fluoro-methylphenylcarbamates of cellulose as f … · 2018-12-18 · fluoro-methylphenylcarbamates...
TRANSCRIPT
Fluoro-methylphenylcarbamates of Cellulose as
Chiral Stationary Phases for Supercritical Fluid
Chromatography
William Farrell1 and Matthew Przybyciel2
1Pfizer Global R&D - La Jolla, CA 2ES Industries - West Berlin, NJ
ABSTRACT
The use of aromatic halogenated substituents within carbohydrate
chiral stationary phases is not a new concept, with early work
dating back to the mid to late 1990’s1,2. Chloro-, bromo-, and
fluoro-methylphenylcarbamates all showed chiral recognition, and
there are several chloro-phenyl substituted versions available
commercially. However, with the resurgence of fluorinated
chemistry being used for drug development, the need for phases
suitable for chiral recognition of fluorinated compounds is
facilitated by re-introduction of these types of halogenated
phases. Introduction of a fluorophilic retention mechanism can be
particularly useful in support of medicinal chemistry drug
discovery efforts, where more than a third of newly approved
small molecule drugs contain fluorine3,4.
Two new fluorinated phases, 4-fluoro-3-methylphenylcarbamate
and 2-fluoro-5-methylphenylcarbamate, were prepared on
cellulose and evaluated for use with drug-like compounds
containing fluorine constituents. Separations of a wide variety of
these compounds will be presented as well as comparative
separations using other halogenated stationary phases.
Introduction
Traditional Phases
Chiral SFC is the primary tool for separation of enantiomers for
medicinal chemistry support. Among the chiral selectors are the
popular polysaccharides phases, which are substituted phenyl
moieties bonded to either amylose or cellulose.
Figure 6: Analytical chromatograms of the separation of
MFCD26517069 using HPLC (top) and SFC (bottom)
conditions.
A non-ideal separation was achieved using HPLC conditions
and a traditional phase (top), but was extremely sensitive to
temperature and thus difficult to scale for purification.
Using a fluorinated phase, complete separation was
achieved using SFC conditions. Significant savings in solvent
and time were achieved upon scale-up to purification. Only a
small percentage of methanol was added post-column to
facilitate collection.
Figure 8: Effect of dipole of moment of stationary phase on
the separation of MFCD265170695,6
Selectivity (α) and resolution (R) values for each separation
are included. Dipole moment values provided reflect the
magnitude differences in an ideal system and were not
measured for each specific phase. a) Cellulose tris-(4-chloro-
3-methylphenylcarbamate) phase (CC4). b) Cellulose tris-(4-
fluoro-3-(trifluoromethyl)phenylcarbamate (CCO-F4-CF3)
phase. c). Cellulose tris-(4-fluoro-3-methylphenylcarbamate)
phase (CCO-F4).
Figure 1: Common phases used for chiral separations
Typical mobile phases used are limited to alcohols combined
with hydrocarbons (e.g. hexanes or heptane in HPLC) or carbon
dioxide (SFC).
Additional phases commonly used include Pirkle-type (such as
the Whelk-O1) and peptide macrocyclic phases. These have a
broader solvent compatibility range, but are usually applied in
reverse phase mode (aqueous/organic) or in polar organic mode
(e.g. 20mM ammonium acetate in methanol), as well as in SFC.
REFERENCES
1. Enantioseparation on fluoro-methylphenylcarbamates of cellulose and amylose as chiral
stationary phases for high performance liquid chromatography, Yashima et.al, Polymer
Journal, Vol 27, No. 8, pp 856-861 (1995).
2. 3-Fluoro-, 3-chloro- and 3-bromo-5-methylphenyl carbamates of cellulose and amylose as
chiral stationary phases for high performance liquid chromatographic enantioseparation, B.
Chankvetadze, et al, J. Chrom. A, 787, (1997) 67-77.
3. Fluorous Reverse Phase Silia Ge: A New Tool for Preparative Separations in Synthetic Organic
and Organofluorine Chemistry, Curran, D. P., Synlett 2001, (9), 1488-1496.
http://dx.doi.org/10.1055/s-2001-16800
4. A Bountiful Year, Jarvis, L. M., Chemical & Engineering News 2013, 91, (5), 15-17
http://dx.doi.org/10.1021/cen-09105-bus1
5. Each column is 4.6mm I.D. x 250mm length containing 5-µm particles maintained at 25°C. The
mobile phase consists of CO2 and percentage co-solvent listed in each chromatogram
delivered at a flow rate of 3.0mL/min with 160 bar outlet pressure.
6. Alkyl Halides. Retrieved from http://crab.rutgers.edu/~alroche/Ch06.pdf
CONCLUSION
The use of fluorine for chiral stationary phases has demonstrated
utility and appears comparable to commercial chlorinated phases.
The magnitude of the selectivity appears to derive from changes in
the dipole of the benzylic functional groups.
Nonetheless, the fluorinated versions offer several advantages in
potential savings in solvent consumption and reduced retention
time.
