Synthesis and anticancer activity of conformationally constrained Smac mimetics containing pseudo β turns

Article abstract of DOI:10.1016/j.tetlet.2018.08.016

Herein, we report synthesis and in vitro anticancer activity of conformationally constrained Smac mimetics containing reverse turn inducing motifs “Ant-Pro” and “^sAnt-Pro”. The synthesis of Smac analogs with diverse hydrophobic groups at the C-

Full text of DOI:10.1016/j.tetlet.2018.08.016

Accepted Manuscript
Synthesis and anticancer activity of conformationally constrained Smac mim-
etics containing pseudo β turns
Sachin B. Baravkar, Mahendra A. Wagh, Debasish Paul, Manas Santra,
Gangadhar J. Sanjayan
PII:
S0040-4039(18)30998-5
https://doi.org/10.1016/j.tetlet.2018.08.016
TETL 50191
DOI:
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 July 2018
7 August 2018
9 August 2018
Please cite this article as: Baravkar, S.B., Wagh, M.A., Paul, D., Santra, M., Sanjayan, G.J., Synthesis and anticancer
activity of conformationally constrained Smac mimetics containing pseudo β turns, Tetrahedron Letters (2018),
doi: https://doi.org/10.1016/j.tetlet.2018.08.016
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Graphical Abstract
Synthesis and anticancer activity of conformationally constrained Smac mimetics containing
pseudo β turns
Sachin B. Baravkar,a Mahendra A. Wagh, a Debasis Paul, b Manas Santra b and Gangadhar J. Sanjayan*a
O
N
N
S
O
O
NH2
NH
O
N
O
N
O
NH
H2N
H
O
O
N
N
H
O
N
H
H
O
NH
CF3
O
N
CF3
F3C
Smac AVPI peptide
F3C
4h
3j
Modified Smac peptide analogs

Tetrahedron Letters
journal homepage: www.elsevier.com
Synthesis and anticancer activity of conformationally constrained Smac
mimetics containing pseudo β turns
Sachin B. Baravkar,a Mahendra A. Wagh,a Debasish Paul,b Manas Santrab and Gangadhar J. Sanjayan*a
a Division of Organic Chemistry, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411 008, Maharashtra, India.
b National Centre for Cell Science, Savitribai Phule Pune University, Ganeshkhind, Pune 411 007, Maharashtra, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Herein, we report synthesis and in vitro anticancer activity of conformationally constrained
Smac mimetics containing reverse turn inducing motifs “Ant-Pro” and “sAnt-Pro”. The synthesis
of Smac analogs with diverse hydrophobic groups at the C-terminus was carried out using
solution phase peptide synthesis. The synthesis of Ant-Pro containing analogs 3a-j was carried
out by ring opening of benzoxazinones 7a-c, whereas, their sulfonamide counterparts 4a-h were
synthesized by using routine acid-amine coupling reaction. In vitro anticancer studies against
breast cancer cell line MDA-MB-231 revealed that some of the new analogs had better
anticancer activity than the standard AVPI Smac tetrapeptide.
Available online
Keywords:
Smac mimetics
Benzoxazinone
AVPI tetrapeptide
Sulfonamide
2009 Elsevier Ltd. All rights reserved.
MDA-MB-231
1. Introduction
XIAP is mostly expressed in human cancers and therefore
Smac mimetics hold promising solution to achieve apoptosis.6 In
Apoptosis, also known as programmed cell death, is a key
process to carry out the normal development and homeostasis in
multicellular organisms. Unusual apoptosis leads to many
common human diseases such as autoimmune diseases,
neurodegenerative disorders, cancer, and others.1 Inhibitors of
apoptosis proteins (IAPs) are a class of proteins responsible for
the inhibition of apoptosis.2 Out of 8 diverse types of known
IAPs; c-IAP1, c-IAP2 and XIAP are the most important and well
investigated. Each IAP contains ~70 amino acid baculovirus IAP
repeat (BIR) domains which mainly inhibits the apoptotic activity
by binding to the members of the caspase family of apoptotic
enzymes.3 The BIR3 domain of human X-linked inhibitor of
apoptosis protein (XIAP) binds to an initiator enzyme caspase-9
whereas BIR2 domain mediates the executer enzymes caspase-3
and caspase-7.4
recent years, several Smac mimetics including peptidic and non-
peptidic backbone as well as monomeric and dimeric small
molecules have been developed and some of which have already
entered the clinical phase trials (figure 1).7 However, there are
limitations for the utility of Smac peptides due to poor cell
permeability coupled with poor in vivo stability and
bioavailability.
