Nimustine hydrochloride [systematic name: 4-amino-5-({[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino}methyl)-2-methylpyrimidin-1-ium chloride], C9H14ClN6O2+·Cl-, is a prodrug of CENU (chloroethylnitrosourea) and is used as a cytostatic agent in cancer therapy. Its crystal structure was determined from laboratory X-ray powder diffraction data. The protonation at an N atom of the pyrimidine ring was established by solid-state NMR spectroscopy.
Supporting information
CCDC reference: 873907
Nimustine hydrochloride, (I), was purchased from Sigma–Aldrich (>98% purity)
and used as received. The TG measurement was performed on a TGA 92 (SETARAM)
device. About 10–15 mg of the sample was filled into corundum crucibles and
measured from room temperature to 473 K at a rate of 3 K min-1 under a
nitrogen atmosphere. The DSC measurement was performed on a DSC 131 (SETARAM)
device in a similar fashion as for the TG measurement. For the solid-state NMR
measurements, 80 mg of crystalline (I) with a natural 15N and 13C isotope
abundance was used. The compound was packed in a 4 mm zirconia rotor. The
measurements were performed in a Bruker 850 MHz instrument at 10 kHz spinning
speed at 290 K and referenced against liquid ammonia. The 13C NMR spectrum
was measured on a Bruker Avance 400 device in tubes filled with
d6-DMSO (reference) and 15–20 mg of (I). The scanning electron
microscopy image in Fig. 1 was recorded using an Amray 1919 ECO
scanning
electron microscope at 20 kV and a low vacuum of 7 Pa. The elemental analysis
(CHNS) was carried out on an Elementar (vario MICRO cube) elemental analyser.
About 1–4 mg of the sample was placed in a tin vessel and measured at 1423 K
under a helium atmosphere with addition of oxygen during the measurement.
Elemental analysis calculated for C9H14Cl2N6O2 (%): C 34.97, H 4.56,
N 27.18; found: C 34.98, H 4.54, N 27.17.
The whole powder pattern out to 1.20 Å resolution was used for the Rietveld
refinement. A total of 86 parameters were refined, namely the background
(fitted using Chebyshev polynomials with 20 refinable coefficients),
zero-point error, scale parameter, atomic coordinates (refined with
restraints), anisotropic peak broadening, lattice parameters, a scale factor
and a common isotropic displacement parameter for the C, N and O atoms. The
isotropic displacement parameter of the H atoms was constrained at 1.2 times
the global isotropic displacement parameter. For the two Cl atoms, individual
isotropic displacement parameters were assigned. 42 suitable chemical
restraints from Mogul (Bruno et al., 2004) were added
(see Table
3), 18 for bond lengths, 23 for bond angles and one for the planarity of the
4-amino-2-methylpyrimidin-1-ium-5-yl group, including the aromatic C and N,
the amino N and the methyl and alkyl C atoms. Finally, the H-atom positions
were adjusted using the DREIDING/X6 force field (Mayo et al.,
1990) within the program package Cerius2 (Accelrys,
2003). During the
optimization, the coordinates of all other atoms and the lattice parameters
were fixed. In the case of the methyl group attached to C2 at the pyrimidine
ring, the H atoms had to be moved to a more suitable orientation with respect
to the amine N1—H1 group. Bond lengths were set manually according to mean
values from the CSD (aromatic C—H = 0.950 Å, methyl C—H = 0.969 Å,
alkyl C—H = 0.985 Å, primary amine N—H = 0.865 Å and secondary amine
N—H = 0.898 Å). The difference electron density was calculated using
SHELXTL (Sheldrick, 2008). The highest residual electron density
(0.41 e Å-3) is located close to the position of the chloride anion. However, a
refinement of split-atom positions for Cl1 did not improve the fit. The final
Rietveld plot for (I) is shown in Fig. 4.
Data collection: WINXPOW (Stoe & Cie, 2004); cell refinement: TOPAS Academic (Coelho, 2007); data reduction: DASH (David et al., 2006); program(s) used to solve structure: DASH (David et al., 2006); program(s) used to refine structure: TOPAS Academic (Coelho, 2007); molecular graphics: Mercury (Macrae et al., 2008); software used to prepare material for publication: publCIF (Westrip, 2010).
