The Use of Chloroformamidine Hydrochloride as a Reagent for the Synthesis
of Guanidines from Electron Deficient Aromatic Amines
Ian Armitage, ‡ Mingkun Fu,‡ Frederick Hicks,† Adiseshu Kattuboina,§ Jennifer S. N. Li,† Ashley
McCarron,*,† and Lei Zhu†
† Chemical
Development Laboratories Boston, Takeda Pharmaceuticals International Company,
35 Landsdowne Street, Cambridge, Massachusetts 02139, United States
‡ Analytical Development Laboratories Boston, Takeda Pharmaceuticals International Company,
35 Landsdowne Street, Cambridge, Massachusetts 02139, United States
§ Chemical Development, Albany Molecular Research Incorporated, 21 Corporate Circle,
Albany, New York 12212, United States
Supporting Information
Table of Contents
Analysis and Stability of Chloroformamidine Hydrochloride ...................................................................... 1
Condition Screening Data ............................................................................................................................. 6
Chloroformamidine Hydrochloride Equivalent Screening Data ................................................................... 7
1
H NMR, 13C NMR, and HRMS Spectra ...................................................................................................... 8
Analysis and Stability of Chloroformamidine Hydrochloride
Analysis
S1
It was found that chloroformamidine HCl (3) reacts rapidly with methanol to give omethylisourea. High resolution mass spectrum (HRMS) of 3 in methanol gave a protonated
molecular ion signal at m/z 75.0550 corresponding to o-methylisourea, with no signal observed
for the protonated chloroformamidine (Figure 1). Furthermore,
13
C NMR gave deuterated o-
methylisourea signals for 3 in CD3OD (Figure 2). Such observations allowed for the utilization
of methanol-induced derivatization of 3 to assess its purity, for which a LC/HRMS quantitation
method was developed.
Figure 1. High Resolution Mass Spectrum of Chloroformamidine HCl (3) in MeOH
LeiMeOH4 #180-210 RT: 1.59-1.82 AV: 10 SB: 18 1.03-1.17 , 2.68-2.98 NL: 1.09E7
F: FTMS + p ESI Full ms [50.00-500.00]
75.0550
10000000
9000000
8000000
Intensity
7000000
6000000
5000000
4000000
3000000
2000000
1000000
76.0583
0
50
100
117.0770 170.0012
150
220.6167
200
271.5452 307.1520
250
300
365.0040 401.4145 432.2280
350
400
m/z
Figure 2. 13C NMR (125 Hz) Spectrum of Chloroformamidine HCl (3) in CD3OD
S2
450
495.7550
500
An ultra high performance liquid chromatography instrument (UHPLC) was utilized for
chromatographic separation. This UHPLC system is comprised of an autosampler, micro mixer
(180μL), high pressure HPLC pump, and vacuum degasser.. The injection volume was set to 1
μL. The column was a 2.6 μm Hilic 100Å, 150 mm x 3.0 mm column. 0.1% TFA in water was
selected for aqueous phase A and 0.1% TFA in acetonitrile was selected for organic phase B.
The flow rate was 1 mL/min and column temperature was set at ambient. The following gradient
was used for a total run of 10 minutes: starting at 95% B and decreasing to 75% B in 4 minutes,
decreasing again to 10% B in 0.1 minute, holding at 10% B for 0.9 minutes, increasing back to
95% B in 0.5 minutes, and subsequently holding at 95% B for 4.5 minutes. About 5% LC flow
elute solution was directed via a “T” splitter to a LTQ mass spectrometer. The instrument was
S3
tuned and calibrated in positive mode by using electrospray ionization (ESI) positive ion
calibration solution. The capillary of heated ESI interface (HESI-II) was set to 5000 V for source
voltage, 100 μA for source current, and 60% for S-Lens RF level. The capillary temperature was
set to 275°C. Sheath gas was set to 40 units. Profile algorithm mass spectrometry data were
acquired with the range of m/z 50 - m/z 500 under mass resolving power (MRP) of 30,000. The
extracted ion chromatography (EIC) signal was extracted at m/z 75.0553 with a mass extraction
window (MEW) of 10 ppm.