O
N
O
O
H
H
N
N
N
N
N
N
H
H
O
O
N
S
O
F
N
Debio 1143
(AT-406)
Debiopharm, Phase I
H
LCL161
Novartis, Phase II
O
Smac (secondary mitochondria derived activator of caspase)
also known as DIABLO (direct IAP-binding protein with low pI)
is an endogenous protein released from mitochondria acting as
XIAP antagonist by binding to its BIR domains to promote
apoptosis (programmed cell death).5 The Smac protein containing
the tetrapeptide sequence AVPI (Ala-Val-Pro-Ile) binds to BIR3
domain of XIAP - thus removing the interaction between XIAP
and caspase-9 which promote apoptosis. AVPI also binds to
BIR2 domain of XIAP thus releasing caspase-3 and caspase-7 for
apoptosis.4
OH
F
O
H
N
N
O
H
N
N
H
O
N
N
NH
NH
H
O
NH
O
O
O
S
H
N
N
N
N
H
N
O
F
OH
CUDC-427
Curis, Phase I
Birinapant Tetralogic
Pharmacauticals, Phase II
 Prof. Dr. Gangadhar J. Sanjayan. Tel.: (+91) 020 25902082;
fax: (+91) 020 2590262; e-mail: gj.sanjayan@ncl.res.in
Figure 1. Selected examples of Smac mimetics entered into clinical trials.

2
Tetrahedron Letters
To address these problems, several groups have made
yields. The final compounds 3a-j were obtained by deprotecting
the Boc group by using TFA/DCM.14 The free amino group of 3i
could be dimethylated using MeI in presence of NaH to get N,N-
dimethylated Smac analog 3j.
considerable efforts and discovered conformationally constrained
peptidic and non-peptidic mimetics of Smac peptide 1 (Figure 2).
Compounds with bicyclic ring system 2 (Figure 2) have been
shown to have moderate to excellent binding affinity towards
XIAP BIR3/BIR2 as well as possess excellent anticancer
activity.8 The crystal structure of BIR3 domain of XIAP
complexed with Smac peptide suggested that the dipeptide motif
valine-proline (V2-P3) forms a reverse turn structure, forcing the
N-termini of tetrapeptide Ala-Val-Pro-Ile to bind to the Smac
binding groove of the XIAP BIR3 domain which plays a pivotal
role for its activity.9
O
COOMe
R2
COOMe
H
O
H2N
Boc
N
i
ii
N
R2
N
R1
O
N
R1
Boc
7a
, R1 = H; R2 = CH3 (74%) (over
6a
6b
6c
, R1 = H; R2 = CH3 (82%)
, R1 = Me; R2 = CH3 (77%)
, R1 = H; R2 = CH(CH3)2 (76%)
two steps)
7b
7c
, R1 = Me; R2 = CH3 (72%)
, R1 = H; R2 = CH(CH3)2 (70%)
H
N
H
OH
O
R3
N
R3
iii
iv
N
Boc
N
N
H
O
O
O
OH
O
O
N
Boc
N
H2N
N
N
8a
9a
9b
9c
9d
, R3 = Ph (88%)
, R3 = Ph (96%)
H2N
O
N
H
N
H
H
O
O
8b
8c
8d
, R3 = 1-Naphthyl (84%)
, R3 = Benzhydryl (85%)
, R3 = Bis(3,5-trifluoromethyl)
, R3 = 1-Naphthyl (99%)
, R3 = Benzhydryl (97%)
, R3 = Bis(3,5-trifluoromethyl) (95%)
H
O
2
1
(90%)
Figure 2. Smac AVPI tetrapeptide 1 and the potent Smac mimetic 2 reported
in the literature8.
