Determination of Impurities Generation in 10–DAB by XRD, 1HNMR and 13C–NMRon Storage for 10 Years Original Research Article Journal of Chemistry and Materials Research Vol. 1 (2), 2014, 44–51 Omprakash H. Nautiyal*

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Journal of Chemistry and Materials Research
Vol. 1 (2), 2014, 45–51
JCMR
Journal of Chemistry and
Materials Research
ORICPublications
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Original Research
Determination of Impurities Generation in 10–DAB by XRD, 1
HNMR
and 13
C–NMRon Storage for 10 Years
Omprakash H. Nautiyal*
Institute of Science and Technology for Advanced Studies & Research, Vallabh Vidya Nagar, Anand 388120, Gujarat, India
Received 14 July 2014; received in revised form 05 August 2014; accepted 09 August 2014
Abstract
10-DAB being very important and is the fifth precursor in the biosynthesis of Paclitaxel in the Taxus species. 10-DAB is isolated from the
Yew tree of American origin namely Taxus baccata, employing methylene dichloride for obtaining the syrupy liquid. The extraction was
carried out at the vapour pressure of the MDC until the matrixes get exhausted for its contents. The syrup is then was admixed with the silica
gel and vacuum dried. The vacuum dried silica admixed syrup is then loaded into the column containing the silica gel of mesh size of 60-120
of acidic pH. It is then subsequently eluted for its resolving the lower and higher fractions of impurities. The pure 10-DAB then successfully
eluted with suitably increasing polarity of acetone: hexane mixture until the pure 10-DAB is obtained. The entire fractions were distilled under
rotary evaporator to obtain the pure residue. It was then subsequently crystallized using hexane to yield 99.5% purity of 10-DAB. It was stored
under ambient conditions of 35±5oC for 10 years and was analyzed for determining the impurities generation employing the XRD, 1HNMR
and 13CNMR. The investigation shows no impurities found to be generated.
Keywords: 10-DAB, storage, impurities generation, XRD, 1
HNMR, 13
NMR.
1. Introduction
Our aim of the investigation was to ascertain the major
changes to be occurred in the molecule of 10–DAB storing it
at genuine conditions over the 10 years. XRD, 1
HNMR and
13C NMR did not exhibit any major changes storing over the
10 years. Only the melting point of the molecule recorded on
the slightly higher side of the value of 235 o C as that reported
of 230–233 o C in the published articles.
10–DAB III is mainly used in the production of anticancer
drugs Paclitaxel and Docetaxel. These drugs are used in cancer
chemotherapy. Paclitaxel is now used to treat patients with
lung, ovarian, breast cancer, head and neck cancer, and
advanced forms of Kaposi's sarcoma. Paclitaxel is also used
for the prevention of rest enosis. Docetaxel is used mainly for
the treatment of breast, ovarian, prostate, and non-small cell
* Corresponding author.
E-mail address: opnautiyalus@yahoo.com (O.H. Nautiyal).
All rights reserved. No part of contents of this paper may be reproduced or
transmitted in any form or by any means without the written permission of
ORIC Publications, www.oricpub.com.
lung cancer. Docetaxel has an FDA approved claim for
treatment of patients who have locally advanced or metastatic
breast or non–small–cell lung cancer who have undergone
anthracycline – based chemotherapy and failed to stop cancer
progression or relapsed and a European approval for use in
hormone-refractory prostate cancer [1,2].
An alternative isolation from the leaves of Taxus baccata
(1g/kg), of Taxol, is ten times greater than the amount of
Taxus brevifolia (0.1g/kg). The removal of the leaves from the
tree has no effect on the health and leaves are regenerated
quickly.
The yew, or Taxus baccata, has been viewed as a
poisonous and mystical tree throughout human civilization.
Though some ancient cultures have used the bark and spiny
leaves of this evergreen conifer for several medicinal
remedies, its benefits were largely overlooked by mainstream
medical institutions until recent generations due to its highly
toxic nature. Much of the focus in the 21st century regarding
the yew tree concerns a chemical derived from its bark called
taxane, the precursor to potent chemotherapy drugs like
Paclitaxel and Docetaxel [3].
Perhaps the most notable medical use for Taxus baccata is
the discovery of its anti-cancerous qualities, particularly in
treating cancers of the ovaries and breasts. Though the bark of

2. 46 O.H. Nautiyal / Journal of Chemistry and Materials Research 1 (2014) 45–51
TABLE 1 XRD data on 10-DAB
No. Visible Data set
Name
Start pos.
