Design and synthesis of taxane derivatives of valproic acid as potent and selective cytotoxic agents

Article abstract of DOI:10.1007/s00044-016-1635-6

Resistance to anticancer agents has important implications for cancer chemotherapy. Small changes in chemical structures of cytotoxic agents can alter their biological interactions that can be beneficial in overcoming the drug resistance problem. Valproic acid, a well-known antiepileptic drug is in advanced clinical studies for cancer treatment. In the present study, valproic acid was incorporated into the taxane moiety at various positions and the new analogs were evaluated for their in vitro cytotoxicity. The novel analog, Valprotaxel showed comparable cytotoxicity in head and neck, and colon cancer cell lines with remarkable improvement in selectivity for cancer cells compared to paclitaxel and docetaxel.

Full text of DOI:10.1007/s00044-016-1635-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1635-6
ORIGINAL RESEARCH
Design and synthesis of taxane derivatives of valproic acid as potent and
selective cytotoxic agents
1
Sunil V. Mali1,2 Moonjit Das1
●
●
Himaja Malipeddi
Received: 6 April 2015 / Accepted: 27 June 2016
© Springer Science+Business Media New York 2016
Abstract Resistance to anticancer agents has important
implications for cancer chemotherapy. Small changes in
chemical structures of cytotoxic agents can alter their bio-
logical interactions that can be beneficial in overcoming the
drug resistance problem. Valproic acid, a well-known
antiepileptic drug is in advanced clinical studies for can-
cer treatment. In the present study, valproic acid was
incorporated into the taxane moiety at various positions and
the new analogs were evaluated for their in vitro cytotoxi-
city. The novel analog, Valprotaxel showed comparable
cytotoxicity in head and neck, and colon cancer cell lines
with remarkable improvement in selectivity for cancer cells
compared to paclitaxel and docetaxel.
human solid tumors such as ovarian, head and neck, blad-
der, lung and breast cancers, and have also been used to
treat malignant glioma and brain metastates (Wani et al.,
1971; Hajek et al., 1996; Lee et al., 1997; Glantz et al.,
1997; Brandes et al., 2000). However, aqueous insolubility,
lack of tumor cell selectivity and acquired and intrinsic
tumor resistance associated with P-glycoprotein (Pgp)
remain as limitations (Sparreboom et al., 1997). Drug
cocktails (combinations) are currently used to overcome
resistance problem in cancer chemotherapy. Small changes
in chemical structure of the taxane moiety can alter inter-
action with Pgp. Cabazitaxel (3), a 7,10 methyl ether analog
of docetaxel is a potent tubulin-binding taxane and a poor
P-gp substrate, approved by US Food and Drug Adminis-
tration in June 2010 for treatment, to prolong survival for
patients with metastatic multidrug resistant (MDR) prostate
cancer in the post docetaxel setting. BMS275183 (4), in
which the C-3 phenyl ring was replaced with t-butyl group
and C-4 acetate was replaced with carbonate enhanced the
oral efficacy in two tumor models (Mastalerz et al., 2003)
(Fig. 1). The C-10 succinate of paclitaxel was reported with
reduced P-gp interactions and improved permeability across
the blood–brain barrier (BBB) (Rice et al., 2003). There has
been a growing interest in the concept of combining two
drugs or pharmacophoric moieties in a single molecule to
create a more medically effective hybrid entity.
●
●
●
Keywords Cytotoxic agents Valproic acid Taxane
Valprotaxel
Introduction
Paclitaxel (1) and docetaxel (2) are widely used che-
motherapeutic agents for the treatment of many types of
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1635-6) contains supplementary material,
which is available to authorized users.
Valproic acid (VPA, 6), an antiepileptic drug is broad-
spectrum inhibitor of histone deacetylase currently studied
for cancer therapy (Göttlicher et al., 2001). The combina-
tion of VPA with other anticancer agents has been con-
sidered as a useful strategy for cancer therapy (Eyal et al.,
2006; Gonzalez et al., 2008; Garcia-Manero et al., 2006;
Soriano et al., 2007). Although VPA is known to induce
P-gp (MDR1) expression, the derivatization of acid group
of VPA results in the loss of the ability to express P-gp
* Himaja Malipeddi
drmhimaja@gmail.com
1
Pharmaceutical Chemistry Division, School of Advanced
Sciences, VIT University, Vellore, Tamil Nadu, India
2
Medicinal Chemistry Division, Piramal Healthcare Ltd.,
Goregaon, Mumbai, India

Med Chem Res
Fig. 1 Structures of taxol analogs and VPA
purchased from Sigma Aldrich chemicals. All reagents and
solvents were of laboratory grade and used without further
purification. Nuclear magnetic resonance (NMR) spectra
were obtained on Bruker AVANCE 300 Instrument. Che-
mical values are reported as δ values in ppm: s = singlet,
d = doublet, dd = doublet of doublet, t = triplet, q = quartet,
m = multiplet, bs = broad singlet. Mass spectrometry and
high-resolution mass spectrometry (HRMS) data were
obtained on Bruker-Micro-Q-TOF instrument.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-
12b-acetoxy-9-(((2R,3S)-3-amino-2-hydroxy-3-
phenylpropanoyl)oxy)-4,6,11-trihydroxy-4a,8,13,13-
tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,12a,12b-
dodecahydro-1H-7,11-methanocyclodeca-[3,4]benzo
[1,2-b]oxet-12-yl benzoate (7)
Fig. 2 Structure of valprotaxel (8)
Docetaxel (0.100 g, 0.123 mmol) was stirred in Tetra-
hydrofuran (THF) (5 ml) in an ice bath. To this was added a
solution of trifluoroacetic acid (0.5 ml) in THF (2 ml). The
reaction mixture was stirred in an ice bath for 2 h under
nitrogen. The reaction mixture was carefully neutralized with
sodium bicarbonate (8 % w/v) addition in an ice bath until pH
8 and extracted with dichloromethane (DCM) (20 ml). Organic
layer was washed with brine (10 ml), dried over sodium sulfate
and concentrated to get free amine compound 7.