The introduction of the CF3 functionality opens a door for further
exploration of commercial phases by substituting the methyl group.
Liquid CO2 can be used to enhance selectivity.
Figure 9: The overall trend in selectivity (α) and resolution (R)
for pharmaceutical compounds supports effectiveness of dipole
moment manipulation on the stationary phase5
As shown in Figures 8 & 9, the addition of electron-withdrawing
functional groups plays a role in the separation mechanism, with
the greatest overall effect achieved with chlorine.
Cellulose tris-(4-chloro-3-methylphenylcarbamate) = 4Cl-3M
Cellulose tris-(4-fluoro-3-(trifluoromethyl)phenylcarbamate = 4F-CF3
Cellulose tris-(4-fluoro-3-methylphenylcarbamate) = 4F-3M
Figure 10: Example chromatograms of metoprolol
enantiomers on each of the commercial fluoro-methyl and
chloro-methyl phases5
Further demonstrating the effect of the phase dipole, selectivity
of metoprolol enantiomers can be enhanced by substituting the
halogen (CCO-F4 to CC4), the methyl group (CCO-F4 to CCO-
F4-CF3), or the relative position of both the halogen and methyl
groups (CCO-F4 to CCO-F2).
18 minutes2 4 6 8 10 12 14 16
mAU
0
10
20
30
40
50
60
70
4.5
01
7.377
14.74015.796
4.5 minutes0.5 1 1.5 2 2.5 3 3.5 4
mAU
0
50
100
150
200
250
300
350
3.099
3.355
Column: Chiralcell OJ-H
Column: 4.6x250mm, 5µm
Mobile Phase: 1% IPA in Heptane
Flow rate: 1.0mL/min
Temp: 0°C
Inj vol: 3µL
Column: CCO-F4
Column: 4.6x250mm, 5µm
Mobile Phase: 100% CO2
Flow rate: 3.0mL/min
Pressure: 200bar
Temp: 10°C
Inj vol: 3µL
O
OR
O*
RORO
*n
R =
O
Commercial OJ-H Cellulose tris(methyl benzoate)
Silica gel
O
OR
O*
RORO
*n
R =
O
N F
Silica gel
Custom Cellulose tris(4-fluoro-3-
methylphenylcarbamate)
O
O
F F
X = C-Cl µ= 1.56D
X = C-F µ= 1.51D
α = 1.19R = 3.97
O
O
F F
5.185
6.182
4.129
4.649
min1 2 3 4 5 6 7
3.0763.320
α = 1.12R = 2.89
α = 1.08R = 2.53
CC44Cl-3M
CCO-F4-CF34F-3CF3
CCO-F44F-3M
MFCD26517069
a)
b)
c)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
R
4F-3M 4F-3CF3 4Cl-3M
1.05
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
alp
ha
Overall trend for selectivity and resolution
4F-3M < 4F-CF3 < 4Cl-3M
Selectivity
Resolution
NH
ONH
OH
CH3
CH3
Pindolol
NH
ONH
OH
CH3
CH3
Propranolol
O O
OH O
CH3
Warfarin
O NH
CH2
OH
CH3
CH3
Alprenolol
NHO
OH
OCH3 CH3
CH3
Metoprolol
N
N
O Cl
Cl
Cl
Cl
N
O
O
O
O
CH3
CH3
CH3
CH3
CH3
CH3
N
CH3
Miconazole
Verapamil
NH2
OONHCH3
CH3
OH
Atenolol
5.387
8.167
4.223
5.757
min2 4 6 8 10 12 14
3.282
4.230
3.948
4.332
CCO-F4-CF3
CCO-F4
CCO-F2
ES CC4
O
OR
O*
RORO
*n
R =
O
N F
Silica gel
Cellulose tris(4-fluoro-3-methylphenylcarbamate)
R =
O
N
F
Cellulose tris(2-fluoro-5-methylphenylcarbamate)
Warfarin20% MeOH
min0.5 1 1.5 2 2.5 3 3.5
1.887
2.395
min0 0.5 1 1.5 2 2.5 3
2.260
2.523PF Compound20% MeOH
Miconazole25% 20mM AMF in MeOH
min0.5 1 1.5 2 2.5
1.799
1.955
min0.5 1 1.5 2 2.5
1.976
2.155Verapamil25% 20mM AMF in MeOH
O O
OH O
CH3
N
O
O
O
O
CH3
CH3
CH3
CH3
CH3
CH3
N
CH3
N
N
O Cl
Cl
Cl
Cl
Miconazole25% 20mM AMF in MeOH
Verapamil25% 20mM AMF in MeOH
N
O
O
O
O
CH3
CH3
CH3
CH3
CH3
CH3
N
CH3
N
N
O Cl
Cl
Cl
Cl
O
OR
O*
RORO*
n
R =
F
CF3
O
Silica gel
Cellulose tris(4-fluoro-3-trifluoromethylphenylbenzoate)
R =
F
F
O
Cellulose tris(3,4-Difluoro-methylphenylbenzoate)
1.366 1.409
1.303
min0.5 1 1.5 2 2.5
1.364
Inj # 1
Inj # 3
Inj # 5
trans stilbene
oxide
O
min0 0.5 1 1.5 2 2.5 3 3.5
2.557
2.808
min0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
3.029
3.499
Figure 3: Example separations using 1st Generation
fluorinated phases5
Expecting to capitalize on fluorophillic interactions, two
prototype phases were made which demonstrate versatility
against a broad selection of compounds.
