7-Nitro-1H-indazole, an inhibitor of nitric oxide synthase

The crystal structure of 7-nitro-1H-indazole, C₇H₅N₃O₂, an inhibitor of nitric oxide synthase, shows the existence of an intramolecular hydrogen bond between an O atom of the nitro group and the NH group of the indazole ring. The crystal packing consists of intermolecular hydrogen bonding and indazole...indazole interactions.

S1. Comment

Nitric oxide (NO) formation by the enzyme nitric oxide synthase (NOS) has been implicated in many neuronal processes, such as regulation of local cerebral blood flow, long-term potentiation, nociception and NMDA receptor-mediated excitotoxicity (Iadecola, 1993; Meller & Gebhart, 1993; Chabrier et al., 1999). The enzyme nitric oxide synthase occurs in three isoforms, an inductible form (iNOS) and two constitutive forms, neuronal (nNOS) and endothelial (eNOS) (Hemmens & Mayer, 1998). All three isoforms catalyse the formation of nitric oxide from L-arginine, O₂ and NADPH, with the production of citrulline, nitric oxide and NADP⁺ (Marletta, 1994). In view of the potential role of NO in various neurological processes, selective inhibitors of nNOS have been used as experimental tools to examine the effect of NO in these systems. Among these compounds, 7-nitro-1H-indazole, (I), has been identified as a potential selective inhibitor of the neuronal NOS activity and is now considered as a very important tool in pharmacological studies. \sch

The affinity of (I) is about 0.9µM for nNOS, about 0.7µM for eNOS and 57µM for iNOS (Moore, Babbedge et al., 1993; Babbedge et al., 1993). However, the precise molecular mechanism of the inhibitory action of (I) remains very unclear. It probably binds to the prosthetic heme moiety, competing with the substrate L-arginine (Moore, Wallace et al., 1993). Therefore, it is most likely that (I) will be bound in the L-arginine binding site, which was identified in the X-ray structure of the complex of iNOS with the L-arginine substrate (Crane et al., 1998). From this structure the important role of Glu 371, close to the heme group, was identified.

In the crystal structure of (I), the O atoms of the nitro group are coplanar with the indazole ring, the angle between the planes of the ring and the nitro group being 3.6 (2)°. This result was expected, but a search of the structures deposited in the Cambridge Structural Database (CSD; release? ref?) indicated that the nitro group attached to the phenyl ring can deviate somewhat from this coplanar arrangement (by as much as 70°; Zinner et al., 1994). Thus one O atom of the nitro group (O11) lies in the proximity of atom H2, which is bound to atom N2 of the indazole ring (Fig. 1). The intramolecular contact distance between O11 and H2 is 2.35 (2) Å, indicating formation of an intramolecular hydrogen bond (Table 1). With this hydrogen bond, the six atoms O11, N10, C9, C1, N2 and H2 form a pseudo-six-membered ring (the r.m.s. deviation of a least-squares plane through these atoms is about 0.021 Å). The observed length of the single bond C9—N10 [1.4427 (16) Å] is shorter than the theoretical length for a C_arom—NO₂ bond of 1.47 Å (Glusker et al., 1994), which indicates the formation of a weak conjugated π-electron system along this bond.

The crystal packing in (I) consists of indazole base pairs made up of two symmetry-equivalent hydrogen bonds, N2—H2···N3, that form across inversion centres (Fig. 2 and Table 1). The H2 atom is therefore implicated in two hydrogen bonds. The coplanar base-pair dimers stack in parallel planes which form columns, with an interplanar spacing of 3.3 Å between the dimers. Two kinds of these columns of stacked dimers are formed by the crystal packing and the difference between them is given by the dimer orientation. The planes of the dimer indazole rings in neighbouring columns are approximately perpendicular to each other, with the shortest C4—H4···Cg1 distance of 3.12 Å (Cg1 is the centroid of the phenyl ring of the indazole at −1/2 − x, 1/2 + y, 3/2 − z). Theoretical simulations show that such an arrangement results in favourable intermolecular attractive forces (Koch & Egert, 1995).

Modelling studies based on the Protein Data Bank (PDB; release? ref?) structure of iNOS complexed with imidazole (Crane et al., 1997), in which we calculated the probable position of (I) in the heme cavity of iNOS, showed that the nitro group can lie in the proximity of Glu 371 and can interact with it through a solvent molecule. In this model, the N3 of the indazole partially covers the Fe ion of the heme.

