Some transformations of tacrolimus, an immunosuppressive drug

Article abstract of DOI:10.1016/j.ejps.2012.12.001

Transformations of the macrocyclic lactone tacrolimus (1), an important immunosuppressive drug produced by Streptomyces species, are described. These transformation products are primarily of interest as reference substances for drug impurity analyses. Upon action of acid (p-toluenesulfonic acid in toluene), tacrolimus is dehydrated by loss of water from the b-hydroxyketone moiety with partial inversion of configuration at C-8, resulting in formation of 5-deoxy-D5,6-tacrolimus and 5-deoxy-δ⁵,β-8-epitacrolimus. The structure of the latter was determined by single-crystal X-ray crystallography. The same products are formed upon action of free radicals (iodine in boiling toluene), along with formation of 8- epitacrolimus. The latter is converted by p-toluenesulfonic acid to 5-deoxy-D5,6-8-epitacrolimus. Treatment of tacrolimus with weak base (1,5-diazabicyclo[4.3.0]nonene) gives, in addition to 8-epitacrolimus, the open-chain acid corresponding to 5-deoxy-Δ⁵,β-tacrolimus, a rare non-cyclic derivative of tacrolimus. Strong base (t-butoxide) causes pronounced degradation of the molecule. Thermolysis of tacrolimus leads to ring expansion by an apparent [3,3]-sigmatropic rearrangement of the allylic ester moiety with subsequent loss of water from the b-hydroxyketone moiety. ¹H and ¹³C NMR spectra of the obtained compounds, complicated by the presence of amide bond rotamers and ketal moiety tautomers, were assigned by extensive use of 2D NMR techniques.

Full text of DOI:10.1016/j.ejps.2012.12.001

European Journal of Pharmaceutical Sciences 48 (2013) 514–522
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Some transformations of tacrolimus, an immunosuppressive drug
Dorthe M. Skytte a, Jerzy W. Jaroszewski a, Kenneth T. Johansen a, Steen Honoré Hansen b,
Liselotte Hansen c, Peter G. Nielsen c, Karla Frydenvang a,
⇑
a Department of Drug Design and Pharmacology, School of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
b Department of Pharmacy, School of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark
c Veloxis Pharmaceuticals A/S, Kogle Allé 4, DK-2970 Hørsholm, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Transformations of the macrocyclic lactone tacrolimus (1), an important immunosuppressive drug pro-
duced by Streptomyces species, are described. These transformation products are primarily of interest
as reference substances for drug impurity analyses. Upon action of acid (p-toluenesulfonic acid in tolu-
ene), tacrolimus is dehydrated by loss of water from the b-hydroxyketone moiety with partial inversion
Received 7 September 2012
Received in revised form 15 November 2012
Accepted 2 December 2012
Available online 10 December 2012
of configuration at C-8, resulting in formation of 5-deoxy-D D
5,6-tacrolimus and 5-deoxy- 5,6-8-epitacrol-
imus. The structure of the latter was determined by single-crystal X-ray crystallography. The same prod-
ucts are formed upon action of free radicals (iodine in boiling toluene), along with formation of 8-
Keywords:
Tacrolimus
Degradation pathway
NMR spectroscopy
Crystal structure
HPLC
epitacrolimus. The latter is converted by p-toluenesulfonic acid to 5-deoxy-
ment of tacrolimus with weak base (1,5-diazabicyclo[4.3.0]nonene) gives, in addition to 8-epitacrolimus,
the open-chain acid corresponding to 5-deoxy-
5,6-tacrolimus, a rare non-cyclic derivative of tacrolimus.
D
5,6-8-epitacrolimus. Treat-
D
Strong base (t-butoxide) causes pronounced degradation of the molecule. Thermolysis of tacrolimus leads
to ring expansion by an apparent [3,3]-sigmatropic rearrangement of the allylic ester moiety with sub-
sequent loss of water from the b-hydroxyketone moiety. 1H and 13C NMR spectra of the obtained com-
pounds, complicated by the presence of amide bond rotamers and ketal moiety tautomers, were
assigned by extensive use of 2D NMR techniques.
Mass spectrometry
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
solvents, a different kind of equilibrium exists between tacrolimus
tautomers with respect to the cyclic ketal moiety (Akashi et al.,
Tacrolimus (1), a 23-membered macrolide lactone, is a natural
product originally isolated from the bacterium Streptomyces tsu-
kubaensis (Tanaka et al., 1987). It is used as an immunosuppressant
to prevent graft rejection after organ transplantations (Bowman
and Brennan, 2008; Busuttil and Lake, 2004; Fortune and Couriel,
2009; Penninga et al., 2010; Scott et al., 2003; Vicari-Christensen
et al., 2009) and also in the treatment of autoimmune diseases
(Ruemmele et al., 2008; Yeoman et al., 2010) and atopic dermatitis
(Kalavala and Dohil, 2011). Furthermore, it is being investigated as
treatment of rheumatoid arthritis (Lee et al., 2010). Tacrolimus is a
highly functionalized molecule with L-pipecolic acid moiety adja-
cent to a masked tricarbonyl functionality, 14 stereocenters, three
double bonds, and a number of free hydroxy groups and other
functionalities. Restricted rotation of the amide bond in the pipeco-
lic acid moiety results in a solvent-dependent equilibrium between
cis and trans rotamers in solution, which can be detected by NMR
spectroscopy (Karuso et al., 1990; Mierke et al., 1991; Petros
et al., 1993; Tanaka et al., 1987). Furthermore, especially in polar
1996; Baumann et al., 1995; Gailliot et al., 1994; Namiki et al.,
1995; Namiki et al., 1993; Schueler et al., 1993).
Tacrolimus acts by binding to the cytosolic immunophilin pro-
tein FKBP12. The bimolecular complex inhibits calcineurin and
thereby hinders the transcription of T-cell activation genes such
as interleukin-2 (Griffith et al., 1995; Kissinger et al., 1995; Moore
et al., 1991; Sewell et al., 1994; Wilson et al., 1995). Tacrolimus is
structurally related to a number of other L-pipecolic acid derived
macrolides such as ascomycin, pimecrolimus, and rapamycin
(sirolimus) (Bulusu et al., 2011; Graziani, 2009; Nghiem et al.,
2002). These compounds all show immunosuppressive activity
by targeting immunophilins. Although the structures of tacrolimus,
ascomycin and pimecrolimus are closely related, their pharmaco-
logical profiles are different. This may be related to the fact, that
even small changes such as inversion of a single stereocenter
engenders considerable changes in the conformation of the mole-
cule (Skytte et al., 2010; Wagner et al., 1996). In this article we de-
scribe a number of derivatives of tacrolimus formed by simple
reactions. These compounds are intended to mimic probable
tacrolimus degradation products formed during manufacturing or
storage. The semi-synthetic compounds can be produced in much
larger amounts compared to degradation products isolated in trace
⇑
Corresponding author. Tel.: +45 35336207; fax: +45 35336041.
