Synthesis, characterization and biological evaluation of bile acid-aromatic/heteroaromatic amides linked via amino acids as anti-cancer agents

Article abstract of DOI:10.1016/j.steroids.2015.12.022

A series of bile acid (Cholic acid and Deoxycholic acid) aryl/heteroaryl amides linked via α-amino acid were synthesized and tested against 3 human cancer cell-lines (HT29, MDAMB231, U87MG) and 1 human normal cell line (HEK293T). Some of the conjugates sh

Full text of DOI:10.1016/j.steroids.2015.12.022

Accepted Manuscript
Synthesis, Characterization and Biological evaluation of Bile acid-aromatic/
heteroaromatic amides linked via amino acids as anti-cancer agents
Devesh S. Agarwal, Hasitha Shilpa Anantaraju, Dharmarajan Sriram, Perumal
Yogeeswari, Shankara H. Nanjegowda, P. Mallu, Rajeev Sakhuja
PII:
S0039-128X(15)00328-1
http://dx.doi.org/10.1016/j.steroids.2015.12.022
STE 7895
DOI:
Reference:
Steroids
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 September 2015
1 December 2015
30 December 2015
Please cite this article as: Agarwal, D.S., Anantaraju, H.S., Sriram, D., Yogeeswari, P., Nanjegowda, S.H., Mallu,
P., Sakhuja, R., Synthesis, Characterization and Biological evaluation of Bile acid-aromatic/heteroaromatic amides
linked via amino acids as anti-cancer agents, Steroids (2015), doi: http://dx.doi.org/10.1016/j.steroids.2015.12.022
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Synthesis,
aromatic/heteroaromatic amides linked via amino acids as anti-cancer agents
Characterization
and
Biological
evaluation
of
Bile
acid-
Devesh S. Agarwal,a Hasitha Shilpa Anantaraju,b Dharmarajan Sriram,b Perumal
Yogeeswari,b Shankara H Nanjegowda,c P Mallu,c Rajeev Sakhujaa,*
aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani 333 031,
Rajasthan, India
bDrug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of
Technology & Science-Pilani, Hyderabad Campus, Hyderabad-500078, India
cDepartment of Chemistry, Sri Jayachamarajendra College of Engineering, Mysore-570006
Email: sakhuja.rajeev@gmail.com
Abstract
A series of bile acid (Cholic acid and Deoxycholic acid) aryl/heteroaryl amides linked via
α-amino acid were synthesized and tested against 3 human cancer cell-lines (HT29,
MDAMB231, U87) and 1 human normal cell line (HEK293T). Some of the conjugates
showed promising results to be new anticancer agents with good in vitro results. More
specifically, Cholic acid derivatives 6a (1.35 µM), 6c (1.41 µM) and 6m (4.52 µM)
possessing phenyl, benzothiazole and 4-methylphenyl groups showed fairly good activity
against the breast cancer cell line with respect to Cisplatin (7.21 µM) and comparable with
respect to Doxorubicin (1 µM), while 6e (2.49 µM), 6i (2.46 µM) and 6m (1.62 µM) showed
better activity against glioblastoma cancer cell line with respect to both Cisplatin (2.60 µM)
and Doxorubicin (3.78 µM) drugs used as standards. Greater than 65% of the compounds
were found to be safer on human normal cell line.
Keywords: cancer; cholic acid; coupling; cytotoxicity; amino acid; heteroaryl amines
1. Introduction
Primary bile acids such as Cholic acid (CA) and Chenodeoxycholic acid (CDCA) are
endogenous steroids that are excreted into the bile canaliculus and the digestive tract after
cholesterol catabolism [1]. Bacterial metabolism of tauryl and glycyl conjugates of CA and
CDCA in distal small intestine and colon produces secondary bile acids such as Deoxycholic
acid (DCA), Ursodeoxycholic acid (UDCA), and Lithocholic acid (LCA) [2]. Slight
differences in the chemical structure of these bile acids make them behave distinctly in the

biological environment. Earlier reports on hydrophobic bile acids indicated their cytotoxic
behaviour towards normal tissues [3], while less hydrophobic bile acid such as UDCA protect
normal cells against apoptosis induced by direct prevention of mitochondrial membrane
perturbation [4]. UDCA and its derivatives have been extensively studied as potent scaffolds
in medicinal chemistry [5]. In vitro and animal studies have suggested the chemopreventive
behaviour of UDCA towards colorectal cancer [6]. UDCA has been used for the prevention
of gastrointestinal disorders in patients having various cancers such as stomach, colon, lung,
breast, and liver cancer [7]. UDCA itself has been evaluated as a drug in clinical phase III
trial therapy to prevent colorectal adenoma recurrence [8]. Recently, Qui and co-workers
reported anticancer effect of UDCA in human oral squamous carcinoma HSC-3 cells through
the caspases [9]. UDCA and its metal complexes decreased the viability and proliferation of
cultured human and animal tumour cells in a time- and concentration-dependent manner [10].
In addition, UDCA expresses an inhibitory effect on the induction of P-glycoprotein
expression and reactive oxygen species by Doxorubicin in HepG2 human hepatoma cells
[11].
In 2001, Powell and co-workers reported a direct correlation between bile acids
hydrophobicity and induction of apoptosis and/or growth arrest in HCT116 cells, however no
apparent link was observed between a particular structural modification and its biological
activity [12]. Nonetheless, reports in the last two decades suggested the induction of
apoptosis by hydrophobic bile acid derivatives in many human cancer cells [13], such as
prostate cancer cells [14], leukemic T cells [15], heaptocellular carcinoma cells [16], colon
cancer cells [17], breast carcinoma cells [18], osteosarcoma cells [19], and cervical
carcinoma cells [20]. However, low cytotoxicity (IC50> 100 µM) and tumour promoter ability
of certain bile acid derivatives has compelled medicinal chemist to design more efficient bile
acid derivatives with potent anti-proliferative and apoptotic properties. Thus, physiological
and carcinogenesis studies of bile acid derivatives have experienced a significant progress in
the last decade.
Many bile acid-amino acid conjugates have been studied for their anti-cancer properties.
Among these, compounds HS-1183, HS-1199 and HS-1200 (Figure 1) showed promising
results in inducing apoptosis with an IC50 values from 25 to 50 µM for SiHa human cervical
carcinoma cells, 30 to 45 µM for MDA-MB-231 cells and 30 to 150 µM for MCF-7. Reports
also suggests unnatural long chain amino acid conjugates of bile acid to possess strong anti-

cancer activity against several tumour cell lines including human breast adenocarcinoma
(ER-, MDA-MB-231), breast adenocarcinoma (ER+, MCF-7), cervix epiteloid carcinoma,
(HeLa S-3) and prostate cancer (PC-3) [21]. Khiel and co-workers reported piperazinyl
linked aryl and heteroaryl substituted bile acid derivatives with apoptosis-inducing activity on
multiple myeloma cancer cell line KMS-11[22]. Bile acid–polyaminocarboxylate conjugates
containing NE3TA, a potential iron chelator displayed significant cytotoxicity in both HeLa
and HT29 colon cancer cells [23]. Other reports on the synthesis and structural studies of bile
acid-4-aminopyridine conjugates [24], N-(2-aminoethyl)amido linked bile-acid–heteroaryl
conjugates [25], bile acid-arene conjugates [26] and bile acid-glutamyl-pyridines [27] have
been described, however no anticancer-activity evaluation studies were reported.
In view of the preceding discussion, we herein describe the design (Figure 1) and synthesis of
new bile acid-aromatic/heteroaromatic amides linked via amino acids. In-vitro anti-cancer
studies were accomplished on human breast adenocarcinoma cell line (MDA-MB-231),
human colorectal adenocarcinoma cell line (HT29) and human glioblastoma cell line (U87).
Figure 1. Design of targeted molecules with reference to known anti-cancer bile acid-amino
acid conjugates
2. Experimental
2.1 General
All the chemicals were purchased from Sigma-Aldich, Alfa Aesar, and Spectrochem India
Pvt. Ltd and used without further purification. The solvents used were purchased from Merck