[2ϴ]
End pos.
[2ϴ]
Step
[2ϴ]
Measured
Date/Time
1 True 2302_10–
DAB
3.01 49.99 0.02 2/23/2013 14.00
No. Pos. [2ϴ] d-spacing
[Å]
Significance Rel. Int.
[%]
Height FWHM [2ϴ]
1 7.126 12.40527 1.295 8.38 48.15 0.1968
2 9.5373 9.27358 3.8255 60.24 314.8 0.2165
3 9.7501 9.0717 1.3756 23.03 220.67 0.1181
4 10.854 8.15142 10.3646 93.29 412.51 0.2558
5 11.502 7.69359 0.8641 4.56 21.84 0.2362
6 12.1983 7.25595 1.6052 18.15 65.2 0.3149
7 12.9741 6.82376 1.6058 41.56 398.18 0.1181
8 13.4034 6.60614 1.9921 12.03 115.24 0.1181
9 14.3161 6.18695 3.7776 40.68 233.81 0.1968
10 15.0525 5.88591 2.2459 12.91 37.11 0.3936
11 16.6754 5.31653 1.397 17.76 127.62 0.1574
12 18.2337 4.86555 0.9867 14.98 107.64 0.1574
13 19.1836 4.6267 3.237 32.82 145.12 0.2558
14 19.7282 4.50019 1.0025 15.96 131.04 0.1378
15 20.7343 4.28405 1.1819 17.07 109.05 0.1771
16 21.766 4.08325 3.0424 70.81 370.01 0.2165
17 22.8858 3.88594 3.2397 100 287.41 0.3936
18 25.2184 3.53154 2.8524 44.38 182.23 0.2755
19 25.9883 3.42865 0.8893 13.45 48.32 0.3149
20 28.4176 3.14084 1.253 31.95 183.65 0.1968
21 29.556 3.0224 0.9395 16.24 93.35 0.1968
22 30.1973 2.95966 1.0096 18.49 75.92 0.2755
23 32.059 2.79191 1.3858 20.38 97.64 0.2362
24 35.5823 2.52313 4.5049 86.94 124.94 0.7872
25 37.0706 2.42518 1.1471 23.27 55.74 0.4723
26 37.8932 2.3744 0.859 9.82 47.06 0.2362
27 38.8853 2.31608 1.8524 22.14 53.04 0.4723
28 41.1473 2.19383 1.212 14.91 17.86 0.9446
29 45.3183 2.00114 0.9774 5.56 26.61 0.2362
30 46.8246 1.93861 1.5103 28.3 30.84 0.768
the tree has been distilled in order to produce taxane and its
chemotherapy by products, such as Docetaxel, many
environmentalists lament the amount of yew bark needed for
just one patient's dose. The yew tree is not in danger of
extinction, but as of 2011 this could result from the increased
demand for the bark. The taxanes concentration (also 10–DAB
III) in yew tissues is much differentiated, and is related to
many factors. These factors can be genetic (differences
between species, origins, and specific trees), epigenetic (time
of material collecting, kind of tissue) or environmental (an
influence of soil type, climate, water) [4].
The removal of the leaves from the tree has no effect on the
“health” of the tree and the leaves are regenerated relatively
quickly, so it is unnecessary to cut down the trees to obtain the
bark. The conversion of 10–DAB to Taxol is thus an excellent
option for the large scale and economic synthesis of Taxol.
The partial synthesis of Taxol is very important when
compared to that of direct isolation of Taxol from the western
yew, since the availability of Baccatin III or 10–DAB is
greater than that of Taxol. Another advantage of the semi-
synthetic approach is that various Taxol analogs, such as
Texotere, which may have improved bioactivity as compared
with Taxol, can also be synthesized [5].

