MAGNETIC RESONANCE IN CHEMISTRY

Magn. Reson. Chem. 37, 393–400 (1999)

Application of LC–NMR to the identification of bulk drug

impurities in GART inhibitor AG2034

Barbara C. M. Potts,1 * Kim F. Albizati,1 Mark O’Neil Johnson2 and Joyce Prosser James2
1
Department of Chemical Development, Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121, USA
2
Bruker Instruments, 47697 Westinghouse Drive, Fremont, California 94539, USA

Received 5 November 1998; revised 4 January 1999; accepted 5 January 1999

ABSTRACT: Liquid chromatography (LC)–NMR spectroscopy was used to obtain detailed information regarding the
structure of a bulk drug impurity present in glycinamide ribonucleotide transformylase (GART) inhibitor AG2034. The
LC–NMR experiments (1D 1 H and 2D TOCSY) were performed in the stop-flow mode on crude retained impurity-
enriched samples of the synthetic precursor to AG2034. Comparative analysis of the data for the parent compound
with those for the impurity indicated that the impurity was nearly a dimer of the parent, and allowed all major
substructures to be delineated. The structural information derived from both LC–NMR and LC–MS data provided
insights into purification methods, which ultimately led to the full characterization of the impurity by high-resolution
NMR spectroscopy. Copyright  1999 John Wiley & Sons, Ltd.
KEYWORDS: NMR; 1 H NMR; 13
C NMR; LC–NMR; bulk drug impurity; pyrimidothiazine

INTRODUCTION and may be adopted as a general strategy for BDI identi-

In an ongoing effort to comply with regulatory guide-
lines for the identification of bulk drug impurities1 (BDIs),
we are currently exploring the utility of high-performance
liquid chromatography (HPLC) coupled with NMR spec-
troscopy (LC–NMR).2 – 4 The difficulties associated with
the identification of BDIs stem from the fact that these
compounds represent low-level components of a mixture.
Even when it is possible to isolate the impurity, sufficient
quantities must be generated to allow for positive iden-
tification. LC–NMR is well suited to meet the challenge
of BDI identification as it rapidly provides detailed struc-
tural information on small quantities of substances which
can be resolved by analytical HPLC, thereby circumvent-
ing purification while providing the requisite sensitivity.
We have recently applied this technique to the identifi-
cation of bulk drug impurities present in manufactured
lots of AG2034 (1), a potent glycinamide ribonucleotide
transformylase (GART) inhibitor currently under clinical
evaluation for antitumor activity.5
Our studies focused on the identification of 2, the major
unidentified impurity in 1. Our goal was to obtain as much
information about the bulk drug impurity profile as pos-
sible from LC–NMR and LC–MS methodologies. The
analyses were performed on retained samples of crude 3,
the synthetic precursor to 1, as these samples were both
enriched in impurities and readily available. The use of
impurity-enriched mother liquors or crude reaction prod-
ucts can overcome problems associated with sensitivity
fication. The major impurity present in crude 3 was 4, the
putative synthetic precursor to 2 (Scheme 1). Thus, the
identification of 2 proceeded through the structure eluci-
dation of 4. Although 3 and 4 were chromatographically
resolved by analytical HPLC, both normal- and reversed-
phased chromatography on a semi-preparative scale failed
to provide pure 4 for structure elucidation by traditional
NMR methods. This made crude 3 an excellent candi-
date for LC–NMR analysis. The analytical HPLC method
and relative quantities and HPLC retention times (Rt) for
the parent compound 3 [47% (UV area % at 280 nm),
Rt D 24.8 min] and 4 [12% (UV area % at 280 nm),
Rt D 29.5 min] were established prior to LC–NMR analy-
sis (Fig. 1); the only modification to the LC–NMR method
was substitution of deuterium oxide for water in the HPLC
method. Crude 3 was injected on to the HPLC column
and the mixture was analyzed by LC–NMR in the stop-
flow mode. As each compound eluted from the analytical
HPLC column, it was detected by its UV absorbance and
subsequently trapped in the LC–NMR flow cell for NMR
analysis. 1 H NMR spectra were acquired with double pre-
saturation of both HOD and acetonitrile resonances. In
order to obtain additional structural information on the
BDI, a 2D TOCSY6 was also acquired. These NMR spec-
tra, together with LC–MS data, provided unique structural
insights into the BDI in a timely manner.
RESULTS AND DISCUSSION