S2. Experimental

Compound (I) was synthesized according to the procedure of Bartsch & Yang (1984). The compound is poorly soluble. Suitable crystals were obtained by slow evaporation from methanol at room temperature. The diameter of the collimator used for the data collection was 1.3 mm.

Computing details

Data collection: CAD-4-PC Software (Enraf-Nonius, 1992); cell refinement: CAD-4-PC Software; data reduction: JANA98 (Petříček & Dušek, 1998); program(s) used to solve structure: SHELXS97 (Sheldrick, 1990); program(s) used to refine structure: SHELXL97 (Sheldrick, 1997); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: SHELXL97.

Figures

	Fig. 1. The molecular view of (I) showing the atom-labelling scheme. Displacement ellipsoids are shown at 50% probability levels and H atoms are drawn as small circles of arbitrary radii. Dashed lines indicate the intramolecular hydrogen bond.
	Fig. 2. The crystal packing of the molecules of (I) projected onto the bc plane and rotated somewhat about the c axis. H atoms are omitted for clarity. Dashed lines indicate the hydrogen bonds and the distances of the centres of mass of the interacting aromatic rings.

7-Nitro-1H-indazole

Crystal data

C7H5N3O2	Dx = 1.560 Mg m^−3
Mr = 163.14	Melting point: 456 K
Monoclinic, P21/n	Mo Kα radiation, λ = 0.71073 Å
a = 5.0186 (5) Å	Cell parameters from 25 reflections
b = 9.6408 (9) Å	θ = 18–25°
c = 14.5082 (14) Å	µ = 0.12 mm^−1
β = 98.21 (1)°	T = 293 K
V = 694.76 (12) Å^3	Prism, dark yellow
Z = 4	1.0 × 0.4 × 0.1 mm
F(000) = 336

Data collection

Enraf-Nonius CAD4 diffractometer	Rint = 0.019
Radiation source: fine-focus sealed tube	θmax = 30.0°, θmin = 2.5°
Graphite monochromator	h = −7→6
θ/2θ scans	k = 0→13
2088 measured reflections	l = 0→20
2022 independent reflections	3 standard reflections every 60 min
1625 reflections with I > 2σ(I)	intensity decay: 1.8%

Refinement

Refinement on F^2	Secondary atom site location: difference Fourier map
Least-squares matrix: full	Hydrogen site location: difference Fourier map
R[F^2 > 2σ(F^2)] = 0.046	All H-atom parameters refined
wR(F^2) = 0.135	w = 1/[σ^2(Fo^2) + (0.0757P)^2 + 0.1137P] where P = (Fo^2 + 2Fc^2)/3
S = 1.06	(Δ/σ)max = 0.003
2022 reflections	Δρmax = 0.38 e Å^−3
129 parameters	Δρmin = −0.16 e Å^−3
0 restraints	Extinction correction: SHELXL97 (Sheldrick, 1997)
Primary atom site location: structure-invariant direct methods	Extinction coefficient: none

Special details

Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes.

Refinement. Refinement of F² against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F², conventional R-factors R are based on F, with F set to zero for negative F². The threshold expression of F² > σ(F²) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F² are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger.

Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Ų)

	x	y	z	Uiso*/Ueq
C1	−0.0843 (2)	0.83074 (11)	0.92647 (8)	0.0348 (2)
N2	−0.2670 (2)	0.90809 (11)	0.96328 (8)	0.0414 (3)
H2	−0.322 (4)	0.9023 (19)	1.0197 (13)	0.061 (5)*
N3	−0.3765 (2)	1.00794 (12)	0.90277 (9)	0.0478 (3)
C4	−0.2637 (3)	0.99367 (14)	0.82698 (10)	0.0466 (3)
H4	−0.318 (3)	1.0536 (18)	0.7718 (12)	0.056 (4)*
C5	−0.0748 (2)	0.88331 (13)	0.83618 (8)	0.0385 (3)
C6	0.1000 (2)	0.82518 (14)	0.77983 (9)	0.0435 (3)
H6	0.102 (3)	0.8626 (18)	0.7197 (12)	0.056 (4)*
C7	0.2618 (3)	0.71625 (15)	0.81404 (9)	0.0464 (3)
H7	0.387 (3)	0.6743 (17)	0.7755 (11)	0.055 (4)*
C8	0.2546 (3)	0.66365 (14)	0.90299 (10)	0.0437 (3)
H8	0.369 (4)	0.5920 (19)	0.9230 (12)	0.058 (5)*
C9	0.0825 (2)	0.71975 (12)	0.95931 (8)	0.0374 (3)
N10	0.0737 (2)	0.66393 (12)	1.05110 (8)	0.0465 (3)
O11	−0.0915 (3)	0.71310 (13)	1.09620 (8)	0.0669 (3)
O12	0.2288 (2)	0.56971 (13)	1.08004 (8)	0.0642 (3)

