가토에서 측뇌실내 4-Aminopyridine의 혈압에 대한 상승효과

임준섭,강선영,백영홍,강삼석,강선영,백영홍,강삼석 대한신경외과학회 1998 Journal of Korean neurosurgical society Vol.27 No.8

어린 흰쥐의 해마 박편에서 Mg-free Medium과 4-Aminopyridine에 의해 유발된 간질양 뇌파에 대한 Lamotrigine의 효과

윤종서(Jong Seo Yoon),이인구(In Goo Lee),최병준(Byung Joon Choi),황경태(Kyung Tai Whang) 대한소아신경학회 2005 대한소아신경학회지 Vol.13 No.2

목 적 : Lamotrigine(LTG)은 최근에 개발된 항경련제 중 하나로 작용 기전에 완전히 밝혀져 있지 않으며, 실험 모델에 따라 항경련 효과를 보이는 적절한 용량 역시 결정되어 있지 않다. 이에 저자는 뇌의 발육이 미성숙한 어린 흰쥐의 해마 박편을 제작하여 Mg-free medium 뇌척수액에서 4-aminopyridine(4-AP)으로 간질양 뇌파를 유발시킨 후 이를 가장 잘 억제할 수 있는 LTG의 농도를 알아보고, 또한 이를 통한 간질양 뇌파의 억제 양상을 관찰함으로써 밝혀진 LTG의 작용 기전 외의 다른 기전의 가능성을 알아보고자 이 실험을 시행하였다. 방 법 : 생후 19-23일 사이의 Sprague-Dawley 흰쥐를 이용하여 대조군(n=12)과 실험군으로 나눈 후 실험군은 LTG 농도에 따라 400(n=9), 800(n=7), 1,000(n=8) µM 투여군으로 분류하였다. 마취 상태에서 뇌를 꺼내어 기본 뇌척수액(NaC1 125 mM; KC1 2.5 mM; NaHPO 2 mM; MgSO 1.25 mM; NaHCO 25 mM; CaC1 mM; glucose 10 mM, pH 7.3-7.4)에 담그고 vibratome으로 400µm 두께의 해마 박편을 만들었다. 대조군은 해마 박편을 Mg-free medium 뇌척수액에 옮겨 놓고 200µM 농도의 4-AP를 첨가한 1시간 후에 CA1 영역의 피라미드 층에서 뇌파를 측정하였고, 실험군은 대조군과 같은 인공 뇌척수액에 각각 400, 800, 1,000 µM의 LTG를 첨가하고 뇌파를 측정하였다. 결 과 : 1) LTG의 간질 간기 뇌파에 대한 효과 : 간질 간기 뇌파는 대조군과 LTG 투여군 모두에서 나타났다. 첫 번째 간질 간기 뇌파가 나타날 때까지의 잠복시간은 대조군에서 52.7±26.9초이었으나 LTG의 농도가 증가함에 따라 점차 길어져 800 µM군에서는 225.0±28.2초, 1,000µM군에서는 322.1±116.4초였다(P<0.05). 간질 간기 뇌파의 지속시간은 대조군에서 64.6±35.6초였으나, LTG 800µM군에서 가장 짧아 39.9±12.6초였다. 2) LTG의 간질 뇌파에 대한 효과:간질 뇌파는 대조군과 LTG 400µM군의 모든 박편에서 나타났으나, LTG의 농도가 증가하면서 발생 빈도가 줄어 800µM군에서는 57.1%, 1,000µM군에서는 12.5%에서만 나타났다. 첫 번째 간질 뇌파가 나타날 때까지의 잠복시간은 대조군에서 142.1±52.6초였으나 400 µM군에서는 304.4±84.5초, 800 µM군에서는 689.8±213.1초였다(각각 P<0.05). 그리고 1,000 µM군에서는 2,071초로 실험군 중 가장 긴 잠복시간을 보였으나 간질 뇌파가 발생한 경우가 1개의 박편 밖에 없어 통계적으로 의의를 구할 수 없었다. 간질 뇌파의 지속시간은 대조군에서 1,534.7±339.3초이었으나 LTG의 농도가 증가함에 따라 점차 짧아져 800 µM군에서는 126.5±76.1초이었고(P<0.05), 1,000 µM군에서는 42초였다. 결 론 : 어린 흰쥐의 해마 박편에서 4-AP와 Mg-free medium에 의해 유발되는 간질양 뇌파를 억제하는 LTG는 800 µM 이상의 농도일 때 가장 효과적인 항경련 효과를 가지고 있었다. 