(India) and were distilled and dried before use. Nuclear magnetic resonance spectra were
1
recorded on Bruker 400 spectrometer. The H NMR experiments were reported in δ units,
parts per million (ppm), and were measured relative to residual chloroform (7.26 ppm) or
DMSO (2.5 ppm) in the deuterated solvent. The 13C NMR spectra were reported in ppm
relative to deuterochloroform (77.0 ppm) or DMSO-d6 (39.5 ppm). All coupling constants J
were reported in Hz. The following abbreviations were used to describe peak splitting
patterns when appropriate: s = singlet, d = doublet, t = triplet, dd = doublet of doublet, m =
multiplet and br s = broad singlet. Melting points were determined on a capillary point
apparatus equipped with a digital thermometer and are uncorrected. Reactions were
monitored by using thin layer chromatography (TLC) on 0.2 mm silica gel F254 plates
(Merck). The chemical structures of final products were confirmed by a high-resolution
ESI/APCI- hybrid quadrupole time-of-flight mass spectrometer. High resolution mass
spectrometry (HRMS) was performed with a waters synapt G2 HDMS instrument using time-
of-flight (TOF-MS) with ESI/APCI- hybrid quadrupole. Optical rotations were recorded
using a Perkin Elmer 343 series polarimeter in methanol.
2.2 General Synthesis of Boc protected aminoacyl aromatic/heteroaromatic amides
To the stirred solution of 1 (1 mmol) in DMF (20 mL), triethyl amine (2.5 mmol) was added
o
at 0 C and subsequently EDC.HCl (1.5 mmol) and HOBt (1 mmol) was added. The reaction
o
mixture was stirred for 15 min. at 0 C, after that aryl/heteroaryl amines (2) (1.1 mmol) was
added and the reaction was stirred at room temperature for 6-8 h. The completion of the
reaction was monitored by TLC. After the completion of the reaction, crushed ice was added.
The resulted precipitate was filtered, washed with cold water and recrystallized with ethyl
acetate/hexanes to yield pure 3a-g.
2.2.1 tert-Butyl ((2S,3R)-3-methyl-1-oxo-1-(phenylamino)pentan-2-yl)carbamate (3a)
1
Off-white solid; yield: 81%, mp: 130-132 °C, H NMR (400 MHz, CDCl3) δ 8.56 (br s, 1H,
NHAr), 7.47 (d, J = 8.0 Hz, 2H, H-3Ar & H-5Ar), 7.21 (t, J = 7.0 Hz, 2H, H-2Ar & H-6Ar), 7.03
(t, J = 7.2 Hz, 1H, H-4Ar), 5.39 (d, J = 8.6 Hz, 1H, NHisoleucine), 4.13 (t, J= 8.0 Hz, 1H,
CHisoleucine), 1.90-1.99 (m, 1H), 1.42 (s, 9H, 3xMeBoc), 1.30 – 1.07 (m, 2H), 0.99 (d, J = 6.8
Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H).13C NMR (101 MHz, CDCl3) δ 170.54 (C=Oamide), 156.30
(C=OBoc), 137.63 (C-1Ar), 128.79 (C-3Ar & C-5Ar), 124.20 (C-4Ar), 119.95 (C-2Ar & C-6Ar),
80.16, 60.07 (NH-CHisoleucine), 37.00, 28.30, 24.89, 15.57, 11.08. IR (KBr, υ, cm-1) 3333,

2970, 2932, 1668, 1605, 1528, 1173, 748. HRMS (ESI): m/z calcd for Chemical Formula:
C17H27N2O3: 307.2021 [M + H]+, found : 307.2009.
2.2.2
tert-Butyl
((2S,3R)-1-(benzo[d]thiazol-2-ylamino)-3-methyl-1-oxopentan-2-
yl)carbamate (3b)
White solid; yield: 68%, mp: 180-182 °C, 1H NMR (400 MHz, CDCl3+DMSO-d6) δ 12.11 (s,
1H, NHAr), 7.75 (d, J = 7.8 Hz, 1H, Hbenzothiazolyl), 7.63 (d, J = 8.0 Hz, 1H, Hbenzothiazolyl), 7.31
(ddd, J = 8.2, 7.3, 1.2 Hz, 1H, Hbenzothiazolyl), 7.19 (td, J = 7.8, 1.1 Hz, 1H, Hbenzothiazolyl), 6.47
(d, J = 9.0 Hz, 1H, NHisoleucine), 4.17 (t, J = 8.2 Hz, 1H, CHisoleucine), 1.81 – 1.72 (m, 1H), 1.34
(s, 9H, 3xMeBoc), 1.21 – 0.99 (m, 2H), 0.84 (d, J = 6.8 Hz, 3H), 0.80 (t, J = 7.4 Hz, 3H).13C
NMR (101 MHz, CDCl3 + DMSO-d6) δ 172.04 (C=Oamide), 157.95 (C=OBoc), 155.63 (C-2Ar),
148.90, 131.98, 126.00, 123.60, 121.34, 120.84, 78.9, 59.39 (NH-CHisoleucine), 37.19, 28.48,
24.83, 15.59, 11.15. IR (KBr, υ, cm-1) 3240, 2970, 1705, 1666, 1536, 1173, 764. HRMS
(ESI): m/z calcd for Chemical Formula: C18H26N3O3S: 364.1694 [M + H]+, found : 364.1676.
2.2.3 (S)-tert-Butyl (1-((1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)amino)-1-
oxopropan-2-yl)carbamate (3c)
Orange solid; yield: 76%, mp: 187-190 °C, 1H NMR (400 MHz, CDCl3) δ 8.98 (s, 1H, NHAr),
7.43 (t, J = 7.8 Hz, 2H, HAr-pyrazolinyl), 7.35 (d, J = 7.6 Hz, 2H, HAr-pyrazolinyl), 7.29 (t, J = 7.4
Hz, 1H, HAr-pyrazolinyl), 5.51 (br s, 1H, NHalanine), 3.91 (d, J = 5.2 Hz, 1H, CHalanine), 3.07 (s,
3H, N-Mepyrazolinyl), 2.18 (s, 3H, C-Mepyrazolinyl), 1.49 – 1.38 (overlapped m, 12H, 3xMeBoc
&
Mealanine).13C NMR (101 MHz, CDCl3) δ 169.29 (C=Oamide), 161.92 (C=Opyrazolinyl), 155.96
(C=OBoc), 150.30, 134.32, 129.27, 127.19, 124.64, 107.74, 79.69, 43.98 (NH-CHalanine), 35.76
(N-CH3pyrazolinyl), 28.35, 12.03, 12.00. IR (KBr, υ, cm-1) 3495, 3310, 2978, 1751, 1606, 1173,
763. HRMS (ESI): m/z calcd for Chemical Formula: C19H27N4O4: 375.2032 [M + H]+, found
: 375.2019.
2.2.4
(S)-tert-Butyl
(1-oxo-3-phenyl-1-((2,4,6-trimethoxyphenyl)amino)propan-2-
yl)carbamate (3d)
White solid; yield: 48%, mp: 151-153 °C, 1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H, NHAr),
7.29 – 7.21 (m, 5H, HAr-phenylalanine), 6.64 (s, 2H, H-3Ar & H-5Ar), 5.24 (d, J = 6.4 Hz, 1H,
NHphenylalanine), 4.56 – 4.47 (m, 1H, CHphenylalanine), 3.77 (s, 3H, OCH3), 3.76 (s, 3H, OCH3),
3.75 (s, 3H, OCH3), 3.06 – 3.16 (m, 2H), 1.39 (s, 9H, 3xMeBoc).13C NMR (101 MHz, CDCl3)

δ 169.70 (C=Oamide), 155.89 (C=OBoc), 153.09 (C-1Ar), 136.56, 134.70, 133.44, 129.21,
128.76, 126.99, 97.62, 80.64, 60.82 (NH-CHphenylalanine), 56.03 (OCH3), 55.99 (OCH3), 55.92
(OCH3), 38.44 (CH2phenylalanine), 28.25 (CH3Boc). IR (KBr, υ, cm-1) 3495, 3333, 2970, 1686,
1528, 1173, 764. HRMS (ESI): m/z calcd for Chemical Formula: C23H31N2O6: 431.2182 [M
+ H]+, found : 431.2166.
2.2.5 (S)-tert-Butyl (3-methyl-1-oxo-1-(pyridin-4-ylamino)butan-2-yl)carbamate (3e)
White solid; yield: 46%, mp: 142-145 °C, 1H NMR (400 MHz, CDCl3) δ 9.24 (s, 1H, NHAr),
8.37 (d, J = 4.6 Hz, 2H, H-3pridyl & H-5pridyl), 7.38 (d, J = 4.4 Hz, 2H, H-2pridyl & H-6pridyl),
5.35 (d, J = 8.5 Hz, 1H, NHvaline), 4.12 – 4.05 (m, 1H, CHvaline), 2.19 – 2.10 (m, 1H), 1.43 (s,
9H, 3xMeBoc), 1.00 (d, J = 5.2 Hz, 3H), 0.98 (d, J = 5.2 Hz, 3H).13C NMR (101 MHz, CDCl3)
δ 171.49 (C=Oamide), 150.41 (C=OBoc), 144.82, 113.60, 113.56, 80.68, 61.02 (NH-CHvaline),
30.50, 28.29, 19.34, 18.12. IR (KBr, υ, cm-1) 3340, 2978, 2932, 1674, 1528, 1165, 818.
HRMS (ESI): m/z calcd for Chemical Formula: C15H24N3O3: 294.1817 [M + H]+, found :
294.1807.
2.2.6 tert-Butyl ((2S,3R)-3-methyl-1-oxo-1-(thiazol-2-ylamino)pentan-2-yl)carbamate (3f)
White solid; yield: 69%, mp: 163-165 °C, 1H NMR (400 MHz, CDCl3) δ 12.49 (s, 1H, NHAr),
7.65 (br s, 1H, Hthiazolyl), 7.01 (d, J = 3.6 Hz, 1H, Hthiazolyl), 5.47 (d, J = 7.7 Hz, 1H,
NHisoleucine), 4.45 – 4.36 (m, 1H, CHisoleucine), 1.88 – 1.81 (m, 1H), 1.41 (s, 9H, 3xMeBoc), 1.26
– 1.07 (m, 2H), 0.90 (d, J = 6.4 Hz, 3H), 0.83 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 170.62 (C=Oamide), 159.10 (C-2Ar), 155.67(C=OBoc), 137.07, 113.79, 79.91,
58.98(NH-CHisoleucine), 38.18, 28.29, 24.53, 15.46, 11.18. IR (KBr, υ, cm-1) 3495, 3340, 2978,
1674, 1520, 1173, 748. HRMS (ESI): m/z calcd for Chemical Formula: C14H24N3O3S:
314.1538 [M + H]+, found : 314.1519.
2.2.7 (S)-tert-Butyl (1-oxo-1-(p-tolylamino)propan-2-yl)carbamate (3g)
1
White solid; yield: 75%, mp: 143-145 °C, H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H,
NHAr), 7.49 (d, J = 8.2 Hz, 2H, H-3Ar & H-5Ar), 7.01 (d, J = 8.1 Hz, 2H, H-2Ar & H-6Ar), 5.51
(br s, 1H, NHalanine), 4.06 (d, J = 6.7 Hz, 1H, CHalanine), 2.20 (s, 3H, MeAr), 1.46 –1.43
(overlapped m, 12H, 3xMeBoc & Mealanine). 13C NMR (101 MHz, CDCl3) δ 170.54 (C=Oamide),
156.30 (C=OBoc), 137.63 (C-1Ar), 133.10 (C-4Ar), 129.97 (C-3/5Ar), 119.95 (C-2/C-6Ar),
80.16, 49.40 (CHalanine), 28.30, 20.91 (CH3Ar), 17.62 (CH3alanine). IR (KBr, υ, cm-1) 3347,