4-amino-5-({[
N-(2-chloroethyl)-
N-nitrosocarbamoyl]amino}methyl)-
2-methylpyrimidin-1-ium chloride
top
Crystal data top
C9H14ClN6O2+·Cl− | F(000) = 640.0 |
Mr = 309.16 | standard setting |
Monoclinic, P21/c | Dx = 1.495 Mg m−3 |
Hall symbol: -P 2ybc | Cu Kα1 radiation, λ = 1.54056 Å |
a = 5.25191 (12) Å | µ = 4.35 mm−1 |
b = 12.2401 (3) Å | T = 293 K |
c = 21.4088 (5) Å | Particle morphology: tablet |
β = 93.2353 (7)° | white |
V = 1374.05 (6) Å3 | cylinder, 10 × 0.7 mm |
Z = 4 | |
Data collection top
Stoe STADI-P with a linear position-sensitive detector diffractometer | Data collection mode: transmission |
Radiation source: X-ray tube | Scan method: step |
Primary focusing, Ge 111 monochromator | 2θmin = 2.0°, 2θmax = 79.99°, 2θstep = 0.01° |
Specimen mounting: 0.7mm glass capillary | |
Refinement top
Refinement on Inet | Profile function: modified Thompson–Cox–Hastings pseudo-Voigt
[Young, R. A. (1993). The Rietveld Method, pp. 115–118. New
York: Oxford University Press Inc.] |
Least-squares matrix: full with fixed elements per cycle | 86 parameters |
Rp = 0.087 | 42 restraints |
Rwp = 0.084 | 0 constraints |
Rexp = 0.061 | H-atom parameters not refined |
RBragg = 0.012 | Weighting scheme based on measured s.u.'s w = 1/σ[Yobs]2 |
χ2 = 1.388 | (Δ/σ)max = 0.001 |
7600 data points | Background function: Bayesian high-pass filter with 20 terms |
Excluded region(s): none | |
Crystal data top
C9H14ClN6O2+·Cl− | V = 1374.05 (6) Å3 |
Mr = 309.16 | Z = 4 |
Monoclinic, P21/c | Cu Kα1 radiation, λ = 1.54056 Å |
a = 5.25191 (12) Å | µ = 4.35 mm−1 |
b = 12.2401 (3) Å | T = 293 K |
c = 21.4088 (5) Å | cylinder, 10 × 0.7 mm |
β = 93.2353 (7)° | |
Data collection top
Stoe STADI-P with a linear position-sensitive detector diffractometer | Scan method: step |
Specimen mounting: 0.7mm glass capillary | 2θmin = 2.0°, 2θmax = 79.99°, 2θstep = 0.01° |
Data collection mode: transmission | |
Refinement top
Rp = 0.087 | 7600 data points |
Rwp = 0.084 | 86 parameters |
Rexp = 0.061 | 42 restraints |
RBragg = 0.012 | H-atom parameters not refined |
χ2 = 1.388 | |
Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) top | x | y | z | Uiso*/Ueq | |
N1 | 0.2496 (3) | 1.00900 (16) | 0.32387 (9) | 0.03447 | |
H1 | 0.36435 | 1.06336 | 0.32703 | 0.04137 | |
Cl1 | 0.6402 (4) | 1.18836 (15) | 0.30215 (9) | 0.04632 | |
C2 | 0.2413 (3) | 0.93001 (14) | 0.36777 (7) | 0.03447 | |
N3 | 0.0697 (3) | 0.84949 (12) | 0.36279 (9) | 0.03447 | |
C4 | −0.1011 (3) | 0.84567 (13) | 0.31280 (7) | 0.03447 | |
C5 | −0.0954 (3) | 0.92776 (12) | 0.26642 (6) | 0.03447 | |
C6 | 0.0829 (3) | 1.00884 (17) | 0.27321 (8) | 0.03447 | |
H6 | 0.08901 | 1.06555 | 0.24301 | 0.04137 | |