O
O
N
v
O
N
O
Our laboratory has been engaged in the development of
conformationally constrained peptides containing diverse
secondary structural features such as helices, β-sheets and reverse
turns.10 Our objective in the present work was to synthesize
conformationally constrained Smac peptidomimetics by
incorporating our reverse turn inducing motifs in the backbone of
the Smac AVPI peptide 1 and explore their bioactivity. Peptides
containing “Ant-Pro” (anthranilic acid-proline)10a,b and “sAnt-
NH
R4
R2
H
O
N
R2
N
R3
N
Boc
R1
R1
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
, R1 = R2 = H; R3 = CH3; R4 = Ph (80%) (over two steps)
, R1 = Me; R2 = H; R3 = CH3; R4 = Ph (76%)
7a
7b
7c
, R1 = H; R2 = CH3
, R1 = R2 = Me
, R1 = H; R2 = CH(CH3)2
, R1 = R2 = H; R3 = CH3; R4 = 1-Naphthyl (71%)
, R1 = Me; R2 = H; R3 = CH3; R4 = 1-Naphthyl (73%)
, R1 = R2 = H; R3 = CH3; R4 = Benzhydryl (79%)
, R1 = Me; R2 = H; R3 = CH3; R4 = Benzhydryl (73%)
, R1 = R2 = H; R3 = CH3; R4 = Bis(3,5-trifluoromethyl) (66%)
, R1 = Me; R2 = H; R3 = CH3; R4 = Bis(3,5-trifluoromethyl) (65%)
, R1 = R2 = H; R3 = CH(CH3)2; R4 = Bis(3,5-rifluoromethyl) (98%)
, R1 = R2 = Me; R3 = CH(CH3)2; R4 = Bis(3,5-trifluoromethyl) (72%)
Pro”
(2-aminobenzenesulfonic
acid-proline)11
dipeptide
sequences have been shown in our earlier studies to form highly
stable 9-membered intramolecular hydrogen bonding (pseudo β-
turn) between amide NH of Ant or sAnt and CO group of proline.
In this context, we thought it worthwhile to incorporate these
reverse turn structures into the native Smac AVPI peptide 1 with
a view to improve its conformational stability. Thus, analogs 3
and 4 containing variable groups were designed and synthesized
(Figure 3).
vi
Scheme 1. Reagents and conditions: (i) Boc-Ala-OH/Boc-NMe-Ala-
OH/Boc-Val-OH, DCC, HOBt, DCM, rt, 24 h; (ii) (a) LiOH, MeOH,
H2O, rt, 6 h; (b) EDC.HCl, DMAP, DCM, rt, 1 h; (iii) R3-NH2,
HBTU, DIEA, DCM, rt, overnight; (iv) TFA, DCM, rt, 1 h; (v) (a)
9a/9b/9c/9d, DBU, 4 Å MS, DMF, rt, 3 h; (b) TFA, DCM, rt, 1 h;
(vi) MeI, NaH, THF, rt, 3h.
O
N
N
Sulfonamide based analogs 4a-h were synthesized as shown
in scheme 2 (vide infra). The earlier synthesized proline amines
9a-d was reacted with 2-nitrobenzenesulfonyl chloride to get the
corresponding nitro derivatives 10a-d in moderate to excellent
yields. The nitro group of 10a-d was reduced by using stannous
chloride to afford the respective amines 11a-d. Amines 11a-d
were coupled with Cbz-LAla-OH or Cbz-NMe-LAla-OH by using
ethylchloroformate to get the corresponding tripeptides, which
were converted to the final analogs 4a-h by deprotecting Cbz
group using HBr/AcOH.15 The final analogs 3a-j, and 4a-h were
purified by using Prep-HPLC before executing biological
activity.