2966, 2932, 1682, 1528, 1165, 818. HRMS (ESI): m/z calcd for Chemical Formula:
C15H23N2O3: 279.1708 [M + H]+, found : 279.1688.
2.3 General Synthesis of aminoacyl-aromatic/heteroaromatic amides hydrochloride salts
A solution of dioxane-HCl (5 mL) was added drop-wise to a solution of 3a-g (1 mmol)
o
dissolved in dioxane at 0 C. The reaction was stirred at room temperature for 2 h and
monitored via TLC. After the completion, the reaction mixture was concentrated, dried under
vacuum, washed with diethyl ether (10 mL x 2) and filtered off to give 4a-g in pure form.
2.3.1 (2S,3R)-2-Amino-3-methyl-N-phenylpentanamide hydrochloride (4a)
1
White solid; yield: 75%, mp: 230-235 °C, H NMR (400 MHz, CDCl3) δ 10.17 (br s, 1H,
+
NHAr), 8.15 (br s, 3H, NH3 ), 7.72–7.47 (m, 2H, H-3Ar & H-5Ar), 7.19 – 7.00 (m, 3H, H-2Ar ,
H-4Ar & H-6Ar), 4.53 – 4.48 (m, 1H, CHisoleucine), 2.16 – 1.92 (m, 1H), 1.70 – 1.50 (m, 1H),
1.20 – 1.06 (m, 1H), 1.01 – 0.90 (br s, 3H, Meisoleucine), 0.80 – 0.57 (br s, 3H). 13C NMR (101
MHz, CDCl3) δ 166.93 (C=Oamide), 137.21 (C-1Ar), 128.86 (C-3/5Ar), 124.82 (C-4Ar), 120.53
(C-2/6Ar), 58.58 (NH-CHisoleucine), 36.98, 24.75, 14.36, 11.36. IR (KBr, υ, cm-1) 3410, 3333,
2970, 1666, 1520, 1173, 1026. HRMS (ESI): m/z calcd for Chemical Formula: C12H19N2O:
207.1497 [M - Cl]+, found : 207.1484.
2.3.2 (2S,3R)-2-Amino-N-(benzo[d]thiazol-2-yl)-3-methylpentanamide hydrochloride (4b)
1
White solid; yield: 88%, mp: 180-183 °C, H NMR (400 MHz, DMSO-d6) δ 8.55 (br s, 3H,
+
NH3 ), 7.79 (d, J = 1.4 Hz, 1H, Hbenzothiazolyl), 7.65 (d, J = 3.9 Hz, 1H, Hbenzothiazolyl), 7.38 –
7.27 (m, 1H, Hbenzothiazolyl), 7.25 – 7.14 (m, 1H, Hbenzothiazolyl), 4.06 – 3.93 (m, 1H, CHisoleucine),
2.09 – 1.92 (m, 1H,), 1.62– 1.45 (m, 1H), 1.21 – 1.10 (m, 1H), 0.94 – 0.82 (overlapped m,
6H).13C NMR (101 MHz, DMSO-d6) δ 168.50 (C=Oamide), 157.39 (C-2benzothiazolyl), 148.45,
131.80, 126.39, 124.15, 121.71, 121.00, 57.27 (NH-CHisoleucine), 36.65, 24.51, 14.87, 11.46 .
IR (KBr, υ, cm-1) 3340, 3055, 2978, 1674, 1528, 1173. HRMS (ESI): m/z calcd for Chemical
Formula: C13H18N3OS: 264.1170 [M - Cl]+, found : 264.1192.
2.3.3
(S)-2-Amino-N-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)propanamide hydrochloride (4c)
Orange solid; yield: 89%, mp: highly hygroscopic,1H NMR (400 MHz, DMSO-d6) δ 9.85 (br
+
s, 1H, NHAr), 8.48 (br s, 3H, NH3 ), 7.62 – 7.58 (m, 2H, HAr-pyrazolinyl), 7.46 – 7.41 (m, 3H,

HAr-pyrazolinyl), 4.15 – 4.07 (m, 1H, CHalanine), 3.17 (s, 3H, N-Mepyrazolinyl), 2.24 (s, 3H, C-
13
Mepyrazolinyl), 1.55 (d, J = 7.0 Hz, 3H, Mealanine). C NMR (101 MHz, DMSO-d6) δ 169.78
(C=Oamide), 160.65 (C=Opyrazolinyl), 151.76, 134.65, 129.67, 127.50, 124.77, 105.96, 48.69
(NH-CHalanine), 36.03 (N-CH3pyrazolinyl), 17.78 (C-CH3pyrazolinyl), 11.36. IR (KBr, υ, cm-1)
3433, 3325, 2986, 1690, 1597, 1165, 756. HRMS (ESI): m/z calcd for Chemical Formula:
C14H19N4O2: 275.1508 [M - Cl]+, found : 275.1496.
2.3.4 (S)-2-Amino-3-phenyl-N-(2,4,6-trimethoxyphenyl)propanamide hydrochloride (4d)
Off-White solid; yield: 85%, hygroscopic, 1H NMR (400 MHz, DMSO-d6) δ 10.54 (br s, 1H,
+
NHAr), 8.38 (br s, 3H, NH3 ), 7.46 – 7.38 (m, 5H, HAr-phenylalanine), 6.99 (s, 2H, H-3Ar & H-
3Ar), 4.31– 4.22 (m, 1H, CHphenylalanine), 3.82 (s, 6H, 2xOCH3), 3.71 (s, 3H, OCH3), 3.21 –
13
3.11 (m, 2H). C NMR (101 MHz, DMSO-d6) δ 166.84 (C=Oamide), 153.14 (C-1Ar), 148.14,
135.18, 134.41, 130.00, 128.96, 127.64, 97.93, 63.19 (NH-CHphenylalanine), 60.52 (OCH3),
56.24 (OCH3), 54.58 (OCH3), 37.33. IR (KBr, υ, cm-1) 3340, 2978, 2932, 1674, 1528, 1173,
818. HRMS (ESI): m/z calcd for Chemical Formula: C18H23N2O4: 331.1657 [M - Cl]+, found
: 331.1668.
2.3.5 (S)-2-Amino-3-methyl-N-(pyridin-4-yl)butanamide hydrochloride (4e)
1
White solid; yield: 96%, mp: 240-242 °C, H NMR (400 MHz, DMSO-d6) δ 12.91 (s, 1H,
+
NHAr), 8.68 (d, J = 3.5 Hz, 2H, H-3pridyl & H-5pridyl), 8.51 (br s, 3H, NH3 ), 8.28 (d, J = 4.0
Hz, 2H, H-2pridyl & H-6pridyl), 4.21 – 4.14 (m, 1H, CHvaline), 1.19 – 1.08 (m, 1H), 1.01 (d, J =
6.7 Hz, 3H), 0.96 (d, J = 6.7 Hz, 3H).13C NMR (101 MHz, DMSO-d6) δ 170.17 (C=Oamide),
152.77, 142.48, 115.35, 58.83 (NH-CHvaline), 30.26, 18.87, 17.80. IR (KBr, υ, cm-1) 3433,
3310, 3078, 2978, 1651, 1597, 1173, 987, 818. HRMS (ESI): m/z calcd for Chemical
Formula: C10H16N3O: 194.1293 [M - Cl]+, found : 194.1312.
2.3.6 (2S,3R)-2-Amino-3-methyl-N-(thiazol-2-yl)pentanamide hydrochloride (4f)
1
White solid; yield: 89%, mp: 238-240 °C, H NMR (400 MHz, CDCl3) δ 12.14 (br s, 1H,
+
NHAr), 8.17 (br s, 3H, NH3 ), 7.70 – 7.58 (m, 1H, Hthiazolyl), , 7.22 – 7.15 (m, 1H, Hthiazolyl),
4.55 – 4.48 (m, 1H, CHisoleucine), 2.06 – 1.97 (m, 1H), 1.64 – 1.47 (m, 1H), 1.12 (m, 1H), 0.91
(brs, 3H), 0.69 (brs, 3H). 13C NMR (101 MHz, CDCl3) δ 170.62 (C=Oamide), 155.67 (C-
2thiazolyl), 137.10, 113.79, 58.98 (NH-CHisoleucine), 38.18, 24.53, 15.46, 11.18. IR (KBr, υ, cm-