C7 | −0.2813 (4) | 0.9303 (2) | 0.20896 (10) | 0.03447 | |
H7A | −0.45178 | 0.90972 | 0.22198 | 0.04137 | |
H7B | −0.29761 | 1.00600 | 0.19363 | 0.04137 | |
N8 | −0.2153 (6) | 0.8603 (2) | 0.15774 (13) | 0.03447 | |
H8 | −0.30248 | 0.79801 | 0.15069 | 0.04137 | |
C9 | −0.0221 (5) | 0.88714 (18) | 0.12384 (12) | 0.03447 | |
O10 | 0.0974 (7) | 0.9721 (2) | 0.12892 (17) | 0.03447 | |
N11 | 0.0358 (4) | 0.80906 (18) | 0.07853 (12) | 0.03447 | |
N12 | −0.1079 (6) | 0.7177 (2) | 0.07355 (17) | 0.03447 | |
O13 | −0.0312 (6) | 0.6446 (3) | 0.03990 (16) | 0.03447 | |
C14 | 0.2722 (5) | 0.8208 (4) | 0.04404 (13) | 0.03447 | |
H14A | 0.36396 | 0.75054 | 0.04452 | 0.04137 | |
H14B | 0.38846 | 0.87426 | 0.06490 | 0.04137 | |
C15 | 0.1973 (5) | 0.8492 (4) | −0.02069 (13) | 0.03447 | |
H15A | 0.09394 | 0.79336 | −0.04356 | 0.04137 | |
H15B | 0.09848 | 0.91744 | −0.01912 | 0.04137 | |
Cl16 | 0.4879 (4) | 0.88295 (15) | −0.06071 (9) | 0.06129 | |
C21 | 0.4272 (5) | 0.9340 (2) | 0.42268 (12) | 0.03447 | |
H21A | 0.39837 | 0.87251 | 0.44993 | 0.04137 | |
H21B | 0.59902 | 0.93044 | 0.40855 | 0.04137 | |
H21C | 0.40540 | 1.00154 | 0.44539 | 0.04137 | |
N41 | −0.2708 (4) | 0.76506 (19) | 0.30853 (12) | 0.03447 | |
H41A | −0.37435 | 0.75975 | 0.27580 | 0.04137 | |
H41B | −0.27387 | 0.71682 | 0.33799 | 0.04137 | |
Geometric parameters (Å, º) top
N1—H1 | 0.898 | C9—O10 | 1.216 (4) |
N1—C2 | 1.351 (3) | C9—N11 | 1.407 (3) |
N1—C6 | 1.355 (2) | N11—N12 | 1.350 (4) |
C2—N3 | 1.336 (2) | N11—C14 | 1.487 (4) |
C2—C21 | 1.486 (3) | N12—O13 | 1.231 (5) |
N3—C4 | 1.358 (2) | C14—H14A | 0.985 |
C4—C5 | 1.414 (2) | C14—H14B | 0.985 |
C4—N41 | 1.330 (3) | C14—C15 | 1.461 (4) |
C5—C6 | 1.367 (3) | C15—H15A | 0.985 |
C5—C7 | 1.527 (3) | C15—H15B | 0.985 |
C6—H6 | 0.950 | C15—Cl16 | 1.839 (4) |
C7—H7A | 0.986 | C21—H21A | 0.969 |
C7—H7B | 0.985 | C21—H21B | 0.969 |
C7—N8 | 1.449 (4) | C21—H21C | 0.970 |
N8—H8 | 0.898 | N41—H41A | 0.865 |
N8—C9 | 1.322 (4) | N41—H41B | 0.865 |
| | | |
H1—N1—C2 | 121.7 | O10—C9—N11 | 120.8 (3) |
H1—N1—C6 | 117.5 | C9—N11—N12 | 118.4 (2) |
C2—N1—C6 | 120.7 (2) | C9—N11—C14 | 119.7 (2) |
N1—C2—N3 | 121.4 (2) | N12—N11—C14 | 121.3 (2) |
N1—C2—C21 | 118.8 (2) | N11—N12—O13 | 116.7 (3) |
N3—C2—C21 | 119.8 (2) | N11—C14—H14A | 109.4 |
C2—N3—C4 | 120.1 (2) | N11—C14—H14B | 110.6 |
N3—C4—C5 | 119.3 (1) | N11—C14—C15 | 107.8 (3) |
N3—C4—N41 | 119.4 (2) | H14A—C14—H14B | 106.6 |
C5—C4—N41 | 121.3 (2) | H14A—C14—C15 | 108.8 |
C4—C5—C6 | 118.9 (1) | H14B—C14—C15 | 113.5 |
C4—C5—C7 | 122.8 (1) | C14—C15—H15A | 114.7 |
C6—C5—C7 | 118.3 (2) | C14—C15—H15B | 106.5 |
N1—C6—C5 | 119.6 (2) | C14—C15—Cl16 | 108.0 (2) |