E-mail address: karla.frydenvang@sund.ku.dk (K. Frydenvang).
0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.12.001

D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
515
amounts from stability studies facilitating the identification and
structure elucidation. Subsequently it will be possible to identify
degradation products observed in stability studies by comparison
(HPLC, NMR) with the synthesized model compounds. The synthe-
sized products were purified by HPLC and identified primarily by
NMR spectroscopy and LC-MS analysis, supported, where neces-
sary, by single crystal X-ray diffraction.
was poured into water (120 ml) containing AcOH (2.4 mmol). The
layers were separated and the organic layer was washed with
water (3 ꢀ 40 ml), dried over Na2SO4, filtered and evaporated in va-
cuo. The product was purified by preparative reversed-phase HPLC
(isocratic elution, MeCN–H2O 60:40) to give 4 as colorless crystals
(147 mg, 15%) and 5 as colorless oil (28.5 mg, 3%).
2.5. Reaction of tacrolimus with sodium t-butoxide
2. Experimental
Tacrolimus (1, 250 mg, 0.30 mmol) was added to sodium t-
butoxide (61 mg, 0.64 mmol) in t-butanol (25 ml), and the solution
was stirred at 65 °C for 1.5 h. The reaction mixture was quenched
with water (40 ml) and extracted with EtOAc (3 ꢀ 25 ml). The
combined organic layer was washed with brine (15 ml), dried over
Na2SO4, filtered, and concentrated in vacuo. The product was
recrystallized from heptane to give 8 (36 mg, 60%) as colorless
crystals.
2.1. Materials and methods
Optical rotations were measured with a Jasco DIP-370 polarim-
eter. UV spectra were recorded with a Thermo Scientific NanoDrop
2000 spectrophotometer, and IR spectra with a Perkin–Elmer Spec-
trum One FTIR spectrophotometer. NMR spectra were recorded at
25 °C on a Bruker Avance 400 or 600 MHz spectrometer using ben-
zene-d6 as solvent and TMS as internal reference. Reactions were
monitored by analytical HPLC on a Shimadzu system consisting
of a LC-10AT liquid chromatography, a SPD-M10A photo-diode ar-
ray detector, a FCV-10AL pump, and a CTO-10AC column oven,
2.6. Thermal rearrangement of tacrolimus
Tacrolimus (1, 500 mg, 0.61 mmol) was dissolved in o-xylene
(25 ml). The solution was refluxed for 24 h, cooled to room temper-
ature, and concentrated in vacuo. Preparative reversed-phase HPLC
(isocratic elution, MeCN–H2O 65:35) yielded 6 as white amorphous
solid (95 mg, 19%) and 7 as colorless oil (25 mg, 5%).
using a Phenomenex Luna C18 column (3
lm, 150 ꢀ 4.6 mm i.d.)
operated at 50 °C, detection wavelength 210 nm. Mobile phases A
and B consisted of 5% of MeCN and 0.1% of formic acid in water,
and 5% of water and 0.1% of formic acid in MeCN, respectively.
Products were purified by preparative HPLC on a Waters system
consisting of a Model 590 pump and a Lambda-Max Model 481
2.7. Iodine-catalyzed transformations of tacrolimus
detector, using
a
Supelco Discovery C18 column (5 lm,
250 ꢀ 21.2 mm i.d.) and mixtures of MeCN and water as mobile
phases. LC-MS was performed on an Agilent 1100 system equipped
with a photo-diode array detector and an Agilent MSD ion-trap
Tacrolimus (1, 500 mg, 0.61 mmol) was dissolved in distilled
toluene (15 ml). Iodine (5 mg, 0.02 mmol) was added, and the reac-
tion mixture was refluxed for 6.5 h in daylight. After cooling and
diluting with toluene (15 ml), the solution was washed with satu-
rated sodium thiosulphate (30 ml), water (30 ml), and brine
(30 ml), dried over Na2SO4, and concentrated in vacuo. Reversed-
phase HPLC (isocratic elution, MeCN–H2O 65:35) gave three com-
pounds: 2 (colorless crystals, 33 mg, 7%), 3 (white amorphous so-
lid, 50 mg, 10%), and 4 (colorless crystals, 41 mg, 8%).
mass spectrometer, using
(3
Bruker micrOTOF-Q II mass spectrometer equipped with an elec-
tro-spray ionization source. Tacrolimus (1) was available from Ve-
loxis Pharmaceuticals.
a Phenomenex Luna C18 column
m, 100 ꢀ 2.0 mm i.d.). HRESIMS spectra were acquired with a
l
2.2. Reaction of tacrolimus with p-toluenesulphonic acid (Tos)
Tacrolimus (1, 500 mg, 0.61 mmol) and Tos (monohydrate,
11.6 mg, 0.061 mmol) were dissolved in toluene (30 ml) and
heated under reflux for 1 h. The solution was washed with water
(30 ml) and then with brine (30 ml), dried over Na2SO4, filtered,
and concentrated in vacuo. The product was purified by preparative
reversed-phase HPLC (isocratic elution, MeCN–H2O 65:35) to give
2 (63 mg, 13%) as colorless crystals and 3 (225 mg, 47%) as white
amorphous solid.
2.8. Compound characteristics
2.8.1. 5-Deoxy-D
5,6-8-epitacrolimus
(3S,4R,8S,9E,12S,14S,15R,16S,18R,19R,26aS)-14,16-Dimethoxy-15,19-
epoxy-19-hydroxy-3-[(1E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclo
hexyl]-1-methylethenyl]-8-(2-propen-1-yl)-8,11,12,13,14,15,16,17,
18,19,24,25,26,26a-tetradecahydro-4,10,12,18-tetramethyl-3H-
pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone (2):
Colorless prisms; mp 186–187 °C (from MeCN–H2O 7:3);
[a
]
D
25 + 50.9° (c 0.32, CHCl3); UV (MeCN) kmax
(e
)
201 nm
2.3. Reaction of 8-epitacrolimus with Tos
(26,300); IR (film) mmax 3432, 2934, 1749, 1650, 1626, 1450,
1193, 1173, 1089, 986, 752 cmꢁ1 1H and 13C NMR, see Table 1;
;
8-Epitacrolimus (4, 44 mg, 0.05 mmol) and Tos (monohydrate,
1.0 mg, 0.005 mmol) was dissolved in toluene (10 ml) and heated
under reflux for 2 h. The solution was diluted with toluene
(5 ml), washed with water (15 ml) and then with brine (15 ml),
dried over Na2SO4, filtered, and concentrated in vacuo. The product
was purified by preparative reversed-phase HPLC (isocratic elution,
MeCN–H2O 65:35) and recrystallized from MeCN–H2O 7:3 to give
2 (21 mg, 49%) as colorless crystals.