To assess the purity of synthesized 3, blank samples (1 mg/mL reference and 1 mg/mL 3) were
first analyzed according to equation [1] (Figure 3a). o-Methylisourea bisulfate (CAS number:
29427-58-5) was purchased and used as the reference standard to quantify 3. The purity of 3 was
investigated by using equation [1], where Area (sample) is the EIC peak area response of 3; MW
(sample) is the molecular weight of 3 (115.0 g/mol); Area (reference) is the EIC peak area
response of o-methylisourea bisulfate; and MW (reference) is the molecular weight of omethylisourea bisulfate (172.2 g/mol) (Figure 3b). The purity of two different lots of 3 were then
determined and found to be 94.4% (batch 1) and 89.3% (batch 2) (Figure 3, c and d respectively).
Furthermore, in order to evaluate the precision of the measured purity value, triplicate
measurements of a separate lot of 3 (Lot E2908-43) were performed by testing three
independently prepared samples and references. The purity values obtained from these analyses
were 80.4%, 83.5%, and 80.8% with a mean of 81.6% and an excellent relative standard
deviation of 2.1%.
Purity 
Area( sample)  MW ( sample)
Area(reference)  MW (reference)
[1]
S4
Figure 3. Purity Assessment of Chloroformamidine HCl (3) using LC/HRMS Analysis
a) Extracted ion chromatogram of blank injection
RT: 0.00 - 10.00
0.29
Relative Abundance
100
0.90
NL: 1.92E4
1.10 1.49
1.74
9.16
80
2.05 2.32
2.82
60
3.44
3.59
4.35
8.45 8.68
6.88 7.06
7.16
4.56
4.75
40
8.31
7.42
6.78
6.41 6.73
5.50
9.81
m/z=
75.0545-75.0561
F: FTMS + p ESI
Full ms
[50.00-500.00]
MS Blank_1
20
0
0
1
2
3
4
5
6
7
8
9
Time (min)
b) Extracted ion chromatogram of reference o-methylisourea bisulfate
RT: 0.00 - 10.01
RT: 1.79
AA: 134922279
Relative Abundance
100
NL: 2.17E7
m/z=
75.0545-75.0561 F:
FTMS + p ESI Full
ms [50.00-500.00]
MS Genesis Ref _1
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
c) Extracted ion chromatogram of chloroformamidine HCl (batch 1)
RT: 0.00 - 10.00
RT: 1.78
AA: 190725012
Relative Abundance
100
NL: 2.33E7
m/z=
75.0545-75.0561 F:
FTMS + p ESI Full
ms [50.00-500.00]
MS Genesis A6_1
80
60
40
20
0
0
1
2
3
4
5
6
7
8
Time (min)
d) Extracted ion chromatogram of chloroformamidine HCl (batch 2)
S5
9
RT: 0.00 - 10.01
RT: 1.80
AA: 180457349
Relative Abundance
100
NL: 2.14E7
m/z=
75.0545-75.0561 F:
FTMS + p ESI Full
ms [50.00-500.00]
MS Genesis A22_1
80
60
40
20
0
0
1
2
3
4
5
6
7
8
9
10
Time (min)
Stability
To assess the stability of 3, samples stored under nitrogen at ambient temperature were analyzed
after 6 months and compared with the initial purity data (t = 0) collected. As can be seen from
the data below, significant degradation occurred over the 6 months of storage and use. Based on
this information, it is recommended that the material be stored under nitrogen at 0-5°C if
possible to prolong the shelf life of 3.
Sample
Batch 1
Batch 2
Purity (%)
t=0
t = 6 mos.
94.4
89.3
76.5
70.5
Condition Screening Data
S6
entrya
1
2
3
4
5
6
7
8
9
10c
11c
12d
13c,d
14c
15c,d
16c,d
17
temp.
conversion (%)b
3
(°C) (equiv) 5a 4a Imp(s).