1) 3310, 3086, 2978, 1678, 1620, 1520, 1196, 694. HRMS (ESI): m/z calcd for Chemical
Formula: C9H16N3OS: 214.1041 [M - Cl]+, found : 214.1063.
2.3.7 (S)-2-Amino-N-(p-tolyl)propanamide hydrochloride (4g)
1
White solid; yield: 79%, mp: 108-110°C, H NMR (400 MHz, CDCl3 + DMSO-d6) δ 10.61
+
(s, 1H, NHAr), 8.31 (br s, 3H, NH3 ), 7.49 (d, J = 8.2 Hz, 2H, H-3Ar & H-5Ar), 7.01 (d, J = 8.1
Hz, 2H, H-2Ar & H-6Ar), 4.10 – 4.02 (m, 1H, CHalanine), 2.20 (s, 3H, MeAr), 1.45 (d, J = 6.8
Hz, 3H, Mealanine). 13C NMR (101 MHz, CDCl3 + DMSO-d6) δ 168.18 (C=Oamide), 136.12 (C-
1Ar), 133.18 (C-4Ar), 129.33 (C-3/5Ar), 119.75 (C-2/6Ar), 49.42 (CHalanine), 20.90 (CH3Ar),
17.66 (CH3alanine). IR (KBr, υ, cm-1) 3402, 3302, 2978, 2901, 1675, 1628, 1528, 1196, 1026,
694. HRMS (ESI): m/z calcd for Chemical Formula: C10H15N2O: 179.1184 [M - Cl]+, found :
179.1176.
2.4 General Synthesis of bile acid-aminoacyl aromatic/heteroaromatic amides
To a stirred solution of 5a-b (0.5 mmol) in DMF, triethyl amine (1.25 mmol) was added at
o
0 C and subsequently EDC.HCl (0.75 mmol) and HOBt (0.5 mmol) was added and the
reaction mixture was stirred for 15 min. at 0 oC. Later the aminoacyl aromatic/heteroaromatic
amides hydrochloride salts (4a-g, 0.6 mmol) was added and the reaction was stirred at room
temperature for 6-8 h. The completion of the reaction was monitored by TLC. After the
completion of the reaction, crushed-ice was added and the resulted precipitate was filtered,
washed with cold water and recrystallized from ethanol to yield pure 6a-m.
2.4.1 (2S,3R)-3-Methyl-N-phenyl-2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-
trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamido)pentanamide (6a)
1
White solid; yield: 85%; mp: 133-135 °C, H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H,
NHAr), 8.09 (d, J = 8.4 Hz, 1H, NHisoleucine), 7.62 (d, J = 7.9 Hz, 2H, H-3Ar & H-5Ar), 7.29 (t,
J = 7.8 Hz, 2H, H-2Ar & H-6Ar), 7.04 (t, J = 7.3 Hz, 1H, H-4Ar), 4.36 – 4.26 (m, 2H,
CHisoleucine & OHCA), 4.06 – 4.00 (m, 1H, H-12CA), 4.03 – 4.15 (m, 1H, H-7CA), 3.78 (br s,
1H, OHCA), 3.60 (br s, 1H, OHCA), 3.21 – 3.15 (m, 1H, H-3CA), 2.23 – 1.92 (m, 6H), 1.82–
1.72 (m, 4H), 1.69 – 1.58 (m, 4H), 1.43 – 1.14 (m, 13H), 0.93 (d, J = 6.2 Hz, 3H, Me-21CA),
13
0.88 – 0.82 (m, 6H, 2xMeisoleucine), 0.80 (s, 3H, Me-19CA), 0.55 (s, 3H, Me-18CA). C NMR
(101 MHz, DMSO) δ 173.35 (C-24CA), 171.16 (C=Oisoleucine), 139.36 (C-1Ar), 129.12 (C-
3/5Ar), 123.70 (C-4Ar), 119.71 (C-2/6Ar), 71.48 (C-12CA), 70.90 (C-3CA), 66.70 (C-7CA), 58.09

(NH-CHisoleucine), 46.68, 46.19, 41.98, 41.80, 36.88, 35.77, 35.66, 35.55, 34.85, 32.84, 32.36,
30.85, 28.99, 27.78, 26.66, 25.04, 23.28, 23.08 (C-19CA), 17.63 (C-21CA), 15.80 & 12.77
(2xMeisoleucine), 11.21 (C-18CA). IR (KBr, υ, cm-1) 3394, 3310, 3094, 2932, 2870, 1651, 1551.
HRMS (ESI): m/z calcd for Chemical Formula: C36H57N2O5 [M + H]+: 597.4267, found :
597.4288. [α]20D = +15.0 (c 1.0, MeOH)
2.4.2 (2S,3R)-2-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)-3-methyl-N-
phenylpentanamide (6b)
1
White solid; yield: 82%; mp : 130-131 °C, H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H,
NHAr), 8.04 (d, J = 8.5 Hz, 1H, NHisoleucine), 7.61 (d, J = 7.8 Hz, 2H, H-3Ar & H-5Ar), 7.30 (t,
J = 7.8 Hz, 2H, H-2Ar & H-6Ar), 7.04 (t, J = 7.3 Hz, 1H, H-4Ar), 4.49 (d, J = 3.7 Hz, 1H,
OHDCA), 4.29 (t, J = 8.2 Hz, 1H, CHisoleucine), 4.20 (br s, 1H, H-12DCA), 3.79 (br s, 1H,
OHDCA), 3.29 – 3.22 (m, 1H, H-3DCA), 2.26 – 2.00 (m, 4H), 1.81 – 1.74 (m, 5H), 1.66 – 1.58
(m, 4H), 1.53 – 1.44 (m, 4H), 1.37–1.29 (m, 8H), 1.22 – 1.10 (m, 4H), 0.93 (d, J = 5.8 Hz,
3H, Me-21DCA), 0.86 – 0.84 (m, 6H, 2xMeisoleucine), 0.81 (d, J = 7.6 Hz 3H, Me-19DCA), 0.56
(s, 3H, Me-18DCA).13C NMR (101 MHz, DMSO-d6) δ 173.31 (C-24DCA), 171.10
(C=Oisoleucine), 139.33 (C-1Ar), 129.10 (C-3/5Ar), 123.69 (C-4Ar), 119.68 (C-2/6Ar), 71.48 (C-
12DCA), 70.42 (C-3DCA), 58.01 (NH-CHisoleucine), 47.89, 46.67, 46.43, 46.13, 42.07, 36.93,
36.74, 36.11, 35.57, 34.26, 33.36, 32.76, 32.30, 30.68, 29.74, 29.04, 27.67, 27.45, 26.57,
25.02, 23.98, 23.53 (C-19DCA), 17.59 (C-21DCA), 15.78 & 12.84 (Meisoleucine), 11.21(C-18DCA).
IR (KBr, υ, cm-1) 3394, 3302, 3140, 3086, 2932, 2870, 1652, 1551. HRMS (ESI): m/z calcd
for Chemical Formula: C36H57N2O4: 581.4318 [M + H]+, found : 581.4306. [α]20D = +25.0 (c
1.0, MeOH)
2.4.3
(2S,3R)-N-(Benzo[d]thiazol-2-yl)-3-methyl-2-((R)-4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-17-yl)pentanamido)pentanamide (6c)
o
White solid; yield: 92%; mp: 139-141 C, 1H NMR (400 MHz, DMSO-d6) δ 12.29 (br s, 1H,
NHbenzothiazolyl), 8.09 (d, J = 7.9 Hz, 1H, NHisoleucine), 7.93 (d, J = 7.5 Hz, 1H, Hbenzothiazolyl),
7.71 (d, J = 8.0 Hz, 1H, Hbenzothiazolyl), 7.40 (t, J = 7.4 Hz, 1H, Hbenzothiazolyl), 7.27 (t, J = 7.6
Hz, 1H, Hbenzothiazolyl), 4.43 (t, J = 8.0 Hz, 1H, CHisoleucine), 4.30 – 4.23 (m, 1H, OHCA), 4.11 –
4.00 (m, 1H, H-12CA), 3.98– 3.90 (m, 1H, H-7CA), 3.74 (br s, 1H, OHCA), 3.55 (m, 1H,