HRESIMS m/z 786.4790 ([M + H]+), C44H68NOþ requires 786.4787
11
(D
M 0.4 ppm).
2.8.2. 5-Deoxy-
D
5,6-tacrolimus
(3S,4R,8R,9E,12S,14S,15R,16S,18R,19R,26aS)-14,16-Dimethoxy-15,19-
epoxy-19-hydroxy-3-[(1E)-2-[(1R,3R,4R)-4-hydroxy-3-methoxycyclo
hexyl]-1-methylethenyl]-8-(2-propen-1-yl)-8,11,12,13,14,15,16,17,
18,19,24,25,26,26a-tetradecahydro-4,10,12,18-tetramethyl-3H-
2.4. Reaction of tacrolimus with 1,5-diazabicyclo[4.3.0]nonene (DBN)
pyrido[2,1-c][1,4]oxaazacyclotricosine-1,7,20,21(4H,23H)-tetrone (3)
25
White amorphous solid; [
(MeCN) kmax
1689, 1648, 1450, 1195, 1173, 1091, 988, 758 cmꢁ1
NMR, see Table 1; HRESIMS m/z 786.4780 ([M + H]+), C44H68NOþ
a
]
ꢁ91.2° (c 0.40, CHCl3); UV
D
Tacrolimus (1, 1.0 g, 1.2 mmol) was dissolved in CH2Cl2
(100 ml). The solution was cooled to 0 °C on an ice bath. A solution
of DBN (302 mg, 2.4 mmol) in CH2Cl2 (40 ml) was added and the
resulting solution was stirred overnight. The reaction mixture
(e
) 197 nm (47300; IR (film) mmax 3446, 2934, 1741,
1H and 13C
;
11
requires 786.4787 (DM 0.8 ppm).

516
D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
Table 1
1H NMR (600 MHz) and 13C NMR (150 MHz) spectroscopic dataa for enones 2 and 3 in benzene-d6.
5-Deoxy-
No.
D
5,6-8-epitacrolimus (2)
1H NMR data
5-Deoxy-D
5,6-tacrolimus (3)
13C NMR data
1H NMR data
13C NMR data
Major rotamer (cis)
Minor rotamer (trans)
Major
rotamer
(cis)
Minor
rotamer
(trans)
Major rotamer (cis)
Minor rotamer (trans) Major Minor
rotamer
rotamer
(cis)
(trans)
1
3
169.8
80.1
169.3
82.0
169.7
80.6
169.3
81.2
5.26 (d, 4.2)
5.26 (d, 4.2)
5.24 (d, 3.0)
5.32 (d, 3.6)
4
2.54–2.57 (m)
2.71–2.75 (m)
38.7
39.7
2.41–2.43 (m)
2.45–2.49 (m)
39.6
40.6
5
6
7
8
9
10
11
6.84 (dd, 15.6, 6.0)
6.26 (dd, 15.6, 1.2)
6.99 (dd, 15.6, 9.0)
6.69 (dd, 15.6, 0.6)
148.6
130.6
200.7
50.1
122.9
137.0
46.5
146.7
128.4
197.7
53.7
124.2
139.4
48.4
6.91 (dd, 15.6, 6.6)
6.25 (dd, 15.6, 1.2)
6.94 (dd, 15.6, 8.4)
6.43 (dd, 15.6, 1.2)
148.1
128.6
199.5
52.3
124.5
138.9
49.5
147.0
129.7
196.8
52.2
124.0
139.5
50.0
3.67 (dt, 9.6, 7.2)
5.02 (dd, 9.6, 1.2)
3.56–3.59 (m)
5.94 (br d, 9.6)
3.64 (m)
5.20 (d, 9.6)
3.37–3.39 (m)
5.11 (d, 10.2)
1.99–2.02 (m), 1.61–
1.63 (m)
2.23–2.26 (m), 1.47–
1.49 (m)
2.04 (dd, 12.6, 3.6),
1.72–1.74 (m)
2.14 (dd, 13.8, 6.0),
1.91–1.94 (m)
12
13
1.70 (m)
1.92–1.95 (m), 0.93–
0.97 (m)
1.90–1.91 (m)
1.77–1.80 (m), 1.51 (m)
28.7
35.0
32.3
37.9
1.56–1.58 (m)
1.70 (m), 0.99 (ddd, 13.8, 1.72 (m), 1.35–1.37
11.4, 2.4)
1.83–1.85 (m)
27.9
34.0
26.3
36.4
(m)
14
3.71 (br d, 9.6)
3.84 (ddd, 10.8, 5.4, 2.4)
76.8
76.3
3.66 (dd, 9.6, 3.6)
3.79 (ddd, 11.4, 4.2,
2.4)
76.7
76.7
15
16
3.89 (d, 9.0)
3.41–3.44 (m)
4.23 (dd, 9.6, 2.4)
3.60 (ddd, 11.4, 9.6, 4.8)
74.2
74.8
72.3
74.1
3.81 (d, 9.0)
3.35–3.37 (m)
4.20 (dd, 9.6, 2.4)
3.59(ddd, 11.4, 9.6,
4.8)
74.1
74.7
72.9
74.5
17
1.85 (dt, 12.0, 4.2), 1.68 1.92–1.95 (m), 1.70 (m)
(m)
35.1
33.5
1.90 (m), 1.59 (m)
1.91–1.94 (m), 1.72
(m)
33.2
33.4
18
19
20
21
23
2.71–2.75 (m)
2.22–2.23 (m)
35.6
99.5
197.9
166.8
39.6
34.1
99.2
191.3
168.5
44.5
2.68 (ddq, 10.8, 6.6, 6.6) 2.47 (m)
35.4
99.2
198.1
165.6
40.0
34.9
99.7
193.4
166.9
44.7
4.71 (br d, 14.4), 3.23
(m)
1.24–1.27 (m), 1.24–
1.27 (m)
1.37–1.40 (m), 1.16–
1.18 (m)
2.19–2.22 (m), 1.95–
1.97 (m)
3.76–3.79 (m), 3.49–
3.52 (m)
1.05–1.08 (m), 0.93–
0.97 (m)
4.65 (br d, 13.2), 3.09
(m)
1.24–1.26 (m), 1.17–
1.18 (m)
3.72 (br d, 15.0),
3.37–3.39 (m)
1.24–1.26 (m), 1.12
(m)
24
25
26
24.9
22.0
27.5
24.9
20.6
26.9
25.0
21.7
27.4
25.1
20.9
27.1
n.a.