(Braiteh et al., 2008). VPA, a relatively small molecule, was
combined with taxane moiety covalently to generate a new
chemical entity. In this paper, we present the synthesis and
the in vitro cytotoxicity study of these taxol analogs. The
novel compound, Valprotaxel (8) has shown interesting
in vitro results (Fig. 2).
1
Materials and methods
Yield 86 % (0.075 g); white solid; H NMR (DMSO-d6,
300 MHz): δ = 7.95 (d, 2H, J = 6.9 Hz, ArH), 7.61–7.75 (m,
3H, ArH), 7.35–7.37 (m, 4H, ArH), 7.19–7.21 (m, 1H, ArH),
5.86 (br s, 1H, CH), 5.40 (d, 1H, J = 7.2 Hz, CH), 5.07 (br s,
Paclitaxel and docetaxel were purchased from Dabur
Pharma Ltd, Nadia, West Bengal, India. VPA was

Med Chem Res
1H, CH), 5.02 (d, 1H, J = 6.9 Hz, CH), 4.96 (s, 1H, CH), 4.88
(d, 1H, J = 8.4 Hz, CH), 4.54 (s, 1H, CH), 4.06 (s, 1H, CH),
3.96–4.03 (m, 3H, CH2 & CH), 3.64 (d, 1H, J = 7.2 Hz, CH),
2.22–2.26 (m, 2H, CH2), 2.11 (s, 3H, CH3), 1.91 (s, 2H, CH2),
1.78–1.87 (m, 2H, CH2), 1.74 (s, 3H, CH3), 1.64–1.68
(m, 4H, CH2), 1.51 (s, 3H, CH3), 1.01 (s, 3H, CH3), 0.97
(s, 3H, CH3); MS: (ES+) m/z 708.2 [M + H]+.
reaction mixture was stirred in an ice bath for 1 h under
nitrogen, and then overnight at RT. The reaction mixture
was quenched with ice water and extracted with DCM (20
ml). The organic layer was washed with brine (10 ml), dried
over sodium sulfate, concentrated and purified by silica gel
(150–300 mesh) column chromatography using acetone:
DCM gradient to get pure compound 10.
Yield 60 % (0.150 g); white solid; 1H NMR (CDCl3, 300
MHz): δ 8.09 (d, 2H, J = 7.2 Hz, ArH), 7.60 (d, 2H, J = 7.5
Hz, ArH), 7.47 (t, 2H, J = 7.5 Hz, ArH), 6.14 (s, 1H, CH),
5.85–5.98 (m, 1H, CH), 5.60-5.64 (m, 1H, CH), 5.26-5.44
(m, 4H, CH2), 4.95 (t, 1H, J = 7.5 Hz, CH), 4.87 (br s, 1H,
CH), 4.69 (d, 1H, J = 5.1 Hz, CH), 4.59-4.64 (m, 2H, CH2),
4.31 (t, 1H, J = 7.2 Hz, CH), 4.01–4.19 (m, 3H, CH2 &CH),
3.84 (d, 1H, J = 7.2 Hz, CH), 2.53–2.64 (m, 2H, CH2), 2.29
(s, 3H, CH3), 2.11 (s, 2H, CH2), 2.07 (s, 3H, CH3), 2.00
(s, 1H, CH), 1.84 (s, 2H, CH2), 1.67 (s, 1H, CH), 1.06–1.13
(m, 6H, CH3); MS: (ES+) m/z 735.3[M + Na]+.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-
12b-acetoxy-4,6,11-trihydroxy-9-(((2R,3S)-2-hydroxy-3-
phenyl-3-(2-propylpentanamido)propanoyl)oxy)-
4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,
12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]
benzo[1,2-b]oxet-12-yl benzoate (8)
Compound 7 (0.070 g, 0.099 mmol) was stirred with VPA
(0.01 g, 0.099 mmol) in DCM (2 ml). DCC (0.024 g, 0.118
mmol) and dimethylaminopyridine (DMAP) (0.006 g,
0.049 mmol) were added and the reaction mixture was
stirred overnight at room temperature (RT). The reaction
mixture was concentrated and purified by silica gel
(150–300 mesh) column chromatography using acetone:
DCM gradient to yield pure compound 8.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-
12b-acetoxy-4,6,11-trihydroxy-4a,8,13,13-tetramethyl-5-
oxo-9-((2-propylpentanoyl)oxy)-2a,3,4,4a,5,6,9,10,11,
12,12a,12b-dodecahydro-1H-7,11-methanocyclodeca
[3,4]benzo[1,2-b]oxet-12-yl benzoate (12)
1
Yield 24 % (0.020 g); off-white solid; H NMR (CDCl3,
300 MHz): δ = 8.13 (d, 2H, J = 7.2 Hz, ArH), 7.53–7.64 (m,
3H, ArH); 7.28–7.40 (m, 5H, ArH), 6.33 (d, J = 9.0 Hz, 1H,
CH), 6.19 (br s, 1H, CH), 5.71 (d, 1H, J = 6.9 Hz, CH), 5.64
(d, 1H, J = 6.9 Hz, CH), 5.21 (s, 1H, CH), 4.95 (d, 1H, J =
8.1 Hz, CH), 4.69 (br s, 1H, CH), 4.33 (d, 1H, J = 8.4 Hz,
CH), 4.23 (br s, 3H, CH & CH2); 3.91 (d, 1H, J = 7.5 Hz,
CH), 3.49 (br s, 1H, CH), 2.58–2.64 (m, 2H, CH2); 2.38
(s, 3H, CH3), 2.29–2.33 (m, 2H, CH2), 2.10–2.14 (m, 1H,
CH), 2.06 (s, 1H, CH), 1.84 (s, 3H, CH3), 1.77 (s, 3H, CH3),
1.54–1.63 (m, 4H, CH & CH2), 1.36–1.38 (m, 3H, CH
&CH2), 1.28 (s, 3H, CH3), 1.25 (s, 3H, CH3), 1.11 (s, 3H,
CH3), 0.82–0.88 (m, 6H, CH3); 13C NMR (CDCl3, 75.4
MHz): δ = 9.8, 11.0, 14.1, 14.3, 20.7, 22.4, 22.6, 24.7, 24.9,
26.3, 31.5, 35.0, 35.1, 35.3, 35.6, 36.8, 43.1, 46.4, 47.5,
54.1, 57.7, 71.8, 72.5, 73.3, 74.5, 74.8, 78.6, 81.1, 84.2,
84.5, 126.8, 128.0, 128.7, 129.1, 129.5, 129.8, 130.1, 133.6,
136.0, 138.3, 166.9, 170.1, 173.3, 175.8, 211.3; HRMS: m/z
ESI (+ve) for C46H60NO13 Calculated; 834.4059 [M + H]+.
