × 10 min); (ii) DIPEA 2 eq., DEPBT 2 eq., DCM/DMF (1/1), 3 h, rt; c, 1% TFA in DCM for (2 × 30 min), rt. 10 min); (ii) DIPEA 2 eq., DEPBT 2 eq., DCM/DMF (1/1), 3 h, rt; c, 1% TFA in DCM for (2 × 30 min), rt. 

Fmoc

Fmoc

N H

COOH

a

Fmoc

a

Fmoc

b

N

NO

O

b

Fmoc

Fmoc

N

COOH

 

COOH N N COOH Scheme 2. Freidinger synthesis of N‐methylated Leucine. Reagents and conditions: a, (CH 2O)n, PTSA  O H O  

(cat.), toluene, azeotropic removal of water; b, Et3SiH, CF3COOH, CHCl3. 

Scheme 2. Freidinger synthesis of N‐methylated Leucine. Reagents and conditions: a, (CH Scheme 2. Freidinger synthesis of N-methylated Leucine. Reagents and conditions: 2O)n, PTSA  a, (CH2 O)n, First aa residue was immobilized on resin using 4.0 eq. N,N‐Diisopropylethylamine (DIPEA) in  3SiH, CF3COOH, CHCl3.  (cat.), toluene, azeotropic removal of water; b, Et PTSA (cat.), toluene, azeotropic removal of water; b, Et3 SiH, CF3 COOH, CHCl3 .

dichloro methane (DCM) for 2 h at room temperature (rt). Subsequent aa coupling including N‐me  aa was performed via 3‐(Diethoxyphosphoryloxy)‐1,2,3‐benzotriazin‐4(3H)‐one (DEPBT) mediated  First aa residue was immobilized on resin using 4.0 eq. N,N‐Diisopropylethylamine (DIPEA) in  First aa residue was immobilized on resin using 4.0 eq. N,N-Diisopropylethylamine (DIPEA) in activation in DCM with DIPEA as base. DEPBT was chosen as more convenient for N‐Me aa coupling  dichloro methane (DCM) for 2 h at room temperature (rt). Subsequent aa coupling including N‐me  dichloro methane (DCM) for 2 h at room temperature (rt). Subsequent aa coupling including N-me and negligible racemization [39]. Solid‐phase reactions were monitored by use of a qualitative Kaiser  aa was performed via 3‐(Diethoxyphosphoryloxy)‐1,2,3‐benzotriazin‐4(3H)‐one (DEPBT) mediated  test  [40]  for  the  detection of  primary amines  and  the  chloranil  test  [41]  for  detection  of  secondary  aa activation in DCM with DIPEA as base. DEPBT was chosen as more convenient for N‐Me aa coupling  was performed via 3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT) mediated

activation in DCM with DIPEA as base. DEPBT was chosen as more convenient for N-Me aa coupling and negligible racemization [39]. Solid‐phase reactions were monitored by use of a qualitative Kaiser  test  [40]  for  the  detection of  primary amines  and  the  chloranil  test  [41]  for  detection  of  secondary 