AcOH
60
1.2
29 2
69
DMF
100
1.2
40 4
56
DMSO
120
1.2
7 66
27
NMP
100
1.2
17 9
74
DMAc
100
1.2
12 59
29
EtOH
100
1.2
51 37
12
IPA
80
1.2
11 89
0
t-butanol
100
1.8
66 25
9
2-butanol
60
1.2
20 80
0
dimethoxyethane
80
1.2
67 16
17
MeCN
80
1.2
65 29
6
MeCN/AcOH
80
1.2
71 1
28
MeCN/sulfolane
80
1.2
>99 <1
0
dioxane
100
1.2
64 36
0
dioxane/AcOH
100
1.4
94 6
0
dioxane/sulfolane
80
1.2
74 26
0
sulfolane
100
1.2
97 2
1
Solvent
a
All reactions conducted on a 2.1 mmol scale in 10 vol. solvent (sampled over 24h - table displays best conversion)
b
Conversion was determined by LC/MS of the in-process reaction mixture
c
Heterogeneous reaction at certain temperatures
d
Solvent ratio 10:1
Chloroformamidine Hydrochloride Equivalent Screening Data
This screen investigated the optimal amount of 3 from 1.2 to 2.0 equivalents. As can be seen in
the table below, improvements in conversion occurred from 1.2 to 1.4 equivalents, with no
S7
significant progress achieved when > 1.4 equivalents was used. Also, the need to remove excess
3 when the larger amounts were utilized made product isolation increasingly more difficult. For
these reasons, 1.4 equivalents of 3 were chosen as optimal for the substrate screen.
entrya
1
2
Solvent
MeCN/AcOH
sulfolane
conversion to 5a (%)b
3
(equiv)
60 °C
80 °C
100 °C
1.2
65
71
33c
1.4
77
77
18c
1.6
82
72
54c
1.8
78
52
65c
2.0
82
77
69c
1.2
90
84
97
1.4
98
97
95
1.6
95
95
93
1.8
97
95
91
2.0
94
94
92
a
All reactions conducted on a 2.1 mmol scale in 10 vol. solvent (sampled over 24h - table displays best conversion)
b
Conversion was determined by LC/MS of the in-process reaction mixture
c Heating
the MeCN/AcOH mixture above the boiling point of MeCN evaporated solvent, decreasing solubility, and
therefore invalidated these results
1H
NMR, 13C NMR, and HRMS Spectra
S8
S9
S10
2
Li2 #72-92 RT: 0.64-0.81 AV: 10 SB: 36 0.27-0.51 , 0.90-1.27 NL: 2.28E6
F: FTMS + p ESI Full ms [101.00-1500.00]
236.1865
2200000
2000000
1800000
1600000
Intensity
1400000
1200000
1000000
800000
118.5967
600000
400000
200000
148.0869 191.1290 219.1604
0
150
200
252.1818
250
303.2039
300
m/z
350.1799
350
402.2133 444.2739 471.3663
400
S11
450
S12
S13
5a
Li1 #67-86 RT: 0.59-0.75 AV: 10 SB: 36 0.26-0.52 , 0.90-1.27 NL: 3.16E6
F: FTMS + p ESI Full ms [101.00-1500.00]
137.0818
3000000
2500000
Intensity
2000000
1500000
1000000
120.0553
152.0677
500000
179.1037 204.0991 235.0000 273.1569 309.1337
0
150
200
250
300
m/z
350.1644 371.1257
350
400
S14
425.0979
465.0789
450
S15
S16
5b
E3329_39_140811150639 #65-71 RT: 0.64-0.70 AV: 4 SB: 11 0.45-0.53 , 0.82-0.94 NL: 5.16E6
F: FTMS + p ESI Full ms [105.00-1000.00]
205.0690
5000000
4500000