Found: 834.4026 [M + H]+ (Mass accuracy: 3.99 ppm).
Compound 10 (0.140 g, 0.196 mmol) was stirred with VPA
(0.141 g, 0.980 mmol) in dry toluene (3 ml). DCC (0.201 g,
0.980 mmol) and DMAP (0.023 g, 0.196 mmol) were
added, and the reaction mixture was heated at 80 °C for
24 h. The reaction mixture was filtered and the filtrate was
concentrated to get crude 11, which was then stirred in
methanol (3 ml) along with ammonium formate and Pd-C
(catalytic) for 2 h at RT. Reaction was quenched with water
(2 ml), extracted with ethyl acetate (20 ml), concentrated
and purified by silica gel (150–300 mesh) column chroma-
tography using acetone: DCM gradient to get pure 12.
Yield 40 % (0.052 g); white solid; 1H NMR (CDCl3, 300
MHz): δ = 8.06 (d, 2H, J = 7.5 Hz, ArH), 7.57 (t, 1H, J =
7.5 Hz, ArH), 7.43 (t, 2H, J = 7.8 Hz, ArH), 6.28 (s, 1H,
CH), 5.57 (d, 1H, J = 7.2 Hz, CH), 5.41-5.50 (m, 1H, CH),
5.24 (d, 1H, J = 10.2 Hz, CH), 4.95–4.98 (m, 1H, CH), 4.78
(t, 1H, J = 7.2 Hz, CH), 4.37–4.39 (m, 1H, CH), 4.26
(d, 1H, J = 9.0 Hz, CH), 4.15 (t, 1H, J = 7.5 Hz, CH), 4.03
(d, 1H, J = 6.6 Hz, CH), 3.84 (d, 1H, J = 6.9 Hz, CH),
2.45–2.53 (m, 2H, CH2), 2.26 (s, 1H, CH), 2.19–2.25
(m, 4H, CH2), 2.01–2.05 (m, 1H, CH), 1.97 (s, 2H, CH2),
1.81 (s, 1H, CH), 1.62 (s, 2H, CH2), 1.33–1.42 (m, 6H,
CH2), 1.17–1.21 (m, 2H, CH2), 1.01–1.07 (m, 6H, CH2),
0.84–0.92 (m, 6H, CH3); MS: (ES+) m/z 671.4 [M + H]+;
13C NMR (CDCl3, 75.4 MHz): δ = 9.4, 10.8, 14.0, 14.9,
15.4, 19.4, 20.4, 20.5, 21.0, 22.5, 26.5, 27.0, 33.4, 34.2,
34.4, 34.5, 35.5, 38.6, 42.6, 45.1, 45.2, 46.1, 46.8, 56.2,
58.7, 67.9, 71.7, 72.3, 74.9, 75.7, 78.7, 79.1, 80.4, 80.8,
Synthesis of 4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,
6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-methano-
cyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate (10)
10-deacetyl baccatin–III (compound 9) (0.200 g, 0.341
mmol) was stirred in dry DCM (5 ml) along with pyridine
(5 ml) in an ice bath. To this was added a solution of allyl
chloroformate (0.102 g, 0.853 mmol) in DCM (2 ml), fol-
lowed by addition of DMAP (0.041 g, 0.341 mmol). The

Med Chem Res
83.8, 84.5, 128.6, 129.3, 130.0, 132.2, 133.6, 142.6, 146.0,
167.0, 170.6, 175.0, 176.5, 203.9, 210.9. HRMS: m/z ESI
(+ve) for C37H54NO11. Calculated; 688.3691 [M + NH4]+.