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and negligible racemization [39]. Solid-phase reactions were monitored by use of a qualitative Kaiser 4 of 21  testMar. Drugs 2016, 14, 161  [40] for the detection of primary amines and the chloranil test [41] for detection of secondary Mar. Drugs 2016, 14, 161  4 of 21  amines. Coupling Coupling reaction reaction was was completed completed  within  aa,  which  amines. within 2 2 h  h except  exceptfor  forcoupling  couplingwith  withN‐Me  N-Me aa, which amines.  Coupling  reaction  was  completed  within  2  h  except  for  coupling  with  N‐Me  aa,  which  generally took 8–10 h. Upon cleavage from solid support with 1% TFA in DCM linear pentapeptide  generally took 8–10 h. Upon cleavage from solid support with 1% TFA in DCM linear pentapeptide generally took 8–10 h. Upon cleavage from solid support with 1% TFA in DCM linear pentapeptide  was obtained in 50%–60% overall yield with 90%–95% purity which was verified by RP‐HPLC.  was obtained in 50%–60% overall yield with 90%–95% purity which was verified by RP-HPLC. was obtained in 50%–60% overall yield with 90%–95% purity which was verified by RP‐HPLC.  The  linear  pentapeptide  was  isolated  by  precipitation  from  diethylether,  collected  by  The linear pentapeptide was isolated by precipitation from diethylether, collected bycollected  centrifugation, The  linear  pentapeptide  was  isolated  by  precipitation  from  diethylether,  by  centrifugation, which can be used for macrocyclisation without further purification. Key challenge  which can be used for macrocyclisation without further purification. Key challenge for macrocyclisation centrifugation, which can be used for macrocyclisation without further purification. Key challenge  for  macrocyclisation  is  lower  yield  due  to  many  intermolecular  reactions,  resulting  into  various  is for  lower yield due to many intermolecular reactions, resulting into various complex oligomeric macrocyclisation  lower  yield  due  to some  many  intermolecular  reactions,  resulting  into  various  complex  oligomeric  is  products.  Recently,  advancement  on  macrocyclisation  reaction  of  products. Recently, some advancement on macrocyclisation reaction of pentapeptide have complex  oligomeric  Recently,  that  some (Benzotriazol‐1‐yloxy)tripyrrolidinophosphonium  advancement  on  macrocyclisation  reaction  been of  pentapeptide  have  products.  been  suggested  suggested that (Benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP) is pentapeptide  have  been  suggested  that  (Benzotriazol‐1‐yloxy)tripyrrolidinophosphonium  hexafluorophosphate  (PyBOP)  is  better  coupling  reagent  for  obtaining  higher  yield  with  short  better coupling reagent(PyBOP)  for obtaining higher yield reagent  with short reaction time [42]. After screening hexafluorophosphate  is  better  coupling  for  obtaining  higher  yield  with  short  reaction time [42]. After screening range of dilution of solvent DCM and base equivalent, employing  range of dilution of solvent DCM and base equivalent, employing PyBOP as a coupling reagent reaction time [42]. After screening range of dilution of solvent DCM and base equivalent, employing  PyBOP  as  a  coupling  reagent  for  macrocyclisation,  promising  reaction  condition  with  0.0007  M for PyBOP  as  a  coupling  reagent  for  macrocyclisation,  promising  reaction  condition  with  0.0007  M  macrocyclisation, promising reaction condition with 0.0007 M dilution of solvent DCM in the presence dilution of solvent DCM in the presence of 2 eq. of base DIPEA and 2 eq. PyBOP have been established  dilution of solvent DCM in the presence of 2 eq. of base DIPEA and 2 eq. PyBOP have been established  of 2(Scheme 3). After 24 h reaction, targeted cyclo pentapeptides were obtained in 44%–66% yield after  eq. of base DIPEA and 2 eq. PyBOP have been established (Scheme 3). After 24 h reaction, targeted (Scheme 3). After 24 h reaction, targeted cyclo pentapeptides were obtained in 44%–66% yield after  cyclo pentapeptides were obtained in 44%–66% yield after preparative RP-HPLC purification. preparative RP‐HPLC purification.  preparative RP‐HPLC purification. 

   

Scheme 3. Macrocylclisation of linear pentapeptide.  Scheme 3. Macrocylclisation of linear pentapeptide. Scheme 3. Macrocylclisation of linear pentapeptide. 

Structure of Macrocyclic Analogues  Structure of Macrocyclic Analogues Structure of Macrocyclic Analogues  We  designed  and  synthesized  three  subsets  of  galaxamide  derivatives.  These  were  mainly  WeWe  designed and synthesized derivatives.These  Thesewere  were mainly designed  and  synthesized three three subsets subsets of of galaxamide galaxamide  derivatives.  mainly  differentiated through N‐Me position, addition to that multi changes have been incorporated with  differentiated through N-Me position, addition to that multi changes have been incorporated with differentiated through N‐Me position, addition to that multi changes have been incorporated with  position and number of  D configuration aa residues, which was common in all three subset (Figure 2).  position and number of DDconfiguration aa residues, which was common in all three subset (Figure 2). position and number of   configuration aa residues, which was common in all three subset (Figure 2). 

Figure 2. Amino acid used at various position for galaxamide derivative.  Figure 2. Amino acid used at various position for galaxamide derivative.  Figure 2. Amino acid used at various position for galaxamide derivative.

   

Some compounds also include two N‐Me aa residue product (Compound 5, 19, and 26). The first  Some compounds also include two N‐Me aa residue product (Compound 5, 19, and 26). The first  subset  compounds also contains  N‐Me  residue  at  third  position  (Compound  Figure  The  Someof compounds include twoaa  N-Me aa residue product (Compound 5,1–5,  19, and 26).3).  The first subset  compounds  aa at  residue  third  position  (Compound  1–5,  Figure  3). third  The  second of  subset  contain contains  N‐Me  aa N‐Me  residue  fourth at  position  (Compounds  6–19,  Figure  4).  The  subset of compounds contains N-Me aa residue at third position (Compound 1–5, Figure 3). The second second  subset  contain  N‐Me  aa  residue  at  fourth  position  (Compounds  6–19,  Figure  4).  The  third  subset contain N‐Me aa residue at fifth position (Compound 20–26, Figure 5).  subset contain N‐Me aa residue at fifth position (Compound 20–26, Figure 5). 