S
O
N
O
O
N
O
NH
R3
NH
R4
H
H
O
O
R2
R2
NH
R1
R3
N
R
1 = H; Me
R1 = R2 = H; Me
R3 = CH3, CH(CH3)2
R4 = Hydrophobic aryl groups
R1
R2 = CH3
R3 = Hydrophobic aryl groups
3
4
Figure 3. Smac analogs 3 and 4 reported in this work containing “Ant-Pro”
(anthranilic acid-proline) and “sAnt-Pro” (2-aminobenzenesulfonic acid-
proline) reverse turn analogs, respectively.
2. Results and Discussion
2.1. Synthesis of Smac analogs:
2.2. In vitro anticancer activity studies:
The synthesis of the designed analogs was carried out in two
different strategies. The anthranilic acid (Ant) based analogs 3a-j
were synthesized by using benzoxazinone ring opening strategy12
as discussed in scheme 1. First, methylanthranilate was coupled
with Boc aminoacids to get the dipeptides 6a-c using DCC as a
coupling agent.13 The methyl esters 6a-c were then hydrolyzed
and free acids obtained were subjected to cyclodehydration to
obtain benzoxazinones 7a-c, by using EDC.HCl (scheme 1). On
the other hand, Boc-LPro-OH was coupled with four different
hydrophobic amines using HBTU coupling conditions and the
Boc group was removed using TFA/DCM to get the free amines
9a-d. These amines were reacted with benzoxazinone 7a-c in
presence of DBU to get the respective tetrapeptides in excellent
The synthesized analogs were tested for their cell growth
inhibition against human breast cancer cell line MDA-MB-231.
Smac peptide (AVPI) was used as a standard (positive control),
which showed IC50 value 80.6 μM (Table 1) (Note: for synthesis
of AVPI see SI page 51). Most of the analogs showed moderate to
anticancer activity compared to the standard Smac peptide
(AVPI). Compounds 3e, 3g, 4e and 4g showed IC50 values 82.5,
78.5, 82.6 and 67.8 μM, respectively, which was comparable or
slightly better than AVPI peptide (results in Table 1).
Compounds 3h, 4d, 4f and 4h were 2-fold more active than
AVPI with IC50 values 32.5, 36.5, 35.8 and 31.5 μM, respectively
(Table 1).

O
S
H
H
N
N
N
R2
Cl
R2
O
O
N
S
N
S
S
i
ii
O
O
iii
O
O
O
N
O
O
O
NH
R3
H
O
NO2
R2
NH
R1
NH2
NO2
4a
4b
4c
4d
4e
, R1 = H; R2 = Me; R3 = Ph (77%)
, R1 = R2 =Me; R3 = Ph (75%)
, R1 = H; R2 = Me; R3 = 1-Naphthyl (68%)
, R1 = R2 = Me; R3 = 1-Naphthyl (69%)
, R1 = H; R2 = Me; R3 = Benzhydryl (84%)
, R1 = R2 = Me; R3 = Benzhydryl (81%)
10a
10b
10c
10d
, R2 = Ph (85%)
, R2= 1-Naphthyl (82%)
, R2= Benzhydryl (80%)
11a
11b
11c
11d
, R2= Ph (91%)
, R2= 1-Naphthyl (94%)
, R2= Benzhydryl (91%)
, R2= Bis(3,5-trifluoromethyl) (76%)
, R2= Bis(3,5-trifluoromethyl) (90%)
4f
4g
4h
, R1 = H R2 = Me; R3 = Bis(3,5-trifluoromethyl) (68%)
, R1 = R2 = Me; R3 = Bis(3,5-trifluoromethyl) (66%)
o
Scheme 2. Reagents and conditions: (i) 9a/9b/9c/9d, DIEA, DCM, rt, 3 h; (ii) SnCl2.2 H2O, 55 C, 6 h; (iii) (a) Cbz-Ala-OH/Cbz-NMe-Ala-
OH, ClCOOEt, Et3N, THF, reflux, 24 h; (b) HBr in AcOH, rt, 20 mins.