3. O.H. Nautiyal / Journal of Chemistry and Materials Research 1 (2014) 45–51 47
Table 2 1
H and 13
C NMR data of 10–deactylbaccatin
Position δC(ppm) δH(ppm) (J in Hz)
1 79.38
2 68.18 5.52 d
3 43.98 3.21 d
4 82.02 –
5 85.99 4.19 d
6 37.57 2.38 m
7 72.80 4.92 dd
8 58.92 –
9 211.82 –
10 77.61 5.52 s
11 131.63 –
12 144.70 –
13 68.18 4.19 br q
14 40.64 2.16 m
15 48.80 –
16 22.73 1.70 s
17 27.22 1.70 s
18 15.30 2.12 d
19 10.37 1.74 s
20 76.28 4.16 d 4.18 d
PhCO 167.77 –
Ph (i) 131.63 –
Ph (o) 129.65 4.92 2H d
Ph (m) 131.12 4.09 2H t
Ph (p) 134.48 4.19 t
1’ 172.05
(167.77)
–
2’ 40.64
(43.98)
2.24 ddd
2.38 dd
3.21 dd
3.20 dd
3’ 68.18
(72.80)
4.68 m
4’ 20.84 1.58 d 1.67 d
AcO 37.57(167.77) 2.24 s
10-deacetylbaccatin III is an intermediate in the biosynthe-
tic pathway of the taxane, Paclitaxel. Taxanes disrupt mitosis
by preventing microtubule destabilization and spindle fibre
formation in animal cells (Molè – Bajer, 1983). As a consequ-
ence of this mechanism, yew trees are slow growing organis-
ms. Without mitosis, cells, especially cells with a high replic-
ation rate such as tumour cells, cannot divide. Taxanes are
currently being investigated as possible treatment for certain
cancers, and Paclitaxel, is currently approved by the FDA for
treatment of AIDS – related Karposi sarcoma, breast cancer,
non-small cell lung cancer, and ovarian cancer (National
Cancer Institute, 2012). 10–DAB is used as the core starting
material in the semi-synthetic manufacturing of Paclitaxel
[Holton, 1991]. 10–DAB is also used as the starting raw
material for Docetaxel (Taxoterel), a potent anticancer
compound [6,7].
The tetra cyclic diterpene moiety of Taxol, 10-
deacetylbaccatin III (10–DAB) (2.1), which is the most
demanding portion of Taxol from the point of view of total
synthesis, is readily available from the renewable leaves of
Taxus baccata (European Yew). 10–DAB can be extracted
from the leaves of this tree in high yields of 1g/kg [8].
2. Materials and methods
The Taxus baccata was imported from USA and the
standard 10-DAB was available in house for interpretational
studies. The extracting solvents, methylene dichloride, chlorof-
orm, methanol were procured from Indian suppliers [9–13].
2.1. Extraction of crude
The Taxus baccata was first of all freed from foreign
particles and air dried. It was then pulverized in the hammer
mill to powder that does not enable the channelling during
extraction process. 1 kg of powder was then successively
charged into the Soxhlet apparatus placed in muslin cloth
thimble. 5 litres of methylene dichloride was charged into the
flask. The extraction was performed over the 12 h until the
matrix completely exhausted. The progress was checked
employing the TLC consisting of MeOH: Hexane (2:8). The
macerated solvent was then concentrated in rotary evaporator
under vacuum to obtain 350 g of dark brown syrupy mass [14].
2.2.Isolation and purification of 10-DAB
350 g of syrupy mass was then admixed with 500 g of 60–
120 mesh silica gels (pH 6.00–6.25). It was then dried under
vacuum until completely dried mass. Initially the column was
preconditioned with the chloroform and the mass was then
charged into the column containing 3.50 kg of same silica gel
by dissolving in chloroform. The lower and higher impurities
were first isolated with the steadily increase in the polarity of
the mobile phase. All fractions were checked with the TLC for
the progress of isolation. All the fractions containing 10–DAB
was collectively concentrated under vacuum in rotary
evaporator. The final concentration yielded the 125 g of 10–
DAB. It was finally crystallized and gave 110 g of 95% assay
of 10-DAB [15,16].
2.3.Analytical interpretation of 10-DAB
The precursor for anti cancer drug like Taxol, Paclitaxel,
Texotere was analyzed by XRD, 1
H–NMR and 13
C–NMR [16–
18].