이는 LTG의 기본 약리 기전이 sodium 통로를 억제하는 것이지만 고농도에서는 potassium 통로에도 작용해 흥분성과 억제성 아미노산의 방출을 억제하고, 억제되었던 GABA의 활동을 증가시키기 때문인 것으로 생각된다. Purpose : In order to elucidate the actual mechanism and the optimal concentration of Lamotrigine(LTG) that suppresses epileptiform discharges, we observed epileptiform discharges from hippocampal slices of immature rat in 4-aminopyridine(4-AP) added Mg -free medium of artificial cerebrospinal fluid(aCSF) with various LTG concentrations. Methods : We divided 19-23 day-old Sprague-Dawley rats into 4 groups; control group(n=12) and 3 LTG group depending on the concentrations of LTG such as 400(n=9), 800(n=7), and 1,000(n=8) µM. The rats were anesthetized and their brains were taken, soaked in aCSF(NaC1 125 mM, KC1 2.5 mM, NaHPO 2 mM, MgSO 1.25 mM NaHCO 25 mM, CaC1 2 mM, Glucose 10 mM, pH 7.3-7.4). And then the brains were cut into 400 µm hippocampal slices by a vibratome. The slices of control group were soaked in 200µM 4-AP added Mg-free medium of aCSF for 1 hour, and then extracellular recordings were performed in hippocampal CA1 pyramidal region. The slices of LTG groups were soaked in the solution containing 400, 800, and 1,000µM LTG, then extracellular recordings were performed. Results : Interictal discharges were observed in all the control and the LTG groups. The latency to the first interictal discharges after 4-AP addition was 52.7±26.9 sec in control group, but was 225.0±28.2 sec in 800 µM and 322.1±116.4 sec in 1,000 µM group of LTG(P<0.05). The duration of interictal discharges was 64.6±35.6 sec in control group, but was the shortest in 800µM group of LTG at 39.3±12.6 sec. Ictal discharges were observed in all of control and 400µM group, but the frequency was decreased as the concentration of LTG increases, 57.1% in 800µM, 12.5% in 1,000 µM group. The latency to ictal discharge after 4-AP addition was 142.1±52.6 sec in control group, but increased as the concentration of LTG increases, 304.4±84.5 sec in 400 µM group and 689.8±213.1 sec in 800 µM group(P<0.05). The duration of ictal discharges was 1,534.7±339.3 sec in control group, but decreased as the concentration of LTG increases, it was 126.5±76.1 sec in 800 µM group(P <0.05) and 42 sec in 1,000 µM group. Conclusion : The antiepileptic effects of LTG were most significant when the concentration, inhibiting epileptiform discharges induced by 4-AP and Mg -free medium in hippocampal slices of immature rats, was 800uM or higher. Although the basic pharmacologic mechanism of LTG is the inhibition of sodium channel, it may also work on potassium channel at higher concentrations.