Table 1. In vitro Cytotoxicity against human breast cancer cell line
MDA-MB-231
Acknowledgments
Sr.
No.
Sr.
No.
Compnd IC50 (μM)
Compnd IC50 (μM)
S.B.B, M.A.W. and D. P. are thankful to CSIR, New Delhi for
research fellowships. GJS thanks BSC0120 drug discovery
program (CSIR, New Delhi) for funding.
1
2
3
4
5
6
7
8
9
10
AVPIa
3a
80.6±5.2
>100
11
12
13
14
15
16
17
18
19
3j
17.5±1.2
>100
4a
4b
4c
4d
4e
4f
3b
3c
>100
>100
Supplementary Material
>100
>100
Full experimental procedures, characterization data, HR-MS,
3d
3e
>100
36.5±1.5
82.6±2.6
35.8±2.4
67.8±3.5
31.5±1.6
1
IR, H and 13C NMR spectra for all compounds. Supplementary
data associated with this article can be found, in the online
version.
82.5±2.7
>100
3f
4. References
3g
78.5±3.4
32.5±0.5
>100
4g
4h
1.
2.
3.
Tsuruo, T.; Naito, M.; Tomida, A.; Fujita, N., Mashima, T.;
Sakamoto, H. Haga, N. Cancer Sci. 2003, 94, 15-21.
(a) Martin, S. J. Cell 2002, 109, 793-796. (b) Verhagen, A. M.;
Vaux, D. L. Apoptosis 2002, 7, 163-166.
Takahashi, R.; Deveraux, Q. L.; Tamm, I.; Welsh, K.; Munt, A.
N.; Salvesen, G. S.; Reed, J. C. J. Biol. Chem. 1998, 273, 7787-
7790.
3h
3i
a AVPI – Smac tetrapeptide was synthesized and used as a positive control for
the comparison.
4.
5.
Ekert, P. G.; Silke, J.; Hawkins, C. J.; Verhagen, A. M.; Vaux, D.
L. J. Cell Biol. 2001, 152, 483-490.
Verhagen, A. M.; Ekert, P. G.; Pakusch, M.; Silke, J.; Connolly,
L. M.; Reid, G. E.; Moritz, R. L.; Simpson, R. J.; Vaux, D. L.
Cell, 2000, 102, 43-53.
Liu, Z.; Sun, C.; Olejniczak, E. T.; Meadows, R. P.; Betz, S. F.;
Oost, T.; Herrmann, J.; Wu, J. C.; Fesik, S. K. Nature 2000, 408,
1004-1008.
(a) Chauhan, D.; Neri,P.; Velankar, M.; Podar, K.; Hideshima, T.;
Fulciniti, M.; Tassone, P.; Raje, N.; Mitsiades, C.; Mitsiades, N.;
Richardson, P.; Zawel, L.; Tran, M.; Munshi, N.; Anderson, K. C.
Blood 2007, 109, 1220-1227. (b) Liu, N.; Tao, Z.; Le Blanc, J. M.;
Zaorsky, N. G.; Sun, Y.; Vuagniaux, G.; Dicker, A. P.; Lu, B. Am.
J. Cancer Res. 2014, 4, 943-951. (c) Flygare, J. A.; Beresini, M.;
Budha, N.; Chan, H.; Chan, I. T.; Cheeti, S.; Cohen, F.; Deshayes,
K.; Doerner, K.; Eckhardt, S. G.; Elliott, L. O.; Feng, B.; Franklin,
M. C.; Reisner, S. F.; Gazzard, L.; Halladay, J.; Hymowitz, S. G.;
La, H.; LoRusso, P.; Maurer, B.; Murray, L.; Plise, E.; Quan, C.;
Stephan, J. P.; Shin, Y. G.; Tom, J.; Tsui, V.; Um, J.;
Varfolomeev, E.; Vucic, D.; Wagner, A. J.; Wallweber, H. J. A.;
Wang, L.; Ware, J.; Wen, Z.; Wong, H.; Wong, J. M.; Wong, M.