LC–NMR and LC–MS analysis

* Correspondence to: B. C. M. Potts, Department of Chemical Deve-
lopment, Agouron Pharmaceuticals, Inc., 3565 General Atomics Court,
San Diego, CA 92121, USA. The 1D 1 H LC–NMR spectrum obtained for 3 [Fig. 2(A)]
E-mail: bpotts@agouron.com was consistent with the 1 H NMR data for pure 3 (Table 1).

Copyright  1999 John Wiley & Sons, Ltd. CCC 0749–1581/99/060393–08 $17.50
394 B. C. M. POTTS ET AL.

Scheme 1

Figure 1. HPLC of crude 3 with UV absorbance monitored at 280 nm showing retention times for 3 and 4 of 24.8 and
29.5 min, respectively.

This spectrum established the chemical shifts of 3 in
the eluent solvent system (acetonitrile–0.1% TFA in
deuterium oxide), allowing a comparative analysis with
unknown compounds present in the mixture. Key reso-
nances present in the 1D 1 H LC–NMR spectrum and
their assignments (by carbon atom, numbered according
to Scheme 1) include (i) a pair of thiophene doublets
at υ 6.95, 7.57 (C-12, C-13); (ii) two ethyl quartets (υ
4.17, 4.06) and methyl triplets (υ 1.22, 1.17) arising from
the two ethyl esters (C-21, C-22, C-24, C-25); (iii) a
multiplet at υ 4.51 assigned to the C˛ proton of the ester-
protected glutamic acid residue (C-17); (iv) resonances
at υ 3.72 and 3.44 arising from the methylene protons
of the pyrimidothiazine (C-7); (v) a broad multiplet at
υ 3.06 encompassing the thiazine methine proton and the
methylene protons adjacent to the thiophene ring (C-6 and

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 393–400 (1999)
 LC–NMR OF BULK DRUG IMPURITIES IN GART INHIBITOR AG2034 395

Figure 2. 1D 1 H LC–NMR spectra of (A) 3 and (B) 4 acquired at 500 MHz on a 4 mm triple-resonance LC–NMR probe in
the stop-flow mode with double presaturation of the HOD and ACN resonances. These data provided the first indication
that 4 was nearly a dimer of 3.

Table 1. 1 H and 13 C NMR assignments for purified samples of 3 and 4 in DMSO-d6

3, Resonance assignments 4, Resonance assignments and correlations
13 1 13
υ C (ppm) at υ H (ppm) at υ C (ppm) at υ1 H (ppm) at
Atom 75 MHz, 500 MHz Atom 75 MHz, 500 MHz Long-range 1 H– 13 C 1
H– 1 H
No. mult. [mult., J (Hz)] No. mult. [mult., J (Hz)] correlationsa correlationsb
2 159.3 s 2 159.5 s
2a 6.04 (s) 2H 2a 6.19 (br s) 1.5H
[6.05, (br s) 2H]
3 10.08 (s) 3 10.51 (br s) 0.1H
[10.1, 0.6H]
4 152.2 s 4 152.3 s
5a 75.8 s 5a 75.8 s H-3, H-8
6 35.3 d 2.88 (m) 6 35.4 d 2.88c H-8, H-9, H-90 H-7, H-70 , H-9, H-90
7d 47.5 t 3.52 (br d, 12.5) 7d 47.6 t 3.51 (m) H-6, H-8, H-9, H-90 H-6, H-70 , H-8
3.22 (br m) 3.24e H-6, H-7, H-8
8 6.65 (s) 8 6.56 (br s) H-7, H-70
[6.65]
8a 155.8 s 8a 155.9 s
9 33.9 t 1.89 (m) 9 33.9 t 1.89 (m) H-6, H-90 , H-10
1.74 (m) 1.74f H-6, H-9
10 27.1 t 2.96 (m) 2H 10 27.2 t 2.95c H-12g H-9, H-90h
11 149.2 s H-9, H-90 , H-10, H-12,
H-13
12 125.6 d 6.89i H-10j , H-13 H-13
13 128.2 d 7.56 (s) H-12 H-12
14 137.2 s H-12k , H-13
15 161.2 s H-13, H-16, H-17, H-170
16 8.51 (br t) H-17, H-170
17 42.8 t 3.45 (m) H-16, H-18 H-16, H-170 , H-18
3.36e H-16, H-17, H-18
18 43.1 d 2.93c H-17, H-170 , H-19, H-17, H-170 , H-19,
H-20, H-200 , H-36 H-190