어린 흰쥐의 해마 박편에서 마그네슘을 제거한 매체와 4-Aminopyridine에 의해 유발된 간질파에 대한 Topiramate의 효과

박소현(So Hyun Park),연영흠(Young Heum Yeon),이인구(In Goo Lee),최병준(Byung Joon Choi),김영훈(Young Hoon Kim),정승연(Seung Yun Chung),황경태(Kyung Tai Whang) 대한소아신경학회 2004 대한소아신경학회지 Vol.12 No.2

목적 : Topiramate(TPM)는 가장 최근에 개발된 항경련제 중 하나로 임상에서 난치성의 부분발작 환자 뿐 아니라 전신성 강직-간대 발작 환자에서도 사용되고 있지만 작용 기전이 완전히 밝혀져 있지 않으며 실험 모델에 따라 항경련 효과를 보이는 가장 적절한 용량 역시 결정되어 있지 않다. 이에 저자들은 어린 횐쥐의 해마 박편을 이용하여 마그네슘을 제거한 뇌척수액에서 4-amino-pyridine(AP)으로 유발되는 간질파를 억제할 수 있는 TPM의 농도와 기전을 알아보기 위하여 이 실험을 시행하였다. 방법 : 생후 19-23일 사이의 Sprague-Dawley 흰쥐를 대조군(n=12)과 실험군으로 나눈 후 실험군의 TPM의 농도에 따라 6(n=11), 20(n=7), 60(n=10), 200(n=14) μM군으로 분류하였다. 마취한 후 뇌를 꺼내어 기본 뇌척수액(NaCl 125 mM; KCl 2.5 mM; NaH2PO4 2 mM; MgSO4 1.25 mM; NaHCO3 25 mM; CaCl2 2 mM, Glucose 10 mM, and pH 7.3-7.4)에 담그고 vibratome으로 400μM 두께의 해마 박편을 만들었다. 대조군에서는 해마 박편을 MgSO4를 제거한 기본 뇌척수액에 담그고 200μM 농도가 되도록 4-AP를 첨가한 1시간 후에 CA1 영역의 피라미드 층에서 뇌파를 측정하였고, 실험군은 대조군과 같은 조건을 유지하면서 TPM을 더 첨가하여 각각 6, 20, 60, 200 μM의 TPM 농도가 되도록 하고 뇌파를 측정하였다. 결과 : 1) 간질 간기 뇌파는 대조군 TPM 6 μM군과 20 μM군의 모든 박편에서 나타났지만 TPM의 농도가 점차 증가함에 따라 출현 빈도가 줄어 60μM군에서는 90%, 200 μM군에서는 35.7%에서 나타났고, 뇌파의 진폭은 대조군에 비해 TPM을 투여한 실험군에서 현저히 작았다. 그리고 4-AP 투여 후 첫 번째 간질 간기 뇌파가 나타날 때까지의 잠복시간은 대조군에서의 52.7±7.5초였으나 TPM의 농도가 증가함에 따라 점차 길어져 60 μM군에서는 64.6±10.3초였고, 200 μM군에서는 568±113.1초였다. 또한 간질 간기 뇌파의 지속시간은 대조군에서 64.6±10.3초였고, TPM 60 μM군에서는 141±38.1초로 연장되었다(P<0.05). 2) 간질 뇌파는 대조군과 TPM 6 μM군의 모든 박편에서 나타났으나, TPM의 농도가 증가함에 따라 출현 빈도가 줄어 60 μM군에서는 55.6%, 200 μM군에서는 28.6%에서 나타났고, 뇌파의 진폭은 대조군에 비해 TPM을 투여한 실험군에서 현저히 작았다. 그리고 4-AP 투여 후 첫 번째 간질 뇌파가 나타날 때까지의 잠복시간은 대조군에서는 142.1±15.2초였으나 TPM의 농도가 증가함에 따라 점차 길어져 60 μM군에서는 431.8±157.4초, 200 투여군에서는 627.8±143.5초였다(P<0.05). 또한 간질 뇌파의 지속시간은 대조군에서 1534.7±97.9초였고, 실험군에서는 TPM의 농도가 증가함에 따라 모든 군에서 의의있게 짧아지는 경향을 보였으며, 이중 60 μM군에서 가장 짧아 155.2±65.5초를 보였다(P<0.05). 3) 간질 중첩 상태는 대조군의 58.3%와 TPM 6 μM군의 27.2%에서만 나타났다. 결론 : 어린 흰쥐의 해마 박편에서 마그네슘을 제거한 매체와 4-AP에 의해 유발된 간질파의 빈도와 잠복시간, 지속시간 및 진폭의 크기는 TPM의 농도가 20 μM일 때부터 억제되기 시작하여, 60 μM일 때 가장 큰 효과를 보이고 있었다. 이는 아마도 TPM이 GABAA 수용체에 대한 GABA의 활동을 증가시키면서, 포타슘 통로의 전도성을 강화시키는 기전에 의한 것으로 추측된다. Purpose: Topiramate(TPM), one of the newest antiepileptic drugs, has been prescribed not only to refractory partial seizures but to generalized tonic-clonic seizures. However, its action mechanisms are not well understood and the optimal dose of antiepileptic efficacy in animal seizure models is not determined yet. In order to elucidate the action mechanisms and the optimal concentration of TPM that suppresses epileptic discharges, we observed ictal and interictal discharges from immature rat hippocampal slices in Mg(2+)-free, and 4-aminopyridine(AP) added artificial CSF