N1—C6—H6 | 119.9 | H15A—C15—H15B | 109.2 |
C5—C6—H6 | 120.5 | H15A—C15—Cl16 | 111.9 |
C5—C7—H7A | 108.5 | H15B—C15—Cl16 | 106.1 |
C5—C7—H7B | 109.1 | C2—C21—H21A | 109.5 |
C5—C7—N8 | 115.4 (2) | C2—C21—H21B | 109.5 |
H7A—C7—H7B | 105.7 | C2—C21—H21C | 109.5 |
H7A—C7—N8 | 108.8 | H21A—C21—H21B | 109.5 |
H7B—C7—N8 | 108.9 | H21A—C21—H21C | 109.5 |
C7—N8—H8 | 119.2 | H21B—C21—H21C | 109.4 |
C7—N8—C9 | 119.5 (2) | C4—N41—H41A | 120.0 |
H8—N8—C9 | 121.2 | C4—N41—H41B | 119.8 |
N8—C9—O10 | 124.9 (3) | H41A—N41—H41B | 120.2 |
N8—C9—N11 | 114.3 (2) | | |
| | | |
H1—N1—C2—N3 | 179.9 | C6—C5—C7—H7B | −24.6 |
H1—N1—C2—C21 | 0.1 | C6—C5—C7—N8 | 98.3 (2) |
C6—N1—C2—N3 | 0.1 (3) | C5—C7—N8—H8 | 105.1 |
C6—N1—C2—C21 | −179.7 (2) | C5—C7—N8—C9 | −72.3 (3) |
H1—N1—C6—C5 | −179.9 | H7A—C7—N8—H8 | −17.1 |
H1—N1—C6—H6 | −0.9 | H7A—C7—N8—C9 | 165.6 |
C2—N1—C6—C5 | −0.1 (3) | H7B—C7—N8—H8 | −131.8 |
C2—N1—C6—H6 | 179.0 | H7B—C7—N8—C9 | 50.8 |
N1—C2—N3—C4 | 0.1 (3) | C7—N8—C9—O10 | −6.0 (4) |
C21—C2—N3—C4 | 179.8 (2) | C7—N8—C9—N11 | 175.5 (2) |
N1—C2—C21—H21A | −180.0 | H8—N8—C9—O10 | 176.7 |
N1—C2—C21—H21B | −60.0 | H8—N8—C9—N11 | −1.9 |
N1—C2—C21—H21C | 60.0 | N8—C9—N11—N12 | 0.7 (4) |
N3—C2—C21—H21A | 0.3 | N8—C9—N11—C14 | −170.6 (3) |
N3—C2—C21—H21B | 120.3 | O10—C9—N11—N12 | −178.0 (3) |
N3—C2—C21—H21C | −119.7 | O10—C9—N11—C14 | 10.8 (4) |
C2—N3—C4—C5 | −0.2 (2) | C9—N11—N12—O13 | −170.1 (3) |
C2—N3—C4—N41 | −179.9 (2) | C14—N11—N12—O13 | 1.0 (4) |
N3—C4—C5—C6 | 0.1 (2) | C9—N11—C14—H14A | 131.7 |
N3—C4—C5—C7 | −179.6 (2) | C9—N11—C14—H14B | 14.6 |
N41—C4—C5—C6 | 179.9 (2) | C9—N11—C14—C15 | −110.0 (3) |
N41—C4—C5—C7 | 0.2 (3) | N12—N11—C14—H14A | −39.3 |
N3—C4—N41—H41A | −176.3 | N12—N11—C14—H14B | −156.4 |
N3—C4—N41—H41B | 1.6 | N12—N11—C14—C15 | 78.9 (3) |
C5—C4—N41—H41A | 3.9 | N11—C14—C15—H15A | −62.4 |
C5—C4—N41—H41B | −178.1 | N11—C14—C15—H15B | 58.4 |
C4—C5—C6—N1 | 0.0 (3) | N11—C14—C15—Cl16 | 172.0 (2) |
C4—C5—C6—H6 | −179.1 | H14A—C14—C15—H15A | 56.2 |
C7—C5—C6—N1 | 179.7 (2) | H14A—C14—C15—H15B | 177.0 |
C7—C5—C6—H6 | 0.7 | H14A—C14—C15—Cl16 | −69.4 |
C4—C5—C7—H7A | 40.4 | H14B—C14—C15—H15A | 174.6 |
C4—C5—C7—H7B | 155.1 | H14B—C14—C15—H15B | −64.5 |
C4—C5—C7—N8 | −82.0 (2) | H14B—C14—C15—Cl16 | 49.1 |
C6—C5—C7—H7A | −139.3 | | |
Hydrogen-bond geometry (Å, º) top
D—H···A | D—H | H···A | D···A | D—H···A |
N1—H1···Cl1 | 0.90 | 2.19 | 3.058 (3) | 161.5 |
N8—H8···Cl1ii | 0.90 | 2.48 | 3.22 (3) | 139.4 |
N41—H41A···Cl1ii | 0.87 | 2.29 | 3.22 (3) | 160.8 |
Experimental details
Crystal data |
Chemical formula | C9H14ClN6O2+·Cl− |
Mr | 309.16 |
Crystal system, space group | Monoclinic, P21/c |
Temperature (K) | 293 |