Wong, S.; Yu, R.; Zobel, K.; Fairbrother W. J. J. Med. Chem.
2012, 55, 4101-4113. (d) Senzer, N. N.; LoRusso, P.; Martin, L.
P.; Schilder, R. J.; Amaravadi, R. K.; Papadopoulos, K. P.; Segota,
Z. E.; Weng, D. E.; Graham, M.; Adjei A. A. J. Clin. Oncol. 2013,
31, 3621-3628.
Compound 3j with N, N-dimethylation at N-terminus showed 4-
fold better activity than AVPI having IC50 value 17.5 μM. This
data suggested that N-methylated analogues 3h, 3j, 4d, 4f and 4h
showed more activity as compared to their free amine (NH2)
counterparts. Compounds having sulfonamide backbone (sAnt-
Pro) were relatively more active than carboxamide (Ant-Pro)
analogues, probably owing to their improved conformational
stability compared to their carboxamide counterparts. Indeed, this
has been observed in many bioactive molecules.16 Furthermore,
compounds having larger hydrophobic groups such as naphthyl,
benzhydril and bis(trifluoromethyl)aniline at C-terminus were
found to be more active than derivatives with phenyl group.
6.
7.
3. Conclusion
In summary, we have successfully synthesized 18 Smac
mimics, containing “Ant-Pro” and “sAnt-Pro” as reverse turn
inducing motifs. The synthesis of “Ant-Pro” containing mimics
has been carried out using benzoxazinone ring opening reaction,
while “sAnt-Pro”-tethered mimics have been synthesized via
standard coupling reaction. In vitro anticancer studies revealed
that most of the compounds show better activity than standard
AVPI tetrapeptide. Further studies are underway in refining the
structural features of the reverse-turn motifs embedded on the
Smac backbone with a view to improve bioactivity.
8.
9.
Sun, H.; Coleska, Z. N.; Lu, J.; Qiu, S.; Quin, D.; Yang, C. Y.;
Gao, W.; Meagher, J.; Stuckey, J.; Wang, S. J. Med. Chem. 2006,
49, 7916-7920.
Fandrich, M.; Fletcher, M. A.; Dobson, C. M. Nature 2001, 410,
165-166.

4
Tetrahedron Letters
10. (a) Prabhakaran, P.; Kale, S. S.; Puranik, V. G.; Rajamohanan, P.




Smac mimetics containing reverse-turn inducing
motifs are reported.
R., Chetina, O. Howard, J. A. K.; Hofmann, H. J.; Sanjayan, G. J.
J. Am. Chem. Soc. 2008, 130, 17743-17754. (b) Nair, R. V.;
Kheria, S.; Rayavarapu, S.; Kotmale, A. S.; Jagadeesh B.;
Gonnade, R. G.; Puranik V. G.; Rajamohanan, P. R.; Sanjayan, G.
J. J. Am. Chem. Soc. 2013, 135, 11477-11480. (c) Nair, R. V.;
Baravkar, S. B.; Ingole, T. S.; Sanjayan, G. J. Chem. Commun.
2014, 50, 13874-13884. (d) Jedhe, G. S.; Kotmale, A. S.;
Rajamohanan, P. R.; Pasha, S.; Sanjayan, G. J. Chem. Commun.,
2016, 52, 1645-1648. (e) Ingole, T. S.; Kale, S. S.; Babu, S. S.;
Sanjayan G. J. Chem. Commun. 2016, 52, 10771-10774.
Reverse-turn incorporation is expected to
improve structural stability.
Some of the analogs show better activity than
the standard AVPI Smac tetrapeptide.
Sulfonamide
counterparts
outperform
11. Vijayadas, K. N.; Davis, H. C.; Kotmale, A. S.; Gawade, R. L.;
Puranik, V. G.; Rajamohanan, P. R.; Sanjayan, G. J. Chem.
Commun. 2012, 48, 9747-9749.
carboxamide analogs, in terms of activity.