1.37–1.39 (m), 1.29–
1.31 (m)
1.37–1.39 (m), 1.17–
1.18 (m)
2.06–2.12 (m), 1.61–
1.63 (m)
2.07–2.08 (m), 2.02–2.03 2.09–2.10 (m), 1.47
(m)
(m)
26a
10
4.48 (br d, 6.6)
5.28 (dd, 6.6, 2.4)
57.0
130.5
132.4
35.5
52.9
129.7
134.8
35.5
4.74 (br d, 4.8)
5.24 (dd, 6.6, 2.4)
57.1
130.6
132.7
35.7
53.4
129.9
134.4
35.7
20
4.97 (br d, 9.0)
2.06–2.12 (m)
1.88–1 90 (m), 0.88–
0.90 (m)
2.85 (ddd, 11.4, 8.4, 4.2) 2.76 (ddd, 11.4, 9.0, 4.2)
3.40–3.42 (m)
n.a.
4.92 (br d, 8.4)
1.97–1.99 (m)
n.a.
5.04 (d, 10.2)
2.07–2.08 (m)
1.91 (m), 0.90–0.94 (m)
5.06 (d, 7.8)
2.02–2.03 (m)
1.83–1.85 (m), 0.85
(m)
30
40
32.2
32.4
35.5
35.1
50
60
70
84.9
74.2
32.2
84.8
74.0
32.1
2.84 (ddd, 11.4, 9.0, 4.2) 2.80 (m)
84.9
74.2
32.2
84.9
74.1
32.2
3.38–3.40 (m)
n.a.
3.40–3.43 (m)
1.95–2.00 (m), 1.35–
1.37 (m)
3.40–3.43 (m)
1.95–2.00 (m), 1.35–
1.37 (m)
80
a
b
1.37–1.40 (m), 0.86–
0.87 (m)
2.71–2.75 (m), 2.38–
2.43 (m)
5.80 (ddt, 17.4, 10.2,
7.2)
5.06 (ddt, 17.4, 1.8, 1.8), 5.10 (ddt, 16.8, 1.8, 1.8), 116.7
4.98 (ddt, 10.2, 1.2, 1.2) 5.00 (ddt, 10.2, 0.6, 0.6)
1.37–1.39 (m), 0.79–
0.83 (m)
2.54–2.57 (m), 2.43–
2.47 (m)
5.76 (ddt, 16.8, 10.2,
7.2)
31.3
36.0
31.0
36.9
1.41 (m), 0.86 (m)
1.41 (m), 0.81 (m)
31.2
37.1
31.0
35.9
2.61 (ddd, 13.8, 7.2, 7.2), 2.77–2.80 (m), 2.39
2.32 (ddd, 13.8, 7.2, 7.2) (ddd, 13.8, 7.2, 7.2)
5.80 (ddt, 17.4, 10.2, 7.2) 5.87 (ddt, 16.8, 10.2,
6.6)
5.08 (dd, 17.4, 1.8), 5.00 5.08 (dd, 17.4, 1.8),
(ddt, 10.2, 1.2, 1.2)
136.9
136.1
117.1
136.5
116.9
137.1
116.6
c
5.00 (ddt, 10.2, 1.2,
1.2)
CH3-4
CH3-10
CH3-12
CH3-18
CH3-10
CH3O-
14
0.74 (d, 7.2)
1.62 (d, 0.6)
0.91 (d, 6.6)
1.21 (d, 6.6)
1.41 (d, 1.2)
3.19 (s)
0.76 (d, 7.8)
1.65 (d, 1.2)
0.86 (d, 6.6)
1.31 (d, 6.6)
1.42 (d, 1.2)
3.31 (s)
13.2
20.7
21.5
16.8
14.5
57.3
15.2
20.8
18.2
17.0
14.6
57.9
0.83 (d, 7.2)
1.65 (s)
0.89 (d, 6.6)
1.16 (d, 6.6)
1.43 (d, 1.2)
3.19 (s)
0.86 (d, 7.2)
1.70 (s)
0.82 (d, 6.6)
1.20 (d, 6.6)
1.47 (d, 1.2)
3.26 (s)
13.8
16.0
21.4
16.8
14.8
57.3
15.0
16.3
19.5
16.8
14.7
57.9
CH3O-
16
CH3O-50
OH-19
3.07 (s)
3.14 (s)
55.9
56.7
56.0
56.5
3.09 (s)
3.08 (s)
3.15 (s)
3.07 (s)
56.0
56.6
56.0
56.5
3.09 (s)
5.58 (d, 1.2)
3.04 (s)
6.49 (d, 1.8)
a
Chemical shifts (d values) with proton multiplicities and coupling constants (Hz) in parentheses, n.a. = not assigned.

D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
517
2.8.3. 2,3-Seco-
D
5,6-tacrolimus (S)-1-[2-[(2R,3R,5S,6R)-6-[(1S,3S,5E,
were located in subsequent difference electron density maps and
included in calculated positions, except for the hydrogen atoms
bonded to methine carbon atoms and to the oxygen atoms. These
hydrogen atoms were refined with fixed isotropic displacement
parameters (Uiso = 1.2Ueq for CH and CH2, Uiso = 1.5Ueq for OH and
CH3). Refinement (570 parameters, 10253 unique reflections)
7R,9E,11R,12S,13E)-7-(2-propen-1-yl)-12-hydroxy-14-[(1R,3R,4R)-4-
hydroxy-3-methoxycyclohexyl]-1-methoxy-3,5,11,13-tetramethyl-8-
oxotetradeca-5,9,13-trien-1-yl]-2-hydroxy-5-methoxy-3-methyltetra
hydro-2H-pyran-2-yl]-2-oxoacetyl]piperidine-2-carboxylic acid (5)
25
Colorless oil; [
a
]
D
ꢁ55.7° (c 0.29, CHCl3); UV (MeCN) kmax
(e)
230 nm (11100); IR (film)
m
max 3384, 2933, 1729, 1641, 1451, 1216,
converged at RF = 0.039, wR2 ¼ 0:081 [9093 reflections with Fo > 4
F
2
1088, 1049, 1027, 753 cmꢁ1
;
1H and 13C NMR, see Table 2; HRE-
r
(Fo); wꢁ1 ¼ ½r2ðF2o Þ þ ð0:0328PÞ þ 1:3370Pꢂ, where P ¼ ðF2oþ
SIMS m/z 804.4887 ([M + H]+), C44H70NOþ requires 804.4893
2F2c Þ=3; S ¼ 1:099ꢂ. The residual electron density varied between
ꢁ0.19 and 0.23 e Åꢁ3. The molecule belongs to a non-centrosym-
metric space group but the absolute configuration could not be
determined [Flack = 0.3(6)] (Flack, 1983). Complex scattering fac-
tors for neutral atoms were taken from International Tables for
Crystallography as incorporated in SHELXL97 (Sheldrick, 2008;
Wilson, 1995). Fractional atomic coordinates, a list of anisotropic
displacement parameters, and a complete list of geometrical data
have been deposited in the Cambridge Crystallographic Data Cen-
tre (CCDC: 844390). Copies of the data can be obtained, free of
charge, on application to the director, CCDC, 12 Union Road, Cam-
bridge CB2 1EZ, UK (fax: +44 1223 336033 or e-mail: deposit@
ccdc.cam.ac.uk).