with various TPM concentrations. Methods: We divided Sprague-Dawley rats of 19 to 23 days old into 5 groups; namely, a control group(n=12) and 4 TPM groups according to the concentration of TPM, 6 (n=11), 20(n=7), 60(n=10), and 200(n=14) micrometer. The rats were anesthetized and their brains were taken, and soaked in artificial CSF(NaCl 125 mM; KCl 2.5 mM; NaH2PO4 2 mM; MgSO4 1.25 mM; NaHCO3 25 mM; CaCl2 2 mM, Glucose 10 mM, and pH 7.3-7.4). Then the brains were cut into 400 micrometer hippocampal slices by a vibratome. The slices of the control group were soaked in 200 micrometer 4-AP added Mg(2+)-free medium for 1 hour, and then extracellular recordings were performed in the hippocampal CA1 pyramidal region. The slices of TPM groups were soaked in solutions containing 6, 20, 60, 200 micrometer TPM, and then extracellular recordings were performed. Results: Interictal discharges were observed in the control group and 6, 20 micrometer groups but the frequency decreased as the concentration of TPM increased:90% in 60 micrometer group, and 35.7% in 200 micrometer group. And the amplitude of TPM groups was much smaller than that of the control group. The latency to the first interictal discharge after 4-AP addition was 52.7±7.5 sec in the control group, 290.2±78 sec in 60 micrometer group, and 568±113.1 sec in 200 micrometer group. Duration of the interictal discharge was 64.6±10.3 sec in the control group, but was prolonged to 141±38.1 sec in 60 micrometer group(P<0.05). Ictal discharges were observed in all of the control and 6 micrometer groups, but the frequency decreased as the concentration of TPM increased:55.6% in 60 micrometer, and 28.6% in 200 micrometer groups. The amplitude of the TPM groups was much smaller than that of the control group. The latency to ictal discharges after 4-AP addition was 141±15.2 sec in the control group, but increased as the concentration of TPM increased:431.8±57.4 sec in 60 micrometer, and 627.8±143.5 sec in 200 micrometer group(P<0.05). The duration of ictal discharges was 1,534.7±97.9 sec in the control group, but decreased as the concentration of TPM increased, the shortest in 60 micrometer group, 155.2±65.5 sec(P<0.05). Status epilepticus was seen in 58.3% of the control and 27.2% of 6 microM groups. Conclusions: TPM suppresses the frequency, latency, and duration of epileptiform discharges induced by Mg(2+)-free, and 4-AP added artificial CSF in immature rat hippocampal slices, starting from 20 micrometer and reaching the maximal effect at over 60 micrometereter. This finding is presumably due to TPM enhancing of GABA receptor currents and/or K+ channel conductance in response to TPM.