(continued overleaf )

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 393–400 (1999)
396 B. C. M. POTTS ET AL.

Table 1. (continued)
3, Resonance assignments 4, Resonance assignments and correlations
13 1 13
υ C (ppm) at υ H (ppm) at υ C (ppm) at υ1 H (ppm) at
Atom 75 MHz, 500 MHz Atom 75 MHz, 500 MHz Long-range 1 H– 13 C 1
H– 1 H
No. mult. [mult., J (Hz)] No. mult. [mult., J (Hz)] correlationsa correlationsb
19 33.4 t 1.94l H-17, H-170 H-18, H-190 , H-20
1.73f H-18h , H-19, H-20
20 26.9 t 3.02c H-22g H-19, H-190 , H-200
2.90c H-20
11 149.5 s 21 150.1 s H-19, H-190 , H-20,
H-200 , H-22, H-23
12 125.5 d 6.92 (d, 2.5) 22 125.5 d 6.88i H-20j , H-200j , H-23 H-23
13 128.9 d 7.69 (d, 3.0) 23 128.9 d 7.67 (d, 3.5) H-22 H-22
14 136.3 s 24 136.3 s H-22k , H-23
15 161.2 s 25 161.4 s H-23, H-26, H-27
16 8.62 (d, 7.5) 26 8.60 (d, 7.5) H-27
17 51.7 d 4.37 (br m) 27 51.8 d 4.36 (br m) H-26, H-28, H-280 , H-29 H-26, H-28, H-280
18 25.7 t 2.07 (m) 28 25.8 t 2.07 (m) H-26, H-27, H-29 H-27, H-28 0 , H-29
1.96 (m) 1.96l H-27, H-28, H-29
19 30.0 t 2.40 (dd, 7.0, 7.5) 2H 29 30.1 t 2.40 (dd, 7.0, 7.5) 2H H-27, H-28, H-280 H-28, H-280
20 172.0 s 30 172.2 s H-28, H-280 , H-29, H-31m
21 59.8 t 4.03 (q, 7.0) 2H 31 60.0 t 4.02n H-32o H-32p
22 13.9 q 1.16 (m) 3H 32q 14.1 q 1.16r H-31s
23 171.5 s 33 171.7 s H-27, H-34
24 60.5 t 4.10 (q, 7.0) 2H 34 60.6tt 4.08n H-35u H-35v
25 13.9 q 1.18 (m) 3H 35q 14.1 q 1.16r H-34w
36 32.5 t 3.26e
37 169.6 s H-36, H-38
38 8.46 (d, 7.0) H-39
39 51.5 d 4.25 (br m) H-38, H-40, H-400 , H-41 H-38, H-40, H-400
40 26.1 t 1.99l H-38, H-39, H-41 H-39, H-400 , H-41
1.83 (m) H-39, H-40, H-41
41 29.8 t 2.35 (dd, 6.5, 7.0) 2H H-39, H-40, H-400 H-40, H-400
42 172.0 s H-40, H-400 , H-41, H-43m
43 60.0 t 4.02n H-44o H-44p
44 14.0 q 1.16r H-43s
45 171.4 s H-39, H-46
46 60.7t t 4.08n H-47u H-47v
47q 14.1 q 1.16r H-46w