12
(DM 0.7 ppm).
2.8.4. Tacrolimus rearrangement product
(3S,4E,6S,7S,10R,11E,14R,16S,17R,18S,20R,21R,28aS)-7,21-Dihy
droxy-16,18-dimethoxy-17,21-epoxy-7,8,10,13,14,15,16,17,18,19,
20,21,26,27,28,28a-hexadecahydro-3-[(1R,3R,4S)-4-hydroxy-3-
methoxycyclohexyl]-4,6,12,14,20-pentamethyl-10-(2-propen-1-yl)-
3H-pyrido[2,1-c][1,4]oxaazacyclopentacosine-1,9,22,23(6H,25H)-
tetrone (6)
25
White amorphous solid; [
a]
ꢁ75.6° (c 0.35, CHCl3); UV
D
(MeCN) kmax (e) 230 nm (94200); IR (film) mmax 3473, 2932, 1717,
1648, 1452, 1380, 1098, 1038, 754 cmꢁ1 1H and 13C NMR, see Ta-
;
ble 3; HRESIMS m/z 804.4887 ([M + H]+), C44H70NOþ requires
12
804.4893 (DM 0.7 ppm).
3. Results and discussion
2.8.5. Tacrolimus rearrangement product, dehydrated
(3S,4E,6S,7S,10R,11E,14R,16S,17R,18S,20R,21R,28aS)-21-Hydroxy-
16,18-dimethoxy-17,21-epoxy-10,13,14,15,16,17,18,19,20,21,26,27,
28,28a-tetradecahydro-3-[(1R,3R,4S)-4-hydroxy-3-methoxycyclo
hexyl]-4,6,12,14,20-pentamethyl-10-(2-propen-1-yl)-3H-pyrido
Reactions described in this work were performed in order to mi-
mic formation of likely transformation products formed in trace
amounts during manufacturing of the drug substance tacrolimus
(1), preparation of pharmaceutical formulations and solid dosage
forms of this drug, and long-time storage of the dosage forms.
The availability of reference samples of tacrolimus transformation
products facilitates impurity identification and quantification in
pharmaceutical products and allows for precautionary steps to-
wards minimizing formation of such impurities. Thus, tacrolimus
was subjected to acid- and base-catalyzed reactions, heat, oxida-
tive conditions and free radicals. These reaction conditions were
expected to lead to different kinds of dehydration reactions, epi-
merizations, rearrangements and isomerization of double bonds.
Of the seven transformation products isolated and identified in this
work, three were characterized for the first time (2, 3 and 5). The
degradation pathways are shown in Fig. 1.
Treatment of tacrolimus (1) with p-toluenesulfonic acid (Tos) in
toluene under reflux for 1 h yielded a mixture of two compounds
less polar than tacrolimus in a ratio of 1:2 (reversed phase HPLC,
relative retention times of 1.57 and 1.53, respectively), resolved
and purified by preparative HPLC. Both products had identical
masses (m/z 808, [M + Na]+, or 784, [M ꢁ H]ꢁ) and similar 1H and
13C NMR spectra. The MS data indicated a dehydration of the
tacrolimus molecule in both cases. This was confirmed by 1H
NMR data (Table 1). Thus, appearance of two new olefinic signals
at low field proved formation of a new double bond next to the car-
bonyl group at C-7 in both products. In addition, treatment with
[2,1-c][1,4]oxaazacyclopentacosine-1,9,22,23(6H,25H)-tetrone (7)
25
Colorless oil; [
a
]
D
ꢁ4.2° (c 0.32, CHCl3); UV (MeCN) kmax
(e)
206 nm (56400); IR (film) mmax 3460, 2936, 1735, 1648, 1453,
1381, 1195, 1096, 1035, 754 cmꢁ1 1H and 13C NMR, see Table 3;
;
HRESIMS m/z 786.4783 ([M + H]+), C44H68NOþ requires 786.4787
11
(DM 0.5 ppm).
2.8.6. (E)-3-[(1R,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-2-methyl-
2-propenal (8)
25
Colorless solid; [
a]
D
ꢁ46.5° (c 0.31, CHCl3); UV (MeCN) kmax
(e) 230 nm (31200); IR (film) mmax 3396, 2933, 1686, 1451, 1246,
1088, 1053, 1034, 913, 772 cmꢁ1 1H and 13C NMR, see Table S1;
;
HRESIMS m/z 199.1329 ([M + H]+), C11H19Oþ requires 199.1329
3
(DM 0.0 ppm).
2.9. X-ray crystallographic analysis of 5-deoxy-
(2)
D
5,6-8-epitacrolimus
Single crystals suitable for X-ray diffraction studies were grown
from a solution in MeCN–H2O 7:3, C44H67NO11, Mr. 786.0, ortho-
rhombic, space group P212121 (No. 19), a = 9.6590(13) Å,
b = 18.766(3) Å, c = 24.5685(14) Å, V = 4453.3(9) Å3, Z = 4, Dc =
1.172 Mg/m3, F(000) = 1704,
0.38 ꢀ 0.15 ꢀ 0.15 mm.
l(Mo Ka
) = 0.083 mmꢁ1, crystal size
acid was expected to cause epimerization at the a-position to the
A single crystal was mounted and immersed in a stream of
carbonyl functionality via reversible acid-catalyzed enolization.
The close similarity of the NMR spectra of the two isomers strongly
suggested that they differed in the configuration of C-8 but the rel-
ative configurations could not be determined from the NMR spec-
tra alone. However, it was possible to crystallize the minor isomer,
which had the longest HPLC retention time, and from single-crystal
X-ray diffractometry the structure was established to be the novel
nitrogen gas [T = 122(1) K]. Data were collected, using graphite-
monochromated Mo K
a radiation (k = 0.71073 Å) on a KappaCCD
diffractometer. Data collection and cell refinement were performed
using COLLECT (COLLECT, 1999) and DIRAX (Duisenberg, 1992).