a, b, c (Å) | 5.25191 (12), 12.2401 (3), 21.4088 (5) |
β (°) | 93.2353 (7) |
V (Å3) | 1374.05 (6) |
Z | 4 |
Radiation type | Cu Kα1, λ = 1.54056 Å |
µ (mm−1) | 4.35 |
Specimen shape, size (mm) | Cylinder, 10 × 0.7 |
|
Data collection |
Diffractometer | Stoe STADI-P with a linear position-sensitive detector diffractometer |
Specimen mounting | 0.7mm glass capillary |
Data collection mode | Transmission |
Scan method | Step |
2θ values (°) | 2θmin = 2.0 2θmax = 79.99 2θstep = 0.01 |
|
Refinement |
R factors and goodness of fit | Rp = 0.087, Rwp = 0.084, Rexp = 0.061, RBragg = 0.012, χ2 = 1.388 |
No. of data points | 7600 |
No. of parameters | 86 |
No. of restraints | 42 |
H-atom treatment | H-atom parameters not refined |
Hydrogen-bond geometry (Å, º) top
D—H···A | D—H | H···A | D···A | D—H···A |
N1—H1···Cl1 | 0.90 | 2.19 | 3.058 (3) | 161.5 |
N8—H8···Cl1ii | 0.90 | 2.48 | 3.22 (3) | 139.4 |
N41—H41A···Cl1ii | 0.87 | 2.29 | 3.22 (3) | 160.8 |
Comparison of the 13C solid-state and 13C liquid NMR measurements of (I)
and the predicted values topAtom | Experimental values | | Predicted values | | |
| Solid state | In d6-DMSO | Neutral | N1—CH3a | N3—CH3a |
C21 | 21.7 | 21.3 | 24.5 | 19.9 | 23.2 |
C7 | 39.0 | 37.9 | 41.2 | 40.2 | 35.3 |
C14 | 40.1 | 39.9 | 40.3 | 40.3 | 40.3 |
C15 | 42.0 | 40.3 | 38.9 | 38.9 | 38.9 |
C5 | 110.9 | 112.1 | 112.7 | 108.0 | 136.0 |
C6 | 148.4 | 141.8 | 158.9 | 145.6 | 121.2 |
C9 | 153.56 | 153.9 | 153.0 | 153.0 | 153.0 |
C2 | 161.1 | 160.9 | 163.9 | 169.6 | 164.0 |
C4 | 164.7 | 163.5 | 161.4 | 164.0 | 164.5 |
Restraints for bond lengths and angles, and flatten and refined values
from the refinement of (I) topBond | Restraint value | Refined value | Angle | Restraint value | Refined value |
N1—C2 | 1.343 | 1.351 (3) | C2—N1—C6 | 118.4 | 120.7 (2) |
N1—C6 | 1.338 | 1.355 (2) | N1—C2—N3 | 121.6 | 121.4 (2) |
C2—N3 | 1.313 | 1.336 (2) | N1—C2—C21 | 119.2 | 118.8 (2) |
C2—C21 | 1.486 | 1.486 (3) | N3—C2—C21 | 119.2 | 119.8 (2) |
N3—C4 | 1.338 | 1.358 (2) | C2—N3—C4 | 120.0 | 120.1 (2) |
C4—C5 | 1.384 | 1.414 (2) | N3—C4—C5 | 120.0 | 119.3 (1) |
C4—N41 | 1.335 | 1.330 (3) | N3—C4—N41 | 120.0 | 119.4 (2) |
C5—C6 | 1.384 | 1.367 (3) | C5—C4—N41 | 120.0 | 121.3 (2) |
C5—C7 | 1.530 | 1.527 (3) | C4—C5—C6 | 120.0 | 118.9 (1) |
C7—N8 | 1.462 | 1.449 (4) | C4—C5—C7 | 120.0 | 122.8 (1) |
N8—C9 | 1.324 | 1.322 (4) | C6—C5—C7 | 120.0 | 118.3 (2) |
C9—O10 | 1.213 | 1.216 (4) | N1—C6—C5 | 120.0 | 119.6 (2) |
C9—N11 | 1.381 | 1.407 (3) | C5—C7—N8 | 112.8 | 115.4 (2) |
N11—N12 | 1.333 | 1.350 (4) | C7—N8—C9 | 120.6 | 119.5 (2) |
N11—C14 | 1.481 | 1.487 (4) | N8—C9—O10 | 126.6 | 124.9 (3) |