Found: 688.3686 [M + NH4]+ (Mass accuracy: 0.76 ppm).
ice bath followed by addition of DCC (0.015 g, 0.0742
mmol) and DMAP (0.003 g, 0.030 mmol). The reaction
mixture was stirred at RT for 16 h under nitrogen. The
precipitated DCU was filtered; the filtrate was concentrated
and purified by silica gel (150–300 mesh) column chroma-
tography using acetone: DCM gradient to get pure com-
pound 14.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-9-
(((2R,3S)-3-benzamido-3-phenyl-2-((2-propylpentanoyl)
oxy)propanoyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-
4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,10,11,12,
12a,12b-dodecahydro-1H-7,11-methanocyclodeca[3,4]
benzo[1,2-b]oxete-6,12b-diyl diacetate (13)
Yield 52 % (0.030 g); white solid; 1H NMR (CDCl3, 300
MHz): δ = 8.11 (d, 2H, J = 7.2 Hz, ArH), 7.59 (t, 1H, J =
7.2 Hz, ArH), 7.49 (t, 2H, J = 7.8 Hz, ArH), 7.34–7.39 (m,
3H, ArH), 7.25–7.29 (m, 2H, ArH), 6.25 (t, 1H, J = 8.7 Hz
CH), 5.68 (d, 1H, J = 7.2 Hz, ArH), 5.47 (br s, 1H, CH),
5.28 (br s, 1H, CH), 5.32 (d, 1H, J = 9.3 Hz, CH), 5.20 (br
s, 1H, CH), 4.96 (d, 1H, J = 7.8 Hz, CH) 4.32 (d, 1H, J =
8.4 Hz, CH), 4.24–4.28 (m, 1H, CH), 4.18 (d, 1H, J = 8.4
Hz, CH), 3.93 (d, 1H, J = 6.9 Hz, CH), 2.53-2.60 (m, 1H,
CH), 2.47 (s, 3H, CH3), 2.28–2.41 (m, 2H, CH2), 2.10–2.16
(m, 2H, CH2), 1.95 (s, 3H, CH3), 1.79–1.89 (m, 1H, CH),
1.74 (s, 3H, CH3), 1.66 (s, 1H, CH), 1.51–1.57 (m, 2H,
CH2), 1.45–1.49 (m, 2H, CH2), 1.37 (s, 9H, CH3), 1.23 (s,
3H, CH3), 1.14–1.19 (m, 2H, CH), 1.11 (s, 3H, CH3),
0.92–1.03 (m, 6H, CH3); 13C NMR (CDCl3, 75.4 MHz): δ
= 10.0, 14.0, 14.2, 20.1, 20.3, 20.9, 22.6, 26.3, 28.1, 34.3,
34.5, 35.5, 36.8, 43.0, 44.9, 46.4, 57.5, 71.6, 71.8, 74.0,
74.4, 78.9, 80.4, 80.9, 84.2, 126.0, 128.0, 128.7, 129.2,
130.2, 133.6, 135.4, 137.4, 139.2, 155.1, 167.0, 167.9,
169.7, 169.7, 175.4, 211.5; MS: (ES+) m/z 934.4 [M + H]+
Paclitaxel (compound 1) (0.050 g, 0.058 mmol) was stirred
with VPA (0.008 g, 0.058 mmol) in dry DCM (2 ml) in an
ice bath, followed by addition of DCC (0.014 g, 0.069
mmol), and DMAP (0.003 g, 0.029 mmol). The reaction
mixture was stirred at RT for 16 h under nitrogen. The
precipitated dicyclohexylurea (DCU) was filtered; the fil-
trate was concentrated and purified by silica gel (150–
300 mesh) column chromatography using acetone: DCM
gradient to get pure compound 13.
1
Yield 61 % (0.035 g); white solid; H NMR (CDCl3,
300 MHz): δ = 8.16 (d, 2H, J = 7.2 Hz, ArH), 7.75 (d, 2H,
J = 7.2 Hz, ArH), 7.61–7.66 (m, 1H, ArH), 7.51–7.57 (m,
3H, ArH), 7.34–7.45 (m, 7H, ArH), 6.88 (d, 1H, J = 9.0 Hz,
CH), 6.31 (s, 1H, CH), 6.27 (d, 1H, J = 9.0 Hz, CH), 5.98
(dd, 1H, J = 9.0 Hz, J = 3.0 Hz, CH), 5.70 (d, 1H, J = 7.2
Hz, CH), 5.50 (d, 1H, J = 3.3 Hz, CH), 5.00 (d, 1H, J = 7.8
Hz, CH), 4.47–4.50 (m, 1H, CH), 4.34 (d, 1H, J = 8.4 Hz,
CH), 4.23 (d, 1H, J = 8.4 Hz, CH), 3.84 (d, 1H, J = 7.2 Hz),
3.45–3.52 (m, 1H, CH), 2.55-2.61 (m, 1H, CH), 2.51 (s,
3H, CH3), 2.35-2.49 (m, 2H, CH2), 2.24 (s, 3H, CH3), 2.19
(s, 1H, CH), 2.10–2.18 (m, 1H, CH), 1.97 (s, 3H, CH3),
1.91–1.95 (m, 2H, CH2), 1.70 (s, 3H, CH3), 1.49–1.56
(m, 2H, CH2), 1.35–1.41 (m, 4H, CH2), 1.27 (s, 3H, CH3),
1.24 (s, 3H, CH3), 1.15 (s, 3H, CH3), 1.09–1.14 (m, 2H,
CH2), 0.76–0.94 (m, 6H, CH3) ; 13C NMR (CDCl3, 75.4
MHz): δ = 9.6, 13.9, 14.8, 20.3, 20.4, 20.8, 22.1, 22.7, 24.9,
25.6, 26.7, 31.5, 33.9, 34.4, 34.6, 35.5, 43.1, 44.9, 45.5,
49.1, 52.8, 58.5, 71.6, 72.1, 73.8, 75.1, 75.6, 79.1, 81.0,
84.4, 126.3, 127.0, 128.3, 128.7, 129.0, 129.2, 130.2,
132.0, 132.7, 133.6, 136.9, 142.8, 167.0, 167.1, 168.0,
169.8, 171.2, 175.4, 211.0; MS: (ES+) m/z 978.4 [M − H]+
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-9-
(((2R,3S)-3-benzamido-2-hydroxy-3-phenylpropanoyl)
oxy)-12-(benzoyloxy)-11-hydroxy-4a,8,13,13-
tetramethyl-5-oxo-4-((2-propylpentanoyl)oxy)-2a,3,4,4a,
5,6,9,10,11,12,12a,12b-dodecahydro-1H-7,11-
methanocyclodeca[3,4]benzo[1,2-b]oxete-6,12b-diyl
diacetate (15)
2′-alcohol of paclitaxel 1 was first protected by stirring
paclitaxel (0.100 g, 0.117 mmol) with methoxyacetic acid
(0.010 g, 0.117 mmol) in DCM (2 ml) in an ice bath, fol-
lowed by addition of DCC (0.026 g, 0.128 mmol) and
DMAP (0.007 g, 0.058 mmol). The reaction mixture was
stirred at RT for 16 h under nitrogen. The precipitated DCU
was filtered; filtrate was concentrated to give 2-mAc
paclitaxel 1a (0.090 g, white solid). This was stirred in
DCM (2 ml) along with VPA (0.014 g, 0.097 mmol), DCC
(0.024 g, 0.116 mmol), and DMAP (0.005 g, 0.048 mmol)
for 24 h. The precipitated DCU was filtered and the filtrate
was concentrated to get the crude compound 1b, which was
then stirred in ammoniated methanol (2 ml) at RT for 2 h.