Figure 7: Example separations on the 2nd Generation
prototype fluorinated phases5
Following on the success of the CCO-F4 phase, two
additional phases were designed to probe different dipole
interactions, of which only one demonstrated versatility
against a broad selection of compounds. The difluorinated
column showed significant bleed and loss of selectivity even
with low percentages of solvent (<5%).
Chiralpak+ AD
Coated -
Amylose
Chiralpak AS
Coated -
Amylose
Chiralpak IC
Immobilized -
Cellulose
Chiralcel OJ
Coated -
Cellulose
Chiralcel OD
Coated -
Cellulose
Lux++ Cellulose-2
Coated -
Cellulose
Lux Cellulose-4
Coated -
Cellulose
Lux Amylose-2
Coated -
Amylose
Whelk-O1Bonded
Pirkle type
ES IndustriesCC4*
Coated -
Cellulose
N
O
R
CH 3
CH 3
CH3
N
O
R
N
O
R
Cl
Cl
O
R
N
O
R
CH 3
CH 3
N
O
R
Cl
CH3
N
O
R
CH3
Cl
N
O
R
Cl
C H3
N
O
R
CH 3
Cl
Si
CH 3CH 3
N
ONO 2
NO 2
O
Column Column type Chiral SelectorColumn Column type Chiral Selector
Figure 2: Chiral phases used in purification support of
medicinal chemistry
As shown in Figure 2, an increasing number of chiral
compounds achieve separation on any of these traditional
phases, especially those compounds with low molecular weight
(i.e. MW < 200 Da) or have limited complexity/functionality near
the chiral center. Gas chromatography is probably best suited for
these compounds; however, it is inconsistent with the goals of
isolating pure enantiomers at any workable scale for medicinal
chemistry support. Ideally, successful separations of the
enantiomers would have a selectivity of >1.1 and a minimum
resolution >1.5.
Figure 4: Example chromatograms of MFCD26517069 using
standard SFC conditions with 1 % acetonitrile as modifier.
Inset: Yellow region represents normal operating conditions
in SFC. Blue region represents rarely used region in SFC.
Using MFCD26517069 as a probe compound, no separation
of enantiomers was initially obtained using any traditional
phases in SFC mode. In addition, the prototype fluorinated
phases also appeared to be ineffective.
Since the compound is very non-polar, mobile phases of
hydrocarbons (HPLC) and carbon dioxide (SFC) were also
explored.
Figure 5: Chromatograms of MFCD26517069 at various
pressure and temperature settings
Borrowing from some the partial success using HPLC (see
Figure 6), modification of the density of the mobile phase in
SFC mode did result in enantiomeric separation. Figure 5
shows the effects of temperature and pressure on the
separation as the conditions move from the yellow to blue
regions indicated in the Figure 4 inset.
25°C
Inlet: 315bar
Outlet: 150bar
Inlet: 370bar
Outlet: 200bar
Inlet: 420bar
Outlet: 250bar
Inlet: 462bar
Outlet: 300bar
Inlet: 505bar
Outlet: 350bar
min0.5 1 1.5 2 2.5 3 3.5
10°C
Inlet: 318bar
Outlet: 150bar
Inlet: 370bar
Outlet: 200bar
Inlet: 420bar
Outlet: 250bar
Inlet: 464bar
Outlet: 300bar
Inlet: 510bar
Outlet: 350bar
Inlet: 550bar
Outlet: 400bar
min0.5 1 1.5 2 2.5 3 3.5
283 298
480
150
323
350
Bar
Kelvin
Celsius
Lines Reflect Density
Region A = Liquid
Yellow Region = “Sweet Spot”
Region C = Low Density
25 5010
PSI
7000
5000
2000
Region B = High Density
Blue Region = Interesting…
min1 2 3 4
mAU
0
20
40
60
80
100
120 1.732
min1 2 3 4
mAU
0
25
50
75
100
125
150
175
200 1.868
O
O
F F
Columns: 4.6x250mm, 5µm
Modifier: 1% MeCN
Flow rate: 3.0mL/min
Pressure: 160bar
Temp: 25°C
Inj vol: 3µL
CCO-F4
CC4
MFCD26517069
Prototype Phases – 1st Generation
Prototype Phases – 2nd Generation
Metoprolol25% 20mM Ammonia in IPA
CCO-F4 CCO-F2
CCO-F4-CF3 CCO-DiF
CCO-F4