4000000
Intensity
3500000
3000000
2500000
2000000
1500000
1000000
500000
0
188.0426
209.0627 308.0174
200
300
471.0522 515.2822 577.0426 644.9294 713.0158 825.3355 905.0208 957.0502
400
500
600
700
m/z
S17
800
900
1000
S18
S19
5c
Li8 #308-336 RT: 2.74-2.98 AV: 14 SB: 42 2.12-2.51 , 3.16-3.49 NL: 3.85E6
F: FTMS + p ESI Full ms [101.00-1500.00]
187.0973
3500000
3000000
Intensity
2500000
2000000
1500000
1000000
500000
170.0709
145.0759
128.0492
0
150
214.0867 237.0981
200
250
285.0658 317.1719
300
m/z
373.1883 400.1800
350
400
S20
471.1549
450
S21
S22
5e
A1 #75-83 RT: 0.76-0.84 AV: 5 SB: 6 0.73-0.75 , 0.92-1.02 NL: 8.84E5
F: FTMS + p ESI Full ms [105.00-1000.00]
204.0742
800000
700000
Intensity
600000
500000
400000
300000
200000
100000
206.0809
187.0477
0
200
250.9728 291.9992 402.2696 446.2962
300
400
568.0550 646.0720 702.1356 786.0845 873.1765
500
600
700
m/z
S23
800
900
956.0384
1000
S24
S25
5f
Li5 #768-786 RT: 6.78-6.92 AV: 9 SB: 49 6.30-6.62 , 7.20-7.73 NL: 1.22E7
F: FTMS + p ESI Full ms [101.00-1500.00]
249.0586
12000000
11000000
10000000
9000000
Intensity
8000000
7000000
6000000
5000000
4000000
3000000
2000000
1000000
0
189.0269
159.0288
200
308.0964
300
419.2013 497.1111 595.0782 649.0507
400
500
600
785.0241
700
m/z
S26
800
897.1029 948.8795
900
1000
S27
S28
5g
Li3 #102-134 RT: 0.91-1.17 AV: 16 SB: 35 0.69-0.85 , 1.45-1.89 NL: 4.54E6
F: FTMS + p ESI Full ms [101.00-1500.00]
152.0813
4500000
4000000
3500000
Intensity
3000000
2500000
2000000
1500000
135.0551 166.0610
1000000
500000
184.0715 210.0873 242.0923
0
150
200
250
301.1407
300
m/z
341.1035
401.1241
350
400
S29
455.0959
450
492.1773
500
S30
S31
S32
S33
5j
Li9 #1005-1036 RT: 8.90-9.17 AV: 16 SB: 29 8.58-8.78 , 9.46-9.74 NL: 2.51E6
F: FTMS + p ESI Full ms [101.00-1500.00]
226.0969
2400000
2200000
2000000
1800000
Intensity
1600000
1400000
1200000
1000000
800000
600000
400000
200000
209.0709
0
200
240.0765
300
347.1463
451.1875
400
577.3664 656.3192
500
600
738.4442 808.7724
700
m/z
S34
800
942.2122
900
S35
S36
5m
A7 #75-82 RT: 0.76-0.82 AV: 4 SB: 9 0.62-0.67 , 0.94-1.04 NL: 2.95E6
F: FTMS + p ESI Full ms [105.00-1000.00]
190.1084
2500000
Intensity
2000000
1500000
1000000
173.0818
500000
0
379.2101
206.1034
200
300
400
493.2027 547.1221
500
600
685.2070 757.9939 807.5592 898.0855 975.7948
700
m/z
S37
800
900
1000
S38
S39
5n
A4 #73-80 RT: 0.74-0.80 AV: 4 SB: 14 0.48-0.60 , 0.93-1.06 NL: 3.51E6
F: FTMS + p ESI Full ms [105.00-1000.00]
192.0586
3500000
3000000
Intensity
2500000
2000000
1500000
195.0577
1000000
150.0370
500000
250.9722
0
200
383.1106
300
400
497.1032 551.0220
500
600
666.0570 742.0737 797.7503
700
m/z
S40
800
926.4885
900
1000