The reaction mixture was concentrated and purified by silica
gel (150–300 mesh) column chromatography using acetone:
DCM gradient to get pure compound 15.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-12b-
acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)amino)-3-
phenyl-2-((2-propylpentanoyl)oxy)propanoyl)oxy)-4,6,11-
trihydroxy-4a,8,13,13-tetramethyl-5-oxo-2a,3,4,4a,5,6,9,
10,11,12,12a,12b-dodecahydro-1H-7,11-methano-
cyclodeca[3,4]benzo[1,2-b]oxet-12-yl benzoate (14)
Docetaxel (compound 2) (0.050 g, 0.0619 mmol) was stir-
red with VPA (0.009 g, 0.0619 mmol) in DCM (2 ml) in an

Med Chem Res
Yield 47 % (0.045 g); white solid; 1H NMR (CDCl3, 300
MHz): δ = 8.13 (d, 2H, J = 7.2 Hz, ArH), 7.78 (d, 2H, J =
7.2 Hz, ArH), 7.64 (t, 1H, J = 7.2 Hz, ArH), 7.49–7.55 (m,
5H, ArH), 7.36–7.46 (m, 5H, ArH), 7.10 (d, 1H , J = 8.7
Hz, ArH), 6.22 (br s, 1H, CH), 6.18 (d, 1H, J = 8.4 Hz,
ArH), 5.82 (d, 1H, J = 6.6 Hz, CH), 5.63–5.69 (m, 2H, CH),
4.96 (d, 1H, J = 8.7 Hz, CH), 4.82 (br s, 1H, CH), 4.33
(d, 1H, J = 8.4 Hz), 4.19–4.22 (m, 2H, CH2), 4.15 (s, 1H,
CH), 3.86–3.94 (m, 2H, CH), 3.69 (br s, 1H, CH), 3.48
(s, 2H, CH2), 2.59–2.69 (m, 1H, CH), 2.40 (s, 3H, CH3),
2.35 (d, 1H, J = 9.0 Hz, CH), 2.19 (s, 3H, CH3), 1.92 (br s,
2H, CH2), 1.83 (d, 1H, J = 4.5 Hz, CH), 1.35 (s, 1H, CH),
1.30 (s, 3H, CH3), 1.27 (br s, 6H, CH3), 1.22 (s, 3H, CH3),
1.18 (s, 3H, CH3), 0.84–0.94 (m, 3H, CH3); 13C NMR
(CDCl3, 75.4 MHz): δ = 10.7, 14.1, 14.6, 20.7, 22.5, 22.6,
26.5, 29.3, 29.7, 31.9, 33.4, 35.5, 43.2, 47.0, 54.8, 56.1,
59.4, 69.5, 71.7, 72.1, 73.1, 74.2, 75.3, 78.4, 81.0, 83.8,
127.0, 128.3, 128.7, 129.0, 130.1, 131.9, 132.8, 133.6,
133.8, 137.9, 140.5, 166.8, 167.0, 168.9, 169.9, 170.4,
172.4, 201.8; MS: (ES+) m/z 980.0 [M + H]+.