4. 48 O.H. Nautiyal / Journal of Chemistry and Materials Research 1 (2014) 45–51
Fig. 2. XRD of 10–DAB
Fig. 1. 10–DAB (10–Deacetyl Baccatin C29H36O10)
2.4.1
H-NMR and 13C-NMR
The nuclear magnetic resonances of protons of 10–DAB
was analyzed using PROB HD of 5 mm, PULPPROG of zg
30, TD 65536, deutoriated methanol as a solvent NS 32, DS
0, SWH 8223.658 Hz, FIDRES 0.125483 Hz, AQ 3.9846387
sec, RG 161, DW 60.800 usec, DE 6.50 usec, TE 292.4 K, D1
1.00000000 sec and TDO 1. Channel f1 consisted of NUC1
1H, P1 11.20 usec, PL1 0.00 dB, PL1W 17.357 W, SFO1
400.132 MHz, SI 32768, SF 400.1300481 MHz, WDW EM,
SSB 0, LB 0.30 Hz, GB 0 and PC 1.00 [16–18].
The nuclear magnetic resonance of carbon of 10-DAB was
analyzed using PROB HD of 5mm PABBO BB-, PULPPROG
zgpg30, TD 65536, deutoriated methanol as a solvent, NS
200, DS 4, SWH26041.666 Hz, FIDRES 0.397364 Hz, AQ
1.2583412 sec, RG 203, DW 19.200 usec, DE 6.50 usec,
TE 293.4 K, D12.00 esc, D110.300 sec, TD0 1. Channel f1
consisted of NUC1 13C, P1 8 usec, PL1 1.60 dB, PL1W
63.255 W, SFO1 100.622 MHz. Channel f2 consisted of
CPDPRG2 waltz 16, NUC2 1H, PCPD2 90.00 usec, PL2
0.00dB, Pl1217.7dB, PL13 20.87 dB, PL2W 17.357 W,
PL12W 0.2834 W, PL13W 0.1420 W, SFO2400.131 MHz, SI
32768, SF 100.612 MHz, WDW EM, SSB 0, LB 1 Hz, GB 0
and PC 1.40 [16–18].
10–DAB was extracted and isolated in the year 2003 and
was hold under ambient conditions at 35±5 o C till 2013
February. Subsequently it was analyzed by XRD, 1H-NMR
and 13CNMR and the interpretation revealed no major
changes as such to occur in the precursor of anti-cancer drugs.
3. Results
10–Deacetyl-baccatin may have been transformed to 10–
oxo –10 – deactyl – baccatin – III, 10 – Oxo – 7 – epi – 10 –
deacetylbaccatin – III over the 10 years of storage. But no
such product formation was anticipated while analyzing
employing the mentioned sophisticated instrument techniques.
Regulatory guidance in ICH Q2A, Q2B, Q3B and FDA 21
CFR section 211, require the development and validation of
stability-indicating impurities method for all pharmaceutical
dosage forms. However, the current guidance documents do
not indicate detailed degradation conditions in stress testing.
The forced degradation conditions, stress agent concentration
and time of stress, are found to effect the % degradation.
Preferably not more than 20% is recommended for active
materials to make the right assessment of stability indicating
nature of the chromatographic methods. The optimisation of
such stress conditions which can yield not more than 20%
degradation is based on experimental study.

5. O.H. Nautiyal / Journal of Chemistry and Materials Research 1 (2014) 45–51 49
Fig. 3. 1
H–NMR of 10–DAB
XRD shown 30 2theta positions, d-spacing, significance,
relative intensity, height and FWHM comparable to literature
cited. (Table 1 and Fig. 1) [19,20].
JK Harper et al. investigated the origins of solid-state
NMR shift differences in polymorphs, carbon NMR chemical
shift tensors are measured for two forms of solid 10–deacetyl
baccatin III: a dimethyl sulfoxide (DMSO) solvate and a un
solvated form. A comparison of ab initio computed tensors
that includes and omits the DMSO molecules demonstrates
that lattice interactions cannot fully account for the shift
differences in the two forms. Instead, conformational
differences in the cyclohexenyl, benzoyl, and acetyl moieties
are postulated to create the differences observed. X-ray
analysis of six Baccatin III analogues supports the suggested
changes in the cyclohexenyl and benzoyl systems. The close
statistical match of the 13 C chemical shifts of both
polymorphic forms with those calculated using the X-ray
geometry of 10-deacetyl Baccatin III supports the contention
that the B, C, and D rings are fairly rigid. Therefore, the
observed tensor differences appear to arise primarily from
conformational variations in ring substituent and the
cyclohexenyl ring.
4. Discussions
1
H and 13
C Nuclear Magnetic Resonance (NMR) Spectra
of Taxol
Despite the complex structure of 10–DAB, its proton NMR
spectrum is relatively simple and can be easily assigned.