Data reduction was performed using EvalCCD (Duisenberg, 1998).
Correction for absorption was performed using Gaussian integra-
tion (Coppens, 1970) as included in maXus (Mackay et al., 1999).
Positions of all non-hydrogen atoms were found by direct meth-
ods (SHELXS) (Sheldrick, 2008). Full-matrix least-squares refine-
ments (SHELXL) (Sheldrick, 2008) were performed on F2,
a,b-unsaturated ketone 2 with inverted configuration at C-8
(Fig. 2). Hence, the major product was the novel ,b-unsaturated
a
ketone 3. The enone 2 was likewise formed as the only product
in the reaction of 8-epitacrolimus (4) with Tos. The epimer 4 is
available by treatment of 1 with 1,5-diazabicyclo[4.3.0]nonene
(DBN) (Skytte et al., 2010).
2
2
minimizing
R
wðF2o ꢁ kFc Þ , with anisotropic displacement parame-
ters of the non-hydrogen atoms. The position of hydrogen atoms

518
D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
Table 2
1H NMR (600 MHz) and 13C NMR (150 MHz) spectroscopic dataa,b for carboxylic acid 5 in benzene-d6.
No.
1H NMR data
13C NMR data
Rotamer/tautomer 1
Rotamer/tautomer 2
Rotamer/tautomer 3
Rotamer/
Rotamer/
Rotamer/
tautomer 1
tautomer 2
tautomer 3
1
160.6 or 164.6 or 165.2 or 165.4 or 166.6 or 167.0 or 168.0 or
168.4 (all isomers)
3
4
5
6
7
3.69–3.72 (m)
3.69–3.72 (m)
3.65 (d, 7.2)
81.1
41.4
149.6
128.4 or 128.5
81.1
41.7
149.7
128.4 or 128.5
81.0
41.0
149.2
130.3
2.40–2.46 (m) (all isomers)
6.89 (dd, 8.4, 15.6)
6.15 (dd, 15.6, 1.8)
6.92 (dd, 7.2, 15.6)
6.08 (br d, 15.6)
6.93 (dd, 6.6, 15.6)
6.39 (br d, 15.6)
195.1 or 197.8 or 198.5 or 199.2 or 199.4 or 199.5 or 201.3 or
201.6 or 205.7 (all isomers)
8
9
3.45–3.57 (m) (all isomers)
5.17 (d, 9.6) or 5.15 (d, 9.6)
51.1 or 51.4
125.3 or 125.4
138.2
51.1 or 51.4
125.3 or 125.4
138.0
51.0
124.9
138.1
5.17 (d, 9.6) or 5.15 (d, 9.6)
5.01–5.02 (m)
1.68–1.71 (m)
10
11
12
13
14
15
16
17
18
2.20–2.22 (m) or 1.98–2.02 (m) or 1.91–1.93 (m) (all isomers)
1.75–1.78 (m) 1.71–1.74 (m)
1.93–1.97 (m) or 1.64–1.74 (m) or 1.52–1.55 (m) (all isomers)
3.80–3.84 (m) (all isomers)
4.03 (dd, 1.8, 9.6)
3.45–3.57 (m)
1.91–1.93 (m) or 1.62–1.65 (m) (all isomers)
49.4 or 50.0 (all isomers)
28.2
37.7
77.2
28.2
37.6
77.0
28.2
38.0
77.5
3.97 (d, 9.6)
3.45–3.57 (m)
3.92 (dd, 3.0, 10.2)
3.40–3.43 (m)
74.1 or 74.2 or 74.3 or 74.5 or 74.8 (all isomers)
74.1 or 74.2 or 74.3 or 74.5 or 74.8 (all isomers)
33.3
34.7
32.9
35.5
33.7 or 34.5
32.6 or 35.4
2.34–2.36 (m)
2.54–2.58 (m)
2.54–2.58 (m) or 2.24–2.26
(m)
19
20
98.9 or 99.3 or 99.6 or 100.8 (all isomers)
195.1 or 197.8 or 198.5 or 199.2 or 199.4 or 199.5 or 201.3 or
201.6 or 205.7 (all isomers)
21
23
160.6 or 164.6 or 165.2 or 165.4 or 166.6 or 167.0 or 168.0 or
168.4 (all isomers)
4.63 (br d, 13.2), 2.37–2.39
4.49 (br d, 11.4), 2.37–2.39
4.69 (br d, 14.4), 2.37–2.39
39.8
43.1
39.9
(m)
(m)
(m)
24
25
26
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
21.5 or 21.7
21.5 or 21.7
28.2
24.2
21.5 or 21.7
27.1
n.a.
21.0
28.7
2.20–2.22 (m), 2.06–2.08
(m)
2.18–2.20 (m), 1.56–1.58
(m)
2.36–2.38 (m), 1.84–1.86
(m)
26a
4.87 (m)
5.49 (br d, 6.0)
5.21 (br d, 4.8)
57.0
135.8
132.3 or 132.1
35.6
35.6
52.2
135.8
132.3 or 132.1
35.6
35.6
n.a.
10
135.8
132.2
35.6
35.6
85.0
20
5.09–5.12 (m) or 5.04–5.06 (m) (all isomers)
2.14–2.20 (m) (all isomers)
1.93–1.97 (m) (all isomers), 0.95–1.03 (m) (all isomers)
2.93–3.02 (m) (all isomers)
3.45–3.57 (m) (all isomers)
1.61–1.65 (m) (all isomers), 1.40–1.43 (m) (all isomers)
30
40
50
84.9
84.6
60
74.1 or 74.2 or 74.3 or 74.5 or 74.8 (all isomers)
n.a.
n.a.
70
n.a.
n.a.
n.a.
n.a.
80
n.a.
n.a.
n.a.
a
b
c
2.61–2.67 (m) (all isomers), 2.32–2.42 (m) (all isomers)
5.78–5.85 (m) (all isomers)
5.09–5.12 (m) or 5.02–5.04 (m) (all isomers)
36.8
36.8
37.0
137.2
116.9
16.3
136.8
116.9
16.1 or 16.2
136.8
116.9
16.1 or 16.2
CH3-4
CH3-10
CH3-12
CH3-18
CH3-10
CH3O-
14
CH3O-
16
CH3O-50
1.12 (d, 6.6)
1.67 (s)
1.11 (d, 6.6)
1.60 (s)
1.06 (d, 7.2)
1.71 (s)
0.84 (d, 6.0)
1.35 (d, 7.2) or 1.33 (d, 7.2)
1.50 (s)
16.9 or 17.0 or 17.1 (all isomers)
0.89 (d, 6.0)
1.24 (d, 6.6)
1.52 (s)
0.92 (d, 5.4)
1.20 (d, 6.0)
1.55 (s)
19.6
16.8
12.3
58.1
19.9
16.7
12.1
58.1
18.9
17.3
12.5
57.6
3.35 (s) or 3.33 (s) or 3.22 (s) (all isomers)
3.17 (s) or 3.13 (s) or 3.12 (s) (all isomers)
3.25 (s) or 3.21 (s) or 3.10 (s) (all isomers)
56.0 or 56.1 (all isomers)
55.9 or 56.6 or 57.6 (all isomers)
a
Chemical shifts (d values) with proton multiplicities and coupling constants (Hz) in parentheses, n.a. = not assigned.