Atom numbers (columns 1, 4, 7 and 8) refer to Scheme 1. Chemical shifts are referenced to DMSO (υ 2.49, 1 H; υ 39.5, 13 C). A prime (0 ) denotes the
upfield-shifted proton of a pair of methylene protons. Brackets indicate exchangeable proton chemical shift observed in more concentrated solution.
1
H multiplicity reported as observed in 1D 1 H NMR. 13 C multiplicity derived from HMQC and DEPT-135 or APT spectra. 1 H resonances in columns
3 and 6 integrate to 1H unless indicated otherewise.
a
Correlation between proton and the 13 C resonance in column 5.
b
Correlation to proton resonance in column 6 observed in 2D 1 H– 1 H DQF-COSY.
c
All resonances denoted by this footnote collectively integrate to 6H.
d
HMQC signals are broadened suggesting conformational exchange.
e
Proximity to HOD signal precludes integration in the 1D spectrum.
f
All resonances denoted by this footnote collectively integrate to 2H.
g,h
Cross peak can be assigned to either one or both correlations.
i
Signal integrates to 2H.
j
Cross peaks not resolved in the 13 C dimension.
k
Cross peak can be assigned to either one or both correlations.
l
All resonances denoted by this footnote collectively integrate to 3H.
m
Cross peak can be assigned to either one or both correlations.
n
All resonances denoted by this footnote collectively integrate to 8H.
o,p
Cross peak can be assigned to either one or both correlations.
q
Degeneracy observed in both the 1 H and 13 C dimensions.
r
All resonances denoted by this footnote collectively integrate to 12H.
s
Cross peak can be assigned to either one or both correlations.
t
Assignments are interchangeable.
u,v,w
Cross peak can be assigned to either one or both correlations.

C-10); and (vi) a triplet at υ 2.47 arising from the C pro-
tons of the diethyl ester-protected glutamic acid residue
(Et2 GLU; C-19). Although the aliphatic protons of C-9
and C-18 were largely obscured by the acetonitrile sol-
vent signal and the exchangeable protons could not be
observed, all of the major functional groups of 3 could be
identified by the presence of signals arising from each of
the spin systems outlined above.
Once data acquisition on 3 was complete, LC–NMR
data were acquired on 4, which was identified by its
UV absorbance and HPLC retention time. The 1D 1 H
LC–NMR spectrum of 4 retained similar features to those
of 3, but a doubling of resonances was immediately
apparent [Fig. 2(B)]. In regions of the spectrum which
could be reliably integrated without concern regarding
attenuation from presaturation of solvent signals, the extra
resonances were determined to be present in a 1 : 1 ratio
with those signals common to 3, suggesting that the
spectrum represented a single compound. For example,
two sets of thiophene resonances were clearly present,

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 393–400 (1999)
 LC–NMR OF BULK DRUG IMPURITIES IN GART INHIBITOR AG2034 397