12. Baravkar, S. B.; Roy, A.; Gawade, R. L.; Puranik, V. G.; G. J.
Sanjayan Synth. Commun. 2014, 44, 2955–2960.
13. Mohapatra, D. K.; Datta, A. Bioorg. Med. Chem. Lett. 1997, 7,
2527-2530.
18.
14. General experimental procedure for the synthesis of compounds
3a-j: The oxazinone 7a-c (1 equiv) were dissolved in dry DMF,
followed by the addition of a pinch of 4 Å molecular sieves. DBU
(1.2 equiv) was added drop wise followed by the addition of
appropriate amines 9a-d (1 equiv) dissolved in DMF (2 mL). The
reaction mixture was stirred at room temperature for 3 h. Reaction
was quenched with ice cold water and the product was extracted
with EtOAc (3 x 25 mL). The organic layer was washed with sat.
KHSO4 (3 x 25 mL) and brine (3 x 25 mL) to remove DMF. The
organic layer was dried with Na2SO4 and concentrated in vacuo to
get the crude product which was further purified using flash
column chromatography to get the Boc protected trimer. The Boc
protected trimer (0.1 g) was reacted with 1 mL TFA/DCM (1:1)
mixture, followed by neutralization with sat. NaHCO3. The aq.
layer was extracted with DCM (3 x 25 mL), DCM layer was dried
using Na2SO4 and solvent evaporated under reduced pressure to
get the corresponding Smac analogs 3a-j.
15. General experimental procedure for the synthesis of compounds
4a-h: Cbz-Ala-OH or Cbz-NMe-Ala-OH (1.5 equiv) was
dissolved in dry THF (50 mL) followed by the addition of
o
ethylchloroformate (1.5 equiv), Et3N (1.5 equiv) at 0 c. Finally,
the amines 11a-d (1 equiv) were added and the reaction mixture
was refluxed for 24 h. The reaction was cooled to rt and solvent
was evaporated under reduced pressure. DCM was added and the
organic layer was washed sequentially with sat. NaHCO3, KHSO4
and brine solution. The organic layer was dried with Na2SO4 and
concentrated in vacuum to get the crude product which was further
purified using flash column chromatography to get the Cbz
protected tripeptide. To the Cbz tripeptide (0.1 g) was added
HBr/AcOH (0.2 mL) and the reaction mixture was stirred at room
temperature for 20 mins. Ice cold water was added and the water
layer was extracted with Et2O to remove side products. The aq.
layer was neutralized by using sat. NaHCO3 and it was extracted
with EtOAc (3 x 25 mL) to get the final products 4a-h.
16. Yoshino, H., Ueda, N., Niijima, J., Sugumi, H., Kotake, Y.,
Koyanagi, N., Yoshimatsu, K., Asada, M., Watanabe, T., Nagasu,
T. and Tsukahara, K., Iijima, A. and Kitoh K J. med. chem.1992,
35, 2496-2497.
17. Cell growth inhibition MTT Assay: The cytotoxic effect of
compounds was evaluated by MTT (3-(4, 5 dimethylthiazol-2-yl)-
2-5 diphenyltetrazolium bromide) assay. 3 X 103 cells per well in
96 well plates were seeded 24 hours prior the drugs treatment. The
cells were treated with different concentrations of the compounds
(0-100μm) for 48 hours in triplicate. Then, MTT assay was used to
determine the cytotoxicity of different drugs in different cell lines.
After 48 hours of drug treatment, MTT solution (20μl of 5mg/ml
stock for each well) was added to the media and cells were further
allowed to be incubated for 3.5 hours in humid 5% CO2 incubator.
Then, media containing MTT solution was replaced by MTT
solvent (iso-propanol, HCl and Triton X-100) and incubated for 15
min at room temperature followed by gentle shaking to ensure the
complete dissolution of Formazan. Finally, absorbance was
measured at 590 nm using a Thermo Pierce Elisa plate reader. All
experiments were carried out at least in triplicate; the percentage
of viable cells was calculated as the mean with respect to the
controls set to 100%.
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