OHCA), 3.21 – 3.08 (m, 1H, H-3CA), 2.21 – 1.94 (m, 5H), 1.86 – 1.57 (m, 7H), 1.49 – 1.35 (m,
5H), 1.31 – 1.08 (m, 10H), 0.91 (d, J = 6.4 Hz, 3H, Me-21CA), 0.84 – 0.78 (m, 6H,
Meisoleucine), 0.75 (s, 3H, Me-19CA), 0.49 (s, 3H, Me-18CA).13C NMR (101 MHz, DMSO-d6) δ
173.58 (C-24CA), 172.42 (C=Oisoleucine), 149.01 (C-1benzothiazolyl), 131.94, 126.47, 123.89,
122.04, 120.90, 71.44 (C-12CA), 70.87 (C-3CA), 66.67 (C-7CA), 57.53 (NH-CHisoleucine), 46.63,
46.16, 41.76, 41.97, 36.60, 35.75, 35.33, 35.51, 34.82, 32.67, 32.22, 31.38, 30.84, 28.96,
27.71, 26.63, 24.99, 23.22, 23.03, 22.49 (C-19CA), 17.67 (C-21CA), 15.72 & 12.70
(2xMeisoleucine), 11.16 (C-18CA). IR (KBr, υ, cm-1) 3394, 3063, 2932, 2870, 1651, 1551.
HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula: C37H56N3O5S: 654.3940, found :
654.3922. [α]20D = +6.0 (c 1.0, MeOH).
2.4.4 (2S,3R)-N-(Benzo[d]thiazol-2-yl)-2-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-
dihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamido)-3-methylpentanamide (6d)
o
White solid; yield: 91%; mp: 119-121 C, 1H NMR (400 MHz, DMSO-d6) δ 12.45 (br s, 1H,
NHbenzothiazolyl), 8.14 (br s, 1H, NHisoleucine), 7.97 (d, J = 6.7 Hz, 1H, Hbenzothiazolyl), 7.74 (d, J =
6.9 Hz, 1H, Hbenzothiazolyl), 7.54 – 7.37 (m, 1H, Hbenzothiazolyl), 7.33 – 7.27 (m, 1H, Hbenzothiazolyl),
4.45 – 4.42 (m, 2H, CHisoleucine & H-12 DCA), 4.21 (br s, 1H, OH DCA), 3.78 (br s, 1H, OH DCA),
3.29 – 3.21 (m, 1H, H-7 DCA), 2.34 – 2.01 (m, 4H), 1.81 – 1.59 (m, 9H), 1.54 – 1.43 (m, 4H),
1.34 – 1.15 (m, 12H), 0.93 (d, J = 4.3 Hz, 3H, Me-21DCA), 0.87 (d, J = 18.6 Hz, 6H,
Meisoleucine), 0.82 (s, 3H, Me-19 DCA), 0.53 (s, 3H, Me-18 DCA).13C NMR (101 MHz, DMSO-
d6) δ 173.66 (C-24DCA), 172.45 (C=Oisoleucine),158.17, 149.03, 131.96, 126.52, 123.95, 122.08,
120.96, 71.47 (C-12DCA), 70.42 (C-3DCA), 57.54, 47.86, 46.66, 46.42, 42.07, 36.73, 36.09,
35.60, 34.25, 33.35, 32.61, 32.18, 30.68, 29.02, 27.67, 27.44, 26.56, 25.01, 23.96, 23.52 (C-
19DCA), 17.62 (C-21DCA), 15.72 & 12.80 (2xMeisoleucine), 11.19 (C-18 DCA). IR (KBr, υ, cm-1)
3394, 3310, 3070, 2932, 2870, 1651, 1551. HRMS (ESI): m/z [M+H]+ calcd for Chemical
Formula: C37H56N3O4S: 638.3991, found : 638.3976. [α]20D = +58.0 (c 1.0, MeOH)
2.4.5
(R)-N-((S)-1-((1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)amino)-1-
oxopropan-2-yl)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (6e)
o
Orange solid; yield: 72 %; mp: 152-154 C.1H NMR (400 MHz, DMSO-d6) δ 9.13 (s, 1H,
NHpyrazolinyl), 8.10 (d, J = 6.9 Hz, 1H, NHalanine), 7.59 (t, J = 7.9 Hz, 2H, HAr-pyrazolinyl), 7.41
(dd, J = 16.6, 8.0 Hz, 3H, HAr-pyrazolinyl), 4.40 (d, J = 4.2 Hz, 1H, OHCA), 4.23 – 4.17 (m , 2H,

CHalanine & H-12CA), 4.10 (d, J = 3.0 Hz, 1H, H-7CA), 3.87 (br s, 1H, OHCA), 3.70 (br s, 1H,
OHCA), 3.38 – 3.35 (m, 1H, H-3CA), 3.12 (s, 3H, N-Mepyrazolinyl), 2.17 (s, 3H, C-Mepyrazolinyl),
2.13 – 2.00 (m, 3H), 1.91– 1.83 (m , 4H), 1.77 – 1.68 (m , 4H), 1.54 – 1.45 (m, 6H), 1.36 –
1.29 (m, 7H), 1.01 (d, J = 5.6 Hz, 3H, Mealanine), 0.98 – 0.91 (m, 3H, Me-21CA), 0.90 (s, 3H,
Me-19CA), 0.67 (s, 3H, Me-18CA). 13C NMR (101 MHz, DMSO-d6) δ 172.98 (C-24CA),
172.63 (C=Oalanine), 171.87 (C=Opyrazolinyl), 162.11, 152.72, 135.46, 129.48, 126.58, 123.80,
107.79, 71.42 (C-12CA), 70.86 (C-3CA), 66.64 (C-7CA), 46.58 (NH-CHalanine), 46.19, 41.80,
36.74, 36.49, 35.74, 35.67, 35.65, 35.48, 35.46, 35.30, 34.81, 32.65, 31.99, 31.83, 31.14,
30.83, 28.96, 27.75, 26.64, 23.24, 23.05(C-19CA), 18.79 & 17.59 (C-Mepyrazolinyl & Mealanine),
17.36 (C-21CA), 11.57 (C-18 CA). IR (KBr, υ, cm-1) 3394, 3086, 3932, 2870, 1751, 1651,
1520. HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula: C38H57N4O6: 665.4278, found
: 665.4242. [α]20D = +18.0 (c 1.0, MeOH)
2.4.6
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-((S)-1-((1,5-dimethyl-3-oxo-
2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)amino)-1-oxopropan-2-yl)pentanamide (6f)
o
1
Orange solid; yield: 67%; mp: 158-160 C, H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H,
NHpyrazolinyl), 8.10 (d, J = 7.3 Hz, 1H, NHalanine), 7.58 (t, J = 7.7 Hz, 2H, HAr-pyrazolinyl), 7.38 –
7.43 (m, J = 16.7, 7.5 Hz, 3H, HAr-pyrazolinyl), 4.47 – 4.55 (m, 2H, CHalanine & H-12DCA), 4.28
(br s, 1H, OHDCA), 3.87 (br s, 1H, OHDCA), 3.39 – 3.33 (m, 1H, H-3DCA), 3.12 (s, 3H, N-
Mepyrazolinyl), 2.17 (s, 3H, C-Mepyrazolinyl), 1.92– 1.83 (m, 5H), 1.75 – 1.66 (m, 4H), 1.61 – 1.50
(m, 4H), 1.48 – 1.30 (m, 8H), 1.27 – 1.10 (m, 5H), 1.01 (d, J = 6.4 Hz, 3H, Mealanine), 0.94 (d,
J = 9.7 Hz, 6H, Me-19DCA & Me-21DCA), 0.66 (s, 3H, Me-18DCA).13C NMR (101 MHz,
DMSO-d6) δ 173.54 (C-24DCA), 172.99 (C=Oalanine), 172.62 (C=Opyrazolinyl), 162.12, 152.68,
135.49, 129.46, 126.56, 123.80, 107.82, 71.44 (C-12DCA), 70.39 (C-3DCA), 48.57 (NH-
CHalanine), 47.89, 46.65, 46.41, 42.06, 36.72, 36.48, 36.10, 35.59, 35.55, 34.24, 33.36, 32.63,
31.95, 31.10, 30.66, 29.03, 27.64, 27.43, 26.55, 23.94, 23.51 (C-19 DCA), 18.77 & 17.55 (C-
Mepyrazolinyl & Mealanine), 17.29 (C-21 DCA), 11.54 (C-18 DCA). IR (KBr, υ, cm-1) 3387, 3063,
2932, 2870, 1750. 1651, 1551. HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula:
C38H57N4O5: 649.4328, found : 649.4364. [α]20D = +58.0 (c 1.0, MeOH)
2.4.7
(R)-N-((S)-1-Oxo-3-phenyl-1-((2,4,6-trimethoxyphenyl)amino)propan-2-yl)-4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-17-yl)pentanamide (6g)

White solid; yield: 68%; mp: 151-153 oC, 1H NMR (400 MHz, DMSO-d6) δ 10.09 (d, J = 9.6
Hz, 1H), 8.24 (d, J = 8.3 Hz, 1H), 7.38 – 7.33 (m, 5H, HAr-phenylalanine), 7.06 (s, 2H, H-2Ar &
H-6Ar), 4.74 – 4.68 (m, 1H, CHphenylanine), 4.40 (d, J = 4.3 Hz, 2H, OH & H-12CA), 4.17 (d, J =
3.5 Hz, 1H, H-7CA), 4.09 (d, J = 3.3 Hz, 2H, 2xOHCA), 3.82 (s, 6H, 2xOCH3), 3.70 (s, 3H,
OCH3), 3.42 – 3.36 (m, 1H, H-3CA), 2.20 – 2.00 (m, 4H), 1.78 (d, J = 9.3 Hz, 3H), 1.63 (s,
2H), 1.27 (m, J = 32.3, 24.7, 11.7 Hz, 11H), 0.93 (d, J = 6.2 Hz, 3H), 0.88 – 0.83 (m, 6H),
0.80 (s, 3H, Me-19 CA), 0.55 (s, 3H, Me-18CA). 13C NMR (101 MHz, DMSO-d6) δ 173.24 (C-
24CA), 170.62 (C=Oalanine), 153.08, 138.26, 135.46, 133.85, 129.57, 128.45, 126.71, 97.43,
71.46 (C-12CA), 70.86 (C-3CA), 66.64 (C-7CA), 60.53 (OCH3), 56.10 (OCH3), 55.20 (OCH3),
46.67 (NH-CHphenylalanine), 46.58, 46.50, 46.25, 46.18, 46.12, 41.94, 41.77, 38.00, 35.81,
35.73, 35.51, 34.81, 32.69, 32.11, 30.83, 28.96, 27.70, 26.62, 23.22, 23.04 (C-19CA), 17.35
(C-21CA), 12.74 (C-18CA). IR (KBr, υ, cm-1) 3394, 3086, 2932, 2870, 1651, 1605. HRMS
(ESI): m/z [M+H]+ calcd for Chemical Formula: C42H61N2O8: 721.4427, found : 721.4396.
[α]20D = +8.0 (c 1.0, MeOH)
2.4.8
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-((S)-1-oxo-3-phenyl-1-
((2,4,6-trimethoxyphenyl)amino)propan-2-yl)pentanamide (6h)
o
1
White solid; yield: 62%; mp: 151-153 C, H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H,
NHAr), 8.26 (d, J = 7.8 Hz, 1H, NHphenylalnine), 7.40 – 7.33 (m, 5H, HAr-phenylalanine), 7.07 (s, 2H,
H-3Ar & H-5Ar), 4.71 – 4.67 (m, 1H, CHphenylalnine), 4.55 (d, J = 4.1 Hz, 2H, H-12DCA
&
OHDCA), 4.29 – 4.21 (m , 1H, OHDCA), 3.82 (s, 6H, 2xOCH3), 3.70 (s, 3H, OCH3), 3.39 –
3.34 (m, 1H, H-3DCA), 1.82 – 1.73 (m, 5H), 1.65 – 1.53 (m, 7H), 1.42 – 1.35 (m, 8H), 1.14 –
1.03 (m, 6H), 0.93 (d, J = 4.7 Hz, 3H, Me-21 DCA), 0.92 (s, 3H, Me-19 DCA), 0.62 (s, 3H, Me-
18 DCA). 13C NMR (101 MHz, DMSO-d6) δ 173.20 (C-24DCA), 170.62 (C=Ophenylalanine),
153.08, 138.26, 135.47, 133.85, 129.56, 128.45, 126.70, 97.44, 71.41(C-12DCA), 70.37 (C-
3DCA), 60.47 (OCH3), 56.10 (OCH3), 47.86 (NH-CHphenylalanine), 46.74, 46.55, 46.49, 46.43,
46.37, 42.03, 36.72, 36.08, 35.57, 35.40, 34.24, 33.33, 32.66, 32.06, 30.66, 29.02,
27.60,27.41 26.53, 23.93, 23.51 (C-19DCA), 17.40 (C-21DCA), 12.81 (C-18DCA). IR (KBr, υ,
cm-1) 3394, 3317, 2932, 2862, 1651, 1543. HRMS (ESI): m/z [M+H]+ calcd for Chemical
Formula: C42H61N2O7: 705.4478 found : 705.4448. [α]20D = +29.0 (c 1.0, MeOH)