and integration of the methyl signals arising from the
ethyl esters was consistent with the presence of four
methyl groups instead of two. These data provided the
first indication that the BDI was nearly a dimer of 3.
Acquisition of a 2D 1 H– 1 H TOCSY experiment allowed
the resonances in the more crowded region of the spectrum
to be analyzed in greater detail (Fig. 3). The multiplet
at υ 3.48 was correlated with a broad doublet at υ 3.78,
and both of these protons showed a correlation with a
signal at υ 3.12. Although some of the aliphatic signals
were obscured by the solvent acetonitrile signal in the
1D 1 H LC–NMR spectrum, the 2D TOCSY made it
possible to identify these resonances. In this manner,
a strong cross peak was observed at ω2 D υ 3.12,
ω1 D υ 1.85. The chemical shifts of this spin system
(υ 3.78, 3.48, 3.12, 1.85 [assigned to protons of C-7 (2H),
C-6 and C-9, respectively] were compared with those of
the LC–NMR spectrum of 3 and found to be consistent
with the substituted pyrimodothiazine, suggesting that this
moiety was also present in 4. A similar spin system
was identified by a pair of coupled spins at υ 3.64,
3.50 (C-17), which were in turn correlated with a proton
at υ 2.98 (C-18), and each of these signals showed a
weak correlation with a proton at ω1 D υ 1.8 (C-19);
these four proton signals were clearly analogous to those
of the substituted thiazine. Four ethyl esters previously
identified by integration of the methyl signals (see above)
strongly suggested the presence of two Et2 GLU residues.
One Et2 GLU spin system was further identified in the
TOCSY spectrum via a correlation of the C˛H at υ 4.55
with the C H2 triplet at υ 2.49 (J D 7.9 Hz). The C H2
triplet of the second Et2 GLU residue was tentatively
assigned from analysis of the 1D spectrum, which showed
a signal of comparable chemical shift, multiplet structure
and intensity (υ 2.40, t, J D 7.9 Hz) to that of its
counterpart at υ 2.49. Although a second C˛H resonance
was not detected in the 1D spectrum, a C˛H–C H2
cross peak was present at ω2 D υ 4.32, ω1 D υ 2.40
in the TOCSY. Hence the inability to observe the C˛H
resonance in the 1D spectrum was clearly a result of
attenuation by presaturation of the HOD line at υ 4.36.
After careful consideration of the data for both the parent
3 and the BDI, it was concluded that for each non-
exchangeable proton present in 3, two such protons were
present in 4.
In order to gain further structural information, LC–MS
analysis was performed on the crude mixture containing
the BDI and the parent drug. The parent ion for the BDI
(4) at m/z 980 (MHC ) suggested that 4 was a dimer of 3
minus C2 N3 H3 , which is equivalent to loss of guanidine
and one additional carbon atom. This interpretation was
consistent with the LC–NMR-derived working hypothesis
that all of the non-exchangeable protons present in 3 were
doubled in 4. A fragment ion observed at m/z 777 was
assigned to loss of an Et2 GLU residue, and an ion at
m/z 642 was consistent with loss of a major fragment

Figure 3. Expanded view of the LC–NMR TOCSY spectrum of 4 highlighting the pyrimidothiazine correlations from H-6
to H-7 and H-70 , the analogous correlations from H-18 to H-17 and H-170 and the two C˛–C H2 correlations arising from
the two Et2 GLU residues. Specific assignment of the 27/29 and 39/41 pair was made by subsequent comparison with the
data for the purified compound (Table 1).

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 393–400 (1999)
398 B. C. M. POTTS ET AL.

Figure 4. Assignment of key LC–MS/MS fragment ions observed for BDI 4.

containing both the pyrimidothiazine and the substituted
thiophene moieties (Fig. 4). These data provided further
confirmation that the major substructural fragments of 3
were present in 4.
The combined LC–NMR and LC–MS data provided
the first structural insights regarding the BDI of inter-
est. The apparent dimerization minus guanidine (and car-
bon) was considered together with the synthetic route to
arrive at potential structures for the BDI. Specifically, the
apparent dimerization minus guanidine suggested that the
BDI might arise from an intermolecular reaction involv-
ing intermediate 6 (Scheme 2). During the synthesis
of AG2034, 6 undergoes an intramolecular nucleophilic
addition to form 7, which subsequently reactions with
guanidine hydrochloride to form the pyrimidothiazine of
AG2034. A possible intermolecular side reaction gener-
ating intermediate 8, which would give rise to 9, could
explain both the dimerization and the incorporation of
only a single guanidine equivalent into the final compound
4. In order to provide complete and unequivocal charac-
terization of this complex BDI, however, isolation and
structure elucidation via high resolution NMR were ulti-
mately required.
Isolation and structure elucidation of purified BDI
The large molecular weight difference between 3 and 4
suggested that size-exclusion chromatography could suc-
ceed where semi-preparative HPLC had failed to provide
pure BDI. As expected, chromatography on Sephadex LH-
20 effectively resolved the BDI from the parent 3. The
final purification was achieved by flash chromatography
and, with pure 4 in hand, it was possible to characterize
fully the BDI by mass spectrometry and traditional high-
resolution NMR methods. The resonance assignments for
4 were made by analysis of 1D 1 H NMR, 2D 1 H– 1 H
DQF-COSY,7 1D 13 C, APT, 2D 1 H– 13 C HMQC8 and 2D