N12—O13 | 1.217 | 1.231 (5) | N8—C9—N11 | 114.5 | 114.3 (2) |
C14—C15 | 1.446 | 1.461 (4) | O10—C9—N11 | 118.9 | 120.8 (3) |
C15—Cl16 | 1.831 | 1.839 (4) | C9—N11—N12 | 118.3 | 118.4 (2) |
| | | C9—N11—C14 | 119.9 | 119.7 (2) |
| | | N12—N11—C14 | 121.8 | 121.3 (2) |
| | | N11—N12—O13 | 114.7 | 116.7 (3) |
| | | N11—C14—C15 | 108.8 | 107.8 (3) |
| | | C14—C15—Cl16 | 106.5 | 108.0 (2) |
| | | | | |
Planar restraint* | | | | | |
N1, C2, C21, N3, C4, C41, C5, C7, C6 | 0.0 | 4.6 (2) | | | |
(*) see TOPAS manual (Coelho, 2007) for formulae. |
The human brain has a special membrane, known as the blood–brain barrier (BBB), that protects it very efficiently from non-self substances. These non-self substances include the vast majority of drugs used in cancer therapy. Drug therapy of brain tumours is thus much more difficult and the range of suitable cytostatics is severely restricted. Every year, most disconcertingly, more than 800 000 new cases in which the central nervous system is afflicted with cancer are registered worldwide. The majority of these cases are concerned with brain tumours, as shown by the latest study by the Robert Koch Institute (Husmann et al., 2010).
A study carried out by Skipper et al. (1961) drew attention to research on the drug class of nitrosoureas. Of all tested cytostatics, a measurable efficiency against brain tumours could only be observed for 1-methyl-1-nitrosourea (the only representative of the nitrosoureas in the aforementioned study). Since then, a range of nitrosourea derivatives have been synthesized and extensively examined (Johnston et al., 1966). Some of them, for example carmustine (or BiCNU or BCNU, from bis-chloroethylnitrosourea) and lomustine (or CeeNU or CCNU, from chloroethylcyclonitrosourea), showed a very high efficacy against tumours and were able to cross the BBB. However, their low solubility in water complicated their application (Kanamaru et al., 1980). In 1972, the Central Research Laboratory, Sankyo Co. Ltd, Tokyo, synthesized further nitrosourea derivatives, including the title nimustine hydrochloride [4-amino-5-({[N-(2-chloroethyl)-N-nitrosocarbamoyl]amino}methyl)-2-methylpyrimidin-1-ium chloride], (I), which represented an improvement, as this derivative is soluble in water and at the same time overcomes the BBB (Nakao et al., 1972). Nimustine hydrochloride is currently marketed under the trade name Nidran (or ACNU, from aminopyrimidinechloroethylnitrosourea). Presently, (I) belongs to the group of standard drugs in the fight against brain tumours, where contemporary therapeutic schemes also include additional radiation and surgical removal of the tumour (Kamiryo et al., 2004).