Ca^(2+) Influx Mediates Apoptosis Induced by 4-Aminopyridine, a K^(+) Channel Blocker, in HepG2 Human Hepatoblastoma Cells

Kim, Jung-Ae,Kang, Young Shin,Jung, Mi-Wha,Kang, Ga-Hwa,Lee, Sun Hee,Lee, Yong Soo 영남대학교 약품개발연구소 2000 영남대학교 약품개발연구소 연구업적집 Vol.10 No.-

Apoptosis appears to be implicated in the pathogenesis and therapeutic applications of cancer. In this study we investigated the induction of apoptosis by 4-aminopyri-dine(4-AP), a K^(+) channel blocker, and its mechanism in HepG2 human hepatoblastoma cells. 4-AP reduced cell viability and induced DNA fragmentation, a hallmark of apoptosis, in a dose-dependent manner. In addition, 4-AP induced a sustained increase in intracellular Ca^(2+) con-centration, which was completely inhibited by the extra-cellular Ca^(2+) chelation with EGTA. 4-AP also induced Mn^(2+) influx, indicating that the 4-AP-induced increased intracellular Ca^(2+) levels were due to activation of Ca^(2+) influx pathway. 4-AP also depolarized membrane poten-tial that was measured by using di-O-C_(5)(3), a voltage-sensitive fluorescent dye. 4-AP-induced Ca^(2+) influx was significantly inhibited not by voltage-operative Ca^(2+) channel blockers(nifedipine or verapamil), but by flufen-amic acid(FA), a known nonselective cation channel blocker. Quantitative analysis of apoptosis by the flow cytometry revealed that treatment with either FA or BAP-TA, an intracellular Ca^(2+) chelator, significant inhibited the 4-AP-induced apoptosis. Taken together, these re-sults suggest that the observed 4-AP-induced apoptosis in the HepG2 cells may result from Ca^(2+) influx through the activation of voltage-sensitive Ca^(2+)-permeable non-selective cation channels. These results further suggest that membrane potential change by modulation of K^(+) channel activity may be involved in the mechanism of apoptosis in human hepatoma cells.

4-Aminopyridine Inhibits the Large-conductance $Ca^{2+}-activated$ $K^+$ Channel $(BK_{Ca})$ Currents in Rabbit Pulmonary Arterial Smooth Muscle Cells

Bae, Young-Min,Kim, Ae-Ran,Kim, Bo-Kyung,Cho, Sung-Il,Kim, Jung-Hwan,Earm, Yung-E The Korean Society of Pharmacology 2003 The Korean Journal of Physiology & Pharmacology Vol.7 No.1

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance $Ca^{2+}$-activated $K^+$ channel ($BK_{Ca}$) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of $BK_{Ca}$. The $BK_{Ca}$ currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single $BK_{Ca}$ currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit $BK_{Ca}$ currentsin the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent $K^+$ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.

Relaxant Effect of 4-Aminopyridine on the Mesenteric Artery of Rat

Se-Hoon Kim,Tae-Im Lee 대한생리학회-대한약리학회 2000 The Korean Journal of Physiology & Pharmacology Vol.4 No.6

<P> It has been well known that 4-aminopyridine (4-AP) has an excitatory effect on vascular smooth muscle due to causing membrane depolarization by blocking K<SUP>⁢</SUP>-channel. However, we observed that 4-AP had an inhibitory effect on the mesenteric artery of rat. Therefore, we investigated the mechanism of 4-AP-induced vasorelaxation. The mesenteric arcuate artery and its branches were isolated and cut into ring. The ring segment was immersed in HEPES-buffered solution and its isometric tension was measured. 4-AP (0.1∼10 mM) induced a concentration-dependent relaxation, which was unaffected by NO synthase inhibitor, N<SUP>G</SUP>-nitro-L-arginine methylester (100μM) or soluble guanylate cyclase inhibitor, methylene blue (10μM). Glibenclamide (10μM), ATP-sensitive K<SUP>⁢</SUP> channel blocker, did not exert any effect on the 4-AP-induced vasorelaxation. 4-AP relaxed the sustained contraction induced by 100 mM K<SUP>⁢</SUP> or Ca<SUP>2⁢</SUP> ionophore, A23187 (10μM) in a dose-dependent manner. In addition, 4-AP significantly decreased the phasic contractile response to norepinephrine in the absence of extracellular Ca<SUP>2⁢</SUP>. However, 4-AP did not block the <SUP>45</SUP>Ca influx of rat aorta. From the above results, we suggest that 4-AP may not block the Ca<SUP>2⁢</SUP> influx through Ca<SUP>2⁢</SUP>-channel, but act as a nonspecific vasorelaxant in arterial smooth muscle.

Xylazine과 pentobarbital로 마취한 개에서 yohimbine과 4-aminopyridine투여가 위십이지장 통과 시간에 미치는 영향

김동준,최민철,성재기,Kim, Dong-jun,Choi, Min-chul,Sung, Jai-ki 대한수의학회 1990 大韓獸醫學會誌 Vol.30 No.1

The present study was undertaken to determine the effect of xylazine-pentobarbital anesthesia on the gastroduodenal transit time of barium sulfate and whether this condition can be antagonized by yohimbine, 4-aminopyridine and yohimbine+4-aminopyridine in dogs. Xylazine-pentobarbital anesthesia prolonged the gastroduodenal transit time to $121.50{\pm}21.25$ minutes compared with $5.25{\pm}0.90$ minutes of control. Yohimbine and yohimbine+4-aminopyridine reversed $121.50{\pm}21.25$ minutes of transit time of anesthetized dog to $25.25{\pm}6.83$ and $63.25{\pm}15.69$ minutes, respectively. 4-aminopyridine alone, $115.75{\pm}$18.35 minutes, was not effective in reversing the xylazine-pentobarbital-induced prolongation of gastroduodenal transit time. Yohimbine was the most effective for reversal of xylazine-pentobarbital-induced prolongation of gastroduodenal transit time in dogs.