Scheme 2

Copyright  1999 John Wiley & Sons, Ltd. Magn. Reson. Chem. 37, 393–400 (1999)
 LC–NMR OF BULK DRUG IMPURITIES IN GART INHIBITOR AG2034
1
H– 13 C HMBC9 data. The process was facilitated by com-
parative analysis with 3, and the 1 H and 13 C assignments
for pure parent 3 and BDI 4 are summarized in Table 1.
Synthetic origin and biological activity of the BDI
The origin of 4 as outlined in Scheme 2 is proposed on the
basis of the synthetic route to AG2034 (1). Deprotection
of intermediate 5 to give the free amine 6 sets the stage
for intramolecular nucleophilic addition to obtain lactam
7. However, if 6 undergoes intermolecular addition to the
ethyl ester of the substituted thiophene, an intermediate
8 is generated which will give rise to 4 upon completion
of the remaining synthetic steps. In order to verify that
4 represented the precursor to the major impurity present
in bulk AG2034 (1), 4 was hydrolyzed under the reaction
conditions which are used to convert 3 to 1. This gave
the free acid, 2, which co-eluted with the major impurity
present in bulk 1 on analytical HPLC. The acid impurity
2 showed a Ki of 69 nM against the GART enzyme.
CONCLUSIONS
LC–NMR and LC–MS provided a window through which
to view BDI 4 prior to performing any purification and
allowed detailed structural information to be obtained in
a timely manner. The combined methodologies provided
key insights into molecular weight, structure and purifi-
cation methods which ultimately allowed this complex
impurity to be fully characterized through high-resolution
NMR of the purified compound.
EXPERIMENTAL
LC–NMR
LC–NMR was performed on a Bruker Avance 500 MHz
NMR spectrometer coupled to a Bruker LC22 pump
equipped with a BPSU-12 (Bruker peak sampling unit).
A 4 mm triple-resonance (1 H, 13 C, 15 N) LC–NMR probe
fitted with triple axis gradients was used. LC–NMR was
performed in the automatic, stop-flow mode. The HPLC
method was initiated with 90% D2 O containing 0.1%
TFA–10% ACN (protonated acetonitrile), held for 10 min,
followed by a gradient to 60% D2 O containing 0.1%
TFA–40% ACN over 20 min (total run time 30 min), with
a flow rate of 0.7 ml min 1 and UV detection at 280 nm.
The sample was prepared as follows. A 5 µl aliquot of a
35 mg sample of crude 3 in 120 µl of CD2 Cl2 and 50 µl
of ACN was diluted to 100 µl of ACN. A 40 µl aliquot
(400 µg total material) was injected on to a Waters Sym-
metry ODS column (4.6 ð 250 mm). It was estimated that
ca 300 µg of 3 (ca 575 nmol) and 60 µg of 4 (61 nmol)
were injected on to the column. NMR data was collected
on the parent compound (3, Rt D 24.8 min) and the BDI
of interest (4, Rt D 29.5 min). A reference spectrum of the
solvent was also collected; in addition to the solvent res-
onances ACN (υ 2.08, internal chemical shift reference)
Copyright  1999 John Wiley & Sons, Ltd.
399
and HOD (chemical shift dependent upon the D2 O/ACN
ratio over the course of the HPLC gradient), impurity
peaks were present at υ 3.56 (q), 3.27 (s), and 1.11 (t). The
1D 1 H spectra were acquired using double presaturation
of both the HOD and ACN resonances. The presatura-
tion was employed during the 1.8 s relaxation delay. Data
were acquired with a 10 kHz sweep width using 32K
time-domain points with an acquisition time of 1.64 s. In
addition, a TOCSY spectrum was collected for 4 using a