Atomic displacement parameters (Ų)

	U^11	U^22	U^33	U^12	U^13	U^23
C1	0.0280 (5)	0.0384 (5)	0.0402 (5)	−0.0036 (4)	0.0120 (4)	−0.0077 (4)
N2	0.0348 (5)	0.0456 (6)	0.0470 (6)	0.0014 (4)	0.0168 (4)	−0.0071 (4)
N3	0.0381 (5)	0.0478 (6)	0.0594 (7)	0.0060 (4)	0.0137 (5)	−0.0041 (5)
C4	0.0394 (6)	0.0490 (7)	0.0526 (7)	0.0042 (5)	0.0102 (5)	0.0020 (6)
C5	0.0323 (5)	0.0434 (6)	0.0415 (6)	−0.0017 (4)	0.0110 (4)	−0.0031 (5)
C6	0.0410 (6)	0.0535 (7)	0.0391 (6)	−0.0035 (5)	0.0158 (5)	−0.0044 (5)
C7	0.0396 (6)	0.0534 (7)	0.0500 (7)	0.0006 (5)	0.0196 (5)	−0.0116 (5)
C8	0.0370 (6)	0.0436 (6)	0.0519 (7)	0.0048 (5)	0.0111 (5)	−0.0063 (5)
C9	0.0344 (5)	0.0396 (6)	0.0394 (5)	−0.0035 (4)	0.0095 (4)	−0.0034 (4)
N10	0.0497 (6)	0.0476 (6)	0.0433 (6)	−0.0042 (5)	0.0099 (4)	−0.0003 (4)
O11	0.0808 (8)	0.0740 (8)	0.0534 (6)	0.0060 (6)	0.0352 (5)	0.0057 (5)
O12	0.0690 (7)	0.0639 (7)	0.0574 (6)	0.0091 (5)	0.0011 (5)	0.0112 (5)

Geometric parameters (Å, º)

C1—N2	1.3499 (13)	C6—C7	1.376 (2)
C1—C9	1.3994 (16)	C7—C8	1.392 (2)
C1—C5	1.4118 (16)	C8—C9	1.3815 (16)
N2—N3	1.3643 (16)	C9—N10	1.4427 (16)
N3—C4	1.3138 (17)	N10—O11	1.2229 (15)
C4—C5	1.4187 (18)	N10—O12	1.2306 (16)
C5—C6	1.3989 (15)
N2—C1—C9	134.01 (11)	C7—C6—C5	118.81 (11)
N2—C1—C5	106.47 (10)	C6—C7—C8	121.36 (11)
C9—C1—C5	119.52 (10)	C9—C8—C7	120.47 (12)
C1—N2—N3	111.67 (10)	C8—C9—C1	119.47 (11)
C4—N3—N2	106.42 (10)	C8—C9—N10	120.26 (12)
N3—C4—C5	111.21 (12)	C1—C9—N10	120.26 (10)
C6—C5—C1	120.36 (11)	O11—N10—O12	123.22 (12)
C6—C5—C4	135.40 (12)	O11—N10—C9	117.49 (12)
C1—C5—C4	104.23 (10)	O12—N10—C9	119.29 (11)

Hydrogen-bond geometry (Å, º)

D—H···A	D—H	H···A	D···A	D—H···A
N2—H2···N3^i	0.902 (18)	2.190 (18)	2.9369 (14)	139.8 (16)
N2—H2···O11	0.902 (18)	2.351 (19)	2.7465 (16)	106.5 (13)

Symmetry code: (i) −x−1, −y+2, −z+2.