To facilitate comparison between spectra, the atom numbering system of tacrolimus (1) was preserved in this compound.
b
Amide bond rotamers of tacrolimus and analogs can be distin-
We found that treatment of 1 with DBN in dichloromethane at
ambient temperature overnight gave not only the previously re-
ported equilibrium mixture of 1 and 4 (Skytte et al., 2010), but also
another product with a considerable shorter retention time on re-
versed-phase HPLC (relative retention time 0.41). Isolation of this
product by repeated preparative HPLC yielded a colorless com-
pound with m/z 826 ([M + Na]+, or 802, [M ꢁ H]ꢁ) and 1H and 13C
NMR data resembling those of the enone 3. From 1D and 2D
NMR data (1H, 13C, DEPT135, COSY, HMBC, HSQC) the structure
was determined as the open-chain form of 3, i.e., the novel carbox-
ylic acid 5. This is based, among others, on the absence of HMBC
correlations between C-1 and H-3, observed for tacrolimus itself
guished by means of differences in 1H NMR and 13C NMR chemical
shifts, primarily in the positions adjacent to the nitrogen atom in
the pipecolic acid moiety (Askin et al., 1990; Karuso et al., 1990;
Schueler et al., 1993). Differences in shielding (Askin et al., 1990)
and torsion angle (Karuso et al., 1990; Schueler et al., 1993) cause
higher chemical shift values of C-26a in the cis rotamers (amide
carbonyl oxygen atom syn to C-1) relative to the trans rotamers
(amide carbonyl oxygen atom anti to C-1), the opposite being true
for the chemical shifts of C-23. For the enone 2 the ratio between
cis and trans rotamers was about 2:1 in benzene-d6, while for the
enone 3 in the same solvent the ratio 6:5 was observed.

Table 3
1H NMR (600 MHz) and 13C NMR (150 MHz) spectroscopic dataa,b for rearrangement products 6 and 7 in benzene-d6.
Rearrangement product 6
Rearrangement product 7
1H NMR data
No.
1H NMR data
13C NMR data
13C NMR data
Major rotamer (trans)
Minor rotamer (cis)
Major rotamer (trans)
Minor rotamer (cis)
trans-Rotamer
trans-Rotamer
1
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
23
24
25
26
169.5
127.9
38.5
71.6
44.9
211.0
53.4
124.3
139.5
50.1
26.5
35.5
76.3
73.0
74.1
33.3
35.3
99.1
198.4
166.3
44.8
25.4
21.3
26.6
52.8
137.5 or 136.6
82.2 or 82.0
40.0
31.8
85.0
74.4
31.8
27.8
35.5
136.6
116.9
17.9
171.4
129.5
38.7
72.4
47.9
212.6
51.9
124.6
137.5 or 136.6
50.6
26.4
34.5
76.3
73.3
74.1
32.7
35.3
99.0
195.2
165.8
39.5
24.6
21.2
26.9
57.2
134.5
82.2 or 82.0
38.9
29.8
85.1
74.5
32.0
27.6
37.5
136.2
117.2
17.8
169.8
129.4
35.0
150.1
124.5
198.6
52.1
125.1
139.3
50.4
26.8
34.4
76.7
73.2
74.3
33.1
35.7
99.1
196.3
165.8
45.7
25.4
22.2
26.3
52.6
137.2
82.6
40.1
32.4
85.2
74.3
31.8
26.5
37.5
136.3
117.0
18.1
15.3
20.1
17.0
18.6
58.9
55.8
56.4
5.38 (dd, 10.2, 1.2)
2.65–2.68 (m)
4.15 (dt, 6.6, 2.4)
4.98–5.01 (m)
2.55–2.58 (m)
3.73–3.76 (m)
3.01–3.04 (m), 2.21–2.23 (m)
5.38 (dd, 10.2, 1.8)
3.00–3.03 (m)
6.94 (dd, 15.6, 4.8)
6.16 (dd, 15.6, 1.8)
2.59–2.60 (m), 2.46–2.51 (m)
3.15–3.17 (m)
4.83 (br d, 10.2)
3.56 (ddd, 9.6, 7.8, 6.0)
5.33 (d, 9.6)
3.32 (dt, 7.8, 7.8)
5.04 (d, 9.0)
1.94–1.96 (m), 1.76–1.77 (m)
1.25–1.27 (m)
1.57–1.59 (m), 1.09–1.12 (m)
3.80 (ddd, 12.0, 5.4, 2.4)
4.13 (dd, 9.6, 2.4)
3.66–3.70 (m)
2.09 (ddd, 12.6, 4.2, 4.2), 1.71–1.75 (m)
2.55–2.58 (m)
2.01–2.05 (m), 1.81–1.86 (m)
1.41–1.45 (m)
1.60–1.62 (m), 1.20–1.21 (m)
3.73–3.76 (m)
3.94 (dd, 9.6, 1.8)
3.41–3.46 (m)
2.00–2.06 (m), 1.66–1.68 (m)
1.72–1.76 (m)
1.36–1.39 (m), 1.01 (dt, 12.6, 3.6)
3.79 (ddd, 12.0, 5.4, 1.8)
4.11 (dd, 9.0, 1.8)
3.66 (ddd, 10.8, 9.6, 4.2)
2.13 (dt, 12.6, 4.2), 1.72–1.76 (m)
2.67 (ddt, 4.2, 6.6, 6.6)
1.81–1.86 (m), 1.57–1.59 (m)
2.55–2.58 (m)
3.66–3.70 (m), 3.34–3.36 (m)
1.26–1.29 (m), 1.26–1.29 (m)
1.41–1.45 (m), 1.26–1.29 (m)
2.18–2.20 (m), 1.26–1.29 (m)
5.23 (br d, 3.6)
4.65 (dd, 13.8, 3.0), 3.29–3.30 (m)
1.26–1.29 (m), 1.24–1.25 (m)
1.41–1.45 (m), 1.26–1.29 (m)
2.18–2.20 (m), 1.68–1.71 (m)
4.62 (br d, 6.6)
3.48 (br d, 13.8), 2.98 (dt, 13.8, 2.4)
1.72–1.76 (m), 1.33 (dddd, 16.2, 12.6, 12.6, 3.6)
1.46–1.48 (m), 1.41–1.43 (m)
2.24 (br d, 13.8), 1.16–1.18 (m)
5.23 (d, 6.0)
26a
10
20
4.72 (d, 8.4)
1.87–1.90 (m)
2.44 (br d, 11.4), 0.79–0.83 (m)
3.01–3.04 (m)
3.41–3.46 (m)
2.01–2.05 (m), 1.37–1.39 (m)
1.41–1.45 (m), 0.79–0.83 (m)
2.62–2.64 (m), 2.25–2.28 (m)
5.74 (ddt, 16.8, 9.6, 7.2)
5.03 (dq, 16.8, 1.8), 4.98–5.01 (m)
0.88 (d, 7.2)
1.53 (d, 0.6)
0.78 (d, 6.6)
1.19 (d, 6.6)
1.64 (d, 1.2)
3.19 (s)
3.31 (s)
3.32 (s)
4.77 (d, 4.8)
1.46–1.48 (m)
4.48 (d, 9.6)
30
1.97 (ddd, 9.0, 3.0, 3.0)