10 kHz sweep width, 180 scans per t1 increment and 160
time-domain points in ω1 , with a 1.5 s relaxation delay for
a total experiment time of 13 hs. The spin-lock was gener-
ating using MLEV-1710 and the spin-lock time was 85 ms.
Data were processed using sin2 ./2/ in both dimensions.
Purification of 4
Crude 3 (1.26 g) containing 4 was dissolved in 30 ml of methanol
and chromatographed on Sephadex LH-20 (5 cm ð 86 cm column, bed
volume 1.69 l), eluting with 100% methanol with a flow rate of
0.7 ml min 1 . Fractions were assayed by TLC and analytical HPLC,
and those containing 4 were pooled to obtain 46 mg of crude 4. This
crude material was further purified by flash chromatography on silica
gel (1 cm ð 14 cm), eluting with 92% dichloromethane–8% methanol
followed by 100% methanol to obtain pure 4 (10 mg). High-resolution
fast atom bombardment MS: m/z calculated for C42 H58 N7 O12 S4 (MHC ),
980.3026; found, 980.2973 (MHC ).
Conversion of 4 to 2
Compound 4 (10 mg) was dissolved in 1 M aqueous NaOH (250 µl) and
stirred at room temperature for 20 min. The solution was acidified with
HCl (1.2 equiv.) to precipitate 2, a yellow solid, which was washed with
water. 1 H NMR (500 MHz, DMSO-d6 ):υ 12.41 [br s, 4H (4 ð COOH)],
10.17 (br s, 1H), 8.49 (m, 2H), 8.34 (d, 1H), 7.66 (d, 1H), 7.56 (d, 1H),
6.88 (m, 2H), 6.71 (br s, 1H), 6.15 (br s, 2H), 4.31 (m, 1H), 4.21 (m,
1H), 3.17–3.58 (m; signal could not be accurately integrated owing to
the presence of HOD), 3.15 (s, 2H), 2.80–3.05 (m, 6H), 2.29 (m, 4H),
2.05 (m, 1H), 1.92 (m, 4H), 1.74 (m, 3H). High-resolution MS: m/z cal-
culated for C34 H41 N7 O12 S4 (MHC ), 868.1774; found, 868.1738 (MHC ).
High-resolution NMR spectroscopy
Purified 4 (5.4 mg) was dissolved in DMSO-d6 . 1D 1 H,
13
C and APT, and 2D gradient 1 H– 13 C HMQC and gradi-
ent 1 H– 13 C HMBC spectra were acquired on a Bruker
Avance 500 MHz NMR spectrometer utilizing a 5 mm
triple-resonance probe equipped with triple axis gradients
and a 5 mm broadband observe probe with a z-axis gra-
dient. In addition, 1D 1 H and 2D 1 H– 1 H DQF-COSY
and 1 H– 13 C HMQC were acquired at 25 ° C on a Var-
ian UnityPlus 500 MHz spectrometer utilizing a 5 mm
triple-resonance (1 H, 13 C, 15 N) probe equipped with z-
axis gradients. Compound 3 (12 mg) was dissolved in
DMSO-d6 . 1D 1 H and 2D 1 H– 1 H DQF-COSY, gradient
1
H– 13 C HMQC and gradient 1 H– 13 C HMBC spectra were
acquired at 25 ° C on a Varian UnityPlus 500 MHz spec-
trometer utilizing a 5 mm triple nucleus (1 H, 13 C, 15 N)
probe equipped with z-axis gradients. 1D 13 C NMR data
were acquired on a GE QE 300 MHz NMR spectrome-
ter equipped with a dual (1 H/13 C) probe. A DEPT-135
spectrum was acquired on a Bruker Avance 500 MHz
spectrometer utilizing a 5 mm broadband inverse probe
equipped with triple axis gradients.
Magn. Reson. Chem. 37, 393–400 (1999)
400 B. C. M. POTTS ET AL.
Acknowledgments
We thank Mary Kay Harper and D. John Faulkner for their contribu-
tions during the initial purification phase of the project, Kanyin Zhang
for performing LC–MS/MS experiments, Steve Margosiak for perform-
ing Ki measurements against GART enzyme, Van Martin for helpful
technical discussions and Nuria Assa-Munt and Xin Jia at The Burn-
ham Institute NMR facility, where several high-resolution NMR data
sets were acquired.
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