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subset contain N-Me aa residue at fourth position (Compounds 6–19, Figure 4). The third subset contain N-Me aa residue at fifth position (Compounds 20–26, Figure 5). Mar. Drugs 2016, 14, 161  Mar. Drugs 2016, 14, 161 

5 of 21  5 of 21 

O O

O O

NH NH NH NH

O O N N

O O

O O

HN H HN N H N O O

Compound 1 Compound 1

NH NH

O O

O O

NH NH NH NH

O O

NH NH

O O N N

O HN O H HN N H N O O Compound 3 Compound 3

NH NH O O

O O

HN H HN N H N O O Compound 4 Compound 4

NH NH O O

O O N N

O O

HN H HN N H N O O Compound 2 Compound 2

NH NH O O

O O

NH NH

O O N N

O O N N N N

HN HN

O O

O O Compound 5 Compound 5

   

Figure 3. Subset 1: N‐Me at third position galaxamide derivative.  Figure 3. Subset 1: N-Me at third position galaxamide derivative. Figure 3. Subset 1: N‐Me at third position galaxamide derivative. 

   

    Figure 4. Subset 2: N‐Me at fourth position galaxamide derivative.  Figure 4. Subset 2: N‐Me at fourth position galaxamide derivative.  Figure 4. Subset 2: N-Me at fourth position galaxamide derivative.

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  Figure 5. Subset 3: N‐Me at fourth position galaxamide derivative.  Figure 5. Subset 3: N-Me at fourth position galaxamide derivative.

2.2. Biological Activity  2.2. Biological Activity 2.2.1. Inhibitory Activity of Galaxamide and Its Analogues  2.2.1. Inhibitory Activity of Galaxamide and Its Analogues Galaxamide and its analogues were evaluated for their cytotoxicity against panel of tumor cell  Galaxamide and its analogues were evaluated for their cytotoxicity against panel of tumor cell lines,  such  as,  Lung  cancer  cell  line  A549,  drug‐resistant  lung  cancer  cell  line  A549/DPP,  human  lines, such as, Lung cancer cell line A549, drug-resistant lung cancer cell line A549/DPP, human hepatocellular  carcinoma  HepG2,  SMMC‐7721  and  also  against  human  normal  liver  cell  line  L02  hepatocellular carcinoma HepG2, SMMC-7721 and also against human normal liver cell line L02 using MTT assay. These compounds selectively differently interact with cancer cell over normal liver  using assay. Theseless  compounds selectively differently interact with overfor  normal cell  MTT line  and  exhibited  cytotoxicity  to  normal  cell  with  >40  μg/mL  IC50cancer   value, cell except  the  liver cell line and exhibited less cytotoxicity to normal cell with >40 µg/mL IC value, except for the 50 Compound 14 and 15 which showed 34.63 and 36.87 μg/mL IC50 value and was also seven fold higher  Compounds 14 and 15 which showed 34.63 and 36.87 µg/mL IC50 value and was also seven fold higher than galaxamide against HepG2. Compound 6, 18 and 22 exhibited interesting activities towards all  than galaxamide against HepG2. Compounds 6, 18 and 22 exhibited interesting activities towards all cell line tested, while Compound 1, 4, 15 and 22 showed strong activity towards SMMC‐7221 cell line  cellin the range of 1–2 μg/mL IC line tested, while Compounds 1, 4, 15 and 22 showed strong activity towards SMMC-7221 cell line 50. Additionally, we have further studied three Compound 6, 18 and 22  in the range of 1–2 µg/mL weunderstand  have further studied threeWe  Compounds 6, 18these  and 22 as  representative  of  all IC these  analogues  to  the  pathway.  have  chosen  50 . Additionally, as of they  have  expressed  activities  towards We all have cell  line  and these had  compounds moderate  as compounds  representative all these analogues interesting  to understand the pathway. chosen as differences with Compound 1, 4 and 15 which showed strongest activity in SMMC‐7721 cell line. The  they have expressed interesting activities towards all cell line and had moderate differences with results are summarized in Figure 6.  Compounds 1, 4 and 15 which showed strongest activity in SMMC-7721 cell line. The results are summarized in Figure 6. 2.2.2. Galaxamide and Its Analogues Caused G0/G1 Arrest  2.2.2. Galaxamide andof Itscancer  Analogues Caused G0/G1 The  cell  cycle  cells  was  assessed  to  Arrest continue  the  investigation  into  the  toxicity  of  galaxamide  and  its  analogues.  7  illustrates  the  representative  experimental  of of The cell cycle of cancer cells Figure  was assessed to continue the investigation into theresults  toxicity propidium  iodide  (PI)  fluorescence  intensity,  which  was  proportional  to  the  DNA  content  and  galaxamide and its analogues. Figure 7 illustrates the representative experimental results of propidium indicated the cell cycle phases. To investigate the mechanism of galaxamide and its analogues on cell  iodide (PI) fluorescence intensity, which was proportional to the DNA content and indicated the growth  inhibition,  SMMC‐7721 the cells  were  exposed  to  representative  compounds  with growth the  cell cycle phases. To investigate mechanism of galaxamide and its analogues on cell concentrations of 4, 8 and 16 μg/mL for 48 h before submitted to cell‐cycle analysis. Result of this  inhibition, SMMC-7721 cells were exposed to representative compounds with the concentrations of 4, experiment  showed  that  treatment  of  SMMC‐7721  cells  with  these  compounds  promoted  a  8 and 16 µg/mL for 48 h before submitted to cell-cycle analysis. Result of this experiment showed considerable increase in the percentage of cells in the G0/G1 phase, which suggested that compounds  that treatment of SMMC-7721 cells with these compounds promoted a considerable increase in the caused  cell‐cycle  arrest  at  G0/G1  phase  in  which  the  cell  grows  and  prepares  to  synthesize  DNA.  percentage of cells in the G0/G1 phase, which suggested that compounds caused cell-cycle arrest Thus,  these  analogues  compounds  directly  interact  with  the  nucleus  of  cancer  cells,  and  did  not  at induce DNA fragmentation in cells, thereby altered the cell‐cycle progression and inhibited growth  G0/G1 phase in which the cell grows and prepares to synthesize DNA. Thus, these analogues compounds directly interact with the nucleus of cancer cells, and did not induce DNA fragmentation of cancer cells.  in cells, thereby altered the cell-cycle progression and inhibited growth of cancer cells.