4-Aminopyridine Improves Lower Urinary Tract Symptoms in a Patient With Benign Prostatic Hyperplasia and Downbeat Nystagmus Syndrome

Michael Strupp,Katharina Feil,Stanislavs Bardins,Raphaela Waidelich 대한배뇨장애요실금학회 2014 International Neurourology Journal Vol.18 No.4

Aminopyridines are potassium channel blockers that increase the excitability of nerve cells and axons; therefore, they arewidely used to treat different neurological disorders. Here we present a patient with idiopathic downbeat nystagmus and lowerurinary tract symptoms (LUTS) due to benign prostatic hyperplasia who was treated with the sustained-release form of4-aminopyridine (4-AP). During treatment with 4-AP, the LUTS improved. This improvement was monitored by using uroflowmetryand the International Prostate Symptom Score. A significant improvement of symptoms was observed in relationto the voided volume. This included an improved emptying of the bladder without an increase in residual urine. In animalstudies, both nonselective K+ channel blockade and selective voltage-sensitive potassium blockade by 4-AP resulted in increasedcontraction on rat detrusor strips. To our knowledge, this is the first clinical observation of the mode of action of 4-APin urological symptoms in humans.

Microwave-assisted Synthesis of Mixed Ligand Complexes of Zn(II), Cd(II) and Hg(II) Derived from 4-aminopyridine and Nitrite Ion: Spectral, Thermal and Biological Investigations

Dhaveethu, Karuthakannan,Ramachandramoorthy, Thiagarajan,Thirunavukkarasu, Kandasamy Korean Chemical Society 2013 대한화학회지 Vol.57 No.3

Zn(II), Cd(II) and Hg(II) complexes with a general composition[$M(L)_2(X)_2$], where L=4-aminopyridine (4AP) and $X=NO_2{^-}$ were prepared under microwave irradiation. The metal complexes were characterized by elemental analyses, molar conductance, IR, Far-IR, electronic, NMR ($^1H$, $^{13}C$), XPS spectral and thermal studies. The spectroscopic studies reveal the composition, different modes of bonding, electronic transition, different chemical environment of C and H atoms and the electronic state of the metal atoms. On the basis of the characterization data, tetrahedral geometry is suggested for all the complexes. The free ligand (4-aminopyridine) and their metal complexes were screened against phytopathogenic fungi and bacteria in vitro and the activities were compared.

Synthesis of 3-Amino-1,4-dihydropyridine Derivative via an Intramolecular Rearrangement of 1,4-Dihydropyridine-3-hydroxamate

Suh, Jung-Jin,Hong, You-Hwa,Bae, Myn The Pharmaceutical Society of Korea 1991 Archives of Pharmacal Research Vol.14 No.4

2,6-Dimethyl-4-(3'-nitrophenyl)-3-methoxylaminocarbonyl-1,4-dihydropyridine-5-carboxylic acid methylester, 3b reacted with 2-cyanoethanol or benzylalcohol to give the corresponding cyanoethylurethane compound 6c in 40.6% yield and benzylurethane compound 6d in 32% yield. The cyanoethylurethane 6c was hydrolized in ethanolic NaOH to give 2,6-dimethyl-4-(3'-nitrophenyl)-1,4-dihydropyridine-3-amino-5-carboxylic acid 5-methyl ester. HCl 8 in 64.8% yield. Another acid hydrolysis of benzylurethane 6d gave 2,6-dimethyl-4-(3'-nitrophenyl)-1,4-dihydropyridine-3-amino-5-carboxylic acid 5-methylester. HBr 11 in 54.7% yield.