2.4.9
(R)-N-((S)-3-Methyl-1-oxo-1-(pyridin-4-ylamino)butan-2-yl)-4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13-dimethylhexadecahydro-
1H-cyclopenta[a]phenanthren-17-yl)pentanamide (6i)
o
1
White solid; yield: 60%; mp: 147-148 C, H NMR (400 MHz, DMSO-d6) δ δ 10.50 (s, 1H,
NHpyridyl), 8.42 (d, J = 4.7 Hz, 2H, H-3pridyl & H-5pridyl), 8.12 (d, J = 7.6 Hz, 1H, NHvaline),
7.58 (d, J = 5.2 Hz, 2H, H-2pridyl & H-6pridyl), 4.35 – 4.33 (m, 1H, CHvaline), 4.29 – 4.23 (m,
1H, OHCA), 4.13 – 4.10 (m, 1H, H-12CA), 4.05 – 4.10 (m, 1H, H-7CA), 3.78 (br s, 1H,
OHCA), 3.60 (br s, 1H, OHCA), 3.24 – 3.13 (s, 1H, H-3CA), 2.24 – 1.99 (m, 7H), 1.80 – 1.62
(m, 7H), 1.40 – 1.21 (m, 11H), 0.94 (d, J = 5.8 Hz, 3H, Me-21CA), 0.92 – 0.86 (m, 6H,
2xMevaline), 0.80 (s, 3H, Me-19CA), 0.55 (s, 3H, Me-18CA). 13C NMR (101 MHz, DMSO-d6) δ
173.66 (C-24CA), 172.37 (C=Ovaline), 150.83, 145.84, 113.70, 71.48 (C-12CA), 70.89 (C-3CA),
66.69 (C-7CA), 59.40 (NH-CHvaline), 46.61, 46.19, 41.97, 41.83, 36.37, 35.35, 35.64, 35.76,
34.84, 32.32, 32.67, 30.70, 30.86, 28.99, 27.75, 26.66, 23.26, 23.09 (C-19CA) 19.62 & 18.94
(2xMevaline), 17.66 (C-21CA), 12.77 (C-18CA). IR (KBr, υ, cm-1) 3387, 3094, 2932, 2870,
1651, 1597. HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula: C34H54N3O5: 584.4063
found : 584.4077. [α]20D = +68.0 (c 1.0, MeOH)
2.4.10
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-((S)-3-methyl-1-oxo-1-
(pyridin-4-ylamino)butan-2-yl)pentanamide (6j)
o
1
White solid; yield: 58%; mp: 143-145 C, H NMR (400 MHz, DMSO-d6) δ 10.53 (s, 1H,
NHpyridyl), 8.42 (d, J = 2.9 Hz, 2H, H-3pridyl & H-5pridyl), 8.15 (d, J = 6.2 Hz, 1H, NHvaline),
7.58 (d, J = 4.2 Hz, 2H, H-2pridyl & H-6pridyl), 4.49 (br s, 1H, CHvaline), 4.17–4.29 (m, 2H, H-
12DCA & OHDCA), 3.79 (br s, 1H, OHDCA), 3.29–3.15 (m, 1H, H-3DCA), 2.27 – 2.15 (m, 2H),
2.09 – 1.99 (m, 2H), 1.83 – 1.69 (m, 4H), 1.69 – 1.56 (m, 3H), 1.53 – 1.45 (m,3H), 1.35 –
1.27 (m, 9H), 1.19 – 1.14 (m, 4H), 0.93 (d, J = 5.3 Hz, 3H, Me-21DCA), 0.91 – 0.87 (m, 6H,
2xMevaline), 0.84 (s, 3H, Me-19DCA), 0.56 (s, 3H, Me-18DCA).13C NMR (101 MHz, DMSO-d6)
δ 173.62 (C-24DCA), 172.36 (C=Ovaline), 150.80, 145.84, 113.71, 71.47 (C-12DCA), 70.41 (C-
3DCA), 59.44 (NH-CHvaline), 47.89, 46.43, 46.66, 42.06, 36.74, 36.10, 35.55, 35.60, 34.27,
33.36, 32.62, 32.28, 30.69, 29.04, 27.65, 27.44, 26.57, 23.97, 23.57 (C-19DCA), 19.63, &
18.95 (2xMevaline), 17.61 (C-21DCA), 12.86 (C-18DCA). IR (KBr, υ, cm-1) 3387, 3325, 3094,
2932, 2870, 1651, 1512. HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula:
C34H54N3O4: 568.4114 found :568.4112. [α]20D = +98.0 (c 1.0, MeOH)

2.4.11 (2S,3R)-3-Methyl-N-(thiazol-2-yl)-2-((R)-4-((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-
3,7,12-trihydroxy-10,13,14-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
yl)pentanamido)pentanamide (6k)
1
White solid; yield: 93%; mp: 158-160 oC, H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 7.1 Hz,
1H, NHthiazolyl), 7.47 (d, J = 3.4 Hz, 1H, Hthiazolyl), 6.99 (d, J = 3.5 Hz, 1H, Hthiazolyl), 4.80 –
4.72 (m, 1H, CHisoleucine), 4.42 (br s, 1H, NHisoleucine), 3.99– 3.92 (m, 1H, H-12CA), 3.83–3.78
(m, 1H, H-7CA), 3.44– 3.35 (m, 1H, H-3CA), 2.28 – 1.95 (m, 5H), 1.92 – 1.69 (m, 5H), 1.65 –
1.51 (m, 6H), 1.47 – 1.33 (m, 6H), 1.30 – 0.99 (m, 5H), 0.97 (s, 3H, Me-21CA), 0.89 (d, J =
5.5 Hz, 3H, Meisoleucine), 0.86 (d, J = 5.9 Hz, 6H, Meisoleucine & Me-19CA), 0.58 (s, 3H, Me-
18CA). 13C NMR (101 MHz, DMSO-d6) δ 173.30 (C-24CA), 171.85 (C=Oisoleucine), 137.90,
113.14, 71.46 (C-12CA), 70.89 (C-3CA), 66.69 (C-7CA), 57.66 (NH-CHisoleucine), 46.64, 46.18,
41.97, 41.80, 36.95, 35.76, 35.62, 35.34, 34.84, 32.80, 32.28, 30.85, 29.00, 27.78, 26.65,
25.01, 23.29, 23.09 (C-19CA), 17.61 (C-21CA), 15.82 & 12.75 (2xMeisoleucine), 11.34 (C-18CA).
IR (KBr, υ, cm-1) 3394, 3078, 2932, 2870, 1651, 1551. HRMS (ESI): m/z [M+H]+ calcd for
Chemical Formula: C33H54N3O5S: 604.3784 found: 604.3765. [α]20D = +18.0 (c 1.0, MeOH)
2.4.12
(2S,3R)-2-((R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13,14-
trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)-3-methyl-N-
(thiazol-2-yl)pentanamide (6l)
o
1
White solid; yield: 89%; mp: 148-150 C, H NMR (400 MHz, CDCl3) δ 7.51 (br s, 1H,
NHthiazolyl), 6.98 (br s, 2H, Hthiazolyl), 4.82 – 4.69 (m , 1H, CHisoleucine), 4.01– 3.90 (m, 1H,
NHisoleucine), 3.65 – 3.54 (m, 1H, H-12DCA), 3.41 – 3.23 (m, 1H, H-3DCA), 2.33 – 2.13 (m, 3H),
1.99 – 1.64 (m, 10H), 1.56 – 1.34 (m, 11H), 1.28 – 1.03 (m, 7H), 0.91 (overlapped s, 6H,
Meisoleucine & Me-21DCA), 0.88 (overlapped s, 6H, Meisoleucine & Me-19DCA), 0.62 (s, 3H, Me-
18DCA). 13C NMR (101 MHz, DMSO-d6) δ 173.49 (C-24DCA), 171.35 (C=Oisoleucine), 138.10,
113.84, 71.47 (C-12DCA), 70.40 (C-3DCA), 57.33(NH-CHisoleucine), 47.90, 46.67, 46.43, 42.05,
36.74, 36.65, 36.10, 35.60, 35.50, 34.27, 33.36, 32.61, 32.21, 30.68, 29.05, 27.67, 27.44,
26.57, 24.99, 23.98, 23.56 (C-19DCA), 17.59 (C-21DCA), 15.71 & 12.84 (2xMeisoleucine), 11.18
(C-18DCA). IR (KBr, υ, cm-1) 3402, 3078, 2932, 2870, 1651, 1551. HRMS (ESI): m/z [M+H]+
calcd for Chemical Formula: C33H54N3O4S: 588.3835 found: 588.3809. [α]20D = +28.0 (c 1.0,
MeOH)