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Subset 1

25

IC50 Value

20 15 10 5 0 1

2

3

4

Galaxamid e

5

DPP

A549

8.74

5.27

5.17

8.89

6.89

10.34

12.34

A549-DPP

27.2

15.01

11.41

19.71

23.61

17.42

16.72

HepG2

12.15

7.76

18.82

16.58

5.23

4.63

8.43

SMMC-7721 35

1.25

5.48

2.77

1.53

2.74

11.21

12.56

 

Subset 2

30

IC50 Value

25 20 15 10 5 0 6

7

8

9

10

11

12

13

14

15

16

17

18

3.63

5.71

7.15

2.76

19

Galax DPP amide

A549

1.73

A549-DPP

5.13

HepG2

7.23 10.41 22.61 23.87 23.02 7.41 13.93 10.22 5.97 16.81 13.49 10.48 6.96

4.25

4.63

SMMC-7721

2.34 14.97 16.29 3.72

2.7

11.21 12.56

Subset 3

6.05 10.62 9.24 16.06 4.47 14

7.54 13.81 6.75

5.21 10.34 12.34

17.34 10.08 10.88 13.24 29.34 27.46 15.39 8.24 22.43 14.53 6.91 17.73 17.42 16.72 6.54

7.54

1.84

7.57

6.05

1.84

7.89

2.9

5.43

8.43

30

 

25

IC50 Value

20 15 10 5 0 20

21

22

23

24

25

26

Galaxam ide

DPP

A549

7.41

6.47

9.4

5.85

3.45

5.18

4.27

10.34

12.34

A549-DPP

16.15

18.6

12.22

9.13

9.26

20.43

11.33

17.42

16.72

HepG2

9.68

15.3

6.41

26.62

7.92

22.88

7.12

4.63

8.43

SMMC-7721

3.35

8.21

1.44

4.32

7.04

6.85

6.65

11.21

12.56

 

Figure  6.  Cytotoxicity  Figure 6. Cytotoxicity of  of compounds  compounds galaxamide  galaxamide and  and its  its three  three subset  subset analogues  analogues against  against A549,  A549, A549/DPP, HepG2 and SMMC‐7721 (IC 50, μg/mL) using MTT assay. The IC50 values are reported as  A549/DPP, HepG2 and SMMC-7721 (IC50 , µg/mL) using MTT assay. The IC50 values are reported as the means ± SD from three independent experiments. Lung cancer cell line A549, drug‐resistant lung  the means ± SD from three independent experiments. Lung cancer cell line A549, drug-resistant lung cancer cell line A549/DPP, human hepatocellular carcinoma HepG2 and SMMC‐7721.  cancer cell line A549/DPP, human hepatocellular carcinoma HepG2 and SMMC-7721.  