Almost all of the signals are well resolved and are distributed
in the region from 1.0 to 8.03 ppm. The strong three–proton
signals caused by the methyl and acetate groups lie in the
region between 1.0 and 2.5 ppm, together with multiplets
caused by certain methylene groups. Most of the protons in the
taxanes skeleton are observed in the region between 2.5 and
7.0 ppm, and the aromatic proton signals caused by the C–2
benzoate, C–3' phenyl and C-3' benzamide groups appear
between 7.0 and 8.03 ppm. The 400 MHz proton and carbon
NMR spectra of 10–DAB are shown in the figure 2 and 3 [21–
25].
As an intermediate for Paclitaxel Appearance; white to off
white crystalline powder Identification 1. FT–IR confirms 2.
HPLC confirm 3. NMR confirms 4. Mass confirm Melting
Point 227–230 degree Specific Rotation: –40.0 to –45.0
degree Lost on Drying NMT 3.0% Residual Solvent (GC)
Total residual solvents: NMT 1.5% Methanol: NMT 0.05%
Acetone: NMT 1% Single largest impurity (except for the
following): NMT 0.2% 10–Deacetyl–19–hydroxybaccatin III:
NMT 1% 10–deacetyl–2–debenzoyl–2–tigloybaccatin III:
NMT 1.0% 10–Deacetyl Paclitaxel: NMT 1.0%(HPLC:
16.447s) 10–Deacetyl Paclitaxel ethyl analogue: NMT 0.3%
10-Deacetyl Paclitaxel propyl analogue: NMT 0.4% 10–
Deacetyl Paclitaxel cephalomannine: NMT 0.5% Assay
(HPLC) NLT 99.0% [9–13,26].
1H NMR (MeOD, 400 MHz) δ 8.10 (d, J=7.6 Hz, 2H),
7.58 (t, J=7.6 Hz, 1H), 7.46 (t, J=7.6 Hz, 2H), 5.61 (d, J=7.1
Hz, 1H), 5.21 (s, 1H), 4.93 (dd, J=1.7, 9.3 Hz, 1H), 4.82 (m,
1H), 4.42 (dd, J=6.6, 10.4 Hz, 1H), 4.27 (d, J=8.2 Hz, 1H),
4.14 (d, J=8.2 Hz, 1H), 3.91 (d, J=6.6 Hz, 1H), 2.53–2.41 (m,
1H), 2.27 (s, 3H), 2.25 (m, 2H), 2.03 (s, 3H), 2.01-1.95 (m,
1H), 1.85 (m, 1H), 1.64 (s, 3H), 1.18 (s, 3H), 1.04 (s, 3H),
1.02-0.85 (m, 18H), 0.69-0.58 (m, 12H);
13C NMR (MeOD,400 MHz) δ 205.7, 170.8, 167.1, 137.7,

6. 50 O.H. Nautiyal / Journal of Chemistry and Materials Research 1 (2014) 45–51
Fig. 4. 13
CNMR of 10–DAB
136.9, 133.4, 130.0, 129.5, 128.5, 84.0, 80.9, 78.7, 75.8, 74.9,
72.8, 67.9, 58.6, 47.2, 42.7, 38.3, 37.3, 26.7, 22.7, 19.4, 14.6,
10.3, 6.9, 6.8, 5.8,5.3.
Since the IR and HPLC are already reported in the
published papers. Hence the specifications are referred from
the published source. Only the melting point of the 10–DAB
was determined to be 235 o C probably storing the sample of
10–DAB for more than 10 years at the ambient temperature in
the dark place. Table 2 displays the chemical shifts and
assignments of proton and carbon respectively. Figure 2 and
figure 3 are the recorded 1H NMR and 13C NMR of the drug
respectively [27–29].
Specification: Appearance: White to off-white crystalline
powder, Identification: Pass (IR, HPLC, NMR) Assay,
(HPLC): 98.0% min, Melting Point: 225 C –230 C, Specific
Rotation: 36.0° ~ –40.0 °, Water (KF): 1.0% max, Residual
Solvent: 1.5% max, Individual impurity: 1.0% max and Total
Impurities: 1.0% max [30–33].
Acknowledgement
Prof. V.S. Patel, Director, SICART deserves a very special
thanks for offering an academic research concessions while
analyzing the moiety employing XRD, 1HNMR and
13CNMR.
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