2.4.13
(R)-N-((S)-1-Oxo-1-(p-tolylamino)propan-2-yl)-4-
((3R,5S,7R,8R,9S,10S,12S,13R,14S,17R)-3,7,12-trihydroxy-10,13,14-
trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide (6m)
o
1
White solid; yield: 93%; mp: 152-154 C, H NMR (400 MHz, DMSO-d6) δ 9.89 (s, 1H,
NHAr), 8.10 (d, J = 7.3 Hz, 1H, NHalanine), 7.48 (d, J = 8.2 Hz, 2H, H-3Ar & H-5Ar), 7.10 (d, J
= 8.2 Hz, 2H, H-2Ar & H-6Ar), 4.38 – 4.33 (m, 2H, CHalanine & OHCA), 4.13 – 4.10 (m, 1H, H-
12CA), 4.04 – 4.01 (m, 1H, H-7CA), 3.78 (br s, 1H, OHCA), 3.61 (br s, 1H, OHCA), 3.21 – 3.17
(m, 1H, H-3CA), 2.25 (s, 3H, MeAr), 2.17 – 2.00 (m, 5H), 1.82 – 1.60 (m, 9H), 1.46 – 1.29 (m,
10H), 1.26 (d, J = 6.9 Hz, 3H, Mealanine), 0.93 (d, J = 6.2 Hz, 3H, Me-21CA), 0.81 (s, 3H, Me-
13
19CA), 0.56 (s, 3H, Me-18CA). C NMR (101 MHz, DMSO-d6) δ 173.13 (C-24CA), 171.70
(C=Oalanine), 137.00, 132.50, 129.49, 119.59, 71.46 (C-12CA), 70.90 (C-3CA), 66.70 (C-7CA),
49.32 (NH-CHalanine), 46.59, 46.19, 41.98, 41.80, 35.76, 35.64, 35.35, 34.84, 32.65, 32.07,
30.86, 29.00, 27.78, 26.66, 23.27, 23.07, 20.90 (CH3alanine), 18.58 (C-19CA), 17.64 (C-21CA),
12.77 (C-18CA). IR (KBr, υ, cm-1) 3394, 3325, 3078, 2932, 2870, 1651, 1520. HRMS (ESI):
m/z [M+H]+ calcd for Chemical Formula: Chemical Formula: C34H53N2O5: 569.3954 found :
569.3937. [α]20D = +28.0 (c 1.0, MeOH)
2.4.14
(R)-4-((3R,5R,8R,9S,10S,12S,13R,14S,17R)-3,12-Dihydroxy-10,13,14-
trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-N-((S)-1-oxo-1-(p-
tolylamino)propan-2-yl)pentanamide (6n)
o
1
White solid; yield: 89%; m.p: 153-155 C, H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H,
NHAr), 8.13 (d, J = 7.1 Hz, 1H, NHalanine), 7.48 (d, J = 8.2 Hz, 2H, H-3Ar & H-5Ar), 7.10 (d, J
= 8.1 Hz, 2H, H-2Ar & H-6Ar), 4.52 – 4.45 (m, 1H, CHalanine), 4.42 – 4.35 (m, 1H, H-12DCA),
4.22 (br s, 1H, OHDCA), 3.79 (br s, 1H, OHDCA), 3.32 – 3.21 (m, 1H H-3DCA), 2.24 (s, 3H,
MeAr), 2.18 – 1.96 (m, 3H),1.83 – 1.71 (m, 6H), 1.69 – 1.54 (m, 6H), 1.52 – 1.29 (m, 13H),
1.25 (d, J = 6.9 Hz, 3H, Mealanine), 0.93 (d, J = 6.2 Hz, 3H, Me-21DCA), 0.84 (s, 3H, Me-
13
19DCA), 0.57 (s, 3H, Me-18DCA). C NMR (101 MHz, DMSO-d6) δ 173.11 (C-24DCA), 171.71
(C=Oalanine), 137.01, 132.48, 129.47, 119.59, 71.47 (C-12DCA), 70.42 (C-3DCA), 49.35 (NH-
CHalanine), 47.90, 46.65, 46.43, 42.07, 37.21, 36.74, 36.11, 35.55, 35.60, 34.26, 33.37, 32.62,
32.03, 30.68, 29.05, 27.45, 27.67, 26.57, 23.97, 23.53, 20.89 (NH-CHalanine), 18.57 (C-19CA),
17.60 (C-21CA), 12.86 (C-18CA). IR (KBr, υ, cm-1) 3394, 3310, 3070, 2932, 2862, 1651, 1512.
HRMS (ESI): m/z [M+H]+ calcd for Chemical Formula: C34H53N2O4: 553.4003 found :
553.4029. [α]20D = +48.0 (c 1.0, MeOH)

2.5 Cytotoxicity Studies
2.5.1 Cell Lines and Cell Culture
The cell lines MDA-MB-231 (human breast adenocarcinoma cell line), HT29 (human
colorectal adenocarcinoma cell line), U87 (human glioblastoma cell line) and HEK293T
(Human normal cell line) were obtained from the National Centre for Cellular Sciences
(NCCS), Pune, India. Cells were cultured either in RPMI -1640 (MDA-MB-231,HEK293T),
DMEM (HT-29) or MEM (U87) media, supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and 1 mM NaHCO3, 2 mM-glutamine, 100 units/ml Penicillin and 100 µg/mL
Streptomycin. All cell lines were maintained in culture at 37 °C in an atmosphere of 5% CO2.
2.5.2 Test Concentration
Initially, stock solutions of each test substances were prepared in 100% DMSO with a final
concentration of 10 mM. Further dilutions were made with culture medium to obtain
experimental stock concentration of 100-0.01 µM. Exactly 100 µL of each diluent was added
to 100 µL of cell suspension (total assay volume of 200 µL) and incubated for 48 h at 37 °C
in 5% CO2.
2.5.3 Methodology
Cytotoxicity was measured using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide] assay, according to the method of Johan van Meerloo [28]. Briefly, the
cells (5 x 103) were seeded in each well containing 0.1 ml of medium in 96 well plates. After
overnight incubation at 37 °C in 5% CO2, the cells were treated with 100 µL of different test
concentrations of test compounds (0.01 to 100 µM) at identical conditions with three
replicates each. The final test concentrations were equivalent to 0.01 µM to 100 µM. The
cell viability was assessed after 48 h, by adding 10 µL of MTT (5 mg/mL) per well. The
plates were incubated at 37 °C for additional 2-4 h. The medium was discarded and the
formazan violet crystals, which were colored for the viable cells, were dissolved with 100 µL
of DMSO. The rate of color formation was measured at 570 nm in a spectrophotometer
(Spectra MAX Plus; Molecular Devices; supported by SOFTmax PRO-5.4). The percent
inhibition of cell viability was determined with reference to the control values (without test
compound). The data were normalised with the lowest and highest values and subjected to
non-linear regression analysis to obtain log (concentration) Vs normalised expression
(variable slope) using graph pad prism 6. The GI50 (inhibition of cell growth) concentrations
were further obtained using the Graph pad prism 6.

3. Results & Discussion
3.1 Chemistry
Synthesis of hybrid molecules between two bioactive moieties have emerged as a successful
approach for the discovery of novel cytotoxic agents with high potency and lower side
effects. In view of this and taking a lead from the existing bile acid amino acid conjugates
(Figure 1), we planned to synthesize bile acid-aryl/heteroaryl amides linked via amino acids.
Two strategies were initially planned for the synthesis of targeted molecules (Figure 2). The
first strategy involves the synthesis of CA-amino acid conjugates [29], followed by coupling
of C-terminus of amino acid residue with an aromatic/heteroaromatic amines, while the
second strategy employs the synthesis of amino acid-aryl/heteroaryl amides, followed by its
coupling with bile acid.
Figure 2. Possible strategies for the synthesis of targeted molecules
We initially attempted the first strategy by synthesizing CA-alanine conjugate in 74% yield
by coupling CA and alanine using DCC/HOBt in DMF:H2O (7:3). However, further coupling
of aniline to CA-alanine conjugate proceeded in poor yields using several coupling reagents
such as DCC, EDC.HCl, EEDQ. We thus switched towards employing the second strategy.
Here, the synthesis commenced with the coupling of protected L-α-amino acids (1a-d) with
different amines (2a-g) using EDC.HCl/HOBt in DMF to yield Boc L-aminoacyl aromatic/
heteroaromatic amides (3a-g) in 46-81% yield. Deprotection of Boc group in 3a-g at room
temperature yielded unprotected aminoacyl aromatic/herteroaromatic amides (4a-g) as
hydrochloride salts in quantitative yields (Scheme 1). The structures of the 3 & 4 were
characterized on the basis of 1H & 13C NMR and mass spectroscopic studies.
Scheme 1. Synthesis of unprotected L-aminoacyl aromatic & heteroaromatic amides