2.59 (br d, 12.6), 0.74 (q, 12.0)
3.07–3.11 (m)
3.46 (ddd, 11.4, 8.4, 4.2)
2.00–2.06 (m), 1.36–1.39 (m)
1.36–1.39 (m), 0.69 (ddt, 13.2, 13.2, 3.6)
2.50 (ddd, 14.4, 7.2, 7.2), 2.28 (ddd, 14.4, 7.2, 7.2)
5.76 (ddt, 16.8, 10.2, 7.2)
5.05 (dd, 16.8, 1.8), 5.00 (ddd, 10.2, 1.2, 1.2)
0.92 (d, 6.6)
1.41 (s)
0.81 (d, 6.0)
1.16 (d, 6.6)
1.67 (d, 1.8)
3.17 (s)
3.34 (s)
3.38 (s)
40
2.12–2.15 (m), 0.91–0.97 (m)
2.75 (ddd, 11.4, 9.0, 4.2)
3.39 (ddd, 11.4, 8.4, 4.8)
1.94–1.96 (m), 1.25–1.27 (m)
1.32–1.35 (m), 1.03–1.05 (m)
2.46–2.51 (m), 2.21–2.23 (m)
5.76 (ddt, 16.2, 9.6, 7.2)
5.03 (dq, 16.8, 1.8), 4.98–5.01 (m)
1.26 (s)
1.63 (d, 0.6)
0.76 (d, 6.6)
1.05 (d, 6.6)
1.57 (d, 1.2)
50
60
70
80
a
b
c
CH3-4
CH3-10
CH3-12
CH3-18
CH3-10
CH3O-14
CH3O-16
CH3O-50
15.6
19.6
16.9
18.7
58.4
56.4 or 56.1
56.4 or 56.1
15.8
19.8
16.5
16.8
57.8
56.1
56.7
3.20 (s)
3.07 (s)
3.18 (s)
a
Chemical shifts (d values) with proton multiplicities and coupling constants (Hz) in parentheses, n.a. = not assigned.
To facilitate comparison between spectra, the atom numbering system of tacrolimus (1) was preserved in these compounds.
b

520
D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
Fig. 1. Overview of the degradation pathways of tacrolimus.
and the macrocyclic compounds mentioned above. However, the
stereochemistry of 5 could not be confirmed from NMR data, but
we propose the structure 5 as the most plausible as NMR data
resembled those of compound 3 more than those of compound 2.
The isolated compound was a mixture of at least four species, pre-
sumably amide bond rotamers as well as tautomers not separable
by HPLC, in a ratio of 1:10:20:24, which complicated interpretation
of the NMR spectra. Nevertheless, extensive use of 2D NMR corre-
lations (COSY, NOESY, HSQC, HMBC) enabled assignment of most of
the signals of three species present in the solution of 5 in benzene-
d6 (Table 2).
tacrolimus the assumed [3,3]-sigmatropic shift was believed to oc-
cur under the relatively mild conditions due to favorable conforma-
tional preorientation of the molecule for this reaction (Ok et al.,
1990). Reflux in toluene (b.p. 111 °C) was not sufficient to induce
the rearrangement reaction. Heating of 1 in refluxing xylene re-
sulted in fact in two main products (Ok et al., 1990), one slightly
more polar and one less polar than tacrolimus (reversed-phase
HPLC, relative retention times 0.83 and 1.55, respectively), along
with a number of minor impurities that were not further analyzed.
The compound with the shortest retention time was identified as
the previously reported rearrangement product 6 based on MS
(pseudomolecular ion isobaric with that of 1, m/z 826, [M + Na]+,
or 802, [M ꢁ H]ꢁ) and NMR data (Table 3). The ratio between cis
and trans amide bond rotamers of 6 was 3:5. In the reaction product
with the relative retention time of 1.55, water had been lost (m/z
A thermal rearrangement of the allylic ester part of tacrolimus to
give 6 was previously reported to take place upon reflux in o-xylene
(b.p. 144 °C) for 25 h. Uncatalyzed thermal rearrangements of
allylic esters usually requires very high temperatures, but for

D.M. Skytte et al. / European Journal of Pharmaceutical Sciences 48 (2013) 514–522
521
lactone group hydrolysis to give the free carboxylic acid 5, whereas
strong bases cause extensive degradation of the molecule. All prod-
ucts were characterized by 1H and 13C NMR spectroscopy, includ-
ing assignment of rotamers of the amide group observed in
solution. Also, 1H and 13C NMR spectra of the previously reported
ring expansion products 6 and 7 were assigned by extensive use
of 2D NMR techniques. These reactions and products are of interest
in connection with production and handling of pharmaceuticals
containing tacrolimus.
Acknowledgments
We thank Drug Research Academy, Faculty of Pharmaceutical
Sciences, University of Copenhagen, for financial support to
D.M.S. and K.T.J. Technical assistance of Niels Vissing Holst, Depart-
ment of Chemistry, University of Copenhagen, with collecting X-
ray data is gratefully acknowledged.
Appendix A. Supplementary material
Fig. 2. ORTEP drawing of the crystal structure of compound 2. Displacement
ellipsoids are drawn at 50% probability level. Hydrogen atoms are drawn as spheres
of arbitrary size. Oxygen atoms are colored red and nitrogen atoms are colored blue
(atoms will appear gray in print). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejps.2012.12.001.
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