2.50–2.62 (m, 1H, CH), 2.39 (s, 2H, CH2), 2.29–2.32 (m,
2H, CH), 1.88-1.90 (m, 2H, CH2), 1.85 (s, 3H, CH3),
1.69–1.75 (m, 2H, CH2), 1.48–1.55 (m, 2H, CH2), 1.37 (s,
6H, CH3 &CH2), 1.22 (s, 1H, CH), 1.12 (s, 3H, CH3),
0.89–1.00 (m, 6H, CH3); 13C NMR (CDCl3, 75.4 MHz): δ
= 10.8, 14.0,14.2, 20.4, 20.6, 22.4, 24.9, 25.6, 26.3, 28.2,
33.4, 33.9, 34.1, 34.4, 34.5, 35.7, 43.1, 45.2, 46.4, 49.1,
56.2, 71.5, 72.3, 73.6, 74.3, 74.5, 78.6, 80.1, 80.7, 83.7,
126.7, 128.0, 128.7, 128.8, 129.1, 130.1, 133.7, 135.5,
138.4, 138.7, 155.3, 167.0, 170.2, 172.5, 175.0, 176.6,
210.4; MS: (ES+) m/z 934.3 [M + H]+.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-
12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)
amino)-2-hydroxy-3-phenylpropanoyl)oxy)-4,11-
dihydroxy-4a,8,13,13-tetramethyl-5-oxo-6-((2-
propylpentanoyl)oxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-
dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-
b]oxet-12-yl benzoate (17)
Docetaxel 2 (0.100 g, 0.123 mmol) was stirred with methoxy-
acetic acid (0.017 g, 0.123 mmol) in DCM (2 ml) in an ice
bath followed by addition of DCC (0.030 g, 0.148 mmol) and
DMAP (0.007 g, 0.061 mmol). The reaction mixture was
stirred at RT for 16 h under nitrogen. The precipitated DCU
was filtered; the filtrate was concentrated to get the crude 2′-
mAc docetaxel 2c (0.090 g, white solid), which was then
stirred in dry DCM (2 ml) along with VPA (0.014 g, 0.102
mmol), DCC (0.025 g, 0.122 mmol) and DMAP (0.006 g,
0.051 mmol) for 24 h. The precipitated DCU was filtered;
filtrate was concentrated to get the crude compound 2d, which
was stirred in ammoniated methanol (2 ml) at RT for 2 h. The
reaction mixture was concentrated and purified by silica gel
(150–300 mesh) column chromatography using acetone:
DCM gradient to get pure compound 17.
Synthesis of (2aR,4S,4aS,6R,9S,11S,12S,12aR,12bS)-
12b-acetoxy-9-(((2R,3S)-3-((tert-butoxycarbonyl)
amino)-2-hydroxy-3-phenylpropanoyl)oxy)-6,11-
dihydroxy-4a,8,13,13-tetramethyl-5-oxo-4-((2-
propylpentanoyl)oxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-
dodecahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-
b]oxet-12-yl benzoate (16)
2′-alcohol and C-10 alcohol of docetaxel were first protected
by stirring docetaxel 2 (0.100 g, 0.123 mmol) with meth-
oxyacetic acid (0.038 g, 0.270 mmol) in dry DCM (4 ml),
followed by addition of DCC (0.055 g, 0.270 mmol) and
DMAP (0.007 g, 0.061 mmol). The reaction mixture was
stirred at RT for 16 h under nitrogen. The precipitated DCU
was filtered; the filtrate was concentrated to get the crude
compound 2a (white solid, 0.095 g), which was stirred in dry
DCM (4 ml) along with VPA (0.014 g, 0.099 mmol), DCC
(0.024 g, 0.118 mmol), and DMAP (0.006 g, 0.049 mmol)
for 24 h. The precipitated DCU was filtered and the filtrate
was concentrated to get the crude compound 2b, which was
further stirred in ammoniated methanol (2 ml) at RT for 2 h.
The reaction mixture was concentrated and purified by silica
gel (150–300 mesh) column chromatography using acetone:
DCM gradient to get pure compound 16.
1
Yield 42 %; (0.040 g); white solid; H NMR (CDCl3,
300 MHz): δ = 8.12 (d, 2H, J = 7.8 Hz, ArH), 7.61–7.63 (m,
1H, ArH), 7.51–7.54 (m, 2H, ArH), 7.40–7.49 (m, 5H,
ArH), 6.18–6.26 (m, 1H, CH), 5.68 (d, 1H, J = 6.9 Hz,
ArH), 5.46 (d, 1H, J = 9.6 Hz, ArH), 5.28 (br s, 1H, CH),
4.95 (d, 1H, J = 8.4 Hz, CH), 4.64 (br s, 1H, CH), 4.34
(d, 1H, J = 8.4 Hz), 4.21 (d, 1H, J = 8.4 Hz), 4.00–4.10 (m,
3H, CH), 3.44–3.55 (m, 1H, CH), 3.43 (d, 1H, J = 5.4 Hz,
CH), 2.49–2.63 (m, 1H, CH), 2.39 (s, 3H, CH3), 2.29–2.31
(m, 2H, CH2), 1.91–1.96 (m, 3H, CH & CH2), 1.88 (s, 6H,
CH3), 1.69–1.73 (m, 4H, CH2), 1.50–1.52 (m, 2H, CH2),
1.35–1.37 (m, 10H, CH3 &CH2), 1.27 (s, 3H, CH3), 1.24
(s, 3H, CH3), 1.17–1.19 (m, 4H, CH2), 1.12 (s, 3H, CH3),
0.89-0.94 (m, 6H, CH3); 13C NMR (CDCl3, 75.4 MHz): δ
= 10.9, 14.0, 14.2, 20.4, 20.6, 22.4, 24.9, 25.6, 26.3, 28.2,
29.7, 33.4, 33.9, 34.1, 35.7, 43.0, 45.2, 46.4, 49.1, 56.2,
71.5, 72.4, 73.6, 74.3, 74.5, 75.3, 78.6, 80.1, 80.7, 83.7,
126.7, 128.7, 128.8, 129.0, 130.1, 133.7, 135.5, 138.7,
Yield 48 % (0.045 g); white solid; 1H NMR (CDCl3, 300
MHz): δ = 8.12 (d, 2H, J = 7.8 Hz, ArH), 7.63 (t, 1H, J =
8.4 Hz, ArH), 7.51 (t, 2H, J = 7.8 Hz, ArH), 7.34–7.40 (m,
4H, ArH), 6.18–6.26 (m, 1H, CH), 5.68 (d, 1H, J = 6.9 Hz,
ArH), 5.46 (d, 1H, J = 9.6 Hz, ArH), 5.28 (br s, 1H, CH),
4.95 (d, 1H, J = 7.8 Hz, CH), 4.64 (br s, 1H, CH), 4.33 (t,
1H, J = 8.4 Hz), 4.21 (t, 1H, J = 8.1 Hz), 4.00-4.06 (m, 2H,
CH2), 3.47–3.55 (m, 1H, CH), 3.39 (d, 1H, J = 5.1 Hz, CH),

Med Chem Res
Scheme 1 Reagents and conditions: (a) TFA, THF, 45 min at 0 °C and NaHCO3, 86 %; (b) VPA, DCC, DMAP, 25 °C, 16 h, 24 %
155.3, 160.0, 166.9, 170.2, 175.0, 210.4; MS: (ES+) m/z
normalized to controls. Each independent experiment was
performed thrice in triplicate.