CA (5a) and DCA (5b) were reacted with unprotected L-aminoacyl aromatic/heteroaroomatic
amides (4a-g) using EDC.HCl/HOBt in DMF at room temperature in 6-8 h to yield their
corresponding amides (6a-n) in excellent yields (Scheme 2). The structures of 6a-n were
characterized on the basis of 1H, C NMR, IR and mass spectroscopic studies. In particular,
13
the relative and concordant integrations of aromatic protons of aryl/heteroaryl moiety to that
of methine proton on C-3, C-12 (and C-6 in case of ) carbons bearing the hydroxyl groups
and the three methyl groups of the CA and DCA moiety confirmed the formation of the
products.
Scheme 2. Synthesis of CA and DCA aryl/heteroaryl amides

3.2 Cytotoxicity Tests
The biological activities of 6a-n were evaluated to investigate their anti-
proliferative/cytotoxic activities in three different types of human cancer cell lines such as
colon, breast and glioblastoma cancers (HT-29, MDA-MB-231 & U87) and percentage
cytotoxicity on normal cell line (HEK293T) (Table 1). Few compounds showed good activity
against breast and glioblastoma cancer cell lines i.e., three (6a, 6c, 6m) for breast and three
(6e, 6i, 6m) for glioblastoma cell lines, out of total fourteen derivatives tested (Table 1). It is
apparent from the results that few derivatives are found to be more potent on breast cancer
cell lines than the other two. Even some derivatives were found to be more potent than
Doxorubicin and Cisplatin against these three cell lines. For instance, compound 6a (1.35
µM), 6c (1.41 µM) and 6m (4.52 µM) possessing phenyl, benzothiazole and 4-methylphenyl
groups showed fairly good activity against the breast cancer cell line with respect to Cisplatin
(7.21 µM) and comparable with to Doxorubicin (1 µM). Compound 6e (2.49 µM), 6i (2.46
µM) and 6m (1.62 µM) showed better activity against glioblastoma cancer cell line with
respect to both Cisplatin (2.60 µM) and Doxorubicin (3.78 µM) drugs used as standards.
Figure 3 display the concentration-responsive curves of the compounds 6e, 6i, 6m on
glioblastoma cell lines and 6a, 6c and 6m on breast cancer cell lines. Further testing the
compounds on a human normal kidney cell line (HEK293T) indicated that >65% compounds
to be safer.
Table -1 In vitro anti-cancer activity evaluatione of 6a-n by MTT assay
%Cytotoxicity
Compound
Code
GI50 [µM]
MDA-MB-231b
1.35
at 25 µM
HEK293Td
46.54
43.27
62.99
34.89
26.21
19.99
13.60
23.28
18.76
56.40
48.33
53.76
52.51
51.32
-
HT-29a
61.76
68.83
36.39
>100
>100
>100
47.43
>100
>100
>100
36.91
44.01
27.86
>100
0.3
U87MGc
66.56
>100
>100
57.17
2.49
6a
17.60
1.41
6b
6c
76.29
>100
6d
6e
70
5.50
6f
54.95
>100
13.19
72.29
2.46
6g
6h
75.76
82.64
36.19
32.64
4.52
6i
23.9
6j
22.15
6.16
6k
6l
6m
1.62
45.44
1.0
9.65
6n
2.60
Doxorubicin
Cisplatin
8.61
7.21
3.78
-

a colon cancer, b breast cancer, c glioblastoma cancer, d human normal kidney cell line
eAll values are with a standard deviation of 0.55 and were analysed using Graph Pad Prism software.
Figure 3. Log concentration-response curve of the most active compounds on cancer cell
lines
3.3 Drug Likliness Properties Evaluation
The synthesized conjugates were evaluated for drug likeliness properties in accordance with
Lipinski rule of five and the results are tabulated Table 2.
Table 2.Drug likeliness properties calculation for the Lipinsky rule using molinspiration.
Compound mi
LogP
TPSA
n
MW
n
n OHNH n viol n rotb Volume
atoms
ON
4.56
5.47
6.40
7.31
3.94
4.86
5.65
6.56
4.05
4.96
4.89
5.81
5.00
5.92
118.88 40
98.65 39
131.77 46
111.54 45
145.82 48
125.59 47
146.58 52
126.35 51
131.77 42
111.54 41
131.77 42
111.54 41
118.88 41
98.65 40
554.77 7
538.55 6
653.93 8
637.93 7
664.89 10
648.89 9
720.95 10
704.95 9
583.81 8
567.82 7
603.87 8
587.87 7
568.80 7
552.80 6
5
4
5
4
5
4
5
4
5
4
5
4
5
4
1
2
2
2
1
1
2
2
1
1
1
2
2
2
7
546.60
538.55
627.33
619.29
640.84
632.80
694.88
686.84
575.83
567.78
583.34
575.30
563.16
555.11
6a
6b
6c
6d
6e
6f
7
9
9
8
8
12
12
8
6g
6h
6i
6j
8
9
6k
6l
9
7
6m
6n
7

The Lipinski rule of five states that a molecule could be a oral drug candidate if it does not
violates more than one rule laid down, the rules are a molecule should not have more than
five hydrogen bond donor, it should not have more than 10 hydrogen bond acceptors, it
should not have molecular weight more than 500 dalton and finally it should not have an
octanol-water partition coefficient more than 5. The drug likeliness properties of the
synthesized conjugates were evaluated by using mol inspiration [30] and it was found that all
the conjugates violated one or more criteria laid down but the compounds which showed
fairly good activity with respect to the standard drugs used against one or the other cancer
lines used for e.g. compound 6a, 6e, 6i violates only 1 rule and could be considered as ideal
candidates for the development of oral drug medications. While compounds 6c and 6m
showed fairly good activity against the evaluated cancer cell lines but violates 2 rules, thus
with further structural modifications and evaluation against other cancer cell lines these
compounds could act as potential drug candidates because many natural products for e.g.
Vincristine, Vinblastine are there which violates one or more criteria of Lipinski rule but still
are potentially active molecules.
3.4 Plausible mode of mechanism
Numerous mechanism have been proposed and proved for the anti-cancer behaviour of bile
acid derivatives. The major mechanism for the action of bile acid derivatives is based on
apoptosis. For example, DCA is known to induce apoptosis via a protein kinase C-dependent
signalling pathway, while CA inhibits cell growth [19]. Reports have also indicated the role
of bile acids in the removal of damaged cells, maintaining homeostasis and thereby DNA
stability and protection against carcinogenesis [5]. Apoptosis has also been induced by bile
acid derivatives in human tumour cells via caspases, p53 and reactive oxygen species
inductions, which thus kills the tumour cells by forming death inducing signalling cascades
and by activating caspases 3 and 9 [31, 32].
Based on the literature reports, the following observations could be inferred from our studies:
1. Lower GI50 values of CA derivatives (6a, 6c, 6e, 6i, 6m) indicated a crucial role of
hydroxyl group at 7th position in the bile acid skeleton in inhibiting the growth of cancer cells
[22].
2. CA conjugates possessed lower lipophilicity than their corresponding DCA derivatives
suggesting that the importance of lipophilicity of the molecule on activity.
3. Among the CA derivatives, presence of aryl group in the side chain (R3) yielded
comparatively better activity against one or other cell lines than substituted aryl or heteroaryl

group. For instance, 6a (R3 = Ph) was found to be most active against breast cancer cell line
as compared to 6c (R3 = benzothiazolyl), 6m (R3 = p-tolyl). Similarly, 6m (R3 = p-tolyl) was
found to be more active against glioblastoma cancer cell line as compared to 6e (R3 =
pyrazolinyl), 6i (R3 = pyridyl). However, these observations cannot be generalized for all
molecules.
3.5 Future Scope of the Present Work
The potent compounds (6a, 6c, 6e, 6i, 6m) will be further studied for their role in growth
inhibition of human tumour cells. Importantly, apoptosis-based assays would be performed to
know the downstream signalling of the potent compounds. Some of the proceedings in future
study include the role of reactive oxygen species, DNA fragmentation and the changes in
caspase gene expression.
4. Conclusion
In summary, we have synthesized a series of bile acid-aminoacyl-aryl/heteroaryl amides in
excellent yields, the cytotoxicity of these were tested against three cancer cell lines. Many of
these derivatives are showing selective activity on one of the cell lines. Though, most of the
derivatives are comparatively less potent than the commercially available drug molecule
Doxorubicin and Cisplatin; nevertheless slight structural modification of these active
derivatives may yield as prospective anticancer drugs. Based on the present results, it is
warranted that these derivatives to be further evaluated on other cancer cell lines.
Acknowledgements
The authors are thankful to UGC, New Delhi for providing startup grant for the research. The
author DA is thankful to DST, New Delhi for providing Junior Research Fellowship.
Appendix A supplementary data
Supplementary data associated with this article can be found, in the online version, at http://
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•
•
A series of bile acid aryl/heteroaryl amides linked via α-amino acid were synthesized
in excellent yields.
Synthesized compounds were tested for their cytotoxic behavior against 3 human
cancer cell-lines (HT29, MDAMB231, U87) and 1 human normal cell line
(HEK293T).
•
•
Three of the conjugates showed promising results to be new anticancer agents with
good in vitro activity (GI50 = 1.35, 1.41, 4.52 µM) against the breast cancer cell line
in comparison to cisplatin (7.21 µM) and doxorubicin (1 µM).
Another three conjugates showed promising results to be new anticancer agents with
good in vitro activity (GI50 = 2.49, 2.46, 1.62 µM) against the Glioblastoma cancer
cell line in comparison to cisplatin (2.60 µM) and doxorubicin (3.78 µM).
• Greater than 65% of the synthesized compounds were found to be safer on human
normal cell line.