The exact mechanism of action is currently still under investigation. However, the preliminary findings indicate that DNA is directly attacked by nimustine by alkylation of the guanine blocks at the O6 position to yield O6-(2-chloroethyl)guanine. This alkylation massively affects the function of DNA, because in contrast with guanine, O6-(2-chloroethyl)guanine prefers to build up base pairs with thymine instead of cytosine. The information stored in DNA is altered and thereby successful replication and transcription are prevented. However, the possibility of repairing the alkylated guanine blocks using the body's own enzyme O6-alkylguanine-DNA alkyltransferase still exists. If this process does not happen, or not fast enough, the affected DNA strands may be damaged almost irreversibly. The O6-(2-chloroethyl)guanine formed is unstable and can react via the intramolecularly formed intermediate N1-O6-ethanoguanine with the complementary DNA strand to form 1-(3-cytosinyl)-2-(1-guanosinyl)ethane. As a consequence, both DNA strands are covalently cross-linked. The resulting double-strand breaks are considerably more difficult to repair, thus usually leading to cell death. However, the body's own mechanisms able to repair even these double-strand breaks are also known. These mechanisms are currently under investigation in the hope that, with the knowledge thus accumulated, the efficiency of agents such as nimustine hydrochloride can be increased (Kobayashi et al., 1994; Margison & Santibáñez-Koref, 2002; Batista et al., 2007; Kondo et al., 2010). The expectation is that the elucidation of the exact crystal structure of nimustine hydrochloride, determined from X-ray powder diffraction and solid-state NMR and reported here, will provide additional momentum to the investigation of the mode of action of nitrosourea derivatives as cytostatics and the body's response to them, as well as to the development of new prodrug candidates.
As a first step, thermal gravimetry (TG) experiments were carried out to determine whether the sample of (I) contained solvent or water molecules. TG showed no mass loss or gain up to 426 K, which proves that the investigated sample of (I) contains no solvent or water molecules in the lattice. Weight loss started at 428 K. Additionally, differential scanning calorimetry (DSC) was carried out, which showed a sharp exothermic signal at 428 K resulting from decomposition.
Four possible positions of protonation are available in the molecule of (I) (N1, N3, N8 and N41). H-atom positions are generally difficult to determine from X-ray powder diffraction, although there have been cases when they have been determined successfully (Schmidt et al., 2011), even from laboratory powder data (Bekö et al., 2010). In this instance, the position of protonation could be determined by solid-state NMR spectroscopy using cross-polarization magic-angle spinning (CPMAS). In the 15N CP MAS spectrum, only the hydrogen-carrying N atoms could be detected. Three 15N peaks were observed with respect to liquid ammonia, which were assigned based on their chemical shifts to the –NH2 (N41, 91.55 p.p.m.), –NH (N8, 102.98 p.p.m.) and aromatic NH+ (N1 or N3, 172.79 p.p.m.) moieties. The corresponding values calculated with respect to nitromethane are -288.68, -277.25 and -207.44 p.p.m., considering that the chemical shift of liquid ammonia is -380.23 p.p.m. with respect to nitromethane. In the literature both reference compounds are used. By comparing the 15N chemical shift values with those obtained by Städeli & von Philipsborn (1980) for aminopyrimidines in different protonation states in solution, it can be unambiguously concluded that the chemical shift value of -207.44 p.p.m. for the aromatic N atom certainly indicates a protonated positively charged state. However, this measurement did not allow a decision to be reached on whether the protonation had occurred at N1 or N3. Solution NMR studies on pyridine and pyrimidine derivatives show that protonation of the ring N atom strongly affects the 13C chemical shifts of the adjacent C atoms (Riand et al., 1977; Liu et al., 1991). Thus, it seemed reasonable to use 13C NMR for the determination of the protonation site on the pyrimidine ring. Due to the low natural abundance of 13C isotopes, no correlation experiments could be performed in the solid state. Therefore, resonance assignments were made based on the 13C solution NMR spectrum recorded in d6-DMSO, since the two spectra (solid state and solution) showed very similar spectroscopic patterns and chemical shifts (see Table 2). The 13C signals in d6-DMSO were assigned using heteronuclear single-quantum correlation (HSQC) and heteronuclear multiple-bond correlation (HMBC) experiments.