956.5 [M + Na]+.
Cell line maintenance
Results and discussion
A panel of six cancer cells representing multiple cancers of
clinical relevance were selected, namely, ACHN (human
renal cell carcinoma, ATCC, CRL-1611), Panc-1 (human
pancreatic adenocarcinoma, ATCC, CRL-1469) cultured
on minimum essential medium (MEM) with 2 mM L-
glutamine and 10 % fetal bovine serum (FBS), H460
(human non small cell lung carcinoma, ATCC, HTB-177),
Calu-1 (human lung carcinoma, ATCC, HTB-54), cultured
on RPMI, 2 mM L-glutamine and 10 % FBS, HCT-116
(human colon cancer, ATCC, CCL- 247) and FADU (Head
and Neck Squmous cell carcinoma, ATCC, HTB- 43) cul-
tured on McCoy’s 5a medium and 10 % FBS and MCF10A
(normal breast epithelium cells) cultured on MEM with 2
mM L-glutamine and 10 % FBS.
VPA was incorporated into the taxane moiety at various
positions viz., C-2′, C-7, C-10, C-13 using ester bond and
amide at C-3′. The synthesis of C-3′ VPA amide (8) was
accomplished by deprotecting the Boc group of docetaxel
using trifluoroacetic acid (TFA), and the free amine was
coupled with VPA using DCC and DMAP as shown in
Scheme 1 (Querolle et al., 2003). The VPA ester at C-13
(12) was synthesized by protecting the C-7 and C-10
hydroxyl groups of 10-DAB-III using allyl chloroformate
and pyridine in chloroform, followed by coupling with VPA
using DCC/DMAP (Heo et al., 2009), and deprotection
using Pd-C and ammonium formate as shown in Scheme 2.
The VPA esters of paclitaxel and docetaxel at C-2′ (13
and 14) were synthesized by using DCC/DMAP as the
coupling agent as shown in Scheme 3. VPA esters at the
C-7 position (15 and 16) were synthesized by protecting the
2′-hydroxy group using base labile methoxyacetate group.
In case of docetaxel both C-2′ and C-10 hydroxyl group
were protected by methoxyacetate. After VPA coupling,
deprotection of the methoxyacetyl group was accomplished
by using ammoniated methanol as shown in Scheme 3.
Similarly C-10 VPA ester of docetaxel (17) synthesis was
accomplished by protecting C-2′hydroxyl using methoxy
acetate. These hybrids were evaluated for their cytotoxicity
using the propidium iodide assay method and their IC50
values were determined.
Cell proliferation assay
Logarithmically growing cells were plated at a density of
5 × 103 cells/well in a 96-well tissue culture grade micro-
plate and allowed to recover overnight. The cells were
challenged with varying concentration of different hybrids
for 48 h. Control cells received standard media containing
dimethyl sulfoxide (DMSO) vehicle at a concentration of
0.2 %. After 48 h of incubation, cell toxicity was determined
by the cell counting kit-8 (CCK-8) reagent (Dojindo
Molecular Technologies, Inc, Maryland, Japan); (WST-1
[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-dis-
ulfophenyl)]-2H-tetrazolium, monosodium salt assay). In
accordance with the manufacturer’s instructions, 10 µl/well
CCK-8 reagents were added and plates were incubated for
2 h. The compound induced anti-proliferation/toxicity was
determined by measuring the absorbance on Tecan Sapphire
multi-fluorescence micro-plate reader (Tecan, Germany,
GmbH) at a wavelength of 450 nm corrected to 650 nm and
Determination of IC50 for different compounds in a
panel of cancer cells
PANC1, ACHN1, CALU1, H460, HCT116, FADU, and
normal breast epithelium cells MCF10A were treated with
different hybrids (Table 1) at an eight-point dilution set of
0.01 µM to 30 µM. After 48 h, the compound-induced

Med Chem Res
Scheme 2 Reagents and conditions: (a) allyl chloroformate, CH2Cl2, pyridine 0 °C and 16 h at 25 °C, 60 %; (b) VPA, DCC, DMAP, 25 °C, 16 h;
(c) Pd-C, ammonium formate, methanol, 25 °C, 3 h, 40 %
toxicity was determined using a CCK-8 live cell dehy-
drogenase non-radioactive assay (Mosmann, 1983).
increase in cytotoxicity compared with 10-DAB-III (9).
Hybrid 12 showed reduced cytotoxicity compared to DAB-
III. The VPA esters at C-2′, C-7 and C-10 (13–16) of
docetaxel and paclitaxel showed reduced cytotoxicity
compared to the parent drugs. However, hybrid 17 showed
better cytotoxicity in the range of 0.15–0.94 µM on the
tested cancer cell lines.