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(a)

(b)

(c)

(d) Figure 7. Galaxamide and its analogues caused G0/G1 phase arrest in SMMC‐7721 cells. SMMC‐7721  Figure 7. Galaxamide and its analogues caused G0/G1 phase arrest in SMMC-7721 cells. SMMC-7721 cells were incubated with or without 4, 8 and 16 μg/mL galaxamide (a) and compounds 6 (b), 18 (c)  cells were incubated with or without 4, 8 and 16 µg/mL galaxamide (a) and compounds 6 (b), 18 (c) and 22 (d) for 48 h. Cell cycle was determined by PI staining using flow cytometry.  and 22 (d) for 48 h. Cell cycle was determined by PI staining using flow cytometry.

2.2.3. Galaxamide and Its Analogues Induced Apoptosis of SMMC‐7721 Cells  2.2.3. Galaxamide and Its Analogues Induced Apoptosis of SMMC-7721 Cells SMMC‐7721 cell apoptosis was examined by flow cytometric analysis using annexin V/PI double  SMMC-7721 cell apoptosis was examined by flow cytometric analysis using annexin V/PI staining after 48 h treatment of galaxamide and its analogues with different concentrations (4, 8 and  double staining after 48 h treatment of galaxamide and its analogues with different concentrations 16 μg/mL). Apoptosis plays an important role in the treatment of cancer, as it is a popular target of  (4, 8 and 16 µg/mL). Apoptosis plays an important role in the treatment of cancer, as it is a cancer treatment strategies. The abundance of literature suggests that targeting apoptosis in cancer  popular target of cancer treatment strategies. The abundance of literature suggests that targeting is feasible. As shown in Figure 8, the lower right quadrants accounted the early apoptotic rate, and  apoptosis in cancer is feasible. As shown in Figure 8, the lower right quadrants accounted the early column statistics of apoptotic rate presented in Figure 9. We found that the representative Compound  apoptotic rate, and column statistics of apoptotic rate presented in Figure 9. We found that the 6, 18 and 22 induced SMMC‐7721 cell apoptosis in dose‐dependent manner. Hence, these analogues  representative Compounds 6, 18 and 22 induced SMMC-7721 cell apoptosis in dose-dependent manner. caused apoptosis in cancer cells through growth arrest during cell‐cycle progression pathway.  Hence, these analogues caused apoptosis in cancer cells through growth arrest during cell-cycle progression pathway.

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(a)

(b)

(c)

(d) Figure 8.8. Galaxamide Galaxamide  and  analogues  induced  apoptosis  in  SMMC‐7721  SMMC‐7721  cells  Figure and itsits  analogues induced apoptosis in SMMC-7721 cells.cells.  SMMC-7721 cells were were incubated with or without 4, 8 and 16 μg/mL galaxamide (a) and compounds 6 (b), 18 (c) and 22  incubated with or without 4, 8 and 16 µg/mL galaxamide (a) and compounds 6 (b), 18 (c) and 22 (d) by  annexin  V/PI  staining.  (the  (d) 48 for  h. apoptosis The  apoptosis  of  SMMC‐7721 cells  was  determined  for h.48  The of SMMC-7721 cells was determined by annexin V/PI staining. B1 (theB1  upper upper left) quadrant represents necrotic cells, B2 (the upper right) quadrant represents late apoptotic  left) quadrant represents necrotic cells, B2 (the upper right) quadrant represents late apoptotic and and dead cells, B3 (the bottom left) represents normal cells, B4 (the bottom right) quadrant represents  dead cells, B3 (the bottom left) represents normal cells, B4 (the bottom right) quadrant represents early early apoptotic cells.  apoptotic cells.

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  Figure 9. Galaxamide and its analogues induced apoptosis in SMMC‐7721 cells. Annexin V positive  Figure 9. Galaxamide and its analogues induced apoptosis in SMMC-7721 cells.   Annexin V positive cells cells of three independent experiments were shown in column statistics. Data expressed as mean ± SD.  of three independent experiments were shown in column statistics. Data expressed as mean ± SD. Figure 9. Galaxamide and its analogues induced apoptosis in SMMC‐7721 cells. Annexin V positive  * p