Through a comparison of the 13C chemical shifts of neutral (in d6-DMSO) and monoprotonated (in TFA) pyrimidine derivatives, Riand et al. (1974) were able to show that the C atoms adjacent to protonated N atoms become more shielded and thus their signals undergo upfield shifts compared with the neutral molecule. These upfield shifts are larger for a C atom attached to H than for one attached to a methyl group. Riand et al. (1974) also determined that, in the case of 4-aminopyrimidines, monoprotonation occurs almost completely on atom N1 para to the amino group. Protonation of N1 in the case of 2-methyl-4-aminopyrimidine has the strongest effect on C6, for which the chemical shift changes from 154.75 to 144.8 p.p.m. The signal of the methyl group at position 2 also undergoes an upfield shift from 25.57 to 21.4 p.p.m. A similar ~10 p.p.m. upfield shift of the C6 resonance was observed for 4-amino-5-methylpyrimidine (from 153.5 to 142.2 p.p.m.) due to protonation at N1. Consequently, the chemical shift of C6 could also be an indicator for the protonation state of N1 in the case of nimustine hydrochloride and its value at 141.8 p.p.m. strongly suggests that N1 is protonated. A prediction of the 13C chemical shifts of the neutral and differently protonated nimustine molecules using the CHEMDRAW Ultra 12.0 software from CambridgeSoft Corporation (Cousins, 2011) provided further substantiation for this finding. Since the software was not able to perform calculations with protonated N atoms, we used the N-methylated form to create the positive charge. The effect of an N-methyl substituent on the 15N, 13C and 1H chemical shifts is very similar to that of an H atom. The results are summarized in the Table 2. The chemical shifts obtained for the N1-methylated derivative show the best agreement with the solid-state NMR data, which again indicates that (I) is protonated at N1.
For the crystal structure determination of nimustine hydrochloride from laboratory X-ray powder diffraction data, the diffractogram was recorded in transmission mode on a Stoe STADI-P diffractometer with a Ge(111) monochromator and a linear position-sensitive detector using Cu Kα1 radiation at 293 K from 2.0 to 79.99° in 2θ. The sample was measured for 52 h in a 0.7 mm capillary in a 2:1 ratio with amorphous SiO2 to minimize preferred orientation, since (I) crystallizes in small tablet-like crystals, as shown in Fig. 1. The Stoe software WinXPOW (Stoe & Cie, 2004) was used for data acquisition.
For indexing and structure solution, the powder pattern was truncated to a real-space resolution of approximately 3.4 Å, which for Cu Kα1 radiation corresponds to the range 3.0–26.5° in 2θ. The background was subtracted with a Bayesian high-pass filter (David & Sivia, 2001). The powder pattern could be indexed without ambiguity with the program DICVOL91 (Boultif & Louër, 1991), resulting in a monoclinic unit cell with V = 1370 Å3. A comparison of the unit-cell volume with Hofmann's volume increments (Hofmann, 2002) led to the estimation that Z = 4. Subsequently, a Pawley refinement (Pawley, 1981) was carried out to extract the integrated intensities and their correlations. The Pawley fit converged with a Pawley χ2 value of 2.29. From the Pawley refinement, the space group was determined to be P21/c using Bayesian statistical analysis (Markvardsen et al., 2001). The crystal structure was solved from the powder pattern in direct space with simulated annealing using the program DASH 3.1 (David et al., 2006). The H atoms were included. The starting molecular geometry was taken from the single-crystal structure of 1-(2-chloroethyl)-3-(trans-4-methylcyclohexyl)-1-nitrosourea, known as semustine (or MeCCNU, from methylchloroethylcyclonitrosourea) [Cambridge Structural Database (CSD; Version 5.32, updated November 2011; Allen, 2002) refcode CEMCNU10; Smith & Camerman, 1978], replacing the 4-methylcyclohexyl group with a 4-amino-2-methylpyrimidin-1-ium-5-yl group, which was constructed with the following bond lengths: C—H = 1.089, C—C alkyl = 1.530, C—C aromatic = 1.384, C—N aromatic = 1.338, C—N amine = 1.468 and N—H amine = 1.015 Å, using the program Mercury (Macrae et al., 2008). Two models were constructed, one with the protonation at N1 and one with it at the N3 position.
The nimustine molecule has seven flexible torsion angles, which were left free during the simulated annealing (rotations around single bonds, except for C—NH2 and C—CH3). In 50 simulated annealing runs, the crystal structure was found approximately ten times for both models. Both models resulted in the same chloride anion position, close to the N1 position, with N1···Cl distances typical for an N—H···Cl hydrogen bond [Reference?]. This confirmed N1 to be protonated, as shown by the NMR experiments, and gave the final indication for further development of this model.
The nimustine molecules in (I) show a zigzag arrangement along the crystallographic c axis. They are bridged by chloride anions via hydrogen bonds formed to the primary amine substituent of the pyrimidine, the secondary amine group of the urea and the protonated N atom of the pyrimidine fragment, leading to a chain-like motif along the crystallographic b axis. The three NH groups form a channel around the chloride anions along the a axis (Fig. 2). Each nimustine molecule is connected to two different chloride anions (Table 1 and Fig. 3). All hydrogen bonds are in the accepted range, as can be seen in Table 1.