The hybrids (13–17) are connected by ester bonds and
are susceptible for ester hydrolysis by esterases. These
hybrids may behave as prodrugs rather than hybrids as these
compounds will get hydrolyzed by esterases in plasma. This
study reveals that molecular hybridization of VPA and taxol
works better at C-3′. Interestingly, hybrid 8, what we called
Analysis of the dose-response curve revealed that the
hybrid (8) showed significant cytotoxicity against colon and
head and neck cancer cell line with IC50 values of 0.01 µM
and 0.08 µM respectively. This is comparable to paclitaxel,
and in normal breast epithelium cells (MCF10A) it showed
an IC50 above 10 µM, which illustrates better therapeutic
index (TI = 1000) for colon cancer cell lines than paclitaxel
(TI = 37.5) and docetaxel (TI = 37.5) by a big margin,
whereas VPA exhibits non-selective cytotoxicity in the
range of 12.1–22.1 µM against the tested cell lines. The C-
13 side chain replacement with VPA (12) did not show any

Med Chem Res
Scheme 3 Reagents and conditions: (a) VPA, DCC, DMAP, 25 °C,
16 h, 52 %; (b) (i) 2.0 eq. methoxyacetic acid, DCC, DMAP, 25 °C,
16 h; (ii) VPA, DCC, DMAP, 25 °C, 16 h; (iii) ammoniated methanol,
25 °C, 2 h, 48 %; (c) (i) 1 eq. methoxyacetic acid, DCC, DMAP, 25 °C,
16 h; (ii) VPA, DCC, DMAP, 25 °C, 16 h; (iii) ammoniated methanol,
25 °C, 2 h, 42 %
as Valprotaxel, showed improvement in therapeutic index
by a big margin compared to paclitaxel. A small change in
structure at C-3′ (where instead of the N-Boc group of
docetaxel we introduced the valpromide) results in
improvement in therapeutic index. This selectivity and
potency of Valprotaxel against various cancer cell lines
makes it good candidate for further evaluation.
cells as compared to the normal cells, particularly in head,
neck and colon cancer cell lines. Valprotaxel holds promise
for further studies in Pgp interactions and permeability
across BBB. It can also be a potential candidate for treat-
ment of brain tumors where therapeutic benefit of paclitaxel
is low and variable.
Acknowledgments The research project was sponsored by Piramal
Healthcare Limited. Authors wish to express their heartfelt thanks to
Dr. Apparao Satyam for his guidance and valuable advice. The authors
are very grateful to Dr. Rajeev Sharma, Dr. Arun Balakrishnan, Dr. H.
Sivaramakrishanan, and Dr. Somesh Sharma for their encouragement
for the research work. Special thanks to Ms. Diana Writer for her
timely support and our colleagues of patents department who spon-
taneously facilitated the authors.
Conclusion
We have demonstrated the synthesis of VPA–taxane
hybrids. The novel drug hybrid Valprotaxel has shown
potency and selectivity towards highly proliferating cancer

Med Chem Res
Table 1 Cytotoxicity of VPA–taxane hybrids (IC50 µM)
% Cytotoxicity (IC50 in µM)
Sample code
aACHN
bPanc1
cCalu1
dH460
eHCT116
fFADU
gMCF1A
Paclitaxel (1)
Docetaxel (2)
VPA (6)
0.082 0.006
0.0075 0.001
16.3 5.0
0.093 0.012
0.0095 0.005
17.5 3.2
0.03 0.040
0.03 0.009
22.1 3.3
0.01 0.05
0.0081 0.01
15.3 2.3
0.22 0.04
2.5 1.2
0.008 0.019
0.0078 0.001
16.5 3.0
0.06 0.012
0.0056 0.001
12.1 3..3
0.08 0.02
2.7 0.30
0.3 0.10
0.21 0.15
15.1 2.0
10.0 2.0
3.3 0.8
Valprotaxel (8)
DAB-III (9)
Hybrid 12
0.12 0.02
1.8 0.20
0.15 0.05
2.2 0.30
1.2 0.02
0.010 0.005
1.9 0.9
10.0 3.00
16.5 3.6
5.2 1.00
6.8 1.6
7.8 1.4
7.0 2.0
3.7 1.6
25 2.0
Hybrid 13
0.93 0.10
1.2 0.02
0.86 0.15
0.98 0.14
1.5 0.35
1.06 0.35
0.86 0.20
0.98 0.15
1.1 0.40
0.96 0.10
0.73 0.15
1.4 0.30
0.78 0.10
0.81 0.20
1.2 0.20
0.76 0.10
0.93 0.10
1.0 0.35
5.7 0.70
5.9 1.0
Hybrid 14
1.1 0.25
Hybrid 15
1.4 0.35
1.2 0.20
10.0 2.0
3.6 0.4.0
7.5 0.50
Hybrid 16
0.76 0.20
0.32 0.05
0.84 0.15
0.45 0.20
0.59 0.10
0.20 0.10
0.45 0.05
0.15 0.05
Hybrid 17
0.94 0.25
Data are mean of three determinations
a
Renal cancer
b
Pancreatic cancer
c
Lung cancer
d
Non-small cell lung cancer
e
Colon cancer
f
Head and neck
g
Normal breast epithelium cells
Compliance with ethical standards
Heo JH, Park SJ, Kang JH, Lee IS, Lee JS, Park YJ, Kim KS, Lee JY
(2009) Development of new efficient methods for docetaxel. Bull
Korean Chem Soc 30:25–26
Conflict of interest The authors declare that they have no conflict of
interest.
Lee JS, Pisters KM, Komaki R, Glisson BS, Khuri FR, Schea R,
Fossella FV (1997) Paclitaxel/carboplatin chemotherapy
as primary treatment of brain metastases in non-small cell lung
cancer: a preliminary report. Sem Oncol 24(4 Suppl. 12):
S12-S55
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